[IOL and aniseikonia calculation combined with documentation of surgical data and IOL inventory]. The exact recording of operation data is a precondition for keeping the standards high in cataract surgery, but surgeons are reluctant to answer a questionnaire after a strenuous operation. We have designed a program easy to use in the Macintosh Hypercard System that covers all aspects of cataract surgery such as: (1) the operating record; (2) a data sheet for recording the various details of the operation; (3) recording of the data on hard disc; (4) managing the IOL stock list; (5) proposal of IOL models that are in stock with regard to IOL power (SRK2) and aniseiconia. This program enables the surgeon to record the operation data with effortless ease and it is well accepted. There is no dictation. The operating record and data sheet are printed immediately. The program is controlled by a "mouse". Selection of the suitable IOL model is facilitated by the link between the calculation of IOL power and the IOL stock list. In special cases the IOL power can be changed to obtain less aniseiconia.


[Drusen in Bruch's membrane. Their significance for the pathogenesis and therapy of age-associated macular degeneration]. The drusen found in Bruch's membrane represent precursors for the development of age-related macular degeneration. The pathogenetic concepts are summarized: 1. As a result of aging changes in the metabolism of the pigment epithelium with age, the normal structure of Bruch's membrane is destroyed. This process is associated with lipid accumulation and the development of drusen. The lipids deposited are predominantly phospholipids or neutral lipids. 2. The clinical appearance of drusen can also vary from one person to another, but individually a significant symmetry of drusen characteristics can be seen, demonstrating that drusen are the results of specific metabolic dysfunctions rather than non-specific aging products. 3. The development of specific forms of age-related macular degeneration corresponds with different drusen. Larger, more confluent and hypofluorescent drusen are associated with the development of pigment epithelium detachments. In eyes with smaller, scattered and hyperfluorescent drusen, choroidal neovascularizations are more likely. 4. Histochemically, larger, hypofluorescent drusen contain predominantly neutral lipids. In contrast, smaller, hyperfluorescent drusen consist predominantly of phospholipids. 5. The accumulation of neutral lipids in Bruch's membrane is therefore associated with Pigment epithelium detachments. These apolar lipids may produce a hydrophobic barrier in Bruch's membrane for the water transport from the retina towards the choroid. A pigment epithelium detachment can develop. 6. The deposition of polar phospholipids predisposes to the development of choroidal neovascularization. These lipids in association with the changed structure of Bruch's membrane may induce an inflammation--like reaction, resulting in the in-growth of choroidal capillaries under the pigment epithelium. The analysis of the relationship between subclinical aging changes in Bruch's membrane and different forms of age-related macular degeneration may help to identify specific risk factors and to predict the future outcome in individual eyes. This may result in differentiated treatment concepts adapted to the specific aging changes in each person.


[Idiopathic proliferative vitreoretinopathy. Activation of microglial cells as the deciding factor]. Mononuclear phagocytes are considered pacemakers in the pathogenesis of proliferative vitreoretinopathy (PVR), but their precise biological origin in preretinal PVR traction membranes has remained obscure. This study presents a combined immunohistochemical protocol for the detection of microglial cells, which was applied to 37 membranes of patients with idiopathic and traumatic PVR and with proliferative diabetic retinopathy (PDR). Microglial cells may be labeled by staining for LN-1, ricinus communis agglutinin-(RCA)-1, vimentin, HLA-DR-II, and nucleoside diphosphatase, but are negative for Leu-M1, Leu-M3, EBM-11, von Willebrand factor, CD22, cytokeratin, and glial fibrillary acidic protein (GFAP). Significant proliferation of microglial cells was found in idiopathic PVR while classical macrophages were typical of traumatic PVR. Only rarely were microglial cells detected in PDR. These findings bring into question previous concepts of the pathobiology of idiopathic PVR and support the hypothesis of idiopathic PVR as a specific disease entity.


[Selective occlusion of ocular neovascularization by photodynamic therapy]. Photodynamic therapy (PDT) has successfully been used to induce vascular occlusion via endothelial damage and subsequent thrombosis. To increase the selective of this method for neovascularizations, characteristics in the ultrastructure of the proliferative vessel wall allow physiological vessels to be spared and predominantly neovascularizations to be occluded: (a) Due to the disturbance of the blood-retina barrier, free dye molecules accumulate within the vascular wall. Using a dye with prolonged retention, such as phthalocyanine (CASPc), it is possible to thrombose neovascularizations 24 h post injection while leaving the physiological vasculature of the anterior segment of the rabbit eye unaffected. (b) Proliferating endothelial cells express high numbers of low-density lipoprotein (LDL) receptors. Chlorin e6 (Ce6), a potent photosensitizer, is covalently bound to LDL. Intravascularly, ce6-LDL complexes selectively label neovascular walls. Since ce6-LDL is incorporated intracellular into enzymatically active lysosomes, photothrombosis is effectively achieved at low drug and light doses in vivo. In addition, the induced damage is spatially confined to the inner vascular lining. We conclude that carrier-mediated PDT may offer a new and sensitive approach for selective treatment of intraocular neovascularizations.


[Early disorders of retinal function in diabetes mellitus]. Thirteen insulin-dependent diabetics, aged 13-30 (mean 19.15) years, entered the study. Patients with retinal ischemia, vascular proliferation, or a Snellen acuity below 0.8 were excluded. Ten normal untrained subjects, aged 20-30 (mean 24) years, provided the normal sample. The battery of special visual function tests consisted of: (1) high luminance Pflüger acuity (acuity/luminance/function, rotatable illiterate "E optotypes, screen luminance 10,000 cd/m2); (2) tests combining color vision with demands on spatial resolution: (a) blue-green chromatic acuity (a Velhagen pseudoisochromatic plate served as optotype, the maximum recognition distance being recorded); (b) standard vs mini-panel D-15 color-arrangement tests; (c) blue preferential computerized perimetry (modified Tübingen automatic perimeter, orange adaptive illumination, Schott cut-off filter OG 550, cupola luminance 5 cd/m2).(ABSTRACT TRUNCATED AT 250 WORDS)


[Model experiments of the outer blood-retinal barrier in vitro]. Because of the difficulty in conducting experiments on the outer blood-retinal barrier in vivo, we developed an in vitro model. Bovine retinal pigment epithelial cells were grown on semipermeable membranes, enabling separate manipulation of the apical and basal medium. As a parameter of barrier function, we measured the transepithelial resistance (TER). Barrier function was also tested with fluorescein. The transepithelial resistance increased under optimal culture conditions, in confluent cultures, by 200 omega and there was no fluorescein leakage. After exposure to trypsin in Ca/Mg-less medium or EDTA or after application of argon laser, we were able to induce a breakdown of the TER and fluorescein leakage. This happened immediately after laser exposure, 1 min after EDTA, and 4 min after trypsin application. We observed no morphological differences after breakdown of the barrier function on the intercellular connections compared to normal confluent cultures following EDTA or trypsin exposure. In all experiments there was a recovery of barrier function after returning the cells to control conditions. These first results demonstrate that our in vitro model is a sensitive method for investigating barrier function in retinal pigment epithelium in cell culture.


[Changes in the invasion front of uveal melanomas]. Central and peripheral cells of 12 uveal melanomas were compared by electron microscopy to determine whether infiltrating melanoma cells have special characteristics (e.g., loss of cell adherence, changes in the cytoskeleton, increase of cell organelles). No significant differences were found. The two theories of tumor invasion ("mechanical" and "locomotive" theory) and the light microscopical findings that occur in the invasion zone of uveal melanomas are discussed.


[Cyclosporin A in therapy of chronic uveitis]. 48 patients with chronic severe uveitis of pressumed noninfectious origin resistent to corticosteroids have been treated with Cyclosporin A. All patients had an initial loading dose of 5 mg/kg/day followed by a dose reduction according to ocular inflammatory activity and tolerability and according the guidelines by BenEzra, Nussenblatt and Timonen. Most of the patients received additional corticosteroids in a low dose. 35 out of these 48 patients (13 suffering from intermediate uveitis, 11 from retinal vasculitis, 5 panuveitis, 4 "pressumed histoplasmosis" and 2 sympathetic ophthalmia) were treated for 1 year and observed after withdrawing of Sandimmun for at least 6 months. The majority of these patients have manifested a positive therapeutic response to Cyclosporine, in particular patients suffering from vasculitis, panuveitis and sympathetic ophthalmia. All 35 patients were treated before for a long time with steroids without success, 19 out of these 35 in addition with cytotoxic agents. The immunosuppressive effect of Cyclosporine was not permanent, frequently the inflammation relapsed on reduction of dosage or withdrawing of the drug. Guidelines for combined regimen (Cyclosporine and corticosteroids and vitrectomy) were given. Although a large variety of side effects were reported the compliance was good.


[Acute necrotizing retinitis with amotio retinae. Surgical and medicamentous antiviral therapy]. This report pertains to the case of a 25-year-old patient suffering from acute necrotizing retinitis (ANR syndrome). Initially, one eye revealed clinical signs of diffuse chorioretinitis accompanied by perivasculitis and heavy keratic precipitates, papillitis, and vitreous infiltrates. After initial improvement under antiphlogistic therapy, however, necrotizing retinitis developed, associated with peripapillar hemorrhages, multiple peripheral retinal holes and eventually complete retinal detachment. The subsequently performed retinal detachment surgery, completed with vitrectomy, cerclage and silicone oil tamponade, was successful. At the same time, the patient was put on systemic therapy based on acyclovir. In the literature, similar developments are usually related to HSV and HZV infections. Although in our case a virological diagnostic test did not reveal the presence of any virus, the characteristic symptoms of the ANR syndrome completely disappeared under the above-mentioned therapy. Visual acuity, previously consisting only in light perception, improved to 0.4.


[Leopard spot chorioretinopathy. Initial manifestation of recurrent acute lymphocytic leukemia]. An 11-year-old girl with a history of acute lymphocytic leukemia of the central nervous system had attained complete remission for almost 3 1/2 years after combination chemotherapy and radiotherapy when she developed iritis and chorioretinopathy of the right eye. Neither an anterior chamber tap nor a diagnostic vitrectomy revealed leukemic cells. Both nonspecific anti-inflammatory therapy and antiviral treatment were unsuccessful. Finally, lymphoblasts were detected in the cerebrospinal fluid and in the bone marrow after repeated lumbar puncture and bone marrow aspiration. Combination chemotherapy alone was resumed, resulting in the resolution of all acute ocular symptoms and bone marrow involvement. Only the leopard-spot-like pigmentary fundus changes persisted. The child has now remained in continuous complete remission for 1 1/2 years.


