A novel ex vivo culture model for inflammatory bone destruction.

Pathological alterations in the balance of bone metabolism are central to the progression of inflammatory bone diseases such as periodontal disease. We have developed and characterized a novel ex vivo murine mandible model of inflammatory bone destruction. Slices of mandible were cultured for 14 days in the presence or absence of P. gingivalis lipopolysaccharide (LPS) or pro-inflammatory cytokines. Following culture, cell viability and tissue histomorphometry were assessed with quantification of matrix proteins, resident osteoclasts, ligament cells, monocytes, macrophages, and neutrophils. In the absence of inflammatory factors, culture viability, osteoclasts, and matrix components were maintained. LPS or TNFα stimulation demonstrated an increase in cellular proliferation, monocyte cells, osteoclast differentiation, and matrix degradation. Pathophysiological bone metabolism can be induced via exposure to LPS and direct influence of TNFα within the model despite the absence of systemic circulation, providing a model for inflammatory bone destruction and investigation of the effects of novel therapeutics.

High- but not low-dose folic acid improves endothelial function in coronary artery disease.

BACKGROUND: While folic acid (FA) reduces plasma homocysteine (Hcy), whether the simultaneous improvement in endothelial function is dependent on Hcy lowering per se is questionable. In the present study the relationship between FA dose, Hcy lowering and endothelial function in patients with coronary artery disease (CAD) was investigated. MATERIALS AND METHODS: Eighty-four patients with CAD received either 400 microg FA or 5 mg placebo daily for a 6-week treatment period. A further 44 patients with CAD received either 100 mg kg(-1) day(-1) of betaine or placebo for a 6-week treatment period. Flow-mediated dilatation (FMD), a measure of endothelial function, was assessed before and after the 6-week periods. Isometric tension and Western blotting were used to investigate the effect of FA on endothelial function and endothelial nitric oxide synthase (eNOS) dimerization in isolated rabbit aortic rings and cultured porcine aortic endothelial cells (PAEC), respectively. RESULTS: Both 400 micro g day(-1) and 5 mg day(-1) FA significantly increased plasma folate and decreased plasma Hcy. The FMD improved significantly after 6 weeks' treatment of 5 mg day(-1) FA but did not correlate with the reduction in Hcy. There was no change in FMD in either the 400 micro g FA or placebo group. In a subgroup analysis of 11 patients in the betaine group, despite a reduced Hcy, a significant impairment in FMD was observed. In the in vitro studies FA, but not betaine, reversed methionine-induced endothelial dysfunction. Moreover, the FA promoted eNOS dimerization in cultured PAEC. CONCLUSIONS: These data suggest that FA dose-dependently improves endothelial function in CAD via a mechanism independently of Hcy lowering. It may involve promotion of eNOS dimerization.

Urinary incontinence in gynecological oncology patients.

The objective of this study was to investigate the prevalence and significance of urinary incontinence (UI) symptoms in gynecological oncology (GO) patients and to test the gynecologists' proficiency in eliciting these symptoms. A prospective survey using questionnaires was designed. Two faculty practice offices in the United States were chosen for the study. Forty GO patients and 40 general gynecology patients were selected from the most recent outpatient appointments of the two hospitals. We used the Urogenital Distress Inventory and the Incontinence Impact Questionnaire to elicit UI symptoms in GO patients and compared the results with their medical records. A control group, selected from a general gynecological practice, was included for comparison. The main outcome measures were to investigate the prevalence and detection rates of UI in GO patients. GO patients were significantly more likely to report UI symptoms on the questionnaire than their gynecologist was able to elicit during a consultation (P < 0.001). The survey found that 60% (24/40) of the GO patients reported at least one symptom of UI, with 23% complaining of "severe" symptoms. Of those patients who reported the symptoms on questionnaire, only 5% (2/40) were detected at the initial physician assessment (P < 0.01). Eighteen percent of the GO patients reported that the UI symptoms adversely affected their quality of life. The prevalence of symptoms was not associated with the primary cancer site. There was no difference in detection rates between the two practice settings. In a multivariate analysis, there was no factor that emerged as the best discriminator for a positive response to the questionnaire. A significant proportion of GO patients report severe UI symptoms that are not detected by gynecologic oncologists or gynecologists during routine consultations.

Cloning of the PYR3 gene of Ustilago maydis and its use in DNA transformation.

The Ustilago maydis PYR3 gene encoding dihydroorotase activity was cloned by direct complementation of Escherichia coli pyrC mutations. PYR3 transformants of E. coli pyrC mutants expressed homologous transcripts of a variety of sizes and regained dihydroorotase activity. PYR3 also complemented Saccharomyces cerevisiae ura4 mutations, and again multiple transcripts were expressed in transformants, and enzyme activity was regained. A 1.25-kilobase poly(rA)+ PYR3 transcript was detected in U. maydis itself. Linear DNA carrying the PYR3 gene transformed a U. maydis pyr3-1 pyrimidine auxotroph to prototrophy. Hybridization analysis revealed that three different types of transformants could be generated, depending on the structure of the transforming DNA used. The first type involved exchange of chromosomal mutant gene sequences with the cloned wild-type plasmid sequences. A second type had integrated linear transforming DNA at the chromosomal PYR3 locus, probably via a single crossover event. The third type had integrated transforming DNA sequences at multiple sites in the U. maydis genome. In the last two types, tandemly reiterated copies of the transforming DNA were found to have been integrated. All three types had lost the sensitivity of the parental pyr3-1 mutant to UV irradiation. They had also regained dihydroorotase activity, although its level did not correlate with the PYR3 gene copy number.

Induction of thermotolerance and mitotic recombination by heat-shock in Ustilago maydiss.

Wild-type cells of Ustilago maydis which have been briefly heated at 42 °C subsequently acquire resistance to the lethal effects of a higher temperature (48 °C). This induced thermotolerance develops over a 2 h period after treatment at 42 °C and is dependent on protein synthesis. It is distinct from the cellular response to DNA-damaging agents, since mutants deficient in this and other repair processes show the same induced thermotolerance as wild-type cells. However, UV-light treatment does induce some resistance to subsequent heat treatment. A single 48 °C heat treatment increases recombination in heteroallelic diploids, but the same treatment of thermotolerant cells does not stimulate recombination. This suggests that heat treatment can damage DNA, but thermotolerant cells are protected from such damage.

New types of mutation affecting formation of alkaline phosphatase by Bacillus subtilis in sporulation conditions.

Mutations defining three new loci, sapA, sapB and phoS, were detected by their ability to overcome the phosphatase-negative phenotype of early-blocked asporogenous mutants in sporulation conditions. Synthesis of alkaline phosphatase by Bacillus subtilis is subject to 'vegetative' and 'sporulation' controls. The phoS mutations resulted in constitutive production of alkaline phosphatase and so could be altered in either the 'vegetative' or the 'sporulation' control system. The sapA and sapB mutations only affected alkaline phosphatase formation in sporulation conditions, and were considered to be sporulation specific. They rendered 'sporulation' alkaline phosphatase formation independent of all the spomutations tested, and so independent of the control of the dependent sequences of spo locus expression; as the enzyme was not formed constitutively, it remained subject to some other sporulation control. The sapA and phoS loci were placed between argC4 and metC3 on the genetic map; the sapB locus was located close to purB6. The three loci mapped separately from all known spo loci.