Hydrolysis of homocysteine thiolactone results in the formation of Protein-Cys-S-S-homocysteinylation

An increased level of homocysteine, a reactive thiol amino acid, is associated with several complex disorders and is an independent risk factor for cardiovascular disease. A majority (>80%) of circulating homocysteine is protein bound. Homocysteine exclusively binds to protein cysteine residues via thiol disulfide exchange reaction, the mechanism of which has been reported. In contrast, homocysteine thiolactone, the cyclic thioester of homocysteine, is believed to exclusively bind to the primary amine group of lysine residue leading to N-homocysteinylation of proteins and hence studies on binding of homocysteine thiolactone to proteins thus far have only focused on N-homocysteinylation. Although it is known that homocysteine thiolactone can hydrolyze to homocysteine at physiological pH, surprisingly the extent of S-homocysteinylation during the exposure of homocysteine thiolactone with proteins has never been looked into. In this study, we clearly show that the hydrolysis of homocysteine thiolactone is pH dependent, and at physiological pH, 1 mM homocysteine thiolactone is hydrolysed to ~0.71 mM homocysteine within 24 h. Using albumin, we also show that incubation of HTL with albumin leads to a greater proportion of S-homocysteinylation (0.41 mol/mol of albumin) than N-homocysteinylation (0.14 mol/mol of albumin). S-homocysteinylation at Cys³⁴ of HSA on treatment with homocysteine thiolactone was confirmed using LC-MS. Further, contrary to earlier reports, our results indicate that there is no cross talk between the cysteine attached to Cys³⁴ of albumin and homocysteine attached to lysine residues.     

The sequence of the major protein stored in ovine ceroid lipofuscinosis is identical with that of the dicyclohexylcarbodiimide-reactive proteolipid of mitochondrial ATP synthase.

The ceroid lipofuscinoses are a group of neurodegenerative lysosomal storage diseases of children and animals that are recessively inherited. In diseased individuals fluorescent storage bodies accumulate in a wide variety of cells, including neurons. Previous studies of these bodies isolated from tissues of affected sheep confirmed that the storage occurs in lysosomes, and showed that the storage body is mostly made of a single protein with an apparent molecular mass of 3500 Da with an N-terminal amino acid sequence that is the same as residues 1-40 of the c-subunit (or dicyclohexylcarbodi-imide-reactive proteolipid) of mitochondrial ATP synthase. In the present work we have shown by direct analysis that the stored protein is identical in sequence with the entire c-subunit of mitochondrial ATP synthase, a very hydrophobic protein of 75 amino acid residues. As far as can be detected by the Edman degradation, the stored protein appears not to have been subject to any post-translational modification other than the correct removal of the mitochondrial import sequences that have been shown in other experiments to be present at the N-terminal of its two different precursors. No other protein accumulates in the storage bodies to any significant extent. Taken with studies of the cDNAs for the c-subunit in normal and diseased sheep, these results indicate that the material that is stored in lysosomes of diseased animals has probably entered mitochondria and has been subjected to the proteolytic processing that is associated with mitochondrial import. This implies that the defect that leads to the lysosomal accumulation concerns the degradative pathway of the c-subunit of ATP synthase. An alternative, but less likely, hypothesis is that for some unknown reason the precursors of subunit c are being directly mis-targeted to lysosomes, where they become processed to yield a protein identical with the protein that is normally found in the mitochondrial ATP synthase assembly, and which then accumulates.     

Influence of potassium solubilizing microorganism (Bacillus mucilaginosus) and waste mica on potassium uptake dynamics by sudan grass (Sorghum vulgare Pers.) grown under two Alfisols

The main aim of this research was to study the dynamics of K release from waste mica inoculated with potassium solubilizing microorganism (Bacillus mucilaginosus) and to investigate its effectiveness as potassic-fertilizer using sudan grass (Sorghum vulgare Pers.) var Sudanensis as test crop grown under two Alfisols. Results revealed that application of mica significantly enhanced biomass yield, uptake and per cent K recoveries by sudan grass than control (no-K). Biomass yield, uptake and per cent K recoveries increased further when mica was inoculated with bacterial strain in both the soils than uninoculated mica. Alfisol from Hazaribag recorded higher yield, uptake and K recoveries than Alfisol from Bhubaneswar. The dynamics of K in soils indicated that K was released from mica to water-soluble and exchangeable pools of K due to inoculation of mica with Bacillus mucilaginosus in both the soils. Significantly greater amounts of water-soluble, exchangeable and non-exchangeable K were maintained in Alfisol from Hazaribag than Bhubaneswar. Release kinetics of K showed significant release of K from mica treated with bacterial strain. Significant correlation between biomass yield, K uptake by sudan grass and different pools of K in soils were observed. X-ray diffraction analysis indicates greater dissolution of mica due to inoculation of Bacillus mucilaginosus strain in both the soils. Thus, bio-intervention of waste mica could be an alternative and viable technology to solubilize insoluble K into plant available pool and used efficiently as a source of K-fertilizer for sustaining crop production and maintaining soil potassium.     

