Calmodulin, poly(ADP-ribose)polymerase and p53 are targets for modulating the effects of sulfur mustard.

We describe two pathways by which the vesicating agent sulfur mustard (HD) may cause basal cell death and detachment: induction of terminal differentiation and apoptosis. Following treatment of normal human epidermal keratinocytes (NHEK) with 10 or 100 microM HD, the differentiation-specific keratin pair K1/K10 was induced and the cornified envelope precursor protein, involucrin, was cross-linked by epidermal transglutaminase. Fibronectin levels were reduced in a time- and dose-dependent manner. The rapid increase in p53 and decrease in Bcl-2 levels was consistent not only with epidermal differentiation but with apoptosis as well. Further examination of biochemical markers of apoptosis following treatment of either NHEK or human papillomavirus (HPV)-immortalized keratinocytes revealed a burst of poly(ADP-ribose) synthesis, specific cleavage of poly(ADP-ribose)polymerase (PARP) in vivo and in vitro into characteristic 89 and 24 kDa fragments, processing of caspase-3 into its active form and the formation of DNA ladders. The intracellular calcium chelator BAPTA suppressed the differentiation markers, whereas antisense oligonucleotides and chemical inhibitors specific for calmodulin blocked both markers of differentiation and apoptosis. Modulation of p53 levels utilizing retroviral constructs expressing the E6, E7 or E6 + E7 genes of HPV-16 revealed that HD-induced apoptosis was partially p53-dependent. Finally, immortalized fibroblasts derived from PARP -/- 'knockout mice' were exquisitely sensitive to HD-induced apoptosis. These cells became HD resistant when wild-type PARP was stably expressed in these cells. These results indicate that HD exerts its effects via calmodulin, 3 and PARP-sensitive pathways.

Color and power Doppler sonography of liver hemangiomas: a dream unfulfilled?

PURPOSE: The aim of this study was to determine whether color Doppler or power Doppler sonography can aid in the diagnosis of hepatic cavernous hemangiomas. METHODS: We imaged 25 hepatic cavernous hemangiomas in 17 patients with gray-scale, color Doppler, and power Doppler sonography. Five malignant lesions were also imaged in the same manner for reference. Hemangiomas had been previously diagnosed by biopsy in 8 patients (15 lesions) and by CT, MRI, and/or tagged red blood cell scanning in 9 patients (10 lesions). RESULTS: Of the 25 hemangiomas, color or power Doppler imaging showed no internal blood flow in 23. Of these 23 lesions, 11 showed a peripheral flow pattern believed to represent flow in displaced blood vessels. This pattern was better visualized with power Doppler imaging in 3 lesions and equally well visualized with color and power Doppler imaging in 8 lesions. Two hemangiomas that had unusual central fibrosis with large vessels in 1 patient showed diffusely increased blood flow on power Doppler study. All 5 malignant lesions showed flow in peripheral vessels, and 1 showed internal vascularity as well. CONCLUSIONS: Neither color nor power Doppler imaging improved the capability of sonography for making a specific diagnosis of benign hepatic cavernous hemangioma.

Appetitive and consummatory sexual behaviors of female rats in bilevel chambers. I. A correlational and factor analysis and the effects of ovarian hormones.

This study investigated measures of sexual behavior displayed by female rats in bilevel chambers, the statistical relationships among the measures, and their dependency on hormone priming. Normative data from a standard 35-min test of sexual behavior were gathered from 82 fully primed sexually experienced Long-Evans females and subjected to multiple correlational and factor analyses. Several consummatory measures of copulation were related significantly, whereas appetitive level changing was statistically independent of consummatory measures. Factor analyses were conducted using orthogonal rotations of correlational matrices derived either from (a) measures of female behavior alone or (b) measures of female and male behavior together. The first analysis revealed five factors that accounted for 84% of the intersubject variance: Receptivity, Pacing, Appetitive Level Changing, Lordosis Reflex, and Solicitation. The second factor analysis with male data included revealed seven factors that accounted for 95% of the intersubject variance: Pacing, Copulatory Rate, Mount Count, Receptivity, Appetitive Level Changing, Solicitation, and Lordosis Reflex. Subsequently, subsets of these females were maintained on different steroid priming regimens (oil, low estrogen, high estrogen, high estrogen and progesterone) prior to a standard test of sexual behavior. Although the expression of all sexual behaviors required estrogen priming, appetitive level changing, solicitation, and pacing required progesterone for their full expression. Finally, appetitive level changing developed following hormone treatment alone, regardless of whether the females received access to sexually active males, inactive castrated males, or other females. Use of bilevel chambers allows complex patterns of sexual behavior to be observed in female rats and may thus facilitate the identification of neurochemical or endocrine mechanisms associated with different aspects of female sexual motivation and performance.

