Characterization and control of endophytic bacterial contaminants in in vitro cultures of Piper spp., Taxus baccata subsp. wallichiana, and Withania somnifera.

Bacterial contamination is a serious problem that causes severe loss of in vitro grown cultures of a number of plants. This problem becomes even more acute if the bacterial contamination is of endophytic origin. In such cases, identification and characterization of the contaminants is essential for achieving specific control of the contaminants through selective use of antibiotic agents, especially if the routinely used contamination control methods practiced elsewhere in tissue culture studies are ineffective. Such is the case with the bacterial contamination observed in the present study. The five endophytic bacteria associated with Piper nigrum and Piper colubrinum, four endophytic bacteria associated with Taxus baccata subsp. wallichiana, two endophytic bacteria associated with Withania somnifera, and two bacteria common to all these plant species were isolated and characterized based on morphological and biochemical tests. Their taxonomic positions based on similarity indices were determined. A control strategy against these bacteria has been developed based on bacteriostatic or bactericidal actions of 12 antibiotics at three different concentrations by solid and liquid antibiogramme assays.

Determination of antidiabetic activity in Allium cepa (onion) tissue cultures.

Seedling, seedling parts and callus cultures of onion were tested for their antidiabetic activity by feeding the tissue-extracts to diabetic rats. The results indicated much higher antidiabetic activity in callus cultures as compared to natural bulbs of onion. These results may be of pharmaceutical significance since the callus can be used as an alternative source for the isolation of antidiabetic compounds.

Apolipoprotein E polymorphism in non-insulin-dependent diabetics of Mumbai, India and its effect on plasma lipids and lipoproteins.

The role of apolipoproteinE (apoE) phenotypes in modulating the plasma lipid and lipoprotein levels was studied in a group of NIDDM patients and healthy individuals residing in Mumbai. The apoE phenotype frequencies were similar in the diabetic and healthy persons. The elevations in the lipid/lipoprotein levels were higher in diabetic subjects (53.3%) than in the controls (8%), showing the frequency of increase to be highest in the apoE4/4 group of diabetics, followed by apoE4/3 and apoE3/2 groups. In the controls as well, a similar trend was observed in different groups, indicating that the susceptibility to changes in lipid concentrations differs among apoE phenotypes. The apoE3/3 bearing individuals in both the categories showed close to normal lipid levels, suggesting it to be the wild type. The occurrence of apoE4 allele was higher than the apoE2 allele in diabetic individuals. Diabetic subjects with the apoE4 allele showed hypercholesterolemia, while those with apoE2 showed the presence of hypertriglyceridemia. One of the striking features of our work is a significant relationship between apoE4/3 phenotype in NIDDM persons and elevated levels of plasma triglyceride, thus suggesting a delayed catabolism of VLDL relative to production. In conclusion, the work suggests that the apoE2 and apoE4 alleles are associated with elevations in lipid levels and these changes are more pronounced in the diabetic individuals in whom most of the lipid levels were higher, indicating a gene environment/disease interaction.

Adventitious shoot regeneration from root, internode, petiole and leaf explants of Piper colubrinum Link.

A single-step, high-frequency regeneration pro-tocol has been standardised for Phytophthora-resistant wild pepper, Piper colubrinum (Link) using root, internode, leaf and petiole explants derived from in vitro plantlets. The effect of BA on shoot-bud induction and elongation was assessed by supplementing half-strength MS medium (macronutrients at half the concentration) with different concentrations of BA, i.e. 0.2-10 mg l-1 in induction media and 0, 0.2 and 0.5 mg l-1 in subculture media. The interaction between culture period and BA concentration was studied by culturing the explants for 8, 15 and 30 days before the first subculture. The elongated shoots were rooted directly in soil and hardened in the greenhouse. The developed protocol would be useful in marker-assisted asymmetric hybridisation programmes involving wild-type Piper colubrinum and the cultivated species P. nigrum.

A simple two-step purification of antidiabetic compounds from Eugenia jambolana fruit-pulp: proteolytic resistance and other properties.

A simple, two-step, procedure has been developed to purify antidiabetic compounds from Eugenia jambolana fruit-pulp. The compounds have been identified as a peptidylglycan and an oligosaccharide with molecular weights of 6.0 and 1.2 kD, respectively. The binding between the sugars and the peptide in the peptidylglycan appears to be covalent. The amino acid and sugar composition of the peptidylglycan have been determined as have the sugars of the oligosaccharide. The intrinsic color of the peptidylglycan was attributed to the compound itself and not to the presence of pigment or metal. The peptidylglycan was resistant to degradation by proteolytic enzymes in vitro, explaining its efficacy in oral feeding.