[Measuring retinal vascular diameter using the scanning laser ophthalmoscope and computer. Initial results]. The diameter of the large retinal vessels is an important factor in retinal microcirculation. A system configuration was developed that uses SLO images to measure the diameter of retinal branch vessels off-line. The place for measurement is defined in the image by the investigator, and the diameter is measured automatically. The mean standard deviation of the diameter of arterial vessels was 2.37% and for veins 3.43% by repeat measurement in sequential pictures by optimal scanner settings. The reproduction of vessel diameters was unsatisfactorily for repeat examinations. The course of these results may be changes in the fundus image connected with changes in the position of the scanner-bulbus (e.g., new examination, poor fixation). The scanner-bulbus position is not yet precisely defined and not reproducible enough.


[Krypton laser therapy. Use in secondary serous detachment of the retinal pigment epithelium and in multiple idiopathic serous detachments of the retinal pigment epithelium]. Two variants of serious detachments of the retinal pigment epithelium (RPE) are demonstrated with reports of typical cases: (1) secondary serous detachment of the RPE; (2) idiopathic serous detachment of the RPE (pigment epithelium detachment not associated with any other retinal or choroidal disease). Fluorescein angiographic criteria for differential diagnosis and the resulting different techniques of krypton laser coagulation are portrayed: (a) focal krypton laser coagulation of the total area of the pigment epithelium detachment; (b) circular, one-row laser coagulation at the detachment borders. The advantages of circular, one-row laser coagulation of idiopathic serous RPE detachment over coagulation of the total area of the RPE detachment are described: first, these are fewer scotomas following coagulation; second, fewer laser foci are required; and third, the risk of subretinal bleeding is lower. These findings emphasize the efficiency of circular, one-row laser coagulation at the detachment borders in the treatment of idiopathic serous RPE detachments.


[Tubulointerstitial nephritis and uveitis (TINU syndrome)]. We report on a case of non-granulomatous bilateral iridocyclitis with tubulointerstitial nephritis in a 13-year-old girl. Complete recovery was achieved after treatment with systemic and topical corticosteroids.


Selenium in the treatment of heavy metal poisoning and chemical carcinogenesis. Selenium (Se) has been shown to counteract the toxicity of heavy metals such as cadmium, inorganic mercury, methylmercury, thallium and to a limited extent silver. Although not as effective as Se, vitamin E significantly alters methylmercury toxicity and is more effective than Se against silver toxicity. Vitamin E is very effective against lead toxicity but Se has little effect. The presumed protective effect of Se against cadmium and mercury toxicity is through the diversion in their binding from low molecular weight proteins to higher molecular weight ones. Se appears effective in counteracting the chemical carcinogens (3-methyl-4-dimethyl-aminoazobenzene, 2-acetylaminofluorene, diethylnitrosamine, aflatoxin, 7,12-dimethylben (a) anthracene, benzopyrene and 3-methylcholanthrene) used to induce skin, liver and mammary tumors, but much less effective against those (dimethylhydrazine, azoxymethane, methylazoxymethanol, bis (2-oxopropyl) nitrosamine, benzopyrene, 1 methyl-1-nitrosourea and n-methyl-n-nitro-nitrosoguanidine) used to produce tumors in the colon, lungs, trachea and pancreas in laboratory animals. In contrast, Se many even increase pancreatic carcinomas in animals treated with bis (2-oxopropyl) nitrosamine. The health implications in humans of Se and heavy metal toxicities and in cancer are discussed.


Concentration of free calcium in erythrocytes and liver mitochondria of zinc-depleted rats. The concentration of Ca(2+)-ions in mitochondria and erythrocytes of zinc-depleted rats was determined with the fluorescent Ca(2+)-indicator fura-2. The zinc-concentration of the diet did not affect the mitochondrial Ca(2+)-concentration. There was an elevation of 5% for the Ca(2+)-concentration in erythrocytes of zinc-depleted and pair-fed rats compared to ad libitum-fed animals. The cause of this elevation could be ATP-depletion because of restricted food intake.


Transplacental passage and fetal deposition of mercury after low-level exposure to methylmercury--effect of seleno-L-methionine. Previous experimental studies on transplacental passage and possible fetotoxicity of methylmercury have almost exclusively used a single dosage or 2-4 repeated doses of mercury on specific days during gestation and often used at relatively high dose levels. In previous studies, selenium supplementation considerably increased the concentration of mercury in the blood of offspring after maternal exposure of rats to methylmercury, whereas whole-body retention and organ deposition of mercury in mice were unaffected. The present study in mice, which involved exposure for 5 weeks to a low dose of methylmercury in the drinking water (1 nmol/ml) before and during pregnancy, demonstrates that mercury is deposited in offspring both in utero and during lactation, and that transplacentally absorbed mercury is not, or only very slowly, excreted. Seleno-L-methionine increased the deposition of mercury in offspring, but the effect was due to slightly higher deposition in the dams. Selenomethionine significantly reduced the kidney deposition of mercury in offspring, whereas liver deposition of mercury was unaffected. These results indicate that interactions between organo-selenium compounds and methylmercury orally administered at low doses is of less importance than previously believed on the basis of experiments with higher doses of selenite injected or supplemented to the diet.


The human selenium status in Hungary.

The human selenium status in Hungary. The selenium concentration of foods and of human blood varies widely between geographical areas depending on the availability of soil Se for plants. The mean serum Se concentration in Europe has been found to vary between 0.80-1.50 mumol/l and the mean Se concentration of wheat between 0.02-0.11 mg/kg. We estimated by various markers the Se status of Hungarians on whom data is lacking. Serum samples (n = 238) from healthy blood donors, and toenails (n = 132) and fingernails (n = 211) from different apparently healthy individuals residing in both urban and rural areas were collected from four districts in Hungary. Wheat samples (n = 29) as grains were obtained directly from farms representing the same areas. Serum Se was determined by electrothermal AAS and the other samples by acid-digestion fluorimetry. The accuracy of the methods was verified by analyzing external reference samples. The mean (+/- SD) serum Se concentration was 0.70 +/- 0.12 mumol/l, ranging between 0.41-1.18 mumol/l. The mean serum Se levels 0.87 mumol/l was significantly (p < 0.001) higher in the East and lower in the South (0.60 mumol/l) compared to the northern area 0.70 mumol/l. The mean (+/- SD) toenail Se concentration was 0.56 +/- 0.18 mg/kg. The Se level in the East was significantly (p < 0.01) higher than the level in the North, 0.68 mg/kg vs. 0.54 mg/kg, respectively, and lowest in the southern area, 0.40 mg/kg. The same pattern applied to fingernails. Wheat Se ranged from 5 to 235 micrograms/kg, with the median 34 micrograms/kg. The lowest Se concentrations were generally found in the South.(ABSTRACT TRUNCATED AT 250 WORDS)


Nickel-, chrom- and cobalt-concentrations in human tissue and body fluids of hip prosthesis patients. The world-wide experience with millions of metallic implantations suggests the biocompatibility of modern alloys, commonly made of cobalt, chromium and nickel. There is, however, little information available on the internal metal exposure resulting from implants. In this study we assessed the metal concentrations in body fluids and tissue samples (muscle, bone) of patients who had undergone total hip replacement. Our patients were divided up into two groups. One group had firmly fixed implants two years after surgery. The other group had loose implants of the same Co-Cr-Mo alloy. Urine analyses revealed an increased renal elimination of nickel, chromium and cobalt. Cobalt and nickel exceeded the upper normal value. In serum the concentrations of nickel and chromium were normal or slightly elevated, the cobalt concentrations were significantly elevated. In some cases tissues adjacent to the implant showed extremely high concentrations of chromium and cobalt. This finding was also obtained in tissues that had no direct contact with the arthroplasty. The findings suggest that alloys of prostheses can undergo corrosion and release metal ions.


Chromatographic separation of serum proteins and estimation of their zinc and copper content. Human serum proteins of blood donors and dialysis patients were separated by means of gel filtration chromatography. The resulting fractions were analyzed for copper and zinc. Separation resulted in 3 zinc peaks with a molecular weight of about 700,000, 300,000, and 75,000 Dalton, with alpha 2-macroglobulin co-eluting in the first and albumin co-eluting in the third zinc peak. The zinc protein(s) of the second peak remained unidentified. The three peaks contained, in succession, 0.72 +/- 0.30 mumol/L (4.8 +/- 1.6%), 1.26 +/- 0.37 mumol/L (8.5 +/- 1.7/1000) and 12.8 +/- 2.1 mumol/L (86.8 +/- 2.8%) of total zinc in the case of blood donors, and 1.19 +/- 1.05 mumol/L (9.2 +/- 7.2%), 0.97 +/- 0.22 mumol/L (8.0 +/- 2.6%), and 10.4 +/- 1.66 mumol/L (82.7 +/- 6.7%) in the case of dialysis patients. Separation followed by copper analysis resulted in the three peaks, as well, with a molecular weight of about 750,000, 140,000, and 75,000 dalton. The copper protein of the first peak remained unidentified, while coeruloplasmin co-eluted in the second and albumin in the third peak. The three peaks contained, in succession, 0.4 +/- 0.16 mumol/L (2.3 +/- 0.95%), 14.6 +/- 0.7 mumol/L (83.9 +/- 4.1%), and 2.4 +/- 0.6 mumol/L (13.7 +/- 3.5%) of total copper in the case of blood donors, and 0.5 +/- 0.73 mumol/L (2.2 +/- 3.2%), 19.5 +/- 1.1 mumol/L (90.5 +/- 4.9%), and 1.6 +/- 0.66 mumol/L (7.3 +/- 3.0/1000) in the case of dialysis patients. Limitation of the method is shown regarding separation of major from minor proteins and albumin from transferrin.


Detection by HPLC-ICP of metallothionein in serum of an epileptic child with valproate-associated hepatotoxicity. A patient who developed valproate-associated hepatotoxicity had significantly lower serum levels of total protein, albumin and selenium than the controls. This study shows that with the beginning of the hepatic coma metallothionein (MT) appeared in the serum mainly in the form of Zn-thionein, which altered the Zn distribution pattern of the serum in a characteristic manner. HPLC-ICP3 was successfully applied to the simultaneous speciation of elements and characterization of MT by the use of one gel permeation column.


Detection of hepatitis B virus DNA in donor blood by the polymerase chain reaction. Blood collected from voluntary donors at local blood banks and blood donation camps was screened for the hepatitis B virus genome using the polymerase chain reaction and for viral markers by standard serological techniques. The sensitivities of the two screening strategies were compared. One hundred and twenty-six blood samples were tested for HBV serological markers--HBsAg, anti-Hbs and anti-HBc--by ELISA: The same samples were also subjected to the polymerase chain reaction using primers made by us. Analysis of the polymerase chain reaction amplified products revealed that 24% of the blood samples which tested negative for HBsAg using the ELISA technique were positive for HBV DNA by the polymerase chain reaction. All the HBsAg positive samples (by ELISA) were also positive by the polymerase chain reaction (which detected additional samples as well). Anti-HBc antibodies showed a much greater concordance with the polymerase chain reaction. The results emphasize that the screening strategies for donor blood need to be re-evaluated in order to check inadvertent transmission of hepatitis B virus during blood transfusion. The ELISA technique to detect HBsAg as the sole serological marker is inadequate to indicate the actual prevalence of hepatitis B virus in the donor blood. The polymerase chain reaction may be a better screening test. If this is not available, the detection of anti-HBc antibodies appear to be a better means of screening blood than HBsAg.


