Cloning and functional expression of an acyl-ACP thioesterase FatB type from Diploknema (Madhuca) butyracea seeds in Escherichia coli.

A cDNA of fatty acyl-acyl carrier protein (ACP) thioesterase (Fat) from developing seed of Madhuca butyracea has been cloned. The deduced amino acid sequence of the cDNA corresponding to the mature polypeptide showed 30-40% and 60-75% identity to the reported FatA and FatB class of plant thioesterases, respectively. This gene, MbFatB, is present as a single copy in M. butyracea genome and the MbFatB protein was detected clearly in seed tissues of this plant but not in that of Indian mustard (Brassica juncea). Heterologous expression of the MbFatB gene driven by different promoters in E. coli wild type and fatty acid beta-oxidation mutant (fadD88) strains resulted production of the recombinant protein with various fusion tags either as biologically inactive (insoluble) or functionally active forms. Expression of functionally active recombinant MbFatB in E. coli affected bacterial growth and cell morphology as well as changed the fatty acid profiles of the membrane lipid and the culture supernatant. Alteration of the fatty acid composition was directed predominantly towards palmitate and to a lesser extent myristate and oleate due to acyl chain termination activity of plant thioesterase in bacteria. Thus, this new MbFatB gene isolated from a non-traditional oil-seed tree can be used in future for transgenic development of oil-seed Brassica, a widely cultivated crop that expresses predominantly oleoyl-ACP thioesterase (FatA) in its seed tissue and has high amount of unwanted erucic acid in edible oil in order to alter the fatty acid profile in a desirable way.

Characterization of gastric mucosal membranes. II. Lipid compositions of purified gastric mitochondria from pig, rabbit and frog.

1. The lipid composition of purified mitochondrial fractions from the fundic mucosa of pig, rabbit and frog were determined. 2. The total lipids expressed as mg lipid per 100 mg mitochondrial protein were approx. the same in pig and rabbit (13.4 and 15.5, respectively) and much higher than in frog (8.5). 3. The levels of phospholipids were about the same in pig and frog (approx. 61% of the total lipid) and lower than rabbit (78%). However, the levels of cholesterol were significantly different in the three species and constituted 22, 9 and 18.2% of the total lipids in pig, rabbit and frog mitochondria, respectively. 4. The glycolipid content in the mitochondrial lipids from pig, rabbit and frog were 7, 5.6 and 10.5%, respectively. 5. Cardiolipin contributed from 5.6 to 7.5% of the total phospholipids in the various species. Phosphatidylethanolamine and phosphatidylcholine together accounted for 80 90% of the total phospholipids in the various species; the contribution of phosphatidylcholine being always higher than that of phosphatidylethanolamine. Small but significant amounts of phosphatidylinositol were present in all species. 6. Generally, the predominant saturated fatty acid in the phospholipids was 16:0 from all species (except in phosphatidylethanolamine from pig and frog), and 18:1 and 18:2 were the predominant unsaturated fatty acids from all species. Sphingomyelin contained the highest amount of saturated fatty acids (over 80%) in both the species (pig and rabbit) studied.

Characterization of partially purified (Na+ + K+)-ATPase from porcine lens.

