Simulation of compartmental models for kinetic data from a positron emission tomograph.

Linear compartmental models are used to describe the disposition of radio-labelled compounds in regions of interest in the mammalian body, based on a time sequence of measurements from a positron emission tomograph (PET). In this paper we show how closed form solutions for the model equations have been incorporated into a computer program for simulation and parameter estimation. A typical PET data example is included to illustrate the implementation and compare the closed form method with a numerical ode solution method.


Development of an expert system advisor for anaesthetic control.

A review is given of numerous approaches which have been taken to provide automated control of depth of anaesthesia. Most of these approaches use a single indicator for anaesthetic state, and do not perform adequately for such a complex system. It is desirable, therefore, to merge a number of qualitative clinical signs and quantitative on-line measurements to provide decision-support for control of anaesthetic depth. The paper describes such an expert system called Resac (Real time Expert System for Advice and Control). Details of the knowledge representation and inference structure are given, together with the method adopted for propagating uncertainty measures, combining fuzzy information and merging quantitative and qualitative indicators. The importance of good human machine interface design is shown via the GEM-based graphics of Resac.


Changes in serum levels of SHBG, endogenous ligands and levonorgestrel induced by ethinylestradiol in Norplant users.

This work was done in search for a model to examine target organ response to fluctuations in serum levels of sex hormone-binding globulin (SHBG) and its ligands. The time course and magnitude of fluctuations of SHBG, levonorgestrel (L-Ng), estradiol (E2), testosterone (T) and dihydrotestosterone (DHT) in serum were examined during and after treatment with 50 ug of ethinylestradiol (EE2) daily for 10 days in 10 volunteer women using NORPLANT implants. Six of these volunteers were also treated with 20 ug of EE2 daily for 7 days and two additional volunteers using a copper-T IUD were treated with 50 ug of EE2 daily for 7 days. In all cases, SHBG and L-Ng levels increased in a close parallel manner several-fold above basal levels during treatment, reaching a maximum around two days after the last EE2 pill. In contrast, the levels of E2 and T increased in one subject, decreased in 5 and remained unchanged in 4, while changes of DHT were unrelated to those of the other ligands. Since the L-Ng "secretion rate" by NORPLANT implants is constant, it follows that the effect of EE2 on its levels is due to a decreased metabolic clearance rate, most likely secondary to the increased binding of L-Ng to SHBG in serum. This interpretation is in agreement with the close parallelism in the fluctuations of L-Ng and SHBG. It is concluded that in NORPLANT users, SHBG and L-Ng, but not the endogenous ligands, behave in a predictable manner in response to EE2. Thus, this model affords the possibility of exploring the influence of SHBG on tissue response to progestins.


Cervical softening with mifepristone (RU 486) after pretreatment with naproxen. A double-blind randomized study.

Pretreatment with the progesterone antagonist mifepristone reduces the stiffness and facilitates mechanical dilatation of the uterine cervix. We studied the influence of the cyclo-oxygenase inhibitor naproxen on the softening effect of mifepristone on the cervix in thirty nulliparae. The patients were randomly allocated to receive 500 mg naproxen (group A) or placebo (group B) orally 60, 48, 36, 24 and 12 hours prior to vacuum aspiration. All patients received 100 mg mifepristone 48 and 36 hours before surgery. We found that the cervical softening effect of mifepristone was not antagonized by naproxen. The study indicates that the effect of mifepristone on the early pregnant cervix is not mediated through an increased production of prostaglandins.


Effects of the prostanoid receptor antagonist, di-4-phloretin phosphate, upon human sperm motility.

The inhibitory effects of the prostanoid receptor antagonist, di-4-phloretin phosphate (DPP), upon washed human sperm motility were determined in vitro (up to 60 min of incubation). Concentrations tested were: 0.001, 0.01, 0.1, 0.25, 0.5, 2.5, 5.0 and 10.0 mM. All concentrations of DPP investigated caused cessation of sperm movement (percentage motility and average forward velocity) and the antimotility effect was essentially irreversible. The concentrations producing 50% inhibition were: percentage motility--1.31 +/- 0.56 mM (mean +/- SEM) and average forward velocity--1.95 +/- 0.68 mM. The mode of antimotility action appears to be multi-faceted. At high (10 mM) and intermediate (2.5 mM) concentrations, changes in fluidity of sperm plasmalemma (hypoosmotic swelling test) and loss of viability (nigrosin-eosin stain technique) constitute the primary means by which motility was disrupted. In contrast, these two parameters of motility remained unaltered at a lower concentration (0.25 mM) of DPP, and the mechanism precipitating the antimotility effect may involve mobilization of stored calcium ions and modulation of cyclic AMP levels.


Effects of multiglycosides of Tripterygium wilfordii (GTW) on rat fertility and Leydig and Sertoli cells.

Primary cultures of rat Leydig and Sertoli cells were used to evaluate the direct effects of GTW on testicular cells and to compare these to the effects of gossypol acetate. Both GTW and gossypol acetate can affect the survival of Leydig and Sertoli cells. But Sertoli cells are much more sensitive than Leydig cells, either to gossypol acetate or GTW. Leydig and Sertoli cells all died when they were exposed to gossypol acetate or GTW at a dose of 3.0 micrograms/ml or 30 micrograms/ml, respectively, for 24 hours. The cell survival-time course demonstrated that the cell numbers were decreased after 2 hours, and especially so after 8 hours. No significant changes were observed in testosterone production in Leydig cells after 24 hours of exposure to 1.0-20 micrograms/ml GTW. The forward motility of epididymal spermatozoa was completely lost and fertility of rat was significantly inhibited after the treatment of GTW in vivo. It is concluded that GTW does affect the fertility of rat and viability of cultured rat Leydig and Sertoli cells.


Intrahepatic cholestasis: a review of biochemical-pathological mechanisms.

Intrahepatic cholestasis involves impaired excretion of bile via the hepatobiliary system as a consequence of one or more lesions within the liver. In humans, intrahepatic cholestasis most often results as a side-effect of drug therapy and the clinical manifestation of this condition, jaundice, has been estimated to account for hospitalization in 2 to 5% of the cases for the general population and approaches as much as 20% in the elderly. With the aging of the population and the common occurrence of poly-drug therapy in geriatric patients, it is to be expected that jaundice due to drug-induced intrahepatic cholestasis will become even more prevalent, and accordingly the need to understand the basic mechanisms of this disease condition will become more urgent. The list of culprit agents implicated in the induction of intrahepatic cholestasis in humans is continually expanding. These include various steroid hormones, bile acids, drugs and other chemicals. Experimentally, a wide spectrum of agents has been shown to precipitate intrahepatic cholestasis. Over the years, a number of hypotheses on the biochemical and pathological mechanisms of intrahepatic cholestasis has emerged, including the following: impaired sinusoidal membrane function; interference with the distribution and binding of cytoplasmic endogenous carrier proteins; interference with mitochondrial energy supply; defects in the canalicular membrane including altered Na+/K+ -ATP-ase activity; impairment of microfilament and microtubule functions; interference with bile secretion involving bile acid dependent and independent fractions, and altered bile acid metabolism due to "hypoactive hypertrophic smooth endoplasmic reticulum". In partial agreement with the latter hypothesis, our studies indicated that impairment of the endoplasmic reticulum might represent one of the early stages in the development of intrahepatic cholestasis. Various experimental conditions that induce intrahepatic cholestasis to different degrees resulted in an interference of the synthesis of microsomal phospholipids and altered microsomal function. The conditions included the administration of various hepatotoxic compounds or steroids, pregnancy, delayed development of the endoplasmic reticulum in neonates, and dietary methyl donor or choline deficiency. This review reports the biochemical-pathological mechanisms postulated to be involved in the genesis of intrahepatic cholestasis with specific reference to experimental models of drug-induced intrahepatic cholestasis. The important practical implications of cholestasis are also briefly surveyed.


Diurnal effect on caffeine acetylation phenotyping: a preliminary report.

The present study examined whether caffeine acetylation phenotype could be altered by its time of administration. Caffeine was given orally to nine healthy subjects at 10 a.m. and 10 p.m. and acetylation phenotype was determined by measuring the major metabolites of caffeine in urine. The results showed that acetylation phenotypes determined in the day trial were not different from those determined during the night trial.


Chlorpromazine enhancement of epirubicin cytotoxicity in vitro: effects on plasma membrane and DNA damage.

The frequently used antiemetic drug chlorpromazine has previously been shown to augment anthracycline-induced toxicity to cultured Chinese hamster fibroblasts measured by cloning /1/. We therefore tested the ability of chlorpromazine to affect the induction of plasma membrane and DNA damage by the anthracycline epirubicin. Plasma membrane damage was determined by the cells' ability to accumulate 86Rb after incubation with 10 mg/l epirubicin, and DNA damage was determined by measuring the amount of DNA precipitation after incubation with 25 mg/l epirubicin. The epirubicin-induced inhibition of 86Rb-accumulation as well as the enhancement of epirubicin-induced DNA damage were markedly enhanced in the presence of chlorpromazine. Chlorpromazine augmentation of epirubicin cytotoxicity, including plasma membrane and DNA damage, may be due to its calmodulin antagonistic action and related to the maintenance integrity. Further studies are justified to evaluate the effects of chlorpromazine influence on antineoplastic drug action in vitro and in vivo.


Use of nitrates in ischemic heart disease.

The organic nitrates have remarkably diverse actions that are or should be beneficial in patients with ischemic heart disease. These drugs are effective in all the important ischemic syndromes. Preliminary data in patients with acute infarction suggest that the drugs may be truly cardioprotective, resulting in improved mortality. This review has not discussed the role of nitrates in congestive heart failure or LV dysfunction, a subject of great importance. The nitrates are useful adjunctive agents in these syndromes, and the two VeHfT trials support the concept that long-term nitrate administration, in conjunction with hydralazine, may favorably alter the natural history of heart failure. This cardioprotective effect is similar to that suggested for the post-MI patient. The data are not strong enough for definitive conclusions at this time. The clinical benefits of nitrates in decreasing subjective (angina) and objective indices of ischemia in stable and unstable angina, as well as limited data in asymptomatic myocardial ischemia, are unequivocal and are as favorable as those for beta blockers or calcium antagonists. Tolerance is an important problem that unfavorably influences the potential benefits of nitrate therapy. I believe that this problem can be avoided with well-designed dosing regimens. Current research into endothelial biology in health and disease has further supported a physiologic role for the organic nitrates in patients with ischemic heart disease. The nitrate-platelet story, while controversial, is promising and offers another positive rationale for nitrate administration. The concept of nitrates replenishing disordered EDRF release or action is an exciting one. Physicians should feel fortunate to have such a remarkable group of drugs available for their patients.


