Isolation of Lactococcus garvieae Strain TRF1 from the Fecal Material of a Timber Rattlesnake

In certain species of fish, such as rainbow trout, infection by the Firmicutes Lactococcus garvieae is problematic. This organism is the causative agent of lactococcosis disease in fish, and it is also considered a potential zoonotic bacterium, since it can cause several opportunistic infections in humans. In this study, L. garvieae strain TRF1 was grown and isolated in pure culture from the fecal material of a Timber Rattlesnake (Crotalus horridus), living in the wild. The presence/absence of several putative virulence factors was identified using staining, PCR amplification, and the construction of a draft genome. Strain TRF1 shared several putative virulence factors with strain Lg2, a known fish pathogen. However, the capsule gene cluster, found in strain Lg2, was not found in strain TRF1. Since this gene cluster is absent in several non-pathogenic strains of L. garvieae, it suggests strain TRF1 may not be pathogenic. However, this hypothesis will have to be tested in an animal model.     

A novel approach for the rapid mutagenesis and directed evolution of the structural genes of west nile virus

Molecular clone technology has proven to be a powerful tool for investigating the life cycle of flaviviruses, their interactions with the host, and vaccine development. Despite the demonstrated utility of existing molecular clone strategies, the feasibility of employing these existing approaches in large-scale mutagenesis studies is limited by the technical challenges of manipulating relatively large molecular clone plasmids that can be quite unstable when propagated in bacteria. We have developed a novel strategy that provides an extremely rapid approach for the introduction of mutations into the structural genes of West Nile virus (WNV). The backbone of this technology is a truncated form of the genome into which DNA fragments harboring the structural genes are ligated and transfected directly into mammalian cells, bypassing entirely the requirement for cloning in bacteria. The transfection of cells with this system results in the rapid release of WNV that achieves a high titer (～10(7) infectious units/ml in 48 h). The suitability of this approach for large-scale mutagenesis efforts was established in two ways. First, we constructed and characterized a library of variants encoding single defined amino acid substitutions at the 92 residues of the "pr" portion of the precursor-to-membrane (prM) protein. Analysis of a subset of these variants identified a mutation that conferred resistance to neutralization by an envelope protein-specific antibody. Second, we employed this approach to accelerate the identification of mutations that allow escape from neutralizing antibodies. Populations of WNV encoding random changes in the E protein were produced in the presence of a potent monoclonal antibody, E16. Viruses resistant to neutralization were identified in a single passage. Together, we have developed a simple and rapid approach to produce infectious WNV that accelerates the process of manipulating the genome to study the structure and function of the structural genes of this important human pathogen.     

Roles of GPR41 and GPR43 in leptin secretory responses of murine adipocytes to short chain fatty acids

GPR41 is reportedly expressed in murine adipose tissue and mediates short chain fatty acid (SCFA)-stimulated leptin secretion by activating Gα_i. Here, we agree with a contradictory report in finding no expression of GPR41 in murine adipose tissue. Nevertheless, in the presence of adenosine deaminase to minimise Gα_i signalling via the adenosine A1 receptor, SCFA stimulated leptin secretion by adipocytes from wild-type but not GPR41 knockout mice. Expression of GPR43 was reduced in GPR41 knockout mice. Acetate but not butyrate stimulated leptin secretion in wild-type mesenteric adipocytes, consistent with mediation of the response by GPR43 rather than GPR41. Pertussis toxin prevented stimulation of leptin secretion by propionate in epididymal adipocytes, implicating Gα_i signalling mediated by GPR43 in SCFA-stimulated leptin secretion.     

Differential regulation of the MAP, SAP and RK/p38 kinases by Pyst1, a novel cytosolic dual-specificity phosphatase.

The Pyst1 and Pyst2 mRNAs encode closely related proteins, which are novel members of a family of dual-specificity MAP kinase phosphatases typified by CL100/MKP-1. Pyst1 is expressed constitutively in human skin fibroblasts and, in contrast to other members of this family of enzymes, its mRNA is not inducible by either stress or mitogens. Furthermore, unlike the nuclear CL100 protein, Pyst1 is localized in the cytoplasm of transfected Cos-1 cells. Like CL100/ MKP-1, Pyst1 dephosphorylates and inactivates MAP kinase in vitro and in vivo. In addition, Pyst1 is able to form a physical complex with endogenous MAP kinase in Cos-1 cells. However, unlike CL100, Pyst1 displays very low activity towards the stress-activated protein kinases (SAPKs) or RK/p38 in vitro, indicating that these kinases are not physiological substrates for Pyst1. This specificity is underlined by the inability of Pyst1 to block either the stress-mediated activation of the JNK-1 SAP kinase or RK/p38 in vivo, or to inhibit nuclear signalling events mediated by the SAP kinases in response to UV radiation. Our results provide the first evidence that the members of the MAP kinase family of enzymes are differentially regulated by dual-specificity phosphatases and also indicate that the MAP kinases may be regulated by different members of this family of enzymes depending on their subcellular location.     

Drosophila ventral furrow morphogenesis: a proteomic analysis

Ventral furrow formation is a key morphogenetic event during Drosophila gastrulation that leads to the internalization of mesodermal precursors. While genetic analysis has revealed the genes involved in the specification of ventral furrow cells, few of the structural proteins that act as mediators of ventral cell behavior have been identified. A comparative proteomics approach employing difference gel electrophoresis was used to identify more than fifty proteins with altered abundance levels or isoform changes in ventralized versus lateralized embryos. Curiously, the majority of protein differences between these embryos appeared well before gastrulation, only a few protein changes coincided with gastrulation, suggesting that the ventral cells are primed for cell shape change. Three proteasome subunits were found to differ between ventralized and lateralized embryos. RNAi knockdown of these proteasome subunits and time-dependent difference-proteins caused ventral furrow defects, validating the role of these proteins in ventral furrow morphogenesis.     

