Phosphorylation and glycosylation interplay: protein modifications at hydroxy amino acids and prediction of signaling functions of the human beta3 integrin family

Protein functions are determined by their three-dimensional structures and the folded 3-D structure is in turn governed by the primary structure and post-translational modifications the protein undergoes during synthesis and transport. Defining protein functions in vivo in the cellular and extracellular environments is made very difficult in the presence of other molecules. However, the modifications taking place during and after protein folding are determined by the modification potential of amino acids and not by the primary structure or sequence. These post-translational modifications, like phosphorylation and O-linked N-acetylglucosamine (O-GlcNAc) modifications, are dynamic and result in temporary conformational changes that regulate many functions of the protein. Computer-assisted studies can help determining protein functions by assessing the modification potentials of a given protein. Integrins are important membrane receptors involved in bi-directional (outside-in and inside-out) signaling events. The beta3 integrin family, including, alpha(IIb)beta3 and alpha(v)beta3, has been studied for its role in platelet aggregation during clot formation and clot retraction based on hydroxyl group modification by phosphate and GlcNAc on Ser, Thr, or Tyr and their interplay on Ser and Thr in the cytoplasmic domain of the beta3 subunit. An antagonistic role of phosphate and GlcNAc interplay at Thr758 for controlling both inside-out and outside-in signaling events is proposed. Additionally, interplay of GlcNAc and phosphate at Ser752 has been proposed to control activation and inactivation of integrin-associated Src kinases. This study describes the multifunctional behavior of integrins based on their modification potential at hydroxyl groups of amino acids as a source of interplay.     

Production of biosurfactant using different hydrocarbons by Pseudomonas aeruginosa EBN-8 mutant

The present investigation dealt with the use of previously isolated and studied gamma-ray mutant strain Pseudomonas aeruginosa EBN-8 for the production of biosurfactant by using different hydrocarbon substrates viz. n-hexadecane, paraffin oil and kerosene oil, provided in minimal medium, as the sole carbon and energy sources. The batch experiments were conducted in 250 mL Erlenmeyer flasks, containing 50 mL minimal salt media supplemented with 1% (w/v) hydrocarbon substrate, inoculated by EBN-8 and incubated at 37 degrees C and 100 rpm in an orbital shaker. The sampling was done on 24 h basis for 10 d. The surface tension of cell-free culture broth decreased from 53 to 29 mN/m after 3 and 4 d of incubation when the carbon sources were paraffin oil and n-hexadecane, respectively. The largest reduction in interfacial tension from 26 to 0.4 mN/m was observed with n-hexadecane, while critical micelle dilution was obtained as 50 x CMC for paraffin oil as carbon source. When grown on n-hexadecane and paraffin oil, the EBN-8 mutant strain gave 4.1 and 6.3 g of the rhamnolipids/L, respectively. These surface-active substances subsequently allowed the hydrocarbon substrates to disperse readily as emulsion in aqueous phase.     

Chromosaponin I stimulates the sugar taste receptor cells of the blowfly, Phormia regina

Chromosaponin I (CSI), a gamma-pyronyl-triterpenoid saponin isolated from pea and other leguminous plants, stimulates the growth of roots in a variety of plants. In the present work, we introduce CSI as a sugar taste substance for the blowfly, Phormia regina. The blowfly has taste chemosensilla on the labellum. The sensory receptor cells in the chemosensillum are highly specialized for the tastes of sugar, salt and water, respectively. Application of CSI induced the feeding response of blowflies including full proboscis extension. CSI also induced impulses of the sugar taste receptor cell in the LL-type sensillum. The optimum concentration of CSI in these responses was 0.1 mM which is much lower than that of sucrose. Based on the comparison of dose-response relationships, CSI is 100 times more effective than sucrose in stimulating the sugar taste receptor cells. CSI-induced impulses appeared after a significant latency compared with sucrose. As far as we know, this is the first report describing that a natural saponin induces sugar responses in insects. CSI is a unique saponin because of its bifunctional property in plants and insects.     

Nicorandil as a novel therapy for advanced diabetic nephropathy in the eNOS-deficient mouse

Nicorandil is an orally available drug that can act as a nitric oxide donor, an antioxidant, and an ATP-dependent K channel activator. We hypothesized that it may have a beneficial role in treating diabetic nephropathy. We administered nicorandil to a model of advanced diabetic nephropathy (the streptozotocin-induced diabetes in mice lacking endothelial nitric oxide synthase, eNOSKO); controls included diabetic eNOS KO mice without nicorandil and nondiabetic eNOS KO mice treated with either nicorandil or vehicle. Mice were treated for 8 wk. Histology, blood pressure, and renal function were determined. Additional studies involved examining the effects of nicorandil on cultured human podocytes. Here, we found that nicorandil did not affect blood glucose levels, blood pressure, or systemic endothelial function, but significantly reduced proteinuria and glomerular injury (mesangiolysis and glomerulosclerosis). Nicorandil protected against podocyte loss and podocyte oxidative stress. Studies in cultured podocytes showed that nicorandil likely protects against glucose-mediated oxidant stress via the ATP-dependent K channel as opposed to its NO-stimulating effects. In conclusion, nicorandil may be beneficial in diabetic nephropathy by preserving podocyte function. We recommend clinical trials to determine whether nicorandil may benefit diabetic nephropathy or other conditions associated with podocyte dysfunction.     

