Early stages of gallstone formation in guinea pig are associated with decreased biliary sensitivity to cholecystokinin.

The purpose of this study was to measure differences in gallbladder sensitivity to cholecystokinin (CCK) in vivo during the early stages of gallstone formation and to correlate these findings to gallbladder CCK receptors. Guinea pigs were placed on either a normal diet or a two-week cholelithogenic diet, after which gallbladder emptying pressure to exogenously administered CCK was measured in vivo, according to the presence or absence of gallstones. At all doses of CCK tested (except 10(-10) mol/kg), the gallbladder response to CCK of guinea pigs that did not develop gallstones (on the cholelithogenic diet) was more sensitive than that of guinea pigs that did develop gallstones. Neither group was different from guinea pigs on a normal diet. In a second experiment, CCK receptors were measured on gallbladder muscularis from guinea pigs after two weeks on the same diet as in the first experiment. Those guinea pigs that did not develop gallstones had greater concentrations of CCK receptors (149 +/- 9 fmol/mg protein) than those that did develop gallstones (70 +/- 23 fmol/mg protein). Neither group was different from normal diet guinea pigs (119 +/- 57 fmol/mg protein). At the time point measured, there were no differences in the lipid chemistry or protein concentrations of gallbladder bile between the guinea pigs on the cholelithogenic diet that did or did not develop gallstones, or those on normal guinea pig chow. We conclude that the early stages of gallstone formation in guinea pigs are associated with decreased gallbladder sensitivity to CCK and that this change may be due to a lower concentration of CCK receptors on the gallbladder smooth muscle.


Response to hepatitis B vaccination by liver transplant candidates.

Liver transplantation (OLTx) is a procedure offered to individuals with advanced liver disease who are expected to live less than a year. Despite improvement in the care of transplant recipients, these patients are exposed to large volumes of blood and, as a result, are at risk to acquire hepatitis. Currently, the only vaccines available for the prevention of hepatitis are those that induce a response to HBsAg. In this study, 144 patients awaiting OLTx and 15 controls were vaccinated three times, once a month, intramuscularly in the deltoid using the Merck Hepatovax plasma-derived vaccine. This schedule was continued regardless of whether or not OLTx occurred before the series was completed. For the 15 controls, the response rate was 93% and for individuals with end-stage liver disease, it ranged from 44 to 54% (P less than 0.004). No difference in the percentage of those developing antibody was detected between groups based upon disease indication or whether the vaccination series was completed before or after OLTx. Of the following: WBC, lymphocytes (percent and number), CD3+ cells (percent and number), CD4+ cells (percent and number), CD8+ cells (percent and number), CD4+/CD8+ ratio, and B cells (percent and number), only the absolute WBC (P less than 0.05) distinguished between those who did and did not develop antibody.(ABSTRACT TRUNCATED AT 250 WORDS)


Hepatitis B vaccine response before and after transplantation in 55 extrahepatic biliary atresia children.

Fifty-five children with cholestatic cirrhosis due to extrahepatic biliary atresia received a course of hepatitis B vaccine. Forty-seven received a plasma-derived vaccine and eight a recombinant vaccine. Antibody determination was evaluated before and after liver transplantation in 30 patients. Twenty-five additional patients had antibody determination after transplantation only. Protection against hepatitis B was observed in 73.3% of the children evaluated prior to transplantation. One to 15 months after transplantation, 54.6% of all children studied showed protective levels of anti HBs. We conclude that hepatitis B vaccination is efficient in inducing immunity in the majority of children with cholestatic cirrhosis. Some patients will loose immunity under immunosuppression, but the protection rate remains higher than reported for patients vaccinated after transplantation.


Reactivation of precore mutant hepatitis B virus leading to fulminant hepatic failure following cytotoxic treatment.

Three hepatitis B virus carriers who were HB(e)Ag negative and having normal liver function developed fulminant hepatitis with evidence of HBV replication following intensive chemotherapy for non-Hodgkin's lymphoma. Each was continuously negative for HB(e)Ag. Analysis of the precore region of HBV isolated from each demonstrated that the HBV of each had a point mutation in the precore region that inhibited the synthesis and the release of hepatitis B(e) antigen. This observation suggests that all HB carriers receiving either immunosuppressive or cytotoxic therapy should be monitored closely even if standard assays suggest that viral replication is not present. Sudden enhanced replication of a HBV mutant as a result of such therapy can be a cause of either very severe hepatitis or occasionally fulminant hepatitis.


Changes in ultrastructure of hepatocytes and liver test results before, during, and after treatment with interferon-beta in patients with HB(e)Ag-positive chronic active hepatitis.

We studied the histological and ultrastructural changes in the liver and alterations in the liver test results before, during, and after treatment with human interferon-beta from five patients with hepatitis B e antigen-positive chronic active hepatitis. A daily dose of 3 x 10(6) to 6 x 10(6) units of interferon-beta was given intravenously for four weeks. The total index of periportal and portal inflammation, intralobular degeneration, and focal necrosis before treatment was decreased significantly six months after treatment (P less than 0.05). Ultrastructurally, the structure of endoplasmic reticulum was irregularly shaped or fragmentally decreased during treatment, but these disappeared six or 12 months after treatment. Glycogen particles diminished greatly during treatment. The alanine aminotransferase concentrations in these patients increased during treatment. Serum albumin and cholinesterase levels decreased significantly at the fourth week of treatment (P less than 0.01) and at the third day (P less than 0.01) to the second week (P less than 0.05) of treatment, respectively. These results suggest that interferon-beta injures endoplasmic reticulum and glycogen areas and damages the cholinesterase activity in the early stage of treatment and protein synthesis in patients with hepatitis B e antigen-positive chronic active hepatitis.


Misoprostol hepatoprotection against ischemia-reperfusion-induced liver injury in the rat.

The hepatoprotective effects of misoprostol, a PGE1 analog, against ischemia-reperfusion liver injury were studied using a rat partial liver ischemia model. Serum ornithine carbamoyltransferase (OCT) and alanine aminotransferase (ALT) levels were determined as biochemical indices of injury. Hepatic cell necrosis was assessed histologically using tetranitroblue tetrazolium (TNBT) and hematoxylin and eosin (H&E) staining. With placebo treatment, 90 min of partial hepatic ischemia followed by 24 hr of reperfusion resulted in increased levels of serum OCT (760 +/- 521 IU/liter) and ALT (4327 +/- 1982 IU/liter), while extensive hepatic necrosis was evident by TNBT and H&E staining. Treatment with two doses of 25 micrograms misoprostol/kg body weight at 1 min before ischemia and 1 min before reperfusion significantly reduced the serum levels of OCT and ALT (207 +/- 189 IU/liter, P less than 0.01 and 2075 +/- 1217 IU/liter, P less than 0.01, respectively) and hepatic necrosis. When a single dose of misoprostol was administered 1 min before reperfusion, similar protective effects were observed. However, when the treatment of misoprostol was delayed to 1 min after reperfusion, significantly less hepatoprotection was seen. Misoprostol exerted no hepatoprotection at all when it was administered at 5 min or later after reperfusion. These results demonstrate that misoprostol partially protects the liver against ischemia-reperfusion injury in the rat. The observation that the protective effect of misoprostol occurs only within the first minute of reperfusion suggests that its mechanism of action involves an early event in reperfusion injury, such as modifying the effects of reactive oxygen metabolites.


Nonessential role of leukotrienes as mediators of acute gastric mucosal injury induced by aspirin in rats.

The present study was designed to determine the role of leukotrienes in aspirin-induced acute gastric mucosal injury in rats. We examined the effects of aspirin, indomethacin, and sodium salicylate on gastric mucosal injury, and on eicosanoid synthesis and content. Aspirin, indomethacin, and acidified salicylate caused significant mucosal injury, while salicylate at pH 7 did not induce significant injury. Aspirin and indomethacin significantly reduced mucosal prostaglandin synthesis and content. No significant changes in mucosal leukotriene C4 synthesis and content were observed. There were no correlations between changes in mucosal leukotriene B4 synthesis and the extent of mucosal injury. We also evaluated the effects of MK-571 (a leukotriene D4 receptor antagonist) and MK-886 (a leukotriene biosynthesis inhibitor) on aspirin-induced gastric mucosal injury. Neither MK-571 nor MK-886 could reduce the mucosal lesions induced by aspirin. These findings suggest that leukotrienes are not involved in aspirin-induced acute gastric mucosal injury in rats.


Acute typhlitis in an immunocompromised host. Report of an unusual case and review of the literature.

A case of acute typhlitis arising in a neutropenic male is described. Light and electron microscopy reveal findings resembling those of an early stage of malakoplakia. The coexistence of these two uncommon lesions suggests a pathogenetic role in the development of septicemia in immunocompromised hosts.


Heterotopic mucinous cystadenoma of the pancreas.

A 46-year-old female who had been experiencing severe diarrhea and marked weight loss underwent exploratory laparotomy because of a mass near the tail of the pancreas noted on CT scan. Pathologic examination revealed a mucinous cystadenoma of the pancreas occurring in heterotopic pancreatic tissue. This is the second reported case of mucinous cystadenoma occurring in heterotopic pancreatic tissue.


Measurement of health status in diabetic patients. Diabetes impact measurement scales.

To develop an instrument to measure health status in adult insulin-dependent (type I) and non-insulin-dependent (type II) diabetic patients. Correlative study to examine psychometric properties of the questionnaire. Test-retest reliability, item-scale correlations, principal-components analysis, correlations with global clinical ratings, and correlations with clinical data extracted from medical records were examined at the diabetes clinics at the University of California, Davis, Medical Center. Patients were volunteer clinic patients able to complete the questionnaire. One hundred thirty patients completed a first administration of the questionnaire, and 52 completed a second administration. Test-retest reliability was satisfactory. Item-scale correlations showed that 40 of 44 questionnaire items were highly correlated with subscale and total scale scores. Principal-components analysis identified one major factor measured by the questionnaire. Cronbach's alpha, a measure of the scales' internal consistency, was of satisfactory magnitude. Global ratings of clinical status by patients and clinicians were highly correlated with scale scores. Correlations of scale scores with clinical data were generally of low magnitude but, where significant, were consistently in the direction hypothesized if the scale truly measures health status or disease impact. The Diabetes Impact Management Scales (DIMS) is an easily administered questionnaire with internal consistency and test-retest reliability. Preliminary correlative analyses support the validity of the instrument as a measure of health status in adult type I and type II diabetic patients. Further work will be necessary to firmly establish the validity of the DIMS and its usefulness in clinical outcomes research.


alpha-Glucosidase inhibition by miglitol in NIDDM patients.