Ear psoriasis.

Psoriasis of the ears occurs in approximately 18 percent of all patients at some time. Prevention by avoidance of picking and scratching and careful cleansing are helpful. Treatment with intralesional injection of triamcinolone acetonide is effective in patients resistant to other therapies.


Trapshooter's stigma.

We report an unusual episode of friction dermatitis combined with residual scarring in a member of the United States trapshooting team.


Mogul skier's palm: traumatic hypothenar ecchymosis.

A unique purpuric--golden lesion on the hypothenar eminence of each palm is described in a mogul skier. These painful lesions, resulting from vigorous repetitive pole planting in the "bumps" at a ski area, are postulated to represent a form of localized soft tissue injury.


Athlete's nodules: sports-related connective tissue nevi of the collagen type (collagenomas).

Sports-related connective tissue nevi of the collagen type (collagenomas) have been referred to as athlete's nodules. Surfers, boxers, marbles players, and football players are some of the athletes in whom these lesions have been observed. The nodules can be found on the dorsal aspect of the feet, knees, or knuckles and can readily be differentiated from other conditions by either clinical history or microscopic features or both. Treatment options include conservative measures or surgical intervention. Recurrent trauma and friction to the involved location are likely causative factors. Although the ultrastructural pathogenesis remains to be established, changes in the molecular metabolism of collagen resulting in enhanced synthesis and/or accumulation of collagen may have a contributory role.


Herpes gladiatorum: a growing problem in sports medicine.

Herpes skin infections among wrestlers--called herpes gladiatorum--occur frequently among high school and college wrestlers nationwide. Contact with opponents with active herpes infections appears to be the strongest risk factor associated with acquisition of infection. Previously acquired antibody to herpes simplex virus 1 infection may offer some protection from acquiring this disease. Morbidity associated with this disease can be significant, and widespread efforts to educate participants in close contact sports, such as wrestling, are advocated as control measures.


Micronuclei induction in pike (Esox lucius) in Swedish lakes contaminated with radiocaesium.

Micronuclei frequency in fish from Swedish lakes with high concentrations of radiocaesium was investigated. The frequency of micronucleus induction in fish varied in different lakes but was not correlated with caesium concentrations.


Interaction of androgens and prolactin on prostatic enzymes of the pyruvate-malate cycle involved in lipogenesis in castrated mature monkey, Macaca radiata.

Effects of androgens, prolactin (Prl) and bromocriptine (Br) on the specific activities of prostatic (caudal and cranial) enzymes of the pyruvate-malate cycle were studied in castrated mature bonnet monkeys. Castration decreased the activity of NADP+ isocitrate dehydrogenase (ICDH), ATP citrate lyase, malate dehydrogenase (MDH), malic enzyme and fatty acid synthase (FAS). Administration of testosterone propionate (TP)/dihydrotestosterone (DHT) increased the activities of all these enzymes in both lobes. Malate dehydrogenase maintained normal activity. Prl also had a stimulatory effect on the enzymes and was further enhanced when Prl was given in combination with TP/DHT. Unlike Prl, bromocriptine treatment inhibited all the enzymes in both lobes. Thus, prolactin was found to have a direct as well as a synergistic effect with androgens on enzymes of the pyruvate-malate cycle in the prostate of castrated mature monkeys.


Effect of cisplatin on lipid peroxidation in pig blood platelets.

The effect of cisplatin (cis-diamminedichloroplatinum II) on the activities of the blood platelet antioxidative enzymes (glutathione peroxidase, superoxide dismutase) as well as on the peroxidation of blood platelet lipids was investigated. Cisplatin was found to cause a significant inhibition of platelet free radical scavering enzyme activity and an increase of malonyldialdehyde levels in blood platelets incubated with cisplatin in vitro.


Utilization of phytohaemagglutinin for in vivo karyological studies in teleost fish.

A simple technique is described for increasing the number of mitoses in circulating blood and in the kidney of teleost fish. This method employed phytohaemagglutinin-P solution, a T-like cell mitogen, which was injected (1 mg/100 g body wt) intraperitoneally, and 53 h later many blasts suitable for karyological studies, blocked in metaphase, were present both in the circulating blood and in the kidney.


A comparison of a solid phase IRC assay and the PSIFT for detection of antibodies to platelets.

A rapid solid phase indicator red cells assay (IRCA) for detection of platelet antibodies was developed and its sensitivity compared with PSIFT. Platelets were attached to the surface of polystyrene microtitre plate wells by means of a sodium carbonate buffer and centrifugation. Uncovered areas were blocked by a gelatin blocking buffer. After serum incubation bound platelet-specific antibodies were made visible by anti-IgG-coated indicator red cells and a brief centrifugation. A positive result, meaning the presence of an anti-platelet antibody was indicated by red cell adherence over the reaction surface. In the absence of serum antibodies to platelets the indicator red cells formed a pellet. The IRCA showed a high sensitivity; the anti-platelet antibody Thrombocyte was detectable until a dilution of 1:1,600 whereas the same antibody in the PSIFT could only be detected until a dilution of 1:400.


Effect of phorbol ester PMA and puromycin on the maturation of mouse oocytes.

The effect of phorbol myristate acetate (PMA) and puromycin (PU) on maturation divisions of mouse oocytes was investigated. PMA changed the morphology and function of the spindle in meiosis I. The spindle had a broad polar region and did not commence karyokinesis in the first maturation division. This persisted in 25% of spindles which were passive up to 30 h of maturation. Half of the oocytes with spindles blocked by PMA between 20 and 30 h of maturation became activated, and 25% of the initial pool underwent abortive karyokinesis consisting of segregation of several chromosomes of the metaphase plate. Cytological observation indicated that the presence of PMA interfered with the elongation of the spindle in anaphase. Under the condition of changed phosphorylation of maturation of proteins by PMA, a reversible interphase was induced by PU. Most of the nuclei induced by PU exhibited pronuclei. A new class of nuclei induced by PU with condensed chromatin and having no nucleoli is described. The mechanism and consequences of interference of PMA on anaphase are discussed.


Penetration of fleroxacin into human and animal tissues.

Fleroxacin concentrations in human and rat tissues were determined by HPLC following extraction with dichloromethane:isopropanol. This method yielded a high recovery of more than 85%. In the investigated tissues the fleroxacin levels were equal to or higher than those concomitantly measured in plasma. The concentration ratios 'tissue/plasma' were 1.6-2.7 for lung, 1.9-2.1 for muscle, 1.1-1.9 for gynaecological tissues and 1.2 for bone. Only in the case of fat and lens/eye lower ratios of 0.05-0.5 were found. The actual measured fleroxacin concentrations in most tissues and in plasma were high. Following oral administration of the recommended therapeutic dose of 400 mg, once daily, peak concentrations of 5-6 mg/l were reached in human plasma at steady state. Even 24 h after drug intake the fleroxacin level was still approximately 1 mg/l and thus in the range of the MIC90 values of susceptible bacteria causative for many types of tissue infections.


Detection of antimicrobial drugs with Bacillus subtilis strain ATCC 6633: an update.

Bacillus subtilis strain ATCC 6633 was examined for susceptibility to 30 additional antimicrobial drugs, the majority of which had been released during the 1980s. The data served to update previous findings with regard to the suitability of this assay strain for the detection of chemotherapeutic agents in fluid clinical specimens.


Saperconazole: in vitro and in vivo anticandidal activity.

The in vitro activity of saperconazole against eight candidal species (81 strains) was determined and compared with fluconazole, Sch 39304 and amphotericin B. Using brain heart infusion broth with an inoculum of 10(4) CFU/ml, the MIC ranges (micrograms/ml) of saperconazole were: less than or equal to 0.015- greater than 32 for Candida albicans, less than or equal to 0.015-16 for C. tropicalis, less than or equal to 0.015-32 for C. glabrata, less than or equal to 0.015-32 for C. parapsilosis, less than or equal to 0.015-0.12 for C. guilliermondii and less than or equal to 0.015-0.06 for C. krusei. Saperconazole was the most active agent tested against Candida species. Saperconazole and 5-fluorocytosine combinations showed synergistic interactions against Candida species, and no antagonistic interaction was demonstrated. In a rat vaginal candidiasis infection model, saperconazole and fluconazole were equipotent producing 75-100% cures at levels of 0.016-0.25%, respectively, when dosed intravaginally. After single oral dosing, saperconazole was 5-fold more potent than fluconazole with an ED50 value of 0.53 mg/kg. These data demonstrate that saperconazole is effective in a rat vaginal candidiasis infection either with a single oral dose or by intravaginal treatment.


In vivo postantibiotic effect of isepamicin and other aminoglycosides in a thigh infection model in neutropenic mice.

The object of this work is to study in neutropenic mice the in vivo postantibiotic effect (PAE) of isepamicin, a new aminoglycoside, gentamicin and netilmicin on Staphylococcus aureus and Escherichia coli and in vivo killing kinetics using two different schedules (A and B) of isepamicin and gentamicin administration against S. aureus: (A) at time zero and every hour up to the 7th or 9th hour and (B) two doses only, at time zero and at the end of the PAE. The PAE of the three aminoglycosides was long (3-5 h), showing that of isepamicin to be the largest, especially on S. aureus. Both A and B treatment models show the same effectiveness for the two tested drugs. These results support the idea that the major significance of the PAE is in its application to dosing regimens.


Temperature influence on life table statistics of the chicken mite Dermanyssus gallinae (Acari: Dermanyssidae).

An age-specific life table for the chicken mite Dermanyssus gallinae (DeGeer, 1778) was based on various observations carried out at 25 degrees C. The generation time was calculated to be 16.8 days; the intrinsic rate of natural increase was 0.12 per day, and the net reproductive rate was 7.2. A nonlinear function was found satisfactory to describe the developmental rate of the different immature life-stages at temperatures below 40 degrees C. The stage-specific survival of the immature life-stages was generally high between 10 and 37 degrees C, but decreased quickly outside this temperature range. Most of the eggs were laid during the first three weeks of the adult life. The proportion of surviving females rapidly decreases after moulting to the adult stage. At a temperature of 30 degrees C, the highest number of eggs (3 eggs per day) was laid.


Regeneration of Haller's sensory organ in the tick Ixodes ricinus L.

