Influence of dietary soybean trypsin inhibitors and DL-ethionine on sulfur amino acid adequacy of diets for young rats.

Weanling male Wistar rats were fed 20% protein diets based on casein or either of two combinations of soy protein isolate and ground raw soy providing three levels of soybean trypsin inhibitors (SBTI; 0, 448 and 808 mg of trypsin inhibited per 100 g of diet respectively). DL-ethionine was included at three levels (0, 0.05% and 0.10%) with each level of SBTI. After 4, 8 and 12 weeks of ad libitum feeding, diets containing SBTI without DL-ethionine were associated with decreases in weight gain, feed efficiency, serum cholesterol and serum urea nitrogen. Higher levels of triglycerides, glutamate pyruvate transaminase (SGPT) and altered serum free amino acid levels were also found. Increased dietary levels of DL-ethionine also resulted in deficits in growth and feed efficiency, decreased serum cholesterol, increased SGPT and similar alterations in serum free amino acids. Combination of dietary SBTI with DL-ethionine resulted in even greater growth deficits and serum cholesterol decreases as well as increases in SGPT and serum triglycerides and changes in serum free amino acid levels. Methionine deficiency in the young rats fed SBTI and DL-ethionine was indicated by the changes in serum amino acids and growth deficits. Moderation of some effects over the 12 week test period suggested decreased methionine requirements in the older rats.


Preparation nutritional value and acceptability of barley rabadi--an indigenous fermented food of India.

Rabadi fermentation of barley flour-buttermilk mixture (fresh and autoclaved) at 30, 35 and 40 degrees C for 6, 12, 18, 24 and 48 h lowered pH, enhanced titratable acidity and did not change fat and total mineral (Ca, Fe, Cu, Zn, Mn and P) content. Protein content of fermented fresh as well as autoclaved barley flour-buttermilk mixture either decreased or remained unchanges. Rabadi prepared from both types of barley flour at different temperatures and time periods was acceptable; but that which was fermented at 40 degrees C for 48 h was less acceptable in terms of taste.


Changes in ascorbic acid and carbohydrate contents in tomato fruits infected with pathogens.

Tomato fruits Var Romer VFN, inoculated with Fusarium equiseti, F. chlamydosporium, Geotrichum candidum, Acremonium recifei, Aspergilus flavus and A. niger showed decline in ascorbic acid content with days of incubation when compared with control (uninoculated) fruits. The total soluble sugars of the inoculated fruits also showed a reduction trend as the storage period was prolonged, but slight increase was observed on the 6th day after inoculation.


ATP-dependent protease in human placenta.

We have found that human placental mitochondria contain ATP-dependent protease in a soluble form. The molecular weight of the protease have been up to 108,000, which is the same as ATP-dependent protease in bovine adrenal cortex. Since this protease has been distributed among steroid hormone-producing tissues such as testis and adrenal cortex and ATP-dependent protease can degrade cytochrome P-450scc, a key enzyme in steroid hormone biosynthesis, we suggest that the protease may have an important role in the regulation of steroid hormone metabolism.


Cellular localization of metallothionein in human term placenta.

Cellular localization of metallothionein (MT) in placenta may provide information on its function as a metal binding protein. Rabbit antibodies to rat liver MT cross-reacted with human MT and were used to localize MT in human term placenta by avidin-biotin peroxidase technique. Serial sections (5 microns) were cut from paraffin-embedded placentae obtained at term from five normal women and incubated with rabbit antibodies to MT. Normal rabbit serum was used as a negative control. The slides were incubated with biotinylated swine anti-rabbit IgG (linking antibody) then with avidin-biotin horseradish peroxidase complex and developed with diaminobenzidine in hydrogen peroxide (0.03 per cent) substrate. The optimum staining of MT was obtained at a 1:800 antibody dilution. MT was identified in fetal amniotic cells, syncytial trophoblasts and villous interstitial cells, and in maternal decidual cells. The presence of MT at specific cellular sites suggests that it may regulate the transplacental transport of metals such as zinc, copper and cadmium. Since the level of cadmium is lower and that of zinc and copper higher in fetal than in maternal blood, this may suggest that placental MT may restrict cadmium while enhancing zinc and copper transport.


Quantitative description of the elaboration and maturation of villi from 10 weeks of gestation to term.

Stereological methods have been applied to a cross-sectional sample of human placentae collected at 10-41 weeks of gestation in order to provide a quantitative description of the growth and maturation of villi. Random tissue sections were analysed to derive volumes, surface areas, lengths, diameters and membrane thicknesses for villi and their fetal capillaries. Expansion of the total volume and surface area of villi can be explained by a dramatic linear growth of terminal villi which begins at about the middle of the second trimester. Growth of intermediate villi also occurs but to a more limited extent. Linear growth is accompanied by villous maturation which involves increases in the relative volume of capillaries and in villous capillarization coupled with decreases in villous diameter, capillary diameter and harmonic thickness of the villous membrane. These findings confirm that placental growth and development depend greatly on growth and maturation of terminal villi. They do not confirm sinusoidal dilation of fetal vessels as a generalized phenomenon. They also support the contention that changes in effective diffusion distances across the villous membrane have real adaptive significance.


Chorionic gonadotrophin and c-myc expression in growing and growth-inhibited (intermediate) trophoblasts.

FEG-3 cells are a clonal line of human choriocarcinoma and resemble villous cytotrophoblasts which are the stem cells for the syncytiotrophoblast in the placenta. FEG-3 cells synthesize and secrete the alpha subunit of human chorionic gonadotrophin (hCG). Treatment of FEG-3 cells with the chemotherapeutic drug (1 microM) methotrexate (MTX) results in an increase in nuclear diameter. Cell division is blocked and a decrease in c-myc mRNA levels in observed. The effects on cell growth and c-myc mRNA expression are reversible, and cells treated with MTX for 48 h retain their proliferative potential. Assessment of placental hormone gene expression reveals that a member of the human growth hormone gene family is expressed at extremely low levels and is unaffected by MTX treatment. Alpha and beta chorionic gonadotrophin (hCG) levels are increased by MTX treatment, but levels decrease following removal of MTX. In contrast to hCG in FEG-3 cells, non-trophoblastic or ectopic production of alpha hCG in human cervical carcinoma cells is inhibited by MTX treatment. These data indicate that MTX will induce morphological and biochemical changes in FEG-3 cells. They reveal an inverse relationship between c-myc and hCG RNA expression, and suggest different mechanisms govern trophoblast versus non-trophoblast production of alpha hCG.


[A battery of neuropsychological tests for severe dementia. An evaluation study].

All patients with Alzheimer's disease progress, more or less rapidly, to a stage where they can no longer be tested with the standard neuropsychological tools. The Severe Impairment Battery (SIB) is a neuropsychological tool used to evaluate patients with severe dementia. Its maximum score is 133. The purpose of this study was to assess the reliability of a French version of SIB. Fifty-four patients with Alzheimer's disease (mean Mini Mental State examination [MMS] 10.11 +/- 6.38) entered the study. Their mean SIB score was 88.4 +/- 33.3. The patients who were most affected (MMS below 5) were distributed by SIB from 7 to 80 points. An inter-examinator study of 36 patients yielded a correlation coefficient r = 0.9957 (P = 0.0001). In an inter-test study of 17 patients the correlation coefficient was also very high (r = 0.874, P = 0.0001). Thus, SIB seems to be a reliable tool to evaluate cognitive functions in cases of severe dementia. The distribution of scores obtained by SIB in patients with an MMS below 10 demonstrates that even within groups of subjects with severe dementia distinctions can be made between their remaining capabilities. Using SIB in these patients should improve our knowledge of the natural course of the disease and give better correlation between clinical data and neuropathological and neurobiochemical tests on the one hand, and enable us to adjust to their condition the departments where these patients are treated on the other hand.


[Microalbuminuria and left ventricular hypertrophy in essential arterial hypertension. A study in non-diabetic patients].

To evaluate the relationship between urinary albumin excretion and left ventricular hypertrophy in essential hypertension, we studied, cross-sectionally, 64 subjects with essential hypertension and no diabetes. Urinary albumin excretion and Sokolow index correlated significantly (r = 0.483; P = 0.0001). Five subjects were positive for microalbuminuria (> 30 mg/24 h) and Sokolow index (> 35 mm); 43 were negative for both, with a concordance rate of 77 percent (chi-squared test 11.1; P = 0.0009). Stepwise multivariate regression analysis indicated two independent determinants for urinary albumin excretion: Sokolow index (F = 18.29), and diastolic blood pressure (F = 12.23). The relationships between urinary albumin excretion, Sokolow index, and blood pressure were not different in the 18 subjects taking angiotensin I-converting enzyme inhibitors and in the 46 others. The close relationship between urinary albumin excretion and Sokolow index observed in this study suggests that left ventricular hypertrophy due to hypertension may account for the increased cardiovascular mortality observed in non diabetic subjects with microalbuminuria.


[Mitochondrial disorder secondary to inflammation in polymyositis. Two cases].

Two cases of polymyositis were followed using phosphorus nuclear magnetic resonance spectroscopy. The spectra recorded during remission were normal, but those collected from the gastrocnemius muscle during the active phase of the diseases showed an increased inorganic phosphate level or a decreased phosphocreatine content. The intracellular pH was normal. These findings may be related to an impairement in mitochondrial metabolism secondary to the inflammatory process. Moreover, the fact that the abnormalities observed disappeared after treatment suggests that phosphorus NMR spectroscopy could be used as a non-invasive method in the follow-up of polymyositis, but this must be confirmed by further studies.


[Serum ferritin assay. Value and limitations].

There is a good correlation between serum ferritin and the amount of iron stored in the body. Reduced levels of serum ferritin are always found in iron deficiency but elevation of serum ferritin can occur without any iron overload. According to pathological situations, red cell ferritin, glycosylated ferritin or acidic ferritin measurements are useful tests for the differential diagnosis of increased serum ferritin concentration.


[Pathogenic role of hyperinsulinism in macroangiopathy. Epidemiological data].