Regulation of dikaryon-expressed genes by FRT1 in the basidiomycete Schizophyllum commune.

The gene FRT1 has previously been shown to induce homokaryotic fruiting in transformation recipients of the basidiomycete Schizophyllum commune. In this paper, we demonstrate by gene disruption experiments that FRT1 is dispensable for dikaryotic fruiting. Nonfruiting homokaryotic FRT1 disruptant strains exhibited enhanced aerial growth of mycelia compared to wild type. Introduction of a functional FRT1 allele into the disruptant restored the wild-type colony morphology. Transcript abundance of the dikaryon-expressed SC1 and SC4 hydrophobin genes and the SC7 gene were greatly elevated in homokaryotic FRT1 disruptant strains. Growth of the disruptant strains under continuous light was found to inhibit the elevation of SC1 and SC4 transcript levels, but not of SC7 mRNA. These data suggest that the role of FRT1 in vegetatively growing homokaryons is to act as a negative regulator of dikaryon-expressed genes.

Prevention of chickenpox in reproductive-age women: cost-effectiveness of routine prenatal screening with postpartum vaccination of susceptibles.

OBJECTIVE: To evaluate economic and clinical outcomes of a program of routine prenatal serotesting for varicella and postpartum vaccination of seronegative women. METHODS: An analytic cost-effectiveness model was constructed to compare the current strategy of no serotesting with 1) selective serotesting of pregnant women without a prior history of chickenpox and 2) serotesting of all pregnant women. In both serotesting strategies, seronegative women were vaccinated postpartum. The model followed a hypothetical cohort of 4 million women over 20 years. Costs and chickenpox disease outcomes during and outside of subsequent pregnancies were considered. The incremental cost-effectiveness (cost per adult chickenpox case prevented) of selective serotesting compared with the current strategy was measured. RESULTS: Compared to no testing, selective serotesting would prevent 43% (48,577 of 112,654) of adult chickenpox cases, save $21.8 million in discounted medical and work loss costs from the societal perspective, and cost $1126 per case prevented from the health payer's perspective (medical costs only). The model was sensitive to varicella seroprevalence and incidence of chickenpox among susceptible women but was relatively insensitive to the cost of serologic testing and vaccination. Compared with selective serotesting, the serotest-all strategy would prevent an additional 15,645 cases, at a societal cost of $7653 per additional case prevented. CONCLUSION: The selective serotesting strategy could prevent nearly half of chickenpox cases among this cohort and is cost-saving from the societal perspective. From the health payer's perspective, it compares favorably with other generally accepted preventive practices. It should be considered for prevention of chickenpox among women of childbearing age.

Histopathologic and immunohistochemical features in human skin after exposure to nitrogen and sulfur mustard.

N-methyl-2,2'-dichlorodiethylamine (HN2)is a topical chemotherapeutic agent used as therapy for cutaneous T-cell lymphomas (CTCL). Di(2-chloroethyl)sulfide (SM), and less often HN2, have been used as chemical weapons, with the skin being a principle target. The mechanisms by which these chemicals produce their therapeutic and toxic effects in skin, however, are not clearly defined. We exposed human skin explants to two doses of HN2 and SM. At 18 hours after exposure, histopathologic features were compared. In addition, immunohistochemical markers to basement membrane proteins were used to evaluate the effects of both chemicals on the basement membrane zone. Gross vesication was not seen. Pyknotic nuclei with or without dyskeratotic changes within epidermal keratinocytes were present at both doses. These changes varied more between skin specimens than they did between doses. Ballooning degeneration was more marked after SM exposures. Diffuse dermal-epidermal separation was present only at high-dose exposures and did not appear to correlate with the degree of changes locally in the overlying epidermis. Antibodies to laminin-5 showed decreased immunoreactivity after exposure to HN2 and SM. Immunoreactivity for laminin- was decreased to a lesser extent, and immunoreactivity for collagen IV and VII was unchanged. HN2 and SM produce similar histopathologic and immunohistochemical features after cutaneous exposure. These features suggest that part of mechanism of action of HN2 and SM is a direct effect on the basement membrane zone. Understanding the effects of HN2 and SM separate from their effect on DNA may be important in designing therapies and in advancing our understanding of the pathophysiologic changes induced by these chemicals when delivered topically.

