A chemotactic peptide from laminin alpha 5 functions as a regulator of inflammatory immune responses via TNF alpha-mediated signaling

Tissue injury triggers inflammatory responses that may result in release of degradation products or exposure of cryptic domains of extracellular matrix components. Previously, we have shown that a cryptic peptide (AQARSAASKVKVSMKF) in the alpha-chain of laminin-10 (alpha5beta1gamma1), a prominent basement membrane component, is chemotactic for both neutrophils (PMNs) and macrophages (Mphis) and induces matrix metalloproteinase-9 (MMP-9) production. To determine whether AQARSAASKVKVSMKF has additional effects on inflammatory cells, we performed microarray analysis of RNA from RAW264.7 Mphis stimulated with AQARSAASKVKVSMKF. Several cytokines and cytokine receptors were increased >3-fold in response to the laminin alpha5 peptide. Among these were TNF-alpha and one of its receptors, the p75 TNFR (TNFR-II), increasing 3.5- and 5.7-fold, respectively. However, the peptide had no effect on p55 TNFR (TNFR-I) expression. Corroborating the microarray data, the protein levels of TNF-alpha and TNFR-II were increased following stimulation of RAW264.7 cells with AQARSAASKVKVSMKF. In addition, we determined that the production of TNF-alpha and TNFR-II in response to AQARSAASKVKVSMKF preceded the production of MMP-9. Furthermore, using primary Mphis from mice deficient in TNFR-I, TNFR-II, or both TNF-alpha receptors (TNFRs), we determined that AQARSAASKVKVSMKF induces MMP-9 expression by Mphis through a pathway triggered by TNFR-II. However, TNF-alpha signaling is not required for AQARSAASKVKVSMKF-induced PMN release of MMP-9 or PMN emigration. These data suggest that interactions of inflammatory cells with basement membrane components may orchestrate immune responses by inducing expression of cytokines, recruitment of inflammatory cells, and release of proteinases.     

Amino acid sequence requirements in the human IgA1 hinge for cleavage by streptococcal IgA1 proteases

All the IgA1 proteases of the different pathogenic species of Streptococcus cleave the hinge of the alpha chain of human IgA1 only at one proline-threonine peptide bond. In order to study the importance of these amino acids for cleavage, several hinge mutant recombinant IgA1 antibodies were constructed. The mutations were found to be without major effect upon the structure or functional abilities of the antibodies. However, they had a major effect upon their sensitivity to cleavage by some of the IgA1 proteases.     

Microbial aspects of atrazine degradation in natural environments

The potential toxicity of thes-triazine herbicide atrazine motivates continuous bioremediation-directed research. Several indigenous soilatrazine-catabolizing microbialassociations and monocultures have been enriched/isolated from compromised sites. Of these, Pseudomonas sp. strain ADP has become a reference strain and has been used to elucidate sequences of the catabolic enzymes atzA, atzB, atzCand atzD involvedin one aerobic degradation pathway and develop probes for the genes which encode these enzymes. Despite this, hitherto unknown or novel microorganisms, with unique sequences and different enzyme-mediated operative pathways, warrant continued investigations for effective site bioremediation. Also, the sustained effectiveness of natural attenuation must be demonstrated continually so regular site evaluations and results analyses, despite the limitations of chemical extraction methodologies, are crucial practices. For both directed and intrinsic bioremediation monitoring, traditional microbial association studies must be complemented by more advanced physiological and molecular approaches. The occurrence of catabolic plasmids, in particular, should be probed with DNA hybridization techniques. Also, PCR-DGGEand subsequent new sequenceelucidation should be used prior to developing new primers for DNA sequences encoding novel catabolic enzymes, and for hybridization probe development, to establish the degradative potential of a compromised site, or adoption of FISH to, for example, monitor bioaugmented remediation.     

Evidence of mitochondrial involvement in the transduction of signals required for the induction of genes associated with pathogen attack and senescence

Using the mRNA differential display technique, seven cDNAs have been isolated that are rapidly induced when cultured tobacco (Nicotiana tabacum) cells are treated with the mitochondrial electron transport inhibitor antimycin A (AA). Interestingly, six of the cDNAs show distinct similarity to genes known to be induced by processes that involve programmed cell death (PCD), such as senescence and pathogen attack. All of the cDNAs as well as Aox1, a gene encoding the alternative oxidase, were found to also be strongly induced by H2O2 and salicylic acid (SA). AA, H2O2 and SA treatment of tobacco cells caused a rapid rise in intracellular ROS accumulation that, when prevented by antioxidant treatment, resulted in inhibition of gene induction. Besides AA, both H2O2 and SA were found to disrupt normal mitochondrial function resulting in decreased rates of electron transport and a lowering of cellular ATP levels. Furthermore, the pre-treatment of tobacco cells with bongkrekic acid, a known inhibitor of the mitochondrial permeability transition pore in animal cells, was found to completely block gene induction when AA, H2O2 or SA were subsequently added. These findings suggest that the mitochondrion may serve an important role in conveying intracellular stress signals to the nucleus, leading to alterations in gene expression.     