Tumor-specific transplantation resistance in mice after treatment of initial tumors with Streptococcus thermophilus.

Antitumor activity observed by treatment with Streptococcus thermophilus was further investigated. The mice cured from fibrosarcoma by treatment with heat-killed preparation of S. thermophilus, when challenged with fibrosarcoma failed to take up the tumor. However, these cured mice when challenged with sarcoma-180 or Ehrlich ascites carcinoma, did not show significant changes in tumor take and/or survival compared to their respective controls. Similarly, mice cured from sarcoma-180 were challenged with fibrosarcoma, sarcoma-180 or Ehrlich ascites carcinoma. Though there was no change in the mean survival time (MST) of the dying mice regarding sarcoma-180 or Ehrlich ascites carcinoma, there was 50 and 30% increase in the number of mice that showed total regression respectively over controls. However, there was no difference in the growth rate of fibrosarcoma. Similar observations were made with mice cured from Ehrlich ascites carcinoma, challenged with these tumors. These findings thus suggest that the antitumor response was tumor-specific and that tumor-associated antigens may have a role in imparting this specificity. Bacterial treatment non-specifically augmented this primary response.

In vitro plant regeneration from leaf callus inPiper colubrinum Link.

Callus-mediated shoot regeneration from leaf explants ofPhytophthora resistant pepper (Piper colubrinum Link.) is described. The effect of basal media composition and growth regulators onin vitro response of explants was evaluated. Shoot buds were induced and elongated on half-strength MS medium containing 2.0 mg l(-1) BA and 0.5 mg l(-1) NAA , as well as 1.0 mg l(-1) BA and 0.5 mg l(-1) 2,4-D. The shoots were rooted in half-strength MS medium with or without IAA or IBA, and then were transferred to soil with 100% survival.

Hypoglycemic activity of Eugenia jambolana and Ficus bengalensis: mechanism of action.

The extract of jaman pulp from fruit of Eugenia jambolana showed hypoplycemic activity. This report is the first evidence of such activity in relation to pulp. The effect of pulp was seen in 30 min, while the seeds of the same fruit required 24 hr. The extracts of bark of Ficus bengalensis caused reduction in blood sugar level. These results were confirmed in streptozotocin-induced diabetic animals. The oral administration of the extract resulted in enhancement in serum insulin levels in normoglycemic and diabetic rats. The incubation of isolated islets of Langerhans from normal as well as from diabetic animals with each of these plant extracts stimulated insulin secretion. These extracts inhibited insulinase activity from liver and kidney.

Antitumor activity of Streptococcus thermophilus against fibrosarcoma: role of T-cells.

Antitumor activity induced by a heat-killed preparation of S. thermophilus against mouse fibrosarcoma was investigated. The treatment of Swiss mice with S. thermophilus prior to transplantation could not prevent tumors. However, animals cured by treatment with a S. thermophilus preparation failed to take up the tumor when rechallenged with fibrosarcoma. S. thermophilus did not induce antitumor activity in animals immunosuppressed by sublethal whole body gamma-irradiation (4 Gy) or hydrocortisone treatment prior to transplantation. Suppression of activity of macrophages by carrageenan had no effect on antitumor activity of the heat inactivated preparation of S. thermophilus. The intravenous administration of sera from cured animals was ineffective in curing the tumours. Spleen cells from cured animals could effectively transfer the antitumor activity to recipients transplanted with the tumor. This effect was abolished when the T-lymphocyte population in the inoculum was specifically depleted. The results thus suggest the involvement of T-lymphocytes in antitumor activity exhibited by S. thermophilus.

Pentose synthesis in glucose-grown cells of Lactobacillus casei.

The pathway of pentose synthesis in glucose-grown cells of Lactobacillus casei was ascertained. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were present in glucose-grown cells, while transaldolase and transketolase were present only in traces. This suggested that only the oxidative arm of this pathway was operative in glucose-grown cells. On the other hand, in ribose-grown cells, transaldolase was induced with a concomitant suppression of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. These results were confirmed by the detection of labelled CO2 produced by L. casei grown on [1-14C]glucose. The activities of the enzymes of the oxidative pentose phosphate pathway as also the rate of CO2 formation were higher in the exponential phase of growth as compared to the stationary phase, when the requirement of the cells for pentoses for the formation of DNA and RNA was higher.

Glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase from Lactobacillus casei: responses with different modulators.

Glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) were separated and partially purified from glucose-grown cells of Lactobacillus casei. The enzymes had similar pH optima, thermosensitivity and molecular weights. They had different net charges and their pI values were 5.38 and 4.52, respectively. Histidine, arginine, lysine and cysteine residues were essential for the activity of G6PD, and all the above amino acids with the exception of lysine were required for 6PGD activity. Mg2+ activated 6PGD up to 15 mM concentration, above which it was inhibitory. It had no effect on G6PD activity. G6PD was specific for NADP+, but 6PGD showed some activity with NAD+ as the cofactor, although it was essentially NADP(+)-preferring. Both the enzymes, were inhibited by NADPH. 6PGD was also inhibited by its product, ribulose 5-phosphate. ATP inhibited 6PGD only at subsaturating concentrations of NADP+. The inhibition was sigmoidal in the absence of Mg2+ and hyperbolic in its presence.

Ribulose 1,5-bisphosphate dependent CO2 fixation in the halophilic archaebacterium, Halobacterium mediterranei.

The cell extract of Halobacterium mediterranei catalyses incorporation of 14CO2 into 3-phosphoglycerate in the presence of ribulose bisphosphate suggesting the existence of ribulose bisphosphate carboxylase activity in this halophilic archaebacterium.

Antitumor activity of lactic acid bacteria on a solid fibrosarcoma, sarcoma-180 and Ehrlich ascites carcinoma.

Antitumor activity of Leuconostoc mesenteroides, Streptococcus cremoris and Streptococcus lactis was investigated in solid mouse fibrosarcoma. Intratumor administration of lyophilised bacterial cells at a dose of 20 mg/kg body wt resulted in the regression of tumors in a maximum number of animals. Intraperitoneal injection at the same dose resulted in a significant increase in the survival time but was ineffective in curing the animals. Intratumor injection of Streptococcus thermophilus at a dose of 20 mg/kg body wt also inhibited the growth of fibrosarcoma accompanied by an increase in the survival time while intraperitoneal administration of S. thermophilus prolonged only the survival time and did not result in cure. In mice bearing the ascitic form of sarcoma-180 or Ehrlich ascites carcinoma, intraperitoneal administration of S. thermophilus resulted in complete cure in a very significant proportion of tumor-bearing mice. S. thermophilus was more effective in sarcoma-180 than in Ehrlich ascites carcinoma. The cured tumors did not recur.

Rat liver uridine diphosphate glucose dehydrogenase: dual mechanism of regulation in vivo.

Rat liver UDPglucose (UDPG) dehydrogenase activity was observed to be decreased after fasting and could be restored to normal levels after refeeding glucose. This could be prevented by prior injection of puromycin, suggesting de novo protein synthesis. Administration of insulin to normal rats on stock diet did not influence the enzyme activity. However, the enzyme activity was decreased in the diabetic condition. Intraperitoneal injection of insulin caused an enhancement of the enzyme activity in diabetic animals. Hepatic UDPG dehydrogenase activity was observed to be decreased on ascorbic acid feeding or intraperitoneal injection of the same. The intraperitoneal injection of either insulin or cAMP to ascorbic acid-treated rats resulted in an increase in enzyme activity reaching normal levels. The insulin-mediated increase could not be prevented by prior injection of puromycin, suggesting a post-translational effect. These results indicate two distinct mechanisms for in vivo regulation of hepatic UDPG dehydrogenase.

Mechanism of aldolase binding to erythrocyte membrane: Part II. Kinetic aspects.

The nature of binding of FDP aldolase to bovine erythrocyte membrane was examined. The Km value of bound and soluble enzyme differed by an order. The absence of time-lag in the velocity-time curves at various concentrations of the substrate and the similar extent of inactivation of bound and soluble enzyme on heat treatment suggested that the enzyme was bound at a point other than the catalytic site. The release of the enzyme by various glycolytic intermediates suggested their involvement in binding to the catalytic site through phosphate linkage. The non-phosphorylated compounds like lactate, reduced glutathione, 2-mercaptoethanol and EDTA were ineffective in eluting the enzyme. On the basis of separate binding sites on the enzyme for membrane and ligands, the mechanism of association dissociation of aldolase has been suggested.