Percutaneous endoscopic gastrostomy for long term enteral nutrition. Percutaneous endoscopic gastrostomy for long term enteral nutrition is often indicated in patients with head, neck and oesophageal cancer but despite its growing popularity elsewhere, it is not widely used in India. Between March 1990 and July 1991, we performed percutaneous endoscopic gastrostomy in 54 patients. The primary sites of tumour were the hypopharynx (11), oral cavity (7), tongue (7), cricopharynx (7), oesophagus (16) and other sites (6). The indications were difficulty in swallowing following treatment (22), preoperative nutritional support (7) and terminal care (21). In 49 patients, it was performed by the 'pull' technique in the endoscopy room under local anaesthesia and mild sedation. Indigenously prepared tubes and blenderised foods were used. Fifteen patients underwent dilatation of the tumour prior to the gastrostomy. The procedure was successful in 50 (93%) patients. Three failures were caused by obstructing tumours and one by a previous gastric resection. Feeding was started 18 to 24 hours after the procedure in 48 patients. No major complications occurred but minor complications were seen in 11 (22%) patients. Fourteen patients had their gastrostomy tube removed after 2 to 6 months of use while 15 patients undergoing therapy or with persistent dysphagia were on gastrostomy feeds for 1 to 6 months. Of the 21 terminally ill patients, 8 died, 6 were lost to follow up and 7 were on feeds for 1 to 6 months. Percutaneous endoscopic gastrostomy is a simple, safe and effective method for long term enteral feeding. Indigenous tubes and home made blenderised foods are adequate substitutes for the more expensive commercial kits and enteral formulations.


HLA and complement C4 studies in psoriasis vulgaris. Psoriasis vulgaris is known to be associated with the presence of certain HLA antigens and complement factors but only studies of Western populations have been reported. We investigated the HLA and complement profile in Indian patients with psoriasis vulgaris. Sixty-seven patients and 132 normal subjects were typed for class I antigens using 262 sera, and 55 patients and 104 normal subjects were typed for class II antigens using 174 sera. The sera from patients and normals were sent to Japanese laboratories for the study of class III antigens. The study revealed that patients had an increased frequency of A1 (p < 0.0025), B17 (p < 0.0025), Cw6 (p < 0.001) DR7 (p < 0.05) and DQw3 (p < 0.005) when compared to normal controls. Analysis of two and three antigen haplotypes revealed a significant increase in the incidence of all haplotypes involving those antigens which showed a high frequency among patients. A significant association was also found with complement factors C4A6.3 and C4A6,X. However, the relative risk was high for C4BQO (10.73). Psoriasis in Indians is associated with the A1, B17 and Cw6 but not with B13 antigens and the complement allotypes mentioned above. The strong association with C4 factors may be as a result of a close linkage between the class III region and Ir genes.


Malarial hepatitis: a heterogeneous syndrome? The incidence of malarial hepatitis in patients with Plasmodium falciparum infection and jaundice is not known and it is not clear whether the condition is a single entity or a heterogeneous syndrome. We prospectively studied the natural history of all patients with falciparum malaria and jaundice admitted to military hospitals in Northeast India from 1988 to 1991. A possible drug or viral cause for the hepatitis was excluded by the history, serological tests and liver histology. Of the 732 patients admitted with falciparum malaria, 39 had jaundice but only 18 had malarial hepatitis indicated by a rise in their serum glutamate pyruvate transaminase levels to more than three times the upper limit of normal and an absence of clinical or serological evidence to suggest drug or viral hepatitis. The liver in these patients was always enlarged. Their mean age was 27.6 years and 85% were males. The mean serum bilirubin was 12.7 +/- 10.3 mg/dl, serum glutamate oxaloacetate transaminase was 212.8 +/- 144.9 IU, serum glutamate pyruvate transaminase was 287.1 +/- 206.2 IU and the serum alkaline phosphatase was 20.4 +/- 10.1 KA. Clinically, 2 groups of patients were seen. Thirteen patients who presented with a severe form of disease had coma, deep jaundice and renal failure. The other 5 patients had a relatively mild illness with only fever, headache and vomiting for 2 days. Four patients with severe disease died. Liver histology (studied in 5 patients) showed Kupffer cell hyperplasia and deposition of malarial pigment. Plasmodium falciparum was demonstrated in sinusoidal red blood cells in only 2 cases. Malarial hepatitis occurred in 18 out of 39 patients with jaundice and falciparum malaria. It is a heterogeneous syndrome with at least two clinical subsets and the severe disease should not be mistaken for fulminant hepatic failure as there is a better response to therapy.


Enteral nutrition in surgical patients.

Enteral nutrition in surgical patients. Malnutrition is common in patients admitted for surgery and is a major cause of increased morbidity and mortality. Nutritional support has been shown to be of help in reducing complications. Parenteral nutrition and commercially available enteral diets are expensive, so the efficacy of a 'home-brew' enteral diet was studied in such patients. Forty malnourished patients, 20 with benign disease and 20 with malignancy, were administered a 'home-brew' enteral diet (1140 calories and 60 g protein per litre) perioperatively for 14 days. They received 2500 to 4000 calories per day according to their requirement. Weight, triceps skinfold thickness, midarm circumference, serum albumin and transferrin, absolute lymphocyte count and creatinine-height index were monitored on days 0, 7 and 14. Nitrogen balance was estimated on alternate days and the results of the two groups were compared. Weight, skinfold thickness and midarm circumference did not change. Serum albumin levels showed a rise in the benign group by day 7, but the rise attained significance in the malignancy group by day 14. A similar pattern was observed in transferrin levels and there was a significant correlation (r = 0.652, p < 0.001) between albumin and transferrin levels. A positive nitrogen balance was attained earlier in the benign group (4.3 v. 5.8 days, p < 0.001). The creatinine-height index showed a rise in both groups by day 7 and a further rise by day 14. Diarrhoea was the commonest complication but was easily controlled with loperamide. The efficacy of the diet was evidenced by the improvement recorded in various objective nutritional parameters and complications were minimal. This cost-effective diet may be used for nutritional support with good results in patients in India and other developing countries.


Molecular cytogenetics of human cancer.

Molecular cytogenetics of human cancer. Cancer may arise from the genetic transformation of a single precursor cell, which proliferates to form a clone. Chromosomal abnormalities are associated with many types of tumours. Some of the chromosomal rearrangements such as translocation, deletion and insertion involve breakage of chromosomes close to known oncogenes. The close linkage between the chromosomal changes, the gene modifications and consequently altered protein function seen in malignant cells suggest that cancer is a genetic disease. Analysis of chromosomal abnormalities and oncogene amplifications in malignant cells have been found to be related to their malignant potential and hence may be utilized in the clinical management of patients with cancer.


Daily diethylcarbamazine for the treatment of Brugia malayi microfilaria carriers. Diethylcarbamazine is effective for the treatment of brugian filaria carriers. However, the 12-day course with a daily dose of 6 mg per kg body weight is rarely completed. The World Health Organization has indicated the need for better regimes to control filariasis. We evaluated the long term effects of three different dosage regimes of diethylcarbamazine--6, 9 and 12 mg per kg body weight--to a total of 72 mg on 93 microfilaria carriers of Brugia malayi. All cases treated with the 6 mg daily dose showed complete clearance of microfilaria immediately after the treatment. However, at the 9 and 12 mg daily doses only 91% and 85% of cases respectively showed complete clearance. There was a close association between the daily dose and the proportion of cases developing side-effects. The mean initial microfilaria counts were significantly higher in those who had side-effects than in those who did not. An 11-month follow up showed that with the 6 mg daily dose the recurrence rate of microfilaraemia was 29% while with the 12 mg daily dose it was 67%. Four per cent of patients on a daily dose of 6 and 9 mg discontinued treatment while 21% of those on the 12 mg daily dose did so. The 6 mg daily dose of diethylcarbamazine was the most effective and best accepted regime.


Iopanoic acid as an adjunct to carbimazole in the management of hyperthyroidism. The thiourea drugs take a few weeks to control the symptoms of hyperthyroidism whilst iodine containing radiographic contrast agents (iopanoic acid and sodium ipodate) have a more rapid effect. There is no report on the use of iopanoic acid administered in conjunction with carbimazole, so we evaluated the efficacy of this combination in the early medical management of patients with hyperthyroidism. Thirty hyperthyroid patients diagnosed by clinical and biochemical criteria were randomized into two treatment groups. Group A (n = 16) received iopanoic acid (500 mg orally twice a day for the first 3 weeks) and carbimazole (30 mg orally in three divided doses) while group B (n = 14) received carbimazole alone. Clinical examination and estimation of serum total T3, total T4 and TSH were done by radioimmunoassay at the start of therapy, weekly for 4 weeks and then at 6, 8 and 12 weeks. In the initial 3 weeks, iopanoic acid induced a significantly greater fall in mean serum total T3 levels (Z = 2.298, p < 0.02) and a slower fall in mean serum total T4 (Z = 2.396, p < 0.05) in group A patients compared to those in group B. This was accompanied by earlier clinical improvement in group A patients. The mean serum total T3 and T4 values rose to higher levels in group A at 4 weeks, one week after discontinuation of iopanoic acid. At the end of 12 weeks, however, there was no significant difference in the mean serum total T3 and T4 levels between the two groups (p > 0.05). Biochemical euthyroidism (i.e. total T3 < 3 nmol/L and total T4 < 170 nmol/L) was achieved later in group A patients than in group B (10.4 +/- 5.0 weeks v. 3.6 +/- 1.2 weeks, p < 0.0001). Iopanoic acid given together with carbimazole induces rapid clinical improvement in hyperthyroid patients than carbimazole alone. However, the delayed achievement of euthyroidism may preclude its routine use in the management of patients with hyperthyroidism except in those with thyrotoxic emergencies.


Total cholesterol and mortality in patients with pre-existing coronary artery disease. A positive correlation exists between serum cholesterol levels and cardiovascular mortality. However, the role of serum cholesterol in persons with pre-existing coronary artery disease is not clear. A cohort of 524 patients with coronary artery disease was divided into four groups based on the total serum cholesterol values. Group I consisted of 68 patients with cholesterol levels of 200 mg/dl or less; Group II of 116 patients with cholesterol levels between 201 and 220 mg/dl; Group III of 187 patients with levels between 221 and 240 mg/dl and Group IV of 153 patients with cholesterol levels greater than 240 mg/dl. Actuarial survival analysis over an 11-year follow up did not show any overall difference in mortality between these groups (Logrank test statistic = 1.89, p > 0.1). Analysis after adjustment of the data also showed that mortality rates were not different (chi (2) = 4.73, p > 0.05). Hazard function analysis indicated that death rates per thousand person years of follow up were 49.97 +/- 8.4 in Group I, 41.38 +/- 8.4 in Group II, 55.39 +/- 4.4 in Group III and 45.38 +/- 6.4 in Group IV. These were also not statistically significant. Comparison of mortality rates in patients with angina pectoris and past myocardial infarction also showed similar results. Total serum cholesterol levels do not influence long term survival in patients with pre-existing coronary artery disease.


