Anticancer actions of carnosine in cellular models of prostate cancer.

Treatments for organ-confined prostate cancer include external beam radiation therapy, radical prostatectomy, radiotherapy/brachytherapy, cryoablation and high-intensity focused ultrasound. None of these are cancer-specific and are commonly accompanied by side effects, including urinary incontinence and erectile dysfunction. Moreover, subsequent surgical treatments following biochemical recurrence after these interventions are either limited or affected by the scarring present in the surrounding tissue. Carnosine (ß-alanyl-L-histidine) is a histidine-containing naturally occurring dipeptide which has been shown to have an anti-tumorigenic role without any detrimental effect on healthy cells; however, its effect on prostate cancer cells has never been investigated. In this study, we investigated the effect of carnosine on cell proliferation and metabolism in both a primary cultured androgen-resistant human prostate cancer cell line, PC346Flu1 and murine TRAMP-C1 cells. Our results show that carnosine has a significant dose-dependent inhibitory effect in vitro on the proliferation of both human (PC346Flu1) and murine (TRAMP-C1) prostate cancer cells, which was confirmed in 3D-models of the same cells. Carnosine was also shown to decrease adenosine triphosphate content and reactive species which might have been caused in part by the increase in SIRT3 also shown after carnosine treatment. These encouraging results support the need for further human in vivo work to determine the potential use of carnosine, either alone or, most likely, as an adjunct therapy to surgical or other conventional treatments.

Measuring the Quality of Personal Care in Patients Undergoing Radiotherapy for Prostate Cancer.

AIMS: To describe the quality of the non-technical component of the care (personal care) of patients receiving radical radiotherapy for prostate cancer and to identify elements of personal care that should be priorities for quality improvement. MATERIALS AND METHODS: One hundred and eight patients undergoing radiotherapy for localised prostate cancer completed a self-administered questionnaire that asked them to rate the importance of 143 non-technical elements of care and to rate the quality of their own care with respect to each element. The elements that a patient rated as both 'very important' and less than 'very good' were deemed to be his priorities for improvement. The priorities of the population were established by ranking the elements based on the percentage of patients who identified them as a priority (importance/quality analysis). RESULTS: The response rate was 65%. The percentage of elements rated 'very good' varied from patient to patient: median 79% (interquartile range 69-92%). The percentage of elements rated either 'very good' or 'good' was higher: median 96% (interquartile range 86-98%). Nonetheless, almost every patient rated at least some elements of his care as less than optimal, regardless of the cut-off point used to define optimal quality. Patients assigned their lowest quality ratings to elements relating to the quality of the treatment environment and comprehensiveness of additional services available to them. However, patients rated most of these elements as relatively unimportant, and importance/quality analysis identified elements of care relating to communication of information about the disease and its treatment as the highest priorities for quality improvement. CONCLUSIONS: Most patients rated most elements of their personal care as very good, but almost all were able to identify some elements that were less than optimal. When ratings of quality were integrated with ratings of importance, elements relating to communication emerged as the patients' highest priorities for quality improvement.

Exploring attitudes toward eating disorders among elite athlete support personnel.

To date, little is known about the beliefs, attitudes, and experiences of athlete support personnel (ASP) working in elite sport toward disordered eating (DE) and eating disorders (EDs). This study seeks to explore this area of mental health, employing an attribution model of stigma as a conceptual lens. Interviews were undertaken with 14 service providers (seven males and seven females) working in high-performance sport in Ireland. In contrast to previous research in the general population, findings revealed that sport-based personnel, in the main, did not hold the individual responsible for the development of their eating disorder. The predominant emotional response of those who had worked with an athlete with a known or suspected eating disorder was anxiety and worry. In line with the findings of previous studies with other health professionals, negative views on the prognosis of those with EDs were expressed by the ASP. Furthermore, confidentiality was found to be a significant barrier to bringing athletes' disclosure of problematic eating or exercise behavior to the fore. The findings of this study add to the limited research exploring attitudes toward EDs in sport and highlights the importance of greater education and openness toward this particular mental health problem.

Assessing the toxic effects of DMSO on cord blood to determine exposure time limits and the optimum concentration for cryopreservation.

