Small Colony variants of Staphylococcus aureus isolated from a patient with infective endocarditis: a case report and review of the literature.

Staphylococcus aureus produces a particular morphological variant called small colony variant (SCV) which is responsible for persistent subclinical infections in predisposed individuals and is usually resistant to aminoglycosides and cell wall active antibiotics. Infections by SCV of S. aureus are an upcoming problem due to difficulty in laboratory diagnosis and resistance to antimicrobial chemotherapy. We here report a case of infective endocarditis caused by SCV of Staphylococcus aureus in a pediatric patient.

STa-induced translocation of protein kinase C from cytosol to membrane in rat enterocytes.

Escherichia coli heat stable enterotoxin (STa) binds to isolated rat intestinal epithelial cells and triggers a cascade reaction including increase of intracellular calcium levels ([Ca(2+)](i)) and membrane bound protein kinase C (PKC) activity. In response to STa, the cytosolic PKC activity falls from 110 to 35 nmol with increase of membrane bound PKC activity from 15 to 78 nmol. Furthermore, the increase of PKC activity induced by STa treatment was always preceded by an increase in [Ca(2+)](i). Cytosolic [Ca(2+)](i) was significantly higher (161 nM) in STa treated cells as compared to untreated cells (51.3 nM). In addition, immunoblot performed on extracts of STa treated rat enterocytes with a monoclonal antibody against PKC alpha showed a prominent band of PKC alpha. Translocation of PKC alpha could be blocked by dantrolene, a drug which inhibits the mobilisation of [Ca(2+)](i) from the intracellular store. Our results, therefore, provide evidence for the role of [Ca(2+)](i) in STa treated cells for the translocation of PKC alpha from cytosol to membrane.

Compensation of the differential floating capacitance between dual microelectrodes.

A circuit for compensating the floating differential capacitance appearing between two recording microelectrodes is presented. It is shown how this floating capacitance can be neutralized so that current in any microelectrode can be injected without any significant crosstalk picked up by the other.

Comparison of cytosolic levels of calcium and G actin in diffuse and localised adherent Escherichia coli-infected HeLa cells.

In the present study we compared the intracellular level of free calcium ([Ca2+]i) and monomeric (G)/total (G+F) actin ratio in HeLa cells infected with diffuse (DAEC) and localised adherent Escherichia coli (LAEC). The level of [Ca2+]i was increased in both DAEC- and LAEC-infected HeLa cells. However, studies with EGTA- and dantrolene-treated cells and also suspension of cells in Ca(2+)-free buffer suggested that the rise of [Ca2+]i in DAEC-infected cells was due to the influx of Ca2+ from extracellular medium, whereas Ca2+ mobilisation from the intracellular stores was responsible for the enhancement of [Ca2+]i in LAEC-infected cells. It was also evident that the infection of HeLa cells with DAEC and LAEC caused alteration of G/G+F actin ratio as compared to that of control cells. The ratio was much lower in LAEC-infected cells than that of DAEC-infected ones. Moreover, cytochalasin B inhibited both DAEC and LAEC invasion to HeLa cells, suggesting further the role of microfilaments in the invasion process.

Prevalence of toxoplasmosis in Calcutta and suburbs--a hospital based study for three years.

A total of 752 subjects were tested randomly for detection of antitoxoplasma antibody and its titre. Of these 752 subjects, 170 i.e. 22.6% showed seropositivity for antitoxoplasma antibody. However the titre varied between 1:2 to 1:1024, but in most cases its was 1:128.

Evidence for stimulation of the inositol triphosphate-Ca2+ signalling system in rat enterocytes by heat stable enterotoxin of Escherichia coli.

