Specific Interaction of the Unmodified Bacteriocin Lactococcin 972 with the Cell Wall Precursor Lipid II

Lactococcin 972 (Lcn972) is a nonlantibiotic bacteriocin that inhibits septum biosynthesis in Lactococcus lactis rather than forming pores in the cytoplasmic membrane. In this study, a deeper analysis of the molecular basis of the mode of action of Lcn972 was performed. Of several lipid cell wall precursors, only lipid II antagonized Lcn972 inhibitory activity in vivo. Likewise, Lcn972 only coprecipitated with lipid II micelles. This bacteriocin inhibited the in vitro polymerization of lipid II by the recombinant S. aureus PBP2 and the addition to lipid II of the first glycine catalyzed by FemX. These experiments demonstrate that Lcn972 specifically interacts with lipid II, the substrate of both enzymes. In the presence of Lcn972, nisin pore formation was partially hindered in whole cells. However, binding of Lcn972 to lipid II could not compete with nisin in lipid II-doped 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes, possibly indicating a distinct binding site. The existence of a putative cotarget for Lcn972 activity is discussed in the context of its narrow inhibitory spectrum and the localized action at the division septum. To our knowledge, this is the first unmodified bacteriocin that binds to the cell wall precursor lipid II.     

Cell delivery systems: Toward the next generation of cell therapies for type 1 diabetes

Immunoprotection and oxygen supply are vital in implementing a cell therapy for type 1 diabetes (T1D). Without these features, the transplanted islet cell clusters will be rejected by the host immune system, and necrosis will occur due to hypoxia. The use of anti‐rejection drugs can help protect the transplanted cells from the immune system; yet, they also may have severe side effects. Cell delivery systems (CDS) have been developed for islet transplantation to avoid using immunosuppressants. CDS provide physical barriers to reduce the immune response and chemical coatings to reduce host fibrotic reaction. In some CDS, there is architecture to support vascularization, which enhances oxygen exchange. In this review, we discuss the current clinical and preclinical studies using CDS without immunosuppression as a cell therapy for T1D. We find that though CDS have been demonstrated for their ability to support immunoisolation of the grafted cells, their functionality has not been fully optimized. Current advanced methods in clinical trials demonstrate the systems are partly functional, physically complicated to implement or inefficient. However, modifications are being made to overcome these issues.     

Increased hepatic fibrosis and JNK2-dependent liver injury in mice exhibiting hepatocyte-specific deletion of cFLIP

Chronic liver disease promotes hepatocellular injury involving apoptosis and triggers compensatory regeneration that leads to the activation of quiescent stellate cells in the liver. The deposition of extracellular matrix from activated myofibroblasts promotes hepatic fibrosis and the progression to cirrhosis with deleterious effects on liver physiology. The role of apoptosis signaling pathways in the development of fibrosis remains undefined. The aim of the current study was to determine the involvement of the caspase-8 homologue cellular FLICE-inhibitory protein (cFLIP) during the initiation and progression of fibrosis. Liver injury and fibrosis from carbon tetrachloride (CCl(4)) and thioacetamide (TAA) were examined in mice exhibiting a hepatocyte-specific deletion of cFLIP (flip(-/-)). Acute liver injury from CCl(4) and TAA were enhanced in flip(-/-) mice. This was accompanied by increased activation of caspase-3 and -9, pronounced phosphorylation of JNK, and decreased phosphorylation of Erk. Deletion of the cJun NH(2)-terminal kinase 2 (JNK2) in flip(-/-) mice protected from injury. Hepatic fibrosis was increased at baseline in 12-wk-old flip(-/-) mice, and progression of fibrosis from TAA was accelerated compared with the wild type. In conclusion, deletion of cFLIP in hepatocytes leads to increased fibrosis and accelerated fibrosis progression. This is accompanied by increased injury involving the activation of caspases and JNK2. Thus predisposition to liver injury involving increased hepatocellular apoptosis is a critical mediator of accelerated fibrogenesis, and prevention of liver injury will be a most important measure for patients with chronic liver disease.     

The 1.3 A crystal structure of the flavoprotein YqjM reveals a novel class of Old Yellow Enzymes

Here we report the crystal structure of YqjM, a homolog of Old Yellow Enzyme (OYE) that is involved in the oxidative stress response of Bacillus subtilis. In addition to the oxidized and reduced enzyme form, the structures of complexes with p-hydroxybenzaldehyde and p-nitrophenol, respectively, were solved. As for other OYE family members, YqjM folds into a (alpha/beta)8-barrel and has one molecule of flavin mononucleotide bound non-covalently at the COOH termini of the beta-sheet. Most of the interactions that control the electronic properties of the flavin mononucleotide cofactor are conserved within the OYE family. However, in contrast to all members of the OYE family characterized to date, YqjM exhibits several unique structural features. For example, the enzyme exists as a homotetramer that is assembled as a dimer of catalytically dependent dimers. Moreover, the protein displays a shared active site architecture where an arginine finger (Arg336) at the COOH terminus of one monomer extends into the active site of the adjacent monomer and is directly involved in substrate recognition. Another remarkable difference in the binding of the ligand in YqjM is represented by the contribution of the NH2-terminal Tyr28 instead of a COOH-terminal tyrosine in OYE and its homologs. The structural information led to a specific data base search from which a new class of OYE oxidoreductases was identified that exhibits a strict conservation of active site residues, which are critical for this subfamily, most notably Cys26, Tyr28, Lys109, and Arg336. Therefore, YqjM is the first representative of a new bacterial subfamily of OYE homologs.     