The partial purification of (Na+ + K+)-ATPase from pig lens has been achieved by treatment with deoxycholate followed by density gradient centrifugation. The specific activity of the final preparation, ranging from 300 to 500 nmol/h per mg protein, is increased approx. 100-fold compared to the homogenate. A parallel increase in rho-nitrophenylphosphatase activity is also observed. Sodium dodecyl sulfate (SDS) gel electrophoresis reveals six major protein bands, one of which is the 93 kDa alpha subunit of (Na+ + K+)-ATPase which can be phosphorylated by reaction with [gamma-32P]ATP. A second band contains a glycoprotein which displays an apparent molecular weight of 51000 and thus appears to be the beta subunit of the enzyme. The enzyme is sensitive to ouabain with the I50 for (Na+ + K+)-ATPase and rho-nitrophenylphosphatase inhibition being 1.2 and 1.3 microM, respectively. Several agents which inhibit (Na+ + K+)-ATPase from other tissues such as oligomycin, Ca2+, vanadate, N-ethylmaleimide, rho-chloromercuribenzenesulfonic acid (PCMBS) and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) also inhibit the lens enzyme. Monovalent cations other than K+ are partially effective in activating the (Na+ + K+)-ATPase and rho-nitrophenylphosphatase activities. The K+ congeners were relatively more effective in supporting (Na+ + K+)-ATPase compared to rho-nitrophenylphosphatase activity. Other kinetic properties of the lens enzyme are also comparable to those of the enzyme from other tissues. Utilizing the partially purified membrane bound enzyme, discontinuities in Arrhenius plots of (Na+ + K+)-ATPase activity, rho-nitrophenylphosphatase activity and fluorescence polarization of the fluidity probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), are observed near the physiological temperature of lens. The possible significance of these observations for the mechanism of cataract formation are discussed.

Studies of gastric Ca2+-stimulated adenosine triphosphatase. I. characterization and general properties.

Gastric microsomes do not contain any significant Ca2+-stimulated ATPase activity. Trypsinization of pig gastric microsomes in presence of ATP results in significant (2-3 fold) increase in the basal (with Mg2+ as the only cation) ATPase activity, with virtual elimination of the K+-stimulated component. Such treatment causes unmasking of latent Mg2+-dependent Ca2+-stimulation ATPase. Other divalent cations such as Sr2+, Ba2+, Zn2+, and Mn2+ were found ineffective as a substitute for Ca2+. Moreover, those divalent cations acted as inhibitors of the Ca2+-stimulated ATPase activity. The pH optimum of the enzyme is around 6.8. The enzyme has a Km of 70 microM for ATP and the Ka values for Mg2+ and Ca2+ are about 4 x 10(-4) and 10(-7) M, respectively. Studies with inhibitors suggest the involvement of sulfhydryl and primary amino groups in the operation of the enzyme. Possible roles of the enzyme in gastric H+ transport have been discussed.

An Na+/K(+)-ATPase inhibitor protein from rat brain cytosol.

A protein isolated from rat brain cytosol is found to inhibit Na+/K(+)-ATPase in rat brain and kidney and H+/K(+)-ATPase from toad gastric mucosa, but has no effect on Ca2+,Mg(2+)-ATPase and Ca(2+)-ATPase isolated either from rat testis or goat spermatozoa. The inhibitor has been partially purified by ammonium sulphate precipitation followed by gel-filtration through Sephadex G-100. The inhibitor seems to bind at or close to the ATP binding site of Na+/K(+)-ATPase, such that the binding of the inhibitor to ATPase is reversible and competitive in nature with respect to the substrate. Optimum inhibition is observed at around the phase transition temperature of brain Na+/K(+)-ATPase and the inhibitory activity is only partially dependent on -SH or -NH2 group(s) of the inhibitor protein.

Unsaturated fatty acid-activated protein kinase (PKx) from goat testis cytosol.