Amputation of legs in nymphs of ticks, obtained from the first laboratory generation, resulted in regeneration of the legs after moulting to adults. Haller's sensory organ on the upper surface of each foreleg tarsus was significantly modified following regeneration. Haller's organ in non-amputated legs of the experimental ticks remained unchanged, being comparable to controls. Pored olfactory sensilla in the anterior pit, in a capsule and on a distal knoll usually increased in number, as well as grooved, thin and conical sensilla. Bordering gustatory and double-walled postcapsular sensilla either decreased or increased in number. All additional sensilla were consistent in their location. Form of the anterior pit and capsule's aperture also deeply changed after the regeneration. The authors distinguish this changes as atavistic. No correlation between changes in different parts of the organ were found. A phenomenon of induction was discovered in our study: if a distal part of tick's gnathosoma was amputated together with the left foreleg, Haller's organ in the right, untreated leg possessed the same changes after moulting as the regenerated organ in the previously amputated left foreleg.


Accuracy of transrectal ultrasound in predicting pathologic stage of rectal cancer before and after preoperative radiation therapy.

Transrectal ultrasound (TRUS) and CT scan staging of rectal cancers before, and TRUS staging after, 45 Gy of irradiation were compared with the pathologic stage of the resected specimen in 19 patients. Accuracy of TRUS before and after irradiation, and of CT scan before irradiation, was 32 percent, 63 percent, and 53 percent, respectively. CT scan before and TRUS after irradiation predicted lymph node involvement in 79 percent and 68 percent of cases, respectively. Positive predictive value for lymph node involvement before irradiation was 60 percent for CT scan and 37.5 percent for TRUS; after irradiation, it was 50 percent for TRUS. Negative predictive value was 100 percent for CT scan and TRUS before radiation and 88 percent for TRUS after irradiation. Preoperative radiation therapy makes TRUS and CT scan less effective as staging techniques. The absence of lymph nodes on TRUS and CT scan before and after irradiation is reliable.


Treatment of rectal prolapse in the elderly by perineal rectosigmoidectomy.

The results and complications of perineal rectosigmoidectomy for complete rectal prolapse in 114 patients have been reviewed. Most patients were elderly and high risk by virtue of other concurrent medical conditions. Fourteen patients (12 percent) developed significant postoperative complications. Hospital stay was short (median, four days). Ten patients were lost to follow-up. The remaining 104 patients were followed for 3 to 90 months. Eleven patients (10 percent) developed recurrent full-thickness rectal prolapse, six of them underwent repeat perineal rectosigmoidectomy. Sixty-seven patients had fecal incontinence prior to surgery. Eleven patients underwent concomitant levatoroplasty; 10 of them either improved or regained full continence of feces postoperatively. Twenty-six of the 56 patients who underwent perineal rectosigmoidectomy alone improved or regained full continence. Rectal prolapse can be successfully treated by perineal rectosigmoidectomy in elderly, high-risk patients with minimal morbidity. Levatoroplasty dramatically improves fecal incontinence occurring in association with rectal prolapse.


Chronic fissure-in-ano: a randomized study comparing open and subcutaneous lateral internal sphincterotomy.

A prospective study comparing open and subcutaneous lateral internal sphincterotomy for chronic anal fissure was conducted. One hundred twelve patients were randomized to open (n = 54) or subcutaneous (n = 58) sphincterotomy. There was no significant difference in acute complications between the subcutaneous (8.6 percent) and open (7.4 percent) groups. Postoperative length of stay was significantly shorter for the subcutaneous group (1.7 +/- 0.2 days) than for the open group (2.3 +/- 0.1 days; P less than 0.001). Although the response rate to a pain questionnaire was less than 50 percent, the data suggest a lower level of postoperative pain in the subcutaneous group. Fissure healing was similar between the subcutaneous (96.6 percent) and open (94.4 percent) groups. We conclude that subcutaneous lateral internal sphincterotomy for chronic fissure-in-ano is effective and may result in significantly less postoperative discomfort, shorter postoperative lengths of stay, and a comparable rate of complications compared with the open technique.


Prognostic significance of anastomotic recurrence from colorectal adenocarcinoma.

A retrospective analysis of the prognostic significance of anastomotic recurrence in 50 patients with colorectal adenocarcinoma was conducted from 1970 to 1987. All primary cancers were located above 10 cm from the anal verge. Forty anastomotic recurrences (80 percent) followed resection of sigmoid or proximal rectal tumors. The overall disease-free interval was 13 months, with 90 percent of recurrences diagnosed within 24 months of the primary resection. Forty-five recurrences (90 percent) were associated with synchronous or metachronous metastases. Overall median survival following the recurrence was 16 months--37 months if the anastomosis was the only recurrence site. Of five patients alive without evidence of disease, all were asymptomatic, and recurrence was confined to the anastomosis. In conclusion, anastomotic recurrence following resection of colorectal adenocarcinoma frequently heralds disseminated disease but can be potentially resected for cure if it is the only site in an otherwise asymptomatic patient.


Functional results of coloanal anastomosis with reservoir.

Functional results in 33 patients who underwent a coloanal anastomosis with reservoir were prospectively evaluated three months after colostomy closure and later (16.2 +/- 5.7 months) and were compared with those of 36 healthy controls. We were unable to demonstrate any significant difference between patients and controls concerning frequency of stools, feeling of the need to defecate, continence of stools and flatus, differentiation between flatus and feces, urgency, and need to wear a protective pad. There was a statistically significant difference concerning the ability to evacuate, which was better in the control group (score = 1.03) than in the patients (score = 1.63) (P less than 0.001). These results suggest that coloanal anastomosis with reservoir provides nearly normal function except for the ability to evacuate.


Relationship between clinical symptoms of anal incontinence and the results of anorectal manometry.

The aim of this work was to analyze clinical symptoms in light of anorectal manometry results. We compared the frequency of clinical symptoms in relation with the presence or absence of functional anomalies. Using this methodology, the following relationships may be suggested: the need to wear a pad, with a decreased resting pressure at the upper part of the anal canal; the inability to delay rectal evacuation, with decreased anal voluntary contraction; interference of incontinence with social activities, with decreased duration of anal voluntary contraction; urinary symptoms, with an increased threshold volume of rectal distention needed to elicit the rectoanal inhibitory reflex; and complete rectal prolapse, with reduced length of the anal canal.


Crohn's disease and carcinoma: increasing justification for surveillance?

Carcinoma of the colon that arises in patients with Crohn's disease is being reported with increasing frequency. To help clarify the nature of this association, records of 25 patients with Crohn's disease and colorectal carcinoma seen from 1957 through 1989 were reviewed. One patient had leiomyosarcoma of the rectum, and two patients had the onset of Crohn's disease after the diagnosis and treatment of colorectal carcinoma. Therefore, 22 patients were available for complete retrospective analysis. The median age at diagnosis of Crohn's disease was 37 years (range, 15-67 years), and the median age at diagnosis of carcinoma was 54.5 years (range, 32-76 years). The median duration of symptoms preceding the discovery of colorectal carcinoma was 18.5 years (range, 0-32 years). Carcinoma arose in colonic segments with known Crohn's disease in 77 percent of patients, and six patients (27 percent) had associated colonic mucosal dysplasia. One lesion was classified as Dukes A, nine lesions were Dukes B, five lesions were Dukes C, and seven lesions were Dukes D. Patients with an onset of Crohn's disease before the age of 40 years had primarily Dukes C or D lesions and consequently poor survival. Most patients presented with nonspecific signs and symptoms, with nothing to distinguish the activity of the Crohn's disease from the presence of colorectal neoplasm. Younger patients with long-standing Crohn's disease should be considered for colonic surveillance to permit earlier diagnosis and treatment of potential colorectal carcinoma.


Influence of loperamide and loperamide oxide on the anal sphincter. A manometric study.

The objective of this study was to investigate the effects of the opioid loperamide and its recently synthesized pharmacologically inactive prodrug loperamide oxide on the anal sphincter. In a double-blind, placebo-controlled crossover study, anorectal manometry was performed in 12 healthy volunteers five hours after oral bolus application of 10 mg of loperamide, loperamide oxide, or placebo. Loperamide significantly increased the threshold volumes for minimal perception and urgency to defecate (P less than 0.05) and raised the volume required to abolish recovery of the rectoanal inhibitory reflex (P less than 0.05). These findings suggest that loperamide has a specific continence-improving action on the anal sphincter. However, anal resting pressure and maximal squeeze pressure were unaffected in our study and do not seem to be responsible for this effect. The effects under loperamide oxide showed a similar tendency but were without statistical significance.


Direct comparison between Czerny-Lembert and circular-stapled anastomotic techniques in colorectal anastomosis: a similar pattern of healing for both.

There are scant experimental data directly comparing the healing of the circular-stapled (CS) anastomotic technique with the standard Czerny-Lembert two-layer hand-sewn (HS) anastomotic technique during the acute and chronic phases of healing. The purpose of this study, therefore, was to serially evaluate wound healing in CS and HS anastomoses in the normal porcine colorectum. Forty-two adult female mixed-breed pigs randomly underwent either HS or CS anastomosis at the sacral promontory. Laser Doppler velocimetry (LDV), was used to measure perianastomotic blood flow. Groups of animals underwent a second surgery at 3, 5, 11, 60, or 120 days postoperatively, and anastomoses were restudied using LDV, gross and microscopic grading of inflammation, bursting pressures, and hydroxyproline content. Additionally, the 60-day and 120-day groups of animals underwent preoperative biplanar barium enemas to identify leaks or stenoses. No significant differences in perianastomotic blood flow between the HS and CS techniques were obtained over the entire 120-day study period. Serial evaluation of wound healing revealed no significant differences between the two anastomotic techniques with respect to bursting pressures, gross or microscopic inflammatory scores, or hydroxyproline content. There were no leaks or stenoses with either technique. Despite earlier reports to the contrary, there appears to be no fundamental difference in the mode of healing in the porcine colorectum comparing the HS technique with the CS technique.


Consequences of delayed ileostomy closure after ileal pouch-anal anastomosis.

Temporary diverting loop ileostomy is a generally accepted component of the ileal pouch-anal anastomosis (IPAA) procedure. Ileostomy closure is usually performed within two to three months but may be delayed because of disruption of the ileonanal anastomosis, suspected leak from the ileal reservoir, concomitant medical problems, or patient convenience. Of 362 patients undergoing IPAA at The Cleveland Clinic Foundation for inflammatory bowel disease, 10 have had their ileostomy closures delayed for more than six months. Clinical and manometric parameters are examined in these patients and compared with those who had earlier closure. There appears to be no significant difference in the functional outcome of IPAA in these patients in terms of number of bowel movements and degree of continence. Reservoir compliance and maximum tolerated volumes are similar. We conclude that delaying ileostomy closure for more than six months after IPAA has no deleterious effect on pouch function.


Rectoanal inhibitory reflex following low stapled anterior resection of the rectum.