The hypothesis of the atherogenic role of endogenous insulin was based on a series of epidemiological studies. Several large-scale prospective studies have demonstrated that diabetes constitutes an independent risk factor for cardiovascular disease. However, neither the duration of diabetes nor the blood glucose level appear to be predictive of the incidence of a cardiovascular accident. More recent prospective studies (Finland, Australia, Paris) in non-diabetic men have shown that hyperinsulinemia, while fasting or after glucose stimulation, constitutes a risk factor for fatal myocardial infarction, but they failed to show whether diabetes or the blood glucose level constituted a risk factor for the disease. Cross-sectional studies have provided similar results. Insulin resistance affects the majority of non-insulin-dependent diabetics and glucose-intolerant patients. It has also been observed in 25 percent of non-obese subjects with a normal glucose tolerance test. Associated hyperinsulinemia prevents the development of diabetes, but diabetes appears when the beta-cell function is altered and can no longer maintain this hyperinsulinemia. However, hyperinsulinemia is not devoid of cardiovascular consequences. Insulin resistance and hyperinsulinemia are also observed in patients with essential hypertension: a correlation between plasma insulin and blood pressure has been reported. These data, together with other experimental arguments, suggest that excessive endogenous insulin may participate in the rise in blood pressure. Furthermore, hypertensive patients have a high risk of coronary heart disease and this risk is not significantly decreased by anti-hypertensive treatments. This is probably related to the presence of other metabolic risk factors associated with insulin resistance: hyperinsulinemia, glucose intolerance, hypertriglyceridemia, decreased HDL cholesterol. These metabolic disorders have been grouped together under the term "syndrome X". All of these risk factors are probably also involved in the development of coronary heart disease in general population. In conclusion, epidemiological studies now suggest that insulin resistance and hyperinsulinemia increase the risk of hypertension and coronary heart disease. A great many experimental studies support this hypothesis. Lastly, it can be proposed that the increased cardiovascular risk in non-insulin-dependent diabetics is related to the fact that they belong to a larger group of insulin-resistant subjects. The management of diabetes, hypertension, and all of the metabolic abnormalities would appear to be the only way of reducing the incidence of cardiovascular disease.


[Arterial hypertension, hyperinsulinism and insulin resistance].

The incidence of hypertension is increased in obesity, a state associated with an insulin resistance syndrome. By using an euglycemic clamp method, Ferrannini et al. demonstrated the existence of an insulin resistance state in patients with essential hypertension. However, the body mass index of the subjects studied appeared to be slightly excessive. This abnormality has not been observed in patients with secondary hypertension. Insulin resistance is probably localized to peripheral tissues such as muscles and may be associated with other cellular abnormalities. Can insulin resistance, characterized by a raised circulating insulin concentration in the presence of normal blood glucose, be responsible for certain "modifications" associated with essential hypertension? Insulin induces sodium retention and increases the aldosterone-secreting effect of angiotensin II. These effects are likely to promote a rise in blood pressure and an increase in the sensitivity of vessels to endogenous substances. Moreover, insulin is a known growth factor and is involved in lipoprotein metabolism. If insulin resistance plays an important role in the maintenance of complications of essential hypertension, it is important that the treatments used tend to correct this anomaly. Thiazide diuretics and beta-blockers aggravate insulin resistance while angiotensin converting-enzyme inhibitors correct this condition.


[Theoretical aspects of the relationship between diabetic macroangiopathy and hyperinsulinism].

Epidemiological and clinical data suggest a relationship between hyperinsulinism and macroangiopathy in non insulin-dependent diabetes. On the other hand, a relationship between the plasma free insulin level and macroangiopathy has not been documented in insulin-dependent diabetes. Other abnormalities in addition to hyperinsulinism and glucose intolerance are frequently associated in the presence of insulin resistance and have been grouped by Reaven under the term syndrome X: raised VLDL triglycerides, decreased HDL, and raised blood pressure. Iatrogenic hyperinsulinism appears to be an arterial risk factor, but by what mechanism may it also constitute an independent risk factor? The following theoretical aspects of a possible atherogenic role of hyperinsulinism are currently being investigated: a) insulin stimulates the proliferation and migration of smooth muscle cells either directly or via a rise in IGF1; b) insulin induces lipogenesis in the intima-media, but it has not been demonstrated that this in situ lipogenesis is atherogenic; c) insulin raises the VLDL production, decreases HDL and modifies the clearance of LDL; d) insulin increases blood pressure by stimulating both the renal reabsorption of sodium and the sympathetic nervous system; insulin resistance may also be expressed at the level of the Na-K-ATPase of vascular smooth muscle cells by decreasing the vasodilator effect of the hormone; e) lastly, insulin induces a defect of fibrinolysis mediated by an increase in the level of plasminogen activator inhibitors (PAI1). In conclusion, the combination of hyperglycemia and hyperinsulinism is probably damaging to the artery. Therapeutic intervention studies are necessary to confirm and define the role of hyperinsulinism in macroangiopathy and to answer the unresolved questions: direct or indirect role? effect of endogenous and/or exogenous hyperinsulinism?


[Mode of action of benfluorex. Recent data].

An increased risk of developing premature atherosclerosis is associated with stress, diabetes, obesity, and hypertension. These conditions are associated with insulin resistance, hyperglycemia, hypertriglyceridemia and hypercholesterolemia. An alternative way of interpreting insulin resistance is to consider that metabolism in this condition would be regulated to a greater extent by stress hormones and in particular by cortisol. Glucocorticoids and fatty acids (which are produced in response to stress) antagonise the actions of insulin in promoting glucose uptake and protein synthesis, in decreasing gluconeogenesis and protein catabolism, and promoting the clearance of intermediate density lipoprotein and low density lipoprotein from the circulation by the liver. They also promote the secretion of very low density lipoprotein thus producing hypertriglyceridemia and hypercholesterolemia. By contrast to this antagonism, cortisol can also facilitate the action of insulin in stimulating the storage of energy via glycogen and fatty acid synthesis and through lipoprotein lipase in adipose tissue. These effects are significant in relation to obesity and to weight gain. An increased control of metabolism by cortisol therefore produces changes in metabolism that are potentially atherogenic and it is associated with insulin resistance and the other risk factors for atherosclerosis. Benfluorex treatment improves insulin sensitivity and has antihyperglycemic and hypolipidemic effects in human beings and in experimental animals. These effects can be observed independently of weight loss, but lowering food intake also produces a metabolic benefit. Long-term treatment with benfluorex can also decrease stress responses in terms of glucocorticoid release and the stimulation of lipolysis probably by its serotoninergic control of the hypothalamic-pituitary-adrenal axis. Such an action provides for an integrated treatment of the obese-diabetic-hyperlipidemic syndrome. Benfluorex produces overall changes in metabolism that tend to normalise the major risk factors associated with premature atherosclerosis. This provides a potential advantage over other therapies for atherosclerosis which may ameliorate a symptom (e.g., hyperlipidemia) without treating the underlying metabolic disturbance that predisposes to atherogenesis.


[Effects of chronic administration of benfluorex on the pharmacokinetics of iodine 123 labelled insulin in Zucker obese rats (fa/fa)].

Zucker obese rats (fa/fa), aged 8 to 10 weeks, were treated orally by benfluorex (2 x 25 mg.kg-1, day-1) for 2 weeks and were compared with a control group of the same age treated with placebo. Benfluorex induced a break in the weight curve, a significant fall in serum triglycerides, blood glucose and plasma insulin and in the insulin content of the pancreas. Following intrajugular injection of iodine 123 labelled insulin, the liver of the treated animals bound 25 percent more insulin than the liver of control animals. Conversely, the renal clearance of insulin of the treated animals was reduced in comparison to the placebo group. These studies confirm that, in an animal model of obesity associated with insulin resistance, benfluorex exerts a marked hypolipidemic effect and improves insulin resistance. They also demonstrate an increased targeting of insulin towards hepatic receptors either due to an increase in the hepatic blood flow or to an increase in the number of hepatic receptors or to an increase in the affinity of these receptors. However, speculative considerations make this last mechanistic hypothesis somewhat improbable.


[Mechanisms of hypoglycemic action of benfluorex].

Benfluorex is a hypolipidemic agent with biguanide-like properties. Its action on glucose metabolism was evaluated in 6 NIDDM patients previously treated with diet alone. Before and after 1 month of benfluorex therapy (450 mg/day p.o.), an euglycemic (100 mg/dl) insulin (40 mU/m2/min) clamp was performed along with 3-3H-glucose infusion and indirect calorimetry. Benfluorex did not affect body weight, while it reduced fasting plasma glucose (144 +/- 16 vs 119 +/- 8 mg/dl; P < 0.05), glycosylated hemoglobin (6.8 +/- 0.8 vs 6.4 +/- 0.4 percent) and fructosamine (2.9 +/- 0.6 vs 2.4 +/- 0.2 mmol/l; P < 0.05). Both triglycerides (2.3 +/- 0.6 vs 1.9 +/- 0.5 mmol/l) and total cholesterol (5.7 +/- 0.7 vs 5.2 +/- 0.6 mmol/l) declined. No changes occurred in plasma fatty acid, insulin, and C-peptide. Basal hepatic glucose production did not change and it was completely suppressed during the clamp studies both before and after benfluorex. Basal oxidation rates of carbohydrates and lipids did not change significantly. During the insulin clamp study, insulin-mediated glucose disposal increased after benfluorex (5.7 +/- 0.3 vs 4.8 +/- 0.2 mg/kg/min; P < 0.01). Lipid oxidation was equally suppressed before and after therapy with benfluorex. Glucose oxidation was not enhanced after benfluorex while non-oxidative glucose metabolism was significantly improved (2.2 +/- 0.7 vs 3.4 +/- 0.4 mg/kg/min; P < 0.05). short-term benfluorex administration a) improves glucose and lipid control, b) improves insulin action by enhancing non-oxidative glucose metabolism, c) does not affect basal insulin secretion. The long-term effect of benfluorex treatment remains to be evaluated.


[Antidiabetic efficacy of benfluorex. Clinical data].

