A single gamma-carboxyglutamic acid residue in a novel cysteine-rich secretory protein without propeptide

Gamma-glutamyl carboxylase catalyzes the modification of specific glutamyl residues to gamma-carboxyglutamyl (Gla) residues in precursor proteins that possess the appropriate gamma-carboxylation recognition signal within the propeptide region. We describe the immunopurification and first biochemical characterization of an invertebrate high molecular weight Gla-containing protein with homologues in mammals. The protein, named GlaCrisp, was isolated from the venom of the marine cone snail Conus marmoreus. GlaCrisp gave intense signals in Western blot experiments employing the Gla-specific antibody M3B, and the presence of Gla was chemically confirmed by amino acid analysis after alkaline hydrolysis. Characterization of a full-length cDNA clone encoding GlaCrisp deduced a precursor containing an N-terminal signal peptide but, unlike other Gla-containing proteins, no apparent propeptide. The predicted mature protein of 265 amino acid residues showed considerable sequence similarity to the widely distributed cysteine-rich secretory protein family and closest similarity (65% identity) to the recently described substrate-specific protease Tex31. In addition, two cDNA clones encoding the precursors of two isoforms of GlaCrisp were identified. The predicted precursor isoforms differed at three amino acid positions (-6, 9, and 25). Analysis by Edman degradation and nanoelectrospray ionization mass spectrometry, before and after methyl esterfication, identified a Gla residue at amino acid position 9 in GlaCrisp. This is the first example of a Gla-containing protein without an obvious gamma-carboxylation recognition site. The results define a new class of Gla proteins and support the notion that gamma-carboxylation of glutamyl residues is phylogenetically older than blood coagulation and the vertebrate lineage.     

Topical thymidine dinucleotide application protects against UVB-induced skin cancer in mice with DNA repair gene (Ercc1)-deficient skin

Topical application of thymidine dinucleotides (pTpT) provides some protection against the effects of UV on the skin, however, many details of the protective mechanism have yet to be elucidated. We have used mice with an epidermis-specific knockout for the nucleotide excision repair gene, Ercc1, to investigate the mechanisms of protection. pTpT offered no protection against the pronounced UV-induced short-term erythema and skin thickening responses that are characteristic of DNA repair-deficient skin. It also had no effect on UV-induced apoptosis in Ercc1-deficient cultured keratinocytes. However, in these short-term experiments in both skin and keratinocyte culture pTpT did cause a slight reduction in proliferation. pTpT application during a chronic UV irradiation protocol provided some protection from UVB-induced skin carcinogenesis in epidermis-specific Ercc1 knockout mice. The median tumour free survival time was increased in the pTpT-treated group and treated animals had fewer tumours. In addition, pTpT-treated animals developed fewer large inwardly growing skin lesions than untreated animals. Furthermore, the proliferation response was reduced in chronically irradiated, non-lesional pTpT-treated skin. We conclude that cancer protection by pTpT in our mice is not modulated by an upregulation of DNA repair, as protection appears to be independent of a functional nucleotide excision repair pathway. We hypothesise instead that protection by pTpT is due to a reduction in epidermal proliferation.     

ERRα regulates osteoblastic and adipogenic differentiation of mouse bone marrow mesenchymal stem cells

The orphan nuclear receptor estrogen-related receptor-α (ERRα) has been reported to have both a positive and a negative regulatory role in osteoblastic and adipocytic differentiation. We have studied the role of ERRα in osteoblastic and adipogenic differentiation of mesenchymal stem cells. Bone marrow mesenchymal stem cells were isolated from ERRα deficient mice and their differentiation capacities were compared to that of the wild-type cells. ERRα deficient cultures displayed reduced cellular proliferation, osteoblastic differentiation, and mineralization. In the complementary experiment, overexpression of ERRα in MC3T3-E1 cells increased the expression of osteoblastic markers and mineralization. Alterations in the expression of bone sialoprotein (BSP) may at least partially explain the effects on mineralization as BSP expression was reduced in ERRα deficient MSCs and enhanced upon ERRα overexpression in MC3T3-E1 cells. Furthermore, a luciferase reporter construct driven by the BSP promoter was efficiently transactivated by ERRα. Under adipogenic conditions, ERRα deficient cultures displayed reduced adipocytic differentiation. Our data thus propose a positive role for ERRα in osteoblastic and adipocytic differentiation. The variability in the results yielded in the different studies implies that ERRα may play different roles in bone under different physiological conditions.     

Anti-malarial market and policy surveys in sub-Saharan Africa

At a recent meeting (Sept 18, 2009) in which reasons for the limited access to artemisinin-based combination therapy (ACT) in sub-Saharan Africa were discussed, policy and market surveys on anti-malarial drug availability and accessibility in Burundi and Sierra Leone were presented in a highly interactive brainstorming session among key stakeholders across private, public, and not-for-profit sectors. The surveys, the conduct of which directly involved the national malaria control programme managers of the two countries, provides the groundwork for evidence-based policy implementation. The results of the surveys could be extrapolated to other countries with similar socio-demographic and malaria profiles. The meeting resulted in recommendations on key actions to be taken at the global, national, and community level for better ACT accessibility.At the global level, both public and private sectors have actions to take to strengthen policies that lead to the replacement of loose blister packs with fixed-dose ACT products, develop strategies to ban inappropriate anti-malarials and regulate those bans, and facilitate technology and knowledge transfer to scale up production of fixed-dose ACT products, which should be readily available and affordable to those patients who are in the greatest need of these medicines.At the national level, policies that regulate the anti-malarial medicines market should be enacted and enforced. The public sector, including funding donors, should participate in ensuring that the private sector is engaged in the ACT implementation process. Research similar to the surveys discussed is important for other countries to develop and evaluate the right incentives at a local level.At the community level, community outreach and education about appropriate preventive and treatment measures must continue and be strengthened, with service delivery systems developed within both public and private sectors, among other measures, to decrease access to ineffective and inappropriate anti-malarial medicines.What was clear during the meeting is that continuing commitment, strengthened interaction and transparency among various stakeholders, with focus on communities, national governments, and evidence-based policy and action are the only way to sustainably address the control of malaria, a disease which continues to have a significant health and socio-economic impact worldwide, particularly in sub-Saharan Africa.Details on the methodology employed in carrying out the studies discussed at this meeting, as well as more detailed results, data analysis and discussion of the studies are soon to be published.     

