A new, unexpected action of olomoucine, a CDK inhibitor, on normal human cells: up-regulation of CLIMP-63, a cytoskeleton-linking membrane protein

Inhibition of cyclin-dependent kinases (CDKs) is a novel strategy in the therapy of human malignancies. The pharmacological CDK inhibitors representing a few distinct classes of compounds exert different target specificity. Considering the fact that dividing and quiescent cells differ in their CDK activity and in the pattern of their expression, one might expect that anti-proliferative efficiency of the pharmacological CDK inhibitors would depend on the mitotic index of treated cells. The present article shows that olomoucine (OLO), a weak CDK2 inhibitor has new, unexpected activity. At concentrations up to 100 microM OLO did not inhibit proliferation of normal human cells, but arrested growth of human HL-60 leukemia cells. The anti-proliferative effect of OLO was clearly weaker than that of roscovitine (ROSC). Surprisingly, OLO at low doses strongly up-regulated a cellular protein with approximately 65 kDa in normal, but not in immortalized and cancer cells. By mass spectrometric analysis CLIMP-63, a cytoskeleton-linking membrane protein was identified as the major component of the up-regulated protein band. These results were subsequently confirmed by immunoblotting. Further experiments revealed that OLO, but not ROSC, strongly up-regulates CLIMP-63 in a dose- and time-dependent manner solely in senescent cells.     

Evaluation of the enteric microflora of captive whooping cranes (Grus americana) and sandhill cranes (Grus canadensis)

The enteric flora of captive whooping cranes (Grus americana) and sandhill cranes (Grus canadensis) has not been well described, despite its potential importance in the understanding of both the normal condition of the intestinal physiology of these animals and the altered colonization within disease states in these birds. Nineteen whooping cranes and 23 sandhill cranes housed currently at the Calgary Zoo or its affiliated Devonian Wildlife Conservation Centre (DWCC) in Calgary, Alberta were sampled from October 2004–February 2005 by collecting aerobic and anaerobic cloacal swabs from each bird. There were seven major groupings of bacteria isolated from both species of crane. Gram‐positive cocci, coliforms, and gram‐negative bacilli were the most prevalent types of bacteria isolated for both crane species, with Escherichia coli, Enterococcus faecalis, and Streptococcus Group D, not Enterococcus the bacterial species isolated most commonly. There was a significant difference in the average number of isolates per individual between the two crane species but no differences between age or gender categories within crane species. Campylobacter sp. were isolated from five whooping cranes. The potential zoonotic pathogen Campylobacter jejuni was isolated from one whooping crane and C. upsaliensis was isolated from a second. Three other isolates were unspeciated members of the Campylobacter genus and likely belong to a species undescribed previously. The evaluation of the enteric cloacal flora of whooping cranes and sandhill cranes illustrates that differences exist between these two closely related crane species, and highlights the potential implications these differences may have for current practices involving captive wildlife. Zoo Biol 0:1–13, 2007. © 2007 Wiley‐Liss, Inc.     

Gene flow between species of Lake Victoria haplochromine fishes

The haplochromine cichlid fishes of Lake Victoria (LV), East Africa, are a textbook example of adaptive radiation-a rapid divergence of multiple morphologically distinguishable forms from a few founding lineages. The forms are generally believed to constitute a "flock" of several hundred reproductively isolated species in a dozen or so genera. This belief has, until now, not been subjected to a test, however. Here, we compare genetic variation at 11 loci in 10 haplochromine populations of 6 different species. Although the genetic diversity in the populations is quite high, using a variety of statistical tests, we find no evidence of genetic differentiation among the populations of LV haplochromines. On genetic distance trees, populations of the same species intermingle with those of different species. At the molecular level, the species are indistinguishable from one another. Genetic comparisons with closely related species in 2 crater lakes indicate that the species within LV continue exchanging genes. These observations have important implications for phylogenetic reconstruction. The approach used in this study is applicable to other instances of adaptive radiation.     

The location and characterisation of the O-linked glycans of the human insulin receptor

O-linked glycosylation is a post-translational and post-folding event involving exposed S/T residues at beta-turns or in regions with extended conformation. O-linked sites are difficult to predict from sequence analyses compared to N-linked sites. Here we compare the results of chemical analyses of isolated glycopeptides with the prediction using the neural network prediction method NetOGlyc3.1, a procedure that has been reported to correctly predict 76% of O-glycosylated residues in proteins. Using the heavily glycosylated human insulin receptor as the test protein six sites of mucin-type O-glycosylation were found at residues T744, T749, S757, S758, T759, and T763 compared to the three sites (T759 and T763- correctly, T756- incorrectly) predicted by the neural network method. These six sites occur in a 20 residue segment that begins nine residues downstream from the start of the insulin receptor beta-chain. This region which also includes N-linked glycosylation sites at N742 and N755, is predicted to lack secondary structure and is followed by residues 765-770, the known linear epitope for the monoclonal antibody 18-44.     