We compared the effect of 3 months of treatment by either benfluorex (B) or metformin (M) on hyperinsulinism and high blood glucose levels in overweight patients with non-insulin-dependent diabetes mellitus (NIDDM). All patients had previously complied with a low-calorie diet that had resulted in loss of more than 1.5 kg in one month, but had failed to ensure metabolic control. Weight changes under treatment were identical in both groups (B and M). Both treatments had a comparable effect on blood glucose levels, and this effect was more marked following an oral glucose load. However, a decrease in serum insulin levels and in the fasting serum insulin/fasting serum glucose ratio was seen with benfluorex only. The differences between the two treatment groups were especially marked among the subgroup of patients who responded poorly to the trial diet (fasting I/fasting G: + 1.82 under M., -3.37 under B, P interaction = 0.029). Clinical tolerance was comparable for both drugs. However, a significant rise in serum lactic acid was seen with metformin. The effect of benfluorex on hyperinsulinism in overweight patients with glucose intolerance or mildly increased fasting blood glucose levels is valuable in these high vascular risk patients. The results of this study open the way to a new approach to the treatment of diabetes which should be confirmed by a longer term study currently underway. However, already, these results encourage the evaluation of the benefit provided by benfluorex in combination with classical hypoglycemic drugs.


Patient characteristics and the effect of three physician-delivered smoking interventions.

This paper investigates individual patient characteristics predicting differential response to each of three physician-delivered smoking interventions after 6 months. Participants were 1,286 currently smoking patients seen by 196 medical and family practice residents in five primary care clinics affiliated with the University of Massachusetts Medical School. Of the participants, 57% were female, 92% were white, their average age was 35 years, and they smoked an average of 23 cigarettes per day. Physicians were trained to provide the following interventions: advice only (AO), a brief (< 10 min) patient-centered counseling intervention (CI), and counseling plus prescription of the nicotine-containing gum Nicorette (CI+NCG). The CI+NCG condition included NCG only when appropriate and if acceptable to the patient. Patients were randomized to one of these three physician-delivered intervention conditions. Overall, stratified univariate analyses revealed that AO produced consistently lower cessation rates across most subgroups (generally 9-13%) but was somewhat more effective for certain groups of lighter smokers. Relative to AO, CI was somewhat more effective (about 20-24%) for less addicted smokers, for those with more previous quit attempts, and for those with fewer close associates who smoke, but generally failed to produce higher quit rates for harder core smokers or for women. CI+NCG had an overall pattern of greater effectiveness for both more addicted and less addicted smokers, with the highest absolute levels of cessation (about 27-30%) among less dependent smokers. Women in this group had cessation rates (20.6%) comparable to those of men (23.6%). Condition-stratified logistic regression analyses, controlling for a wide range of covariates, revealed associations similar to those observed in the univariate analyses: An overall logistic model in which intervention conditions were fitted as dummy variables produced the following significant main effects: sex, years smoked, contact with other smokers, symptoms, and CI+NCG condition. Significant interactions were observed for both CI and CI+NCG and smoking when feeling too ill to continue normal activities and CI+NCG and amount smoked. We observed significant main effects on cessation of variables related to addiction, sex, social factors, and physician counseling interventions. Specific interactions were observed between reported smoking when feeling ill and each of the counseling interventions as well as by amount smoked in the CI+NCG condition.


Views of a general population on mass screening for colorectal cancer: the Burgundy Study.

The success of a screening program depends on a number of factors, including the validity of the test, its efficacy, its feasibility, and the rate of compliance. Thus, it is important to determine the factors influencing compliance to the screening test in order to obtain a high participation rate. A mass screening campaign for colorectal cancer using the Hemoccult test was conducted in Burgundy, France. One year later, a questionnaire was mailed to a random sample of the population to assess the views of the general public (870 persons who had not completed the Hemoccult test, 750 persons who had a negative test, and 100 persons who had a positive test). The compliance rate of this survey was 32.2% among persons who had not completed the screening test, 88.2% among persons with a negative result, and 98.0% among persons with a positive result. The main reason for not participating was not wanting to know more about their health status (34.3%). Of the factors credited with encouraging persons to perform the test, the most important ones were the practitioner's explanations (55.7%) and the leaflet sent by mail (42.5%). The impact of the media campaign was weak. A small portion of the population (4.3%) said that they were very anxious while waiting for the results. Among persons with a positive screening test, only 1.2% regretted having taken it. Among those who took the test, 94.2% said they were ready to do it again; and among those who did not take it, 36.7% would accept it at a second screening. The data suggest that overall the Hemoccult is well accepted, that the campaign did not upset the population, and that it is worthwhile at the second screening to include those who did not participate in the first screening test.


Comparison of recruitment strategies and associated disease prevalence for health promotion in rural elderly.

Although interest in health promotion for the elderly is increasing, the issues of recruitment into such programs and self-selection have not been well explored. While clinical studies require high participation levels and expensive recruitment, community efforts are satisfied with recruiting small numbers of volunteers from poorly defined populations. These small samples may not be representative of the populations at risk. As part of the Rural Health Promotion Project, a Medicare demonstration, community-based recruitment methods were evaluated and participant characteristics were compared. A total of 3,884 individuals ages 65-79 were recruited in northwestern Pennsylvania, using four sequential recruitment strategies, varying in aggressiveness. The methods were: (A) mail only, (B) mail with phone recruitment follow-up, (C) mail with phone recruitment and scheduling, and (D) mail with aggressive phone recruitment and scheduling. Recruitment yields were Method A, 13.5%; B, 21.1%; and C, 31.6%. The most aggressive Method (D) yielded 37.0% participation. More aggressive methods (C and D) recruited more educated individuals. No other demographic or health status differences were noted. These data show that large numbers of the elderly can be recruited into a health promotion program using aggressive methods and professional interviewers.


Trends in cardiovascular disease risk factors by educational level: the Stanford Five-City Project.

Trends in blood pressure, smoking, and cholesterol were examined from 1979-1980 through 1985-1986 in four cities in California by level of education (< high school, high school graduate, some college, college or postgraduate). Four biennial cross-sectional surveys (n = 6,580) were conducted in two treatment and two control cities to evaluate a 6-year community health education intervention, conducted as part of the Stanford Five-City Project. Over the 8-year study period, men and women ages 25-74 from each educational group in the treatment cities showed significant declines in smoking prevalence and levels of blood pressure and cholesterol (with the exception of cholesterol in women). In general, declines in the least educated group (< high school) were stronger than declines in the most educated group (college or postgraduate). Similar declines occurred in each educational group in control cities. These results illustrate that persons from all educational levels can modify their risk for CVD and are of particular importance because of the higher prevalence of CVD risk factors among those with less education. The similarity of time trends in treatment as well as control cities suggests that the broad-based, multisource health education efforts in the United States are succeeding across the educational spectrum.


Reduction of ischemic heart disease risk markers in the teenage children of heart attack patients.

Families of people with ischemic heart disease (IHD) are likely to share high levels of risk markers and to have similar lifestyle patterns. Teenage children in such families were the focus of a behavioral intervention program. Families were randomly allocated to either an "early" or a "late" advice group. The late group and a third "control" group (consisting of families with no cardiovascular disease) received the intervention only after baseline measurements were repeated in all three groups at the end of 12 months. Pedigree analysis was used to compare changes across groups. The decrease in self-reported total fat intake was greater among teenagers in the early group (mean = -3.38, SE = 1.00) than among those in the late group (mean = -0.58, SE = 1.06), as was the decrease in saturated fat intake (mean = -2.46, SE = 0.56; mean = -0.54, SE = 0.60, respectively). These differences were statistically significant (P < 0.05). There were no differences between these groups for changes in total or high-density lipoprotein cholesterol levels. There was a statistically significant difference between the change in total cholesterol levels in the control group (mean = +0.08, SE = 0.07) and that in the late group (mean = -0.14, SE = 0.11). The intervention program appears to have made the teenagers aware of high-fat foods, as stated fat intake was significantly lowered, but it was not successful in decreasing blood cholesterol levels. Having a parent with IHD did, however, influence the teenagers' risk-related behavior, leading to a significant reduction in blood cholesterol levels.


A survey of physicians' attitudes and practices related to exercise promotion.

One hundred sixty-eight physicians responded to a survey to determine their attitudes and practices related to exercise and the development of exercise prescriptions. The mean age was 45.5 +/- 10.8 years, with the majority being male (86.7%). The survey found that 48% of the physicians required an exercise history as part of their initial examination and 91% encouraged their patients to participate in regular exercise programs. Seventy percent of the physicians did not develop exercise prescriptions and only 23% were familiar with the American College of Sports Medicine guidelines related to the development of exercise prescriptions. Only a small number of physicians (3%) had ever taken a college-level course related to exercise physiology and the development of exercise programs. The majority (78%) felt that there was a definite need in medical school for a course related to the medical aspects of exercise. Results of this investigation indicate that while physicians support exercise for health promotion and rehabilitation, greater emphasis needs to be placed on physician involvement in promoting and/or prescribing exercise.


Melanoma prevention: behavioral and nonbehavioral factors in sunburn among an Australian urban population.

To determine the independent contribution of behavioral factors to the occurrence of sunburn, sun protection behavior was assessed over 13 successive summer weekends in a total of 1,655 adults in Melbourne, Australia. Telephone survey respondents provided detailed accounts of activities engaged in, time spent outside, and hat, clothing, and sunscreen coverage in the 4 hr around the solar midday on both weekend days, as well as skin type, sociodemographic descriptors, and degree of sunburn experienced. Independent measures of atmospheric temperature and ambient ultraviolet radiation (UVR) were added to individual records. The (mostly recreational) weekend sunburn in this urban sample was strongly associated with UVR, as expected. Temperature at 3 PM, sensitive skin type, youthfulness, and being male were also independently associated with sunburn. After all other predictors were controlled for, the body exposure index (which took into account time outside and hat, clothing, and sunscreen coverage) made a strong independent contribution to the explanation of sunburn (P < 0.001). It was concluded that behavior change strategies to prevent malignant melanoma of the skin are warranted.


Total indoor smoking ban and smoker behavior.

To assess smoking policy support and effects, 1,083 hospital employees (203 smokers) were surveyed by anonymous questionnaire 1 year after the announcement (5 months after implementation) of a new total indoor smoking ban. A second follow-up, limited to smoker respondents only, was conducted 2 years postannouncement. A total indoor smoking ban was supported by the vast majority of nonsmokers (89%) and ex-smokers (86%) and by nearly half of the then-smoking population (45%). Consistent with previous reports, the smoking ban was associated with a significant decrease in cigarette use during work hours, particularly among moderate to heavy smokers. However, the ban did not result in increased institutional quit rates. Light smokers (< 10 cig/day), compared with heavy smokers (> or = 30 cig/day), were more likely to support the no-smoking policy and had fewer problems observing the ban. They were also less apt to report a decrease in work productivity. A total indoor smoking ban had little effect on overall institutional quit rates. Heavy smokers will, predictably, experience the greatest difficulty complying with a total indoor nonsmoking policy.