BACKGROUND AND OBJECTIVES: Advantages of using cord blood (CB) over other sources of haematopoietic progenitor cells, such as bone marrow, include the ability to cryopreserve and bank the samples until requested for a transplant. Cryopreservation requires the addition of a cryoprotectant to prevent the formation of intracellular ice during freezing. Dimethyl sulphoxide (DMSO) is commonly used at a concentration of 10% (v/v); however, there is evidence to suggest this chemical is toxic to cells as well as to patients after infusion. MATERIALS AND METHODS: The toxic effects of DMSO were assessed through cell viability and in vitro functional assays in fresh and post-thaw CB samples before determining the maximum exposure time and optimal concentration for cryopreservation. RESULTS: A dose-dependent toxicity of DMSO was observed in fresh samples with 40% removing all viable and functional haematopoietic progenitor cells (HPC). In fresh and post-thaw analysis, minimal toxic effect was observed when cryopreservation was delayed for up to 1 h after 10% DMSO addition. After thawing, DMSO washout was superior to dilution or unmanipulated when maintained for long periods (advantage observed 1 h after thawing). Finally, the optimum concentration for cryopreserving CB was found to be 7.5 to 10% with detrimental effects observed outside of this range. CONCLUSION: These results support the use of 7.5-10% as the optimal DMSO concentration and the maximum exposure time should be limited to <1 h prior to freezing and 30 min post-thaw.

HAGE (DDX43) is a biomarker for poor prognosis and a predictor of chemotherapy response in breast cancer.

BACKGROUND: HAGE protein is a known immunogenic cancer-specific antigen. METHODS: The biological, prognostic and predictive values of HAGE expression was studied using immunohistochemistry in three cohorts of patients with BC (n=2147): early primary (EP-BC; n=1676); primary oestrogen receptor-negative (PER-BC; n=275) treated with adjuvant anthracycline-combination therapies (Adjuvant-ACT); and primary locally advanced disease (PLA-BC) who received neo-adjuvant anthracycline-combination therapies (Neo-adjuvant-ACT; n=196). The relationship between HAGE expression and the tumour-infiltrating lymphocytes (TILs) in matched prechemotherapy and postchemotherapy samples were investigated. RESULTS: Eight percent of patients with EP-BC exhibited high HAGE expression (HAGE+) and was associated with aggressive clinico-pathological features (Ps<0.01). Furthermore, HAGE+expression was associated with poor prognosis in both univariate and multivariate analysis (Ps<0.001). Patients with HAGE+did not benefit from hormonal therapy in high-risk ER-positive disease. HAGE+and TILs were found to be independent predictors for pathological complete response to neoadjuvant-ACT; P<0.001. A statistically significant loss of HAGE expression following neoadjuvant-ACT was found (P=0.000001), and progression-free survival was worse in those patients who had HAGE+residual disease (P=0.0003). CONCLUSIONS: This is the first report to show HAGE to be a potential prognostic marker and a predictor of response to ACT in patients with BC.

Expression of transglutaminase-2 isoforms in normal human tissues and cancer cell lines: dysregulation of alternative splicing in cancer.

The multiple enzymatic activities and functions of transglutaminase type 2 (TG2) may be attributed to alternative TG2 molecules produced by differential splicing of TG2 mRNA. Different RNA transcripts of the human TG2 gene (TGM2) have been identified, but the expression of TG2 multiple transcripts has never been systematically addressed. We have confirmed and rationalized the main TG2 variants and developed a screening assay for the detection of alternative splicing of TG2, based on real-time reverse-transcription PCR. We have quantified the multiple TG2 transcripts in a wide range of normal tissues and in cancer cell lines from four different sites of origin. Our data show a significant correlation in the expression of canonical and alternative TG2 isoforms in normal human tissue, but differences in alternative splicing of TG2 in cancer cell lines, suggesting that in cancer cells the alternative splicing of TG2 is a more active process.

ELISPOT assays provide reproducible results among different laboratories for T-cell immune monitoring--even in hands of ELISPOT-inexperienced investigators.