Prenatal diagnosis and control strategies for beta-thalassaemia on the Indian subcontinent. The control of beta-thalassaemia by prenatal diagnosis is an important option in India. The disease is common and resources are scarce for the treatment of affected children. Presently the most suitable approach for prenatal diagnosis is DNA analysis of a chorion villus sample by direct mutation detection. For the implementation of such a prenatal diagnosis programme it is essential to know the spectrum of beta-thalassaemia mutations in the population. The beta-thalassaemia mutations of 708 unrelated carriers from seven different regions of the Indian subcontinent were characterized by the allele-specific polymerase chain reaction and DNA sequencing. Sixteen different beta-thalassaemia mutations were identified which could be divided into three groups according to their frequencies. Five common mutations accounted for 93% of alleles. Significant regional differences in the frequency and distribution of mutations were observed. The spectrum of mutations defined forms the basis for a beta-thalassaemia prenatal diagnosis programme in India. A strategy has been outlined for mutation detection which could be successfully applied. Since prenatal diagnosis has become comparatively simple, recommendations are made for a community-based thalassaemia control programme.


The use of diagnostic bronchoscopy in lung cancer. The role of fibreoptic bronchoscopy in the diagnosis of bronchogenic carcinoma is well established. However, in developing countries, where the burden of illness is large and diagnostic facilities are limited, only a small number of patients are diagnosed at a stage when they might benefit from operation. We felt it would be desirable to identify subsets of patients suspected of harbouring lung cancers, in whom bronchoscopy would have not only a high diagnostic yield but also provide useful information which might influence treatment, the patient's quality of life and duration of survival. We analysed the records of 588 patients, who had been bronchoscoped for suspected lung cancer, over a period of 8 years at a tertiary referral centre in north India. The patients were divided into different clinical subgroups on the basis of their clinical and radiological presentation, and the diagnostic yield from bronchoscopy in each group was calculated. A decision analysis model was constructed and the expected value of clinical information was determined for each group. A tissue diagnosis was established by bronchoscopy in 177 (30%) patients and by additional investigations in 43 (7.3%) patients. A positive tissue diagnosis was obtained most often in patients with clinical and radiological evidence of pulmonary collapse (50%) and mass lesions (38-42%). Only 12% of patients with malignancy underwent resection and 70% of them belonged to the above two groups. The expected value of clinical information was greatest in patients with collapse (0.077) or mass lesions (0.067-0.065). A diagnostic work-up including fibreoptic bronchoscopy is indicated early in patients with collapse or mass lesions of the lung. Patients with non-resolving pneumonia, pleural effusion, metastatic disease and non-specific lesions on chest X-ray should receive a low priority for bronchoscopy.


T cell subpopulations in amoebic liver abscess. Cellular immunity may play a major role in the pathogenesis of amoebic liver abscess but there is little data on the effect of treatment on T cell subpopulations in such patients. We performed a prospective, controlled study of the T lymphocyte subpopulations in 17 patients with amoebic liver abscess before, and at 4 and 8 weeks after treatment with metronidazole (30 mg/kg/day). T4 and T8 cells were studied using monoclonal antibodies by the alkaline phosphatase anti-alkaline phosphatase staining technique. The mean T4 cell percentages in the acute stage of illness and at 4 and 8 weeks after treatment were 27, 26 and 27 respectively and the mean T8 cell percentages were 19, 24 and 29. The T4:T8 ratio at the acute stage was 1.7, and 1.1 and 1.2 at 4 and 8 weeks of therapy. The T4:T8 ratio at the acute stage did not differ significantly (p > 0.05) from that in the control group. However, at 4 and 8 weeks after therapy there was a significant increase (p < 0.05) in the T8 cells with no significant change in the T4 cells. We suggest that sensitization of the T8 cells occurs in patients with amoebic liver abscess in the later phase of the disease. This may be responsible for the elimination of the parasite from the human host.


Control of breathing by endogenous opioid peptides: possible involvement in sudden infant death syndrome. Experiments have been performed in order to evaluate the respiratory consequences of a suppression or accumulation of endogenous opioid peptides, in the neuronal network which generates the motor respiratory activity. Iontophoretic application of naloxone onto respiratory neurons increases their firing activity and increases their respiratory modulation. On the other hand the local injection of kelatorphan (an enkephalinase inhibitor) decreases the firing of respiratory neurons and thus reduces the respiratory modulation. This effect of kelatorphan mimics the effect on respiratory neuron of an iontophoretic application of met-enkephalin. Furthermore the local injection of kelatorphan reduces the frequency of the respiratory output recorded from the phrenic nerve. This effect is reversed by systemic administration of naloxone. The results demonstrate the involvement of endogenous opioid peptides in the control of breathing suggesting that in Sudden Infant Death Syndrome a possible dysregulation in opioidergic system could occur.


In vitro study of newborn rat brain maturation: implication for sudden infant death syndrome. We have used slice preparation from newborn rats to study the development of the nucleus tractus solitarius neuronal network and brain intracellular phosphorus metabolites. As shown previously on adults, the newborn preparation retains local excitatory and inhibitory synaptic connections and enables study of intrinsic electrical properties in the nucleus tractus solitarius. Electrophysiological investigation of inhibitory synaptic transmission demonstrated a maturational step at days 4-6 after birth. Nuclear magnetic resonance spectroscopy of brain slices revealed a metabolic maturation between postnatal days 11 and 17. Results emphasize the differential maturation steps during the postnatal development of rat central nervous system. Possibly, Sudden Infant Death Syndrome may result from the abnormal timing in the occurrence of these steps.


Expression of recombinant human nerve growth factor in Escherichia coli. Nerve growth factor (NGF) is a neurotrophic factor for basal forebrain cholinergic neurons and may be of benefit in neurodegenerative diseases of humans. A method is described to obtain significant amounts of biologically active recombinant human NGF (rhNGF) in one step. RhNGF was expressed in E. coli and the majority of the protein accumulated in inclusion bodies. It was immunoprecipitated by a serum against mouse NGF. Solubilization of the inclusion bodies was done in 3M guanidine HCl and renaturation was effected by dilution and air oxidation in the presence of 6 microM CuSO4. Recoveries were 10-12 micrograms of rhNGF per ml of bacterial suspension. Its biological activity was tested in a bioassay system employing sympathetic chick embryo ganglia and was inhibited by the monoclonal antibody 27/21 against mouse NGF.


Regulatory mechanisms in melatonin biosynthesis in retina. The vertebrate retina produces melatonin in a light-dependent rhythmic fashion, synchronized with, but independent from the rhythm of the hormone formation in the pineal gland. This review summarizes the current status of our knowledge on regulatory mechanisms involved in controlling the retinal melatonin biosynthesis. Special emphasis is given to the role and mode of action of dopamine and GABA, two established retinal neurotransmitters, as well as that of second messengers (cyclic AMP, calcium ions). Comparisons are made between lower vertebrates and mammals.


Potent in vivo but not in vitro inhibition of monoamine oxidase by 3-chloro-alpha-phenylpyrazinemethanol. 3-Chloro-alpha-phenylpyrazinemethanol (3-CPM) inhibited monoamine oxidase (MAO) types A and B in vivo in mouse brain, heart and liver. The inhibition was dose-dependent at doses of 0.3-32 mg/kg i.p. and occurred within 1 h after the compound was injected. 3-CPM was a very weak inhibitor of mouse brain mitochondrial MAO activity in vitro, even when preincubated with the enzyme; MAO-A was inhibited only about 50% at a high concentration of 3-CPM (1 mM), and MAO-B was inhibited even less. After a 10 mg/kg i.p. dose of 3-CPM in mice, both MAO-A and MAO-B were inhibited at day 1, but activity had largely recovered within a few days in brain, liver and heart. 3-CPM at doses of 1, 3, 10 and 32 mg/kg i.p. caused dose-dependent antagonism of the depletion of striatal dopamine and of cortical norepinephrine by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. 3-CPM is therefore a potent inhibitor of MAO-A and of MAO-B in mice in vivo despite its weak effect on the enzyme in vitro. A metabolite of the drug may be involved in the in vivo effects.


Calcium dependent release of acetylcholine and gamma-aminobutyric acid from the rabbit retina. The concurrent release of endogenous ACh and GABA from the retina (in the presence of physostigmine) was measured using either an eye-cup preparation in rabbits anaesthetized with urethane or isolated rabbit retinas. There was a spontaneous resting release of ACh and GABA from the dark adapted retina of ca 5 and 160 pmol min-1 respectively. Stimulation of the initially dark adapted retina in vivo with flickering light (0.1-20 Hz) increased the release of ACh by up to 5 times the spontaneous resting release but did not cause a detectable increase in GABA release. The maximum light-evoked release of ACh was about 24 pmol min-1/retina and occurred at a frequency of 10 Hz. However, the maximum release of ACh per flash occurred at 0.1 Hz at which frequency the average ACh release per flash from one amacrine cell was ca 2.35 x 10(-18) mol. Exposure of the retina to the potent inhibitors of GABA uptake, SKF89976A and SKF100330A markedly reduced the resting release of ACh and abolished the light-evoked release of ACh but did not enable a light-evoked release of GABA to be detected. Bicuculline blocked the inhibitory actions of both SKF89976A and SKF100330A on ACh release but the combination of bicuculline and uptake inhibitor did not result in a light-evoked release of GABA. In contrast, KCl (20 mM) applied locally to the retina in vivo resulted in the release of both ACh and GABA (61 and 2.6-fold respectively). KCl (20 mM) also evoked large increases in ACh and GABA release from isolated rabbit retinas in room light (13.5 and 3.4-fold respectively). The K-evoked release of ACh and GABA from the rabbit retina both in vivo and in vitro was calcium dependent. These experiments are the first in which endogenous ACh and GABA release from the retina have been simultaneously measured and suggest that the release mechanisms for these transmitters are fundamentally similar.


