Growth-dependent photoinactivation kinetics of Enterococcus faecalis.

AIMS: To investigate how the growth stage of Enterococcus faecalis affects its photoinactivation in clear water. METHODS AND RESULTS: Enterococcus faecalis were grown in batch cultures to four different growth stages or grown in chemostats set at four different dilution rates, then harvested and exposed to full spectrum or UVB-blocked simulated sunlight. Experiments were conducted in triplicate in clear water with no added sensitizers. Decay curves were shoulder-log linear and were generally not statistically different in experiments conducted under full spectrum light. Shoulders were longer and first order inactivation rates smaller when experiments were seeded with cells grown to stationary as compared to exponential phase, and for slower growing cells when experiments were done under UVB-blocked light. Chemostat-sourced bacteria generally showed less variability among replicates than batch-sourced cells. CONCLUSIONS: The physiological state of cells and the method via which they are being generated may affect the photoinactivation experimental results. SIGNIFICANCE AND IMPACT OF THE STUDY: Photoinactivation experiments conducted with exponential phase cells may overestimate the photoinactivation kinetics in the environment, particular if UVB-independent mechanisms predominate. Chemostat-sourced cells are likely to provide more consistent experimental results than batch-sourced cells.

The novel estrogen-responsive B-box protein (EBBP) gene is tamoxifen-regulated in cells expressing an estrogen receptor DNA-binding domain mutant.

We have identified a 2.6-kb mRNA whose steady state levels are increased 2- to 4-fold by treatment of human mammary epithelial cells (HMEC) stably expressing an estrogen receptor (ER) transgene with either estrogen (E) or the antiestrogen, 4-hydroxy-tamoxifen (HT). The cDNA corresponding to this mRNA encodes a 564-amino acid protein, named estrogen-responsive B box protein (EBBP), that is a new member of a subfamily within the B box zinc finger protein family, which includes transcription factors (e.g. TIF1), tumor suppressor proteins (e.g. PML), and proteins implicated in development (e.g. ret finger protein, XNF7). The EBBP mRNA is detectable by Northern blot analysis in most tissues, with the exception of liver and peripheral blood lymphocytes, and the gene has been mapped to human chromosome 17p11.2. In contrast to most B box family members, EBBP has a predominantly cytoplasmic localization. Studies of the estrogenic regulation of EBBP expression demonstrated that the E-dependent increase in EBBP mRNA levels in the ER-transfected HMEC is an early, ER-mediated, and cycloheximide-insensitive process. In HMEC stably transfected with an ER mutant containing a deletion in the second zinc finger of the DNA-binding domain, E and HT had different effects on EBBP gene expression; EBBP regulation by E was dramatically reduced while the effects of HT were augmented. These data indicate that HT can modulate EBBP mRNA expression through a mutated ER, which has little activity when bound by E, and suggest that different molecular mechanisms control the E and HT responsiveness of the EBBP gene.

Cloning of a cDNA encoding a receptor related to the formyl peptide receptor of human neutrophils.

We cloned a cDNA (RFP) encoding a receptor (RFP) related (70% overall nucleotide homology) to the formyl peptide receptor of human neutrophils (hFPR). RFP is a seven-transmembrane-domain receptor and its distribution is limited to myeloid cells. Domain sequence comparison with hFPR reveals highly conserved regions and provides clues to putative domains involved in ligand binding and receptor desensitization.

Cloning and genetic characterization of an evolutionarily conserved human olfactory receptor that is differentially expressed across species.

We have cloned the full-length cDNA and genomic region of a human prostate specific G-protein coupled receptor with properties characteristic of an olfactory receptor. A partial cDNA sequence of this gene, called PSGR, was recently cloned. The gene contains two exons and one intron of 14.9 kb in its 5'untranslated region, and was mapped to human chromosome 11p15.2. A cluster of transcription initiation sites for the 2.8 kb PSGR mRNA was identified. Cloning of the homologous gene from the mouse revealed 93% amino acid homology between the human and mouse or rat (previously cloned as RA1c) proteins, and 99% identity between the rat and mouse homologs. Although northern analysis indicated expression of the human PSGR homolog was prostate specific, its mRNA could also be detected in the olfactory zone and the medulla oblongata of the human brain. In the mouse, the PSGR gene is predominantly expressed in the brain and colon. In the rat, the PSGR homolog is expressed in the liver in addition to the brain. These data add to the growing body of evidence suggesting that olfactory receptors may have functional roles in tissues other than the olfactory organ, and further, suggest that these functions may vary across species.

Efficient site-directed in vitro mutagenesis using phagemid vectors.

Several methods have been developed that enhance the efficiency of in vitro, site-directed mutagenesis. Kunkel (8,9) has developed a method which uses a strong selection for the mutated strand and, hence, is highly efficient, but yet simple and rapid. This method originally used M13 phage as the vector. In this paper, we describe a refinement of this method using phagemid vectors, which combine the advantages of plasmids (such as high copy number and stability of cloned DNA) with the single-stranded DNA generating capability of M13 phage. We demonstrate that high efficiency of mutant production can be obtained with these vectors. We also analyzed by sequencing 11 mutated clones and found no second-site mutations, suggesting that alterations other than the site-directed mutation rarely occur in our system.

Bst DNA polymerase permits rapid sequence analysis from nanogram amounts of template.