In Escherichia coli heat stable enterotoxin (STa)-treated rat enterocytes, the rise of inositol triphosphate (IP3) preceded the rise of [Ca2+]i. Chelation of extracellular Ca2+ with EGTA and suspension of cells in Ca2+ free buffer both demonstrated the enterotoxin-induced initial rise of [Ca2+]i with a concomitant loss of sustained phase. Furthermore, pretreatment of cells with dantrolene resulted in a decrease of the early response of [Ca2+]i, indicating the initial effect of the rise of [Ca2+]i was mostly due to its mobilization from some IP3-sensitive intracellular stores.

Alteration of the microenvironment in plasma membranes of rat enterocytes after Escherichia coli heat stable enterotoxin treatment: effect on protein kinase C activity.

Plasma membranes isolated from Escherichia coli heat stable enterotoxin (STa) treated rat enterocytes were studied in respect to protein kinase C activity and fluidity change. Pretreatment of enterocytes with STa increased the membrane bound protein kinase C activity about 5 fold as compared to control. STa treatment made the membrane more fluid as evident from a higher phospholipid/cholesterol ratio and greater unsaturated fatty acid levels. Moreover, the phase transition temperature of the STa treated membrane appeared to be significantly lower than that of the corresponding control membrane, thereby further indicating a rise in fluidity of the membrane in the former case. Our results, therefore, suggested that following STa enterotoxin treatment an appropriate fluid environment in the rat intestinal cell membrane was essential for the activation of protein kinase C.

Ultrastructural changes in HeLa cells associated with enteroadherent Escherichia coli isolated from infants with diarrhoea in Calcutta.

In the present study, we compared two enteroadherent Escherichia coli (EAEC) not belonging to the classical enteropathogenic O serotypes with respect to their attachment to and invasion of HeLa cells. Depending on the pattern of adherence to HeLa cells, one of the strains showed localized adherence (LA), and the other showed diffuse adherence (DA). Electronmicroscopic study showed that LA-EAEC produced the intimate attaching and effacing lesions and intracellular penetration in cultured HeLa cells. In contrast, DA-EAEC exhibited fimbrially-mediated adhesion to HeLa cells. Both LA and DA possessed morphologically distinct fimbriae. LA-EAEC expressed rod-like fimbriae, whereas fibrillar fimbriae were observed in DA strain. Ultrastructural study showed that the mechanisms of invasion by both the strains were different. LA-EAEC strains invaded the cells after pedestal formation and were enclosed in membrane-bound vacuoles. In contrast, DA-EAEC showed no membrane dissolution and pedestal formation during internalization of HeLa cells. A carefully controlled study will be necessary to establish the pathogenic role of DA-EAEC in diarrhoea.

Fine needle aspiration cytology of cervical lymphadenopathy with special reference to tuberculosis.

One hundred eighty cases of cervical lymphadenopathy have been studied by fine needle aspiration cytological examination followed by histopathologic examination of the excised lymph nodes. The diagnostic accuracy was 84.4% for tuberculous lymphadenitis by fine needle aspiration cytological examination. Observation of caseous necrosis (84.2%) and epithelioid cells (73.6%) were the most characteristic diagnostic features in the aspirated smears. Acid-fast bacilli were observed in 45.6% cases. Metastatic carcinoma also yielded a high diagnostic accuracy ie, 89%. Fine needle aspiration cytology has been found to be safe, quick, inexpensive with high diagnostic accuracy in cervical lymphadenopathy.

Serological study of human toxoplasmosis in Calcutta.

A total of 248 randomly selected subjects from urban, semiurban and rural areas of Calcutta was studied serologically for the prevalence of antibody to Toxoplasma gondii using latex agglutination technique. Fifty-nine (23.79%) out of these 248 subjects were found to possess anti-toxoplasma antibody. Seropositivity was found to be higher in females (25%) as compared to males (22.32%). Agewise highest positivity (30.5%) for toxoplasma antibody was observed in the third decade and lowest in the first decade of life, though all the age groups were involved by this protozoal infection. Sexwise distribution of anti-toxoplasma antibody showed highest positivity rate in the third decade in males and in the fourth decade in females. Twenty-five per cent of the subjects had history of contact with cat and/or soil and most of the subjects belonged to the middle and low income groups.