In vitro binding of isolated synaptic vesicles to presynaptic plasma membranes: activation by Ca2+ and protein kinase C. An in vitro model to study the molecular control of binding of highly purified synaptic vesicles to presynaptic plasma membranes has been developed. Presynaptic plasma membranes were immobilized by dotting onto nitrocellulose, and binding of iodinated synaptic vesicle membranes was studied under varying experimental conditions. Synaptic vesicles bind to presynaptic plasma membranes in the presence of Ca2+ and ATP. Binding is reduced in the presence of EGTA and abolished by the calmodulin antagonist trifluoperazine. Vesicle binding is stimulated 5-fold after incubation--prior to dotting--of presynaptic plasma membranes with ATP in the presence of the phorbol-ester 12-O-tetradecanoylphorbol-13-acetate (1 microM) and 2.5-fold after preincubation with Ca2+ (50 microM). Pretreatment of plasma membranes with alkaline phosphatase strongly reduces vesicle binding. Microsomes prepared from bovine liver did not bind to presynaptic plasma membranes. Our results suggest that activation of protein kinase C and Ca2+ stimulate binding of synaptic vesicles to the presynaptic membrane. In the intact nerve terminal this interaction may represent an initial step in synaptic vesicle exocytosis.


Inhibition of evoked neurotransmitter release from rat hippocampus by a polypeptide toxin isolated from the marine snail Conus distans. The active fraction, isolated and partially purified from the crude venom of the marine snail Conus distans, with a molecular mass of about 25 kDa, inhibits neurotransmitter release in rat hippocampus. This toxin (distans Toxin) inhibits the electrically evoked tritium labelled noradrenaline release from rat hippocampal slices in a dose and time dependent manner. The neurotransmitter release is mainly regulated by N-type of voltage sensitive Ca(2+)-channels. The distans toxin behaves as a partial antagonist of calcium in the buffer, possibly by competing with calcium for this type of voltage sensitive Ca(2+)-channels.


Homocysteine-induced alterations in extracellular amino acids in rat hippocampus. The effects of DL-homocysteine, and DL-homocysteate, on extracellular levels of amino acids in the rat hippocampus have been studied using brain microdialysis. Hippocampal electroencephalogram activity was monitored simultaneously using an electrode attached to the dialysis probe. DL-Homocysteine (1200 mg/kg; i.p. injection) produced epileptic activity in hippocampus in an inconsistent manner. Alterations in electroencephalogram activity were not observed in urethane anaesthetized animals, whereas 50% of Hypnorm anaesthetized animals exhibited epileptic activity. DL-Homocysteate (2 mu mol; i.c.v.) induced epileptic activity in a majority of animals anaesthetized using urethane. Dialysate levels of aspartate were significantly elevated by homocysteine in both groups of animals. Conversely, dialysis levels of GABA were reduced. Dialysate levels of other amino acids measured (glutamate, glutamine, taurine, alanine and valine) were not affected significantly. Dialysate levels of taurine were increased significantly in animals injected with homocysteate. These data suggest that the imbalance in excitatory:inhibitory neurotransmission in the hippocampus caused by these alterations in extracellular levels of neuroexcitatory (i.e. aspartate) and neuroinhibitory (i.e. GABA) transmitters could underly the epileptic effect of homocysteine.


Increase in cortical and midbrain tryptophan hydroxylase activity by intracerebroventricular administration of corticotropin releasing factor: block by adrenalectomy, by RU 38486 and by bilateral lesions to the central nucleus of the amygdala. Corticotropin releasing factor (CRF) infused bilaterally into the lateral ventricles of awake, chronically cannulated, male Sprague-Dawley rats produced a dose-dependent increase in the in vitro activity of cortical and midbrain tryptophan hydroxylase after 60 min. The maximal increase in enzyme activity of 60% over that of vehicle-treated controls was reached 45 min after an infusion of 3 micrograms CRF. The increase in enzyme activity after a single dose of CRF resembled that seen after exposure of rats to an acute sound stress: it was reversed by preincubation of the enzyme preparation with alkaline phosphatase and was nonadditive with the increase in activity obtained in the presence of phosphorylating conditions. The response to intracerebroventricularly administered CRF was abolished by bilateral adrenalectomy, but restored by repeated daily systemic administration of the synthetic glucocorticoid, dexamethasone (500 micrograms/day, i.p. for 3 days), to the adrenalectomized rats. Intracerebroventricular administration of the glucocorticoid antagonist, RU 38486 (200 micrograms/day for 4 days), also blocked the acute increase in tryptophan hydroxylase activity in response to CRF. Finally, bilateral lesions to the central nucleus of the amygdala, a region involved in mediating behavioral, endocrine and autonomic responses to stressful stimuli, abolished the increase in enzyme activity in response to intraventricular CRF. The glucocorticoid sensitivity of the response to CRF, as well as the involvement of the central nucleus of the amygdala support the view that CRF may have a role in mediating the enhancement of tryptophan hydroxylase activity by acute sound stress.


Beta-endorphin: regional levels profile in the brain of the human infant. Immunoradiometrical determinations of beta-endorphin (beta-EP) levels in 29 discrete brain regions from a series of victims of "Sudden Infant Death Syndrome" yielded a uniformly low levels profile in various areas of telencephalon, thalamus, pons, cerebellum and medulla oblongata. This low levels profile was interrupted by intermediate and high beta-EP levels in the midbrain and in two diencephalic zones. This study provides, for the first time, a comprehensive, neurochemically determined regional profile of beta-EP levels in the brain of the human infant.


Beta-endorphin and neurotensin in brainstem and cerebrospinal fluid in the sudden infant death syndrome. Beta-endorphin (BE) and neurotensin (NT) are two neuropeptides which induce apneas. In infants who died of Sudden Infant Death Syndrome (SIDS) we measured, in brainstem and CSF, BE and NT by IRMA and RIA respectively. BE and NT levels are compared to same aged infant and adult controls. CSF BE level was significantly higher in SIDS than in the two control groups (86 +/- 14 vs 33 +/- 13 and 16 +/- 5 pmol/l). In 6 SIDS victims NT and BE were assayed in 5 brainstem sections, each of them divided in median, intermediate and lateral parts. We found high levels of BE in every fragment (3-11 pmol/mg protein) while NT elevated values were restricted to the mesencephalic regions (1.4-12 pmol/mg), the medial pons (6 pmol/mg) and the intermediate parts of the medulla (including the olive: 1.3-1.6 pmol/mg). These results support the hypothesis that NT and/or BE could induce or participate to the fetal issue of SIDS.


Rapid kinetics of depolarization-induced changes in intrasynaptosomal calcium concentrations. The rapid kinetics of depolarization-evoked calcium influxes in isolated nerve terminals from rat cortex were monitored by stopped-flow spectrofluorimetry using specific indicators (Fluo-3, Indo-1). A very rapid increase in the intrasynaptosomal Ca(2+)-level was detected within the subsecond time range after depolarizing synaptosomes by mixing with physiological saline containing elevated K(+)-concentrations. About 15 mM [K+]o was determined as threshold concentration for inducing Ca(2+)-influx, which increased with higher concentration and saturated at [K+]o-concentrations of about 40 mM [K+]o.


Inhibition of high affinity choline uptake in the rat brain by neurotoxins: effect of monosialoganglioside GM1. Mustard derivatives of ethyl-choline and hemicholinium-3 have been suggested as possible specific cholinergic neurotoxins. In this study a structural analog of hemicholinium-3, a,a'-bis[di(2-chloroethyl)amino]-4,4'-2-biacetophenone (toxin 7), was added to synaptosomes prepared from the cortex, striatum or hippocampus of rat brain. Synaptosomal high affinity choline uptake (HACU) was significantly decreased in a dose-dependent manner by addition of toxin 7, while synaptosomal uptake of GABA or dopamine was not changed. Incubation of cortical synaptosomes with the monosialoganglioside GM1 prevented the decrease in HACU seen following administration of toxin 7. This preventative effect of GM1 was greater if GM1 was added prior to or concomitant with toxin 7, than if GM1 was added following toxin 7. Two newly synthesized hemicholinium-3 analogs, 4-[3'-di(2-chloroethyl)aminopropionyl]biphenyl (toxin 5) and 4-[3'-di(2-bromoethyl)aminopropionyl]biphenyl (toxin 6) caused a large decrease in HACU when added to cortical synaptosomes, this decrease was significantly greater than that seen with the same dose of toxin 7 or ethyl-choline aziridinium (AF64A). Ultrastructural changes in the synaptosomal membrane following incubation with toxin 7 or toxin 7 with GM1 were examined by electron microscopy. Development of a compound which is both a potent neurotoxin, and is specific for cholinergic neurons will allow new insights into the normal function of the cholinergic system in the CNS and provide animal models of disease states in which cholinergic degeneration is an important element.


A minimal model to account for the response of N-Methyl-D-aspartate receptors expressed in Xenopus oocyte injected with rat brain mRNA. N-Methyl-D-aspartate (NMDA) receptors were expressed in Xenopus oocytes by injecting rat brain mRNA. NMDA-elicited responses in the oocytes were measured by the voltage-clamping method. The following measurements were made in the presence of 50 microM glycine (Gly) to establish the relationship between the NMDA concentration and the current: (1) the NMDA-induced membrane current before desensitization; (2) the NMDA-induced membrane current after desensitization equilibrium; (3) the fraction of the active form of the receptor after desensitization equilibrium in the presence and absence of 50 microM Gly; (4) the rate of the recovery of desensitized receptors upon removal of NMDA. Gly was essential for not only the activation of NMDA receptors but also their desensitization. These results were analyzed on the basis of a minimal model where one agonist and one Gly binding site were assumed. The equilibrium and rate constants of the model were evaluated for NMDA in the presence of saturating amounts of Gly. This model will be useful for systematically explaining the complicated responses of NMDA receptors.


Specific ganglioside-cell protein interactions: a study performed with GM1 ganglioside derivative containing photoactivable azide and rat cerebellar granule cells in culture. The incubation of cultured rat cerebellar granule cells with a photoreactive derivative of radiolabeled GM1 ganglioside, [3H]GM1(N3), followed by illumination, led to the specific association of ganglioside to cell proteins. After 30 min of incubation only a few out of the cell proteins became radiolabeled. Two of these, at apparent molecular weights of 95 and 112 kDa, are interacting with the portion of associated ganglioside that is released by trypsin treatment; others, in the region between 31 and 44 kDa, are probably bound to molecules of ganglioside inserted into the outer membrane layer, thus showing that the ganglioside association to the cell surface is a selective phenomenon, involving specific proteins. Increasing the incubation time up to 24 h resulted in a larger number of radiolabeled proteins, probably as a consequence of the internalization and metabolic processing of administered [3H]GM1(N3). In fact, photoreactive and radioactive metabolic derivatives of [3H]GM1(N3) can also interact with a number of proteins. After 24 h incubation, some radioactivity was also associated to cytosolic proteins. Again in this case the interaction with proteins seems to be a specific process involving only a few out of the total cytosolic proteins.