Measurements of antibodies in bodily fluids (e.g., by ELISA) have provided robust and reproducible results for decades and such assays have been validated for monitoring of B-cell immunity. In contrast, measuring T-cell immunity has proven to be a challenge due to the need to test live cells in functional assays ex vivo. Several previous efforts looking into the reproducibility of ex vivo T-cell assays between different laboratories, or even within the same laboratory, have provided rather discouraging results. The hypothesis we tested in this study is that those poor results are due to the lack of assay and data analysis standardization, rather than the inherent complexity of T-cell assays. In this study, 11 laboratories across Europe and the United States were provided identical reagents and were asked to follow the same protocol while testing aliquots of the same three cryopreserved peripheral blood mononuclear cells (PBMC) in an interferon-gamma (IFNgamma) ELISPOT assay measuring the antigen-specific T-cell response to a CMV peptide. All individuals performing the assays were ELISPOT novices. At their first attempt, while three of these individuals failed with the basic logistics of the trial, eight detected the peptide-specific CD8+ T-cells in frequencies approximating the values established by the Reference Laboratory. The data show that ELISPOT assays provide reproducible results among different laboratories when the assay procedure and data analysis is standardized. Since ELISPOT assays have been qualified and validated for regulated studies, they are ideal candidates for robust and reproducible monitoring of T-cell activity in vivo.

Cancer/testis antigens for therapeutic use.

Since van der Bruggen and colleagues first identified specific human tumour-associated antigens of the MAGE family, numerous potential immunotherapeutic targets have been discovered, often belonging to the so-called cancer/ testis gene family. Several members of this group have been described as immunogenic and have been utilised in clinical trials. In a search for interesting targets within this family, our laboratory has focussed its works for a number of years on two novel cancer/testis antigens called T21 and HAGE. In this article, we will focus our discussion on their levels of expression in a wide variety of both normal and cancer tissues, their possible role in tumour cell development and proliferation, and their immunogenic potential.

CTL responses to Leishmania mexicana gp63-cDNA vaccine in a murine model.

Immunity to Leishmania is believed to be strongly dependent upon the activation of Th1 immune responses, although the exact role of cytotoxic T lymphocytes (CTLs) has not yet been determined. The aims of this study were to establish a suitable cytotoxicity assay to measure CTL activity and to compare immunity induced by Leishmania mexicana gp63 cDNA via i.m. injection and gene gun immunization in the BALB/c mouse model. The CTL activity was evaluated by short-term (51)Cr-release cytotoxicity assays against CT26 tumour cells transfected with L. mexicana gp63 cDNA and dendritic cells (DCs) loaded with soluble Leishmania antigen (SLA) as targets. The results clearly demonstrated that higher protection to L. mexicana infection was induced by gene gun DNA-immunization vs. i.m. injection. Cytotoxic T lymphocyte activity of splenocytes was observed in mice immunized either with L. mexicana gp63 cDNA or SLA and long-lived CTL activity was observed in immunized and/or re-challenged mice but not naïve mice infected with the parasite.

DNA demethylation and histone deacetylation inhibition co-operate to re-express estrogen receptor beta and induce apoptosis in prostate cancer cell-lines.

INTRODUCTION: Epigenetic silencing mechanisms are increasingly thought to play a major role in the development of human cancers, including prostate cancer. Promoter CpG island hypermethylation and histone hypoacetylation, catalyzed by DNA methyltransferase (DNMT) and histone deacetylase (HDAC), respectively, are associated with transcriptional repression in a number of cancers. Evidence is accumulating the two mechanisms are dynamically linked, yet few studies have examined a potential interaction in prostate cancer. METHODS: LNCaP, DU-145, and PC-3 prostate cancer cells were co-treated with a DNMT inhibitor, 5'-aza-2'-deoxycytidine (5-AZAC), and an HDAC inhibitor, trichostatin A (TSA). Following treatment cells were processed for cell proliferation/apoptosis assays, or harvested for real-time RT-PCR. Assessed target genes were estrogen receptor beta (ERbeta), estrogen receptor alpha (ERalpha), androgen receptor (AR), progesterone receptor (PGR), and prostate specific antigen (PSA). RESULTS: In all cell-lines, co-treatment was associated with reduced cell proliferation compared with control groups (P<0.05). A reciprocal rise in caspase activation was identified, indicating apoptosis was the major mechanism of cell death. Most marked effects were seen in the androgen-dependent, AR-positive LNCaP cell-line. In all cell-lines, an additive re-expression of ERbeta was identified in the co-treatment group, a finding not seen for either AR or PSA. CONCLUSION: At concentrations associated with gene re-expression, the DNA demethylating agent 5-AZAC and the HDAC inhibitor TSA co-operate to induce apoptosis in prostate cancer cell-lines. Increased apoptosis in the co-treatment group was associated with marked re-expression of ERbeta, raising the possibility of further targeting of prostate cancer cells with ERbeta-selective agents.

