Inhibition of the growth of cariogenic bacteria in vitro by plant falvanones

Phytoalexins, defensive compounds produced by plants against microbial infections, were purified fromSophora exigua (Leguminosae) and their growth inhibitory effects on oral cariogenic bacteria were determined in vitro. Among three isolated compounds, 5,7,2,4-tetrahydroxy-8-lavandulylflavanone completely inhibited the growth of oral bacteria including primary cariogenic mutans streptococci, other oral streptococci, actinomycetes, and lactobacilli, at concentrations of 1.56 to 6.25 g/ml.     

Collagenolytic activity of eosinophilic granuloma in vitro.

Lytic activity of eosinophilic granuloma and other tumours was studied in vitro on collagen substrate. Collagen degradation was measured through the release of hydroxyproline-rich peptides into the medium. The in vitro lytic action was at a maximum in the case of EG and was correlated with the presence of histiocytic cells.     

Regulating the human HECT E3 ligases

Ubiquitination, the covalent attachment of ubiquitin to proteins, by E3 ligases of the HECT (homologous to E6AP C terminus) family is critical in controlling diverse physiological pathways. Stringent control of HECT E3 ligase activity and substrate specificity is essential for cellular health, whereas deregulation of HECT E3s plays a prominent role in disease. The cell employs a wide variety of regulatory mechanisms to control HECT E3 activity and substrate specificity. Here, we summarize the current understanding of these regulatory mechanisms that control HECT E3 function. Substrate specificity is generally determined by interactions of adaptor proteins with domains in the N-terminal extensions of HECT E3 ligases. These N-terminal domains have also been found to interact with the HECT domain, resulting in the formation of inhibitory conformations. In addition, catalytic activity of the HECT domain is commonly regulated at the level of E2 recruitment and through HECT E3 oligomerization. The previously mentioned regulatory mechanisms can be controlled through protein–protein interactions, post-translational modifications, the binding of calcium ions, and more. Functional activity is determined not only by substrate recruitment and catalytic activity, but also by the type of ubiquitin polymers catalyzed to the substrate. While this is often determined by the specific HECT member, recent studies demonstrate that HECT E3s can be modulated to alter the type of ubiquitin polymers they catalyze. Insight into these diverse regulatory mechanisms that control HECT E3 activity may open up new avenues for therapeutic strategies aimed at inhibition or enhancement of HECT E3 function in disease-related pathways.     

The structure and function of Escherichia coli penicillin-binding protein 3

Escherichia coli penicillin-binding protein PBP3 is a key element in cell septation. It is presumed to catalyse a transpeptidation reaction during biosynthesis of the septum peptidoglycan but, in vitro, its enzymatic activity has only been demonstrated with thiolester analogues of the natural peptide substrate. It has no detectable transglycosylase activity with lipid II as substrate. This tripartite protein is constructed of an N-terminal membrane anchor-containing module that is essential for cell septation, a non-penicillin-binding (n-PB) module of unknown function and a C-terminal penicillin-binding (PB) module exhibiting all the characteristic motifs of penicilloyl serine transferases. The n-PB module, which is required for the folding and stability of the PB module, may provide recognition sites for other cell division proteins. Initiation of septum formation is not PBP3-dependent but rests on the appearance of the FtsZ ring, and is thus penicillin-insensitive. The control of PBP3 activity during the cell cycle is briefly discussed.     

Collagen treated with (+)-catechin becomes resistant to the action of mammalian collagenase

Treatment of radioactively labeled guinea-pig skin soluble collagen or calf skin collagen with the flavonoid (+)-catechin makes the collagen resistant to the action of mammalian collagenase but not to the action of bacterial collagenase. Complete resistance to the action of the mammalian enzyme may be achieved by incubating 0.6 mg of collagen (dry weight) with 0.1 mM (+)-catechin, followed by dialysis to remove the unbound flavonoid. Since incubation of the mammalian enzyme with (+)-catechin does not inhibit its activity, it is postulated that (+)-catechin binds tightly to collagen and modifies its structure sufficiently to make it resistant to enzyme degradation.     