Large-scale expression and purification of high-molecular-weight glutenin subunits

The high-molecular-weight glutenin subunits (HMW-GSs) are considered to be one of the most important components of wheat gluten, contributing to the unique viscoelastic properties of wheat dough. The HMW-GSs are highly homologous in sequence and structure and a mixture of subunits is usually present in wheat flours. Consequently, it is difficult to purify these proteins separately in appreciable amounts. Expression in heterologous systems provides a clear opportunity to produce large amounts of single HMW-GS proteins, amounts (up to 100 mg) which are required for in vitro analysis of these proteins. However, since the first expression studies of HMW-GSs, over 10 years ago, this technology has not been widely utilized. Previous studies have been analytical or small scale (5-100 ml) and in most cases only partial purity was obtained. In the present paper, we describe in detail the expression of the HMW-GSs Glu1-Dx2, Dx5, Dy10, and Dy12 for the first time on a large scale, producing up to 100 mg of target protein from a 2-liter bacterial culture, using a Biostat fermenter. Our results include optimization of expression conditions to increase yield and stability of proteins. Results also include localization, differences between x- and y-type expression and small-scale versus large-scale expression. We also developed a large-scale purification procedure. The bacterially expressed proteins have the same molecular weight on SDS-PAGE and the same retention times on RP-HPLC as their native counterparts extracted from flour. Functionality tests, on the bacterially produced proteins, have shown a clear correlation with the equivalent native proteins from flour. These results provide a clear opportunity to produce protein in amounts necessary for more detailed studies of the structure and function of the HMW-GSs and glutenin polymers on dough development and quality.     

Micro-scale environmental variation amplifies physiological variation among individual mussels

The contributions of temporal and spatial environmental variation to physiological variation remain poorly resolved. Rocky intertidal zone populations are subjected to thermal variation over the tidal cycle, superimposed with micro-scale variation in individuals'' body temperatures. Using the sea mussel (Mytilus californianus), we assessed the consequences of this micro-scale environmental variation for physiological variation among individuals, first by examining the latter in field-acclimatized animals, second by abolishing micro-scale environmental variation via common garden acclimation, and third by restoring this variation using a reciprocal outplant approach. Common garden acclimation reduced the magnitude of variation in tissue-level antioxidant capacities by approximately 30% among mussels from a wave-protected (warm) site, but it had no effect on antioxidant variation among mussels from a wave-exposed (cool) site. The field-acclimatized level of antioxidant variation was restored only when protected-site mussels were outplanted to a high, thermally stressful site. Variation in organismal oxygen consumption rates reflected antioxidant patterns, decreasing dramatically among protected-site mussels after common gardening. These results suggest a highly plastic relationship between individuals'' genotypes and their physiological phenotypes that depends on recent environmental experience. Corresponding context-dependent changes in the physiological mean–variance relationships within populations complicate prediction of responses to shifts in environmental variability that are anticipated with global change.     

The cytokine and chemokine expression profile of nucleus pulposus cells: implications for degeneration and regeneration of the intervertebral disc

Introduction

The aims of these studies were to identify the cytokine and chemokine expression profile of nucleus pulposus (NP) cells and to determine the relationships between NP cell cytokine and chemokine production and the characteristic tissue changes seen during intervertebral disc (IVD) degeneration.

Methods

Real-time q-PCR cDNA Low Density Array (LDA) was used to investigate the expression of 91 cytokine and chemokine associated genes in NP cells from degenerate human IVDs. Further real-time q-PCR was used to investigate 30 selected cytokine and chemokine associated genes in NP cells from non-degenerate and degenerate IVDs and those from IVDs with immune cell infiltrates (‘infiltrated’). Immunohistochemistry (IHC) was performed for four selected cytokines and chemokines to confirm and localize protein expression in human NP tissue samples.

Results

LDA identified the expression of numerous cytokine and chemokine associated genes including 15 novel cytokines and chemokines. Further q-PCR gene expression studies identified differential expression patterns in NP cells derived from non-degenerate, degenerate and infiltrated IVDs. IHC confirmed NP cells as a source of IL-16, CCL2, CCL7 and CXCL8 and that protein expression of CCL2, CCL7 and CXCL8 increases concordant with histological degenerative tissue changes.

Conclusions

Our data indicates that NP cells are a source of cytokines and chemokines within the IVD and that these expression patterns are altered in IVD pathology. These findings may be important for the correct assessment of the ‘degenerate niche’ prior to autologous or allogeneic cell transplantation for biological therapy of the degenerate IVD.     

Antibacterial and antitubercular activity of fosmidomycin, FR900098, and their lipophilic analogs

The nonmevalonate pathway (NMP) of isoprene biosynthesis is an exciting new route toward novel antibiotic development. Inhibitors against several enzymes in this pathway are currently under examination. A significant liability of many of these agents is poor cell penetration. To overcome and improve our understanding of this problem, we have synthesized a series of lipophilic, prodrug analogs of fosmidomycin and FR900098, inhibitors of the NMP enzyme Dxr. Several of these compounds show improved antibacterial activity against a panel of organisms relative to the parent compound, including activity against Mycobacterium tuberculosis (Mtb). Our results show that this strategy can be an effective way for improving whole cell activity of NMP inhibitors.