The presence of icaADBC is detrimental to the colonization of human skin by Staphylococcus epidermidis.

Previous studies have demonstrated that Staphylococcus epidermidis isolates colonizing the skin of healthy humans do not typically encode icaADBC, the genes responsible for the production of polysaccharide intercellular adhesin or biofilms. It was therefore hypothesized that the presence of icaADBC was deleterious to the successful colonization of human skin by S. epidermidis. Using a human skin competition model, it was determined that the strong biofilm-producing S. epidermidis strain 1457 was outcompeted at 1, 3, and 10 days by an isogenic icaADBC mutant (1457 ica::dhfr), suggesting a fitness cost for carriage of icaADBC.

Staphylococcus epidermidis saeR is an effector of anaerobic growth and a mediator of acute inflammation.

The saeRS two-component regulatory system regulates transcription of multiple virulence factors in Staphylococcus aureus. In the present study, we demonstrated that the saePQRS region in Staphylococcus epidermidis is transcriptionally regulated in a temporal manner and is arranged in a manner similar to that previously described for S. aureus. Studies using a mouse foreign body infection model demonstrated that the virulence of strain 1457 and the virulence of a mutant, strain 1457 saeR, were statistically equivalent. However, histological analyses suggested that the polymorphonuclear neutrophil response at 2 days postinfection was significantly greater in 1457-infected mice than in 1457 saeR-infected mice, demonstrating that SaeR influences the early, acute phases of infection. Microarray analysis demonstrated that a saeR mutation affected the transcription of 65 genes (37 genes were upregulated and 28 genes were downregulated); in particular, 8 genes that facilitate growth under anaerobic conditions were downregulated in 1457 saeR. Analysis of growth under anaerobic conditions demonstrated that 1457 saeR had a decreased growth rate compared to 1457. Further metabolic experiments demonstrated that 1457 saeR had a reduced capacity to utilize nitrate as a terminal electron acceptor and exhibited increased production of lactic acid in comparison to 1457. These data suggest that in S. epidermidis SaeR functions to regulate the transition between aerobic growth and anaerobic growth. In addition, when grown anaerobically, 1457 saeR appeared to compensate for the redox imbalance created by the lack of electron transport-mediated oxidation of NADH to NAD+ by increasing lactate dehydrogenase activity and the subsequent oxidation of NADH.

Fluorescence detection of plant extracts that affect neuronal voltage-gated Ca2+ channels.

Structurally novel compounds able to block voltage-gated Ca2+ channels (VGCCs) are currently being sought for the development of new drugs directed at neurological disorders. Fluorescence techniques have recently been developed to facilitate the analysis of VGCC blockers in a multi-well format. By utilising the small cell lung carcinoma cell line, NCI-H146, we were able to detect changes in intracellular Ca2+ concentration ([Ca2+](i)) using a fluorescence microplate reader. NCI-H146 cells have characteristics resembling those of neuronal cells and express multiple VGCC subtypes, including those of the L-, N- and P-type. We found that K+-depolarisation of fluo-3 loaded NCI-H146 cells causes a rapid and transient increase in fluorescence, which was readily detected in a 96-well plate. Extracts of Australian plants, including those used traditionally as headache or pain treatments, were tested in this study to identify those affecting Ca2+ influx following membrane depolarisation of NCI-H146 cells. We found that E. bignoniiflora, A. symphyocarpa and E. vespertilio caused dose-dependent inhibition of K+-depolarised Ca2+ influx, with IC(50) values calculated to be 234, 548 and 209 microg/ml, respectively. This data suggests an effect of these extracts on the function of VGCCs in these cells. Furthermore, we found similar effects using a fluorescence laser imaging plate reader (FLIPR) that allows simultaneous measurement of real-time fluorescence in a multi-well plate. Our results indicate that the dichloromethane extract of E. bignoniiflora and the methanolic extract of E. vespertilio show considerable promise as antagonists of neuronal VGCCs. Further analysis is required to characterise the function of the bioactive constituents in these extracts and determine their selectivity on VGCC subtypes.

Prostate brachytherapy: comparison of dose distribution with different 125I source designs.