To determine the efficacy of the alpha-glucosidase inhibitor miglitol (BAYm 1099) regarding the starch content of food. Thirty-six non-insulin-dependent diabetes mellitus (NIDDM) subjects were studied in a double-blind randomized study comparing treatment with a single dosage of 100 mg miglitol or placebo and a single-blind crossover comparison of three test meals in which the carbohydrate contained either 30, 50, or 70% starch, and quantities of fat and protein were kept constant. Postprandial blood glucose excursions were reduced by approximately 50% with miglitol after all test meals. In contrast, after miglitol treatment, maximum postprandial serum C-peptide and insulin values reached the same levels as after placebo treatment, although the time to reach these maximum levels was delayed. Free fatty acid values decreased after both miglitol and placebo similarly. Twenty-eight untoward events in 15 patients were reported in the miglitol treatment group and 11 events in 7 patients in the placebo treatment group. Miglitol reduces postprandial blood glucose excursions independent of the starch content of the meal. Because no effects were found on incremental postprandial maximal levels of serum insulin and C-peptide, it may be that miglitol exerts, in addition to a delay of intestinal carbohydrate absorption, extraintestinal effects as well, particularly effects on disposition of glucose or anti-insulin counterregulatory factors.


Prevalence and health-care features of hyperglycemia in semiurban-rural communities in southern Saudi Arabia.

To determine the prevalence of hyperglycemia in semiurban-rural Saudi Arabian communities and to assess some of its important health-care related aspects. A cluster of 12 villages near the city of Abha, the capital of the southern region in Saudi Arabia, with a total population of 2150 (290 families) has been included in this study. The family head was interviewed about diagnosed diabetes among family members. Also, the capillary blood glucose level was measured in subjects greater than or equal to 10 yr (the target population, n = 1419) with a reflectance meter after 2-h fasting after the main midday meal. The 87% of the target population who responded for screening showed a prevalence of 4.6% of diabetes, which was higher among men (5.5%, age adjusted, P less than 0.05) than among women (3.6%). Estimates were based on 57 cases ascertained by positive history of the disease (n = 49) or blood glucose level greater than 11.1 mM (200 mg/dl) without previous diagnosis of diabetes (n = 8). The prevalence of impaired glucose tolerance (blood glucose between 7.8 (140 mg/dl) and 11.0 mM (199 mg/dl) with no history of diabetes) was 3.7%, being higher among women than among men (P less than 0.01). Eighty-eight percent of the previously diagnosed cases were receiving treatment at the time of screening. However, in only 12.2% of those receiving treatment was glucose found to be controlled (level less than 7.8 mM [140 mg/dl]). Diabetes frequency in this Saudi community is relatively high. There is a pressing need to promote public awareness about the disease and to improve the competency of the health-care team for achieving better control and early detection of the disease.


Urinary excretion of IGF-I and growth hormone in children with IDDM.

To compare the urinary output of insulinlike growth factor I (IGF-I) and growth hormone (GH) in prepubertal and pubertal children with insulin-dependent diabetes mellitus (IDDM) versus nondiabetic subjects and to analyze the relationship between the urinary excretion of these peptides and degree of metabolic control. Group 1 included 30 IDDM patients who had had diabetes for 4.9 +/- 0.7 yr and had normal renal function (mean age 11.6 +/- 0.9 yr); group 2 consisted of 31 control subjects (mean age 9.2 +/- 0.6 yr). Sensitive radioimmunoassays were used to measure IGF-I and GH in urine aliquots from 12-h timed overnight collections that had been dialyzed, concentrated 50-fold, and lyophilized. Significantly lower IGF-I and GH outputs per kilogram body weight per 12 h were observed in IDDM subjects compared with control subjects. When data were expressed per kilogram of body weight, no difference was observed between the urinary output of IGF-I and GH between prepubertal and pubertal subjects within group 1 or group 2. The prepubertal children had significantly lower HbA1 than the pubertal population; however, no correlation was found between urinary output of IGF-I or GH and HbA1. A positive correlation was observed between urinary IGF-I and GH (r = 0.85, P less than .001). Patients with long-standing IDDM excrete significantly lower urinary levels of IGF-I and GH compared with normal subjects. Serial measurements of these peptides from onset of IDDM are needed to define whether the changes observed are present at diagnosis or are secondary to duration of disease.


Microalbuminuria in type I diabetic patients. Prevalence and clinical characteristics.

To estimate the prevalence of microalbuminuria, overnight urinary albumin excretion rate (AER) greater than or equal to 30 and less than or equal to 250 micrograms/min, in a large sequential sample of nonhypertensive insulin-dependent (type I) diabetic patients attending hospital diabetic clinics, to identify micro- and normoalbuminuric patients in this sample for subsequent intervention and natural history follow-up studies, and to compare the clinical characteristics of the micro- and normoalbuminuric patients identified. Screening was conducted in two phases. In phase 1, all eligible patients were asked to provide an early morning urine specimen for measurement of albumin concentration and albumin-creatinine ratio. In phase 2, all patients with an albumin concn greater than or equal to 15 mg/L and/or an albumin-creatinine ratio greater than or equal to 3.5 mg/mmol and a random sample of those with an albumin concn less than 15 mg/L and albumin-creatinine ratio less than 3.5 mg/mmol were asked to collect a timed overnight urine specimen for determination of AER. Among 1888 patients (16-60 yr old, diabetes onset less than 40 yr, and duration of diabetes less than 35 yr) who were screened, the prevalence of microalbuminuria was approximately 3.7% (95% confidence interval (CI) 2.7-7.6%). Duration of diabetes was significantly longer in micro- than normoalbuminuric patients (20 vs. 15 yr, respectively; P less than 0.001), and in no patient with microalbuminuria was the duration of diabetes less than 5 yr. Systolic and diastolic blood pressures, higher in micro- than normoalbuminuric patients (132 vs. 122 mmHg, P less than 0.01; 77 vs. 72 mmHg, P less than 0.01), were strongly associated with AER. Microalbuminuria in type I diabetes, which appears to represent an earlier phase in the development of clinical nephropathy, is associated with elevated blood pressure and a longer duration of diabetes.


Glycemic control and peripheral nerve conduction in children and young adults after 5-6 mo of IDDM. Wisconsin Diabetes Registry.

A cohort of people (n = 86) was examined in the first few months after insulin-dependent diabetes mellitus (IDDM) diagnosis to evaluate the effect of hyperglycemia on nerve conduction velocities and latencies. Unselected cases with IDDM, who were 6-29 yr of age, were identified at diagnosis from a large, geographically defined area of southern Wisconsin. Peripheral nerve conduction was measured on a sample from this cohort. Peroneal nerve conduction velocity was significantly inversely related to glycosylated hemoglobin (P less than 0.05, age and height adjusted). All other nerve conduction velocities and latencies (median motor, median sensory, and sural) showed the same tendency, but the associations were not statistically significant. Twenty-four-hour urine C-peptide and duration of diabetes (3-11 mo) were not consistently related to nerve conduction parameters after controlling for age and height. These findings suggest that as early as 5-6 mo after diabetes diagnosis, and at a time frequently characterized by partial remission of IDDM, hyperglycemia has a role in the acute slowing of nerve conduction velocity. Other factors such as residual endogenous insulin production do not appear to influence these early changes.


Impact of associated conditions on glycemic control of NIDDM patients.

To assess the impact of associated conditions (obesity, dyslipidemia, and hypertension) on the glycemic control of non-insulin-dependent diabetes mellitus (NIDDM) patients under home-life conditions. We analyzed the metabolic data of 271 NIDDM patients (89% Mexican American) screened in a population-based survey (the San Antonio Heart Study). Obesity was present in 77% of the patients, hypertension in 23%, hypertriglyceridemia (serum triglycerides greater than 2.9 mM) in 23%, and hypercholesterolemia (serum total cholesterol greater than 6.5 mM) in 14%. Forty percent of the patients had two or more comorbid conditions. With the use of a multiple linear regression model, which was adjusted for age, sex, ethnicity, distribution of body fat (waist-hip ratio), plasma insulin, and treatment (of both diabetes and hypertension), we found that the presence of higher serum triglyceride concentrations was associated with significantly higher plasma glucose levels both in the fasting state (1.4 mM, P less than 0.001) and 2 h after an oral glucose load (1.2 mM, P = 0.003). The presence of obesity, hypertension, or high serum cholesterol levels was not associated with significant changes in glycemic control. When the entire group was stratified by diabetes treatment (untreated n = 89, diet n = 75, oral agents n = 82, insulin n = 25) and after adjusting for age, sex, ethnicity, and waist-hip ratio, only fasting and 2-h plasma glucose and insulin concentrations were significantly different across treatment groups, with diet and oral agents being associated with higher fasting (P less than 0.001) and postglucose (P less than 0.005) plasma glucose levels and lower plasma insulin concentrations (P less than 0.005) compared with newly diagnosed patients. Neither serum lipids nor blood pressure differed across treatment. In NIDDM patients under home-life conditions, higher serum triglycerides are associated with higher fasting and postglucose hyperglycemia regardless of antidiabetic treatment. The presence of obesity, hypertension, or high serum cholesterol levels is not associated with significant changes in glycemic control.


Incidence of IDDM in Western Australia in children aged 0-14 yr from 1985 to 1989.

To document the incidence of insulin-dependent diabetes mellitus (IDDM) in Western Australia in children aged 0-14 yr between 1985 and 1989 and to test for differences in incidence by year of diagnosis, age of diagnosis, and sex. A population-based register that used a primary source of case ascertainment (diabetes clinics at teaching hospitals and direct approach to general practitioners and general physicians) and a secondary source (Western Australian Hospital Morbidity Data System) established numerator data. Denominator data were obtained from the Australian Bureau of Statistics. From 1985 to 1989 inclusive, 235 children in the 0- to 14-yr age-group were diagnosed with IDDM in Western Australia. Case ascertainment was estimated at 99% complete. The mean age-adjusted (developed-world population) annual incidence of IDDM was 13.2 per 100,000 person-yr and there was no evidence of an increasing incidence over the 5 yr. However, girls were more likely than boys to be diagnosed with IDDM in this period (P = 0.006). The incidence of IDDM in Western Australia is in the middle range of IDDM incidence in countries throughout the world. The unexpected finding of an increased incidence of IDDM in girls compared with boys needs to be confirmed in a future study.


Psychosocial state of patients with IDDM prone to recurrent episodes of severe hypoglycemia.

The aim of this study was to investigate the psychosocial situation in patients with insulin-dependent diabetes mellitus (IDDM) with recurrent attacks of severe hypoglycemia (SH). The study group consisted of 17 adult patients with SH and 17 patients matched to the study group with regard to sex, age, and duration of diabetes without severe attacks. The psychosocial situation was measured by means of self-rated questionnaires and an observer's rating scale. Parameters such as social support, life events, type A behavior, neuroticism, and vital exhaustion were not different, although a higher anxiety rating (P less than 0.05) and a lower rating of happiness (P less than 0.01) were found in the SH group. We conclude that the anxiety level is increased and that experienced happiness is decreased in patients prone to recurrent severe hypoglycemia.


Pittsburgh Epidemiology of Diabetes Complications Study. Measuring diabetic neuropathy follow-up study results.

This project evaluated the utility of quantitative sensory techniques in predicting the development of neuropathy for subjects participating in a prospective study. Distal symmetric polyneuropathy was evaluated in 77 insulin-dependent diabetes mellitus individuals via quantitative sensory testing, nerve conduction studies, and clinical examination. Although the specificity and positive predictive value were low for the quantitative sensory techniques as predictors of neuropathy diagnosed on clinical exam approximately 2 yr later, the sensitivity for vibratory thresholds was high (100%). Variability over the 2-yr interval was shown on follow-up testing for each of the objective assessment modalities and it was not explained by differences for potential risk factors measured at baseline. Despite a cross-sectional relationship between the assessment modalities and clinically overt neuropathy at baseline, these follow-up data suggest that the potential for the objective modalities as predictors of clinically diagnosed neuropathy may be limited.