Motivational characteristics of smokers at the workplace: a public health challenge.

Few studies have focused on the motivational characteristics of smokers who do not volunteer for cessation. This study examined the relationship between demographic and selected psychosocial factors and motivation and intention to quit smoking among employed smokers at five worksites. In addition, the distributions of smokers who are at different stages of readiness to change their smoking behavior are presented. Results indicate that, overall, less than 8% of employed smokers are currently ready to quit smoking and that blue-collar workers are lower in motivation than white-collar workers. Predictors of higher levels of motivation to quit smoking included higher socio-economic status, maleness, lower levels of self-reported nicotine dependence, and stronger perceptions that smoking was against the social norms of the workplace. Implications for intervention, evaluation, and policy are discussed in the context of the challenge of making a public health impact on reducing overall smoking prevalence.


Worksite smoking control activities: prevalence and related worksite characteristics from the COMMIT Study, 1990.

There is a pressing need for current information on worksite smoking policies and stop-smoking activities. To address these needs and identify worksite characteristics associated with such activities, a computer-assisted telephone interview was administered to 793 worksites having at least 50 employees. Within each of the 11 COMMIT intervention communities, attempts were made to survey 30 worksites within each of three size strata: 50-99 employees, 100-249 employees, and 250+ employees. Response to the survey was good, with a screening rate of 95% and a response rate of 90%. Overall, 69% of responding worksites reported having written smoking policies, but only 15% banned all indoor smoking. Smaller worksites and manufacturing and wholesale/retail companies were less likely than other organizations to be smoke-free or have representatives attend smoking policy workshops. Thirty-five percent of worksites reported offering smoking cessation resources or assistance for employees, but only 20% offered both cessation resources and restrictive or smoke-free policies. Smaller worksites as well as wholesale/retail sales and manufacturing worksites were less likely to offer cessation resources or to participate in community wide stop-smoking events. Although the level of worksite smoking control activities was higher than that reported in earlier surveys, there is still a substantial need to increase the level of such activities, especially at smaller worksites and in manufacturing and sales organizations.


Public objections to environmental tobacco smoke.

METHODS. A randomly selected sample of Californian adults was surveyed by telephone to assess their knowledge, attitudes, and behaviors with respect to tobacco use and its control. The questions asked of this sample were whether they had recently (in the past 12 months) asked someone not to smoke in certain situations and whether they would ever consider doing such a thing. RESULTS. Results of the survey indicated that 59.1% of nonsmokers and 44.4% of smokers had asserted themselves within the past 12 months, while another 28.2 and 29.1% of the nonsmokers and smokers, respectively, indicated that they would consider asking someone not to smoke. Only 12.7% of the nonsmokers and 26.4% of the smokers indicated that they would not consider doing such a thing. Those more likely to object publicly to tobacco smoke are generally younger, are more educated, and have anti-tobacco attitudes related to either a specific health belief or social influence.


Factors associated with hypertension in Nigerian civil servants.

Study of hypertension in segments of West African populations in transition toward Westernization may lead to better understanding of the high risk for hypertension among Westernized blacks. Five hundred fifty-nine urban civil servants, ages 25-54, were recruited from six ministries of Bendel State, Nigeria. Blood pressure, physical measurements, urinary protein and glucose, fasting blood glucose, and demographic data were collected at the workplace. Subjects were classified as senior staff (professionals or administrators) or junior staff (non-administrators). Among 172 male senior staff, the age-adjusted rate of hypertension (diastolic blood pressure > or = 90 mm Hg, systolic blood pressure > or = 140 mm Hg, or on an antihypertensive medication) was 43% and occurrence rose dramatically from 21 to 63% across age groups 25-34 to 45-54, respectively. Among 266 male junior staff, the age-adjusted rate of hypertension was 23%, and occurrence did not rise with age. Logistic regression showed that body mass index (kg/m2), age, alcohol drinking, and being senior staff were all independently related to hypertension in men. On the other hand, the age-adjusted rate of hypertension in 121 women was 20% and was significantly related only to body mass index. Male urban civil servants appeared to have a risk for hypertension similar to that of U.S. black males. Age, body mass index, alcohol drinking, and other unidentified factors related to higher socioeconomic status were strong determinants of hypertension in this population.


Smoking, exercise, and physical fitness.

Research on smoking and physical activity provides strong evidence of smoking's negative impact and physical activity's positive impact on long-term health. However, evidence regarding the association between smoking and exercise activity and the independent effects of these factors on fitness is lacking. The associations among exercise activity, smoking behavior, and physical fitness were examined in 3,045 Navy personnel. Exercise and smoking behaviors were measured using a lifestyle survey. Physical fitness was assessed using scores on the Navy's Physical Readiness Test. Analyses of variance were conducted to examine the relationships among smoking status, exercise activity, and PRT performance. Multiple regression procedures were used to examine the relationship between smoking and physical fitness after statistically controlling for the effects of exercise. Smoking was associated with lower exercise levels and lower physical endurance--both cardiorespiratory (1.5-mile run) and muscular (sit-ups). After controlling for exercise activity, smoking remained significantly associated with lower physical endurance but was not related to overall body strength (lean body mass) or percentage body fat. Smoking is a detriment to physical fitness even among relatively young, fit individuals. Study findings suggest that smokers will have lower physical endurance than nonsmokers, even after differences in the average exercise levels of smokers and nonsmokers are taken into account. Cigarette smokers should be given strong encouragement to stop smoking as part of any effort to improve physical fitness.


Cholesterol-related counseling by registered dietitians in northern California.

An estimated 40 million Americans have serum cholesterol levels that warrant medically supervised dietary intervention. Although registered dietitians are expected to play an important role in treating these patients, current treatment practices in the community are largely unknown. A questionnaire concerning treatment practices was mailed to all 377 registered dietitians listed in the directories of the American Dietetic Association for two large California districts. Number of patients seen and length and content of dietary counseling were ascertained for three types of patients: (a) hypercholesterolemic outpatients without heart disease, (b) hypercholesterolemic outpatients who have heart disease, and (c) inpatients with myocardial infarction. A return rate of 59% (n = 252) was obtained for the questionnaire. A total of 44% of the registered dietitians counseled hypercholesterolemic patients in any of the categories surveyed. About 30% of the respondents counseled hypercholesterolemic outpatients without heart disease. They saw an average of 4.9 such patients a week, spent an average of 53 min in an initial session, and usually did not see the patient again in follow-up. Fewer than 10% of patients had as many as four sessions. About 27% of the respondents saw hypercholesterolemic outpatients with heart disease, averaging 3.5 such contacts per week. The reported practices were similar to those provided to noncardiac outpatients. About 22% of registered dietitians worked with hospitalized myocardial infarction patients. They spent an average of a total of 41 min over 2.5 visits with each patient. Currently, outpatient registered dietitian counseling for hypercholesterolemia appears to be limited in both the number of patients reached and the duration of the counseling. Further research into the impact of, barriers to, and efficacy of alternative delivery methods of dietary counseling is needed.


Compensation strategies in sun protection behaviors by a population with nonmelanoma skin cancer.

Initiation of sun protection strategies can be promoted, to some extent, by educational efforts, but little is known about the merit of continuing education interventions in sustaining the desired behaviors or adding new behaviors. This prospective study clarifies the choices individuals make among the four strategies that allow them to maintain lifestyle changes. From 1983 to 1987, the population received education about sun protection coupled with the removal of a nonmelanoma skin cancer. One year later, their choice of sun protection methods was determined. Then annually from 1985 to 1989, they received written recommendations about sun protection for a period of 2-6 years after the initial education. The maintenance, cessation, and addition to the initial sun protection behaviors were ascertained by a questionnaire, as was the intention to change. Frequency of physician visits and development of subsequent nonmelanoma skin cancer were evaluated by medical chart review for the 2-6 year phase of continued education. One percent of the population consisting mostly of women described ceasing tanning after 2-6 years of education. The population related a greater use of protective clothing and/or sunscreen with an SPF of 15 or greater as their reported restrictions on outdoor activities ceased. An emerging new strategy of some of the population (n = 185) was the use of sunscreens with an SPF less than 15 in association with attempts to deliberately tan and longer daily outdoor exposure. Neither frequency of physician visits nor numbers of subsequent nonmelanoma skin cancers influenced continuation or addition of sun protection behaviors. While the greatest reported change in behavior was temporarily associated with educational intervention linked to removal of the skin cancer, continued educational efforts may have recruited some individuals to cease tanning and encouraged others to adopt the use of protective clothing or more frequent sunscreen use as they were unable to maintain the limitations on outdoor activities. It is not possible to structure a control group restricted from mass media education; therefore, the effectiveness of specific behavior-directed education cannot be precisely determined. Nonetheless, the population described using knowledge to develop compensation sun protection strategies that preserved lifestyle.


The importance of physician encouragement in breast cancer screening of older women.

The relationship between physician encouragement and breast cancer screening is examined with a population-based survey of 630 women between the ages of 45 and 75. Although the women interviewed were selected on the basis of their noncompliance with mammography guidelines, nearly half had previously had at least one mammogram. Women reported having received more physician encouragement of breast self-examination than of mammography. Older women reported less encouragement of both screening modalities than younger women. Multivariate analyses revealed physician encouragement to be more strongly associated with screening mammography than with health status, health care utilization, attitudes, and sociodemographic characteristics: those who reported having received physician encouragement were nearly four times more likely to have ever had screening mammography. These and related findings are used to highlight the critical importance of physician behavior in the secondary prevention of breast cancer in older women and to identify types of patients whose needs for screening are most likely to be overlooked by physicians.


[Current pattern of anthracosilicosis, its complications and correlation with other diseases (evaluation of 300 legal autopsies 1977-1988)].