PURPOSE: To evaluate the interchangeability of various commercially available iodine 125 ((125)I) sources and to assess the dosimetric effect of a change in source. MATERIALS AND METHODS: A modified peripherally loading prostate brachytherapy plan to deliver 145 Gy was devised by using a model (125)I source, which until recently was the only available (125)I source. A dose-volume histogram was generated. By using the available radial dose functions and anisotropy distributions for eight other currently commercially available sources, the same implant placement was planned and dose-volume histogram distributions tabulated. This exercise was performed for 15-, 45-, and 60-cm(3) glands. No implants were placed, and no physical radiation measurements were made. Dose calculations were theoretic: They were generated by using a widely available treatment planning system. RESULTS: There was little difference in dose distribution to the volume receiving 100% of the prescribed dose (<6%); only one source showed a difference greater than 2%. Large differences, up to -40% to +60%, were seen in the volume of tissue encompassed within internal high-dose regions receiving 150% or 200% of the prescribed dose. These findings held true, irrespective of gland size, within a clinically relevant range (15-60 cm(3)) and for a uniformly loaded radionuclide distribution. CONCLUSION: Reviewing only peripheral dose at or near the prescription dose of 145 Gy revealed little difference in doses for various source designs. Marked differences in high-dose regions were seen and may affect the dose received by internal sites. Effects of these changes on cure and/or complications remain speculative.

Modulation of in vitro platelet 5-HT release by species of Erythrina and Cymbopogon.

Extracts of Australian plants were screened to detect constituents affecting adenosine di-phosphate (ADP) induced platelet aggregation and [14C]5-hydroxytryptamine (5-HT) release. Extracts of four tested plants including, Eremophila gilesii, Erythrina vespertilio, Cymbopogon ambiguus, and Santalum acuminatum, were found to cause significant inhibition of platelet 5-HT release. Inhibition levels ranged from 56-98%, and was not due to the non-specific effects of protein binding tannins. These extracts, and those we have previously identified as being active, were examined further to determine if they affect epinephrine (EPN), arachidonic acid (A.A) or collagen stimulated platelet aggregation and 5-HT release. Among those extracts investigated, we found that both the methanolic extract of E. vespertilio and the dichloromethane (DCM) extract of C. ambiguus were most potent and caused significant inhibition of platelet activation induced by EPN, A.A and to a lesser extent by collagen. Inhibition of ADP induced platelet 5-HT release by both of these extracts, was dose-dependent, with IC50 values for E. vespertilio and C. ambiguus estimated to be 20.4 microl (1.855 mg/ml) and 8.34 microl (0.758 mg/ml), respectively. Overall, C. ambiguus exhibited most activity and also caused dose-dependent inhibition of A.A induced platelet activation. These results indicate that inhibition may occur specifically at a site within the A.A pathway, and suggest the presence of a cyclo-oxygenase inhibitor. Both E. vespertilio and C. ambiguus are reported to be traditional headache treatments, with the present study providing evidence that they affect 5-HT release.

Antithrombotic effect of LB-30057 (CI-1028), a new synthetic thrombin inhibitor, in a rabbit model of thrombosis: comparison with inogatran.

LB-30057 (CI-1028) is a novel, orally bioavailable, direct thrombin inhibitor with a Ki of 0.38 nM against human thrombin. The effects of LB-30057 on thrombus formation and hemostasis were evaluated in a veno-venous shunt model of thrombosis in rabbits, and compared with inogatran, another direct inhibitor of thrombin. Each compound was studied at 5 or 6 different doses with 5 or 6 rabbits in each group. After administration as a bolus i.v. injection followed by continuous infusion, both LB-30057 and inogatran dose-dependently inhibited thrombus formation, which was measured as an increase in time to occlusion (TTO) and a decrease in thrombus weight. Both compounds also improved vena caval blood flow and reduced the overall incidence of thrombotic occlusion. LB-30057 significantly prolonged TTO from 23 +/- 4 min (before dose) to 110 +/- 10 min at the highest dose (0.7 mg/kg + 47 microg/kg/min) (p < 0.001), and reduced thrombus weight from 57 +/- 2 mg to 15 +/- 5 mg (p < 0.001). Occlusive thrombus formed in only one of six rabbits that received the highest dose of LB-30057 (vs. 13/13 in the control group, p < 0.01). At the dose that produced the maximum antithrombotic effect (0.7 mg/kg + 47 microg/kg/min), LB-30057 increased aPTT and bleeding time approximately 2-and 2.5-fold above baseline, respectively. On a gravimetric basis, LB-30057 and inogatran displayed comparable in vivo antithrombotic efficacy. When compared to equally effective anti thrombotic doses of inogatran, LB-30057 caused less prolongation in aPTT, had no effect on PT, and tended to have less of effect on bleeding time. These results indicate that LB-30057 is an effective antithrombotic compound and it appears to have a better benefit/risk profile than inogatran in this experimental model.