Microalbuminuria associated with diabetic neuropathy.

To test the hypothesis that microalbuminuria may show an independent statistical association with diabetic neuropathy. An observational study of a prospectively identified cohort was conducted at the University Medical Center. The cohort consisted of 78 consecutive diabetic patients who fulfilled the criteria of having diabetes for greater than 10 yr, a normal serum creatinine, urine negative for macroalbuminuria by a commonly used dipstick method, a blood glucose less than 13.8 mM (less than 250 mg/dl), and an HbA1 less than 11% (normal range 5.5-8.5%). Medical record review established the presence of chronic complications of diabetes. Urine albumin level was measured by radioimmunoassay. Albumin concn greater than or equal to 15 mg/L was used as a cutoff value for microalbuminuria. Twenty-five of 78 patients (32%) showed microalbuminuria. Of these, 51% had neuropathy, 39% had retinopathy, 35% arterial hypertension, 17% peripheral vascular disease, and 15% ischemic heart disease. After adjusting for age, sex, and type and duration of diabetes, diabetic neuropathy and hypertension showed a significant association with microalbuminuria. After adjusting for other diabetic complications, diabetic neuropathy showed a significant association with microalbuminuria. Microalbuminuria is independently associated with diabetic neuropathy. This association lends support to the theory of a vascular etiology for diabetic distal symmetrical neuropathy.


Accuracy of home blood glucose monitors.

To determine the accuracy of five home blood glucose monitors (HBGM) in reference to a standard laboratory reference method. The study took place in the laboratory of a 350-bed private acute-care hospital. Subjects were a sample of convenience of 207 diabetic and nondiabetic adult and pediatric patients scheduled for fasting blood work that included a blood glucose test. Venous blood samples were collected for laboratory determination of blood glucose level. A separate sample was collected for testing on two each of five HBGMs: AccuChek II M, Tracer II, ExacTech, Glucometer II with Memory, and One Touch. Multiple regression analysis showed that all 10 monitors could be used to predict laboratory blood glucose values. The monitors with the highest predictabilities were One Touch, Tracer II, and AccuChek II M. Consistency between monitors of the same brand was lowest with One Touch. AccuChek II M had the smallest SD between the 2 monitors used in the study. HBGM can be used to predict actual laboratory values of blood glucose. However, the controlled environment of the study should be considered and patient education made a high priority when recommending monitors.


Somatosensory conduction delay in central and peripheral nervous system of diabetic patients.

Fifty-four diabetic patients with or without clinical evidence of neuropathy and with no clinical evidence of CNS dysfunction were studied by somatosensory-evoked potentials after electrical stimulation of the median nerve at the wrist and recorded from the scalp electrode against a noncephalic reference. Peripheral conduction index, calculated as the distance from the wrist to the C7 spinous process divided by the P9 latency, was significantly decreased (P less than 0.01) in diabetic patients (69.81 +/- 6.47 m/s) compared with 28 age-matched nondiabetic subjects (76.85 +/- 5.65 m/s). The P11-P13 interpeak latency, representative of the transit time from the dorsal column at the level of the sensory input into the cervical cord to the brain stem along the somatosensory pathways (CCT1), and the P13-N19 interpeak latency, representative of the transit time from the brain stem to the somatosensory cortex (CCT2), were significantly increased in diabetic patients (CCT1, 2.51 +/- 0.63 ms; CCT2, 5.76 +/- 0.92 ms) compared with nondiabetic subjects (CCT1, 2.28 +/- 0.36 ms, P less than 0.05; CCT2, 5.18 +/- 0.51 ms, P less than 0.01). We conclude that, in diabetic patients, neurophysiological abnormalities may be present in two distinct parts of the CNS and the peripheral nervous system.


Comparison of combined therapies in treatment of secondary failure to glyburide.

To compare the effectiveness of alternative combined treatments in patients with non-insulin-dependent diabetes mellitus (NIDDM) with secondary failure to sulfonylureas. A crossover study was carried out by randomly assigning 16 NIDDM patients to a combined treatment with the addition of either a single low-dose bedtime injection of 0.2 U/kg body wt NPH insulin or an oral three times a day administration of 1.5 g/day metformin to the previously ineffective glyburide treatment. Both combined therapies significantly (P less than 0.01) reduced fasting plasma glucose (FPG), postprandial plasma glucose (PPPG) and percentage of HbA1. The addition of metformin was more effective than the addition of insulin (P less than 0.01) in improving PPPG in the 8 patients with higher post-glucagon C-peptide levels. In contrast, the efficacy of neither combined therapy was related to patient age, age of diabetes onset, duration of the disease, percentage of ideal body weight, and FPG. The addition of insulin but not metformin caused a significant (P less than 0.01) increase of mean body weight. Neither combined treatment caused changes in serum cholesterol and triglyceride levels. No symptomatic hypoglycemic episode was reported in any of the 16 patients. The addition of bedtime NPH insulin or metformin was effective in improving the glycemic control in most NIDDM patients with secondary failure to glyburide. The combination of metformin and sulfonylurea was more effective in reducing PPPG and did not induce any increase of body weight.


Erythrocyte spermidine levels in IDDM patients.

To evaluate whether erythrocyte levels of polyamines spermidine and spermine (expressed in nmol/ml packed erythrocytes [PRBCs]) are modified in insulin-dependent diabetes mellitus (IDDM) and are associated with the presence of retinopathy or nephropathy. We studied erythrocyte spermidine and spermine levels in 38 IDDM patients with or without persistent microalbuminuria (urinary albumin excretion rate [AER] between 20 and 200 micrograms/min), macroalbuminuria (AER greater than 200 micrograms/min), or retinopathy compared with 60 sex- and age-matched control subjects. Mean +/- SD erythrocyte spermine content was similar in both diabetic (9.7 +/- 5.5 nmol/ml PRBCs) and control (8.8 +/- 3.5 nmol/ml PRBCs) subjects, whereas spermidine was higher in diabetic (19.1 +/- 7.2 nmol/ml PRBCs) than in control (14.5 +/- 4 nmol/ml PRBCs, P = 0.0007) subjects. Moreover, spermidine was significantly higher in the groups with microalbuminuria (n = 11, 22.5 +/- 9.2 nmol/ml PRBCs) and macroalbuminuria (n = 4, 22.2 +/- 5.7 nmol/ml PRBCs) than in both normoalbuminuric (n = 23, 16.9 +/- 5.6 nmol/ml PRBCs) and control (F = 9.78, P = 0.0001) subjects, and correlated with log AER (r = 0.41, P = 0.009). Similarly, proliferative retinopathy was associated with a significant increase in spermidine (n = 5, 20 +/- 7 nmol/ml PRBCs compared with control subjects [P = 0.0009]). Our data suggest that erythrocyte spermidine content is increased in IDDM patients associated with both diabetic nephropathy and advanced retinopathy.


Short-term effect of red wine (consumed during meals) on insulin requirement and glucose tolerance in diabetic patients.

To determine the effect of wine on insulin requirement or glucose tolerance. Five men with insulin-treated diabetes and 10 men with non-insulin-treated diabetes ate the same lunch with the same volume of either water or red wine (2 glasses). Insulin requirement was determined with an artificial pancreas (Biostator). Glucose tolerance was evaluated from the postprandial glycemic level. There was no significant difference in insulin requirement determined with an artificial pancreas in the insulin-treated patients after the two meals (31.5 +/- 4.21 U with water and 31.8 +/- 4.3 U with wine). Glucose tolerance in the non-insulin-treated patients was lower after the meal with wine. Moderate prandial wine consumption has no adverse effect on the glycemic control of diabetic patients. Thus, it appears unnecessary to proscribe the consumption of red wine in moderation with meals to diabetic patients. Wine contains tannins and phytates that can explain its action.


Role of industry representatives in providing assistance to certified diabetes educators.

As diabetes educators have become more frequently involved in decisions regarding medications, equipment, and supplies, industry representatives have increasingly provided various types of assistance. The major objectives of this survey were to determine 1) the types of assistance being provided to certified diabetes educators (CDEs) by industry representatives, 2) whether product recommendations are based on assistance provided, and 3) the types of assistance that CDEs consider appropriate. We developed the survey instrument and it was mailed to every fifth person listed in the 1989 directory of CDEs. Three hundred twenty-five (51%) of the active surveys were returned. Those responding indicated that 1) the types of support provided most frequently by industry representatives include instructional materials, samples, supplies, and equipment for patient care and education; 2) the number of types of support provided is directly related to the number of industry representatives seen on a regular basis and the frequency of interaction; 3) 22% of the educators state that they make product recommendations based on support provided; and 4) the role of the industry reps should be to continue support for patient care and education, to provide more support for public and professional education, and to minimize support for personal items, e.g., gifts or meals. The survey indicates that CDEs are receiving many types of assistance from industry representatives and that they prefer that such assistance be targeted toward patient and professional education.


Vascular endothelial cell antibodies in diabetic patients. Association with diabetic retinopathy.

To determine the incidence of antiendothelial cell antibodies in diabetic patients with and without retinopathy. The study consisted of 70 insulin-dependent diabetic (IDDM) subjects, 36 non-insulin-dependent diabetic (NIDDM) subjects, and 40 nondiabetic control subjects. Blood samples were obtained from diabetic patients and control subjects and patients with background and proliferative retinopathy were identified. Vascular endothelial cell (VEC) antibodies were examined in the sera of 36 NIDDM subjects, 70 IDDM subjects, and 40 nondiabetic control subjects by indirect immunofluorescence. VEC antibodies were present in 5 of 40 (12%) control subjects, 7 of 23 (30%) newly diagnosed IDDM patients, 6 of 17 (35%) IDDM patients without retinopathy, 12 of 18 (67%) IDDM patients with background retinopathy (P less than 0.05), and 9 of 12 (75%) IDDM patients with proliferative retinopathy (P less than 0.01). Three of 13 (23%) NIDDM patients with retinopathy and 6 of 23 (26%) without retinopathy were VEC antibody positive. No associations were observed between the presence of VEC antibodies and either the quality of glycemic control or the duration of diabetes. A significant association between VEC antibodies and large-vessel disease was found in IDDM patients with retinopathy (P less than 0.05). Antibodies directed against vascular endothelial cells may play a role in the development of microvascular, and possibly macrovascular, disease in diabetes.


Relationship between plasma insulin and blood pressure in South African black women in Johannesburg.

