Near ultraviolet radiation induced changes in goat lens.

The possibility that the ultraviolet radiation from sunlight and other ambient sources as a major causative factor for the onset of cataract processes and photolytic changes of the eye lens constituents was studied. Normal goat lenses exposed in vitro to near UV radiation in the region of 315-400 nm (UV-A) revealed distinct morphological changes in the ultrastructure, increase in the inorganic elements; C, H, N, and a sharp shift in the intrinsic fluorescence spectra. UV exposure resulted in an alteration in the lambda max of the excitation spectra, a red shift in the emission absorption maxima and also an increase in the absolute fluorescence intensity. Scanning electron microscopic study showed a significant increase in the interfibrillar distances of the lens structural proteins. It is argued that the UV light induced covalent modifications of the lens proteins and their aggregations might have occurred due to the generation of photolytic products which then lead to oxidative damages.

Isolation and characterization of the basal cell membranes of the human term placenta.

A relatively simple and rapid method is described for the isolation of basal cell membranes (BCM) from the human placenta at term which showed considerable improvement in the yield, purity and membrane characteristics as compared to the earlier described methods. The method is based on thorough washings of the syncytium in balanced salt solution, selective grinding, hypotonic lysis, sonication, incubation with EDTA and then more conventional differential centrifugation and ultracentrifugation. The isolated material showed smooth surfaced vesicular structure of various sizes as revealed by both positive and negative staining and transmission electron microscopic analysis. The membrane was highly enriched in Na+/K+, Ca2+ and Mg2+ dependent ATPase activities while the cross contamination with brush border surfaces was low as revealed by the marker enzyme assays specific for the brush border membrane (BBM) such as the disaccharide hydrolases, aminopeptidase and alkaline phosphatase. The membrane showed a relatively low lipid/protein ratio and the lipid composition represented by a variety of phospholipids (phosphatidyl choline, phosphatidyl ethanolamine, sphingomyelin, phosphatidyl inositol and phosphatidyl serine), neutral lipids (cholesterol, triacyl glycerol, free fatty acids) and glycosphingolipids (ganglioside, cerebroside and sulfatide). It also contained plasmalogens. On SDS-PAGE analysis and Coomassie blue staining reaction, the isolated membrane showed 14 major bands with as many minor ones with a molecular weight ranging between 30-110 kDa.

Ca(2+)-transport across brush border and basal surfaces of human term [correction of terms] placenta.

Brush border microvillous (BBM) and basal cell membranes (BCM) were isolated from syncytiotrophoblast of human term placenta by homogenization, sonication, prolonged stirring and differential centrifugation. Uptake of 45Ca(2+)-CaCl2 in membrane vesicles in morpholino propane sulphonic acid (MOPS) buffer was studied up to 60 min. Maximum uptake of the radioisotope was recorded at 10 and 15 min of incubation of the BBM and BCM, respectively. Radioisotopic uptake was also dependent on the Ca(2+)-concentration, linear up to 3 mu mole and then assuming hyperbolic substrate saturation kinetics. The Lineweaver-Burk transformation of the data gave Kt value for BBM and BCM, 0.85 and 1.08 microM, respectively while the Vmax of uptake (Jmax) in the same were 105.26 and 188.68 pmole Ca2+/microgram protein/20 min. Ca(2+)-Uptake in placental BBM and BCM vesicles was inhibited by two Ca(2+)-channel blockers, nifedipine and verapamil to as much as 50% while Ca(2+)-ionophore A23187 enhanced the uptake process significantly.

Effect of Ca(2+)-channel blockers, Ca(2+)-ionophore and increased pyrene excimer formation on the microsomal glucose-6-phosphatase.

The dependence of microsomal glucose-6-phosphatase (G-6-Pase) activity on Ca2+ as well as the membrane lipid microviscosity was studied by the effect of Ca(2+)-channel blockers (namely verapamil and nifedipine), Ca(2+)-ionophore, A23187 and pyrene excimer formation. Channel blockers depressed the G-6-Pase and Ca(2+)-ATPase while the ionophore increased these activities. Dimethyl sulfoxide, a known membrane surface active agent showed no change. Ca(2+)-uptake into the membrane has expectedly been lowered by the channel blockers while the ionophores facilitated the ion flux. Excimer formation of the fluorescent probe, pyrene as an indicator of increased membrane fluidity, and microviscosity calculated from there on, showed that Ca(2+)- and lipid microenvironment in the membrane significantly influenced the activity of G-6-Pase. Membrane lipid composition such as phospholipid/cholesterol molar ratio which also indicates an increased membrane fluidity is markedly increased with the ionophore but decreased with the channel blockers, while protein/phospholipid ratio remained unchanged. Microsomal G-6-Pase is a multicomponent multifunctional protein. It is argued that Ca2+ may play the role of an obligatory cofactor not only for the hydrolysis of G-6-P (catalytic part of the enzyme) but also involved in the regulation of substrate and product transport in or out of the endoplasmic reticulum lumen.