Inhibition of platelet aggregation and 5-HT release by extracts of Australian plants used traditionally as headache treatments.

To identify potential migraine therapeutics, extracts of eighteen plants were screened to detect plant constituents affecting ADP induced platelet aggregation and [14C]5-hydroxytryptamine (5-HT) release. Extracts of the seven plants exhibiting significant inhibition of platelet function were reanalysed in the presence of polyvinyl pyrrolidone (PVP) to remove polyphenolic tannins that precipitate proteins. Two of these extracts no longer exhibited inhibition of platelet activity after removal of tannins. However, extracts of Crataegus monogyna, Ipomoea pes-caprae, Eremophila freelingii, Eremophila longifolia, and Asteromyrtus symphyocarpa still potently inhibited ADP induced human platelet [14C]5-HT release in vitro, with levels ranging from 62 to 95% inhibition. I. pes-caprae, and C. monogyna also caused significant inhibition of ADP induced platelet aggregation. All of these plants have been previously used as traditional headache treatments, except for C. monogyna which is used primarily for protective effects on the cardiovascular system. Further studies elucidating the compounds that are responsible for these anti-platelet effects are needed to determine their exact mechanism of action.

Effects of a factor Xa inhibitor, DX-9065a, in a novel rabbit model of venous thrombosis.

The objective of this study was to evaluate the effects of DX-9065a, a nonpeptide, direct inhibitor of factor Xa (FXa), in a novel experimental model of venous thrombosis. The experiments were conducted on anesthetized rabbits in which a veno-venous shunt with cotton threads was inserted into the vena cava. DX-9065a was administered intravenously to the rabbits as an initial bolus followed by a maintenance infusion using the following dosing schedules: DX-I: 0.25 mg/kg + 3 micrograms/kg/min.; DX-II: 0.75 mg/kg + 9 micrograms/kg/min.; DX-III: 1.5 mg/kg + 18 micrograms/kg/min.; DX-IV: 3.0 mg/kg + 36 micrograms/kg/min.; DX-V: 6.0 mg/kg + 72 micrograms/kg/min. DX-9065a induced a dose-dependent increase in the time to occlusion and a dose-dependent decrease in thrombus weight. Because of the unique character of the model, we were also able to show a dose-dependent increase in blood flow through the shunt. In addition, there were dose-dependent increases in prothrombin time (PT) and activated coagulation time (ACT) with more variable responses in the activated partial thromboplastin time (APTT). DX-9065a had little effect on thrombin time (TT) or bleeding time at all doses tested. In conclusion, dose-dependent antithrombotic efficacy was documented with DX-9065a in this new model of venous thrombosis. Although the in vivo potency of the compound was not striking, the results support the utility of FXa inhibition in venous thrombosis and demonstrate the utility of this experimental model for evaluating the efficacy of novel anticoagulants.

A novel model of venous thrombosis in the vena cava of rabbits.

The objective of this study was to develop and validate a new experimental model of venous thrombosis in the rabbit. A 3-cm length of siliconized PE tubing was used as a veno-venous shunt inserted into the abdominal vena cava of anesthetized rabbits. The PE tubing contained six cotton threads which helped to restrict blood flow through the tubing and served as a foreign, thrombogenic surface upon which a thrombus could develop. By continuously measuring blood flow through the vena cava, the rate of thrombus development can be monitored until zero flow is achieved indicating that a completely occlusive thrombus is present. The shunt can be removed making it possible to weigh the thrombus and/or determine its composition. A second shunt can be placed in the vena cava to make a second determination of time to occlusion and thrombus weight, using the data from the first shunt as an internal control standard for comparison. Reproducibility of the technique was demonstrated in a control group (n = 7) in which two successive shunts were used without an antithrombotic intervention. In studies with the first and second shunts, time to occlusion averaged 20.6+/-5.2 min and 20.2+/-5.7 min (pNS), respectively. The net thrombus weights (less the wet weight of the cotton threads) were 49.0+/-3.5 mg and 47.0+/-3.3 mg (pNS). Histologic examination of the thrombi indicated that they were largely composed of fibrin and red blood cells, consistent with the characteristics of venous thrombi. The low molecular weight heparin (LMWH) enoxaparin was used as an antithrombotic intervention to validate the model. Dose-dependent changes in time to occlusion and thrombus weight were achieved which paralleled alterations in coagulation parameters (thrombin time and activated partial thromboplastin time) and bleeding time determined with an ear bleeding technique. The veno-venous shunt model is easy to use, reproducible, and responds appropriately to an antithrombotic intervention, indicating that it should be useful for experimental evaluation of antithrombotic agents designed for venous thromboembolic disorders.