To examine the relationship between fasting plasma insulin and blood pressure (BP) in 40 urbanized normotensive South African black women aged 24-60 yr, and to assess the effects of body mass index (BMI) and fasting plasma glucose on BP. The women comprised equal numbers of young nonobese nondiabetic subjects, middle-aged nonobese nondiabetic subjects, middle-aged obese nondiabetic subjects, and middle-aged obese newly diagnosed non-insulin-dependent diabetic subjects. Systolic and diastolic BPs were recorded (in duplicate) after 15 min of recumbency, and fasting plasma glucose and insulin levels were determined thereafter. The data were analyzed by simple and multivariate regression. There was a wide distribution of individual physical and biochemical features. With simple correlations, systolic BP correlated significantly with age, BMI, and fasting glucose but not with insulin. Diastolic BP correlated significantly with all four variables (r = 0.37, P less than 0.05). When adjusted for age, BMI, and glucose, however, the significant correlation between diastolic BP and insulin diminished (r = -0.04). As in other nonwhite communities, plasma insulin does not appear to play a major role in regulating the BP of South African black women.


Sex differences in secondary attack rate of IDDM to siblings of probands through older ages. Pittsburgh Etiology of IDDM Study.

To determine the descriptive epidemiological patterns of the secondary attack rate of insulin-dependent diabetes mellitus (IDDM) among siblings of probands through older ages. A family history analysis was performed on 1774 IDDM probands who were diagnosed or seen within 1 yr of diagnosis at Children's Hospital of Pittsburgh from 1 January 1950 through 31 December 1981. The probands were discharged on insulin and were diagnosed at less than 17 yr of age. The time frame permitted the risk of IDDM for siblings of probands to be calculated over a broad spectrum of age. Risk estimates for the 3966 full natural siblings through 10, 20, and 30 yr of age were 1.6, 4.1, and 6.3%, respectively. Secondary attack rates were equivalent for male and female siblings through 15 yr of age (3%); however, the risk to males increased an additional 4% between 16 and 30 yr of age compared with 2.5% for females (P = 0.01). There was no evidence of an excess sex concordance among affected sibling pairs. Males have a greater secondary attack rate of IDDM at older ages than females. This may be due to an increased exposure to environmental agents among males or protective influences operating among females.


Beneficial effect of low-glycemic index diet in overweight NIDDM subjects.

To determine whether low-glycemic index (GI) diets have clinical utility in overweight patients with non-insulin-dependent diabetes mellitus (NIDDM). Six patients with NIDDM were studied on both high- and low-GI diets of 6-wk duration with metabolic diets with a randomized crossover design. Both diets were of similar composition (57% carbohydrate, 23% fat, and 34 g/day dietary fiber), but the low-GI diet had a GI of 58 compared with 86 for the high-GI diet. Small and similar amounts of weight were lost on both diets: 2.5 kg on high-GI diet and 1.8 kg on low-GI diet. On the low-GI diet, the mean level of serum fructosamine, as an index of overall blood glucose control, was lower than on the high-GI diet by 8% (P less than 0.05), and total serum cholesterol was lower by 7% (P less than 0.01). In overweight patients with NIDDM, reducing diet GI improves overall blood glucose and lipid control.


[Myelodysplastic syndromes. The epidemiological and etiological aspects].

From a total of 18,416 bone-marrow biopsy reports entered into the Düsseldorf Bone Marrow Registry between 1975 and 1990 those of patients diagnosed as having preleukaemia, myeloid dysplasia, panmyelopathy with hypercellular bone marrow, refractory anaemia, sideroblastic anaemia or smoldering leukaemia were reanalyzed together with patient-data. If the diagnosis of myelodysplastic syndrome (MDS) was confirmed, the original blood and bone-marrow smear was re-examined and classified, 584 cases in all (3.2%). During the same period acute myeloid leukaemia had been diagnosed in 506 patients (2.8%). The average annual incidence of MDS in the Düsseldorf area was 3.65 per 100,000 inhabitants. Over 80% of cases occurred from the age of 60 years, while 7% were younger than 50 years. The sex ratio was the same in all subgroups of MDS, except chronic myelomonocytic leukaemia (male:female ratio 1:1.57). 31 patients (5.3%) had received ionizing radiation and/or cytostatic or immunosuppressive treatment for various underlying diseases before MDS had been diagnosed (secondary MDS). Preceding occupational contact with organic solvents could not be excluded with certainty in 12 patients. These data suggest that MDS is a relatively frequent disease of hematopoiesis among the older age groups. The proportion of secondary (treatment-induced) forms is small and does not explain the recently observed increased incidence.


[The solitary pulmonary histoplasmoma].

A 63-year-old man who grows orchids as a hobby, fell ill with weakness and pain in his hips and legs 2 months after his latest trip to South America (Ecuador). The WBC count was 9900/microliters with unremarkable differential count while blood sedimentation rate was raised to 60/100 mm. The chest X-ray demonstrated in the right upper lobe a well-circumscribed coin lesion (3 cm diameter) of soft-tissue density, uncalcified and without cavitation. Computed tomography in addition revealed an enlarged lymph-node at the lower hilar pole, but no mediastinal lymphoma. Bronchoscopy demonstrated narrowing of a subsegmental ostium of the 6th segment on the right. An attempt at transbronchial biopsy failed. As a peripheral bronchial carcinoma was suspected, a posterolateral thoracotomy was performed (4 months after the trip to Ecuador). Rapid histological examination was negative for tumour and the lesion was therefore enucleated. Histologically (Grocott silver staining) a histoplasmoma was diagnosed. Several serum samples were positive for precipitating (M-band) and complement-binding antibodies (titre 12 days preoperatively was 1:16). The postoperative course was without complication. No anti-histoplasma antibodies were demonstrable 1 year postoperatively.


[Toxic myopathy with kidney failure as a colchicine side effect ifn familial Mediterranean fever].

A 24-year-old woman with familial Mediterranean fever (FMF) had for one year been treated with colchicine, 1 mg daily, for repeated bouts of fever, abdominal pain and arthritis. She was also known to have renal amyloidosis. Lately she had developed gastrointestinal symptoms, muscle pain and obvious, predominantly proximal muscular weakness in both legs. The cause of the symptoms was rhabdomyolysis with an increased creatinine activity of 1000 U/l and marked myoglobinuria (1600 micrograms/l), as well as renal failure with normal uric acid and a creatinine clearance of 3 ml/min per 1.73 m2. Serum creatinine concentration was 970 mumol/l, urea 34 mmol/l. Muscle biopsy corresponded to a subacute necrotizing myopathy with vacuole formation, signs typical of toxic damage. Renal biopsy confirmed advanced amyloidosis. The colchicine dose was reduced to 0.5 mg/d. The renal failure responded to conservative treatment. The myopathy symptoms receded within 4 weeks, creatinine clearance rising to 25 ml/min per 1.73 m2. 12 months after reduction of the colchicine dose the patient was without any FMF-related symptoms.


An association between complete and incomplete stress fractures of the humerus in racehorses.

Twenty-one horses had a complete unilateral humeral fracture during race training or racing at a California racetrack during the period 24 February 1990 to 10 July 1991. Fractures occurred approximately equally in left and right limbs, and in males and females. Most fractures occurred during training, and in 2- and 3-year-old horses. Only 5 of 16 Thoroughbred horses with known racing records had previously raced more than once, and their mean time between races was less than the time between their last race and fracture (P = 0.07). Ten of 13 humeri studied further had gross evidence of periosteal callus bridging one portion of the fracture line, indicative of a pre-existing stress fracture.


Clinical and radiographic features, treatment and outcome in 15 horses with fracture of the medial aspect of the patella.

A sagittal fracture of the medial aspect of the patella was identified in 15 horses, 2 of which had been kicked and 12 of which had hit a fixed fence while jumping. Eight horses showed concurrent fragmentation of the base of the patella, and 2 had sustained a concurrent fracture of the distal end of the lateral trochlear ridge of the femur. A cranioproximal-craniodistal oblique radiographic view was essential to identify the site of the medial patellar fracture and to determine its configuration. Fourteen horses were treated by surgical removal of the medial patellar fracture fragment(s). Of 12 horses (83%) without evidence of pre-existing degenerative joint disease, 10 were treated successfully with return to full athletic function.


Microwave thermography: a non-invasive technique for investigation of injury of the superficial digital flexor tendon in the horse.

Microwave thermographs were recorded from 77 normal horses. In 51% the lowest temperature was recorded in the mid-metacarpal region, and in 41% it was in the distal metacarpal region. The mean temperature of the normal limbs ranged from 25.04 to 37.4 degrees C. Maximum temperature differences between symmetrical points in both forelimbs ranged from 0 to 5.33 degrees C and differences in mean limb temperatures between both forelimbs ranged from 0 to 2.91 degrees C. In 48 horses with acute (less than 4 weeks' duration) injury of the superficial digital flexor tendon (SDFT) (36 unilateral, 12 bilateral) and 12 horses with acute injury of the soft tissues of the palmar metacarpal region other than the SDFT (all unilateral) 66% of forelimbs had acute SDFT injury, and 50% of those with other soft tissue injuries, had elevations of the temperature in the mid- or distal metacarpal region. Abnormal values for mean limb temperature, difference in mean limb temperature and maximum temperature difference between locations in opposite forelimbs were detected in 75% of the horses with SDFT injury and in only 16% of the horses with other soft tissue injury. The sensitivity of microwave thermography for the detection of SDFT injury was 81% and the specificity 74%. When 30 horses in National Hunt training were examined weekly for 5 weeks, 2 horses sustained SDFT injury during that period. The microwave thermographs recorded from these 2 horses, at 1 and 2 weeks before the onset of clinical signs, were abnormal. However, 16 horses which did not develop tendon injury also displayed thermographic abnormalities.


Videoendoscopic evaluation of the mare's uterus: I. Findings in normal fertile mares.

The new generation of videoendoscopes uses an electronic, instead of an optical, system for image transmission. Advantages over conventional fibre-optic endoscopes include increased image quality, handling robustness and direct display of the image on a TV monitor for multiple simultaneous viewing. In the present study, hysteroscopy was performed on 14 normal fertile Welsh Pony and Thoroughbred mares at various times during the annual and ovarian breeding cycles. Oestrus was characterised by an oedematous, relaxed cervix lying on the floor of the vagina, diffuse oedema of the endometrium and the occasional accumulation of small amounts of clear secretions in the uterine lumen. In dioestrus, the cervix appeared tight and pale and was located in the centre of the vaginal fornix. The endometrium appeared thinner due to the reduction in interstitial fluid, which allowed the underlying blood vessels to become visible, but it still had a glistening surface as a result of endometrial gland secretions. The appearance of the uterotubal papillae reflected the changes in the cervix, being relaxed, pinkish and oedematous during oestrus but remaining pale, tight and erect in dioestrus (between Days 7 and 12 after ovulation). A flexible polythene cannula was passed into the uterine lumen through the working channel of the endoscope for directed collection of uterine fluid for cytological and bacteriological examinations. Site-directed biopsies of the endometrium were taken, with flexible forceps inserted through the working channel of the endoscope, or with rigid forceps passed through the cervix alongside the endoscope. Both methods enabled accurate visual selection of the sampling area but the rigid forceps were preferred due to the larger size of the piece of endometrium recovered.


Videoendoscopic evaluation of the mare's uterus: II. Findings in subfertile mares.