Gene expression profiles of inflammatory breast cancer reveal high heterogeneity across the epithelial-hybrid-mesenchymal spectrum

Inflammatory breast cancer (IBC) is a highly aggressive breast cancer that metastasizes largely via tumor emboli, and has a 5-year survival rate of less than 30%. No unique genomic signature has yet been identified for IBC nor has any specific molecular therapeutic been developed to manage the disease. Thus, identifying gene expression signatures specific to IBC remains crucial. Here, we compare various gene lists that have been proposed as molecular footprints of IBC using different clinical samples as training and validation sets and using independent training algorithms, and determine their accuracy in identifying IBC samples in three independent datasets. We show that these gene lists have little to no mutual overlap, and have limited predictive accuracy in identifying IBC samples. Despite this inconsistency, single-sample gene set enrichment analysis (ssGSEA) of IBC samples correlate with their position on the epithelial-hybrid-mesenchymal spectrum. This positioning, together with ssGSEA scores, improves the accuracy of IBC identification across the three independent datasets. Finally, we observed that IBC samples robustly displayed a higher coefficient of variation in terms of EMT scores, as compared to non-IBC samples. Pending verification that this patient-to-patient variability extends to intratumor heterogeneity within a single patient, these results suggest that higher heterogeneity along the epithelial-hybrid-mesenchymal spectrum can be regarded to be a hallmark of IBC and a possibly useful biomarker.     

Evidence for interference, coinfections, and intertypic virus enhancement of infection by ovine-caprine lentiviruses.

The ovine-caprine lentiviruses share nucleotide homology and serological properties in their gag-pol genes and gene products but constitute two distinct biological groups represented by ovine visna virus of Icelandic origin and by caprine arthritis-encephalitis and ovine progressive pneumonia viruses of U.S. origin. Two members of each group, visna 1514 and its antigenic variant LV1-1 in the first group and CAEV/CO and S93, a field isolate virus from a local arthritic sheep, in the second group, were examined in the present study in competitive-binding studies in fibroblast and macrophage cell cultures. The cultures were preinoculated with each of the four viruses and then reinoculated with either 1514 virus or CAEV/CO, labeled with [35S]methionine. Both 1514 and CAEV/CO caused homologous interference. LV1-1 and S93 viruses shared the interference patterns of 1514 and CAEV/CO, respectively. 1514 and LV1-1 did not interfere with binding of CAEV/CO. Similarly, CAEV/CO and S93 did not interfere with binding of 1514. Remarkably, certain combinations, such as S93 plus 1514, resulted in enhanced binding of the second virus. Other experiments showed that the enhancement in binding extended to enhancement in replication of the second virus. These latter data suggested that individual cells supported replication of both viruses. Further testing of this phenomenon showed that goats could be doubly infected with two noninterfering viruses, 1514 and CAEV/CO. The ability of noninterfering related lentiviruses to infect the same cell and also the same host animal may be important in the natural history of these viruses in providing ideal conditions for the development of new recombinant viruses.     

Cytotoxic turrianes of Kermadecia elliptica from the New Caledonian rainforest

In the course of an automated screening for small molecules presenting cytotoxic activity, eight new cyclophanes named kermadecins A-H (1-8), have been isolated from the bark of a New Caledonian plant, Kermadecia elliptica, Proteaceae. A LC/APCI-MS study performed on kermadecins A, B and C, provided a reliable method for the detection of other analogues existing in small quantities in the extract. This led to the isolation of five other members of this chemical series. The structures were elucidated by extensive mono- and bi-dimensional spectroscopy and mass spectrometry. The cytotoxic activity of four of them was evaluated on various cell lines.     

Hybrid Spreading Mechanisms and T Cell Activation Shape the Dynamics of HIV-1 Infection

HIV-1 can disseminate between susceptible cells by two mechanisms: cell-free infection following fluid-phase diffusion of virions and by highly-efficient direct cell-to-cell transmission at immune cell contacts. The contribution of this hybrid spreading mechanism, which is also a characteristic of some important computer worm outbreaks, to HIV-1 progression in vivo remains unknown. Here we present a new mathematical model that explicitly incorporates the ability of HIV-1 to use hybrid spreading mechanisms and evaluate the consequences for HIV-1 pathogenenesis. The model captures the major phases of the HIV-1 infection course of a cohort of treatment naive patients and also accurately predicts the results of the Short Pulse Anti-Retroviral Therapy at Seroconversion (SPARTAC) trial. Using this model we find that hybrid spreading is critical to seed and establish infection, and that cell-to-cell spread and increased CD4+ T cell activation are important for HIV-1 progression. Notably, the model predicts that cell-to-cell spread becomes increasingly effective as infection progresses and thus may present a considerable treatment barrier. Deriving predictions of various treatments’ influence on HIV-1 progression highlights the importance of earlier intervention and suggests that treatments effectively targeting cell-to-cell HIV-1 spread can delay progression to AIDS. This study suggests that hybrid spreading is a fundamental feature of HIV infection, and provides the mathematical framework incorporating this feature with which to evaluate future therapeutic strategies.     

Effect of osmolytes and chaperone-like action of P-protein on folding of nucleocapsid protein of Chandipura virus.

Amino acid sequences of nucleocapsid proteins are mostly conserved among different rhabdoviruses. The protein plays a common functional role in different RNA viruses by enwrapping the viral genomic RNA in an RNase-resistant form. Upon expression of the nucleocapsid protein alone in COS cells and in bacteria, it forms large insoluble aggregates. In this work, we have reported for the first time the full-length cloning of the N gene of Chandipura virus and its expression in Escherichia coli in a soluble monomeric form and purification using nonionic detergents. The biological activity of the soluble recombinant protein has been tested, and it was found to possess efficient RNA-binding ability. The state of aggregation of the recombinant protein was monitored using light scattering. In the absence of nonionic detergents, it formed large aggregates. Aggregation was significantly reduced in the presence of osmolytes such as d-sorbitol. Aggregate formation was suppressed in the presence of another viral product, phosphoprotein P, in a chaperone-like manner. Both the osmolyte and phosphoprotein P also suppressed aggregation to a great extent during refolding from a guanidine hydrochloride-denatured form. The function of the phosphoprotein and osmolyte appears to be synergistic to keep the N-protein in a soluble biologically competent form in virus-infected cells.