Modulation of Bcl-2 expression: a potential component of estrogen protection in NT2 neurons.

Neuroprotective effects of estrogen have been demonstrated against a variety of cytotoxic insults. We present data here addressing a possible mechanism of estrogen neuroprotection in the human teratocarcinoma cell line NT2 terminally differentiated to a neuronal phenotype. Cell death induced by H2O2 or glutamate results in a dose-dependent cell death of NT2 neurons, while 24 h of estrogen pretreatment significantly enhances neuronal viability. Bcl-2 expression has been shown to reduce oxidative stress and prevent cell death. In NT2 neurons, Bcl-2 levels are dramatically elevated upon differentiation and are further enhanced with estrogen treatment. These results suggest that neuroprotective effects of estrogen may be related to increases in Bcl-2 expression.

The therapeutic potential of novel anticoagulants.

Conventional anticoagulant therapy has been based on indirect inhibition of coagulation factors with heparin and warfarin. These agents display liabilities prompting the development of new anticoagulants over the last two decades. The first to be developed was a series of low molecular weight heparins(LMWHs). Their favourable pharmacokinetic profiles and risk/benefit ratios led to widespread use in Europe and, more recently, approval for their use in the USA. Paralleling the development of LMWHs has been the pursuit of a different strategy focused on direct rather than indirect inhibition of enzymes in the coagulation cascade. In contrast to heparin, LMWHs, or other glycosaminoglycans, direct inhibitors exert their effects independent of either antithrombin III (ATIII) or heparin cofactor II (HCII) and more effectively inhibit clot-bound thrombin or FXa. Highly potent, selective (versus other serine proteases)direct thrombin and FXa inhibitors have been identified and isolated from natural sources, such as leeches, ticks and hookworms. The recombinant forms and analogues of the senatural proteins have been produced using molecular biology techniques, i.e., rHirudin, Hirulogs, recombinant tick anticoagulant peptide (rTAP), recombinant antistasin (rATS) and recombinant nematode anticoagulant peptide-5 (rNAP-5). The design of novel structures or the modification of existing chemicals has led to the synthesis of many non-peptide, low molecular weight inhibitors of thrombin and FXa. Some of them are orally active and may be suitable for long-term clinical use. In addition, considerable progress has been made in developing specific TF/VIIa complex inhibitors. The anticoagulation properties of the new agents are being characterised in experimental studies. Some of them have been advanced to large scale clinical trials and their effectiveness, and sometimes relative ineffectiveness,in arterial and venous thromboembolic disorders has been demonstrated. They are being tested for their potential as new antithrombotic agents that act via direct enzyme inhibition. Thus,the clinician should in future be able to target different thrombotic conditions with proven, specific anticoagulant interventions.

Squamous cell carcinoma of the midventral abdominal pad in three gerbils.

Squamous cell carcinoma of the midventral abdominal pad was diagnosed in 3 male gerbils. Two of the gerbils had raised, ulcerated masses on the midventral portion of the abdomen. The first gerbil was 2 years old, and an excisional biopsy was performed. The gerbil survived 23 months after surgery without evidence of metastasis or clinical signs of local recurrence. At necropsy, neoplastic squamous cells were seen on histologic examination of the surgery site. The second gerbil was 4 years old, and surgical excision of the tumor with concurrent castration was curative. The third gerbil was moribund on admission, perhaps because ulceration of the tumor may have allowed bacteria to invade the tissue, resulting in septicemia and disseminated intravascular coagulation. These gerbils illustrated that hematologic, radiographic, and biochemical testing in rodents can be useful and that excision of squamous cell carcinoma tumors of the midventral abdominal pad of gerbils can be an effective treatment.