Videoendoscopy of the reproductive tract was performed in 87 Thoroughbred mares with histories of reduced fertility. During hysteroscopy samples for cytological, microbiological and histological examinations were obtained under visual control. Common findings in these broodmares included: (a) endometrial degeneration, as assessed by an uneven distribution or atrophy of endometrial folds and/or a scarred appearance of the endometrium (49 mares, 56%); (b) endometrial cysts of various sizes and locations within the uterus with the most common location being at the base of the uterine horns (48 mares, 55%); (c) fluid accumulation in the uterine lumen (28 mares, 32%). A few mares had transluminal adhesions (7 mares, 8%) and in 2 mares the adhesions appeared to obstruct one uterine horn completely. A solitary discrete lump was detected in the wall of the uterine body in one mare and the suspicion of it being a leiomyoma was confirmed histologically with the aid of a visually directed biopsy sample. Free intraluminal structures were present in the uterine lumen in 3 mares, including one inspissated blood clot and two suspected remnants of resorbing pregnancies. Flexible biopsy forceps and scissors passed through the working channel of the endoscope were used to sever small thin adhesions, but this method proved inadequate for multiple adhesions or cysts. Solitary endometrial cysts were removed by means of conventional rigid biopsy forceps passed alongside the endoscope, although bleeding from the operation site usually limited this type of intervention.


Videoendoscopic evaluation of the mare's uterus: III. Findings in the pregnant mare.

During a 5-year period 65 Pony and 20 Thoroughbred pregnant mares were subjected to videoendoscopic hysteroscopy from 10 to 266 days of gestation. The aims of these examinations were to 1) observe foetal and placental development in vivo (60 Pony and 10 Thoroughbred mares); 2) eliminate one of unicornuate twin conceptuses (9 Thoroughbred mares); 3) recover embryonic foetal and placental tissues non-surgically for experimental purposes (47 Pony mares); 4) induce focal separation of the placenta in late gestation as an experimental model of placentitis (5 pony mares and 1 Thoroughbred mare). It was possible to view the embryo and all constituent extra-embryonic membranes of the conceptus between Days 10 and 87 of gestation without having to perforate the allantochorion. This allowed study of physiological processes such as the coordinated uterine contractions responsible for conceptus motility between Days 7 and 17, active foetal movements, which began as early as Day 34, and invasion of chorionic girdle cells into the endometrium, which occurred between Days 34 and 38. From Day 90, vision of the foetus was reduced or prevented by the increased thickness of the allantochorion. Transendoscopic recovery of the conceptus was successful in all of 10 mares under 30 days of gestation, whereas only 10 of 18 attempts between Days 30 and 45 produced the conceptus without resorting to uterine lavage after initial rupture of the allantochorion. All 9 attempts to eliminate one of unilateral twin conceptuses were unsuccessful and the technique was abandoned as clinically unsuitable. Nevertheless, 9 of 22 (41%) single conceptuses remained viable after one or more hysteroscopic examinations.(ABSTRACT TRUNCATED AT 250 WORDS)


Skeletal muscle characteristics in young trained and untrained standardbred trotters.

Muscle biopsies were taken from the middle gluteal muscle of 28 Standardbred trotters, 3-4 years of age. The 13 horses in Group T were trained consistently from 18 months of age, whereas the 15 horses in Group UT were not exposed to any systematic training before 3 years of age. Group T horses had a lower percentage of Type IIB fibres (31%) than did Group UT horses (39%). Citrate synthase (CS) activity, representing oxidative capacity, was higher in Group T (72 mmol kg-1 min-1) than in Group UT (47 mmol kg-1 min-1). Biopsies were taken from 4 horses in each group when they were foals and then annually until 3-4 years of age. Results from this study indicate that regular training of Standardbreds from 18 months of age resulted in increased CS activity and a decrease in the percentage of Type IIB fibres. This study shows that training, not growth, is the main factor that induces a high oxidative capacity and a high Type IIA/IIB fibre ratio in muscle of Standardbred trotters.


Milk and water intakes of foals sucking grazing mares.

Intakes of milk and milk nutrients were determined for 8 foals at 11-18 days of age and for 10 foals at 30-44 days and 60-74 days of age while sucking grazing mares. Water intakes (sources other than milk) of the foals were determined at 30-44 days and 60-74 days of age. Five of the 10 mares were fed a protein supplement (24% crude protein) in addition to grazing during the stud season. The protein supplement did not influence foal intakes of milk and milk nutrients, milk composition, weight gains of the mares or the growth rate of the foals. Foal milk intakes increased (P less than 0.05) from 16.9 kg/day at 11-18 days to 18.1 kg/day at 60-74 days of age. The water intakes of the foals increased (P less than 0.01) from 3.9 kg/day at 30-44 days to 5.5 kg/day at 60-74 days of age. Total fluid intakes per kg foal liveweight were 246, 202 and 172 g at 11-18, 30-44 and 60-74 days of age, respectively. For each kg of weight gain, foals consumed 12.8, 15.7 and 16.4 kg milk at 11-18, 30-44 and 60-74 days of age. Stage of lactation had a significant effect on the total solids, lactose and protein content of milk. The fat and gross energy content of milk remained constant.


Treatment of respiratory infections in horses with ceftiofur sodium.

Ceftiofur sodium was evaluated as a therapy for respiratory infections in horses. This cephalosporin antimicrobial was administered intramuscularly every 24 h and at a dose of 2.2 mg/kg (1.0 mg/lb) of body weight. The efficacy of ceftiofur sodium was compared with that of a positive control drug, ampicillin sodium (recommended dose of 6.6 mg/kg [3 mg/lb], given every 12 h). Both treatments were continued for 48 h after clinical symptoms were no longer evident (maximum of 10 days). Fifty-five (55) horses with naturally acquired respiratory infections were included in the study; 28 were treated with ceftiofur and 27 with ampicillin. Clinical improvement was recorded for 92.9% of the patients treated with ceftiofur and 92.6% of the animals receiving ampicillin. Both therapies reduced body temperatures to an afebrile level after 2 days of treatment. Complete recovery/cure was noted for 78.6% of the ceftiofur patients and 59.3% of the horses treated with ampicillin. Supporting variables (depression/malaise, respiration/dyspnoea, nasal discharge) were assessed and these also substantiated the effectiveness of the treatments. Both antibiotics were well tolerated. Neither pain nor swelling were noted at the ceftiofur injection site(s). None of the animals developed diarrhoea. Data from this study indicated that ceftiofur sodium is an effective and safe treatment for respiratory infections in horses.


The incidence of abnormal limb development in the Irish thoroughbred from birth to 18 months.

A two part survey was carried out in Irish Thoroughbred horses in 1988 and 1989 to establish the incidence and prevalence of developmental skeletal problems, particularly possible manifestations of developmental orthopaedic disease (DOD). Survey One was a retrospective study based on a questionnaire involving the foal crops of 46 stud farms for 3 successive seasons; the 1711 animals initially documented represented 10.46% of Irish foal registrations. The second survey involved repeated monitoring of the 1988 foal crop from birth to 18 months of age on 17 stud farms. The 248 foals initially examined represented 4.24% of foal registrations. Treatment for DOD was deemed necessary for 11.3% of the animals in Survey One. Angular limb deformities and physeal dysplasia ("epiphysitis") together constituted 72.9% of the cases treated. The peak incidence of DOD problems occurred between weaning and the end of December. More than half the animals treated (53.9%) recovered completely, ie achieved expected sale value as yearlings, 27.5% of those treated showed incomplete recovery and mild to moderate loss of sale value and the remaining 18.7% were killed or lost much of their sale value. In the second survey, while 67% of animals exhibited some form of DOD, the incidence and severity of problems treated were not significantly different from those of farms in Survey One. Again, physeal dysplasia and angular limb deformity were the predominant clinical conditions and their severity was greatest during the early winter. During the suckling phase colts had a significantly greater incidence of DOD. At all other stages the incidence and severity of DOD was similar in both sexes.(ABSTRACT TRUNCATED AT 250 WORDS)


Radiographic anatomy of soft tissue attachments in the equine metacarpophalangeal and proximal phalangeal region.

The sites of bony attachment of the tendons, ligaments, and fibrous portion of the joint capsules of the equine metacarpophalangeal (fetlock) joint region were determined by gross dissection. These sites were transposed to standard radiographic views of the fetlock joint to yield illustrations that can be used as an aid in the diagnosis of soft tissue pathology from radiographs. Evidence of direct attachment of the common digital extensor tendon to the proximal phalanx was not found. Branches of the superficial digital flexor tendon were found to insert only on the middle phalanx. The recently described sites of insertion of the branches of the superficial digital flexor tendon to the proximal phalanx were found to be sites for attachment of the deep axial palmar ligaments of the proximal interphalangeal joint.


Inhibition of calcium-dependent release of noradrenaline from PC12 cells by botulinum type-A neurotoxin. Long-term effects of the neurotoxin on intact cells.

(a) Clostridium botulinum type-A neurotoxin (BoNTA) inhibited the calcium-dependent release of noradrenaline from PC12 cells in a dose-dependent manner. Under conditions in which intact PC12 cells were incubated with BoNTA for 20 h at 37 degrees C, a neurotoxin concentration of approximately 0.12 +/- 0.03 microM was required to inhibit 50% of the calcium-dependent noradrenaline release. (b) PC12 cells, differentiated in the presence of nerve growth factor for 14 days, showed a similar dose-dependent inhibition of noradrenaline release by BoNTA with unchanged sensitivity. No specific saturable binding of 125I-labelled BoNTA was observed to either differentiated or undifferentiated PC12 cells, suggesting a lack of high-affinity acceptors on the cell surface for the neurotoxin. It is proposed that BoNTA enters PC12 cells either by non-specific binding to the cell membrane or via a low-concentration low-affinity acceptor molecule. (c) A study of the long-term effects of BoNTA on noradrenaline release from PC12 cells showed that the neurotoxin remains active within the growing cells for several days. Noradrenaline release from PC12 cells exposed to BoNTA (0.3 microM) for 24 h was reduced to less than 20% of control values over a subsequent 4-day period. After 8 days, release levels were significantly lower (60-65%) than control values, despite a more than 10-fold increase in the cell mass. (d) Investigation of the subcellular distribution of BoNTA after incubation with PC12 cells for 96 h revealed the bulk of the toxin (94-98%) to be associated with the cell membrane fraction. Of this, 50-80% of the BoNTA was associated with the nuclear and cell debris fraction and 11-25% was recovered in the large-granule-vesicle fraction; the specific binding of the neurotoxin to these membrane fractions was found to be similar. (e) Examination of the form of the cell-associated BoNTA after incubation for 96 h with PC12 cells revealed no evidence of any significant degradation of either neurotoxin subunit. This suggests that the neurotoxin adopts a relatively stable form within the cell. On SDS/PAGE under non-reducing conditions, no trace of protein bands corresponding to either of the BoNTA subunits were observed, suggesting that little or none of the neurotoxin subunits exists in a monomeric form within the cells.(ABSTRACT TRUNCATED AT 400 WORDS)


Identification of the sites in the eukaryotic elongation factor 1 alpha involved in the binding of elongation factor 1 beta and aminoacyl-tRNA.