ADAMTS-2 functions as anti-angiogenic and anti-tumoral molecule independently of its catalytic activity

ADAMTS-2 is a metalloproteinase that plays a key role in the processing of fibrillar procollagen precursors into mature collagen molecules by excising the amino-propeptide. We demonstrate that recombinant ADAMTS-2 is also able to reduce proliferation of endothelial cells, and to induce their retraction and detachment from the substrate resulting in apoptosis. Dephosphorylation of Erk1/2 and MLC largely precedes the ADAMTS-2 induced morphological alterations. In 3-D culture models, ADAMTS-2 strongly reduced branching of capillary-like structures formed by endothelial cells and their long-term maintenance and inhibited vessels formation in embryoid bodies (EB). Growth and vascularization of tumors formed in nude mice by HEK 293-EBNA cells expressing ADAMTS-2 were drastically reduced. A similar anti-tumoral activity was observed when using cells expressing recombinant deleted forms of ADAMTS-2, including catalytically inactive enzyme. Nucleolin, a nuclear protein also found to be associated with the cell membrane, was identified as a potential receptor mediating the antiangiogenic properties of ADAMTS-2.     

Collagenase-like peptidase activity in serum from patients with rheumatoid arthritis

Summary The activities of collagenase-like peptidase, estimated by using (succinyl-Gly-Pro-Leu-Gly-Pro-Leu-Gly-Pro)-4-methyl-coumaryl-7-amide as substrate, and of dipeptidyl-aminopeptidase IV were decreased in the sera from patients with rheumatoid arthritis. Both enzymes bring about the degradation of peptides derived from collagen. A significant positive correlation was observed between the activities of the two serum peptidases.     

Behaviour of the prolyl hydroxylase to collagen synthesis in KB cells]

KB cells, which synthetized collagen at a low rate, shown a prolyl hydroxylase activity at the same rate that fibroblast. The relationship between collagen synthesis and prolyl hydroxylase activity in these cells was discussed.     

Collagenase-like peptidase activity in serum from patients with rheumatoid arthritis

The activities of collagenase-like peptidase, estimated by using (succinyl-Gly-Pro-Leu-Gly-Pro-Leu-Gly-Pro)-4-methylcoumaryl-7-amide as substrate, and of dipeptidyl-aminopeptidase IV were decreased in the sera from patients with rheumatoid arthritis. Both enzymes bring about the degradation of peptides derived from collagen. A significant positive correlation was observed between the activities of the two serum peptidases.     

Determination of the kinetic parameters of enzyme inhibition of the K-type mechanism: application to the control of alpha-glucosidase activity of honeybee hemolymph by glucose]

The alpha-glucosidasic activity of emerging honeybees haemolymph is submitted to a feed-back inhibition by glucose, according to a mechanism of the "K" type (competitive). The "resulting affinity-constant" (measured in the presence of the enzyme both with substrate and inhibitor) is linear function of the inhibitor concentration. The affinity constants between enzyme and pure substrate on one hand, and between enzyme and pure inhibitor on the other hand, were determined by means of this relation, which led to respectively equivalent values after determinations under in vitro or in vivo inhibitions.     

The diversity of the DnaJ/Hsp40 family, the crucial partners for Hsp70 chaperones

DnaJ/Hsp40 (heat shock protein 40) proteins have been preserved throughout evolution and are important for protein translation, folding, unfolding, translocation, and degradation, primarily by stimulating the ATPase activity of chaperone proteins, Hsp70s. Because the ATP hydrolysis is essential for the activity of Hsp70s, DnaJ/Hsp40 proteins actually determine the activity of Hsp70s by stabilizing their interaction with substrate proteins. DnaJ/Hsp40 proteins all contain the J domain through which they bind to Hsp70s and can be categorized into three groups, depending on the presence of other domains. Six DnaJ homologs have been identified in Escherichia coli and 22 in Saccharomyces cerevisiae. Genome-wide analysis has revealed 41 DnaJ/Hsp40 family members (or putative members) in humans. While 34 contain the typical J domains, 7 bear partially conserved J-like domains, but are still suggested to function as DnaJ/ Hsp40 proteins. DnaJA2b, DnaJB1b, DnaJC2, DnaJC20, and DnaJC21 are named for the first time in this review; all other human DnaJ proteins were dubbed according to their gene names, e.g. DnaJA1 is the human protein named after its gene DNAJA1. This review highlights the progress in studying the domains in DnaJ/Hsp40 proteins, introduces the mechanisms by which they interact with Hsp70s, and stresses their functional diversity. Received 27 April 2006; received after revision 5 June 2006; accepted 19 July 2006     

Lysyl oxidase: an oxidative enzyme and effector of cell function

Lysyl oxidase (LOX) oxidizes the side chain of peptidyl lysine converting specific lysine residues to residues of alpha-aminoadipic-delta-semialdehyde. This posttranslational chemical change permits the covalent crosslinking of the component chains of collagen and those of elastin, thus stabilizing the fibrous deposits of these proteins in the extracellular matrix. Four LOX-like (LOXL) proteins with varying degrees of similarity to LOX have been described, constituting a family of related proteins. LOX is synthesized as a preproprotein which emerges from the cell as proLOX and then is processed to the active enzyme by proteolysis. In addition to elastin and collagen, LOX can oxidize lysine within a variety of cationic proteins, suggesting that its functions extend beyond its role in the stabilization of the extracellular matrix. Indeed, recent findings reveal that LOX and LOXL proteins markedly influence cell behavior including chemotactic responses, proliferation, and shifts between the normal and malignant phenotypes.     