Isolation & chemical composition of lung surfactant from human amniotic fluid.

Lung surfactant was isolated from human amniotic fluid collected at term and studied with reference to the material isolated from human and rabbit lung lavage. The isolated material showed 58 per cent lipid by dry weight, 29 per cent protein and relatively smaller amounts of nucleic acids, sialic acid and hexose. Phosphatidyl choline was the predominant phospholipid species and accounted for 46 per cent of the total lipid by weight, followed by phosphatidyl glycerol (7%) and phosphatidyl ethanolamine (5%). Cholesterol was the major neutral lipid fraction present (10%) and was almost entirely in the free form. Other lipid fractions present in minor quantity were triglycerides, esterified cholesterol, phosphatidyl serine, phosphatidyl inositol and sphingomyelin. The material contained a very high degree of alkaline phosphatase activity, while other enzymes such as acid phosphatase, glucose-6-phosphatase, ATPases, 5'-nucleotidase and beta-N-acetyl glucosaminidase were also present.

Interaction of pulmonary surfactant-associated protein (SP-A) with surfactant lipids.

A pulmonary surfactant-associated protein complex with components of 36, 32 and 28 kDa was isolated from human lung homogenates and reassembled with surfactant lipids prepared as small unilamellar liposomes. The role of divalent cations in the assembly of this recombinant lipoprotein complex was studied by monitoring the changes in turbidity, intrinsic tryptophanyl fluorescence and surface activity. The protein-facilitated lipid aggregation was promoted on addition of 5 to 20 mM Ca2+. Intrinsic fluorescence measurements on SP-A (28-36 kDa) indicated that the tryptophan side chains were in a relatively hydrophobic environment, that the wavelength of maximum fluorescence emission and also the relative fluorescence, were changed upon the binding of lipid. Tryptophanyl fluorescence of the lipoprotein assembly was quenched as indicated by a reduction in the effective Stern-Volmer constant. These results suggest that Ca2+ lipid-protein interactions are involved in the structure and function of extracellular lung surfactant assembly.

Analysis of the major surfactant-associated proteins in human lung.

The protein component of human pulmonary surfactant was analysed. A purified surfactant preparation, after delipidation, was subjected to gel filtration on Sephadex G-200. The proteins present in the surfactant were categorised by means of SDS-polyacrylamide gel electrophoresis into serum and non-serum components. Molecular masses determination showed the presence of three sub-groups with molecular masses of 60-68 kDa, 28-36 kDa and 10-18 kDa, respectively. Antiserum generated against 28-36 kDa protein strongly reacted with the purified surfactant and amniotic fluid, while it did not show any cross reactivity with other groups of proteins and serum in a double diffusion immunoprecipitation assay. We propose that this protein is the major non-serum surfactant-associated protein present in human lung surfactant.

Effect of H2-receptor antagonists, cimetidine and ranitidine on reproductive functions in male mice.

Na+,K(+)-ATPase and Ca(2+)-ATPase in testis were inhibited with an oral administration of cimetidine and ranitidine. Cimetidine at dose level of 100 and 30 mg while ranitidine at 70 and 10 mg per kg body wt inhibited the enzyme activities, 24 hr after single administration or daily administration for 15-days. Mg(2+)-ATPase activity was increased with cimetidine while ranitidine inhibited the enzyme. Michaelis-Menten kinetic characteristics revealed mixed type of inhibition for Na+,K(+)-ATPase with cimetidine, whereas it was noncompetitive for Ca(2+)-ATPase with cimetidine as well as ranitidine administration. Inhibition of Na+,K(+)-ATPase with ranitidine was also of noncompetitive type. Mg(2+)-ATPase behaved differently with administration of ranitidine at both the time points used i.e. noncompetitive type of inhibition after 24 hr and mixed type after 15-days. Histologically, signs of degeneration of testicular elements appeared after administration of cimetidine with a significant decrease in tubular diameter and germinal epithelial cell height. Ranitidine administration did not produce any change in the seminiferous tubules of testis. Scanning electron microscopy of spermatozoa from cimetidine-treated mice exhibited distinct departure from the normal morphology such as, (i) breaks at various places along distal portion of the tail, (ii) roughening, wrinkling and disorganization of plasma membrane of the head region, (iii) decapitation of the head and (iv) changes in shape of cytoplasmic droplet. Ranitidine administration showed normal morphology of the spermatozoa.

Interaction of H2-receptor antagonists, cimetidine and ranitidine with microsomal drug metabolizing and other systems in liver.