In this article we report the identification of the sites which are involved in the binding of the GDP-exchange factor EF-1 beta and aminoacyl tRNA to the alpha-subunit of the eukaryotic elongation factor 1 (EF-1) from Artemia. For this purpose the polypeptide chain of EF-1 alpha, having 461 amino acid residues, was proteolytically cleaved into large fragments by distinct proteases. Under well defined conditions, a mixture of two large fragments, free from intact EF-1 alpha and with molecular masses of 37 kDa and 43 kDa, was obtained. The 37-kDa and 43-kDa fragments comprise the residues 129-461 and 69-461, respectively. However, in aqueous solution and under non-denaturing conditions, the mixture still contained a short amino-terminal peptide, encompassing the residues 1-36, that remained tightly bound. The ability of the mixture of the 37+43-kDa fragments, including this amino-terminal peptide 1-36, to bind GDP or to facilitate aminoacyl tRNA binding to salt-washed ribosomes was severely reduced, compared to intact EF-1 alpha. However, both of these complexes were able to bind to the GDP-exchange-stimulating subunit EF-1 beta. A 30-kDa fragment, comprising the residues 1-287, was generated after treatment of the protein with endoproteinase Glu-C. This fragment contained the complete guanine nucleotide binding pocket. Although it was able to bind GDP and to transport aminoacyl tRNA to the ribosome, no affinity towards EF-1 beta was observed. We propose that the guanine-nucleotide-exchange stimulation by EF-1 beta is induced through binding of this factor to the carboxy-terminal part of EF-1 alpha. As a result, a decreased susceptibility towards trypsin of the guanine-nucleotide-binding pocket of EF-1 alpha, especially in the region of its presumed effector loop is induced.


Stimulation of a membrane tyrosine phosphatase activity by somatostatin analogues in rat pancreatic acinar cells.

A phosphoryl protein tyrosine phosphatase (PTPase) activity has been characterized in rat pancreatic acinar membranes using 32P-labeled poly(Glu,Tyr) as substrate. Acinar membranes exhibited a high affinity for the substrate, with an apparent Km of 0.46 microM and an apparent Vmax of 0.9 nmol.mg protein-1.min-1. Acinar membrane PTPase activity displayed specific characteristics of other PTPases; it was inhibited by the inhibitors Zn2+, orthovanadate and by the divalent cations Mn2+ and Mg2+, and was stimulated by the reducing-agent dithiothreitol. It was also inhibited by soybean trypsin inhibitor and stimulated by trypsin. Gel permeation of pancreatic acinar membranes gave a single peak of enzyme activity with an apparent molecular mass of 70 000 Da. Further purification by HPLC on DEAE revealed two peaks of PTPase activity at 120 mM and 180 mM NaCl. These two peaks reacted in a Western-blot procedure with anti-(peptide) serum directed towards conserved domain of PTPase as a common 67-kDa form associated with lower-molecular-mass proteolytic fragments (31-56 kDa). Incubation of pancreatic acini with somatostatin analogues, SMS 201-995 or BIM 23014, resulted in a stimulation of membrane PTPase activity. The stimulation was rapid and transient, with a maximal level reached within 15 min of addition. The two analogs stimulated PTPase activity in a dose-dependent manner with half-maximal activation occurring at 7 pM and 37 pM and maximal activation at 0.1 nM and 0.1-1 nM for SMS 201-995 and BIM 23014, respectively. The stimulated-membrane PTPase activity also eluted at an apparent molecular mass of 70 kDa in gel-permeation chromatography. The two analogs inhibited the binding of [125I-Tyr3]SMS 201-995 to pancreatic acinar membranes with similar relative potencies to that observed on stimulation of PTPase activity. We conclude that pancreatic acinar membranes possess a low-molecular-mass PTPase which is stimulated by somatostatin analogs at concentrations involving activation of membrane somatostatin receptors.


Purification and characterization of a thermostable proteinase isolated from Thermus sp. strain Rt41A.

Thermus sp. strain Rt41A produces an extracellular thermostable alkaline proteinase. The enzyme has a high isoelectric point (10.25-10.5) which can be exploited in purification by using cation-exchange chromatography. The proteinase was purified to homogeneity and has a molecular mass of 32.5 kDa by SDS/PAGE. It is a glycoprotein, containing 0.7% carbohydrate as glucose equivalents, and has four half-cystine residues present as two disulphide bonds. Maximum proteolytic activity was observed at pH 8.0 against azocasein and greater than 75% of this activity was retained in the pH range 7.0-10.0. Substrate inhibition was observed with casein and azocasein. The enzyme was stable in the pH range 5.0-10.0 and maximum activity, in a 10-min assay, was observed at 90 degrees C with 5 mM CaCl2 present. No loss of activity was observed after 24 h at 70 degrees C and the half-lives at 80 degrees C and 90 degrees C were 13.5 h and 20 min, respectively. Removal of Ca2+ reduced the temperature for maximum proteolytic activity against azocasein to 60 degrees C and the half-life at 70 degrees C was 2.85 min. The enzyme was stable at low and high ionic strength and in the presence of denaturing reagents and organic solvents. Rt41A proteinase cleaved a number of synthetic amino acid p-nitrophenol esters, the kinetic data indicating that small aliphatic or aromatic amino acids were the preferred residue at the P1 position. The kinetic data for the hydrolysis of a number of peptide p-nitroanilide substrates are also reported. Primary cleavage of the oxidized insulin B chain occurred at sites where the P1' amino acid was aromatic. Minor cleavage sites (24 h incubation) were for amino acids with aliphatic side chains at the P1' position. The esterase and insulin cleavage data indicate the specificity is similar for both the P1 and P1' sites.


Ribonucleases of diverse specificities in rabbit brain nuclei.

A salt extract of rabbit brain nuclei contains three endoribonucleases, designated RNases Y, A and R, which produce acid-soluble products when incubated at near-neutral pH in the absence of metal ions. RNases Y and A yield products with the monoesterified phosphate at the 3' position, through 2',3'-(cyclic)phosphate intermediates. Oligonucleotides terminating with a 2',3'-(cyclic)phosphate are the end-products of the action of RNase R. Double-stranded substrates are highly resistant to the action of all enzymes. On the basis of limited hydrolysis of end-labelled 5S RNA, the three enzymes differ in their preference for the susceptible phosphodiester bond. Thus, RNase Y hydrolyses preferentially the YpN bond, RNase A the ApN bond and RNase R the RpU bond where R is guanosine in most cases. The advantages and disadvantages of using homopolyribonucleotides and dephosphorylated dinucleotides and trinucleotides in determining various aspects of the specificity of RNases are discussed.


Glycosphingolipids in cestodes. Chemical structures of ceramide monosaccharide, disaccharide, trisaccharide and tetrasaccharide from metacestodes of the fox tapeworm, Taenia crassiceps (Cestoda: Cyclophyllidea).

The presence of glycosphingolipids in the metacestodes of the fox tapeworm, Taenia crassiceps, has been established. The normal-phase TLC pattern of the neutral-fraction glycolipids revealed groups of bands corresponding to homologous components of increasing sugar chain length. The three simplest glycolipid components have been isolated and their chemical constitution determined as being of the neogala series: Gal beta 1Cer, Gal beta 6Gal beta 1Cer and Gal beta 6Gal beta 6Gal beta 1Cer. The ceramide tetrasaccharide fraction has been found to consist of a mixture of neogalatetraosylceramide, as an elongation of the neogala series, Gal beta 6Gal beta 6Gal beta 6Gal beta 1Cer and the component Gal alpha 4Gal beta 6Gal beta 6Gal beta 1Cer (both occurring in approximately equimolar proportions). The long-chain bases of the ceramide monogalactoside, digalactoside, trigalactoside and tetragalactosides contain, as well as small amounts of sphingosine, predominantly dihydrosphingosine/phytosphingosine in the approximate ratios 1.7:1, 1.4:1, 1:1 and 2.3:1, respectively. The major ceramide fatty acids have particularly long chains, with hexacosanoic and octacosanoic acids predominating. Upon reverse-phase TCL, the glycolipid components ceramide monogalactoside, digalactoside and trigalactoside were each separable into five component bands. Parent glycolipid components therefore show component band distributions comparable to one another in being governed by similar ceramide constitutions.


The structure of pneumococcal lipoteichoic acid. Improved preparation, chemical and mass spectrometric studies.

Pneumococcal lipoteichoic acid was extracted and purified by a novel, quick and effective procedure. Structural analysis included methylation, periodate oxidation, Smith degradation, oxidation with CrO3, and fast-atom-bombardment mass spectrometry. Hydrolysis with 48% (by mass) HF and subsequent phase partition yielded the lipid anchor (I), the dephosphorylated repeating unit of the chain (II) and a cleavage product of the latter (III). The proposed structures are: (I) Glc(beta 1----3)AATGal(beta 1----3)Glc(alpha 1----3)acyl2Gro, (II) Glc(beta 1----3)AATGal(alpha 1----4)GalNAc(alpha 1----3)GalNAc(beta 1----1)ribitol and (III) Glc(beta 1----3)AATGal(alpha 1----4)GalNAc(alpha 1----3)GalNAc, where AATGal is 2-acetamido-4-amino-2,4,6-trideoxygalactose, and all sugars are in the pyranose form and belong to the D-series. Alkaline phosphodiester cleavage of lipoteichoic acid, followed by treatment with phosphomonoesterase, resulted in the formation of II and IV, with IV as the prevailing species: [sequence: see text] The linkage between the repeating units was established as phosphodiester bond between ribitol 5-phosphate and position 6 of the glucosyl residue of adjacent units. The chain was shown to be linked to the lipid anchor by a phosphodiester between its ribitol 5-phosphate terminus and position 6 of the non-reducing glucosyl terminus of I. The lipoteichoic acid is polydisperse: the chain length may vary between 2 and 8 repeating units and variations were also observed for the fatty acid composition of the diacylglycerol moiety. Preliminary results suggest that repeating units II and IV are enriched in separate molecular species. All species were associated with Forssman antigenicity, albeit to a various extent when related to the non-phosphocholine phosphorus. Owing to its unique structure, the described macroamphiphile may be classified as atypical lipoteichoic acid.


Sequence analysis of photoaffinity-labelled peptides derived by proteolysis of photosystem-2 reaction centres from thylakoid membranes treated with [14C]azidoatrazine.

Photosystem-2 reaction centres were prepared from pea thylakoid membranes that had been photoaffinity labelled with [14C]-azidoatrazine (2-azido-4-ethylamino-6-isopropylamino-s-triazine), a derivative of the herbicide atrazine which binds to the secondary plastoquinone electron-acceptor site of photosystem 2. SDS/PAGE of the 14C-labelled reaction centres followed by fluorography revealed photoaffinity-labelled proteins of apparent molecular masses 30 kDa and 55 kDa, which corresponded to the D1 polypeptide and to an SDS-stable heterodimer of the D1 and D2 polypeptides, respectively. To obtain sequence information on the site of photoaffinity labelling, an 8-kDa photoaffinity-labelled peptide, generated by proteolysis of the reaction-centre material with trypsin, was isolated and purified to apparent homogeneity using reverse-phase and size-exclusion HPLC techniques. The amino terminus of the photoaffinity-labelled peptide was determined to be Leu-Gly-Met-Arg-Pro-Xaa-Ile-Ala-Val-Ala-Tyr by Edman sequencing. This corresponds to the amino terminus of a predicted tryptic peptide of D1 and confirms that azidoatrazine photolabels the D1 polypeptide of photosystem 2 in the region Leu137-Arg225. Chymotrypsin/trypsin digestion of photoaffinity-labelled reaction centres followed by reverse-phase HPLC was used to isolate a smaller photoaffinity-labelled peptide. On Edman sequencing, Ser-Ala were identified as the first two residues and 14C was released on the third cycle, after which further degradation was blocked. The two potential peptide fragments with Ser-Ala at the amino terminus in the region Leu137-Arg225 are Ser148-Ala-Pro and Ser212-Ala-Met. Proline is an unlikely target for reaction with the nitrene of the photoactivated azidoatrazine, and the data are thus consistent with Met214 as the site of photoaffinity labelling on D1 when thylakoid membranes are illuminated with ultraviolet irradiation in the presence of [14C]azidoatrazine.


