RAB-6.1 and RAB-6.2 Promote Retrograde Transport in C. elegans

Retrograde transport is a critical mechanism for recycling certain membrane cargo. Following endocytosis from the plasma membrane, retrograde cargo is moved from early endosomes to Golgi followed by transport (recycling) back to the plasma membrane. The complete molecular and cellular mechanisms of retrograde transport remain unclear. The small GTPase RAB-6.2 mediates the retrograde recycling of the AMPA-type glutamate receptor (AMPAR) subunit GLR-1 in C. elegans neurons. Here we show that RAB-6.2 and a close paralog, RAB-6.1, together regulate retrograde transport in both neurons and non-neuronal tissue. Mutants for rab-6.1 or rab-6.2 fail to recycle GLR-1 receptors, resulting in GLR-1 turnover and behavioral defects indicative of diminished GLR-1 function. Loss of both rab-6.1 and rab-6.2 results in an additive effect on GLR-1 retrograde recycling, indicating that these two C. elegans Rab6 isoforms have overlapping functions. MIG-14 (Wntless) protein, which undergoes retrograde recycling, undergoes a similar degradation in intestinal epithelia in both rab-6.1 and rab-6.2 mutants, suggesting a broader role for these proteins in retrograde transport. Surprisingly, MIG-14 is localized to separate, spatially segregated endosomal compartments in rab-6.1 mutants compared to rab-6.2 mutants. Our results indicate that RAB-6.1 and RAB-6.2 have partially redundant functions in overall retrograde transport, but also have their own unique cellular- and subcellular functions.     

Development of sporeless/low sporing strains of Pleurotus through mutation

Summary The sporophores of Pleurotus are gymnocarpous and continuously release spores in the atmosphere causing respiratory allergies like hay fever and farmer’s lung disease among workers. The allergy is caused by the antigens present on the walls of the spores. Apart from this, during commercial production, these spores settle on the fruit bodies, germinate and form a velvety film which gives an unpleasant appearance to the mushrooms. The spores emitted may include new genotypes likely to attack wood or trees. Spore allergy is one of the most important limiting factors for the large scale cultivation of this species. Different approaches are being adopted at IIHR for the production of commercial sporeless/low-sporing strains of Pleurotus to alleviate the spore allergy problem. Attempts were made during the present investigation to produce sporeless or low-sporing mutants through u.v. mutation. Mutation of the mycelium did not yield the desired results. Mutation of the spores of Pleurotus sajor-caju yielded an extremely low-sporing mutant after 75 min exposure. The character has been found to be stable for more than 10 generations of subculturing.     

Effects of prostaglandins E-2 and F-2 alpha on the metabolism of [U-14C]glucose by mouse morulae-early blastocysts in vitro

During 5-h culture in the presence of radioactive glucose, PGE-2 (10 micrograms/ml) significantly inhibited incorporation of glucose into the acid-soluble glycogen pool. PGE-2 at 1 and 10 micrograms/ml and PGF-2 alpha at 1 microgram but not 10 micrograms/ml stimulated incorporation of glucose into non-glycogen macromolecules during culture. However, the utilization of acid-soluble glycogen and other biochemical pools was not affected by the presence of PGs in the medium during 24-h chase culture of pulse-labelled embryos. Carbon dioxide production was significantly suppressed in the presence of PGs but accumulation of lactate was not affected. The results indicate that PGE-2 and PGF-2 alpha, in physiological concentrations, directly influence the metabolism of glucose by preimplantation embryos.     

TGF-Beta inhibits the in vitro induction of lymphokine-activated killing activity

Summary Employing serum-free media, human peripheral blood mononuclear cells, and purified recombinant interleukin-2 (IL-2), conditions were observed in which the development of IL-2-driven cytotoxic activity was suppressed. The cytotoxic activity of such IL-2-generated lymphokine activated killing (LAK) was tested against natural killer-resistant cultured tumor cells (Daudi, Raji, and a glioma). LAK generation was inhibited by addition of some normal sera, normal platelets, or some tumor cells. Because recent reports have indicated that transforming growth factor-beta (TGF-beta)-like factors are often secreted by tumors and the acidic alpha granules of platelets and can be present in sera, we tested the effect of purified human TGF-beta on the activation of LAK. Our results indicated that TGF-beta is very suppressive for LAK induction, and can completely prevent both the IL-2-driven proliferation and cytotoxicity at concentrations as low as 5 ng/ml. Titrations of IL-2 and of TGF-beta indicated that the suppression is dose-dependent and can be avoided by employing higher levels of IL-2. It was also found that the suppressive effect of TGF-beta can be overcome by washing suppressed cell populations and further culture in low levels of IL-2. Collectively, these data indicate that TGF-beta can be a potent inhibitor of LAK generation under standard activation conditions, but that this effect is regulated by the relative level of IL-2 and may be overcome and/or reversed in vitro.     

