Ultrastructural localization of an extracellular protease in Pseudomonas fragi by using the peroxidase-antiperoxidase reaction.

An extracellular protease, which previously has been found to correlate with the appearance of bleblike evaginations on the cell wall of Pseudomonas fragi ATCC 4973, was purified 38-fold by ammonium sulfate precipitation and Sephadex chromatography to yield a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyclonal rabbit antiserum raised against the purified enzyme had an enzyme-linked immunosorbent assay titer of 4 X 10(7). The peroxidase antiperoxidase method was used to localize the neutral protease in P. fragi at the ultrastructural level. Electron microscopy of cell sections of this organism revealed that high concentrations of positive immunoperoxidase reaction product were located near the cell wall, whereas control sections stained with preimmune or heterologous serum did not show similar deposits to be present. These results are consistent with the hypothesis that blebs appearing on P. fragi contain high concentrations of neutral protease.     

PIASy-mediated Tip60 sumoylation regulates p53-induced autophagy

Posttranslational modifications of p53 integrate diverse stress signals and regulate its activity, but their combinatorial contribution to overall p53 function is not clear. We investigated the roles of lysine (K) acetylation and sumoylation on p53 and their relation to apoptosis and autophagy. Here we describe the collaborative role of the SUMO E3 ligase PIASy and the lysine acetyltransferase Tip60 in p53-mediated autophagy. PIASy binding to p53 and PIASy-activated Tip60 lead to K386 sumoylation and K120 acetylation of p53, respectively. Even though these two modifications are not dependent on each other, together they act as a “binary death signal” to promote cytoplasmic accumulation of p53 and execution of PUMA-independent autophagy. PIASy-induced Tip60 sumoylation augments p53 K120 acetylation and apoptosis. In addition to p14^ARF inactivation, impairment in this intricate signaling may explain why p53 mutations are not found in nearly 50% of malignancies.     

“The Good into the Pot,the Bad into the Crop!”—A New Technology to Free Stem Cells from Feeder Cells

A variety of embryonic and adult stem cell lines require an intial co-culturing with feeder cells for non-differentiated growth, self renewal and maintenance of pluripotency. However for many downstream ES cell applications the feeder cells have to be considered contaminations that might interfere not just with the analysis of experimental data but also with clinical application and tissue engineering approaches. Here we introduce a novel technique that allows for the selection of pure feeder-freed stem cells, following stem cell proliferation on feeder cell layers. Complete and reproducible separation of feeder and embryonic stem cells was accomplished by adaptation of an automated cell selection system that resulted in the aspiration of distinct cell colonies or fraction of colonies according to predefined physical parameters. Analyzing neuronal differentiation we demonstrated feeder-freed stem cells to exhibit differentiation potentials comparable to embryonic stem cells differentiated under standard conditions. However, embryoid body growth as well as differentiation of stem cells into cardiomyocytes was significantly enhanced in feeder-freed cells, indicating a feeder cell dependent modulation of lineage differentiation during early embryoid body development. These findings underline the necessity to separate stem and feeder cells before the initiation of in vitro differentiation. The complete separation of stem and feeder cells by this new technology results in pure stem cell populations for translational approaches. Furthermore, a more detailed analysis of the effect of feeder cells on stem cell differentiation is now possible, that might facilitate the identification and development of new optimized human or genetically modified feeder cell lines.     

Spice phenolics inhibit human PMNL 5-lipoxygenase

A wide variety of phenolic compounds and flavonoids present in spices possess potent antioxidant, antimutagenic and anticarcinogenic activities. We examined whether 5-lipoxygenase (5-LO), the key enzyme involved in biosynthesis of leukotrienes is a possible target for the spices. Effect of aqueous extracts of turmeric, cloves, pepper, chili, cinnamon, onion and also their respective active principles viz., curcumin, eugenol, piperine, capsaicin, cinnamaldehyde, quercetin, and allyl sulfide were tested on human PMNL 5-LO activity by spectrophotomeric and HPLC methods. The formation of 5-LO product 5-HETE was significantly inhibited in a concentration-dependent manner with IC(50) values of 0.122-1.44 mg for aqueous extracts of spices and 25-83 microM for active principles, respectively. The order of inhibitory activity was of quercetin>eugenol>curcumin>cinnamaldehyde>piperine>capsaicin>allyl sulfide. Quercetin, eugenol and curcumin with one or more phenolic ring and methoxy groups in their structure showed high inhibitory effect, while the non-phenolic spice principle allyl sulfide showed least inhibitory effect on 5-LO. The inhibitory effect of quercetin, curcumin and eugenol was similar to that of synthetic 5-LO inhibitors-phenidone and NDGA. Moreover, the inhibitory potency of aqueous extracts of spice correlated with the active principles of their respective spices. The synergistic or antagonistic effect of mixtures of spice active principles and spice extracts were investigated and all the combinations of spice active principles/extracts exerted synergistic effect in inhibiting 5-LO activity. These findings clearly suggest that phenolic compounds present in spices might have physiological role in modulating 5-LO pathway.     