Molecular cloning and characterization of the Saccharomyces cerevisiae CYT2 gene encoding cytochrome-c1-heme lyase.

Cytochrome c1, a subunit of the mitochondrial ubiquinol--cytochrome-c reductase, is synthesized on cytosolic ribosomes as a precursor protein of 37 kDa. Maturation to the mature 31-kDa form involves two proteolytic processing steps of the amino-terminal presequence. After removal of the amino-terminal part by the matrix-localized processing peptidase, the carboxy-terminal part of the presequence is cleaved off by an unknown intermembrane space protease. This step depends on covalent linkage of heme to the apoprotein. At least two complementation groups (I and II) can be distinguished among mutants of the yeast Saccharomyces cerevisiae, which are defective in this second proteolytic processing, i.e. they accumulate the intermediate-sized form of cytochrome c1 instead of the mature form. Recently, it was shown that complementation group II defines the structural gene for cytochrome c1 [Sadler, I., Suda, K., Schatz, G., Kaudewitz, F. & Haid, A., (1984) EMBO J. 3, 2137-2143]. We report on the molecular cloning and characterization of the CYT2 gene representing complementation group I. It maps on chromosome XI and encodes a mitochondrial protein of about 26 kDa. Extensive similarity to Neurospora crassa and S. cerevisiae cytochrome-c--heme lyase, as well as the phenotype of cyt2 mutants, strongly suggest that we have identified the gene for cytochrome-c1--heme lyase.


Molecular characterization of a human anti-Rh(D) antibody with a DH segment encoded by a germ-line sequence.

The lambda-light-chain and lambda-heavy-chain variable-region genes of an anti-Rh(D) (Rh, Rhesus; D, heavy-chain diversity region) human monoclonal antibody secreted by lymphocytes transformed by the Epstein-Barr virus have been cloned and sequenced. Sequence comparison of the anti-Rh(D)mAb lambda-chain variable region with those of the other available human lambda chains revealed that it belonged to the human V lambda I (V lambda, variable region of lambda chain) subgroup. The greatest sequence similarity (80%) was observed with that of another anti-Rh antibody lambda-chain directed against the Rh(c) antigen. For the VH (VH, variable region of heavy chain) sequence, the highest similarity (86%) was observed with the germline VHG3 gene which belongs to the VHI subgroup. The expressed DH sequence of the anti-Rh(D) antibody is also of germline origin and complementarity-determining region 3 is thus produced by VH-DH and DH-JH (J, joining region) joining without recombination of multiple DH gene segments.


The effect of amino acid substitution at position 219 of Citrobacter freundii cephalosporinase on extension of its substrate spectrum.

The cephalosporinase of Citrobacter freundii GN346 is a class-C beta-lactamase comprising 361 amino acids. The substitution of the glutamic acid at position 219 in the enzyme by lysine was previously shown to broaden its substrate specificity to unfavorable substrates such as oxyimino cephalosporins [Tsukamoto, K., Ohno, R. & Sawai, T. (1990) J. Bacteriol. 172, 4348-4351]. To investigate the cause of this phenomenon, Glu219 was changed to glutamine, cysteine or tryptophan. All the resultant enzymes showed higher cefuroxime-hydrolytic activities than the wild type, the order of increasing cefuroxime-hydrolytic activity being as follows: Trp greater than Lys greater than Cys greater than Gln greater than Glu. The rate of hydrolysis of cefuroxime by the Trp219 enzyme was approximately 3 x 10(4) times that of the wild-type enzyme. The order of increasing cefuroxime hydrolysis was approximately proportional to the molecular volume of the amino acid substituted and independent of the ionic character of the amino acids. The cysteine residue at position 219 in the Cys219 enzyme allowed its complete reaction with an SH-blocking reagent, 4-chloromercuriphenylsulfonic acid. The modified enzyme with the bulkier residue showed a 45% higher cefuroxime-hydrolytic activity than the untreated enzyme. These results suggested that extension of the substrate spectrum may be attributed to alteration in the configuration of the enzyme around position 219.


The protein of M(r) 21,000 constituting the prosome-like particle of duck erythroblasts is homologous to apoferritin.

In duck erythroblasts, two major populations of untranslated messenger (m) RNP can be separated by sucrose gradient centrifugation in low ionic strength. One of these contains globin mRNA associated to protein factors, among them the prosomes. The other, sedimenting in the 35S zone, contains non-globin mRNA. From this '35S' mRNP, a new RNP particle called the prosome-like particle was isolated and characterized [Akhayat, O., Infante, A. A., Infante, D., Martins de Sa, C., Grossi de Sa, M.-F. & Scherrer, K. (1987) Eur. J. Biochem. 170, 23-33]. The PLP is a multimer of a protein of M(r) 21,000, and contains small RNA species. The particle is tightly associated with repressed mRNA and inhibits in vitro protein synthesis. We show here that the protein of M(r) 21,000, constituting the prosome-like particle, is apoferritin. Different approaches confirm the RNP character of this particle and provide evidence that some of its RNA species are tRNA. The hypothesis is discussed as to whether (apo-)ferritin might serve other functions in addition to iron storage.


The primary structure of the alpha subunit of a starfish guanosine-nucleotide-binding regulatory protein involved in 1-methyladenine-induced oocyte maturation.

Starfish-oocyte maturation induced by 1-methyladenine (MeAde) was inhibited by microinjection of pertussis toxin (PTX). The inhibition appeared to result from PTX-catalyzed ADP-ribosylation of a 39-kDa guanosine-nucleotide-binding regulatory protein (G protein) in the oocyte. These results strongly support the hypothesis that the MeAde-induced signals operate via a membrane receptor and are carried by the PTX-sensitive G protein. When PTX-injected oocytes were treated with dithiothreitol, 85% of them reinitiated meiosis, suggesting that dithiothreitol did not act on the MeAde receptor. We constructed a cDNA library from the immature ovary of starfish, Asterina pectinifera, and screened it with the cDNA of the alpha subunit of an inhibitory rat G protein (Gi-2). A positive cDNA clone contained an open reading frame of 1062 bases which had 74% identity with the rat Gi-2 cDNA. The deduced amino acid sequence was 85% and 89% identical to rat Gi-2 and rat Gi-1, respectively. The alpha subunit of the G protein purified from cortices of starfish oocytes was digested by trypsin and the resulting four peptides were microsequenced. Comparison of these amino acid sequences with the predicted one indicated that the isolated cDNA clone encoded the alpha subunit of the PTX-sensitive G protein in oocytes. The C-terminal sequence, KNNLKDCGLF, was identical to that of Gi, suggesting that the cysteine residue is the site of ADP-ribosylation.


Sequence of the tufA gene encoding elongation factor EF-Tu from Thermus aquaticus and overproduction of the protein in Escherichia coli.

The sequence of the tufA gene from the extreme thermophilic eubacterium Thermus aquaticus EP 00276 was determined. The GC content in third positions of codons is 89.5%, with an unusual predominance of guanosine (60.7%). The derived protein sequence differs from tufA- and tufB-encoded sequences for elongation factor Tu (EF-Tu) of Thermus thermophilus HB8, another member of the genus Thermus, in 10 of the 405 amino acid residues. Three exchanges are located in the additional loop of ten amino acids (182-191). The loop, probably involved in nucleotide binding, is absent in EF-Tu of the mesophile Escherichia coli. Since EF-Tu from E. coli is quite unstable, the protein is well-suited for analyzing molecular changes that lead to thermostabilization. Comparison of the EF-Tu domain I from E. coli and Thermus strains revealed clustered amino acid exchanges in the C-terminal part of the first helix and in adjacent residues of the second loop inferred to interact with the ribosome. Most other exchanges in the guanine nucleotide binding domain are located in loops or nearest vicinity of loops suggesting their importance for thermostability. The T. aquaticus EF-Tu was overproduced in E. coli using the tac expression system. Identity of the recombinant T. aquaticus EF-Tu was verified by Western blot analysis, N-terminal sequencing and GDP binding assays.


Biogenesis of the yeast vacuole (lysosome). Proteinase yscB contributes molecularly and kinetically to vacuolar hydrolase-precursor maturation.

The vacuolar proteinase yscB (PrB) has been implicated in the final maturation of procarboxypeptidase yscY (pro-CpY) to the mature wild-type form CpYb of 61 kDa. In PrB-deficient mutants, only the proteinase yscA processed form CpYa of 62 kDa is found [Mechler, B., Müller, H. & Wolf, D. H. (1987) EMBO J. 6, 2157-2163]. We report now that, akin to CpY, two forms of mature proteinase yscA (PrA) can be distinguished. In PrB-deficient mutant cells, PrAa, migrating at about 43 kDa in SDS/PAGE, is found, whereas PrAb, found in wild-type cells, had the known molecular mass of 42 kDa. In the PrB-deficient strain, pro-PrA and pro-CpY matured only to the higher-molecular-mass forms, PrAa and CpYa, and the maturation of both precursors was slower than in the isogenic wild-type strain. Pulse-labeling experiments indicated that the mature forms, PrAb or CpYb, are generated directly in the PrB-containing wild-type strain in vivo. In vitro experiments showed that PrB is able to trigger maturation of its 42-kDa pro-PrB precursor to mature PrB in the absence of PrA. Mature PrB and its proteolytic activity, however, shows a higher stability in the presence of mature PrA. The data indicate a molecular and kinetic participation of proteinase yscB in vacuolar hydrolase precursor maturation.


Primary structures of ribosomal proteins L3 and L4 from Bacillus stearothermophilus.

Ribosomal proteins L3 and L4 were purified to homogeneity from total protein isolated from the 50S subunit of Bacillus stearothermophilus by reversed-phase high-performance liquid chromatography (RP-HPLC). Amino acid sequences of both proteins were determined by automated N-terminal sequence analysis and sequencing of internal peptides. Using oligonucleotides deduced from the N-terminal region of protein L3 as hybridization probes, a DNA fragment coding for proteins L3, L4 and the N-terminal part of protein L23 has been identified, cloned and sequenced. The organization of the genes is identical to that found in the S10 operon of Escherichia coli. Comparison of the sequences of proteins L3 and L4 with those of other organisms revealed that all proteins of the L3 family are highly conserved. On the other hand, the archaebacterial L4 proteins show no significant sequence similarity to the E. coli L4 protein whereas the L4 protein of B. stearothermophilus is significantly similar to all of the L4 proteins and thus justifies the membership of all the L4 proteins in one protein family. The results are discussed with respect to the phylogenetic relationship between eubacteria, archaebacteria and eukaryotes and possible functional domains of proteins L3 and L4.


alpha 1(VIII)-collagen gene transcripts encode a short-chain collagen polypeptide and are expressed by various epithelial, endothelial and mesenchymal cells in newborn mouse tissues.

