Differential alphav integrin-mediated Ras-ERK signaling during two pathways of angiogenesis

Antagonists of alphavbeta3 and alphavbeta5 disrupt angiogenesis in response to bFGF and VEGF, respectively. Here, we show that these alphav integrins differentially contribute to sustained Ras-extracellular signal-related kinase (Ras-ERK) signaling in blood vessels, a requirement for endothelial cell survival and angiogenesis. Inhibition of FAK or alphavbeta5 disrupted VEGF-mediated Ras and c-Raf activity on the chick chorioallantoic membrane, whereas blockade of FAK or integrin alphavbeta3 had no effect on bFGF-mediated Ras activity, but did suppress c-Raf activation. Furthermore, retroviral delivery of active Ras or c-Raf promoted ERK activity and angiogenesis, which anti-alphavbeta5 blocked upstream of Ras, whereas anti-alphavbeta3 blocked downstream of Ras, but upstream of c-Raf. The activation of c-Raf by bFGF/alphavbeta3 not only depended on FAK, but also required p21-activated kinase-dependent phosphorylation of serine 338 on c-Raf, whereas VEGF-mediated c-Raf phosphorylation/activation depended on Src, but not Pak. Thus, integrins alphavbeta3 and alphavbeta5 differentially regulate the Ras-ERK pathway, accounting for distinct vascular responses during two pathways of angiogenesis.     

Association of the calpain/calpastatin network with subcellular organelles

The calcium-activated cysteine protease calpain is intimately involved in modulating cell adhesion and migration. The two ubiquitous isoforms of this protease, calpain I and II, are considered to be cytosolic proteins that can translocate to both focal complexes/adhesions or the plasma membrane. Using confocal microscopy and isopycnic density centrifugation, the results demonstrate that calpain I and II, the 30kDa regulatory subunit, and calpastatin associate with the endoplasmic reticulum and Golgi apparatus. Confocal microscopy reveals that calpain II colocalizes with the subcellular proteins calnexin and Rab6 in cells bound to laminin. To further verify this association, cell lysates prepared from laminin stimulated and unstimulated cells were subjected to isopycnic density centrifugation. The results reveal an increased association of calpain I, II, calpastatin, and the 30kDa regulatory subunit with the endoplasmic reticulum and Golgi apparatus as evidenced by their position in the gradient relative to calnexin, Rab6, caveolin, and beta1 integrin after laminin stimulation. This correlates with the accumulation of inducible calpain activity at the endoplasmic reticulum-Golgi apparatus interface. Further experiments established that calpain II colocalizes with phosphatidylinositol 4,5-bisphosphate. Finally, calpain II associates with membrane lipid rafts. These results provide new insights into how the calpain/calpastatin network is spatially and temporally regulated in cells binding to the extracellular matrix.     

Identification of a lipopolysaccharide alpha-2,3-sialyltransferase from Haemophilus influenzae

We have identified a gene for the addition of N-acetylneuraminic acid (Neu5Ac) in an alpha-2,3-linkage to a lactosyl acceptor moiety of the lipopolysaccharide (LPS) of the human pathogen Haemophilus influenzae. The gene is one that was identified previously as a phase-variable gene known as lic3A. Extracts of H. influenzae, as well as recombinant Escherichia coli strains producing Lic3A, demonstrate sialyltransferase activity in assays using synthetic fluorescent acceptors with a terminal galactosyl, lactosyl or N-acetyl-lactosaminyl moiety. In the RM118 strain of H. influenzae, Lic3A activity is modulated by the action of another phase-variable glycosyltransferase, LgtC, which competes for the same lactosyl acceptor moiety. Structural analysis of LPS from a RM118:lgtC mutant and the non-typeable strain 486 using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy confirmed that the major sialylated species has a sialyl-alpha-(2-3)-lactosyl extension off the distal heptose. This sialylated glycoform was absent in strains containing a lic3A gene disruption. Low amounts of sialylated higher molecular mass glycoforms were present in RM118:lgtC lic3A, indicating the presence of a second sialyltransferase. Lic3A mutants of H. influenzae strains show reduced resistance to the killing effects of normal human serum. Lic3A, encoding an alpha-2,3-sialyltransferase activity, is the first reported phase-variable sialyltransferase gene.     

Characterization of the Japanese pufferfish (Takifugu rubripes) T-cell receptor α locus reveals a unique genomic organization

Polymerase chain reactions with degenerate V gene segment primers were used to isolate the putative T-cell receptor alpha-chain gene (TCRA) from Japanese pufferfish (Takifugu rubripes). The putative TCRA chain cDNA is composed of an N-terminus leader peptide followed by the variable region and the constant region. The variable portion of the TCRA gene is encoded by V and J gene segments separated in the germline. As in mammals, the V-J junction sequences are GC rich and highly diversified. Amino acid residues that are required to maintain the function and structural integrity of the TCRA polypeptide, including the conserved Trp-Tyr-Lys and Tyr-Tyr-Cys motifs in the V gene segments, the Lys-Leu-X-Phe-Gly-X-Gly-Thr-X-Leu motif in the J gene segment, the three cysteine residues in the constant region and the charged residues in the transmembrane region are all preserved in the pufferfish. These conserved features suggest that the TCRA gene families in fish and mammals have evolved from a common ancestor.     