The cytosolic fraction of goat cauda epididymis possesses a protein kinase (PKx) activity which is stimulated by a number of unsaturated fatty acids of which arachidonic acid is the best activator in absence of cAMP or Ca(2+). Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and diacylglycerol have no effect either alone or in combination. The membrane fraction does not show any appreciable kinase activity even after detergent treatment. PKx migrates as a single band of apparent molecular mass of 116 kDa on 10% SDS-PAGE after sequential chromatographic separation on DEAE-cellulose, phenyl-Sepharose, high-Q anion exchange and protamine-agarose affinity column. PKx phosphorylates histone H1, histone IIIs and protamine sulfate, but not casein. However, the best phosphorylation was obtained with a substrate based on PKC pseudosubstrate sequence (RFARKGSLRQKNV). The kinase phosphorylates two endogenous cytosolic proteins of 60 and 68 kDa. Ser residues are primarily phosphorylated although a low level of phosphorylation is observed on Thr residues also. Ca(2+) and Mn(2+) inhibit PKx activity in the micromolar range. Staurosporine is found to inhibit the PKx activity to a significant level at sub-nanomolar concentration. Lyso-phosphatidylcholine and certain detergents at very low concentrations (<0.05%) stimulate enzyme activity to some extent. The immuno-crossreactivity study with antibody against different PKC isotypes suggests that the protein kinase under study is not related to any known PKC family. Even the antibody against PKN (a related protein kinase reported in rat testis found to be activated by arachidonic acid) does not cross-react with this protein kinase. Hence we believe that the protein kinase (PKx) reported here is different even from the PKN of rat testis. The phosphorylation of endogenous proteins by the protein kinase may be involved in cell regulation including fertility regulation and signal transduction.

Na(+)-K(+)-ATPase activity of different regions of rat brain in response to immunization and bone marrow cytokine treatment.

The bone marrow cytokine (Bio-Immuno Modulator, BIM or BM-Fr1) has been suggested to correct immunoincompetence by modulating brain Na(+)-K(+)-ATPase. The brain region affected and mechanism of action of BM-Fr1 are unknown, however. Here we report that immunization of immunocompetent rats indirectly inhibited Na(+)-K(+)-ATPase activity (59%) in the left cerebral lobe (LC) and irrespective of BM-Fr1 treatment, stimulation of the enzyme was observed in the LC at the peak of immune response. BM-Fr1 treatment, which corrected immunoincompetence in malnourished rats, also modulated a different LC Na(+)-K(+)-ATPase profile to that seen in immunocompetent animals. Immunogen and BM-Fr1 seem to exert their influence in brain via a cytosolic inhibitor protein of Na(+)-K(+)-ATPase. Thus we suggest that (1) BM-Fr1 plays an important role in immune homeostatasis by modulating Na(+)-K(+)-ATPase activity of LC and (2) Na(+)-K(+)-ATPase is not the receptor for either immunogen or BM-Fr1.

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.

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.

Enteropathogenicity of Aeromonas hydrophila and Plesiomonas shigelloides.

Aeromonas hydrophila was enteropathogenic in ligated ileal loops of rabbits, causing a fluid accumulation of 1-0 - 2-0 ml per cm of gut length. Gut reaction could be produced with an inoculum as low as 10-4 viable bacteria. There was no difference in the nature of the positive reactions given by strains isolated from diarrhoeal and non-diarrhoeal children and adults and from water. Plesiomonas shigelloides, on the other hand, did nt cause a significant gut reaction. A. hydrophila multiplied in the ileal loop by about 10-5 wheras P. shigelloides did so at only 10-2-3. These experiments on a animal model thus indicated the enteropathogenic nature of A. hydrophilia, but no definite conclusion could be drawn from this study on P. shigelloides.

Differential effect of bone marrow protein on brain ATPase of immunized control and malnourished rats.

After sheep erythrocyte (SRBC) immunization in balanced-diet-fed (BDF) rats, the brain microsomal ATPase activity for the first 48 h was suppressed compared with preimmunization level, then stimulated at the peak of the immune response. In contrast, rats malnourished by deprivation of vitamin B complex and ascorbic acid and with significantly lower pre- and postimmunization enzyme activity than the BDF rats, had stimulated enzyme activity only on immunization, and their immune response was found to be suppressed. A bone marrow-derived bioimmunomodulator (BIM) revived the immunocompetence of the malnourished immunized animals, and brain ATPase activity in these animals after BIM injection followed a pattern similar to that of the immunized BDF rats. We suggest that, after SRBC immunization under the influence of BIM, suppression followed by stimulation of the brain microsomal ATPase might be related to immune response.

Cellular immunity status in anaemia in pregnancy.

