In vitro activity of mastoparan-AF alone and in combination with clinically used antibiotics against multiple-antibiotic-resistant Escherichia coli isolates from animals

The in vitro activity of mastoparan-AF, an amphipathic antimicrobial peptide isolated from the hornet venom of Vespa affinis, alone and in combination with various clinically used antibiotics, was investigated against 21 Escherichia coli isolates/strains. Most E. coli isolates tested were detected containing multiple-antimicrobial resistance genes. Antimicrobial activity of mastoparan-AF was measured by MIC, MBC, time-kill kinetic assay and chequerboard titration method. Mastoparan-AF exhibited potent antimicrobial activity against most multiple-antibiotic-resistant E. coli isolates at the concentrations ranging from 4 to 16 μg/ml. Combination studies showed that mastoparan-AF acts synergistically with certain antibiotics, i.e., cephalothin or gentamicin, against some multiple-antibiotic-resistant E. coli isolates. In conclusion, mastoparan-AF alone or in combination with other antibiotics could be promising as alternatives for combating multiple-antibiotic-resistant E. coli in future clinical applications.     

Comparative peptide binding studies of the PABC domains from the ubiquitin-protein isopeptide ligase HYD and poly(A)-binding protein. Implications for HYD function

The PABC domain is a peptide-binding domain that is specifically found in poly(A)-binding protein (PABP) and a HECT ubiquitin-protein isopeptide ligase (E3) known as HYD (hyperplastic discs), EDD (E3 isolated by differential display), or Rat100. The PABC domain of PABP recruits various regulatory proteins and translation factors to poly(A) mRNAs through binding of a conserved 12-amino acid peptide motif, PAM2 (PABP-interacting motif 2). In contrast, little is known about the specificity or function of the domain from HYD. Here, we used isothermal calorimetry and surface plasmon resonance titrations to show that the PABC domain of HYD binds PAM2 peptides with micromolar affinity. NMR chemical shift perturbations were used to map the peptide-binding site in the PABC domain of HYD. The structural features of binding are very similar to those of the interactions with the domain of PABP, which explains the overlapping peptide specificity and binding affinity. We identified the anti-proliferative Tob proteins as potential binding partners of HYD. This was confirmed by glutathione S-transferase pulldown and immunoprecipitation experiments demonstrating the interaction with full-length Tob2. Altogether, our results point to a role of the PABC domain as a protein-protein interaction domain that brings together the processes of translation, ubiquitin-mediated protein degradation, and cell cycle control.     

Short-term synaptic plasticity in the nociceptive thalamic-anterior cingulate pathway

Background

ATP-sensitive potassium (K_ATP) channels in neurons regulate excitability, neurotransmitter release and mediate protection from cell-death. Furthermore, activation of K_ATP channels is suppressed in DRG neurons after painful-like nerve injury. NO-dependent mechanisms modulate both K_ATP channels and participate in the pathophysiology and pharmacology of neuropathic pain. Therefore, we investigated NO modulation of K_ATP channels in control and axotomized DRG neurons.

Results

Cell-attached and cell-free recordings of K_ATP currents in large DRG neurons from control rats (sham surgery, SS) revealed activation of K_ATP channels by NO exogenously released by the NO donor SNAP, through decreased sensitivity to [ATP]i. This NO-induced K_ATP channel activation was not altered in ganglia from animals that demonstrated sustained hyperalgesia-type response to nociceptive stimulation following spinal nerve ligation. However, baseline opening of K_ATP channels and their activation induced by metabolic inhibition was suppressed by axotomy. Failure to block the NO-mediated amplification of K_ATP currents with specific inhibitors of sGC and PKG indicated that the classical sGC/cGMP/PKG signaling pathway was not involved in the activation by SNAP. NO-induced activation of K_ATP channels remained intact in cell-free patches, was reversed by DTT, a thiol-reducing agent, and prevented by NEM, a thiol-alkylating agent. Other findings indicated that the mechanisms by which NO activates K_ATP channels involve direct S-nitrosylation of cysteine residues in the SUR1 subunit. Specifically, current through recombinant wild-type SUR1/Kir6.2 channels expressed in COS7 cells was activated by NO, but channels formed only from truncated isoform Kir6.2 subunits without SUR1 subunits were insensitive to NO. Further, mutagenesis of SUR1 indicated that NO-induced K_ATP channel activation involves interaction of NO with residues in the NBD1 of the SUR1 subunit.

Conclusion

NO activates K_ATP channels in large DRG neurons via direct S-nitrosylation of cysteine residues in the SUR1 subunit. The capacity of NO to activate K_ATP channels via this mechanism remains intact even after spinal nerve ligation, thus providing opportunities for selective pharmacological enhancement of K_ATP current even after decrease of this current by painful-like nerve injury.     