Identification of immunogenic MHC class II Tyrosinase-derived peptides using HLA-DR1 and HLA-DR4 transgenic mice.

The immunogenicity of "novel" MART-1 and Tyrosinase class-II peptides was assessed in transgenic mice. Tyrosinase(141-161) peptide was found to be immunogenic and endogenously processed in the HLA-DRbeta1*0101 and HLA-DRbeta1*0401 transgenic mice with peptide specific production of IFNgamma or IL-5 respectively. The MART-1(29-43) peptide was only found immunogenic in HLA-DRbeta1*0101 mice.

Fiction, poetry and mental health: expressive and therapeutic uses of literature.

People with experience as mental health clients, mental health nurses, writers and other professionals have used literature to benefit mental health service users in various ways. These include expressive writing, as well as applications in psychotherapy and counselling and to deal with specific problems and symptoms. In addition, therapeutic story-telling, bibliotherapy and poetry therapy have been used. Various benefits have been described, but some accounts do not include evidence of clinical effectiveness. However, positive treatment outcomes have been reported in research papers and other literature, with particular evidence of clinical effectiveness in some studies of bibliotherapy, therapeutic writing and poetry therapy. Further work is needed to clarify and measure the effectiveness of various expressive and therapeutic uses of literature. The authors also recommend collaboration among practitioners and the need for supporting evidence for proposals for increased resources in this field.

Induction of human cytotoxic T lymphocytes that preferentially recognise tumour cells bearing a conformational p53 mutant.

The tumour-suppressor gene p53 is pivotal in the regulation of apoptosis, and point mutations within p53 are the commonest genetic alterations in human cancers. Cytotoxic T lymphocytes (CTL) recognise peptide-MHC complexes on the surface of tumour cells and bring about lysis. Therefore, p53-derived peptides are potential candidates for immunisation strategies designed to induce antitumour CTL in patients. Conformational changes in the p53 protein, generated as a result of point mutations, frequently expose the 240 epitope, RHSVV (amino acids 212-217), which may be processed differently from the wild-type protein resulting in an altered MHC-associated peptide repertoire recognised by tumour-specific CTL. In this study 42 peptides (37 overlapping nonameric peptides, from amino acids 193-237 and peptides 186-194, 187-197, 188-197, 263-272, 264-272, possessing binding motifs for HLA-A2) derived from the wild-type p53 protein sequence were assayed for their ability to stabilise HLA-A2 molecules in MHC class I stabilisation assays. Of the peptides tested, 24 stabilised HLA-A2 molecules with high affinity (fluorescence ratio >1.5) at 26 degrees C, and five (187-197, 193-200, 217-224, 263-272 and 264-272) also stabilised the complexes at 37 degrees C. Peptides 188-197, 196-203 and 217-225 have not previously been identified as binders of HLA-A2 molecules and, of these, peptide 217-225 stabilised HLA-A2 molecules with the highest fluorescence ratio. Peptide 217-225 was chosen to generate HLA-A2-restricted CTL in vitro; peptide 264-272 was used as a positive control. The two primary CTL thus generated (CTL-217 using peptide 217 225; and CTL-264 using peptide 264-272) were capable of specifically killing peptide-pulsed T2 or JY cells. In order to determine whether these peptides were endogenously processed and to test the hypothesis that mutants expressing different protein conformations would generate an alternative peptide repertoire at the cell surface, a panel of target cells was generated. HLA-A2+ SaOs-2 cells were transfected with p53 cDNA containing point mutations at either position 175 (R-->H) or 273 (R-->H) (SaOs-2/175 and SaOs-2/273). Two HLA-A2-negative cell lines, A431 and SKBr3, naturally expressing p53 mutations at positions 273 and 175 respectively, were transfected with a cDNA encoding HLA-A2. The results showed that primary CTL generated in response to both peptides were capable of killing SaOs-2/175 and SKBr3-A2 cells, which possess the same mutation, but not SaOs-2/273, A431-A2 or SKBr3 cells transfected with control vector. This suggests that these peptides are presented on the surface of SaOs-2/175 and SKBr3-A2 cells in a conformation-dependent manner and represent potentially useful target peptides for immunotherapy.