Regulation of chondrocyte differentiation by ADAMTS-12 metalloproteinase depends on its enzymatic activity

ADAMTS-12, a metalloproteinase that belongs to ADAMTS family, is strongly upregulated during chondrogenesis and demonstrates prominent expression in the growth plate chondrocytes. ADAMTS-12 potently inhibits chondrocyte differentiation, as revealed by altered expression of both early and later genes critical for chondrogenesis. In addition, ADAMTS-12-mediated inhibition of chondrogenesis depends on its enzymatic activity, since its point mutant lacking enzymatic activity completely loses this activity. Furthermore, the C-terminal four thrombospondin motifs known to bind COMP substrate is necessary for its full proteolytic activity and inhibition of chondrocyte differentiation. Mechanism studies demonstrate that ADAMTS-12 induces PTHrP, whereas it inhibits IHH during chondrogenesis. Furthermore, PTHrP induces ADAMTS-12 and ADAMTS-12 is hardly detectable in PTHrP-/-growth plate chondrocytes. Importantly, knocking down ADAMTS-12 mRNA levels or blocking ADAMTS-12 activity almost abolishes the PTHrP-mediated inhibition of type X collagen expression. Collectively, these findings demonstrate that ADAMTS-12, a downstream molecule of PTHrP signaling, is a novel regulator of chondrogenesis. X. H. Bai, D.W. Wang: These two authors contributed equally to this work.     

Unveiling molecular mechanisms of bacterial surface proteins: <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> as a model organism for structural studies

Bacteria present a variety of molecules either on their surface or in a cell-free form. These molecules take part in numerous processes in the interactions with their host, with its tissues and other molecules. These molecules are essential to bacterial pathogenesis either during colonization or the spread/invasion stages, and most are virulence factors. This review is focused on such molecules using Streptococcus pneumoniae, a Gram-positive bacterium, as an example. Selected surface proteins are introduced, their structure described, and, whenever available, their mechanisms of function on an atomic level are explained. Such mechanisms for hyaluronate lyase, pneumococcal surface protein A, pneumolysin, histidine-triad and fibronectin-binding proteins are discussed. Elucidation of molecular mechanisms of virulence factors is essential for the understanding of bacteria and their functional properties. Structural biology appears pivotal for these studies, as structural and mechanistic insights facilitate rational approach to the development of new treatments. Received 12 March 2007; received after revision 28 June 2007; accepted 18 July 2007     

From MDR to MXR: new understanding of multidrug resistance systems, their properties and clinical significance

The ATP binding cassette (ABC) superfamily of membrane transporters is one of the largest protein classes known, and counts numerous proteins involved in the trafficking of biological molecules across cell membranes. The first known human ABC transporter was P-glycoprotein (P-gp), which confers multidrug resistance (MDR) to anticancer drugs. In recent years, we have obtained an increased understanding of the mechanism of action of P-gp as its ATPase activity, substrate specificity and pharmacokinetic interactions have been investigated. This review focuses on the functional characterization of P-gp, as well as other ABC transporters involved in MDR: the family of multidrug-resistance-associated proteins (MRP1-7), and the recently discovered ABC half-transporter MXR (also known as BCRP, ABCP and ABCG2). We describe recent progress in the analysis of protein structure-function relationships, and consider the conceptual problem of defining and identifying substrates and inhibitors of MDR. An in-depth discussion follows of how coupling of nucleotide hydrolysis to substrate transport takes place, and we propose a scheme for the mechanism of P-gp function. Finally, the clinical correlations, both for reversal of MDR in cancer and for drug delivery, are discussed.     