Right latissimus dorsi cardiomyoplasty improves left ventricular energetics.

BACKGROUND: The mechanism by which cardiomyoplasty appears to enhance left ventricular (LV) function is not well understood. We applied the time-varying elastance model to study the effect of cardiomyoplasty on LV function, ventriculovascular coupling, and LV energetics in an acute canine model. METHODS: Right latissimus dorsi cardiomyoplasty was performed in 5 dogs. The end-systolic pressure-volume relation was generated by using brief caval occlusions. End-systolic elastance, effective arterial elastance, stroke work, internal work, total mechanical work, and stroke work efficiency (stroke work/total mechanical work) were calculated from these pressure-volume data. Myocardial oxygen consumption and overall mechanical efficiency (stroke work/myocardial oxygen consumption) were predicted using the myocardial oxygen consumption-total mechanical work relation. RESULTS: Skeletal muscle contraction significantly increased end-systolic elastance, an index of contractility. Although stroke work did not change significantly, the increase in end-systolic elastance led to a 29% decrease in total mechanical work, a 50% decrease in internal work, and an increase in stroke work efficiency from 53% to 66%. This was consistent with the observed 29% decrease in effective arterial elastance/end-systolic elastance, an indicator of ventriculovascular coupling that is related inversely to stroke work efficiency. Predicted myocardial oxygen consumption decreased by at least 22%, and predicted overall mechanical efficiency increased at a minimum from 16.1% to 18.4%. CONCLUSIONS: These results support the theory that cardiomyoplasty unloads the LV by decreasing LV volumes and increasing contractility. These effects appear to improve LV energetics by decreasing total mechanical work without significantly affecting stroke work, resulting in improved stroke work efficiency. The decrease in total mechanical work strongly suggests a decrease in myocardial oxygen consumption and an increase in overall mechanical efficiency.

Tail pinch induces fos immunoreactivity within several regions of the male rat brain: effects of age.

Brief, intermittent stressors, such as low-level foot shock or tail pinch, induce a general excitement and autonomic arousal in rats that increases their sensitivity to external incentives. Such stimulation can facilitate a variety of behaviors, including feeding, aggression, sexual activity, parental behavior, and drug taking if the appropriate stimuli exist in the environment. However, the ability of tail pinch to induce general arousal and incentive motivation appears to diminish with age. Here we report on the ability of tail pinch to induce Fos immunoreactivity within several brain regions as a function of age. Young (2-3 months) and middle-aged (12-13 months) male rats were administered either five tail pinches (one every 2 min), one tail pinch, or zero (sham) tail pinches (n = 4 per stimulation condition). Rats were sacrificed 75 min following the onset of stimulation, and their brains were prepared for immunocytochemical detection of Fos protein. Fos immunoreactivity was induced by one and five tail pinches in several brain regions, including the anterior medial preoptic area (mPOA), paraventricular nucleus of the hypothalamus (PVN), paraventricular nucleus of the thalamus (PV-Thal), medial amygdala (MEA), basolateral amygdala (BLA), lateral habenula (LHab), and ventral tegmental area (VTA), of young rats compared with those that received zero tail pinches. In contrast to young rats, middle-aged rats had significantly less Fos induced by one and five tail pinches in the mPOA, PVN, MEA, BLA, and VTA, but an equivalent amount induced in the LHab. Fos immunoreactivity was not found within the medial prefrontal cortex, nucleus accumbens, striatum, lateral septum, or locus coeruleus in either young or old rats. Tail pinch appears to activate regions of the brain known to be involved in behavioral responses to both incentive cues and stressors. The lower level of cellular reactivity to tail pinch in middle-aged rats suggests a diminished neural responsiveness to incentives and stressors.

L-oxothiazolidine 4-carboxylate pretreatment of isolated human peripheral blood lymphocytes reduces sulfur mustard cytotoxicity.

Sulfur mustard (HD, mustard gas) is a vesicant chemical warfare agent for which there is no specific medical countermeasure. A potential approach to combating the debilitating effects of this agent is the use of compounds that can react with this material before it interacts with critical macromolecules. Glutathione (GSH), a tripeptide that exists in high concentrations in cells, reacts with HD and is involved in HD detoxification. Pretreatment of human peripheral blood lymphocytes (PBL) with 10 mmol/L L-oxothiazolidine-4-carboxylate (OTC), a "masked" cysteine precursor, increased intracellular glutathione levels 25-50% over control values. Pretreated PBL were harvested, washed, and exposed to 10, 50, or 100 mumol/L HD. Flow cytometry was used to measure cytotoxicity by propidium iodide uptake. Pretreatment of PBL with OTC led to small decreases in cytotoxicity after HD exposure. However, treatment of cells with OTC after HD exposure was not beneficial. Compounds that can modulate GSH levels within the cell may help to reduce the cytotoxicity of HD when used a pretreatment.