The rectoanal inhibitory reflex plays an important role in the normal mechanisms of anorectal continence. Anterior resection abolishes the reflex, but whether it recovers, particularly after inverted stapled anastomosis, is not clear. Anal manometry was performed on patients undergoing low anterior resection for carcinoma. Maximum anal resting pressure and the rectoanal inhibitory reflex were assessed preoperatively and up to two years postoperatively. The reflex was present in 43 of 46 patients (93 percent) preoperatively, in 8 of 45 patients (18 percent) on the 10th postoperative day, and in 6 of 29 patients (21 percent) between six months and one year following surgery. Twenty patients were studied more than two years postoperatively, and in 17 (85 percent) the reflex was demonstrated. In the majority of low anterior resection patients, the rectoanal inhibitory reflex is abolished by surgery, remains absent throughout the first year, and has recovered by the end of the second postoperative year. This may be important in the recovery of anorectal function in these patients.


Proliferative activity of colonic mucosa at different distances from primary adenocarcinoma as determined by the presence of statin: a nonproliferation-specific nuclear protein.

The field change is one hypothesis concerning the development of colorectal carcinoma. Removal of a carcinoma without its entire surrounding altered mucosa may result in the development of a recurrence. S44, a monoclonal antibody directed against statin, a nuclear protein expressed in nonproliferating cells in either a quiescent or senescent state, was used to determine the rate of cell growth in colorectal mucosa at different distances from carcinomas. The specimens of 18 patients undergoing resection of a colorectal carcinoma were immediately opened after operation, and strips of mucosa were taken at distances of 1 cm, 5 cm, and 10 cm from the carcinoma. For each location, 10 longitudinally oriented crypts were evaluated for statin-positive cells identified by the presence of a dark brown peroxidase-conjugated antibody reaction product. The average percentage of statin-positive cells per crypt was significantly lower at a 1-cm distance from the carcinoma compared with the mucosa located 5 and 10 cm from the carcinoma (20.89 +/- 4.33 at 1 cm, 32.41 +/- 5.27 at 5 cm, and 34.23 +/- 6.45 at 10 cm). None of the calculated parameters showed any significant difference between the 5-cm and 10-cm locations. The fact that the proliferation rate of the mucosal cells returns to the normal level at 5 cm from the margin of the carcinoma suggests that cells located within this distance still retain proliferative potential even though they are morphologically indistinguishable from their normal counterparts. We conclude that failure to remove this transitional, potentially proliferative mucosa may result in subsequent development of anastomotic or perianastomotic recurrences.


Healing of ischemic colonic anastomosis: fibrin sealant does not improve wound healing.

Fibrin adhesives have been advocated as a protective sealant in high-risk colonic anastomoses to prevent leakage. To assess the effect of fibrin glue sealing on the healing ischemic anastomosis, we compared the healing of sutured colonic anastomoses in the rat, with and without fibrin adhesive (Groups IA and IB), and ischemic anastomoses with and without fibrin adhesive (Groups IIA and IIB). On days two, four, and seven, 10 animals in each group were sacrificed. Adhesion formation was scored, and the in situ bursting pressure was measured. The collagen concentration and degradation were estimated by measuring hydroxyproline. Adhesion formation was more prominent in Groups IB, IIA, and IIB on day four only; abscesses were noted in the ischemic group in four rats. Anastomotic bursting pressure was significantly lower in sealed (IB) and ischemic anastomoses (IIA) than in normal anastomoses (IA) on day four. Sealing of ischemic anastomoses did not change bursting pressures on days two, four, and seven. The relative decrease of collagen in the sealed anastomoses is significantly higher on day four only. It is concluded that sealing of normal colonic anastomoses in the rat has a negative effect on wound healing. Ischemia at the anastomotic site results in weaker anastomotic strength on day four postoperatively. Also in ischemic anastomoses, fibrin sealant does not improve wound healing during the first seven days. Adhesion formation on ischemic intestinal anastomoses was not prevented by fibrin sealing.


Are intersecting staple lines a hazard in intestinal anastomosis?

To determine the safety of intersecting staple lines, 22 pigs were operated upon with a functional end-to-end enteroanastomosis 40 cm distal to the ligament of Treitz using linear stapling devices. The procedure was repeated on the colon, where a colocolostomy was created. The blood flow at intersecting staple lines and single-row staple lines for each anastomosis was studied with the reference organ method 24 hours after the first operation. The purpose was to evaluate whether there is a reduction in blood flow at the site of intersecting staple lines, causing an increased risk for anastomotic leakage. The reduction in mean blood flow in crossing compared with noncrossing staple lines was 6 percent (-5-17 percent) for small bowel anastomoses and 7 percent (-6-19 percent) for colonic anastomoses. An equivalence test showed that, if a reduction in blood flow exists between crossing and noncrossing staple lines, it is most likely less than 30 percent (P less than 0.001) for both small bowel and colonic anastomoses. This experimental study demonstrates that intersecting staple lines in small bowel and colonic anastomoses do not reduce anastomotic blood flow to a dangerous level.


Experimental carcinogenesis at sutured and sutureless colonic anastomoses.

This study explores the role of sutures and the healing colonic wound in experimental carcinogenesis. One hundred sixty rats underwent surgery with colotomy and repair using silk, steel, or Vicryl (Ethicon, Somerville, NJ) sutures or a sutureless technique. Forty rats had a sham procedure. All animals received azoxymethane for 12 weeks at a dose of 10 mg/kg/week. Half the rats commenced carcinogen before surgery, and half commenced it eight weeks after surgery. Animals with anastomotic tumors were found in 46 percent of the sham group (P less than 0.05 cf. sutured), 41 percent of the sutureless group (P less than 0.02 cf. sutured), and 68 percent of the sutured group. The corresponding figures for anastomotic carcinoma were 9 percent (P less than 0.001 cf. sutured), 22 percent, and 38 percent. No significant differences in tumor yield were noted among the different sutures. However, several differences were noted between the two carcinogen models. In those animals that received surgery first, there was a higher incidence of anastomotic tumors (P less than 0.002) and cancers (P less than 0.0001) in the sutured and sutureless groups, and those tumors that occurred in the sutured group were considerably larger than in those that had carcinogen first (15.9 mm cf. 4.9 mm; P less than 0.0001). Overall, all sutures seem to enhance anastomotic tumor formation, and we would suggest that a sutureless anastomosis may diminish this risk.


Rectosigmoid stent for obstructing colonic neoplasms.

Attempting proctoscopic placement of a rectosigmoid stent is proposed as a first step in treating obstructing rectosigmoid neoplasms. If stent placement is successful, elective colon resection can be performed following treatment of any coexisting medical problems that would complicate an emergency colon resection and after routine mechanical bowel preparation.


The diabetic foot problem--a failed system of health care?

It is evident that there is excess morbidity and mortality as a consequence of foot problems in patients with diabetes mellitus. Most of the data relative to foot lesions is taken from the study of subsets of diabetic patients undergoing lower extremity amputation. Such data probably do not provide accurate information relative to the incidence and/or prevalence of diabetic foot problems. Available evidence does suggest that diabetic foot care may be inadequate and the efficacy of various diagnostic and therapeutic modalities has not been proven. Advances have been made in obtaining a better understanding of the microbiology of lower extremity infected lesions both in hospitalized patients and in those subjects being followed in the outpatient setting. Attention should be directed at developing a systematic classification of foot lesions which can be universally applied. We need to understand and relate to those risk factors contributing to the development and progression of lower extremity lesions in the diabetic patient. In evaluating the efficacy of diverse diagnostic and therapeutic approaches, well-controlled clinical investigations need to be undertaken. Attention should be focussed upon both measures of process and outcome where appropriate. Finally, there needs to be recognition that the problem of the diabetic foot lesion represents a major public health challenge.


First phase insulin release in the non-obese diabetic mouse: correlation with insulitis, beta cell number and autoantibodies.

The ontogenic variation of beta cell function and its relationship with the degree of islet damage and levels of autoantibodies have been studied in the non-obese diabetic (NOD) mouse model. We conducted in vivo first phase insulin release (FPIR) in response to intravenous glucose and studied its correlation with the degree of insulitis, islet cell antibody (ICA) and insulin autoantibody (IAA) levels in female NOD mice cross-sectionally at days 40 (n = 19), 90 (n = 21), 150-160 (n = 21) and day 250 (n = 20). The mean +/- SEM FPIR values showed an age-related decline from day 40 (46.2 +/- 5.3 microU/ml) to day 150-160 (17.8 +/- 2.5 microU/ml) and then doubled at day 250 (34.5 +/- 5 microU/ml), while the mean +/- SEM insulitis scores increased progressively until day 150-160 (61.7 +/- 6.1%) and then declined slightly at day 250 to 50.2 +/- 6.2%. In female NOD mice with spontaneous diabetes (n = 4) and streptozotocin-induced diabetic Swiss mice (n = 5) FPIR was either absent or greatly attenuated. A statistically significant inverse correlation between FPIR and insulitis was found among NOD mice at days 90 (P = 0.02; r = -0.52) and 150-160 (P = 0.03; r = -0.48). However, no statistically significant correlation was observed at days 40 and 250. Morphometric techniques applied to day 150-160 pancreatic sections showed a statistically significant negative correlation between insulitis and beta cell number per unit area of islet tissue (P = 0.0001; r = - 0.75). At this age some islet beta cells showed different intensities of staining by immunofluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)


Periodicity of insulin secretion comprises multiple cycles with different duration in perfused rat islets.

Insulin secretion from pancreatic islets has been found to be periodic by in vivo and in vitro experiments. The pacemaker which regulates the periodicity may be localized in the central nervous system or in the pancreas, though the precise location and the mechanisms of generating pacing have not been determined. In order to solve these problems, we examined the period of secretory cycles of insulin in isolated islets using a prolonged perfusion system, and investigated the effects of glucose and other agents on these periods. Isolated islets from male Wistar rats were enclosed in a millipore holder and were perfused with MEM containing 1 mg/ml glucose at a flow rate of 0.3 ml/min for 240 min. The effluent was collected at 1-min intervals to measure insulin secretion. The results were analyzed by the maximum entropy method to demonstrate the periodicity of insulin secretion. When islets were perfused with 1 mg/ml glucose, the periodicity comprised five cycles with different duration: 71.5 +/- 14.6 min, 29.8 +/- 3.4 min, 19.2 +/- 1.5 min, 11.6 +/- 2.1 min and 4.3 +/- 0.4 min. This indicates the presence of a pacemaker within the islets, although, in vivo, participation of a higher center to control periodicity has to be taken into account. Further, the presence of a long cycle (71.5 +/- 14.6 min) of insulin secretion which previously has only been observed in vivo was first demonstrated in this in vitro study. The cycles were consistent even in islets which were desensitized to glucose by cultivating in a high glucose medium for 5 days before perfusion.(ABSTRACT TRUNCATED AT 250 WORDS)


Lack of specificity of islet cell surface antibodies (ICSA) in IDDM.