Purification and regulation of glutamine synthetase in a collagenolytic Vibrio alginolyticus strain

Glutamine synthetase (EC 6.3.1.2) has been purified from a collagenolytic Vibrio alginolyticus strain. The apparent molecular weight of the glutamine synthetase subunit was approximately 62,000. This indicates a particle weight for the undissociated enzyme of 744,000, assuming the enzyme is the typical dodecamer. The glutamine synthetase enzyme had a sedimentation coefficient of 25.9 S and seems to be regulated by a denylylation and deadenylylation. The pH profiles assayed by the -glutamyltransferase method were similar for NH₄-shocked and unshocked cell extracts and isoactivity point was not obtained from these eurves. The optimum pH for purified and crude cell extracts was 7.9. Cell-free glutamine synthetase was inhibited by some amino acids and AMP. The transferase activity of glutamine synthetase from mid-exponential phase cells varied greatly depending on the sources of nitrogen or carbon in the growth medium. Glutamine synthetase level was regulated by nitrogen catabolite repression by (NH₄)₂SO₄ and glutamine, but cells grown, in the presence of proline, leucine, isoleucine, tryptophan, histidine, glutamic acid, glycine and arginine had enhanced levels of transferase activity. Glutamine synthetase was not subject to glucose, sucrose, fructose, glycerol or maltose catabolite repression and these sugars had the opposite effect and markedly enhanced glutamine synthetase activity.Abbreviations GS glutamine synthetase - SMM succinate minimal medium - ASMM ammonium/succinate minimal medium - GT -glutamyl transferase - SVP snake venom phosphodiesterase     

Floral Induction in Wolffia microscopica by Salicylic Acid and Related Compounds under Non-inductive Long Days

Flowering in Wolffia microscopica, a short-day plant, couldbe induced with salicylic acid (SA), under long days. Aspirin,benzoic acid and salicylaldoxime were also effective for inductionof flowering in this duckweed. Amonsgt these, SA is the mosteffective compound, as it could induce flowering even at 10^–7M. Flowering was further enhanced when Wolffia fronds were subjectedto short days, in the presence of SA. However, SA neither showedany effect on flowering ofW. microscopica in the absence ofEDTA in the nutrient medium, nor could it, by itself, supporteven the vegetative growth. The probable mechanism of actionof SA has also been discussed. It appears that the effect cannotbe due simply to chelation of metal ions and perhaps the salicylmoiety itself exerts a specific effect. (Received March 15, 1983; Accepted May 6, 1983)     

Molecular cloning and expression of a xylanase gene from Cellulomonas sp. into Escherichia coli

Summary The xylanase gene of Cellulomonas sp. NCIM 2353 was cloned in pUC 18 and selected by growth on xylan as the sole carbon source. The functional clone harboured the recombinant plasmid with an insert of 1.42 kbp, as determined by restriction mapping and Southern hydridization. The clone secreted a xylanase of 45 000 mol. wt. as determined by Western blot analysis using specific antixylanase antibodies. The DNA insert carried the full structural gene along with its promoter and possibly regulatory sequences, since xylanase activity in the clone Cs11 was inducible by xylan. Offprint requests to: D. N. Deobagkar     

High Frequency Divisions in Leaf Base Protoplasts of Wheat (Triticum aestivum L.)

Protoplasts were isolated from the basal meristematic region of leaves from 6-day-old seedlings of wheat (Triticum aestivum). Protoplasts divided when cultured on MS medium (as agarose beads) in presence of nurse tissue. Donor seedlings when grown on BAP-supplemented MS medium were found to be considerably superior for protoplast isolation and culture than when grown on MS basal medium, in terms of protoplast viability, cell wall formation and cell division frequency. In addition, reduction of ammonium content of the culture medium, together with a dark Incubation, led to a high protoplast division frequency of about 70%. Microcolonies of 10-to 12-celled stages were obtained in Triticum aestivum, varieties HD 2329, HD 2285, Kalyan Sona, Arjun and CPAN 1676.     

Role of DL α-lipoic acid in gentamicin induced nephrotoxicity

The effect of DL -lipoic acid on the nephrotoxic potential of gentamicin was examined. Intraperitoneal injection of gentamicin (100 mg/kg/day) to rats resulted in decreased activity of the glycolytic enzymes-hexokinase, phosphoglucoisomerase, aldolase and lactate dehydrogenase. The two gluconeogenic enzymes—glucose-6-phosphatase and fructose-1, 6-diphosphatase, the transmembrane enzymes namely the Na⁺, K⁺-ATPase, Ca²⁺-ATPase, Mg²⁺-ATPase and the brushborder enzyme alkaline phosphatase, also showed decreased activities. This decrease in the activities of ATPases and alkaline phosphatase suggests basolateral and brush border membrane damage. Decreased activity of the TCA cycle enzymes isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH) and malate dehydrogenase (MDH), suggests a loss in mitochondrial integrity. These biochemical disturbances were effectively counteracted by lipoic acid administration. Lipoic acid administration by gastric intubation at two different concentrations (10 mg and 25 mg/kg/day) brought about an increase in the activity of the glycolytic enzymes, ATPases and the TCA cycle enzymes. The gluconeogenic enzymes however showed a further decrease in their activities at both the concentrations of lipoic acid administered. These observations shed light on the nephroprotective action of lipoic acid against experimental aminoglycoside toxicity and the protection afforded at 25 mg/kg/day of lipoic acid was noted to be higher than that at 10 mg level.