Evidence for protein kinase C stimulation in rat enterocytes pretreated with heat stable enterotoxin of Escherichia coli.

Rat intestinal epithelial cells were isolated and the activity of the calcium- and phospholipid-dependent protein kinase C (PKC) was investigated. The stimulation of activity by Escherichia coli heat stable enterotoxin (STa) was about 5-fold compared to control activity (16.91 +/- 1.69 vs 93.56 +/- 10.40 nmol/mg protein/min) and was dose dependent. Maximum enzyme activity was observed after incubation for 1 min with 6 ng of purified STa. The synergistic effects of calcium, phosphatidylserine and diolein on the enzyme activity were noted both in control and STa-treated cells. Staurosporine, a potent PKC inhibitor, significantly reduced the enzyme activity. Autoradiographic analysis of polyacrylamide gel electrophoresis revealed that pretreatment of the cells with STa also resulted in the phosphorylation of specific membrane proteins each with a molecular mass of 37 kDa, 100 kDa and 140 kDa. However, STa had no direct role on the enzyme activity. Our results, therefore, provide evidence for the involvement of PKC in STa-induced signal transduction in rat enterocytes.

Biochemical characterization of a calcium ion stimulated-ATPase from goat spermatozoa.

The goat spermatozoa membranes isolated after treatment with octa (ethylene glycol) mono n-dodecyl ether (C12E8) followed by discontinuous sucrose density gradient centrifugation have been found to contain an ATPase that is stimulated by externally added Ca2+ only. The membrane fraction has also found to contain Mg(2+)-dependent Ca(2+)-ATPase activity, however the former activity is about 2 fold higher than the latter. The molecular weight of the enzyme is found to be about 97,000 on SDS-polyacrylamide gel. The optimum concentration of Ca2+ required for maximum activity is 3 mM for both Mg(2+)-dependent and Mg(2+)-independent Ca(2+)-ATPase. Histidine and imidazole buffers are found to be the most suitable for dependent and independent enzyme activities respectively. ATP with an optimum concentration of 4 mM is observed to be the best substrate than any other nucleotides. The inhibitors like trifluoperazine and vanadate and group specific probes e.g. DTNB and TNBS inhibit these two enzymes but at different rates. Ca(2+)-uptake study shows that the uptake in the presence of Ca2+ and ATP is higher than in the presence of Mg2+, Ca2+ and ATP. The findings lead us to believe that the Mg(2+)-independent Ca(2+)-ATPase has some role in Ca2+ transport like Mg(2+)-dependent enzyme.

Expression of virulence and antibiotic resistance in an Escherichia coli transconjugant carrying a large plasmid pCAT120 of Shigella dysenteriae type I and its spontaneous fragmentations.

The role of a 120-kb plasmid in relation to virulence and drug resistance factor in Shigella dysenteriae was studied. For characterization of plasmids, the mating system is a useful and efficient means of transferring both large and small plasmids to a new host. The conjugative transfer of a 120-kb (pCAT120) ampicillin-resistant plasmid of S. dysenteriae to E. coli K-12 was not successful. Introduction of an E. coli fertility factor plasmid F, did not help to mobilize the plasmid. Low transfer frequencies of antibiotic markers to E. coli were achieved by treatment of the donor S. dysenteriae with N-methyl-N'-nitro-N-nitrosoguanidine. The transconjugants showed resistance to ampicillin, chloramphenicol, tetracycline and cadmium. A transconjugant carrying the 120-kb plasmid of S. dysenteriae produced keratoconjunctivitis in guinea pigs. Repeated subculture of Clmr transconjugant (pCAT120) on tryptic soya agar plates became Clms and showed four distinct DNA bands ranging from 3 to 10 kb in size on agarose gel electrophoresis. Utilization of organic acids, metal resistance (Cd), dye-binding properties (Crb+, Ebr+) and drug resistance (Amp, Tet) were identified on 10, 7, 4 and 3-kb plasmid DNA fragment of pCAT120 respectively. Crb+ 4-kb DNA fragment of pCAT120 was isolated, purified and transferred to an avirulent E. coli K12 by transformation. However, transformant (pET4) showed poor growth on solid media and its growth in liquid culture was only possible after supplementation of the unknown low-molar-mass thermolabile factor(s) secreted by the recipient strain.(ABSTRACT TRUNCATED AT 250 WORDS)