Human islets were isolated by collagenase digestion and tissue culture from pancreata obtained from organ donor subjects and dispersed islet cells were prepared from hand-picked islets. Islet cell surface antibodies (ICSA), detected by indirect immunofluorescence on isolated islet cells, were present in sera from nine of 22 (41%) subjects with recent-onset insulin-dependent diabetes mellitus (IDDM) and three of 11 (27%) control subjects. Sera had been heat inactivated, adsorbed against a human B lymphoblastoid cell line (IM-9) and tested in the presence of 4% bovine serum albumin. However, with a double labelling technique, we were unable to show that ICSA were specific for beta cells. Of the nine ICSA-positive IDDM sera, three stained both beta and non-beta cells, three beta cells only and three non-beta cells only; the three ICSA-positive control sera stained both beta and non-beta cells. There was no apparent relationship between ICSA and standardised measurements of islet cell antibodies (ICA) and insulin autoantibodies (IAA). These results lead us to question whether, despite previous reports, ICSA are specific for beta cells or indeed for IDDM.


Nasal glucagon in the treatment of hypoglycaemia in type 1 (insulin-dependent) diabetic patients.

The aim of this study was to compare the effect of nasally administered glucagon in doses of 1 (A) and 2 mg (B), with 1 mg glucagon administered intramuscularly (C) in 12 C-peptide-negative IDDM patients. Spontaneous recovery (D) from insulin-induced hypoglycaemia in the same patients was used as reference. The mean age was 31.1 (21-48) years, diabetes duration 10.8 (2.7-31) years and HbA1c 7.7 (6.5-9.8)%. Hypoglycaemia was induced by i.v. insulin infusion. When blood glucose (BG) reached about 2 mmol/l either glucagon was administered or the patients recovered spontaneously. BG nadir was 1.6 (1.1-2.3) mmol/l. BG increments during the first 15 min after glucagon administration were: (A) 1.9 +/- 0.7 (0.4-3.0); (B) 2.5 +/- 0.7 (1.5-3.5); (C) 2.5 +/- 1.0 (1.2-4.7); and (D) 0.3 +/- 0.4 (0-1.0) mmol/l, respectively. All treatments were more effective, measured as increments in BG, than spontaneous recovery, P less than 0.00001. There was no difference between nasal treatment with 2 mg (B) and i.m. treatment (C), both being more effective than 1 mg (A) nasal treatment, P less than 0.1. BG continued to increase up to 10 mmol/l 90 min after i.m. glucagon administration, whereas it stabilized at a level of 4.6-6 mmol/l, 30-45 min after nasal administration. Eighty percent of the patients had side-effects to nasal administration - local irritation, rhinitis or sneezing. Half of the patients sneezed, without correlation with the delivered dose of glucagon. None of the patients had side-effects which would preclude further treatment.(ABSTRACT TRUNCATED AT 250 WORDS)


Reliability of glucose measurement by glucose test strips in tropical conditions.

The reliability of glucose test strips and reflectance meters after storage in tropical conditions was assessed. In a first experiment, sealed bottles containing glucose test strips were stored for 1 month in closed sterilising ovens at 40 degrees C, one in dry (0% humidity) and the other in wet (75% humidity) conditions. Results were compared to measurements obtained with glucose test strips stored under standard Western conditions, in non-diabetic and diabetic patients, using the same standard reflectance meter. A second experiment was performed similarly, comparing the values obtained with reflectance meters stored in different conditions, using standard glucose test strips. Reproducibility was assessed on the basis of duplicate measurements for each condition of storage. We observed that temperature and humidity did not significantly affect the glucose test strips (1st experiment), while a slight underestimation of capillary blood glucose was found using reflectance meters stored at 50 degrees C, irrespective of the humidity (2nd experiment). All the correlation coefficients between duplicates were greater than 0.975 irrespective of the conditions of storage for glucose test strips and reflectance meters. Therefore, glucose test strips and reflectance meters appear fairly reliable at the grass-root level in the tropics, if used carefully.


Urinary albumin excretion rate (AER) in newly-diagnosed type 2 Indian diabetic patients is associated with central obesity and hyperglycaemia.

Urinary albumin excretion rate (AER) was measured in non-diabetic controls (n = 143) and newly diagnosed impaired glucose tolerant (IGT, n = 64) and non-insulin-dependent (type 2) diabetic patients (n = 146). AER progressively increased from non-diabetic [3.7 (1.1-51.3) micrograms/min, median (5-95th centile)] to IGT [4.8 (1.3-53.7)] and diabetic [7.3 (1.4-91.6)] groups. Eight percent of non-diabetic, 19% of IGT and 23% of type 2 diabetic patients showed 'microalbuminuria' (AER, 20-200 micrograms/min) (non-diabetic vs diabetic P less than 0.01, non-diabetic vs IGT NS, IGT vs diabetic NS). AER was directly related to waist-hip ratio (P less than 0.001) and HbA1 (P less than 0.01) in diabetic patients; 80% of diabetic patients with microalbuminuria were men (P less than 0.06 compared to 'normoalbuminuric' diabetic patients). Association of AER with waist-hip ratio was present in men as well as women. Thus, in the newly diagnosed type 2 Indian diabetic patients AER is associated with central obesity in addition to its well known association with hyperglycaemia. Our findings offer a possible explanation for the increased risk of proteinuria in diabetic men than in women because men are centrally more obese. It could also explain previous reports of higher AER in migrant Asian diabetic patients in the U.K. compared to native white Caucasian diabetic patients because Asians are known to be more centrally obese.


Hypertension in newly diagnosed non-insulin-dependent diabetic subjects.

Medical records of newly diagnosed non-insulin-dependent diabetic patients attending a Diabetic Centre in Modena, northern Italy, during the period 1985-88 were reviewed to analyze the prevalence of hypertension at age of diagnosis of diabetes and its association with selected risk factors. The prevalence of hypertension was also determined in a representative sample of control subjects. In the multivariate analysis, greater body mass index and older age, but not smoking, were strongly associated with increased rates of hypertension both in control and in diabetic subjects. In diabetic patients, family history of diabetes, defined as presence of diabetes in close relatives, was not significantly associated with hypertension, while rates of hypertension were significantly lower in patients who reported at least one parent affected by diabetes. After adjustment for age and body mass index, newly diagnosed non-insulin-dependent diabetes was not an independent risk factor for hypertension. These findings seem to be consistent with the hypothesis that diabetes and hypertension are not linked by a common genetic background.


Physiologic concentrations of inorganic phosphate accelerate fructosamine synthesis.

The effect of physiologic concentrations of inorganic phosphate (Pi) on fructosamine (FRA) synthesis was studied. After 75 g oral glucose administration (OGTT), 'delta FRA/24 h', defined as delta FRA after incubating serum or other specimens at 37 degrees C for 24 h after adding 1000 mg/dl glucose, was significantly decreased in parallel to the decrease of plasma Pi concentrations. 'The FRA index', defined as the FRA value divided by the corresponding glucose concentration, both at fasting, correlated significantly with plasma Pi concentrations. In vitro incubation of serum total protein (TP), albumin (ALB), gamma-globulin (GLB), free lysine (Lys), and free valine (Val) with glucose at different concentrations of Pi showed a Pi-dependent increase of FRA synthesis throughout 48 h of incubation. The accelerating effect of 5 mg/dl Pi on FRA synthesis from TP, ALB, GLB, Lys, and Val at pH 7.4 was, respectively, as great as 48, 20, 24, 13 or 25% of those without Pi. Increase of pH from 6 to 10 logarithmically increased delta FRA/24 h in contrast to a logarithmic decrease of the accelerating effect of Pi on delta FRA/24 h. These data show that physiologic concentrations of Pi accelerate protein glycation by accelerating dehydrogenation during the Amadori rearrangement through the negative charge of Pi. Because this accelerating effect of physiologic Pi presumably exists in vivo, Pi concentration must be taken into account as an accelerating factor for FRA synthesis in evaluating diabetic control, and further studies must be carried out to elucidate whether hyperphosphatemia accelerates glycation-induced diabetic complications.


Current needs for increased accuracy and precision in measurements of low levels of lead in blood.

Needs for accurate and precise measurements of low levels of lead in blood are discussed. Current methodologies are sufficient for measuring blood lead (PbB) levels greater than 0.5 microM, but the accuracy and precision of those methodologies are inadequate for measuring lower (less than 0.5 microM) PbB concentrations. These analytical limitations are primarily due to contamination bias incurred during sample collection, processing, and analyses. Consequently, trace metal-clean procedures should be adopted to elucidate the contribution of environmental lead to the PbB levels of the U.S. population and to estimate the threshold(s) of subclinical lead toxicity.


Neurobehavioral effects of formaldehyde and solvents on histology technicians: repeated testing across time.

Neurobehavioral functions were studied by periodic testing of 318 histology technicians and by a single session testing 494 of such technicians from 1982 through 1986. Tests included immediate recall of stories, of drawings, and of number series from the Wechsler Memory Scale, block designs from the Wechsler Adult Intelligence Scale (WAIS), slotted pegboard, trail making A and B, embedded figures, number writing on the fingers, visual simple and two-choice reaction times, balance (speed of body sway), and the profile of mood state (POMS) score. Mean test scores of four initial "first test" groups were not different from 1982 to 1986. Immediate recall of stories and drawings improved on the first retest as did the block design score. Other test scores showed insignificant variation over the 4 years, including memory for numbers forward and backward, trails A and B, number writing on fingers, radius of body sway, and choice reaction time. Only placing of pegs in the pegboard took longer. POMS score was lower in the second year in naive subjects and decreased from 39 to 13 in those retested. Variations in results of neurobehavioral tests given at intervals across 4 years were small. No cumulative effects of occupational exposures or of aging were found. However, small decreases may have been offset by increases due to test familiarity or learning.


Residential exposure to chromium waste--urine biological monitoring in conjunction with environmental exposure monitoring.

To determine whether a population living on or adjacent to four sites of chromate production waste was measurably exposed to environmental chromium, spot samples of urine were collected along with wipe samples of household dust and lifestyle/activity interview data. Findings were compared to those from a control population in two communities with no significant chromium use or waste sites. Urine samples were collected and analyzed, employing measures to minimize background chromium contamination. The average Cr mass in dust was 3.7 times that in control houses. The mean creatinine-corrected urine Cr (Cr/c) level of the exposed subgroup residing in households in the 75th percentile of Cr mass in wipe samples was significantly greater than that of the control population. This subgroup was primarily located at a single exposure location. Using lifestyle/activity data, significantly elevated Cr/c urine levels were identified in other exposed subgroups defined by employment location and by outside play time. These data show an association between elevated exposure to chromium in household dust and elevated urine levels of chromium, consistent with residential exposure to chromate production waste. These data also suggest an association between chromium exposure and activities outside the home which are consistent with exposure to chromate production waste.