Type-VIII collagen is a major constituent of Descemet's membrane and contains two genetically distinct alpha chains, alpha 1(VIII) and alpha 2(VIII). We have previously cloned the human alpha 1(VIII) and alpha 2(VIII) genes and determined that they are located on chromosomes 3 and 1, respectively. Comparison of the alpha 1(VIII) and alpha 2(VIII) genes with the alpha 1(X)-collagen gene showed that the structure of the three genes and the sequence of their collagen polypeptides were strikingly similar. Therefore, we have grouped the three genes in a common subclass of collagens which we have named the short-chain collagens because of the relatively small size of their triple-helical domains. In the present study, we have isolated and characterized a mouse gene fragment encoding the entire mouse alpha 1(VIII)-collagen chain and determined the complete primary structure of the polypeptide chain. The size of mouse alpha 1(VIII)-collagen mRNA, as estimated by Northern-blot analysis, was 4.2 kb, compared with 2.8 kb previously reported for the corresponding rabbit mRNA. By cloning and sequencing of four overlapping cDNA, we demonstrate that this larger size of mouse alpha 1(VIII) mRNA is due to a larger 3' untranslated region in the mouse transcript. Using the gene fragment as a probe, we performed Northern-blot hybridization analysis of RNA prepared from newborn mice and demonstrated that alpha 1(VIII) collagen mRNA is expressed at high levels in the calvarium, eye and skin. In situ hybridization revealed that alpha 1(VIII) RNA is present in skin keratinocytes, corneal epithelial and endothelial cells, lens epithelial cells, as well as mesenchymal cells surrounding cartilage and calvarial bone and in the meninges surrounding the brain.


Purification and characterization of a recombinant murine interleukin-6. Isolation of N- and C-terminally truncated forms.

Murine interleukin-6 (IL-6), when expressed in Escherichia coli using the pUC9 vector, accumulated as insoluble aggregates or 'inclusion bodies'. After selective urea washing of the inclusion bodies, to remove extraneous proteins, murine IL-6 was solubilized with 8 M guanidine hydrochloride and then rapidly purified to homogeneity by gel-permeation chromatography followed by reversed-phase HPLC. It was demonstrated that complete disulfide bond formation in murine IL-6 occurred during the early urea washing/guanidine hydrochloride extraction steps, so no refolding step was required. When fully reduced murine IL-6 was dissolved in 8 M guanidine hydrochloride and allowed to air-oxidize, complete disulfide bond formation, monitored by analytical reversed-phase HPLC, was shown to occur within 13 h at 6 degrees C. About 25 mg pure protein was obtained from 37 g wet cells. This recombinant murine IL-6 had a specific activity in the hybridoma growth factor assay of 2 x 10(8) U/mg, which is equivalent to that of native murine IL-6. During the purification procedure, a number of variant forms of murine IL-6 were isolated and partially characterized. Two of these forms, T1 and T3, were C-terminal deletants of murine IL-6 lacking about 60 and 20 amino acids from the C-terminus, respectively, while the other form, T2, was an N-terminal deletant lacking 37 amino acids from the N-terminus. None of these variant forms of murine IL-6 bound to the murine IL-6 receptor and, consequently, all were inactive in the hybridoma growth factor assay.


Structural and functional characterisation of recombinant human haemoglobin A expressed in Saccharomyces cerevisiae.

Recombinant human HbA, produced by co-expressing alpha-globin and beta-globin chains in the yeast Saccharomyces cerevisiae, has been characterised extensively both physically and functionally. Structural studies using N-terminal sequence analysis, peptide mapping, amino acid composition analysis and electrospray MS demonstrated that the recombinant protein was identical to standard HbA purified from erythrocytes. The functional properties of the recombinant protein were assessed using equilibrium and kinetic measurements of oxygen and carbon monoxide binding. The oxygen-binding studies demonstrated that the yeast-derived HbA behaved as a fully functional, cooperative tetramer (Hill coefficient, 2.9), exhibited a normal Bohr effect and response to phosphate, and displayed a rate of oxygen dissociation identical to that of the native human molecule. The recombinant protein also showed the same characteristics of carbon monoxide combination as the standard protein. These studies demonstrate that yeast provides an ideal system for the production of Hb for structural and functional analysis and a potentially useful source of HbA for formulation into a Hb-based oxygen carrier.


Urokinase binding to laminin-nidogen. Structural requirements and interactions with heparin.

Recently we have shown that heparin and related sulfated polyanions are low-affinity ligands of the kringle domain in the amino-terminal region (ATF) of human urokinase (u-PA), and proposed that this may facilitate loading of u-PA onto its receptor at the focal contacts between adherent cells and their matrix. We have now tested other components of the cell matrix (fibronectin, vitronectin, thrombospondin and laminin-nidogen) for u-PA binding, and found that laminin-nidogen is also a ligand of the u-PA ATF. Direct binding assays and competition binding assays with defined fragments of laminin-nidogen showed that there are u-PA binding sites in fragment E4 of laminin as well as in nidogen. The long-arm terminal domain of laminin (fragment E3), which contains a heparin-binding site, competed for binding of u-PA to immobilised heparin. However nidogen, which does not bind to heparin, also inhibited binding of u-PA to heparin, and this effect was also observed with recombinant nidogen and with a fragment of nidogen lacking the carboxy-terminal domain. Direct binding assays confirmed that u-PA binds to nidogen through a site in the u-PA ATF. We conclude that u-PA binds to laminin-nidogen by interactions involving the ATF region of u-PA, the E4 domain of laminin and the rod or amino-terminal regions of nidogen. Since nidogen is suggested to be an important bridging molecule in the maintenance of the supramolecular organization in basement membranes, the presence of a binding site for u-PA in nidogen indicates a role for plasminogen activation in basement membrane remodelling.


Penicillin-binding protein 2x of Streptococcus pneumoniae. Expression in Escherichia coli and purification of a soluble enzymatically active derivative.

A 2.5-kb DNA fragment including the structural gene coding for the penicillin-binding protein 2x (PBP 2x) of Streptococcus pneumoniae has been cloned into the vector pJDC9 and expressed in Escherichia coli. Mapping of RNA polymerase binding sites by electron microscopy indicated that the pbpX promoter is well recognized by the E. coli enzyme. However, high-level expression occurred mainly under the control of the lac promoter upstream of the pJDC9 multiple cloning site. After induction with isopropyl beta-d-thiogalactopyranoside, PBP 2x was expressed as one of the major cellular proteins. PBP 2x produced in E. coli corresponded to the pneumococcal PBP 2x in terms of electrophoretic mobility, fractionation with the cytoplasmic membrane, and penicillin-binding capacity. Deletion of 30 hydrophobic N-terminal amino acid residues at positions 19-48 resulted in high-level expression of a cytoplasmic, soluble PBP 2x derivative (PBP 2x*) which still retained full beta-lactam-binding activity. A two-step procedure involving dye affinity chromatography was established for obtaining large amounts of highly purified enzymatically active PBP 2x*.


Regulation of the mammalian carbamoyl-phosphate synthetase II by effectors and phosphorylation. Altered affinity for ATP and magnesium ions measured using the ammonia-dependent part reaction.

We have measured the 'core' mammalian carbamoyl-phosphate synthetase II (CPSII) activity, using NH4Cl as the nitrogen-donating substrate and trapping carbamoyl phosphate as urea through its reaction with ammonium ions. When ATP and magnesium ion concentrations are close to those found in the cell, the substrate saturation curves for ammonia and bicarbonate are hyperbolic, giving Km (NH3) values of 166 microM at high ATP concentrations and 26 microM at low ATP concentrations, while the Km (bicarbonate) is 1.4 mM at both ATP concentrations used. These values for the Km (NH3) are lower than previously reported for CPS II, and closer to the values for the mitochondrial counterpart. The Km for ammonia and bicarbonate are not altered by phosphorylation of the multienzyme polypeptide CAD, which contains the first three enzyme activities of pyrimidine biosynthesis. The CPS II activity is lower with an excess of either ATP or magnesium ions, causing the apparently sigmoid dependence of activity upon ATP concentration to be enhanced at low concentrations of free magnesium ions. The feedback inhibitor, UTP, acts by stabilising a state with a low affinity for magnesium ions and for ATP. In the presence of the activator, 5-phosphoribosyl diphosphate (PRibPP), the enzyme has a higher affinity for magnesium ions and thus the ATP dependence of the activity is hyperbolic. Phosphorylation of CAD similarly activates the CPS II enzyme by increasing the affinity for magnesium ions and by pushing the equilibrium away from the low-affinity UTP-stabilised state. Using our improved assay procedure, we observe a very large activation by PRibPP of carbamoylphosphate synthesis at low concentrations of magnesium ions, and we find that unlike UTP, the activator PRibPP is able to act on the phosphorylated enzyme.


Biosynthesis of vitamin B12. Transformation of riboflavin 2H-labeled in the 1'R position of 1'S position into 5,6-dimethylbenzimidazole.

The 5,6-dimethylbenzimidazole moiety of vitamin B12 is formed from riboflavin in aerobic and some aerotolerant bacteria. Thereby C1' of riboflavin is transformed into C2 of the vitamin B12 base. In the present publication a study on this transformation with riboflavin 2H-labeled in the 1'R or 1'S position is described. This study was undertaken in order to find out if one of the two hydrogens at C1' is transferred to C2 of 5,6-dimethylbenzimidazole. The 2H-labeled riboflavin samples were synthesized starting from unlabeled or 1-2H-labeled ribose and 3,4-dimethylaniline yielding N-beta-D-ribopyranosyl-3,4-dimethylaniline. The unlabeled riboside was reduced to N-D-ribityl-3,4-dimethylaniline with sodium cyanoborotrideuteride, the 2H-labeled riboside with sodium cyanoborohydride. The ribityl derivatives were transformed into N-D-ribityl-2-phenylazo-4,5-dimethylaniline, and condensed with barbituric acid to riboflavin. The reduction of the ribosyl compound to the ribityl derivative is only partially stereospecific. Thus the riboflavin synthesized from unlabeled ribose had a 2H ratio of 3/1 (1'R/1'S), the riboflavin obtained from D-[1-2H1]ribose of 1/3 (1'R/1'S). The 2H content in these positions was determined from the 1H-NMR spectra. These spectra showed also that 1 mol 2H/mol riboflavin was present in position 1'. The deuterated riboflavin samples were incubated under aerobic conditions with broken cell preparations of Propionibacterium shermanii. The deuterium content of the 5,6-dimethylbenzimidazole isolated was determined by mass spectrometry and by 1H NMR. These measurements revealed that the hydrogen in the pro-S position at C1' of riboflavin is retained during 5,6-dimethylbenzimidazole formation, and is thus found at C2 of this base.