Collagen treated with (+)-catechin becomes resistant to the action of mammalian collagenase

Summary Treatment of radioactively labeled guinea-pig skin soluble collagen or calf skin collagen with the flavonoid (+)-catechin makes the collagen resistant to the action of mammalian collagenase but not to the action of bacterial collagenase. Complete resistance to the action of the mammalian enzyme may be achieved by incubating 0.6 mg of collagen (dry weight) with 0.1 mM (+)-catechin, followed by dialysis to remove the unbound flavonoid. Since incubation of the mammalian enzyme with (+)-catechin does not inhibit its activity, it is postulated that (+)-catechin binds tightly to collagen and modifies its structure sufficiently to make it resistant to enzyme degradation.Acknowledgments. This work was supported by grants from the Easter Seal Research Foundation of the National Easter Seal Society for Crippled Children and Adults (R-7821), and the National Institutes of Health (HL-20447). (+)-Catechin was a generous gift from Zyma SA, CH-1260 Nyon, Switzerland.     

Hexosaminidase and alkaline phosphatase activities in articular chondrocytes and relationship to cell culture conditions.

Hexosaminidase and alkaline phosphatase activities in rabbit articular chondrocytes have been studied under different cell culture conditions. Chondrocytes were cultured in monolayer primary culture, monolayer subcultured to the fifth passage (in vitro aging) and cultured within a collagen gel; enzymatically released cartilage cells were used as control. Under these conditions, the two enzymes behave quite differently in relationship to alteration of the chondrocyte phenotype in culture. Increased lysosomal hexosaminidase activity could be considered to be a marker of the dedifferentiated phenotype in monolayer subculture; membrane alkaline phosphatase activity could be used as a marker of non-proliferating cells.     

Hexosaminidase and alkaline phosphatase activities in articular chondrocytes and relationship to cell culture conditions

Hexosaminidase and alkaline phosphatase activities in rabbit articular chondrocytes have been studied under different cell culture conditions. Chondrocytes were cultured in monolayer primary culture, monolayer subcultured to the fifth passage (in vitro aging) and cultured within a collagen gel; enzymatically released cartilage cells were used as control. Under these conditions, the two enzymes behave quite differently in relationship to alteration of the chondrocyte phenotype in culture. Increased lysosomal hexosaminidase activity could be considered to be a marker of the dedifferentiated phenotype in monolayer subculture; membrane alkaline phosphatase activity could be used as a marker of non-proliferating cells.     

The growth of human fibroblasts and A431 epidermoid carcinoma cells on gamma-irradiated human amnion collagen substrata

Summary Human fibroblasts and A431 human epidermoid carcinoma cells were cultured on gamma-irradiated human amnion collagen as well as on plastic dishes and non-irradiated collagen coated dishes. The morphology, attachment, growth and short-term cytotoxicity of these culture conditions have been determined. Both irradiated and non-irradiated amnion collagen enhanced the attachment and proliferation of fibroblasts as compared to the plastic dishes. No differences in these properties were observed for A431 cells cultured on irradiated collagen when compared with culture on non-irradiated collagen substrates. Cytotoxicity assays showed that irradiated and non-irradiated collagens were not cytotoxic for either fibroblasts or A431 cells. The results demonstrated that amnion collagen irradiated at doses of 0.25–2.0 Mrads is optimal for cell growth.     

Substrate ambiguity among the nudix hydrolases: biologically significant, evolutionary remnant, or both?

Many members of the nudix hydrolase family exhibit considerable substrate multispecificity and ambiguity, which raises significant issues when assessing their functions in vivo and gives rise to errors in database annotation. Several display low antimutator activity when expressed in bacterial tester strains as well as some degree of activity in vitro towards mutagenic, oxidized nucleotides such as 8-oxo-dGTP. However, many of these show greater activity towards other nucleotides such as ADP-ribose or diadenosine tetraphosphate (Ap₄A). The antimutator activities have tended to gain prominence in the literature, whereas they may in fact represent the residual activity of an ancestral antimutator enzyme that has become secondary to the more recently evolved major activity after gene duplication. Whether any meaningful antimutagenic function has also been retained in vivo requires very careful assessment. Then again, other examples of substrate ambiguity may indicate as yet unexplored regulatory systems. For example, bacterial Ap₄A hydrolases also efficiently remove pyrophosphate from the 5′ termini of mRNAs, suggesting a potential role for Ap₄A in the control of bacterial mRNA turnover, while the ability of some eukaryotic mRNA decapping enzymes to degrade IDP and dIDP or diphosphoinositol polyphosphates (DIPs) may also be indicative of new regulatory networks in RNA metabolism. DIP phosphohydrolases also degrade diadenosine polyphosphates and inorganic polyphosphates, suggesting further avenues for investigation. This article uses these and other examples to highlight the need for a greater awareness of the possible significance of substrate ambiguity among the nudix hydrolases as well as the need to exert caution when interpreting incomplete analyses.