Asbestos in organs and placenta of five stillborn infants suggests transplacental transfer.

Digests of lungs, liver, and placenta from five stillborn infants of 22 to 38 weeks gestational age were examined for asbestos and other fibers using light and electron microscopy, energy dispersive X-ray analysis, and selected area diffraction analysis. Uncoated chrysotile asbestos fibers were found in the digests of at least one of the three tissues examined from each stillborn infant. The asbestos fiber burdens ranged from 71,000 to 357,000 fibers/g wet tissue. Most of the fibers were small, with the mean length ranging from 0.83 to 2.53 microns. While appreciable numbers of uncoated chrysotile fibers were present, no coated asbestos fibers were found in any of the stillborns. Both coated and uncoated nonasbestos fibers were found in at least one of the tissue digests of all five stillborns. The uncoated nonasbestos fibers were characterized as aluminum silicates, diatomaceous earth fragments, or other fibers. The coated nonasbestos fibers or ferruginous bodies were consistent with being formed on diatomaceous earth fragments, black carbon cores, or sheet silicate cores. Since the placenta is the only route of communication between the fetus and the outside environment, our findings strongly suggest a transplacental transfer of asbestos and other fibers in humans.


The effect of ozone associated with summertime photochemical smog on the frequency of asthma visits to hospital emergency departments.

A retrospective study using ambient ozone, temperature, and other environmental variables and their effect on the frequency of hospital visits for asthma was conducted in New Jersey, an area that often exceeds the allowable national standard for ozone. Data on emergency department visits for asthma, bronchitis, and finger wounds (a nonrespiratory control) were analyzed for the period May through August for 1988 and 1989. Asthma visits were correlated with temperature while the correlation between asthma visits and ozone concentration was nonsignificant. However, when temperature was controlled for in a multiple regression analysis, a highly significant relationship between asthma visits and ozone concentration was identified. Between 13 and 15% of the variability of the asthma visits was explained in the regression model by temperature and ambient ozone levels. This association, when compared to similar studies in Canada, shows the contribution of ozone to asthma admissions to be stronger in areas with higher ozone concentrations. Thus, among regions with periodic accumulations of ozone in the ambient atmosphere, an exposure-response relationship may be discernible. This supports the need to attain air quality standards for ozone to protect individuals in the general population from the adverse health effects caused by ambient ozone exposure.


Sugarcane exposure and the risk of lung cancer and mesothelioma.

A case-control study was conducted of the risk of lung cancer and mesothelioma from environmental and occupational exposures associated with sugarcane production. A slight, not statistically significant, excess risk of lung cancer was observed among participants who reported working in the sugarcane industry (odds ratio 1.8, 95% confidence interval 0.5-7.5). No increased risk was observed among our population, associated with living near sugarcane growing areas. Little difference was observed between cases and controls in years employed in the industry or jobs performed. Only one mesothelioma case and no controls reported working in the sugarcane industry.


Elementary school absences and PM10 pollution in Utah Valley.

This study assessed the association between school absenteeism and respirable particulate pollution (PM10) in Utah Valley for the six school years of 1985 to 1990. Weekly absenteeism data from the Provo School District and daily data from a single elementary school in the Alpine School District were analyzed for kindergarten through sixth grade. PM10 concentrations during the study period averaged approximately 50 micrograms/m3 with the 24-hr maximum equal to 365 micrograms/m3. Absenteeism was regressed on PM10 pollution levels, temperature, snowfall, and variables indicating day of week, month of school year, and days preceding and following holidays and extended weekends. Estimated associations between absenteeism and PM10 pollution in both data sets were positive, statistically significant (P less than 0.01), and robust to different model specifications. PM10 effects persisted for up to 3 or 4 weeks. Regression results from both data sets indicated that an increase in 28-day moving average PM10 equal to 100 micrograms/m3 was associated with an increase in the absence rate equal to approximately two percentage points, or an increase in overall absences equal to approximately 40%. Similar relationships were observed for all grade levels, although the response of absences to air pollution was generally greater for grades 1-3 compared with grades 4-6. Associations between absenteeism and PM10 pollution were observed even for levels below 150 micrograms/m3.


Experimental studies on the pathogenesis of the gastric mucosal lesions induced by whole-body vibration.

In order to determine the pathogenesis of gastric mucosal lesions induced by whole-body vibration (WBV), the effects of WBV (3.0 G, 10 Hz, 90 min) on gastric mucosal blood flow (GMBF), plasma corticosterone (COR) and catecholamines (CA), and gastric ulcer formation were investigated in relation to the effects of forced water-immersion stress (FWI) upon the same parameters. While WBV increased GMBF during the exposure, FWI decreased it both during and after the stress. No difference in the severity of ulcer formation between the WBV and the FWI groups was seen. Both WBV and FWI increased plasma COR and CA, although the degree of the increase in COR that accompanied WBV was less than that associated with FWI. Truncal vagotomy attenuated the reduction of GMBF and the extent of ulcer formation that occurred with FWI, but promoted a reduction of GMBF with WBV. These findings indicate that ulceration induced by WBV may be caused primarily by its direct and specific mechanical actions and not by indirect, central nervous system effects known to be important in the pathogenesis of ulcerations produced by mental stress.


Styrene in adipose tissue of nonoccupationally exposed persons.

Given a styrene tissue/blood partition coefficient of ca. 39 and a relatively low perfusion rate of ca. 0.03 ml/min-g tissue, adipose tissue provides a useful physiologically damped integrative measure of environmental exposure. Styrene in the adipose tissue of nonoccupationally exposed individuals was measured for the first time. Tissue samples obtained from elective surgery patients and postmortem donors were analyzed by capillary gas chromatography and found to contain 1.12 +/- 1.06 (mean +/- SD) ppm styrene. Using these measured tissue levels and an apparent clearance of styrene (defined as the ratio of blood clearance to adipose tissue/blood partition coefficient), environmental intake of styrene was estimated to be 2.23 mg/hr, corresponding to an inhaled concentration of 1.96 mg/m3 (476 ppb). This value is two to three orders of magnitude higher than typical breathing zone air measurements, indicating additional undiscovered sources of styrene exposure.


Postnatal lead exposure affects motor skills and exploratory behavior in rats.

The present study was undertaken to investigate the behavioral effects of postnatal lead exposure. Newborn male Sprague-Dawley rats were given 1 or 8 mg/kg lead acetate intraperitoneally daily for 20 days. Control rats received 1 mg/kg sodium acetate, or 8 mg/kg sodium acetate in oversized litters. The high dose lead acetate group and the high dose, oversized sodium acetate group showed impaired weight and length increment during the end of the treatment. Rats treated with the higher dose of lead showed delayed eye opening. The time required to turn in a negative geotaxis test was transiently longer in rats treated with the higher dose of lead. A tendency of reduced forepaw grasping ability was seen in lead-treated rats during the end of the lead exposure. Ambulation and rearing in an open field were lower for the rats treated with the higher dose of lead acetate during certain periods of development. Impaired performance in a balancing rod test was also seen in the rats treated with the higher dose of lead at the adult stage, while no difference was seen in ambulation or gnawing activity during tail pinch-induced stress. Thus, lead intoxication in rats during the early postnatal period, with doses that approximate those in children, induced transient as well as persistent dysfunctions in exploratory behavior and motor skills. These observed actions of lead may be related to impaired maturation of sensitive brain regions which develop postnatally.


A method for the generation of monoclonal antibodies against rare cell-surface molecules.

Monoclonal antibodies provide a powerful tool for the identification and analysis of novel cell-surface molecules. We present here a method for antigen preparation and an immunization protocol that facilitates generation of mAb reactive with cell-surface molecules of low abundance and/or low antigenicity. The procedure involves isolation and extensive fractionation of cell-surface and detergent-soluble extracellular-matrix molecules prior to immunization. Cell-surface proteins on intact tissue are biotin-labeled using a reagent that does not penetrate cells. Avidin affinity chromatography is then used to purify these biotinylated molecules. Size-exclusion HPLC is used to separate these surface molecules on the basis of apparent molecular mass. Finally, immunization with antigen coupled to keyhole-limpet hemocyanin is combined with long-term booster immunizations to generate a hyperimmune response resulting in high-affinity IgG. A test application of this approach was aimed at the generation of mAb against cell-surface molecules of approximately 135 kDa in the developing chicken retinotectal system. Immunochemical analyses using antibodies produced by this approach which showed restricted patterns of tissue staining reveal that mAb were generated against all previously identified immunoglobulin superfamily molecules of this size in this system. Furthermore, we produced many additional antibodies that labeled retinotectal tissue in novel staining patterns. In the two cases analyzed in detail, these new patterns reflect the distributions of previously uncharacterized members of the immunoglobulin superfamily. The success of this initial study suggests that this method may represent a broadly applicable approach towards the preparation of extensive libraries of antibodies against cell-surface molecules expressed on cells from numerous sources.


Molecular dynamics study of a complex between the human histocompatibility antigen HLA-A2 and the IMP58-66 nonapeptide from influenza virus matrix protein.

The structure of the influenza-virus-matrix-protein (IMP) 58-66 nonapeptide, bound to the major-histocompatibility-complex-encoded human leukocyte antigen (HLA) A2 protein was studied by molecular dynamics simulation. Starting from the extra electron density map of peptides co-crystallized with HLA-A2, the nonapeptide IMP58-66 was docked residue by residue in the protein binding cleft. The complex was simulated for 100 ps in a shell of 1372 water molecules. The averaged simulated HLA-A2 conformation was found to be similar to the crystal structure (0.182 nm RMS deviation, for the backbone atoms of the alpha 1-alpha 2 domain). Nine out of the 14 hydrogen bonds observed in the antigen-binding site were reproduced in the simulation. The IMP58-66 peptide exhibits an extended conformation with kinks at positions 3 and 5. The side chains of residues 2, 3 and 9 develop van der Waals' interactions with hydrophobic pockets of HLA-A2, corresponding to polymorphic residues of the major-histocompatibility-complex-encoded proteins. Both the N-terminus and C-terminus of the nonapeptide were anchored in the antigen-binding groove by hydrogen bonds with conserved amino acids. The N-terminus was more flexible and contacts four HLA-A2 conserved tyrosines (Tyr7, Tyr59, Tyr159 and Tyr171) and Glu63 by direct or water-relayed hydrogen bonds. Water intercalation occurred only around the N-terminus of the peptide, the C-terminal carboxylate forming strong hydrogen bonds with polar residues (Tyr84 and Thr143) and a salt bridge with Lys146 all over the molecular dynamics simulation. This model is fully compatible with the recently published crystal structure of the HLA-B27 protein, complexed by a mixture of self nonapeptides.