Fifty pregnant women (25 anaemic and 25 non-anaemic) and 20 non pregnant women (10 anaemic and 10 non-anaemic) were studied. All pregnant women delivered full term (37-41 wk) singleton babies. Maternal blood lymphocyte stimulation indices (SI) at 0 and 24 h were lower in anaemic and non-anaemic pregnant women, compared to anaemic and non-anaemic non-pregnant women. This difference was more marked in anaemic pregnant women, as compared to non pregnant anaemic women at 0 and 24 h respectively. The SI of maternal and cord blood lymphocytes were significantly lower in severely anaemic mothers both at 0 and 24 h and in those with maternal serum iron levels below 50 micrograms/dl or maternal per cent transferrin saturation was below 15 per cent. The anaemic mothers and their offspring were found to have significantly lower blastogenic response to PHA added at 24 h indicating depression of T-suppressor cell function.

Studies on the mechanism of action of Albizzia lebbeck, an Indian indigenous drug used in the treatment of atopic allergy.

Studies were conducted on the decoction of the bark of Albizzia lebbeck which has been in use by Ayurvedic physicians for bronchial asthma and eczema. The effect of A. lebbeck was studied on the degranulation rate of sensitized peritoneal mast cells of albino rats when challenged with antigen (horse serum). Triple vaccine was used as adjuvant. Disodium cromoglycate (DCG) and prednisolone were used for comparison. Drugs were given during the first or second week of sensitization and the mast cells studied at the end of the second or third week. Serum from these rats was used to passively sensitize recipient rats whose peritoneal mast cells were then studied. The in vitro effects of A. lebbeck and DCG on the degranulation rate of the sensitized mast cells were also studied. The results show that A. lebbeck has a significant cromoglycate-like action on the mast cells. In addition, it appears that it inhibits the early processes of sensitization and synthesis of reaginic-type antibodies. If A. lebbeck is given during the first week of sensitization it markedly inhibits the early sensitizing processes, while if given during the second week it suppresses antibody production during the period of drug administration. The active ingredients of the bark appear to be heat-stable and water-soluble.

A correlative study of serum zinc and in vivo cell mediated immune status in rheumatic heart disease.

This study was conducted to determine the zinc status and assess relationship between serum zinc and in vivo cell mediated immunity (CMI) in patients with rheumatic heart disease (RHD). The study comprised 22 patients with active rheumatic heart disease (ARHD), 15 patients with chronic rheumatic heart disease without activity (CRHD) (selection based on Jone's Criteria--Revised), and 15 age and sex matched healthy control. Zinc estimation was done by atomic absorption spectrophotometer. To assess CMI in vivo, phytohaemagglutinin skin test and skin window test were done. Serum zinc and in vivo CMI in patients with ARHD and CRHD compared with controls. Mean serum zinc was significantly decreased in patients with ARHD and CRHD, more pronounced in the former (P less than 0.001); and mean 24 h urinary zinc was significantly increased in patients with ARHD (P less than 0.001) as compared to controls. A significant depression in CMI in vivo was observed in patients with ARHD and CRHD (P less than 0.001). A significant positive correlation was seen among serum zinc and markers of in vivo CMI (P less than 0.001). In conclusion, depletion of zinc, observed in RHD, probably causes immune alterations and suggest role of zinc in immunopathogenesis of RHD. Zinc supplementation may alter the course of rheumatic fever and RHD.

Delayed cutaneous hypersensitivity ad peripheral blood lymphocyte counts in gastrointestinal cancer.

Cell-mediated immune responses as expressed by delayed cutaneous hypersensitivity (DCH) reactions to specific recall antigens (PPD and BCG) and non-specific contact antigen (DNCB), and peripheral blood lymphocyte counts were studied in 60 patients of gastrointestinal cancer and 20 control subjects. The pattern of these immune responses in gastrointestinal cancer was significantly different from tumour-free control subjects of comparable age and sex distribution and apparently indicates a depressed immunologic function in cancer patients. Our study suggests that DNCB skin testing and peripheral blood lymphocyte counts are relatively simple and reliable investigations for predicting the advanced stage of gastrointestinal cancer (and BBY implication prognosis).