Differential induction of Fos in the female rat brain following different amounts of vaginocervical stimulation: modulation by steroid hormones.

Vaginocervical stimulation (VCS), produced either by copulation with intromission or by manual stimulation of vagina and cervix with a glass rod, induces neuroendocrine and behavioral responses that are critical for female reproduction in many species. We and others have shown that Fos mRNA and protein are induced within different estrogen-concentrating and -non-concentrating regions of the female rat brain following copulation with intromission and manual VCS. In the present study, we investigated the amount of distributed VCS required to induce Fos immunoreactivity within estrogen-concentrating regions of the medial preoptic area, lateral septum, bed nucleus of the stria terminalis, ventromedial hypothalamus, medial amygdala, and mesencephalic central gray, and whether estrogen and progesterone could alter the threshold or pattern of induction. Ovariectomized rats were administered estradiol benzoate (10 micrograms) 48 h and progesterone (500 micrograms) 4 h before receiving either 0, 1, 5, 10, 20, 30, 40, or 50 manual VCSs with a lubricated glass rod. Ovariectomized hormone control rats received injections of the sesame oil vehicle 48 and 4 h before VCS. All rats were sacrificed 75 min after the first VCS. Fos immunoreactivity was induced differentially by VCS within the different regions, and the hormones either augmented, inhibited, or had no effect on the induction. These data demonstrate that cells within different estrogen-concentrating regions of the female rat brain are differentially sensitive to VCS, and that steroid hormones can either increase or decrease the amount of Fos induced by different amounts of VCS. Different brain regions may participate in gating the sensory information of VCS into different behavioral and neuroendocrine events.

Complications of iliac crest bone graft harvesting.

Autologous bone grafts harvested from the iliac crest are commonly used in reconstructive orthopaedic surgery. Autologous bone is used to help promote bone healing in fractures and to provide structural support for reconstructive surgery. The results of autologous bone grafting are more predictable than the use of xenografts, cadaveric allografts, or synthetic bone substitutes because autologous bone grafts provide osteoinductive and osteoconductive properties, are not immunogenic, and are usually well incorporated into the graft site. In a retrospective review of 414 consecutive cases of iliac crest bone graft procedures performed at Brooke Army Medical Center from 1983 to 1993, 41 (10%) minor and 24 (5.8%) major complications were identified. Minor complications included superficial infections, superficial seromas, and minor hematomas. Major complications included herniation of abdominal contents through massive bone graft donor sites, vascular injuries, deep infections at the donor site, neurologic injuries, deep hematoma formation requiring surgical intervention, and iliac wing fractures. Harvesting of iliac crest bone graft can be associated with significant morbidity. However, with adequate preoperative planning and proper surgical technique, the incidence of these complications can be reduced.

Ca2+ homeostasis and Ca2+ signalling in sulphur mustard-exposed normal human epidermal keratinocytes.

To investigate the role of increased cytoplasmic free calcium levels ([Ca]i) in sulphur mustard-induced cellular toxicity, the [Ca]i of human epidermal keratinocytes exposed to sulphur mustard concentrations between 50 and 800 microM were measured. No early increase in [Ca]i was observed during the first 20 min after application of the agent. Furthermore, at 3, 6 and 24 h after exposure to sulphur mustard, the [Ca]i of sulphur mustard-exposed cells differed only slightly from that of control cells. However, sulphur-mustard exposed cells responded with a weaker rise of [Ca]i upon stimulation with histamine and ATP than control cells. These results suggest that sulphur mustard can induce small increases in [Ca]i, which reflect abnormal cellular physiology rather than acute toxicity. In addition, it has been shown that sulphur mustard disturbs cellular Ca2+ signalling in human epidermal keratinocytes.

Recovery and viability of an acute myocardial infarct after transmyocardial laser revascularization.