A fast SCOP fold classification system using content-based E-Predict algorithm

Background  

Domain experts manually construct the Structural Classification of Protein (SCOP) database to categorize and compare protein structures. Even though using the SCOP database is believed to be more reliable than classification results from other methods, it is labor intensive. To mimic human classification processes, we develop an automatic SCOP fold classification system to assign possible known SCOP folds and recognize novel folds for newly-discovered proteins.     

Mesenchymal stem cells are superior to angiogenic growth factor genes for improving myocardial performance in the mouse model of acute myocardial infarction

Summary Both cell therapy and angiogenic growth factor gene therapy have been applied to animal studies and clinical trials. Little is known about the direct comparison between cell therapy and angiogenic growth factor gene therapy. The goal of this study was to compare the effects of human bone marrow-derived mesenchymal stem cells (hMSCs) transplantation and injection of angiogenic growth factor genes in a model of acute myocardial infarction in mice. The hMSCs were obtained from adult human bone marrow and expanded in vitro. The purity and characteristics of hMSCs were identified by flow cytometry and immunophenotyping. Immediately after ligation of the left anterior descending coronary artery in male severe combined immunodeficient (SCID) mice, culture-expanded hMSCs or angiogenic growth factor genes were injected intramuscularly at the left anterior free wall. The engrafted hMSCs were positive for cardiac marker, desmin. Infarct size was significantly smaller in the hMSCs-treated group than in the angiopoietin-1 (Ang-1) or vascular endothelial growth factor (VEGF)-treated group at day 28 after infarction. hMSCs transplantation was better in decreasing left ventricular end-diastolic dimension and increasing fractional shortening than Ang1 or VEGF gene therapy. Capillary density was markedly increased after hMSCs transplantation than Ang1 and VEGF gene therapy. In conclusion, intramyocardial transplantation of hMSCs improves cardiac function after acute myocardial infarction through enhancement of angiogenesis and myogenesis in the ischemic myocardium. hMSCs are superior to angiogenic growth factor genes for improving myocardial performance in the mouse model of acute myocardial infarction. Transplantation of MSCs may become the future therapy for acute myocardial infarction for myocardial regeneration.     

A single gene encoding vitellogenin in the sea urchin Strongylocentrotus purpuratus: sequence at the 5'' end.

The synthesis of vitellogenin (yolk protein precursor) in the sea urchin, Strongylocentrotus purpuratus, is unique in that both males and females produce a high level of the protein. In this paper we show that this organism also is unique in possessing only a single vitellogenin gene. Like the genes that encode analogous proteins in vertebrates, the sea urchin gene is large, about 19 kb in length. The sequence surrounding the 5' end of the gene revealed several other similarities to vertebrate vitellogenin genes: the signal sequence is exceptionally short and has a sequence similar to those from frog and chick; there is a canonical TATA box at -32; and there is a sequence closely resembling the estrogen-responsive element at -207.     

Searching and mining visually observed phenotypes of maize mutants

There are thousands of maize mutants, which are invaluable resources for plant research. Geneticists use them to study underlying mechanisms of biochemistry, cell biology, cell development, and cell physiology. To streamline the understanding of such complex processes, researchers need the most current versions of genetic and physical maps, tools with the ability to recognize novel phenotypes or classify known phenotypes, and an intimate knowledge of the biochemical processes generating physiological and phenotypic effects. They must also know how all of these factors change and differ among species, diverse alleles, germplasms, and environmental conditions. While there are robust databases, such as MaizeGDB, for some of these types of raw data, other crucial components are missing. Moreover, the management of visually observed mutant phenotypes is still in its infant stage, let alone the complex query methods that can draw upon high-level and aggregated information to answer the questions of geneticists. In this paper, we address the scientific challenge and propose to develop a robust framework for managing the knowledge of visually observed phenotypes, mining the correlation of visual characteristics with genetic maps, and discovering the knowledge relating to cross-species conservation of visual and genetic patterns. The ultimate goal of this research is to allow a geneticist to submit phenotypic and genomic information on a mutant to a knowledge base and ask, "What genes or environmental factors cause this visually observed phenotype?".     

Whither biological database research?

We consider how the landscape of biological databases may evolve in the future, and what research is needed to realize this evolution. We suggest today's dispersal of diverse resources will only increase as the number and size of those resources, driving the need for semantic interoperability even more strongly. Because the complexity of the questions biologists want answered automatically continues to rapidly escalate, we will need to draw upon high-performance computing resources such as the GRID to process complex queries. Finally, we still need data, and our ways of acquiring and curating data must improve by orders of magnitude.