Demonstration of histamine receptors on the surface of Entamoeba histolytica.

Histamine receptors on the surface of E. histolytica could be demonstrated by histochemical method using three isolates of the protozoa grown and maintained in modified Boeck and Drbohlav's medium. Prior treatment of E. histolytica with cimetidine a H2 blocker, blocked the histamine uptake. Similar treatment with mepyramine maleate, a H1 blocker, did not prevent histamine uptake by the protozoa. It is postulated that E. histolytica has H2 receptors on its surface.

Enhancement of virulence of Entamoeba histolytica by histamine in vitro.

Three isolates of E. histolytica isolated and maintained in modified Boeck and Drbohlav's medium and the axenic nonpathogenic strain NIH-200 maintained in Diamond's TPS-1 medium were used to assess the effect of histamine added to the cultures on their pathogenicity in just weaned rats of Charles Foster strain. Initially when the three polyxenic isolates were examined for their Pathogenicity after growing them with graded concentrations of histamine viz. M-2 through M-6 (log dilutions of the molar concentration of histamine dihydrochloride) the caecal scores were significantly enhanced in cultures grown with M-2 and M-3 concentrations. Subsequently NIH-200 strain was examined similarly after growing it with 20 micrograms/ml of histamine dihydrochloride through three generations. The Neal's caecal score of NIH-200 increased significantly stepwise up to second subculture and became stabilised at third generation with histamine.

In vitro effect of 5-hydroxytryptamine on concanavalin-A agglutinability and multiplication of Entamoeba histolytica.

Six isolates of E. histolytica isolated and maintained by serial passage in modified Boeck and Drbohlav's medium were used in this study. When five of the isolates were grown in the above medium with 5-hydroxytryptamine (5-HT) added to the overlay, the agglutinability of the amoebae by concanavalin- A (con-A) was significantly increased compared to corresponding control cultures. Four isolates of E. histolytica grown with 5-HT had 1.5 to 2 times higher counts than the control cultures. Similarly the con-A agglutinability and counts of cultures of NIH-200 (an axenic strain of E.histolytica) were enhanced when grown in association with 5-HT.

Effect of exogenous 5-hydroxytryptamine on pathogenicity of Entamoeba histolytica in experimental animals.

In an effort to find out the mechanism(s) operative in enhancing the pathogenicity of E. histolytica in hosts under heat stress reported earlier, effect of 5-hydroxytryptamine (5-HT) on the virulence of the parasite was examined in just weaned Charles Foster strain of albino rats. Pathogenicity of 10 strains of E. histolytica, from various forms of intestinal amoebiasis, grown in modified Boeck and Drbohlav's medium was assessed by caecal scoring. Administration of 5-HT in infected animals significantly enhanced the pathogenicity of all the seven strains tested. Treatment of the host with the 5-HT precursor L-tryptophan also increased the caecal scores examined with three strains of E. histolytica. Prior blocking of tissue 5-HT receptors by administration of methysergide almost completely abolished the pathogenicity enhancing effect of 5-HT treatment. This suggested that 5-HT itself and not any of its metabolites was responsible for the observed increase in pathogenicity of E. histolytica on 5-HT treatment of the host.

Immuno-stimulatory effect of levamisole in borderline leprosy cases.

Eleven cases of borderline-borderline leprosy were subjected to levamisole therapy for 3 months. Levamisole was given 150 mg/day on three consecutive days every fortnight along with dapsone (DDS). Immuno-stimulation was assessed by lepromin test using Lepromin-A supplied by W.H.O. containing 4.0 X 10(7) bacilli per milli litre. It was found that there was statistically significant change in lepromin reaction after levamisole therapy.