Identification of serine proteases from Leishmania braziliensis

Leishmania (V) braziliensis is one of the most important ethiologic agents of the two distinct forms of American tegumentary leishmaniasis (cutaneous and mucosal). The drugs of choice used in leishmaniasis therapy are significantly toxic, expensive and are associated with frequent refractory infections. Among the promising new targets for anti-protozoan chemotherapy are the proteases. In this study, serine proteases were partially purified from aqueous, detergent and extracellular extracts of Leishmania braziliensis promastigotes by aprotinin-agarose affinity chromatography. By zymography, the enzymes purified from the aqueous extract showed apparent activity bands of 60 kDa and 45 kDa; of 130 kDa, 83 kDa, 74 kDa and 30 kDa from the detergent extract; and of 62 kDa, 59 kDa, 57 kDa, 49 kDa and 35 kDa from the extracellular extract. All purified proteases exhibited esterase activity against Nalpha-benzoyl-L-arginine ethyl ester hydrochloride and Nalpha-p-tosyl-L-arginine methyl ester hydrochloride (serine protease substrates) and optimal activity at pH 8. 0. Proteases purified from the aqueous and extracellular extracts were effectively inhibited by benzamidine (trypsin inhibitor) and those from the detergent extract were inhibited by N-tosyl-L-phenyl-alanine chloromethyl ketone (chymotrypsin inhibitor) indicating that all these enzymes are serine proteases. These findings indicate that L. braziliensis serine proteases display some biochemical similarities with L. amazonensis serine proteases, demonstrating a conservation of this enzymatic class in the Leishmania genus. This is the first study to report the purification of a serine protease from Leishmania braziliensis.     

Long-Term Consequences of Grazing and Burning on Northern Peatland Carbon Dynamics

Abstract Using a 50-year-old field experiment, we investigated the effects of the long-term land management practices of repeated burning and grazing on peatland vegetation and carbon dynamics (C). Plant community composition, C stocks in soils and vegetation, and C fluxes of CO₂, CH₄ and DOC, were measured over an 18-month period. We found that both burning and grazing reduced aboveground C stocks, and that burning reduced C stocks in the surface peat. Both burning and grazing strongly affected vegetation community composition, causing an increase in graminoids and a decrease in ericoid subshrubs and bryophytes relative to unburned and ungrazed controls; this effect was especially pronounced in burned treatments. Soil microbial properties were unaffected by grazing and showed minor responses to burning, in that the C:N ratio of the microbial biomass increased in burned relative to unburned treatments. Increases in the gross ecosystem CO₂ fluxes of respiration and photosynthesis were observed in burned and grazed treatments relative to controls. Here, the greatest effects were seen in the burning treatment, where the mean increase in gross fluxes over the experimental period was greater than 40%. Increases in gross CO₂ fluxes were greatest during the summer months, suggesting an interactive effect of land use and climate on ecosystem C cycling. Collectively, our results indicate that long-term management of peatland has marked effects on ecosystem C dynamics and CO₂ flux, which are primarily related to changes in vegetation community structure.     

VSGdb: a database for trypanosome variant surface glycoproteins,a large and diverse family of coiled coil proteins

Background  

Trypanosomes are coated with a variant surface glycoprotein (VSG) that is so densely packed that it physically protects underlying proteins from effectors of the host immune system. Periodically cells expressing a distinct VSG arise in a population and thereby evade immunity. The main structural feature of VSGs are two long α-helices that form a coiled coil, and sets of relatively unstructured loops that are distal to the plasma membrane and contain most or all of the protective epitopes. The primary structure of different VSGs is highly variable, typically displaying only ~20% identity with each other. The genome has nearly 2000 VSG genes, which are located in subtelomeres. Only one VSG gene is expressed at a time, and switching between VSGs primarily involves gene conversion events. The archive of silent VSGs undergoes diversifying evolution rapidly, also involving gene conversion. The VSG family is a paradigm for α helical coiled coil structures, epitope variation and GPI-anchor signals. At the DNA level, the genes are a paradigm for diversifying evolutionary processes and for the role of subtelomeres and recombination mechanisms in generation of diversity in multigene families. To enable ready availability of VSG sequences for addressing these general questions, and trypanosome-specific questions, we have created VSGdb, a database of all known sequences.