A study of the hinge-bending mechanism of yeast 3-phosphoglycerate kinase.

The hinge-bending mechanism proposed as part of the catalytic mechanism for phosphoglycerate kinase (PGK) has been investigated using yeast PGK and the site-directed mutant [H388Q]PGK, where His388 is replaced by Gln. The emission and quenching of fluorescence, supported by the aromatic CD band, show that the mutation in the waist region affects the tryptophan environment in the C-terminal domain. The mutant is also less stable to guanidine denaturation and less cooperative in its unfolding. The effect of substrates on the conformation of PGK was studied using 8-anilino-1-naphthalenesulphonic acid (ANS), a competitive inhibitor of ATP binding to the C-terminal domain, and 8-(2-[(iodoacetyl)ethyl]amino)naphthalene (I-AEDANS), attached to Cys197 on the N-terminal domain. Under the influence of substrates the novel anisotropy decay curves for ANS indicate a 1-5 degrees change in the orientation of the probe, interpreted as a small reorientation of the domains about the waist region. The experimental data are interpreted as a small swivelling of the domains about the waist region under the influence of substrate. The results with AEDANS anisotropy decay are consistent with those for ANS. The enzyme activity of PGK shows a break in the Arrhenius plot at 20 degrees C mirrored by a break in the temperature dependence of tryptophan ellipticity. This is interpreted as a change in protein dynamics associated with destabilisation of the waist region. This destabilisation is shown to have already taken place in the mutant enzyme and in the wild type at pH 5.6, both of which exhibit linear Arrhenius plots. NMR titration curves show that the pH effect must be due to a group other than histidine. The results give further support to the permissive model of hinge bending previously proposed by one of the authors, in which binding of substrate destabilises the waist region. This loosens the hinge which can then swing slightly to bring the domains closer together to make favourable interactions between the domains and the substrates, with the exclusion of water.


Role and environment of the conserved Lys27 of snake curaremimetic toxins as probed by chemical modifications, site-directed mutagenesis and photolabelling experiments.

The positive charge of Lys27 was suppressed by chemical means in two short-chain curaremimetic toxins, namely erabutoxin a (Ea) from Laticauda semifasciata and toxin alpha from Naja nigricollis. This modification leads to a decrease in the binding affinity of the toxins for the nicotinic acetylcholine receptor, which range 6-15-fold, as judged from both the data reported here and those previously described in the literature. A negatively charged glutamate residue has been introduced at position 27 of erabutoxin a by site-directed mutagenesis. This change provokes a 120-fold decrease in the affinity, which reflects a major alteration of toxin-receptor cognate events. Using toxin-alpha derivative harbouring a photoactive group at Lys27, we probed the toxin local environment in a receptor-bound state by photocoupling experiments. The delta chain was the predominant coupling target, in contrast to previous observations indicating that a photoactive probe on Lys47 predominantly labelled the alpha chain. The toxin derivative weakly labelled the alpha and gamma chains but not the beta chain. The toxin may therefore interact with subunits other than the alpha chain, at least in the vicinity of Lys27.


Thermodynamics of cation binding to Nereis sarcoplasmic calcium-binding protein. Direct binding studies, microcalorimetry and conformational changes.

Nereis sarcoplasmic calcium-binding protein contains three functional EF-hand sites which bind Ca2+ or Mg2+ competitively. Here it was confirmed over a large range of [Mg2+] that the positive cooperativity in binding of Ca2+ (nH = 2) is the result of allostery in Mg2+ dissociation. At pH 7.5, Ca2+ or Mg2+ binding provokes the release of 1.4 mol proton/mol protein, whereas no protons are released during Ca(2+)-Mg2+ exchange. The enthalpy change as a function of Ca2+ binding yields a two-step curve with an inflection point at 1 mol Ca2+/mol protein and a maximum of -66 kJ/mol at 3 mol Ca2+/mol protein. Binding of three Mg2+ ions is cooperative (nH = 1.8) with a maximal enthalpy change of -15.1 kJ/mol protein. Difference spectroscopy led to the conclusion that, in the metal-free protein, the structure around the aromatic residues is well organized, but that Tyr and Trp residues are still solvent-exposed. Upon Ca2+ binding Tyr and Trp spectra are blue-shifted, but some Trp residues are confined to a positively charged pocket. Examination of the Ca(2+)-saturated three-dimensional crystal structure confirmed that Trp4 and Trp57 are located in such pockets or clefts, close to the surface. During the allosteric T----R transition, promoted by binding of the first Mg2+, the Trp residues move to a hydrophobic environment. For both Ca2+ and Mg2+, the enthalpy change and the conformational change in the environment of the aromatic residues is much more pronounced in the first, than in the subsequent two binding steps. In this respect, the latter seem to be equivalent.


Regulation of the final phase of mammalian melanogenesis. The role of dopachrome tautomerase and the ratio between 5,6-dihydroxyindole-2-carboxylic acid and 5,6-dihydroxyindole.

The regulation of the final steps of the melanogenesis pathway, after L-2-carboxy-2,3-dihydroindole-5,6-quinone (dopachrome) formation, is studied. It is shown that both tyrosinase and dopachrome tautomerase are involved in the process. In vivo, it seems that tyrosinase is involved in the regulation of the amount of melanin formed, whereas dopachrome tautomerase is mainly involved in the size, structure and composition of melanin, by regulating to the incorporation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) into the polymer. Moreover, using L-3,4-dihydroxyphenylalanine (dopa) and related compounds, it was shown that the presence of dopachrome tautomerase mediates an initial acceleration of melanogenesis since L-dopachrome is rapidly transformed to DHICA, but that melanin formation is inhibited because of the stability of this carboxylated indole compared to 5,6-dihydroxyindole (DHI), its decarboxylated counterpart obtained by spontaneous decarboxylation of L-dopachrome. Using L-dopa methyl ester as a precursor of melanogenesis, it is shown that this carboxylated indole does not polymerize in the absence of DHI, even in the presence of tyrosinase. However, it is incorporated into the polymer in the presence of both tyrosinase and DHI. Thus, this study suggests that DHI is essential for melanin formation, and the rate of polymerization depends on the ratio between DHICA and DHI in the medium. In the melanosome, this ratio should be regulated by the ratio between the activities of dopachrome tautomerase and tyrosinase.


Kinetic behaviour of alpha-chymotrypsin in reverse micelles. A stopped-flow study.

At the aim of investigating whether the early rapid phase of enzyme turnover is different in reverse micelles compared with bulk water, the kinetic properties of alpha-chymotrypsin have been studied in reverse micelles formed by sodium bis(2-ethylhexyl)sulfosuccinate in isooctane. Pre-steady state and steady-state kinetic constants, in water and in reverse micelles, have been determined by stopped-flow spectrophotometry for the hydrolysis of two substrates, namely acetyl-L-tryptophan-p-nitrophenyl ester and p-nitrophenyl acetate. It has been shown that, for both substrates, the acylation rate constant (k2) is very much lower in reverse micelles than in water. However, the deacylation rate constant (k3) and the turnover number (kcat) are not significantly changed in reverse micelles with respect to bulk water. Therefore, despite considerable rate changes in the acylation step, deacylation is rate limiting both in water as well as in reverse micelles, under the experimental conditions used.


Differential scanning calorimetry of chicken erythrocyte nuclei.

Investigation of structural features of native chromatin requires the use of intact nuclei, a turbid material which cannot be analyzed by optical methods. Differential scanning calorimetry does not require optically clear samples and has been proved by a number of authors to be a powerful tool in this field of study. By this technique, chicken erythrocyte nuclei were found to undergo at least four thermal transitions, centered at 59, 74, 88 and 98 degrees C. The highest temperature transition is strongly dependent on age and storage conditions of the nuclei. Adequate storage conditions overcame this problem and reproducible scans were obtained over a period of several months. This technical improvement has permitted the reconsideration of the occurrence of the fourth calorimetric transition, previously believed to be displayed only in replicating nuclei. Evidence gathered in the presence of perturbants and possible ligands allows the assignment of the four transitions to a nuclear protein scaffold, histones, nucleosomal DNA and a superstructured form of DNA. Moreover, it suggests that the higher-order structure is stabilized by fibronectin-like proteins.


Formation of homogeneous carbohydrate-lectin cross-linked precipitates from mixtures of D-galactose/N-acetyl-D-galactosamine-specific lectins and multiantennary galactosyl carbohydrates.

Quantitative precipitation studies have shown that the Man/Glc-specific lectin concanavalin A (ConA) forms homogeneous (homopolymeric) cross-linked precipitates with individual asparagine-linked oligomannose and bisected hybrid-type glycopeptides in the presence of binary mixtures of the carbohydrates [Bhattacharyya, L., Khan, M. I. & Brewer, C. F. (1988) Biochemistry 27, 8762-8767]. The results indicate that the ConA-glycopeptide precipitates are highly organized cross-linked lattices that are unique for each carbohydrate. Using similar techniques, the present study shows that the Gal-specific lectins from Erythrina indica and Ricinus communis (agglutinin I) form homogeneous cross-linked complexes with individual carbohydrates in binary mixtures of triantennary and tetraantennary complex-type oligosaccharides with terminal Gal residues. Conversely, binary mixtures of Gal/GalNAc-specific lectins from E. indica, Erythrina cristagalli, Erythrina flabelliformis, R. communis, soybean (Glycine max), and Wistaria floribunda (tetramer) in the presence of a naturally occurring or synthetic branched-chain oligosaccharide with terminal GalNAc or Gal residues provide evidence for the formation of separate cross-linked lattices between each lectin and the carbohydrate. The present results therefore demonstrate the formation of homogeneous lectin-carbohydrate cross-linked lattices in (a) a mixture of branched-chain complex-type oligosaccharides in the presence of a specific Gal/GalNAc-binding lectin, and (b) a mixture of lectins with similar physicochemical and carbohydrate binding properties in the presence of an oligosaccharide. These findings show that lectin-carbohydrate cross-linking interactions provide a high degree of specificity which may be relevant to their biological functions as receptors.


Integration of early light-inducible proteins into isolated thylakoid membranes.