The spectrum of silicosis of coal miners has changed during the past decades. The life expectancy of the miners suffering from silicosis has been successfully adapted to that of the non-miners as a result of a consistent therapy. Morphologically, the processes involving large callosities have receded markedly; in their place, there has been an increased incidence of generalised focal dust emphysema that are difficult to differentiate clinically from the common chronic obstructive pulmonary diseases. Chronic bronchitis and emphysema are the most important concomitant pulmonary diseases; the incidence of tuberculosis is still enhanced and carcinomas of the lung are only rarely to be acknowledged as so-called carcinomas in scar tissue. Other major diseases that coincide with silicosis are seen in p.m. statistics with customary frequency of incidence (cardiovascular diseases almost 50%, malignant tumours including those of the lung about 25%, other major diseases without respiratory organs about 10%). The main problem in expertising is to differentiate the influence of these diseases from those of silicosis; expertising must employ the legally prescribed terminology. In our own investigations based on 300 postmortem expertises, death as a result of a professionally acquired disease was acknowledged in 48% of all cases of silicosis of severity grades I to III, silicosis being the sole major disease in only half of the cases and in the other cases an essential partial contributor to the cause of death. The significance of the extended generalised dust emphysema as a special type of pneumoconioses that must be classified as grave, is emphasised in contrast to previously compiled statistics.


[Public patient education in pneumology].

Since February 1990 public information evenings pertaining to respiratory tract diseases have been held every month in the cities of Fellbach, Backnang and at times in Waiblingen. These sessions have met with a great show of interest from patients, doctors and the public in general, as indicated by the consistently large number of over 100 participants. This continual educational work with its accompanying measures, such as, asthma-sport, breathing exercises, and books and videos on loan, represents an effective alternative to the formally structured, patient training programs limited to a specific time period. A broad range of effectiveness is attained by engaging public media in the informative process.


[Pulmonary infiltrations with eosinophilia (pulmonary eosinophilia)].

The pulmonary eosinophilias are characterised by radiographic lung shadows with either a peripheral blood eosinophilia of more than 450/microliter or histologic abnormalities consisting of both interstitial and intraalveolar accumulations of eosinophils and macrophages. We describe the clinical features, radiographic changes, results of bronchoscopy and follow-up studies of three women with chronic eosinophilic pneumonia of unknown aetiology. In all patients the illness resolved rapidly after treatment with corticosteroids, however one patient experienced a second episode after treatment withdrawal. We demonstrate the wide differential diagnosis of the syndrome of pulmonary eosinophilia.


[Bronchiectasis with bronchopleural fistula].

We report on a female patient who is now 56 years of age suffering from bronchiectasis disease. Due to the bronchopleural fistula in the middle lobe we decided to perform an operation despite left-sided bronchiectases and in spite of the obstructive airways disease.


A comparative ultrastructural and histochemical study of the metacercarial cyst walls of four species of Paragonimus (Troglotrematidae: Trematoda).

The metacercarial cyst walls of Paragonimus westermani, P. miyazakii, P. ohirai, and P. iloktsuenensis were examined using ultrastructural and histochemical techniques. The cyst walls of P. westermani, P. miyazakii, and P. ohirai were found to have five distinct layers, whereas the wall of P. iloktsuenensis had only two. The so-called outer cyst wall recognized on light microscopy by Miyazaki (1961) was composed of layers I-III and the so-called inner cyst wall comprised layers IV and V. The outermost layer (I) consisted of collagen fibers and probably originated from the host. Layer III detached easily from layer IV on removal of the cysts from the host tissues. Layer IV was composed of proteoglycans, and layer V consisted of protein alone. A regular hexagonal pattern in layer V appeared to be characteristic of P. westermani. Although the basic morphological patterns observed in the layers constituting the cyst walls were similar in all species examined, the thickness of each layer was apparently different. This characteristic seems to be a suitable criterion for the identification of metacercarial cysts of Paragonimus species.


Electrophoretic analysis of a natural population of the Thai Paragonimus heterotremus and its genetic relationship to the three Japanese species P. miyazaki, P. ohirai and P. westermani.

A total of 18 enzymes (encoded by 20 loci) from Paragonimus heterotremus collected at a local area in Saraburi, Thailand, were electrophoretically compared with those from the three Japanese species P. ohirai, P. miyazaki and P. westermani using horizontal starch-gel electrophoresis. Relative genetic distances between these species were quantified. The Thai P. heterotremus was found to be most closely related to P. miyazaki and rather distantly related to P. westermani. On the other hand, among the three Japanese species, the closest relationship was observed between P. miyazaki and P. ohirai.


Scanning electron microscopy of cercariae, metacercariae and adults of Pygidiopsis ardeae Køie, 1990 (Digenea, Heterophyidae).

The penetration apparatus of the cercaria of Pygidiopsis ardeae Køie, 1990 (Heterophyidae) is provided with five large preoral hooklets. Various types of presumably sensory structures surround the small oral aperture. Small, pointed spines protrude throughout the cercarial body. After parasite penetration and encystment in the fish intermediate host, the metacercarial tegument increases its absorptive area by developing irregular projections. Concurrently the pointed spines become scale-like and serrated. The tegumental outgrowths appear to have regressed in infective metacercariae. The external surface of mature worms removed from the intestine of domestic chickens does not differ from that of infective excysted metacercariae. Adults taken from experimentally infected chickens were identical to specimens obtained from naturally infected herons.


High osmotic pressure for Pneumocystis carinii London Resin White embedding enables fine immunocytochemistry studies: I. Golgi complex and cell-wall synthesis.

A method for embedding Pneumocystis carinii in hydrophilic resin (London Resin White) has been developed for immunocytochemistry studies. Using high osmotic pressure (about 850 mosmol) from fixation to embedding, this method improved the preservation of the fine structure as well as the antigenicity of rabbit- and SCID mouse-derived P. carinii. Cytochemistry studies were performed using colloidal gold-conjugated lectins (concanavalin A, glycine max, Ulex europaeus) that reacted with the cytoplasmic components (endoplasmic reticulum, Golgi vesicles). Colloidal gold-conjugated streptavidin was also tested and was found to be reactive with the parasite cell wall and cytoplasmic components, which precludes its indiscriminate use in P. carinii immunocytochemistry studies.


Effects of calcium channel blockers on the contractility of the filariid Acanthocheilonema viteae.

The role of calcium in muscle contractility was explored in the filarial nematode Acanthocheilonema viteae (Dipetalonema viteae). The parasite was slit open longitudinally and mounted in a smooth-muscle chamber that had been filled with aerated (95% N2/5% CO2) physiological solution at 37 degrees C. Nifedipine (10(-6) M) and cadmium (3 x 10(-5) M) reduced the spontaneous isotonic contractions of A. viteae, whereas verapamil (10(-5) M) and diltiazem (10(-5) M) enhanced them. The effects of nifedipine and verapamil did not appear to be due to the solvent ethanol. All of the drugs reduced the maximal contraction induced by acetylcholine (ACh, 10(-5) M), although nifedipine was the most potent. After the exposure of worm preparations to a calcium-free medium containing ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, 10(-4) M) for 1 h, application of ACh (10(-5) M) induced a small, transient contraction. Subsequent applications of ACh in this medium had no effect. Thus, the nematode muscle contraction appears to depend on extracellular calcium. Nifedipine, diltiazem, and verapamil could act by reducing the calcium influx across the muscle membrane.


Histone-DNA interactions in the chromatin of procyclic Trypanosoma brucei brucei.

The dissociation of histone proteins a-d from the chromatin of Trypanosoma brucei brucei procyclic culture forms was investigated by removing the proteins from the DNA by centrifugation of soluble chromatin through isokinetic sucrose gradients in the presence of NaCl. The dissociation of the T. b. brucei histones was compared with that of their higher-eukaryote counterparts H3, H2A, H2B and H4. All four histones of T. b. brucei remained bound to the DNA at 500 mM NaCl, were partially released at 750 mM NaCl and were completely dissociated from the DNA at 1 M NaCl. These interactions of histones a-d with the DNA were comparable with those of the H2 histones in the chromatin of higher eukaryotes, and histones a and d interacted with the DNA more weakly than did their higher-eukaryote counterparts H3 and H4. Substoichiometric amounts of an additional protein were recovered in the top fractions of the gradients under all dissociation conditions. This protein migrated in the H1 region of rat-liver chromatin in various gel systems. Its early release from the DNA also indicated a resemblance to histone H1. The presence of only small amounts of this protein and the relatively weak interactions of histones a and d with the DNA suggest that the mechanisms involved in chromatin compaction in T. b. brucei are different from those in higher eukaryotes.


Studies on the developmental cycle of Trichospirura leptostoma (Nematoda: Thelaziidae). Experimental infection of the intermediate hosts Blatella germanica and Supella longipalpa and the definitive host Callithrix jacchus and development in the intermediate hosts.

The cockroaches Blatella germanica and Supella longipalpa can act as intermediate hosts of Trichospirura leptostoma as demonstrated by experimental infestation. The parasite developed from the embryonated egg into the infective larval stage (L3) in cockroaches within 5-6 weeks. After experimental infection of marmosets (Callithrix jacchus), eggs were first found in faecal samples (prepatency) at 8-9 weeks post-infection. Patency lasts about 2 years. Despite the presence of living adult worms in the marmosets' pancreas, no additional eggs were observed in their faeces after the patent period.


Litomosoides carinii: extraction of the microfilarial sheath components and antigenicity of the sheath fractions.

Microfilarial sheaths of Litomosoides carinii were isolated and extracted with 2% sodium dodecyl sulfate (SDS) and 5% 2-mercaptoethanol (2ME). Extraction with SDS alone did not alter the ultrastructure of the sheaths and yielded five polypeptides (27-67 kDa) that were not recognized by antibodies of infected hosts but reacted with antibodies to host-serum proteins. 2ME treatment caused partial solubilization of the sheaths (45% as determined by amino acid analysis), which could be further improved by combining 2ME with SDS. The remainder showed filamentous/threadlike structures on electron microscopic examination. As compared with whole sheaths, the insoluble proportion was markedly enriched in alanine and cysteine but contained less galactosamine, serine, and threonine. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of 2ME/SDS-extractable components showed 12-16 bands of 14- greater than 120 kDa. A predominant component had an apparent molecular mass of 22 kDa. Two bands (42 and 120 kDa) could be stained with Coomassie blue but showed "negative" staining when gels were stained with silver. Several components (but not the 22-kDa polypeptide) bore phosphocholine epitopes. Apart from the negatively staining bands, most of the 2ME-soluble sheath components were recognized by antibodies of L. carinii-infected Mastomys coucha. Except for several polypeptides that had been unspecifically recognized by IgM, the antibody response to sheath components started at the end of the prepatent period.