OBJECTIVES: The short- and long-term effectiveness of transmyocardial laser revascularization was evaluated in the setting of an acute myocardial infarction. BACKGROUND: Theoretically, transmyocardial laser revascularization allows direct perfusion of the ischemic area as ventricular blood flows through the channels to the myocardium. METHODS: Infarcts were created by coronary occlusion in 30 sheep. Eighteen of these sheep were studied to assess short-term efficacy. The infarct was reperfused after 1 h by either removing the occlusion or by laser drilling using a high power carbon dioxide laser. The occlusions were left in place for the control group. To monitor regional recovery, percent systolic shortening was measured. To evaluate long-term effectiveness, 12 additional sheep underwent creation of an infarct. Six were treated with the laser, and six were untreated. The animals were restudied 30 days later. RESULTS: In the short-term experiment, the control and reperfusion groups exhibited no recovery of regional contractility. The laser group demonstrated improvement throughout the recovery period. There was a significant difference in the area of necrosis within the same area at risk (reperfusion group 44 +/- 6% and control group 39 +/- 5% vs. laser group 6 +/- 2%). After 30 days, none of the control animals showed evidence of contraction in the infarct, whereas the laser-treated animals did. Histologic analysis of the laser-treated infarcts revealed patent channels surrounded by viable myocardium. The control-group infarcts were necrotic and scarred. CONCLUSIONS: On the basis of both short- and long-term improved contractility, as well as diminished necrosis in the area at risk, these results indicate that transmyocardial laser revascularization may be an alternative method of treating ischemic heart disease.

Prevention of complement-induced pulmonary hypertension and improvement of right ventricular function by selective thromboxane receptor antagonism.

The effect of complement activation on the pulmonary vascular system and on right ventricular function was studied in sheep (n = 12) by injection of cobra venom factor. Animals were instrumented for measurement of pulmonary flow, mean pulmonary artery pressure, right ventricular stroke work, arterial blood gases, and systemic vascular resistance. Blood was sampled from the left atrium and pulmonary artery to measure thromboxane B2, the metabolite of thromboxane A2, by radioimmunoassay. After baseline measurements, animals were randomly assigned to receive a selective thromboxane receptor antagonist SQ30741 as a 10 mg/kg bolus with an infusion of 10 mg/kg per hour or else to receive vehicle. Cobra venom factor was then injected (30 U/kg) in all animals, and data were recorded at 15, 30, 60, 90, and 120 minutes. In control animals there was a 2.4-fold increase in mean pulmonary artery pressure and a 76% increase in right ventricular stroke work at 15 minutes from baseline (p < 0.05); these values remained elevated for 30 minutes and returned to baseline by 1 hour with no change in systemic vascular resistance. Arterial oxygenation decreased by 124% at 15 minutes and remained depressed through the experiment, but in treated animals oxygen tension remained unchanged from baseline. Thromboxane B2 increased 95% from baseline in the control group and 1.5 fold in treated animals and followed a similar time course as the functional measurements (p < 0.05). A pulmonary vascular thromboxane B2 gradient of approximately 1000 pg/ml was measured at 15 and 30 minutes in both control and treated groups. (p < 0.05) We conclude that after complement activation in this model pulmonary hypertension and decreased oxygen tension are mediated by thromboxane release from the pulmonary vascular bed. This increased afterload causes a stress on the right ventricle as demonstrated by the increased right ventricular stroke work. Selective thromboxane receptor antagonism may be a beneficial therapy for pulmonary hypertension in patients after cardiopulmonary bypass.

Sulfur mustard-increased proteolysis following in vitro and in vivo exposures.

The pathologic mechanisms underlying sulfur mustard (HD)-induced skin vesication are as yet undefined. Papirmeister et al. (1985) postulate enhanced proteolytic activity as a proximate cause of HD-induced cutaneous injury. Using a chromogenic peptide substrate assay, we previously reported that in vitro exposure of cell cultures to HD enhances proteolytic activity. We have continued our investigation of HD-increased proteolytic activity in vitro and have expanded our studies to include an in vivo animal model for HD exposure. In vitro exposure of human peripheral blood lymphocytes (PBL) to HD demonstrated that the increase in proteolytic activity is both time- and temperature-dependent. Using a panel of 10 protease substrates, we established that the HD-increased proteolysis was markedly different from that generated by plasminogen activator. The hairless guinea pig is an animal model used for the study of HD-induced dermal pathology. When control and HD-exposed PBL and hairless guinea pig skin where examined, similarities in their protease substrate reactivities were observed. HD-exposed hairless guinea pig skin biopsies demonstrated increased proteolytic activity that was time-dependent. The HD-increased proteolytic response was similar in both in vitro and in vivo studies and may be useful for elucidating both the mechanism of HD-induced vesication and potential treatment compounds.