Identification of early microbial colonizers in human dental biofilm

AIMS: To elucidate the first colonizers within in vivo dental biofilm and to establish potential population shifts that occur during the early phases of biofilm formation. METHODS AND RESULTS: A 'checkerboard' DNA-DNA hybridization assay was employed to identify 40 different bacterial strains. Dental biofilm samples were collected from 15 healthy subjects, 0, 2, 4 and 6 h after tooth cleaning and the composition of these samples was compared with that of whole saliva collected from the same individuals. The bacterial distribution in biofilm samples was distinct from that in saliva, confirming the selectivity of the adhesion process. In the very early stages, the predominant tooth colonizers were found to be Actinomyces species. The relative proportion of streptococci, in particular Streptococcus mitis and S. oralis, increased at the expense of Actinomyces species between 2 and 6 h while the absolute level of Actinomyces remained unaltered. Periodontal pathogens such as Tannerella forsythensis(Bacteroides forsythus), Porphyromonas gingivalis and Treponema denticola as well as Actinobacillus actinomycetemcomitans were present in extremely low levels at all the examined time intervals in this healthy group of subjects. CONCLUSION: The data provide a detailed insight into the bacterial population shifts occurring within the first few hours of biofilm formation and show that the early colonizers of the tooth surface predominantly consist of beneficial micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: The early colonizers of dental plaque are of great importance in the succession stages of biofilm formation and its overall effect on the oral health of the host.     

Mechanosensing role of caveolae and caveolar constituents in human endothelial cells

A variety of evidence suggests that endothelial cell functions are impaired in altered gravity conditions. Nevertheless, the effects of hypergravity on endothelial cell physiology remain unclear. In this study we cultured primary human endothelial cells under mild hypergravity conditions for 24-48 h, then we evaluated the changes in cell cycle progression, caveolin1 gene expression and in the caveolae status by confocal microscopy. Moreover, we analyzed the activity of enzymes known to be resident in caveolae such as endothelial nitric oxide synthase (eNOS), cycloxygenase 2 (COX-2), and prostacyclin synthase (PGIS). Finally, we performed a three-dimensional in vitro collagen gel test to evaluate the modification of the angiogenic responses. Results indicate that hypergravity shifts endothelial cells to G(0)/G(1) phase of cell cycle, reducing S phase, increasing caveolin1 gene expression and causing an increased distribution of caveolae in the cell interior. Hypergravity also increases COX-2 expression, nitric oxide (NO) and prostacyclin (PGI2) production, and inhibits angiogenesis as evaluated by 3-D collagen gel test, through a pathway not involving apoptosis. Thus, endothelial cell caveolae may be responsible for adaptation of endothelium to hypergravity and the mechanism of adaptation involves an increased caveolin1 gene expression coupled to upregulation of vasodilators as NO and PGI2.     

A HERG current sustains a cardiac-type action potential in neuroblastoma S cells

From the adrenergic SH-SY5Y human neuroblastoma clone, we isolated a subclone (21S) endowed with a glial-oriented phenotype. At difference from the parental clone, 21S cells responded to depolarizing stimuli with overshooting action potentials, whose repolarization phase was composed of an initial rapid episode, followed by a long-lasting plateau and a slow return to the resting potential (V(REST)). The action potential depolarization phase was sustained by a TTX-sensitive Na(+) current, while the first repolarizing episode was produced by the scanty delayed rectifier potassium current (I(KDR)) expressed in 21S cells. The bulk of repolarization, including the after-hyperpolarization, was sustained by the human eag related (HERG) potassium current (I(HERG)) that also governs V(REST) in 21S cells. This double role of I(HERG), together with the poor expression of I(KDRs), represents a novel finding in electrophysiology, as well as gives a clue to identify a new excitable element of the complex cellular population of neuroblastoma.     

Characterization of an ectonucleoside triphosphate diphosphohydrolase 1 activity in alkaline phosphatase-depleted rat osseous plate membranes: possible functional involvement in the calcification process

An ectonucleoside triphosphate diphosphohydrolase 1 (NTPDase1) activity present in alkaline phosphatase-depleted rat osseous plate membranes, obtained 14 days after implantation of demineralized bone particles in the subcutaneous tissue of Wistar rats, was characterized. At pH 7.5, NTPDase1 hydrolyzed nucleotide triphosphates at rates 2.4-fold higher than those of nucleotide diphosphates, while the hydrolysis of nucleotide monophosphates and non-nucleotide phosphates was negligible. NTPDase 1 hydrolyzed ATP and ADP following Michaelis-Menten kinetics with V=1278.7+/-38.4 nmol Pi/min/mg and K(M)=83.3+/-2.5 microM and V=473.9+/-18.9 nmol Pi/min/mg and K(M)=150.6+/-6.0 microM, respectively, but in the absence of magnesium and calcium ions, ATP or ADP hydrolysis was negligible. The stimulation of the NTPDase1 by calcium (V=1084.7+/-32.5 nmol Pi/min/mg; and K(M)=377.8+/-11.3 microM) and magnesium (V=1367.2+/-41.0 nmol Pi/min/mg and K(M)=595.3+/-17.8 microM) ions suggested that each ion could replace the other during the catalytic cycle of the enzyme. Oligomycin, ouabain, bafilomycin A(1), theophylline, thapsigargin, ethacrynic acid, P(1),P(5)-(adenosine-5')-pentaphosphate and omeprazole had negligible effects on the hydrolysis of ATP and ADP by NTPDase1. However, suramin and sodium azide were effective inhibitors of ATP and ADP hydrolysis.To our knowledge this is the first report suggesting the presence of NTPDase1 in rat osseous plate membranes. Considering that the ectonucleoside triphosphate diphosphohydrolase family of enzymes participates in many regulatory functions, such as response to hormones, growth control, and cell differentiation, the present observations raise interesting questions about the participation of this activity in the calcification process.     