Gangliosides and neuronal differentiation. Using the GD3-specific mAb R24 we demonstrate by immunohistochemistry that the first embryonic cells of chicken expressing GD3 represent heavily proliferating cells of mesodermal origin (mesenchymal stem and endothelial cells). At this developmental stage (E1-1.5) neuroectodermal cells of the forming neural tube are not stained by R24 or any other available anti-ganglioside antibodies. These cells of the neural tube start to express GD3 at around E1.5 in parallel with increasing proliferative activity. Likewise proliferating and migrating neuronal crest derivates as well as undifferentiated retinal cells, the forming lens and otic placodes increasingly express GD3 in an organ-specific pattern following the spatiotemporal increase in mitotic activity. Immunostaining of GD1b (mAb D21b) or c-pathway polysialogangliosides (mAb Q211) is not obtained before E2.5, is nervous tissue specific and restricted to "new-born" neurons, which start to migrate and form first neurites. This striking change in ganglioside synthesis and expression also occurs in primary cell cultures (after or without previous Q211-mediated complement kill of neurons) during differentiation of mitotic progenitor cells to neurons (neurogenesis). In cell culture, the fluorescence staining is evenly distributed over the whole neuronal surface including filopodia at the growth cones. Monensin (10(-8) M) prevents expression of GD1b and c-polysialogangliosides and simultaneously differentiation of neuronal morphology (neurogenesis). The presence of exogenous gangliosides from bovine brain leads to a decrease of the monensin effect or even abolishes it.


Disposition of exogenous tritium-labelled GM1lactone in the rat. The disposition of labelled [3H]GM1lactone, the inner ester of ganglioside GM1, was studied in the rat. After i.v. administration [3H]GM1lactone was quickly converted to its corresponding open form most likely by plasma esterases, and then displayed a pharmacokinetic profile identical to [3H]GM1. Following intramuscular administration of [3H]GM1lactone [3H]GM1 levels in plasma and in tissues were higher than those obtained after the administration of an equivalent dose of [3H]GM1. This increased bioavailability means that GM1lactone can be considered as a potential prodrug of GM1.


Study of brain and vascular plexus gangliosides in meningoencephalitis of various etiology. The content of brain and vascular plexus gangliosides and their composition have been studied in 15 cases of meningoencephalitis of various etiology and degree of disease. The most pronounced decrease of ganglioside concentration was found in brain of children, who died from herpes virus infection. Decrease of ganglioside content was revealed in brain grey matter of patients with influenza virus or meningococcus infection, but not in cases of mycoplasma infection. These data provide evidence of nerve cell destruction due to meningoencephalitis of various etiology. The biochemical data obtained are in good agreement with the results of the brain of children infection by herpes virus (increase of GD1b content in grey matter). Ganglioside content in vascular plexes of patients with meningoencephalitis was found to be, on the contrary, much higher (ca 4 times) than in the controls. The higher the lesion of choroid plexes in meningoencephalitis is, the higher the ganglioside content in them is.


Gangliosides and regeneration of the goldfish optic nerve in vivo and in vitro. One to forty days after optic nerve transection, goldfish received an i.p. injection of [3H]proline (proteins), 3HNAcGluc (gangliosides) or [3H]thymidine (DNA). After 1 or 2 days of incorporation, both optic systems were analyzed by biochemical and autoradiographical procedures. In the regenerating retina an enhanced retinal mitotic activity, protein synthesis (up to 2-fold) and ganglioside synthesis (up to 1.5-fold) was found. Simultaneously, a transiently enhanced accumulation (up to 4.5-fold) of axonally transported protein- and ganglioside-bound radioactivity in the regenerating optic nerve stump occurred. These regeneration-related proliferative and metabolic changes were found to be maximal at 6-8 days post lesion, but still measurable after 40 days. Concerning the endogenous ganglioside metabolism, in the regenerating retina no obvious change in ganglioside synthesis and composition could be observed, while in the regenerating optic nerve there was an enhanced accumulation of the ganglioside GP1c. Daily i.p. application of a ganglioside mixture from bovine brain (GMix) or of the monosialoganglioside GM1, did not alter significantly the degree and time course of the above regeneration induced metabolic changes or the regain of visual acuity. Sprouting activity of goldfish retinal explants was found to strongly depend upon a conditioning lesion of the optic nerve, reaching a maximum 8 days after nerve transection. This result strictly coincided with the profile of metabolic changes observed in vivo. Again, daily i.p. or i.o. injection of exogenous gangliosides did not influence the lesion induced increase of retinal sprouting activity. However, in normal, not regenerating animals, a local i.o. injection of GMix or GM1 led to a significant enhancement of the "basal" sprouting activity, normally occurring after lesion of the retina after injection of 0.9% NaCl. This ganglioside related stimulation was maximal at low concentrations (3 micrograms/eye) and did not occur at high concentrations (> 30 micrograms/eye). Injection of the phospholipid phosphatidylcholine or phosphatidylserine had no or a slightly inhibitory effect, when compared to NaCl controls. These data suggest an involvement of gangliosides in the complex process of induction of axonal sprouting.


Lasting effects of postnatal hypoxia and saline injection on the striatal dopamine transport and their modification by gangliosides. Hypobaric hypoxia (10 h daily, pO2 10 kPa) and saline administration (2.5 microliters/g body wt) from the 2nd till the 11th day of life both induced a long-lasting increase of the low-affinity dopamine (DA) uptake capacity in S1-fractions of the rat striatum. Additionally, the potassium-stimulated DA release was enhanced in adult control rats postnatally injected with saline. The administration of a mixture of bovine brain gangliosides (30 micrograms/g body wt) was found to prevent these effects. However, the kinetic constants of the DA uptake of hypoxic rats treated with gangliosides were reduced in comparison to untreated controls. Thus, the effects of gangliosides appear to differ between hypoxic and control conditions. The modification of the dopaminergic activity during brain development is discussed as a possible mechanism of the preventive effects of gangliosides against long-term cerebral dysfunctions following hypoxia or stress.


24R,25-dihydroxyvitamin D3 prevents aluminum-induced alteration of brain gangliosides in uremic rats by keeping the metal within perivascular astrocytes of the blood-brain barrier. The administration of aluminum (Al) to uremic rats leads to Al accumulation in different brain regions with subsequent alteration of brain gangliosides. Addition of 24R,25-dihydroxyvitamin D3[24R,25-(OH)2D3] did not influence the brain Al content determined by plasma argon emission spectrometry, but prevented the decrease in brain gangliosides. By using electron microscopy and laser microprobe mass analysis, it was demonstrated that in rats given 24R,25-(OH)2D3 together with Al, the metal was mainly kept within perivascular astrocytes of the blood-brain barrier. On the contrary, in rats given Al only, the metal was evenly distributed throughout the brain areas causing extensive demyelination, chromatolysis of nerve cells in some brain regions (hippocampus) and brain edema. Our results could find application in the prevention of Al-induced encephalopathy in patients on hemodialysis.


Ganglioside-dependent factor, inhibiting lipid peroxidation in rat brain synaptosomes. Preincubation of rat brain synaptosomes with GM1, GD1a or GT1b (10(-10)-10(-6) M), as well as with phorbol 12-myristate, 13-acetate (10(-10)-10(-6) M) was found to have dose dependent inhibitory effect on Fe(2+)-ascorbate induced lipid peroxidation, while penetrating analogue of c-AMP markedly decreased the inhibitory effect of these compounds. In liposomes made of lipids isolated from synaptosomal membranes the degree of inhibition of induced LPO by gangliosides was practically absent. The inhibitory effect of GM1 on lipid peroxidation could not be revealed after thermal denaturation of synaptosomes or after treatment with polymyxin B (inhibitor of lipid-dependent protein kinases). These results and some other data provide evidence for the existence of ganglioside-dependent factor inhibiting lipid peroxidation in brain tissue. It may be suggested to be a protein kinase modulated by gangliosides.


Cell surface distribution of endogenous and effects of exogenous gangliosides on neuronal survival, cell shape and growth in vitro. In vitro immunostaining of neurons from spinal cord or brain of embryonic chicken by means of monoclonal anti-ganglioside antibodies (Q211, D21b) revealed a fluorescence-labeling of c-polysialogangliosides and GD1b evenly distributed over the entire neuronal surface including filopodia at the growth cones. On electronmicroscopical level the gold-stained ganglioside-antigens were found more or less densely packed in small adjacent areas suggesting a concentration in local "domains". Survival in serum-free or serum-containing medium of embryonic spinal cord motoneurons, which normally died if not cultivated in muscle conditioned medium or in contact to myotubes, was remarkably improved in the presence of a ganglioside mixture (10 microM) from bovine brain. If embryonic neurons from optic lobes were cultivated at low Ca(2+)-concentration (< 20 microM) they developed flat, broad cell bodies with many filopodia and only a few flat-shaped short processes. A very weak cytoskeleton-staining by means of rhodamine-linked phalloidine indicated that polymerization of actin was impaired in these neurons. At the same low Ca(2+)-concentration of < 20 microM but in the presence of ganglioside GM1 (up to 100 microM) most of the neurons developed a "normal" cell shape with rounded perikarya and thin neurites with "normal" shaped growth cones. In this case rhodamine-linked phalloidine revealed a much more intense staining mainly concentrated within the growing tips. The morphology and growth of the ganglioside-treated neurons resembled that of neurons cultivated at a higher Ca(2+)-concentration of at least 600 microM.


Gangliosides in the human brain development and aging. In this study, brain gangliosides in prenatal and postnatal human life were analyzed. Immunohistochemically, the presence of "c"-pathway of gangliosides (GQ1c) in embryonic brain was only recorded at 5 weeks of gestation. Biochemical results indicated a twofold increase in human cortex ganglioside concentration between 16 and 22 weeks of gestation. The increasing ganglioside concentration was based on an increasing GD1a ganglioside fraction in all regions analyzed except cerebellar cortex, which was characterized by increasing GT1b. In this developmental period, GD3 was found to be localized in the ventricular zone of the cortical wall. After birth, GD1b ganglioside in neuropil of granular cell layer corresponding to growing mossy fibers was expressed in cerebellar cortex. Between birth and 20/30 years of age, a cerebral neocortical difference of ganglioside composition was observed, characterized by lowest GD1a in visual cortex. Analyzing the composition of gangliosides in cortical regions during aging, they were observed to follow region-specific alterations. In frontal cortex, there was a greater decrease in GD1a and GM1 than in GT1b and GD1b, but in occipital (visual) cortex there was no change in individual gangliosides. In hippocampus, GD1a moderately decreased, whereas other fractions were stable. In cerebellar cortex, GD1b and GT1b fractions decreased with aging.


Cortical distribution of gangliosides in Alzheimer's disease. In Alzheimer's disease, all ganglio-series gangliosides (GM1, GD1a, GD1b and GT1b) were found to be decreased in temporal and frontal cortex, and nucleus basalis of Meynert. In addition, in Alzheimer's disease simple gangliosides (GM2, GM3) were elevated in frontal and parietal cortex, possibly correlating to accelerated lysosomal degradation of gangliosides and/or astrogliosis occurring during neuronal death.


