Synthesis, characterization, and antitumor activity of 1,2-bis(diphenylphosphino) ethane platinum(II) and palladium(II) complexes

A number of 1,2-bis(diphenylphosphino)ethane monomeric platinum(II) and palladium(II) complexes have been synthesized in light of their potential antitumor activity. The metal center is coordinated with a number of carboxylate anions in the cis-configuration. These complexes have been characterized by elemental analysis, conductivity measurement, and various spectroscopic techniques [IR and 195Pt NMR]. In vivo screening tests for activity of these complexes were performed against the L1210/0 murine leukemia cancer model, but none displayed a significant level of antitumor activity.     

Respiratory Syncytial Virus (RSV) RNA loads in peripheral blood correlates with disease severity in mice

Background

Respiratory Syncytial Virus (RSV) infection is usually restricted to the respiratory epithelium. Few studies have documented the presence of RSV in the systemic circulation, however there is no consistent information whether virus detection in the blood correlates with disease severity.

Methods

Balb/c mice were inoculated with live RSV, heat-inactivated RSV or medium. A subset of RSV-infected mice was treated with anti-RSV antibody 72 h post-inoculation. RSV RNA loads were measured by PCR in peripheral blood from day 1-21 post-inoculation and were correlated with upper and lower respiratory tract viral loads, the systemic cytokine response, lung inflammation and pulmonary function. Immunohistochemical staining was used to define the localization of RSV antigens in the respiratory tract and peripheral blood.

Results

RSV RNA loads were detected in peripheral blood from day 1 to 14 post-inoculation, peaked on day 5 and significantly correlated with nasal and lung RSV loads, airway obstruction, and blood CCL2 and CXCL1 expression. Treatment with anti-RSV antibody reduced blood RSV RNA loads and improved airway obstruction. Immunostaining identified RSV antigens in alveolar macrophages and peripheral blood monocytes.

Conclusions

RSV RNA was detected in peripheral blood upon infection with live RSV, followed a time-course parallel to viral loads assessed in the respiratory tract and was significantly correlated with RSV-induced airway disease.     

Heat Stress-Induced Life Span Extension in Yeast

The yeastSaccharomyces cerevisiaehas a limited life span that can be measured by the number of times individual cells divide. Several genetic manipulations have been shown to prolong the yeast life span. However, environmental effects that extend longevity have been largely ignored. We have found that mild, nonlethal heat stress extended yeast life span when it was administered transiently early in life. The increased longevity was due to a reduction in the mortality rate that persisted over many cell divisions (generations) but was not permanent. The genesRAS1andRAS2were necessary to observe this effect of heat stress. TheRAS2gene is consistently required for maintenance of life span when heat stress is chronic or in its extension when heat stress is transient or absent altogether.RAS1,on the other hand, appears to have a role in signaling life extension induced by transient, mild heat stress, which is distinct from its life-span-curtailing effect in the absence of stress and its lack of involvement in the response to chronic heat stress. This distinction between theRASgenes may be partially related to their different effects on growth-promoting genes and stress-responsive genes. Theras2mutation clearly hindered resumption of growth and recovery from stress, while theras1mutation did not. TheHSP104gene, which is largely responsible for induced thermotolerance in yeast, was necessary for life extension induced by transient heat stress. An interaction between mitochondrial petite mutations and heat stress was found, suggesting that mitochondria may be necessary for life extension by transient heat stress. The results raise the possibility that theRASgenes and mitochondria may play a role in the epigenetic inheritance of reduced mortality rate afforded by transient, mild heat stress.     

A clean, more efficient method for in-solution digestion of protein mixtures without detergent or urea

Proteolytic digestion of a complicated protein mixture from an organelle or whole-cell lysate is usually carried out in a dilute solution of a denaturing buffer, such as 1-2 M urea. Urea must be subsequently removed by C18 beads before downstream analysis such as HPLC/MS/MS or complete methylation followed by IMAC isolation of phosphopeptides. Here we describe a procedure for digesting a complicated protein mixture in the absence of denaturants. Proteins in the mixture are precipitated with trichloroacetic acid/acetone for denaturation and salt removal and resuspended in NH4HCO3 buffer. After trypsinolysis, the resulting peptides are not contaminated by urea or other nonvolatile salts and can be dried in a SpeedVac to remove NH4HCO3. When this protocol was applied to an extract of A431 cells, 96.8% of the tryptic peptides were completely digested (i.e., had no missed cleavage sites), in contrast to 87.3% of those produced by digestion in urea buffer. We successfully applied this digestion method to analysis of the phosphoproteome of adiposomes from HeLa cells, identifying 33 phosphorylation sites in 28 different proteins. Our digestion method avoids the need to remove urea before HPLC/MS/MS analysis or methylation and IMAC, increasing throughput while reducing sample loss and contamination from sample handling. We believe that this method should be valuable for proteomics studies.     

Squaring up to DNA: pentapeptide repeat proteins and DNA mimicry

Pentapeptide repeats are a class of proteins characterized by the presence of multiple repeating sequences five amino acids in length. The sequences fold into a right-handed β-helix with a roughly square-shaped cross section. Pentapeptide repeat proteins include a number of examples which are thought to function as structural mimics of DNA and act to competitively bind to the type II topoisomerase DNA gyrase, an important antibacterial target. DNA gyrase-targeting pentapeptide repeat proteins can both inhibit DNA gyrase—a potentially useful therapeutic property—and contribute to resistance to quinolone antibacterials (by acting to prevent them forming a lethal complex with the DNA and enzyme). Pentapeptide repeat proteins are therefore of wide interest not only because of their unusual structure, function, and potential as an antibacterial target, but also because knowledge of their mechanism of action may lead to both a greater understanding of the details of DNA gyrase function as well as being a useful template for the design of new DNA gyrase inhibitors. However, many puzzling aspects as to how these DNA mimics function and indeed even their ability to act as DNA mimics itself remains open to question. This review summarizes the current state of knowledge regarding pentapeptide repeat proteins, focusing on those that are thought to mimic DNA, and speculates on potential structure-function relationships which may account for their differing specificities.     