A simple, rapid method is presented for the enzymatic sequence analysis of nanogram amounts of single-stranded or double-stranded DNA. This approach employs the thermostable DNA polymerase from Bacillus sterothermophilus and exploits its ability to efficiently extend all of the template-primer complex, even at low substrate concentrations. The procedure requires few pipetting steps, no preannealing step and very short reaction time. This method can significantly reduce the cost associated with DNA polymerase and the amount of template and time required to perform the enzymatic sequencing reactions. As little as a 10-ng aliquot of such sequencing reactions can be analyzed on a fluorescence-based capillary gel electrophoresis instrument recently developed in our laboratory. This highly sensitive detection, in conjunction with the ability to efficiently sequence nanogram amounts of template, strongly suggests the feasibility of direct DNA sequencing of single bacteriophage M13 plaques without prior amplification.

Sequencing with the large fragment of DNA polymerase I from Bacillus stearothermophilus.

The large fragment of DNA polymerase I, isolated from Bacillus stearothermophilus, was used for dideoxy sequencing. This heat-stable enzyme permits performing sequencing reactions at high temperature to melt secondary structure and results in uniform band intensities and low background on the autoradiogram. The enzyme can be used in the standard Sanger one-step protocol or in a two-step protocol which separates the labeling reaction from the elongation-termination reaction. The enzyme can be used in double-stranded sequencing. 35S-labeled nucleotides may be used instead of 32P-labeled nucleotides. Both 7-deaza-dGTP and dITP can be used during the reaction in order to minimize band compression on the gel. Results presented here indicate that this enzyme should be a useful tool for sequence determination.

Plaque regrowth effects of a triclosan/pyrophosphate dentifrice in a 4-day non-brushing model.

Triclosan is a lipophilic antimicrobial agent which, when present in an aqueous dentifrice vehicle, is complexed by or in close contact with polymers and surface-active molecules, emulsifying agents, flavoring oils and other hydrophobic ingredients. Because of this, dentifrice products containing triclosan may not have triclosan in a bioavailable state and, hence, the products themselves can not be assumed to possess antimicrobial activity. In order to determine the antimicrobial effects on dental plaque of a triclosan/pyrophosphate dentifrice relative to a negative control (without triclosan or pyrophosphate), a crossover 4-day non-brushing study was conducted. Thirty-four subjects were enrolled in this randomized two-period, double-blind crossover investigation with thirty-three subjects completing all aspects. Following a baseline plaque examination and complete plaque removal at the start of the first 4-day treatment period, subjects initiated a twice-daily supervised dosing regimen, during which they rinsed with their first assigned dentifrice in slurry form while refraining from tooth-brushing and all other oral hygiene procedures. Evaluations to quantify test product effects on plaque were conducted on Day 5. After a week-long interim washout period, subjects repeated the twice daily rinsing regimen over Days 1-4 of Treatment Period 2 with their second assigned product, again with examinations on Day 5. Analysis of data demonstrated subjects had significantly (p = 0.0296) less plaque when rinsing with the triclosan/pyrophosphate dentifrice slurry as compared to the negative control dentifrice slurry; the relative treatment difference as determined by the primary examiner was 12.7%. A trainee examiner observed a 16.0% reduction on a subset of subjects (p = 0.0139). This efficacy result compares favorably with results from other studies of triclosan-containing products. The examinations for oral safety demonstrated no meaningful clinical differences between the triclosan/pyrophosphate dentifrice and control dentifrice.

Cloning of the gene coding for a human receptor for formyl peptides. Characterization of a promoter region and evidence for polymorphic expression.

Recently we reported that, in HL-60 cells, transcription of the formyl peptide receptor (FPR) gene can be up- and downregulated by agents that induce differentiation of HL-60 cells into neutrophils. To begin studying the mechanisms involved in regulation of FPR gene expression, we cloned two human cDNAs and the gene coding for FPR. The genomic clone (pINF14) contained a 14.5-kb insert. A 2.7-kb EcoRI fragment was obtained from pINF14 that hybridized with an FPR open reading frame probe. The EcoRI fragment was sequenced and found to contain an intronless FPR open reading frame. Sequence alignment of the EcoRI genomic fragment with the FPR cDNA revealed that the first 31 bases of 5' untranslated FPR cDNA were not represented in the genomic fragment. Furthermore, a splicing consensus sequence was present in the genomic fragment at the site of divergence with the cDNA sequence. Restriction mapping and Southern blot analysis identified a 121-bp fragment that contained the sequence corresponding to the first 31 bases of 5' untranslated FPR cDNA. An additional (previously undescribed) 15-bp cDNA sequence in the 5' end of FPR were identified using an anchored polymerase chain reaction. This sequence was also contained in the genomic 121-bp fragment. This 121-bp fragment was located 5.2 kb (intron) upstream of the FPR open reading frame. It contained an unusual TATA box and displayed transcriptional activity in vitro and in vivo. Potential binding sites for AP-1 and glucocorticoid receptor were identified upstream of the putative TATA box.(ABSTRACT TRUNCATED AT 250 WORDS)

RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100/calgranulin polypeptides.

S100/calgranulin polypeptides are present at sites of inflammation, likely released by inflammatory cells targeted to such loci by a range of environmental cues. We report here that receptor for AGE (RAGE) is a central cell surface receptor for EN-RAGE (extracellular newly identified RAGE-binding protein) and related members of the S100/calgranulin superfamily. Interaction of EN-RAGEs with cellular RAGE on endothelium, mononuclear phagocytes, and lymphocytes triggers cellular activation, with generation of key proinflammatory mediators. Blockade of EN-RAGE/RAGE quenches delayed-type hypersensitivity and inflammatory colitis in murine models by arresting activation of central signaling pathways and expression of inflammatory gene mediators. These data highlight a novel paradigm in inflammation and identify roles for EN-RAGEs and RAGE in chronic cellular activation and tissue injury.