NMR structure of oxidized Escherichia coli glutaredoxin: comparison with reduced E. coli glutaredoxin and functionally related proteins. The determination of the NMR structure of oxidized Escherichia coli glutaredoxin in aqueous solution is described, and comparisons of this structure with that of reduced E. coli glutaredoxin and the related proteins E. coli thioredoxin and T4 glutaredoxin are presented. Based on nearly complete sequence-specific 1H-NMR assignments, 804 nuclear Overhauser enhancement distance constraints and 74 dihedral angle constraints were obtained as the input for the structure calculations, for which the distance geometry program DIANA was used followed by simulated annealing with the program X-PLOR. The molecular architecture of oxidized glutaredoxin is made up of three helices and a four-stranded beta-sheet. The three-dimensional structures of oxidized and the recently described reduced glutaredoxin are very similar. Quantitative analysis of the exchange rates of 34 slowly exchanging amide protons from corresponding series of two-dimensional [15N,1H]-correlated spectra of oxidized and reduced glutaredoxin showed close agreement, indicating almost identical hydrogen-bonding patterns. Nonetheless, differences in local dynamics involving residues near the active site and the C-terminal alpha-helix were clearly manifested. Comparison of the structure of E. coli glutaredoxin with those of T4 glutaredoxin and E. coli thioredoxin showed that all three proteins have a similar overall polypeptide fold. An area of the protein surface at the active site containing Arg 8, Cys 11, Pro 12, Tyr 13, Ile 38, Thr 58, Val 59, Pro 60, Gly 71, Tyr 72, and Thr 73 is proposed as a possible site for interaction with other proteins, in particular ribonucleotide reductase. It was found that this area corresponds to previously proposed interaction sites in T4 glutaredoxin and E. coli thioredoxin. The solvent-accessible surface area at the active site of E. coli glutaredoxin showed a general trend to increase upon reduction. Only the sulfhydryl group of Cys 11 is exposed to the solvent, whereas that of Cys 14 is buried and solvent inaccessible.


Direct observation by X-ray analysis of the tetrahedral "intermediate" of aspartic proteinases. We report the X-ray analysis at 2.0 A resolution for crystals of the aspartic proteinase endothiapepsin (EC 3.4.23.6) complexed with a potent difluorostatone-containing tripeptide renin inhibitor (CP-81,282). The scissile bond surrogate, an electrophilic ketone, is hydrated in the complex. The pro-(R) (statine-like) hydroxyl of the tetrahedral carbonyl hydrate is hydrogen-bonded to both active-site aspartates 32 and 215 in the position occupied by a water in the native enzyme. The second hydroxyl oxygen of the hydrate is hydrogen-bonded only to the outer oxygen of Asp 32. These experimental data provide a basis for a model of the tetrahedral intermediate in aspartic proteinase-mediated cleavage of the amide bond. This indicates a mechanism in which Asp 32 is the proton donor and Asp 215 carboxylate polarizes a bound water for nucleophilic attack. The mechanism involves a carboxylate (Asp 32) that is stabilized by extensive hydrogen bonding, rather than an oxyanion derivative of the peptide as in serine proteinase catalysis.


Lysine-21 of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase participates in substrate binding through charge-charge interaction. Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase (G6PD) was isolated in high yield and purified to homogeneity from a newly constructed strain of Escherichia coli which lacks its own glucose 6-phosphate dehydrogenase gene. Lys-21 is one of two lysyl residues in the enzyme previously modified by the affinity labels pyridoxal 5'-phosphate and pyridoxal 5'-diphosphate-5'-adenosine, which are competitive inhibitors of the enzyme with respect to glucose 6-phosphate (LaDine, J.R., Carlow, D., Lee, W.T., Cross, R.L., Flynn, T.G., & Levy, H.R., 1991, J. Biol. Chem. 266, 5558-5562). K21R and K21Q mutants of the enzyme were purified to homogeneity and characterized kinetically to determine the function of Lys-21. Both mutant enzymes showed increased Km-values for glucose 6-phosphate compared to wild-type enzyme: 1.4-fold (NAD-linked reaction) and 2.1-fold (NADP-linked reaction) for the K21R enzyme, and 36-fold (NAD-linked reaction) and 53-fold (NADP-linked reaction) for the K21Q enzyme. The Km for NADP+ was unchanged in both mutant enzymes. The Km for NAD+ was increased 1.5- and 3.2-fold, compared to the wild-type enzyme, in the K21R and K21Q enzymes, respectively. For the K21R enzyme the kcat for the NAD- and NADP-linked reactions was unchanged. The kcat for the K21Q enzyme was increased in the NAD-linked reaction by 26% and decreased by 30% in the NADP-linked reaction from the values for the wild-type enzyme. The data are consistent with Lys-21 participating in the binding of the phosphate group of the substrate to the enzyme via charge-charge interaction.


Beta beta homodimers exist in native rabbit skeletal muscle tropomyosin and increase after denaturation-renaturation. Native tropomyosin from rabbit skeletal muscle (RSTm) consists mainly of alpha alpha and alpha beta coiled coils (alpha/beta approximately 3-4/1). In some extant studies, no beta beta molecules have been found. In this study, RSTm from several different preparations was disulfide cross-linked, both preparation and cross-linking being done under nondenaturing conditions. The cross-linked product was assayed for the presence of beta beta molecules cross-linked at both C36 and C190 (beta = beta). In such cross-linked RSTm, 3-8% beta = beta is detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, C4 reversed-phase high-performance liquid chromatography, and a free-solution capillary electrophoresis experiment. This percentage becomes approximately 4-10% beta beta when corrected for incomplete double cross-linking and is independent of protein concentration (0.1-10.0 mg/mL), indicating that the observed beta beta species are not artifacts due to intermolecular cross-linking. Upon denaturation and subsequent renaturation either by heating to 55 degrees C or by incubating at 45 degrees C followed by quenching to room temperature, or by guanidine hydrochloride exposure followed by phased renaturation by dialysis, the fraction of beta beta increases, indicating that the reassociation favors homodimer formation somewhat over random association. This result differs from the random association observed when the sulfhydryl on one of the chains is carboxyamidomethylated (Holtzer, M.E., Breiner, T., & Holtzer, A., 1984, Biopolymers 23, 1811-1833), and from the overwhelming heterodimer preferences reported for tropomyosins from other organisms (Lehrer, S.S., Qian, Y., & Hvidt, S., 1989, Science 246, 926-928; Lehrer, S.S. & Qian, Y., 1990, J. Biol. Chem. 265, 1134-1138).


A functional protein hybrid between the glucose transporter and the N-acetylglucosamine transporter of Escherichia coli. The glucose and N-acetylglucosamine-specific transporters (IIGlc/IIIGlc and IIGlcNAc) of the bacterial phosphotransferase system mediate carbohydrate uptake across the cytoplasmic membrane concomitant with substrate phosphorylation. The two transporters have 40% amino acid sequence identity. Eight chimeric proteins between the two transporters were made by gene reconstruction. All hybrid proteins could be expressed, some inhibited cell growth, and one was active. The active hybrid transporter consists of the transmembrane domain (residues 1-386) of the IIGlc subunit and the two hydrophilic domains (residues 370-648) of IIGlcNAc. The N-terminal hydrophilic domain of IIGlcNAc contains the transiently phosphorylated cysteine-412. The hybrid protein is specific for glucose, which indicates that the sugar specificity determinant is in the transmembrane domain and that the cysteine from which the phosphoryl group is transferred to the substrate is not part of the binding site. The protein sequence (LKTPGRED) at which the successful fusion occurred has the characteristic properties of an interdomain oligopeptide linker (Argos, P., 1990, J. Mol. Biol. 211, 943-958).


Mast cell tryptases: examination of unusual characteristics by multiple sequence alignment and molecular modeling. Tryptases are trypsin-like serine proteinases found in the granules of mast cells. Although they show 40% sequence identity with trypsin and contain only 20 or 21 additional residues, tryptases display several unusual features. Unlike trypsin, the tryptases only make limited cleavages in a few proteins and are not inhibited by natural trypsin inhibitors, they form tetramers, bind heparin, and their activity on synthetic substrates is progressively inhibited as the concentration of salt increases above 0.2 M. Unique sequence features of seven tryptases were identified by comparison to other serine proteinases. The three-dimensional structures of the tryptases were then predicted by molecular modeling based on the crystal structure of bovine trypsin. The models show two large insertions to lie on either side of the active-site cleft, suggesting an explanation for the limited activity of tryptases on protein substrates and the lack of inhibition by natural inhibitors. A group of conserved Trp residues and a unique proline-rich region make two surface hydrophobic patches that may account for the formation of tetramers and/or inhibition with increasing salt. Although they contain no consensus heparin-binding sequence, the tryptases have 10-13 more His residues than trypsin, and these are positioned on the surface of the model. In addition, clustering of Arg and Lys residues may also contribute to heparin binding. Putative Asn-linked glycosylation sites are found on the opposite side of the model from the active site. The model provides structural explanations for some to the unusual characteristics of the tryptases and a rational basis for future experiments, such as site-directed mutagenesis.


Quantitative analysis of cyclic beta-turn models. The beta-turn is a frequently found structural unit in the conformation of globular proteins. Although the circular dichroism (CD) spectra of the alpha-helix and beta-pleated sheet are well defined, there remains some ambiguity concerning the pure component CD spectra of the different types of beta-turns. Recently, it has been reported (Hollósi, M., Kövér, K.E., Holly, S., Radics, L., & Fasman, G.D., 1987, Biopolymers 26, 1527-1572; Perczel, A., Hollósi, M., Foxman, B.M., & Fasman, G.D., 1991a, J. Am. Chem. Soc. 113, 9772-9784) that some pseudohexapeptides (e.g., the cyclo[(delta)Ava-Gly-Pro-Aaa-Gly] where Aaa = Ser, Ser(OtBu), or Gly) in many solvents adopt a conformational mixture of type I and the type II beta-turns, although the X-ray-determined conformation was an ideal type I beta-turn. In addition to these pseudohexapeptides, conformational analysis was also carried out on three pseudotetrapeptides and three pseudooctapeptides. The target of the conformation analysis reported herein was to determine whether the ring stress of the above beta-turn models has an influence on their conformational properties. Quantitative nuclear Overhauser effect (NOE) measurements yielded interproton distances. The conformational average distances so obtained were interpreted utilizing molecular dynamics (MD) simulations to yield the conformational percentages. These conformational ratios were correlated with the conformational weights obtained by quantitative CD analysis of the same compounds. The pure component CD curves of type I and type II beta-turns were also obtained, using a recently developed algorithm (Perczel, A., Tusnády, G., Hollósi, M., & Fasman, G.D., 1991b, Protein Eng. 4(6), 669-679). For the first time the results of a CD deconvolution, based on the CD spectra of 14 beta-turn models, were assigned by quantitative NOE results. The NOE experiments confirmed the ratios of the component curves found for the two major beta-turns by CD analysis. These results can now be used to enhance the conformational determination of globular proteins on the basis of their CD spectra.