Estrogen protects primary cortical neurons from glutamate toxicity.

The gonadal steroid estrogen has been shown to affect neuronal growth, differentiation and survival. We examined the ability of estrogen to protect primary cortical neurons from toxicity induced by the excitatory neurotransmitter glutamate. In these experiments, a 24-h pretreatment with 15 and 50 nM 17 beta-estradiol significantly reduced cellular lactate dehydrogenase (LDH) release from primary cortical neurons, indicating that neurons treated with 17 beta-estradiol were protected from a toxic glutamate exposure. Pretreatment with related steroids such as progesterone, dihydrotestosterone, dexamethasone or cholesterol did not significantly decrease LDH release. The anti-estrogen tamoxifen blocked the protective effects of 17 beta-estradiol suggesting that a classical steroid hormone receptor may be involved in the mechanism subserving estrogen neuroprotection during glutamate toxicity.

Effect of beta agonists on protein turnover in isolated chick skeletal and atrial muscle.

Various beta-adrenergic agonists were found to inhibit rates of protein degradation and net protein breakdown in isolated chick extensor digitorum communis (EDC) and atrial muscles. Rates of protein synthesis were not altered by these compounds. The beta-agonist cimaterol inhibited rates of protein degradation in EDC muscles incubated with or without amino acids and insulin. Cimaterol also inhibited the increased proteolysis induced by injury to muscle or by incubating muscles at body temperature (42 degrees C) versus 37 degrees C. Thus, beta-agonists may help promote skeletal muscle accretion in vivo even under conditions of severe negative nitrogen balance by slowing muscle proteolysis.

K(+)-stimulated amino acid release from cultured cerebellar neurons: comparison of static and dynamic stimulation paradigms.

The release of several endogenous amino acids and adenosine from rat cerebellar neuronal cultures following elevated K+ exposure in the presence and absence of added Ca2+ was studied. The amino acids aspartate (ASP), glutamate (GLU) and GABA were released from the cultures in a dose- and Ca(2+)-dependent manner. Taurine (TAU) and the nucleoside adenosine (ADN) efflux rates were dose-dependent but Ca(2+)-independent, and basal levels increased in the absence of Ca2+. The K+ depolarization induced release of serine (SER), alanine (ALA) and proline (PRO), was not dose-dependent and in the absence of extracellular Ca2+ (with added Mg2+) higher basal release of SER and ALA, but not PRO, was noted. These findings demonstrate that in addition to known cerebellar neurotransmitters, other neuroactive and neutral amino acids are released from cultured cerebellar neurons in response to K+ depolarization. Their observed efflux suggests they may have as yet unidentified roles in neuronal function with different classes of efflux corresponding to: neurotransmitter-type release (ASP, GLU, GABA), an osmoregulatory, possibly neuromodulatory-type release (TAU), a Ca(2+)-insensitive, possibly neuromodulatory-type release (ADN), and a depolarization-sensitive release (SER, ALA, PRO) of which SER and ALA are partially Ca(2+)-sensitive.

Effect of beta agonists on protein turnover in adipose tissue.

Chronic administration (21 days) of the beta agonist cimaterol to rats decreased epididymal fat by 27%, and inhibited in vitro rates of protein synthesis by 34% and net protein breakdown by 71% in adipose tissue. Likewise, incubation of rat adipose tissue with cimaterol and isoproterenol stimulated lipolysis, and inhibited protein synthesis and degradation. Thus, in addition to affecting muscle mass and lipid metabolism, beta agonists appear to slow rates of protein turnover in adipose tissue.

The synthesis of 3-phosphonoalanine, phosphonopyruvic acid and phosphonolactic acid. Scission of the C-P bond during diazotization of phosphonoalanine.

3-Phosphonoalanine has been made by the Strecker synthesis from phosphonoacetaldehyde, which is easily prepared from vinyl acetate. It gives phosphonopyruvate by transamination when treated with glyoxylate. Phosphonolactate, an analogue of phosphoglycerate, is prepared by reducing phosphonopyruvate. Diazotization of phosphonoalanine was investigated as a route for making phosphonolactate: addition of NaNO2 to the isoelectric form of phosphonoalanine gave much scission of the C-P bond with release of phosphate; addition of HBr prevented this release and gave largely the bromo acid. The supplement reports the synthesis of arsonolactate, a similar analogue, by treating chlorolactate with alkaline arsenite.