An in-vitro system has been established to study the integration of early light-inducible proteins (ELIP) into isolated thylakoid membranes. The in-vitro-expressed ELIP precursor proteins exist in two forms, a high-molecular-mass aggregate which is accessible to trypsin but no longer to the stromal processing protease and a soluble form which is readily cleaved to the mature form by the stromal protease. The mature form of ELIP is integrated into thylakoid membranes; its correct integration can be deduced from the observation that the posttranslationally transported products and the in-vitro integrated ELIP species are cleaved by trypsin to products of the same apparent molecular mass. Trypsin-resistant fragments of high-molecular-mass and low-molecular-mass ELIP appear to have the same size. The processed ELIP species, as well as an engineered mature form of ELIP, are integrated into isolated thylakoid membranes. Integration of the mature protein occurs in the absence of stroma, into sodium-chloride-washed, and trypsin-treated thylakoid membranes. The process of integration is almost temperature independent over 0-30 degrees C. Analysis of the time course of integration leads to the conclusion that, under in-vitro conditions, processing but not integration into membranes is the rate-limiting step. In the absence of stroma, the ELIP precursor is bound to the thylakoid membranes, however, it is no longer accessible to the stromal maturating protease when added after binding has occurred. In conclusion, integration of ELIP differs in many essential details from that of its relatives, the light-harvesting chlorophyll a/b protein family.


The three-dimensional structure of guanine-specific ribonuclease F1 in solution determined by NMR spectroscopy and distance geometry.

Two-dimensional 1H-NMR studies have been performed on ribonuclease F1 (RNase F1), which contains 106 amino acid residues. Sequence-specific resonance assignments were accomplished for the backbone protons of 99 amino acid residues and for most of their side-chain protons. The three-dimensional structures were constructed on the basis of 820 interproton-distance restraints derived from NOE, 64 distance restraints for 32 hydrogen bonds and 33 phi torsion-angle restraints. A total of 40 structures were obtained by distance geometry and simulated-annealing calculations. The average root-mean-square deviation (residues 1-106) between the 40 converged structures and the mean structure obtained by averaging their coordinates was 0.116 +/- 0.018 nm for the backbone atoms and 0.182 +/- 0.015 nm for all atoms including the hydrogen atoms. RNase F1 was determined to be an alpha/beta-type protein. A well-defined structure constitutes the core region, which consists of a small N-terminal beta-sheet (beta 1, beta 2) and a central five-stranded beta-sheet (beta 3-beta 7) packed on a long helix. The structure of RNase F1 has been compared with that of RNase T1, which was determined by X-ray crystallography. Both belong to the same family of microbial ribonucleases. The polypeptide backbone fold of RNase F1 is basically identical to that of RNase T1. The conformation-dependent chemical shifts of the C alpha protons are well conserved between RNase F1 and RNase T1. The residues implicated in catalysis are all located on the central beta-sheet in a geometry similar to that of RNase T1.


Purification and properties of aminopeptidase H from chicken skeletal muscle.

Aminopeptidase H was purified from fresh chicken breast muscle by ammonium sulfate fractionation and successive chromatographies on DEAE-cellulose, Ultrogel AcA 34, activated thiol-Sepharose 4B, phenyl-Sepharose CL-4B and DEAE-cellulose again. The purified enzyme migrated as a single band on SDS/PAGE. Aminopeptidase H exhibits activity against both L-leucine beta-naphthylamide and alpha-N-benzoyl-DL-arginine beta-naphthylamide. The molecular mass of this enzyme was found to be 52 kDa on SDS/PAGE and 400 kDa on Sepharose 6B column chromatography. The optimum pH for the hydrolysis of both substrates was 8.0 and this activity was remarkably enhanced by reducing agents. The enzyme was strongly inhibited by monoiodoacetate and leupeptin, but not affected by EDTA, phenylmethylsulfonyl fluoride, pepstatin, bestatin or puromycin. Aminopeptidase H has been shown to hydrolyze di-, tri- and tetrapeptides in the manner of an aminopeptidase, as well as the beta-naphthylamide derivatives of amino acids. However, the enzyme has not been shown to hydrolyze proteins such as hemoglobin, bovine serum albumin, myofibrillar proteins or sarcoplasmic proteins.


Purification, phosphorylation and control of the guanine-nucleotide-exchange factor from rabbit reticulocyte lysates.

A simple, improved procedure for the isolation of guanine-nucleotide-exchange factor (GEF) and for eukaryotic initiation factor 2 (eIF-2) from rabbit reticulocyte lysates has been developed using ion-exchange chromatography on S-Sepharose, Q-Sepharose, Mono Q and Mono S. The majority of the eIF-2 is separated from GEF at an early stage in the procedure and the remaining small amount of eIF-2.GEF complex is separated from the bulk of the GEF by FPLC on Mono S. The procedure yields approximately 2 mg each of eIF-2 and GEF, of 90% and greater than 80% purity, respectively, from the blood of ten rabbits. All fractions of purified GEF contain four subunits of molecular masses 84, 66, 54 and 39 kDa, with various amounts of a fifth, 30-kDa subunit. The modulation of GEF activity was investigated using the highly purified factor in a guanine-nucleotide-exchange assay. The activity of GEF was stimulated by physiological concentrations of the polyamines, spermine and spermidine, but was unaffected by another polycationic compound, polylysine. Activity was also found to be inhibited by 1 mM NADP+ or NAD+, and this inhibition was overcome by the presence of 1 mM NADPH. Stoichiometric amounts of GEF were unable to release GDP from eIF-2.GDP complexes in the absence of free guanine nucleotides, suggesting that GEF operates by a ternary-complex mechanism. Casein kinase 1 or casein kinase 2 can each phosphorylate the largest subunit (84 kDa) of GEF. These enzymes both phosphorylate serine residues in GEF but they phosphorylate distinct sites, as demonstrated by phosphopeptide mapping following proteolytic or cyanogen bromide digestion. Neither of these kinases phosphorylated any of the other subunits of GEF to any significant extent and several other kinases were inactive against GEF. No effect of phosphorylation on activity could be demonstrated.


The polygalacturonases of Aspergillus niger are encoded by a family of diverged genes.

Aspergillus niger produces several polygalacturonases that, with other enzymes, are involved in the degradation of pectin. One of the two previously characterized genes coding for the abundant polygalacturonases I and II (PGI and PGII) found in a commercial pectinase preparation was used as a probe to isolate five more genes by screening a genomic DNA library in phage lambda EMBL4 using conditions of moderate stringency. The products of these genes were detected in the culture medium of Aspergillus nidulans transformants on the basis of activity measurements and Western-blot analysis using a polyclonal antibody raised against PGI. These transformants were, with one exception, constructed using phage DNA. A. nidulans transformants secreted high amounts of PGI and PGII in comparison to the previously characterized A. niger transformants and a novel polygalacturonase (PGC) was produced at high levels by A. nidulans transformed with the subcloned pgaC gene. This gene was sequenced and the protein-coding region was found to be interrupted by three introns; the different intron/exon organization of the three sequenced A. niger polygalacturonase genes can be explained by the gain or loss of two single introns. The pgaC gene encodes a putative 383-amino-acid prepro-protein that is cleaved after a pair of basic amino acids and shows approximately 60% amino acid sequence similarity to the other polygalacturonases in the mature protein. The N-terminal amino acid sequences of the A. niger polygalacturonases display characteristic amino acid insertions or deletions that are also observed in polygalacturonases of phytopathogenic fungi. In the upstream regions of the A. niger polygalacturonase genes, a sequence of ten conserved nucleotides comprising a CCAAT sequence was found, which is likely to represent a binding site for a regulatory protein as it shows a high similarity to the yeast CYC1 upstream activation site recognized by the HAP2/3/4 activation complex.


A strong protein-tyrosine kinase activity is associated with a baculovirus-expressed chicken tkl gene.

We have previously described a gene named tkl (tyrosine kinase related to lck). It belongs to the src family of protein-tyrosine kinases and among these it has significant homology to the lck gene (lymphoide cell kinase). The tkl gene product may represent the avian homolog of Lck, which is believed to participate in a lymphocyte-specific signal transduction pathway by association with a membrane receptor. To study the biochemical properties of the protein, a nearly complete tkl gene (isolated from a cDNA library from chicken spleen cells) was expressed in a baculovirus system. Approximately 10% of the extracted protein consisted of the soluble 51-kDa Tkl protein (p51tkl) at 40 h post-infection. This protein was found to be phosphorylated on tyrosine and serine residues at a ratio of 5:1. As expected, glycosylation or myristoylation could not be detected. Immunocomplex kinase assays indicated strong autophosphorylation of p51tkl at tyrosine residues and phosphorylation of exogenous substrates such as D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histones H2b and H4, and casein. This protein-tyrosine kinase activity also exhibited a marked preference for Mn2+ compared to Mg2+. The high level expression of enzymatically active Tkl should provide an excellent tool to further study the biological functions of this class of enzymes.


Cytochemical and immunocytochemical characterization of a fibrillar network (GP2) in pancreatic juice: possible role as a sieve in the pancreatic ductal system.

The secretory product of the exocrine pancreas contains sedimentable and non-sedimentable materials. Electron microscopy of the pellet obtained after ultracentrifugation reveals two major components: microvesicles (pancreasomes) and a fibrillar network of small mesh size. Negative staining of an unfixed pellet demonstrated that these structures are not fixation artifacts. Cytochemical analysis showed that pancreasomes are reactive to osmication and uranyl acetate staining, whereas the fibrillar network was unreactive thereby indicating that the latter does not contain lipids; however, lead citrate staining reveals the network. Alcian blue, known to bind sulfate groups of mucosubstances, reacted strongly with the fibrillar network. The pellet was also characterized by immunocytochemistry with specific antibodies to amylase and glycoprotein 2 (GP2). Both antibodies were located only on the fibrillar network. Washing of the pellet with 100 mM KCl-250 mM NaBr had little effect on GP2 content, but reduced considerably alpha-amylase associated with the reticular matrix. It appeared that GP2 was the major component of the scaffolding that gives rise to the fibrillar network and that other proteins such as alpha-amylase could reversibly bind to it. When double-labeling immunocytochemistry was carried out on the unwashed pellet, labeling of the first antigen reduced the labeling of the second. Removal of amylase by washing the pellet increased the GP2 signal. These results indicate that amylase is bound on the GP2 network. Although the function of the GP2 network is still not clearly defined several possibilities could be envisaged at the level of the pancreatic duct system: 1) The network could drain off any aggregates or precipitates forming in small ducts. 2) The small mesh of the network would present a physical barrier to infecting bacteria that could enter into the duct system from the intestine, especially in conditions of low flow rates. 3) The network may exert a mechanical pressure on the membranes bordering the acinar lumen and small ducts thereby preventing their collapse in basal conditions.