Inclusion of solvation free energy with molecular mechanics energy: alanyl dipeptide as a test case. A combined force field of molecular mechanics and solvation free energy is tested by carrying out energy minimization and molecular dynamics on several conformations of the alanyl dipeptide. Our results are qualitatively consistent with previous experimental and computational studies, in that the addition of solvation energy stabilizes the C5 conformation of the alanyl dipeptide relative to the C7.


An optimization approach to predicting protein structural class from amino acid composition. Proteins are generally classified into four structural classes: all-alpha proteins, all-beta proteins, alpha + beta proteins, and alpha/beta proteins. In this article, a protein is expressed as a vector of 20-dimensional space, in which its 20 components are defined by the composition of its 20 amino acids. Based on this, a new method, the so-called maximum component coefficient method, is proposed for predicting the structural class of a protein according to its amino acid composition. In comparison with the existing methods, the new method yields a higher general accuracy of prediction. Especially for the all-alpha proteins, the rate of correct prediction obtained by the new method is much higher than that by any of the existing methods. For instance, for the 19 all-alpha proteins investigated previously by P.Y. Chou, the rate of correct prediction by means of his method was 84.2%, but the correct rate when predicted with the new method would be 100%! Furthermore, the new method is characterized by an explicable physical picture. This is reflected by the process in which the vector representing a protein to be predicted is decomposed into four component vectors, each of which corresponds to one of the norms of the four protein structural classes.


Selection of representative protein data sets. The Protein Data Bank currently contains about 600 data sets of three-dimensional protein coordinates determined by X-ray crystallography or NMR. There is considerable redundancy in the data base, as many protein pairs are identical or very similar in sequence. However, statistical analyses of protein sequence-structure relations require nonredundant data. We have developed two algorithms to extract from the data base representative sets of protein chains with maximum coverage and minimum redundancy. The first algorithm focuses on optimizing a particular property of the selected proteins and works by successive selection of proteins from an ordered list and exclusion of all neighbors of each selected protein. The other algorithm aims at maximizing the size of the selected set and works by successive thinning out of clusters of similar proteins. Both algorithms are generally applicable to other data bases in which criteria of similarity can be defined and relate to problems in graph theory. The largest nonredundant set extracted from the current release of the Protein Data Bank has 155 protein chains. In this set, no two proteins have sequence similarity higher than a certain cutoff (30% identical residues for aligned subsequences longer than 80 residues), yet all structurally unique protein families are represented. Periodically updated lists of representative data sets are available by electronic mail from the file server "netserv@embl-heidelberg.de." The selection may be useful in statistical approaches to protein folding as well as in the analysis and documentation of the known spectrum of three-dimensional protein structures.


The molecular mechanism for the tetrameric association of glycogen phosphorylase promoted by protein phosphorylation. The allosteric transition of glycogen phosphorylase promoted by protein phosphorylation is accompanied by the association of a pair of functional dimers to form a tetramer. The conformational changes within the dimer that lead to the creation of a protein recognition surface have been analyzed from a comparison of the crystal structures of T-state dimeric phosphorylase b and R-state tetrameric phosphorylase a. Regions of the structure that participate in the tetramer interface are situated within structural subdomains. These include the glycogen storage subdomain, the C-terminal subdomain and the tower helix. The subdomains undergo concerted conformational transitions on conversion from the T to the R state (overall r.m.s. shifts between 1 and 1.7 A) and, together with the quaternary conformational change within the functional dimer, create the tetramer interface. The glycogen storage subdomain and the C-terminal subdomain are distinct from those regions that contribute to the dimer interface, but shifts in the subdomains are correlated with the allosteric transitions that are mediated by the dimer interface. The structural properties of the tetramer interface are atypical of an oligomeric protein interface and are more similar to protein recognition surfaces observed in protease inhibitors and antibody-protein antigen complexes. There is a preponderance of polar and charged residues at the tetramer interface and a high number of H-bonds per surface area (one H-bond per 130 A2). In addition, the surface area made inaccessible at the interface is relatively small (1,142 A2 per subunit on dimer to tetramer association compared with 2,217 A2 per subunit on monomer-to-dimer association).


Design, synthesis, and characterization of a protein sequencing reagent yielding amino acid derivatives with enhanced detectability by mass spectrometry. We report the design, chemical synthesis, and structural and functional characterization of a novel reagent for protein sequence analysis by the Edman degradation, yielding amino acid derivatives rapidly detectable at high sensitivity by ion-evaporation mass spectrometry. We demonstrate that the reagent 3-[4'(ethylene-N,N,N-trimethylamino)phenyl]-2-isothiocyanate is chemically stable and shows coupling and cyclization/cleavage yields comparable to phenylisothiocyanate, the standard reagent in chemical sequence analysis, under conditions typically encountered in manual or automated sequence analysis. Amino acid derivatives generated with this reagent were detectable by ion-evaporation mass spectrometry at the subfemtomole sensitivity level at a pace of one sample per minute. Furthermore, derivatives were identified by their mass, thus permitting the rapid and highly sensitive determination of the molecular nature of modified amino acids. Derivatives of amino acids with acidic, basic, polar, or hydrophobic side chains were reproducibly detectable at comparable sensitivities. The polar nature of the reagent required covalent immobilization of polypeptides prior to automated sequence analysis. This reagent, used in automated sequence analysis, has the potential for overcoming the limitations in sensitivity, speed, and the ability to characterize modified amino acid residues inherent in the chemical sequencing methods that are currently used.


The refined 1.9-A X-ray crystal structure of D-Phe-Pro-Arg chloromethylketone-inhibited human alpha-thrombin: structure analysis, overall structure, electrostatic properties, detailed active-site geometry, and structure-function relationships. Thrombin is a multifunctional serine proteinase that plays a key role in coagulation while exhibiting several other key cellular bioregulatory functions. The X-ray crystal structure of human alpha-thrombin was determined in its complex with the specific thrombin inhibitor D-Phe-Pro-Arg chloromethylketone (PPACK) using Patterson search methods and a search model derived from trypsinlike proteinases of known spatial structure (Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S.R., & Hofsteenge, J., 1989, EMBO J. 8, 3467-3475). The crystallographic refinement of the PPACK-thrombin model has now been completed at an R value of 0.156 (8 to 1.92 A); in particular, the amino- and the carboxy-termini of the thrombin A-chain are now defined and all side-chain atoms localized; only proline 37 was found to be in a cis-peptidyl conformation. The thrombin B-chain exhibits the characteristic polypeptide fold of trypsinlike serine proteinases; 195 residues occupy topologically equivalent positions with residues in bovine trypsin and 190 with those in bovine chymotrypsin with a root-mean-square (r.m.s.) deviation of 0.8 A for their alpha-carbon atoms. Most of the inserted residues constitute novel surface loops. A chymotrypsinogen numbering is suggested for thrombin based on the topological equivalences. The thrombin A-chain is arranged in a boomeranglike shape against the B-chain globule opposite to the active site; it resembles somewhat the propeptide of chymotrypsin(ogen) and is similarly not involved in substrate and inhibitor binding. Thrombin possesses an exceptionally large proportion of charged residues. The negatively and positively charged residues are not distributed uniformly over the whole molecule, but are clustered to form a sandwichlike electrostatic potential; in particular, two extended patches of mainly positively charged residues occur close to the carboxy-terminal B-chain helix (forming the presumed heparin-binding site) and on the surface of loop segment 70-80 (the fibrin[ogen] secondary binding exosite), respectively; the negatively charged residues are more clustered in the ringlike region between both poles, particularly around the active site. Several of the charged residues are involved in salt bridges; most are on the surface, but 10 charged protein groups form completely buried salt bridges and clusters. These electrostatic interactions play a particularly important role in the intrachain stabilization of the A-chain, in the coherence between the A- and the B-chain, and in the surface structure of the fibrin(ogen) secondary binding exosite (loop segment 67-80).(ABSTRACT TRUNCATED AT 400 WORDS)


Structural analysis of wild-type and mutant yeast calmodulins by limited proteolysis and electrospray ionization mass spectrometry. Calmodulin from Saccharomyces cerevisiae was expressed in Escherichia coli and purified. The purified protein was structurally characterized using limited proteolysis followed by ESI mass spectrometry to identify the fragments. In the presence of Ca2+, yeast calmodulin is sequentially cleaved at arginine 126, then lysine 115, and finally at lysine 77. The rapid cleavage at Arg-126 suggests that the fourth Ca(2+)-binding loop does not bind Ca2+. In the presence of EGTA, yeast calmodulin is more susceptible to proteolysis and is preferentially cleaved at Lys-106. In addition, mutant proteins carrying I100N, E104V or both mutations, which together confer temperature sensitivity to yeast, were characterized. The mutant proteins are more susceptible than wild-type calmodulin to proteolysis, suggesting that each mutation disrupts the structure of calmodulin. Furthermore, whereas wild-type calmodulin is cut at Lys-106 only in the presence of EGTA, this cleavage site is accessible in the mutants in the presence of Ca2+ as well. In these ways, the structural consequence of each mutation mimics the loss of a calcium ion in the third loop. In addition, although wild-type calmodulin binds to four proteins in a yeast crude extract in the presence of Ca2+, the mutants bind only to a subset of these. Thus, the inability to adopt the stable Ca(2+)-bound conformation in the third Ca(2+)-binding loop alters the ability of calmodulin to interact with yeast proteins in a Ca(2+)-dependent manner.


Guanidine derivatives restore activity to carboxypeptidase lacking arginine-127. Arg-127 stabilizes the oxyanion of the tetrahedral intermediate formed during Zn2+ carboxypeptidase A-catalyzed hydrolysis. Mutant carboxypeptidases lacking Arg-127 exhibit substantially reduced rates of hydrolysis with the change manifest almost entirely in kcat (kcat/Km is decreased by 10(4) for R127A). Therefore, Arg-127 stabilizes the enzyme-transition state complex but not the ground state enzyme-substrate complex (Phillips, M.A., Fletterick, R., & Rutter, W.J., 1990, J. Biol. Chem. 265, 20692-20698). The addition of guandine, methylguanidine, or ethylguanidine to R127A increases the kcat for hydrolysis of Bz-gly(o)phe by 10(2) without changing the Km. Dissociation constants (Kd) for the guanidine derivatives range from 0.1 to 0.5 M. The binding affinity for the transition state analog Cbz-phe-alaP(o)ala is increased similarly by 10(2); in contrast, the binding affinity of the ground state inhibitor benzylsuccinic acid is not altered. Thus, guanidine derivatives mimic Arg-127 in stabilizing the rate-limiting transition state. Hydrolysis of Bz-gly-(o)phe by wild-type carboxypeptidase, R127K, or R127M is not substantially affected by guanidine derivatives. Additionally, primary amines do not change the activity of R127A. These observations imply that guanidine binds in the cavity vacated by Arg-127 specifically and in a productive conformation for catalysis.