Experimental infection of pigs with a Taenia species from Korea: parasitological and serological aspects.

Belgian Landrace piglets were experimentally infected with eggs of a Taenia sp. of Korean origin. At autopsy, metacestodes were present only in the livers. The proportion of degenerated metacestodes increased from 12%-39% at 5 weeks to 94%-100% at 10 weeks after infection. A sandwich enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies raised against the excretory-secretory products of T. saginata metacestodes detected circulating antigen in the sera of the pigs at 1 week post-infection. A good correlation was found between the presence of viable metacestodes and the detection of circulating antigen; the latter disappeared as the metacestodes died off. However, the antibodies were detected only after 3 weeks of infection and onwards until the necropsy of the pigs.


Ultrastructure of sperm and spermatogenesis of Rugogaster hydrolagi, Schell 1973 (Platyhelminthes, Trematoda, Aspidogastrea, Rugogastridae).

Mature sperm of Rugogaster hydrolagi exhibit the structure typical of Trematoda, with 2 axonemes of the 9+ "1" pattern being incorporated into the sperm body, dorsal and ventral microtubules occurring in the principal region that contains the nucleus and mitochondrion, and a complete row of peripheral microtubules lying in the proximal region surrounding the two axonemes. During spermiogenesis, a zone of differentiation develops that comprises a row of microtubules along the cell membrane and an adjacent dense region along the nuclear membrane. The intercentriolar body and centrioles appear between the microtubules and the nucleus. Free flagella with striated rootlets grow out in opposite directions, the cell membrane protrudes and lengthens into a median cytoplasmic process (MCP), the nucleus and mitochondrion move into the MCP and the flagella rotate and eventually fuse with the process. A dense region develops in the spermatid shaft some distance from the proximal end, but it is not present in the mature sperm. The spermatid elongates and is eventually pinched off from the residual body at the level of the arching membranes after the rootlets and intercentriolar body have disappeared. Thus, spermatogenesis and sperm in the aspidogastrean Rugogaster hydrolagi correspond to those in digenean Trematoda.


Interaction between Ascaris suum and Pasteurella multocida in the lungs of mice.

In an experiment including 8 groups of 15 mice, the effect of migrating Ascaris suum larvae in the lungs on the establishment and pathogenicity of aerosol exposure to Pasteurella multocida was investigated. Following aerosol exposure to P. multocida, mice with migrating A. suum in their lungs developed more severe pneumonia and septicaemia than did parasite-free mice. The parasite-induced effect on bacterial pathogenicity was more marked for a non-toxin-producing P. multocida as compared with a toxin-producing strain of P. multocida, possibly due to the higher spontaneous pathogenicity of the non-toxigenic strain of P. multocida. The present results should encourage controlled experiments on possible interactions between A. suum and various airborne microbial infections in pigs.


Detection and quantitation of cell-surface sugar receptor(s) of Leishmania donovani by application of neoglycoenzymes.

Promastigote culture forms of the log growth phase of Leishmania donovani stock LRC L 51 were investigated for expression of cell-surface carbohydrate-binding sites using 15 types of a chemically glycosylated enzyme termed neoglycoenzyme. Carbohydrate conjugation and coupling yield were kept constant to ensure that the type of carbohydrate moiety was the only variable feature of the applied tools. Para-aminophenyl derivatives of the following carbohydrate residues were used for the glycosylation of beta-galactosidase from Escherichia coli: beta-D-lactose, beta-D-thiogalactose, alpha-D-mannose, alpha-L-rhamnose, alpha-D-N-acetylgalactosamine, beta-D-N-acetylgalactosamine, beta-D-N-acetylglucosamine, the alpha- and beta-glucosides maltose and cellobiose, beta-D-xylose, alpha-D-mannose-6-phosphate, the alpha-galactoside melibiose, alpha-L-fucose, and beta-D-glucuronic acid as well as sialic acid. Only melibiose, fucose, and glucuronic acid showed no binding affinity for the cultured flagellates; this served as an internal control reaction to exclude any binding to the linker group. This result demonstrates that many but not all sugar types can be recognized by appropriate receptor structure(s) on the surface of the promastigote Leishmania. Transformation of the binding data for neoglycoenzymes exposing lactose, mannose, rhamnose, and N-acetylated hexose residues, which was carried out to obtain the dissociation constants and to estimate the number of binding sites at saturation, revealed KD values of around 100 mM and around 10(4) binding sites for the polyvalent ligands.


Phosphorylation of Toxoplasma gondii major surface antigens.

The phosphorylation of the major surface proteins of Toxoplasma gondii tachyzoites was investigated. Metabolic labeling of intracellular tachyzoites with [32P]-orthophosphate followed by immunoprecipitation with specific monoclonal antibodies showed that all five major surface proteins were labeled as expected for GPI-anchored proteins. More detailed analysis of the phosphorylated sites revealed that surface proteins P30 and P22 contained phosphoserine residues in addition to the phosphorylated molecules that are presumably localized in the GPI-membrane anchors.


A cytochemical study of the interaction between Tritrichomonas foetus and mouse macrophages.

Light and electron microscopy were used to analyse the process of interaction of normal and antibody-coated Tritrichomonas foetus with resident and activated mouse peritoneal macrophages. Activated macrophages ingest more parasites than do resident macrophages. Previous incubation of the parasites in the presence of sub-agglutinating concentrations of a polyclonal anti-T. foetus antibody significantly increased their ingestion by the macrophages. Adherence of the parasites to the surface of activated macrophages triggers the respiratory oxidative burst as revealed by reduction of nitroblue tetrazolium. This process was more evident in antibody-coated parasites. Transmission electron microscopy showed the presence of reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]-oxidase in the portions of the macrophage plasma membrane that were in contact with the parasites as well as in the phagocytic vacuoles. Fusion of macrophage lysosomes with parasite-containing phagocytic vacuoles was observed in macrophages labeled with Lucifer yellow and gold-labeled peroxidase as well as by localisation of acid phosphatase.


Changes in atrial natriuretic factor and plasma renin activity in dogs infected with Trypanosoma brucei.

When beagle dogs were infected with Trypanosoma brucei, a marked reduction in the plasma concentration of atrial natriuretic factor (ANF) occurred in the terminal stage of the disease during weeks 3 and 4. At the same time there was an increase in plasma renin activity (PRA) after infection. Ultrastructural studies of the atria of these dogs demonstrated a reduction in ANF granules. The changes in ANF and PRA occurred in association with severe pancarditis and the development of heart failure. By impairing the ability of the heart and kidneys to regulate blood volume, the alterations in ANF and PRA could be involved in the pathogenesis of heart failure in T. brucei-infected dogs.


Sarcocystis jorrini sp. nov. from the fallow deer Cervus dama.

Sarcocystis jorrini sp. nov. isolated from the muscle of naturally infected fallow deer (Cervus dama) from the Doñana National Park (Spain) is described by light and electron microscopy. Sarcocysts were macroscopic (2.7 x 1.4 mm) and were found in all striated muscle, including that of the esophagus and the heart. As visualized by light microscopy, the cyst walls were formed by mammilated protrusions surrounded by a coating of connective tissue measuring 10-35 mm in thickness. Electron microscopy revealed that the parasitophorous vacuole was serrated in appearance, with numerous pinocytic vesicles running along its whole length. Villar protrusions were of varying shape and were square, rectangular, or linguiform in cross section. The core of the villi contained small dense granules and microfilaments, which were more abundant in peripheral areas. Ground substance contained larger dense granules clustered around the base of villi. The sarcocyst was surrounded by a secondary cyst wall composed of collagen fibrils and fibrocytes. S. jorrini is the first macroscopically visible sarcocyst of the genus Cervus reported to have a secondary cyst wall.


Plasmodium falciparum and P. berghei: detection of sporozoites and the circumsporozoite proteins in the saliva of Anopheles stephensi mosquitoes.

Sporozoites and free circumsporozoite (CS) protein were stained immunoenzymatically in 1-min saliva samples collected from Anopheles stephensi mosquitoes infected with either Plasmodium berghei or P. falciparum. The number of sporozoites in 1-min saliva-streak samples significantly increased as the salivary gland index rose from 3+ to 4+. For P. berghei-infected mosquitoes from which saliva had been collected before 30 days postfeed, the median sporozoite counts for 3+ and 4+ gland indexes were 4.5 and 116, respectively. For P. falciparum-infected mosquitoes, the median counts obtained in two experiments were 4.5 and 14.5 (3+) and 97 and 107 (4+), respectively. The frequency of sporozoite detection in the saliva of mosquitoes containing < 100 salivary-gland sporozoites was low (0.1), whereas that in the saliva of mosquitoes with > 100 sporozoites was high (0.96). In highly infected 4+ P. berghei-infected mosquitoes from which saliva had been collected after 30 days postinfection, both the volume of saliva collected and the median number of sporozoites recovered decreased significantly.


Scanning and transmission electron microscopy of host cell pathology associated with penetration by Eimeria papillata sporozoites.

Scanning and electron microscopy was used to study the pathogenesis that occurred in mouse epithelial cells that had been penetrated by Eimeria papillata sporozoites. Optimal penetration of parasites injected into nonligated and ligated mouse intestine was found to occur at 4-15 min post-inoculation. During initial penetration, the parasite caused disruption of the microvilli of the intestinal cells, which led to detachment of the microvilli from the plasma membrane of the penetrated cell. Host cells penetrated by the parasite showed extensive destruction of the internal cellular organization together with blebbing of host-cell cytoplasm and release of internal organelles such as mitochondria. Ultimately, the penetrated cells completely broke down, leaving vacuolated areas next to ultrastructurally normal epithelial cells.


Immunocytochemical localization of a cysteine protease in adult worms of the liver fluke Fasciola sp.