Matrix metalloproteinase-9 (gelatinase B) deficiency leads to increased severity of Staphylococcus aureus-triggered septic arthritis

Matrix metalloproteinases constitute a family of structurally related endopeptidases that are crucial for the normal turnover of the extracellular matrix. Elevated levels of MMP-9 have been demonstrated in synovial fluids of rheumatoid arthritis patients, and a correlation with the severity of the disease has been described. The aim of this study was to explore the impact of MMP-9 expression on joint inflammation and destruction in a model of bacterially induced septic arthritis. MMP-9 knock-out mice and C57Bl6 congenic controls were inoculated intravenously or intra-articularly with Staphylococcus aureus. Arthritis was evaluated clinically and by means of histology. Zymographic analyses were performed to study ex vivo induction of MMP-9 following exposure to S. aureus. The MMP-9 knock-out mice displayed a significantly higher frequency and severity, but not destructivity, of arthritis than did the wild-type mice. The knock-out mice also proved to harbour an increased number of bacteria locally in joints and systemically in kidneys, possibly by impaired extravasation and recruitment of leukocytes and a deficient early defence against infection. Our findings indicate that deficiency in MMP-9 increases the degree of joint inflammation due to decreased bacterial clearance.     

Distribution of sibling species of the Anopheles annulipes complex (Diptera: Culicidae) in the Townsville region of Australia

Abstract  As members of the Anopheles annulipes species complex have been implicated as possible malaria vectors in the past, their response to dams and urban development is of medical interest. We collected An. annulipes sensu lato (s.l.) Walker from the Ross River Dam and environs of tropical north Queensland, and scored them for 28 allozyme loci. Allozyme genotype data were analysed by the Unweighted Pair Group Method of Analysis applied to percentage fixed allelic differences (%FD) and by a Bayesian clustering approach. Both approaches revealed four groups, which differed by more than 25%FD, suggesting four species. Specimens sequenced for the Internal transcribed spacer subunit 2 of rDNA suggested that three of these groups were An. annulipes species D, species I and species O. Anopheles annulipes species D occurred in the Ross River and the Ross River Dam, An. annulipes species O inhabited rock pools, and An. annulipes species I inhabited both rock pools and artificial containers in domestic situations. Construction of the Ross River Dam appears to have benefited at least An. annulipes species D, and nearby housing development An. annulipes species I. Although An. annulipes s.l. does not appear to be a major medical risk, mosquito monitoring around Townsville may need to take into account the multispecies status of this taxon.     

Phenobabelomics--mouse phenotype data resources.

An essential aspect to understanding the functional significance of individual genes in the mouse genome is an understanding of the phenotypic consequences of gene mutations. A wide variety of online sites exist that provide different types of phenotypic information on the laboratory mouse. In this review, we describe the major resources that are currently available and discuss some of the bioinformatics requirements that will be necessary to make more seamless searching, comparison and analysis of these various data types possible.     

Increased beta-oxidation in muscle cells enhances insulin-stimulated glucose metabolism and protects against fatty acid-induced insulin resistance despite intramyocellular lipid accumulation

Skeletal muscle insulin resistance may be aggravated by intramyocellular accumulation of fatty acid-derived metabolites that inhibit insulin signaling. We tested the hypothesis that enhanced fatty acid oxidation in myocytes should protect against fatty acid-induced insulin resistance by limiting lipid accumulation. L6 myotubes were transduced with adenoviruses encoding carnitine palmitoyltransferase I (CPT I) isoforms or beta-galactosidase (control). Two to 3-fold overexpression of L-CPT I, the endogenous isoform in L6 cells, proportionally increased oxidation of the long-chain fatty acids palmitate and oleate and increased insulin stimulation of [(14)C]glucose incorporation into glycogen by 60% while enhancing insulin-stimulated phosphorylation of p38MAPK. Incubation of control cells with 0.2 mm palmitate for 18 h caused accumulation of triacylglycerol, diacylglycerol, and ceramide (but not long-chain acyl-CoA) and decreased insulin-stimulated [(14)C]glucose incorporation into glycogen (60%), [(3)H]deoxyglucose uptake (60%), and protein kinase B phosphorylation (20%). In the context of L-CPT I overexpression, palmitate preincubation produced a relative decrease in insulin-stimulated incorporation of [(14)C]glucose into glycogen (60%) and [(3)H]deoxyglucose uptake (40%) but did not inhibit phosphorylation of protein kinase B. Due to the enhancement of insulin-stimulated glucose metabolism induced by L-CPT I overexpression itself, net insulin-stimulated incorporation of [(14)C]glucose into glycogen and [(3)H]deoxyglucose uptake in L-CPT I-transduced, palmitate-treated cells were significantly greater than in palmitate-treated control cells (71 and 75% greater, respectively). However, L-CPT I overexpression failed to decrease intracellular triacylglycerol, diacylglycerol, ceramide, or long-chain acyl-CoA. We propose that accelerated beta-oxidation in muscle cells exerts an insulin-sensitizing effect independently of changes in intracellular lipid content.