Cimetidine has been demonstrated to impair microsomal oxidative drug metabolizing and other enzyme systems in mouse liver. The inhibition is rapid, occurring after a single administration and also found to be dose-dependent. It is more significant after daily administration for 15 days. Enzyme inhibition by ranitidine, another H2-receptor antagonist was comparatively less at all the concentrations of the drug tested. An increased activity of alkaline phosphatase, glutamate-pyruvate and glutamate-oxaloacetate transaminase was observed in liver with cimetidine administration, whereas that of lactate and succinate dehydrogenase was inhibited only after administration of 2000 mg cimetidine per kg body weight. Except alkaline phosphatase other enzymes were unaffected after ranitidine administration. Analysis of lipid classes in liver showed that phospholipid, triglycerides and free fatty acid contents were significantly decreased in drug administration while cholesterol level showed very little or no change. Microsomal and soluble protein contents were significantly increased which probably indicate that the inhibition in the enzyme activity by histamine H2-receptor antagonists may be a lipid mediated process and not resulted from the reduced availability of the enzyme protein.

Non-sporing anaerobes in hospital sepsis.

In comparison to normal controls, the non-sporing anaerobes were often isolated from orodental sepsis (42% to 44.4%), chronic suppurative otitis media (40%), septic abortion (40.3%), uterocervical wound (45.4%), vaginitis (50%) and cancer cervix (50%). This was true (40%) in perforating ulcers of foot in leprosy. These organisms were less frequently noted in abdominal (11%) and episiotomy (22.8%) wounds and leucorrhoea (33.3%). The role of non-sporing anaerobes was also suggested by the high percentage ratio of number of isolates to number of cases and by its primary isolation in moderate to heavy number. Barring the cases of cancer cervix, the aerobic bacteria were the most common (78.8% to 100%) in all other conditions.

Sensitivity of the different tests of enterotoxigenicity of Escherichia coli and their correlation.

Enterotoxigenicity of E. coli isolates was tested in 136 cases of acute gastroenteritis. Heat labile toxin (LT) produced in-vitro was tested in rabbit ileal loop (RIL); vero cell line and Biktn plate. The results of live cultures were evaluated in RIL. The overall data of these four models were not statistically different. Elaboration of LT in these four models ranged from 14-21.4%. Out of the 20 LT producing strains 14 (70%) also revealed ST. Of the 6 positive reactors on vero cell line, appeared to produce vero toxin (VT) only. Out of 29 LT positive E. coli, 1 (3.45%) and 2 (6.89%) strains respectively revealed colonising factor antigen (CFA) I and II. The high incidence of ETEC showing both LT and ST has been highlighted in the age group 0-4 years, and its impact on nutritional status is discussed.

Etiology of acute childhood diarrhoea in Calcutta.

Of the 152 cases of acute diarrhoea, 124 (81.5%) revealed potential pathogens. Altogether 27 (21.2%) out of 127 strains of Escherichia coli, Klebsiella pneumoniae, Enterobacter, Proteus and Acinetobacter produced enterotoxin. Single pathogenic bacteria (40 cases 26.3%), parasite (6; 6%), rota virus (6; 6%), toxigenic bacteria (19; 12.5%) and mixed agents (37; 24.24.3%) were recorded in 108 cases (71.0%). Another 14 (9.2%) cases exclusively revealed moderate to heavy growth of suspected enteric pathogens like K. pneumoniae, Proteus, Enterobacter, Pseudomonas aeruginosa, anaerogenic E. coli and Citrobacter and 2 (1.3%) had high counts of T'. hominis. Of the known pathogens, the preponderance of A. hydrophila (24.4%), rota virus (15.7%) and Aeromonas hydrophila (14.0%) in 1-4 y, Vibrio cholerae (45.6%) and Trichuris trichiura (13.0%) in 4-14 y age group is highlighted. Other pathogenic bacteria were non-01 V. cholerae (3.2%), V. parahaemolyticus (2.6%), V. fluvialis (0.6), Plesiomonas shigelloides (3.9%), Salmonella (2.6%), Shigella (1.9%), EPEC (1.9%), EEC (5.2%) and Campylobacter jejuni (3.9%) and the parasites were Entamoeba histolytica (2.6%) and Giardia intestinalis (2.6). Comparative study of age matched controls with those of diarrhoea suggested the pathogenic role of E. histolytica and T. hominis.

Effect of selected metal ions on the motility and carbohydrate metabolism of ejaculated human spermatozoa.

Zinc, lead and cadmium in the form of chloride salts when added to a standard assay system containing 80 X 10(-6) ejaculated washed human spermatozoa caused a dose and duration-dependent inhibition of their motility. The activity of certain key enzymes of carbohydrate and energy metabolism, viz, glycogen phosphorylase, glucose-6-phosphatase, fructose-1, 6-diphosphatase, glucose-6-phosphate isomerase, amylase, Mg2+- dependent ATPase and lactic and succinic acid dehydrogenases were also found to be inhibited. The order of inhibitory effects of the heavy metals were zinc less than lead less than cadmium. The metal chelating agent, ethylene diamine tetra-acetic acid (EDTA, disodium salt) also interfered with the spermatozoal motility and inhibited the enzyme activities.