The intracellular localization of a cysteine protease from Fasciola sp. that hydrolyzes host hemoglobin as a nutritional source was examined by light and electron microscopic immunocytochemistry using a monoclonal antibody specific to the enzyme. Immunoperoxidase staining was predominantly restricted to large numbers of granules in the parasite intestinal epithelial cells and to host erythrocytes present in the intestinal lumens. In immunogold electron microscopy, the gold particles were consistently deposited on the electron-dense secretory granules in intestinal epithelial cells and on the intestinal contents. These findings suggest that the Fasciola cysteine protease in the secretory granules is secreted as a digestive enzyme into the intestinal lumen, where it may play an important role in the extracellular degradation of host proteins, including hemoglobin.


Neuroanatomy of Cysticercus cellulosae (Cestoda) as revealed by acetylcholinesterase and nonspecific esterase histochemistry.

The nervous system of Cysticercus cellulosae, the metacestode stage of the tapeworm Taenia solium, was delineated using histochemical methods for the localization of the enzyme markers; nonspecific esterase and acetylcholinesterase. The main features of the nervous system include a pair of cerebral ganglia, a circumcerebral nerve ring, a rostellar nerve ring, and anterior and posterior nerves and their branches. The posterior nerves form a subtegumental network in the strobila and the bladder wall. A nerve network around excretory tubules could also be demonstrated, suggesting neuronal control of excretion in the metacestode. No sheath was observed around the nervous system. The morphological features described suggest "cephalization" of the nervous system in this parasite.


Summer-arrested development of abomasal trichostrongylids in Awassi sheep in semi-arid areas of north-west Syria.

Inhibition in trichostrongylids was studied in Awassi sheep in North-west Syria, which has cold winters and hot summers. On six occasions during a 1-year period, five ewes with natural helminth infections were slaughtered after being held in pens for 3 weeks. The percentage of inhibited larvae was lowest in January (6%); it increased during the spring to 76.7% in April and to 84.6% in June and then decreased during the autumn (57.1% in September, 26.1% in December). Inhibition occurred mainly during the early fourth stage in mucosa but was also observed during the late fourth stage in digesta. Teladorsagia circumcincta was the main species involved, but it seemed that this phenomenon also occurred in Marshallagia marshalli.


Transmission electron microscopy of sensory receptors of Echinostoma revolutum (Froelich 1802) cercaria (Digenea: Echinostomatidae).

Nonciliate, uniciliate, and multiciliate receptors were shown by transmission electron microscopy to occur in the body tegument of Echinostoma revolutum (Froelich 1802) cercariae. Detailed studies revealed three types of nonciliate, four types of uniciliate, and one type of multiciliate sensory endings. Most receptors were singly distributed. Only a few of them formed paired receptor complexes in three regions of the cercarial body. These receptor complexes were found to possess nonciliate, uniciliate, and multiciliate sensory endings.


In vitro culture of the strobilar stage of Echinococcus granulosus of sheep and donkey origin from Jordan.

Protoscolices of Echinococcus granulosus isolated from hydatid cysts of sheep and donkeys in Jordan were cultured in vitro using a modified diphasic culture system. Protoscolices from these two sources manifested differences in the mode of development, evagination and growth rates. Protoscolices isolated from sheep cysts grew in vitro in the polyzoic direction up to the three- to four-segmented mature worms, reaching a length of 2.9 mm. In contrast, donkey protoscolices failed to develop beyond the early stages, even after 67 days of culturing. On prolonged culturing, few worms of donkey origin reached the banding and segmentation stages, attaining a maximal length of 1.6 mm at periods ranging between 81 and 114 days of culturing. None of these segmented worms showed genital differentiation or analgen. The evagination and growth rates of protoscolices isolated from donkey liver cysts were compared with those obtained from sheep liver or lung cysts. The most significant difference in these rates occurred at the commencement of the segmentation stage. Differences in the development, growth and evagination rates observed between the donkey and sheep forms may reflect the strain variation of E. granulosus in this country.


Host specificity of Giardia muris isolates from mouse and golden hamster.

One isolate of Giardia muris from a naturally infected laboratory mouse (Mus musculus) and one from a naturally infected golden hamster (Mesocricetus auratus) were passaged three times by the inoculation of ten cysts (the minimal infectious dose) into barrier-maintained homologous hosts. Both of the resultant isolates were tested for infectivity by intragastric inoculation of 3-5 x 10(5) cysts into 40 mice (2 inbred strains), 40 rats (2 inbred strains), and 19 golden hamsters (1 outbred strain). Rats were not susceptible to infection with either isolate. Mice and golden hamsters did develop infections following their inoculation with the heterologous isolates. The mean intensity of heterologous infections with the hamster isolates was significantly lower than that of homologous infections. The mouse isolate induced a higher mean intensity of infection in hamsters as compared with homologous recipients. The mean intensity of infections induced by both isolates was greater in male hamsters than in females.


The solution structure of echistatin: evidence for disulphide bond rearrangement in homologous snake toxins.

The solution structure of the fibrinogen antagonist, echistatin, has been determined by a combination of NMR and simulated annealing methods. While the structure of the disulphide-linked core is well-defined by the NMR data, the N- and C-termini and the loop bearing the RGD sequence (which is responsible for the fibrinogen antagonist properties) are poorly defined. The pattern of disulphide bridges, which could not be determined by classical methods, was predicted by a statistical analysis of the simulated annealing structures. This pattern is distinct from that for the homologous protein kistrin, leading to the novel suggestion that homologous proteins possess non-conserved patterns of disulphide bridges.


Construction of a dictionary of sequence motifs that characterize groups of related proteins.

An automatic procedure is proposed to identify, from the protein sequence database, conserved amino acid patterns (or sequence motifs) that are exclusive to a group of functionally related proteins. This procedure is applied to the PIR database and a dictionary of sequence motifs that relate to specific superfamilies constructed. The motifs have a practical relevance in identifying the membership of specific superfamilies without the need to perform sequence database searches in 20% of newly determined sequences. The sequence motifs identified represent functionally important sites on protein molecules. When multiple blocks exist in a single motif they are often close together in the 3-D structure. Furthermore, occasionally these motif blocks were found to be split by introns when the correlation with exon structures was examined.


Ligand requirements for Ca2+ binding to EGF-like domains.

Site-specific mutagenesis studies of the first epidermal growth factor-like (EGF-like) domain of human clotting factor IX suggest that the calcium-binding site present in this domain (dissociation constant Kd = 1.8 mM at pH 7.5 and ionic strength I = 0.15) involved the carboxylate residues Asp47, Asp49 and Asp64. To further characterize the ligands required for calcium binding to EGF-like domains, two new mutations, Asp47----Asn and Asp49----Asn, were introduced into the domain by peptide synthesis. 1H-NMR spectroscopy was used to obtain the dissociation constants for calcium binding to these mutations. Calcium binding to the Asp49----Asn modified domain is only mildly affected (Kd = 6 mM, I = 0.15), whereas binding to the Asp47----Asn modified domain is severely reduced (Kd = 42 mM, I = 0.15). From these data, it is proposed that the anionic oxygen atoms of the side chains of residues 47 and 64 are essential for calcium binding, whereas the side chain ligand for calcium at residue 49 can be a carboxyamide oxygen. As a control, the introduction of the modification Glu78----Asp in a region of the domain not believed to be involved in calcium binding had very little effect on the Kd for calcium (Kd = 2.6 mM, I = 0.15). Finally, the effect of an Asp47----Gly substitution found in the natural haemophilia B mutant, factor IXAlabama, was investigated. This peptide has a markedly reduced affinity for calcium (Kd = 37 mM, I = 0.15), suggesting that the defect in factor IXAlabama is due to impaired calcium binding to its first EGF-like domain.


Comparison of the modelled thyroxine binding site in TBG with the experimentally determined site in transthyretin.

The structure of cleaved thyroxine-binding globulin (TBG) has been modelled on the crystal structure of cleaved alpha 1-antitrypsin (a member of the serine proteinase inhibitor, serpin, superfamily) based on the high sequence homology exhibited by the two proteins. Particular attention was paid to the identification and modelled characteristics of the thyroxine binding site. The primary aim of the study was to compare the site qualitatively with the crystallographically determined binding site of transthyretin, the other major transporter of thyroxine, in an attempt to explain the higher binding affinity of the site compared with the known thyroxine binding site in transthyretin (10(10) versus 10(8) M-1). The proposed binding site shares some similar characteristics with the transthyretin binding site but also includes a cluster of aromatic residues which are entirely absent in transthyretin. It is proposed that this might account for the substantial difference in binding affinities.


Effects of signal peptide changes on the secretion of bovine somatotropin (bST) from Escherichia coli.

Bovine somatotropin (bST) was secreted from Escherichia coli at moderate levels of 1-2 micrograms/ml/OD using expression vectors in which the bST gene was fused to the lamB secretion signal. To study the secretion properties of bST in E.coli further, two approaches for modifying the secretion signal were employed. In the first case, fusion proteins were constructed with six alternative bacterial secretion signals: three from E.coli proteins (HisJ, MalE and OmpA), two from bacteriophage proteins (M13 coat protein and PA-2 Lc) and one from the chitinase A protein of Serratia marcescens. The results, as monitored by Western blot analysis of both total cell protein and the periplasmic fraction, showed that these changes in the secretion signal did not significantly affect the secretion properties of bST. In the second approach, a library of random mutations was created in the lamB secretion signal and 200 independent clones were screened. The level of secreted bST was determined by growing individual clones in duplicate in microtiter wells, inducing protein expression and measuring the bST released by osmotic shock using a particle concentration fluorescent immunoassay. The secretion properties of several novel variants in the LamB signal peptide are presented.


Improved insulin stability through amino acid substitution.

Insulin analogs designed to decrease self-association and increase absorption rates from subcutaneous tissue were found to have altered stability. Replacement of HB10 with aspartic acid increased stability while substitutions at B28 and/or B29 were either comparable to insulin or had decreased stability. The principal chemical degradation product of accelerated storage conditions was a disulfide-linked multimer that was formed through a disulfide interchange reaction which resulted from beta-elimination of the disulfides. The maintenance of the native state of insulin was shown to be important in protecting the disulfides from reduction by dithiothreitol and implicitly from the disulfide interchange reaction that occurs during storage. To understand how these amino acid changes alter chemical stability, the intramolecular conformational equilibria of each analog was assessed by equilibrium denaturation. The Gibbs free energy of unfolding was compared with the chemical stability during storage for over 20 analogs. A significant positive correlation (R2 = 0.8 and P less than 0.0005) exists between the conformational stability and chemical stability of these analogs, indicating that the chemical stability of insulin's disulfides is under the thermodynamic control of the conformational equilibria.