Diacylglycerol breakdown in plasma membrane of rat intestinal epithelial cells. Effect of E. coli heat-stable toxin.

Rat intestinal epithelial cells were isolated and the activity of the enzyme diacyglycerol lipase (DG lipase, EC 3.1.1.3) was investigated. When cells were treated with Escherichia coli heat-stable toxin (ST) liberation of endogenous glycerol and fatty acids was observed. The enzyme responsible for this effect could be demonstrated to be a DG lipase by using specific substrates. It was found that the activity of DG lipase was increased 5-6-fold with the substrates diolein and 1,2-dioleyl-rac-glycerol and triolein being neutral lipid insensitive to DG lipase. ST had no direct effect on the DG lipase. The enzyme DG lipase was activated via a chain reaction due to the hydrolysis of phosphatidylinositol (PI) by the enzyme PI-specific phospholipase C stimulated by ST.

Stimulation of phosphoinositides breakdown by the heat stable E. coli enterotoxin in rat intestinal epithelial cells.

Rat intestinal epithelial cells were labelled with [32P]Pi and extracted, and the phospholipids were analysed by thin-layer chromatography. 32P-incorporation in phosphatidylinositol (PI) and phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-phosphate (PIP2) were measured in control and heat stable enterotoxin (ST)-treated cells. ST was found to induce rapid degradation of PIP and PIP2. The degradation of inositol lipids was accompanied by an increase of water soluble inositol phosphate (IP1, IP2, IP3) compounds. There was a two-fold increase of radioactivity in IP2 and IP3 but no significant change was observed in IP1. Phospholipase C activity was increased tenfold with substrate PIP2 in ST-pretreated cells. The present study indicates that ST triggers another second messenger system by increasing the PIP2 hydrolysis with the enzyme phospholipase C.

The protein receptor for cholerabacteriophage phi 149.

Choleraphage phi 149 receptor activity was found in the outer membrane (OM) protein of Vibrio cholerae 154. Receptor protein for phage phi 149 was separated from trypsin-treated OM on a Sephadex G-100 column. Of the three peaks obtained, phage receptor activity was noted only in peak II. SDS-PAGE showed that the Mr of the protein was 35,000. The protein was heat-labile and protease-sensitive. The specificity of this protein as choleraphage phi 149 receptor was investigated by carrying out a protection experiment by anti-protein (peak II) rabbit sera.

Tryptophanase activity in different toxigenic and nontoxigenic strains of Vibrio cholerae: effect of glucose.

Tryptophanase activity was measured in eight different toxigenic and nontoxigenic strains of Vibrio cholerae (V. cholerae) in presence and absence of inducer tryptophan (2 mM). Stimulation of enzyme activity was observed in both toxigenic and nontoxigenic strains of V. cholerae in presence of inducer. Tryptophanase activity remained much higher in toxigenic strains than that in nontoxigenic strains. Low levels of enzyme activity in nontoxigenic strains could be increased by the addition of exogenous cyclic AMP. A lower concentration of glucose (0.25 gm%) in culture medium produced no inhibitory effect on enzyme activity. But a higher concentration of glucose (3 gm%) repressed the tryptophanase activity. The repressive effect of glucose could be reversed by the addition of exogenous cyclic AMP.