Selective inhibition of trypsins by insect peptides: role of P6-P10 loop

PMP-D2 and HI, two peptides from Locusta migratoria, were shown to belong to the family of tight-binding protease inhibitors. However, they interact weakly with bovine trypsin (K(i) around 100 nM) despite a trypsin-specific Arg at the primary specificity site P1. Here we demonstrate that they are potent inhibitors of midgut trypsins isolated from the same insect and of a fungal trypsin from Fusarium oxysporum (K(i) 相似文献   

W206R]-procaspase 3: an inactivatable substrate for caspase 8.

We report here the cloning and high-level expression of a soluble proform of human caspase 3 (Ser(24)-H(277)) engineered to contain a short stretch of N-terminal sequence (MTISDSPREQD) from the prosegment of procaspase 8 and a C-terminal heptahistidine tag. The precursor protein isolated from extracts of recombinant Escherichia coli by immobilized metal-ion affinity chromatography was predominantly unprocessed and migrated as a 32-kDa polypeptide on sodium dodecyl sulfate-polyacrylamide gels. Incubation of this protein with recombinant human caspase 8 produced fragments characteristic of the properly processed caspase 3, but the product was inactive. Amino-terminal sequence analysis of the caspase 3 polypeptides proved that caspase 8 had specifically cleaved the Asp(175)-Ser(176) bond to yield the expected p18 and p12 subunits, with partial cleavage at the Asp(28)-Ser(29) bond to release the prosegment. The lack of caspase 3 activity was found to be the result of a fortuitous mutation in which Trp(206) in the S4 subsite was replaced by arginine (W206R). This mutant procaspase 3, which we call m-pro3, serves as a useful reagent with which to test the efficacy of caspase 8 inhibitors in blocking processing of the natural polypeptide substrate of this enzyme and may be valuable as a source of "proenzyme" for crystallographic analysis.     

Inhibition of pig liver and Zea mays L. polyamine oxidase: a comparative study

Polyamine oxidase (PAO) is involved in polyamine metabolism and production of hydrogen peroxide in animal and plants, thus representing a key system in development and programmed cell death. In the present study, the inhibitory effect of amiloride, p-aminobenzamidine, clonidine, 4',6-diamidino-2-phenyl-indole (DAPI), gabexate mesylate, guazatine, and N,N'-bis(2,3-butadienyl)-1,4-butane-diamine (MDL72527) on the catalytic activity of pig liver and Zea mays L. PAO, Lens culinaris L. and Pisum sativum L. and swine kidney copper amine oxidase, bovine trypsin, as well as neuronal constitutive nitric oxide synthase (NOS-I) was investigated. Moreover, agmatine and N(3) -prenylagmatine (G3) were observed to inhibit pig liver and Zea mays L. PAO, bovine trypsin, and NOS-I action, but were substrates for Lens culinaris L., Pisum sativum L. and swine kidney copper amine oxidase. Guazatine and G3 inhibited selectively Zea mays L. PAO with K(i) values of 7.5 x 10(-9) M and 1.5 x 10(-8) M, respectively (at pH 6.5 and 25.0 degrees C). As a whole, the data reported here represent examples of enzyme cross-inhibition, and appear to be relevant in view of the use of cationic L-arginine-and imidazole-based compounds as drugs.     

Expanding distribution of human serotype G6 rotaviruses in Australia

Serotype G6 rotaviruses are common pathogens of cattle but are rarely found in humans. In Australia, human G6 isolates have previously been detected in two major southern population centres. A new isolate, ASG6.02, was detected in central Australia (Alice Springs) in 1997. Comparison of the deduced amino acid sequence of the major neutralizing antigen, VP7, indicated that ASG6.02 was related to human G6 viruses isolated from children in Italy and Australia. Phylogenetic analysis supported the close relationship between ASG6.02 and other Australian isolates and indicated that G6 VP7 sequences generally clustered according to the species of origin (human, bovine or porcine). The VP4 type of ASG6.02 was determined as P-type [14], in common with other isolates from Australia and Italy. The detection of ASG6.02 indicates that the distribution of this serotype is increasing in this country and may have implications for successful vaccine development.     

Distribution of surnames and identities in the Cimbro-Mòcheno communities of Italy

The surnames of populations of the municipalities with Cimbro and Mòcheno origins are compared with each other and with other municipalities of the neighbourhood. This study starts from the supposition that a community of surnames shares a common cultural origin, maintained by reciprocal mobility. The analysis has been carried out by using estimates of the similarities between populations, the topological representations obtained by them and the spatial autocorrelation. On the whole, this research shows no evidence of peculiar distinctions between the populations that share Cimbro and Mòcheno origins compared to the neighbouring ones. Moreover, there is not any evident process of undifferentiated diffusion along all the directions. On the contrary, it is emphasized that belonging to the same geographic region and to the same administrative subdivision mostly influences the similarity between populations. The exception is the Cimbro municipality of Luserna, which presents a peculiar structure of surnames different from other municipalities of the same territory.