YeeI, a novel protein involved in modulation of the activity of the glucose-phosphotransferase system in Escherichia coli K-12

The membrane-bound protein EIICB(Glc) encoded by the ptsG gene is the major glucose transporter in Escherichia coli. This protein is part of the phosphoenolpyruvate:glucose-phosphotransferase system, a very important transport and signal transduction system in bacteria. The regulation of ptsG expression is very complex. Among others, two major regulators, the repressor Mlc and the cyclic AMP-cyclic AMP receptor protein activator complex, have been identified. Here we report identification of a novel protein, YeeI, that is involved in the regulation of ptsG by interacting with Mlc. Mutants with reduced activity of the glucose-phosphotransferase system were isolated by transposon mutagenesis. One class of mutations was located in the open reading frame yeeI at 44.1 min on the E. coli K-12 chromosome. The yeeI mutants exhibited increased generation times during growth on glucose, reduced transport of methyl-alpha-d-glucopyranoside, a substrate of EIICB(Glc), reduced induction of a ptsG-lacZ operon fusion, and reduced catabolite repression in lactose/glucose diauxic growth experiments. These observations were the result of decreased ptsG expression and a decrease in the amount of EIICB(Glc). In contrast, overexpression of yeeI resulted in higher expression of ptsG, of a ptsG-lacZ operon fusion, and of the autoregulated dgsA gene. The effect of a yeeI mutation could be suppressed by introducing a dgsA deletion, implying that the two proteins belong to the same signal transduction pathway and that Mlc is epistatic to YeeI. By measuring the surface plasmon resonance, we found that YeeI (proposed gene designation, mtfA) directly interacts with Mlc with high affinity.     

A novel technique for the identification of CpG islands exhibiting altered methylation patterns (ICEAMP)

Aberrant CpG methylation changes occurring during tumour progression include the loss (hypomethylation) and gain (hypermethylation) of methyl groups. Techniques currently available for examining such changes either require selection of a region, then examination of methylation changes, or utilise methylation-sensitive restriction enzymes to identify an alteration. We describe here a novel method that identifies genomic regions as a consequence of altered methylation during tumourigenesis. A methyl-CpG binding domain column isolates methylated GC-rich sequences from both tumours and surrounding normal tissue. Subsequent subtractive hybridisation removes sequences common to both, leaving only methylated sequences unique to the tumour. Libraries of sequences generated using DNA derived from a breast tumour (histological grade; poorly differentiated) as ‘tester’ and from matched normal tissue as ‘driver’ were examined; 26% of clones had the sequence criteria of a CpG island (CGI). Analysis using the bisulfite technique revealed that a number of these sequences were methylated in tumour DNA relative to the normal control. We have therefore demonstrated the ability of this technique, the identification of CGI exhibiting altered methylation patterns (ICEAMP), to isolate tumour-specific methylated GC-rich sequences. This will allow a comprehensive identification of methylation changes during tumourigenesis and will lead to a better understanding of the processes involved.     

A new quantitative LC tandem mass spectrometry assay for serum 25-hydroxy vitamin D

BackgroundThe accurate measurement of 25-hydoxy vitamin D (25OH-D) in serum has been a challenge for many years. We developed a liquid chromatography tandem mass spectrometry (LC Tandem MS) assay for the quantitative determination of 25OH-D₂ and 25OH-D₃ in serum. The new method was compared with two widely used commercially available immunoassays.MethodsSample preparation involved protein precipitation with acetonitrile containing deuterated forms of the target species as internal standards. An API 5000 mass spectrometer coupled with a photoionization source was used for quantitation. The performance of the new LC Tandem MS assay was compared with a radioimmunoassay (RIA, Diasorin) and a chemiluminescence immunoassay (ECLIA, Roche Diagnostics), analysing serum obtained from 152 individuals.ResultsUsing 100 μl of serum, the LC Tandem MS assay had a limit of quantitation of 1.3 nmol/L for both 25OH-D₂ and 25OH-D₃ with a linear response between 1.3 and 625 nmol/L and accuracy of between 95 and 124%. Intra- and inter-assay precision were ≤7% and ≤4%, respectively. Measurement of 25OH-D levels in 152 serum samples gave run averages of 71, 56 and 62 nmol/L for LC Tandem MS, ECLIA and RIA, respectively. Correlations between the various methods were: LC Tandem MS vs. RIA: r = 0.931; LC Tandem MS vs. ECLIA: r = 0.784; RIA vs. ECLIA: r = 0.787. The LC Tandem MS method had a positive proportional bias of 26% over the RIA, whereas the ECLIA showed variable differences.ConclusionThe new LC Tandem MS assay is accurate and precise at physiologically relevant 25OH-D concentrations, and compares favourably with the RIA. In contrast, the ECLIA shows variable bias with the other assays tested.     

GOstat: find statistically overrepresented Gene Ontologies within a group of genes

SUMMARY: Modern experimental techniques, as for example DNA microarrays, as a result usually produce a long list of genes, which are potentially interesting in the analyzed process. In order to gain biological understanding from this type of data, it is necessary to analyze the functional annotations of all genes in this list. The Gene-Ontology (GO) database provides a useful tool to annotate and analyze the functions of a large number of genes. Here, we introduce a tool that utilizes this information to obtain an understanding of which annotations are typical for the analyzed list of genes. This program automatically obtains the GO annotations from a database and generates statistics of which annotations are overrepresented in the analyzed list of genes. This results in a list of GO terms sorted by their specificity. AVAILABILITY: Our program GOstat is accessible via the Internet at http://gostat.wehi.edu.au     

Ecological tracers reveal resource convergence among prey fish species in a large lake ecosystem

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