Water balance and osmoregulation in Stenocara gracilipes,a wax-blooming tenebrionid beetle from the Namib Desert

Dehydration (10 days at 27 degrees C) of the Namib tenebrionid Stenocara gracilipes resulted in a rapid weight loss (17.5%), and a substantial decline in haemolymph volume (72%). Although the lipid content decreased significantly, metabolic water production was insufficient to maintain total body water (TBW). Rehydration (no food) resulted in increases in haemolymph volume, body weight (sub-normal), and TBW to normality. Haemolymph osmolality, sodium, potassium, chloride, amino acids, and sugars (trehalose and glucose), were all subject to osmoregulatory control during both dehydration and rehydration. Major osmolar effectors in this species are sodium, chloride, and amino acids, with most of the contribution to regulation of haemolymph osmolality coming from changes in the levels of these constituents. Changes in amino acid levels are not the result of interchange with soluble protein during dehydration (the possibility exists during extended rehydration, however). Despite faecal losses of sodium being low (8.2% of that removed from the haemolymph during dehydration), sodium concentrations do not return to normal during rehydration. Chloride concentrations increase supra-normally when access to water is allowed, and remain elevated throughout the rehydration period. Although faecal loss of potassium greatly exceeded the amount removed from the haemolymph (by approximately 1.8 times), haemolymph potassium levels were strongly regulated during rehydration. S. gracilipes demonstrates an exquisite capacity to regulate haemolymph osmolality under conditions of both acute water-shortage and -abundance. Together with an efficient water economy (drinking when fog-water is available, and a superb water conservation mechanism in the form of wax-bloom production), this must serve to contribute to long-term survival of this species in an otherwise harsh abode.     

In vitro culture of coffee zygotic embryos for germplasm preservation

Immature zygotic embryos of Coffea arabica L. Cv. Cauvery (Catimor) were cultured on Murashige and Skoog (1962) (MS) medium supplemented with abscisic acid (ABA) at 0, 0.4, 3.8, 18.9, 37.8 and 75.6 μM., L-cystein hydrochloride at 50 mg 1^-1 and sucrose at 3%. Cultures were preserved in parafilm sealed Petri dishes in dark at a temperature of 25 ± 1 °C for up to two years. The preserved embryos were taken out from the media at 6 month intervals in order to test their viability by germination on MS + NAA (0.5 μM) + BA (4.4 μM). On the preservation media devoid of ABA or with a low concentration (0.4 μM) of ABA, the embryos germinated and showed higher mortality with increasing duration of storage. In contrast, the embryos became increasingly dormant with increasing concentrations of ABA and a 74.2% survival was found even after 2 years on medium supplemented with 18.9 μM or 37.8 μM of ABA. The results suggest that embryos can be preserved with a little loss of viability in the presence of ABA even at the normal room temperature (25 + 1 °C) up to two years without any transfer. Application of this technique for germplasm preservation of coffee is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Acute and chronic effects of insulin on lactate dehydrogenase (LDH) activity in tissues of albino rat (Wistar strain)

Male albino rats were treated with insulin for one week (acute) and four weeks (chronic). The lactate dehydrogenase (LDH) activity, lactate and pyruvate levels were estimated in the tissues of experimental and control animals. LDH activity decreased in all the tissues of acute- and chronic treated animals whereas the lactate content is elevated. Pyruvate content also showed increment except in heart and pancreas with reference to acute treatment where it is decreased. The hyperinsulinaemia effect in relation to lacticacidaemia and its influence on energy demand and ammonia secretion is discussed.     

Seasonal pattern of photosynthetic rate and its relationship with chlorophyll content,ribulose-1,5-bisphosphate carboxylase activity and biomass production

Net photosynthetic rate (P_N), ribulose-1,5-bisphosphate carboxylase (RuBPC) activity, chlorophyll (Chl) content and biomass production were estimated at monthly intervals inChukrasia tabularis, Dolichandrone atrovirens, Eugenia jambolana, Gmelina arborea, Lannea coromandelica, Terminalia arjuna andTerminalia bellerica from September 1990 to August 1991. The leaves of all the seven tree species showed significantly higher P_N during summer than in winter and these rates differed from one species to the other. A positive correlation was found between P_N of different tree species and their Chl content or biomass production. There was no significant correlation between ribulose-1,5-bisphosphate carboxylase activity and P_N when these were expressed on leaf area basis.     

Gene flow from the Indian subcontinent to Australia: evidence from the Y chromosome

Phenotypic similarities between Australian Aboriginal People and some tribes of India were noted by T.H. Huxley during the voyage of the Rattlesnake (1846-1850). Anthropometric studies by Birdsell led to his suggestion that a migratory wave into Australia included populations with affinities to tribal Indians. Genetic evidence for an Indian contribution to the Australian gene pool is contradictory; most studies of autosomal markers have not supported this hypothesis (; and references therein). On the other hand, affinities between Australian Aboriginal People and southern Indians were suggested based on maternally inherited mitochondrial DNA. Here, we show additional DNA evidence in support of Huxley's hypothesis of an Indian-Australian connection using single-nucleotide polymorphisms (SNPs) and short tandem repeats (STRs) on the nonrecombining portion of the Y chromosome (NRY). Phylogenetic analyses of STR variation associated with a major Australian SNP lineage indicated tight clustering with southern Indian/Sri Lankan Y chromosomes. Estimates of the divergence time for these Indian and Australian chromosomes overlap with important changes in the archaeological and linguistic records in Australia. These results provide strong evidence for an influx of Y chromosomes from the Indian subcontinent to Australia that may have occurred during the Holocene.