Lack of fitness costs associated with acetamiprid resistance in Bemisia tabaci (Hemiptera: Aleyrodidae)

Sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), is a devastating pest that can cause severe damage to a range of crops by direct feeding and by plant virus transmission. Because of indiscriminate use of insecticides, this whitefly has developed resistance to several insecticides, including neonicotinoids. Our objectives were to determine fitness components affected by acetamiprid resistance in B. tabaci. Assay results showed that selection with acetamiprid had removed heterozygotes from the field population because the survival rate of the resistant population was significantly greater than that of the field population at a very high dose. Comparison of various life traits between the acetamiprid-selected (Aceta-SEL) population and three other populations showed that the numbers of eggs laid by acetamiprid Aceta-SEL population were significantly lower compared with that of other populations but that the proportions of eggs hatched were significantly higher. However, the time taken by nymphal stages of the Aceta-SEL population to develop was significantly higher than that of the susceptible populations. The intrinsic rate of increase, net reproductive rate, mean generation time, and doubling time of Aceta-SEL was significantly higher than Lab-PK and UNSEL populations, but the growth index was similar for all populations. The growth index and high intrinsic value of Aceta-SEL population suggest that the resistance allele may not have detrimental impact. The lack of fitness costs in B. tabaci could promote the rapid development of resistance to acetamiprid and other neonicotinoids. This resistance could threaten the sustainability of whitefly management program on genetically engineered cotton (Gossypium hirsutum L.) where neonicotinoids are being sprayed to manage sucking pests in the field.     

Inhibitory effect of antibodies against human chorionic gonadotropin on the growth of colorectal tumour cells

Human chorionic gonadotropin (hCG) was initially believed to be secreted exclusively by the embryo with its primary function being "rescue" of the corpus luteum. However, recently it has been found that the hormone (or its individual subunits) is also secreted by many cancers and that in many cases secretion is associated with poor patient prognosis. In this study, we assessed the presence of hCG in colorectal cancer cells (CCL-253) and evaluated the anti-tumour effects of anti-hCG antibodies in vitro and in vivo. Anti-hCG antibodies were reactive with CCL-253, as revealed by confocal immunoflourescence microscopy; both cell surface and intracellular expression were observed. Western blot analysis showed that antibodies appeared to interact with several moieties, indicating a level of cross-reactivity. Anti-hCG antiserum specifically reduced the viability of tumor cells and the addition of complement increased in vitro anti-tumor effects. In nude mice implanted with CCL-253 cells, administration of anti-hCG antiserum caused a significant reduction in tumor volume; all treated animals survived, while mortality was observed in control animals. Results suggest that anti-hCG antibodies can mediate significant anti-tumor activity both in vitro and in vivo and lend support to the rationale of anti-hCG immunization in the therapy of gonadotropin- sensitive cancers.     

Two independent replicons can support replication of the anthrax toxin-encoding plasmid pXO1 of Bacillus anthracis

The large pXO1 plasmid (181.6kb) of Bacillus anthracis encodes the anthrax toxin proteins. Previous studies have shown that two separate regions of pXO1 can support replication of pXO1 miniplasmids when introduced into plasmid-less strains of this organism. No information is currently available on the ability of the above two replicons, termed RepX and ORFs 14/16 replicons, to support replication of the full-length pXO1 plasmid. We generated mutants of the full-length pXO1 plasmid in which either the RepX or the ORFs 14/16 replicon was inactivated by TargeTron insertional mutagenesis. Plasmid pXO1 derivatives containing only the RepX or the ORFs 14/16 replicon were able to replicate when introduced into a plasmid-less B. anthracis strain. Plasmid copy number analysis showed that the ORFs 14/16 replicon is more efficient than the RepX replicon. Our studies demonstrate that both the RepX and ORFs 14/16 replicons can independently support the replication of the full-length pXO1 plasmid.     

Use of flux pre-analysis to enable ¹³C tracer studies in pyruvate kinase-deficient Escherichia coli

Pyruvate kinase-deficient Escherichia coli (PB25) is a low by-product-producing yet fast-growing mutant that has been shown to have technological potential. Determining the flux limits through finding the extreme point flux sets was previously reported to identify alternate metabolite trafficking scenarios. Previously, the extreme point flux sets were used to design tracer experiments; however, variation in extracellular measurements was not considered, and reaction reversibility was assumed to be low to moderate. In this study, we examined the utility of limiting the fluxes and predetermining the trafficking scenarios in PB25, including confirmation of quasi-linearity between extreme points to ensure sensitivity is maintained. The effects of variation in extracellular measurements and reaction reversibilities were also examined. Tightened flux limits reduced the nonlinearity between label distribution and fluxes. For low to moderate reversibility, contrast was also preserved. However, for highly reversible phosphoglucoisomerase activity, information from common analytes could lead to a flux solution that is biased towards one extreme point. Based on the PB25 model, some suggestions are provided for how predetermining flux limits and trafficking scenarios could enable flux identification in larger network problems.