Functional genomics of pathogenic bacteria

Microbial diseases remain the commonest cause of global mortality and morbidity. Automated-DNA sequencing has revolutionized the investigation of pathogenic microbes by making the immense fund of information contained in their genomes available at reasonable cost. The challenge is how this information can be used to increase current understanding of the biology of commensal and virulence behaviour of pathogens with particular emphasis on in vivo function and novel approaches to prevention. One example of the application of whole-genome-sequence information is afforded by investigations of the pathogenic role of Haemophilus influenzae lipopolysaccharide and its candidacy as a vaccine.     

Series of incidents of Listeria monocytogenes non-invasive febrile gastroenteritis involving ready-to-eat meats

AIMS: A series of cases and outbreaks of febrile noninvasive gastrointestinal disease involving 31 identified cases was investigated in terms of the numbers and types of Listeria monocytogenes present in the suspect foods (ready-to-eat meats) and clinical samples from cases. METHODS AND RESULTS: Foods and faecal samples involved in the incidents were tested for the presence and number of L. monocytogenes. Isolates were typed by macrorestriction analysis using pulsed-field gel electrophoresis. The foods contained high levels of L. monocytogenes, in one case 1.8 x 10(7) g-1. Faecal samples contained L. monocytogenes for up to 15 d after the contaminated food was consumed. All isolates from the food and faecal samples were of serotype 1/2 and were indistinguishable from one another by macrorestriction typing. CONCLUSIONS: It is likely that the meats were contaminated either during their manufacture after they had been cooked or by underprocessing. The long shelf lives on these products would have allowed the contaminating L. monocytogenes to grow to the high numbers measured in this study, causing food poisoning as described. SIGNIFICANCE AND IMPACT OF THE STUDY: Outbreaks of febrile noninvasive listeriosis are relatively rare. This report adds ready-to-eat meats to the range of foods that have acted as vehicles for such outbreaks.     

Identification and characterization of two novel low-molecular-weight dual specificity phosphatases

We have cloned and characterized two novel human low molecular weight dual specificity phosphatases (LMW-DSPs). Both genes are expressed exclusively in the testis, but are not altered in any of several disease states examined. Transfection into COS cells indicates that both proteins are expressed in the nucleus and the cytoplasm. Both proteins are able to dephosphorylate the phosphotyrosine analog pNPP in vitro and can be inhibited by sodium orthovanadate. In vitro experiments also demonstrate that both DSPs can dephosphorylate single and diphosphorylated synthetic MAPK peptides, with preference for the phosphotyrosine and diphosphorylated forms over phosphothreonine. However, when co-transfected with MAPKs into COS cells, the novel DSPs exhibited no detectable in vivo activity against MAPKs under our conditions. Our data suggest that these novel LMW-DSPs might belong to a new subclass of testis-specific proteins that act independently of the MAPK signal transduction cascade and do not depend on N-terminal docking regions for substrate binding.     

Examination of the kinetic mechanism of mitogen-activated protein kinase activated protein kinase-2

The kinetic mechanism of mitogen-activated protein kinase activated protein kinase-2 (MAPKAPK2) was investigated using a peptide (LKRSLSEM) based on the phosphorylation site found in serum response factor (SRF). Initial velocity studies yielded a family of double-reciprocal lines that appear parallel and indicative of a ping-pong mechanism. The use of dead-end inhibition studies did not provide a definitive assignment of a reaction mechanism. However, product inhibition studies suggested that MAPKAPK2 follows an ordered bi-bi kinetic mechanism, where ATP must bind to the enzyme prior to the SRF-peptide and the phosphorylated product is released first, followed by ADP. In agreement with these latter results, surface plasmon resonance measurements demonstrate that the binding of the inhibitor peptide to MAPKAPK2 requires the presence of ATP. Furthermore, competitive inhibitors of ATP, adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP) and a staurosporine analog (K252a), can inhibit this ATP-dependent binding providing further evidence that the peptide substrate binds preferably to the E:ATP complex.     

Predicting demographic group structures based on DNA sequence data

The ability to infer relationships between groups of sequences, either by searching for their evolutionary history or by comparing their sequence similarity, can be a crucial step in hypothesis testing. Interpreting relationships of human immunodeficiency virus type 1 (HIV-1) sequences can be challenging because of their rapidly evolving genomes, but it may also lead to a better understanding of the underlying biology. Several studies have focused on the evolution of HIV-1, but there is little information to link sequence similarities and evolutionary histories of HIV-1 to the epidemiological information of the infected individual. Our goal was to correlate patterns of HIV-1 genetic diversity with epidemiological information, including risk and demographic factors. These correlations were then used to predict epidemiological information through analyzing short stretches of HIV-1 sequence. Using standard phylogenetic and phenetic techniques on 100 HIV-1 subtype B sequences, we were able to show some correlation between the viral sequences and the geographic area of infection and the risk of men who engage in sex with men. To help identify more subtle relationships between the viral sequences, the method of multidimensional scaling (MDS) was performed. That method identified statistically significant correlations between the viral sequences and the risk factors of men who engage in sex with men and individuals who engage in sex with injection drug users or use injection drugs themselves. Using tree construction, MDS, and newly developed likelihood assignment methods on the original 100 samples we sequenced, and also on a set of blinded samples, we were able to predict demographic/risk group membership at a rate statistically better than by chance alone. Such methods may make it possible to identify viral variants belonging to specific demographic groups by examining only a small portion of the HIV-1 genome. Such predictions of demographic epidemiology based on sequence information may become valuable in assigning different treatment regimens to infected individuals.