Ricin A Chain from <Emphasis Type="Italic">Ricinus sanguineus</Emphasis>: DNA Sequence,Structure and Toxicity

The gene coding for the ricin A-chain from Ricinus sanguineus (R_sTA) was isolated and sequenced (GB: DQ661048). Comparison of R_sTA with the ricin A-chain from Ricinus communis (RTA) revealed the presence of five differences in the gene sequence. At the protein level only two differences were noticed, the replacement of Asn136 by Ser (N136S) and Ile173 by Val (I174 V). From the R_sTA structure model (PMDB: PM0074652), the N136S mutation was predicted to have no effect on R_sTA structure. The I174V mutation is believed to have no effect on the R_sTA structure or on toxicity since this replacement was found in the Ricinus agglutinin’s A-chain and the latter has a comparable toxicity to RTA. The Ser221 of the putative lipase active site was found. Toxicity experiments showed that R_sTA and RTA have similar toxicities. This finding proves that the N136S and I174 V mutations have no effect on R_sTA toxicity.     

Efficient production of L-ribose with a recombinant Escherichia coli biocatalyst

A new synthetic platform with potential for the production of several rare sugars, with l-ribose as the model target, is described. The gene encoding the unique NAD-dependent mannitol-1-dehydrogenase (MDH) from Apium graveolens (garden celery) was synthetically constructed for optimal expression in Escherichia coli. This MDH enzyme catalyzes the interconversion of several polyols and their l-sugar counterparts, including the conversion of ribitol to l-ribose. Expression of recombinant MDH in the active form was successfully achieved, and one-step purification was demonstrated. Using the created recombinant E. coli strain as a whole-cell catalyst, the synthetic utility was demonstrated for production of l-ribose, and the system was improved using shaken flask experiments. It was determined that addition of 50 to 500 microM ZnCl(2) and addition of 5 g/liter glycerol both improved production. The final levels of conversion achieved were >70% at a concentration of 40 g/liter and >50% at a concentration of 100 g/liter. The best conditions determined were then scaled up to a 1-liter fermentation that resulted in 55% conversion of 100 g/liter ribitol in 72 h, for a volumetric productivity of 17.4 g liter(-1) day(-1). This system represents a significantly improved method for the large-scale production of l-ribose.     

Isolation and characterization of immunoglobulin of the Indian major carp, rohu [Labeo rohita (Ham.)

Information on the structure and character of immunoglobulin of fishes is essential in health management. A study was carried out to characterize the serum immunoglobulin (IgM) of the Indian major carp, rohu Labeo rohita (Ham.). Rohu (500g) were immunised with bovine serum albumin (BSA) and the anti-BSA antibody was purified employing BSA-CL agarose affinity column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified Ig in a 3% gel under non-reduced conditions revealed a single protein having a molecular weight of 850kDa. Analysis of the purified serum in 10% SDS-PAGE under reduced conditions revealed that the immunoglobulin contained heavy and light chains with molecular weights of 85 and 23kDa, respectively. A polyclonal mouse anti-rohu IgM was prepared and used in an immunodot test which showed a specific reaction of the crude rohu anti-BSA antiserum and the purified anti-BSA IgM with BSA. Results indicate that the immunoglobulin of L. rohita is tetrameric IgM, similar to that of other fishes.     

SERCA2 regulates non-CF and CF airway epithelial cell response to ozone

Calcium mobilization can regulate a wide range of essential functions of respiratory epithelium, including ion transport, ciliary beat frequency, and secretion of mucus, all of which are modified in cystic fibrosis (CF). SERCA2, an important controller of calcium signaling, is deficient in CF epithelium. We conducted this study to determine whether SERCA2 deficiency can modulate airway epithelial responses to environmental oxidants such as ozone. This could contribute to the pathogenesis of pulmonary exacerbations, which are important and frequent clinical events in CF. To address this, we used air-liquid interface (ALI) cultures of non-CF and CF cell lines, as well as differentiated cultures of cells derived from non-CF and CF patients. We found that ozone exposure caused enhanced membrane damage, mitochondrial dysfunction and apoptotic cell death in CF airway epithelial cell lines relative to non-CF. Ozone exposure caused increased proinflammatory cytokine production in CF airway epithelial cell lines. Elevated proinflammatory cytokine production also was observed in shRNA-mediated SERCA2 knockdown cells. Overexpression of SERCA2 reversed ozone-induced proinflammatory cytokine production. Ozone-induced proinflammatory cytokine production was NF-κB- dependent. In a stable NF-κB reporter cell line, SERCA2 inhibition and knockdown both upregulated cytomix-induced NF-κB activity, indicating importance of SERCA2 in modulating NF-κB activity. In this system, increased NF-κB activity was also accompanied by increased IL-8 production. Ozone also induced NF-κB activity and IL-8 release, an effect that was greater in SERCA2-silenced NF-κB-reporter cells. SERCA2 overexpression reversed cytomix-induced increased IL-8 release and total nuclear p65 in CFTR-deficient (16HBE-AS) cells. These studies suggest that SERCA2 is an important regulator of the proinflammatory response of airway epithelial cells and could be a potential therapeutic target.