Altering the association properties of insulin by amino acid replacement.

The importance of ProB28 and LysB29 on the self-association of insulin was established by systematically truncating the C terminus of the B chain. The relationship between structure and association was further explored by making numerous amino acid replacements at B28 and B29. Association was studied by circular dichroism, size-exclusion chromatography and ultracentrifugation. Our results show that the location of a prolyl residue at B28 is critical for high-affinity self-association. Removal of ProB28 in a series of C-terminal truncated insulins, or amino acid replacement of ProB28, greatly reduced association. The largest disruption to association was achieved by replacing LysB29 with Pro and varying the amino acid at B28. Several of the analogs were predominantly monomers in solutions up to 3 mg/ml. These amino acid substitutions decreased association by primarily disrupting the formation of dimers. Such amino acid substitutions also substantially reduced the Zn-induced insulin hexamer formation. The formation of monomeric insulins through amino acid replacements was accompanied by conformational changes that may be the cause for decreased association. It is demonstrated that self-association of insulin can be drastically altered by substitution of one or two key amino acids.


Selection of a thermostable variant of chloramphenicol acetyltransferase (Cat-86).

The moderate thermophile Bacillus stearothermophilus was used as a host in which to detect more thermostable variants of the B.pumilus chloramphenicol acetyltransferase (Cat-86) protein. Seventeen mutants were isolated and detected by their ability to grow in the presence of chloramphenicol at a previously restrictive temperature (58 degrees C). The genes encoding these proteins were sequenced; all 17 mutants carried the same C to T transition that conferred an amino acid substitution of alanine by valine at position 203 of the protein sequence. The wild-type and one mutant Cat-86 protein were purified to homogeneity using affinity chromatography, and kinetic and thermal stability studies were undertaken. Both enzymes had similar sp. act. in the region of 215 U/mg, with Km values for chloramphenicol in the range 13.8-15.4 microM and for acetyl CoA in the range 13.6-15.5 microM. The A203V mutant shows greater stability than the wild-type Cat-86 protein at temperatures above 50 degrees C and appears to pass through a transition state between 48 and 50 degrees C.


Cumulative stabilizing effects of glycine to alanine substitutions in Bacillus subtilis neutral protease.

Oligonucleotide-directed mutagenesis has been used to replace glycine residues by alanine in neutral protease from Bacillus subtilis. One Gly to Ala substitution (G147A) was located in a helical region of the protein, while the other (G189A) was in a loop. The effects of mutational substitutions on the functional, conformational and stability properties of the enzyme have been investigated using enzymatic assays and spectroscopic measurements. Single substitutions of both Gly147 and Gly189 with Ala residues affect the enzyme kinetic properties using synthetic peptides as substrates. When Gly replacements were concurrently introduced at both positions, the kinetic characteristics of the double mutant were roughly intermediate between those of the two single mutants, and similar to those of the wild-type protease. Both mutants G147A and G189A were found to be more stable towards irreversible thermal inactivation/unfolding than the wild-type species. Moreover, the stabilizing effect of the Gly to Ala substitution was roughly additive in the double mutant G147A/G189A, which shows a 3.2 degrees C increase in Tm with respect to the wild-type protein. These findings indicate that the Gly to Ala substitution can be used as a strategy to stabilize globular proteins. The possible mechanisms of protein stabilization are also discussed.


A structural role of histidine 15 in human glutathione transferase M1-1, an amino acid residue conserved in class Mu enzymes.

His15 is a conserved amino acid residue in all known class Mu glutathione transferases. This His residue in human glutathione transferase M1-1 has been mutated into 17 different amino acid residues by means of site-directed random mutagenesis to determine if any substitutions are compatible with catalytic activity. The majority of the mutant proteins appeared unstable and could not be isolated in reasonable quantities by heterologous expression in Escherichia coli. Five mutant enzymes, H15C, H15K, H15N, H15Q and H15S were purified and more extensively characterized. The mutant proteins shared the same size as that of the wild-type enzyme but could be separated from the parental enzyme by reverse phase HPLC. For all the mutant forms except H15N, the sp. act. with 1-chloro-2,4-dinitrobenzene was less than 3% of the wild-type value--the H15N mutant enzyme displayed 29% of the wild-type activity. None of the catalytically active mutant enzymes showed any major alteration of the binding affinity for the substrate analog S-hexylglutathione, suggesting that His15 is not part of the active site of the enzyme. The high activity of the mutant H15N, also reflected in the kcat/Km, V and S0.5 values, rules out the possibility that His15 in the native enzyme contributes to catalysis by serving as a base. The role of His15, largely replaceable by Asn in the same position, appears to be structural, probably involving hydrogen bonds that maintain the protein in a stable and catalytically active conformation. A critical structural role of His15 in a buried position may explain the evolutionary conservation of this residue in the class Mu glutathione transferases.


Analysis of disulphide bridge function in recombinant bovine prolactin using site-specific mutagenesis and renaturation under mild alkaline conditions: a crucial role for the central disulphide bridge in the mitogenic activity of the hormone.

We have previously described a method for isolating Escherichia coli-produced methionyl bovine prolactin (Met-bPRL) and its renaturation using thioredoxin. This report describes an alternative renaturation procedure in which extracted Met-bPRL is incubated in air at pH 10 and 20 degrees C. Within 1 h of such treatment essentially all of the reduced Met-bPRL was converted to the oxidized form; this was accompanied by an increase to full mitogenic activity in the Nb2 cell bioassay. It was also found that, to minimize contamination by high mol. wt Met-bPRL derivatives, it is essential to have a reducing agent (dithiothreitol) present during disruption of the bacteria and to extract the protein at neutral pH. The contribution of each of the three disulphide bridges in bPRL to its bioactivity was studied with Met-bPRL variants, prepared via site-specific mutagenesis, in which cysteines were replaced by serines to prevent disulphide bond formation. Variants lacking the C4-C11 bridge, the C191-C199 bridge or both these terminal bridges were as mitogenic as authentic bPRL. (Variants lacking the C191-C199 bridge had markedly increased solubility in the presence of deoxycholate.) In contrast, variants lacking the C58-C174 bridge had greatly reduced bioactivity, indicating that integrity of the large disulphide loop is crucial to the hormone's mitogenic activity.


Specificity determinants of rat tissue kallikrein probed by site-directed mutagenesis.

Site-specific mutagenesis was employed to study structure-function relationships at the substrate binding site of rat tissue kallikrein. Four kallikrein mutants, the Pro219 deletion (P219del), the 34-38 loop Tyr-Tyr-Phe-Gly to Ile-Asn mutation [YYFG(34-38)IN], the Trp215----Gly exchange (W215G) and the double mutant with Tyr99----His and Trp215----Gly exchange (Y99H:W215G) were created by site-directed mutagenesis to probe their function in substrate binding. The mutant proteins were expressed in Escherichia coli at high levels and analyzed by Western blot. These mutant enzymes were purified to apparent homogeneity. Each migrated as a single band on SDS-PAGE, with slightly lower molecular mass (36 kDa) than that of the native enzyme, (38 kDa) because of their lack of glycosylation. The recombinant kallikreins are immunologically identical to the native enzyme, displaying parallelism with the native enzyme in a direct radioimmunoassay for rat tissue kallikrein. Kinetic analyses of Km and kcat using fluorogenic peptide substrates support the hypothesis that the Tyr99-Trp215 interaction is a major determinant for hydrophobic P2 specificity. The results suggest an important role for the 34-38 loop in hydrophobic P3 affinity and further show that Pro219 is essential to substrate binding and efficient catalysis of tissue kallikrein.


pH-sensitive interactions between IgG and a mutated IgG-binding protein based upon two B domains of protein A from Staphylococcus aureus.

A fusion protein, consisting of the N-terminal 81 amino acids from an inactive bovine DNase I (Q38,E39-E38,Q39) and two sequential synthetic IgG-binding domains based upon domain B of Protein A from Staphylococcus aureus has been shown to bind to porcine IgG with a similar affinity and pH profile to Protein A. The same residue in each B domain (Tyr111 and Tyr169) has been mutated by cassette mutagenesis to Ser, Glu, His, Lys or Arg and the effect of the mutation on binding interactions with porcine IgG investigated. The evidence presented suggests that the interactions at the B domain are highly sensitive to the presence of a charged residue.


Overexpression and structure--function analysis of a bioengineered IL-2/IL-6 chimeric lymphokine.

A synthetic chimeric IL-2/IL-6 gene was synthesized to engineer a bifunctional lymphokine which was overproduced in Escherichia coli. Following denaturation of the inclusion bodies in 6 M guanidine and refolding and reoxidation in the presence of a redox system, the fusion protein (rIL-2/IL-6) was purified to homogeneity and shown to react with both monospecific anti-IL-2 and anti-IL-6 antisera. A collagen-like spacer was introduced between the two cytokine moieties to generate IL-2 and IL-6 molecules upon collagenase digestion. After cleavage, the two subunits, purified in a single-step procedure, were found to be correctly reoxidized and functionally as active as their native counterparts. Circular dichroism studies of rIL-2/IL-6 revealed that both cytokine subunits refolded independently and exhibited the alpha-helical structures characteristic of the corresponding wild-type lymphokines. The chimera displayed full IL-2 activity in the CTLL-2 cell proliferation assay. It also retained the IL-6 property to enhance IgM synthesis in SKW6.4 cells, induce the proliferation of B-cell hybridomas and stimulate the production of fibrinogen in hepatocytes. Because IL-2 amplifies the cellular immune response and IL-6 up-regulates the humoral response, this bifunctional lymphokine represents a potentially useful therapeutic adduct and may serve as an immunomodulator to enhance the host's response to vaccination.


The structure of the complex between influenza virus neuraminidase and sialic acid, the viral receptor.

Crystallographic studies of neuraminidase-sialic acid complexes indicate that sialic acid is distorted on binding the enzyme. Three arginine residues on the enzyme interact with the carboxylate group of the sugar which is observed to be equatorial to the saccharide ring as a consequence of its distorted geometry. The glycosidic oxygen is positioned within hydrogen-bonding distance of Asp-151, implicating this residue in catalysis.