Binding of the transition state analog MgADP-fluoroaluminate to F1-ATPase

Escherichia coli F1-ATPase from mutant betaY331W was potently inhibited by fluoroaluminate plus MgADP but not by MgADP alone. beta-Trp-331 fluorescence was used to measure MgADP binding to catalytic sites. Fluoroaluminate induced a very large increase in MgADP binding affinity at catalytic site one, a smaller increase at site two, and no effect at site three. Mutation of either of the critical catalytic site residues beta-Lys-155 or beta-Glu-181 to Gln abolished the effects of fluoroaluminate on MgADP binding. The results indicate that the MgADP-fluoroaluminate complex is a transition state analog and independently demonstrate that residues beta-Lys-155 and (particularly) beta-Glu-181 are important for generation and stabilization of the catalytic transition state. Dicyclohexylcarbodiimide-inhibited enzyme, with 1% residual steady-state ATPase, showed normal transition state formation as judged by fluoroaluminate-induced MgADP binding affinity changes, consistent with a proposed mechanism by which dicyclohexylcarbodiimide prevents a conformational interaction between catalytic sites but does not affect the catalytic step per se. The fluorescence technique should prove valuable for future transition state studies of F1-ATPase.     

Crystallization of 5-aminolaevulinic acid dehydratase from Escherichia coli and Saccharomyces cerevisiae and preliminary X-ray characterization of the crystals.

5-Aminolaevulinic acid dehydratase (ALAD) catalyzes the formation of porphobilinogen from two molecules of 5-aminolaevulinic acid. Both Escherichia coli and Saccharomyces cerevisiae ALADs are homo-octameric enzymes which depend on Zn2+ for catalytic activity and are potently inhibited by lead ions. The E. coli enzyme crystallized in space group I422 (unit cell dimensions a = b = 130.7 A, c = 142.4 A). The best crystals were obtained in the presence of the covalently bound inhibitor laevulinic acid. The yeast enzyme (expressed in E. coli) crystallized in the same space group (I422) but with a smaller unit cell volume (a = b = 103.7 A, c = 167.7 A). High resolution synchrotron data sets were obtained from both E. coli and yeast ALAD crystals by cryocooling to 100 K.     

Preliminary investigation of the influence of pH on the solid-state refuse methanogenic fermentation

Laboratory refuse columns and bottle cultures were used to examine the responses of the interrelated metabolic processes of acid metabolism, solvent metabolism and methanogenesis of the solid-state refuse fermentation to single initial additions of exogenous buffering capacity. Refuse samples poised at neutral and alkaline pH values, by the addition of 0.2 mol/1 phosphate buffer, were found to promote acid-ogenesis to such an extent that methanogenesis was inhibited. The same inhibition was also apparent in the presence of acid pH regimes which were initially characterized by the accumulation of solvents. The possible implications of controlling pH by adding buffer to promote and exploit the refuse fermentation are discussed.     

Monoclonal antibodies identify a group of nuclear pore complex glycoproteins

Using monoclonal antibodies we identified a group of eight polypeptides of rat liver nuclear envelopes that have common epitopes. Most or all of these proteins are structurally distinct, as shown by tryptic peptide mapping and analysis with polyclonal antibodies. While these polypeptides are relatively tightly bound to nuclear membranes, only one is an integral membrane protein. The eight antigens cofractionate with the nuclear pore complex under various conditions of ionic strength and detergent. It can be seen by immunofluorescence microscopy that the monoclonal antibodies reacting with these antigens stain the nuclear surface of interphase cells in a finely punctate pattern. When the nuclear envelope is disassembled and subsequently reformed during mitosis, the proteins are reversibly dispersed throughout the cytoplasm in the form of minute foci. By EM immunogold localization on isolated nuclear envelopes, the monoclonal antibodies label exclusively the nuclear pore complex, at both its nucleoplasmic and cytoplasmic margins. Considered together, our biochemical and localization data indicate that the eight nuclear envelope polypeptides are pore complex components. As shown in the accompanying paper (Holt, G. D., C. M. Snow, A. Senior, R. S. Haltiwanger, L. Gerace, and G. W. Hart, J. Cell Biol., 104:1157-1164) these eight polypeptides contain a novel form of glycosylation, O-linked N-acetylglucosamine. The relative abundance and disposition of these O-linked glycoproteins in the pore complex are consistent with their having a role in nucleocytoplasmic transport.     

A novel fimbrial haemagglutinin produced by a strain of Salmonella of serotype Salinatis

A strain of Salmonella of serotype Salinatis, that produced a mannose-resistant and eluting haemagglutinin (MREHA) when cultured at 37 degrees C but not at 18 degrees C, was examined by electron microscopy after negative staining. Production of this MREHA, previously described as being non-fimbrial, was correlated with the presence of thin fimbriae which had an external diameter of 3.6 nm. The purified Salinatis thin fimbriae had an estimated Mr of 19 kDa. This fimbrial MREHA was not produced by strains of the antigenically related serotypes Duisburg and Sandiego.