Characterization of the effects of hepatitis C virus nonstructural 5A protein expression in human cell lines and on interferon-sensitive virus replication.

The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy in clinical studies. Biochemical studies have demonstrated that NS5A interacts in vitro with and inhibits the IFN-induced, RNA-dependent protein kinase, PKR, and that NS5A interacts with at least one other cellular kinase. The present study describes the establishment and characterization of various stable NS5A-expressing human cell lines, and the development of a cell culture-based assay for determining the inherent IFN resistance of clinical NS5A isolates. Human epithelioid (Hela) and osteosarcoma (U2-OS) cell lines were generated that express NS5A under tight regulation by the tetracycline-dependent promoter. Maximal expression of NS5A occurred at 48 hours following the removal of tetracycline from the culture medium. The half-life of NS5A in these cell lines was between 4 to 6 hours. NS5A protein expression was localized cytoplasmically, with a staining pattern consistent with the location of the Golgi apparatus and endoplasmic reticulum. In the majority of cell lines, no obvious phenotypic changes were observed. However, three genotype 1b NS5A-expressing osteosarcoma cell lines exhibited cytopathic effect and severely reduced proliferation as a result of high-level NS5A expression. Full-length NS5A protein isolated from a genotype 1b IFN-nonresponsive patient (NS5A-1b) was capable of rescuing encephalomyocardititis virus replication during IFN challenge up to 40-fold, whereas a full-length NS5A-1a and an interferon sensitivity determining region (ISDR) deletion mutant (NS5A-1a-triangle upISDR) isolated from a genotype 1a IFN-nonresponsive patient showed no rescue activity. The NS5A-1b and NS5A-1a proteins also rescued vesicular stomatitis virus replication during IFN treatment by two- to threefold. These data cummulatively suggest that NS5A expression alone can render cells partially resistant to the effects of IFN against IFN-sensitive viruses, and that in some systems, these effects may be independent of the putative ISDR. A scenario is discussed in which the NS5A protein may employ multiple strategies contributing to IFN resistance during HCV infection.

Evolution of hepatitis C virus quasispecies in hypervariable region 1 and the putative interferon sensitivity-determining region during interferon therapy and natural infection.

To study hepatitis C virus (HCV) genetic mutation during interferon (IFN) therapy, the temporal changes in HCV quasispecies heterogeneity were compared before and after treatment for nine patients infected with HCV genotype 1, including four nonresponders, four responders who relapsed after therapy, and one responder who experienced a breakthrough of viremia during therapy. Nine untreated patients with an average time between specimens of 8.4 years served as controls. Sequences from the second envelope glycoprotein gene hypervariable region 1 (HVR1) and the putative IFN sensitivity-determining region (ISDR) of the nonstructural NS5A gene were analyzed by heteroduplex mobility assays and nucleotide sequencing. A strong positive correlation was found between the percent change in a heteroduplex mobility ratio (HMR) and percent change in nucleotide sequence (r = 0.941, P < 0.001). The rate of fixation of mutations in the HVR1 was significantly higher for IFN-treated patients than for controls (6.97 versus 1.31% change in HMR/year; P = 0.02). Similarly, a higher rate of fixation of mutations was observed in the ISDR for IFN-treated patients than for untreated controls, although the result was not significant (1.45 versus 0.15 amino acid changes/year; P = 0.12). On an individual patient basis, IFN therapy was associated with measurable HVR1 and ISDR mutation in nine of nine (100%) and two of nine (22.2%) patients, respectively. Evolution to IFN-resistant ISDR sequences was observed in only one of nine IFN-treated patients. These data suggest that IFN therapy frequently exerts pressure on the HCV envelope region, while pressure on the ISDR was evident in only a subset of patients. Thus, the selection pressures evoked on HCV genotype 1 quasispecies during IFN therapy appear to differ among different patients.

Baculovirus expression and purification of human and rat cytochrome P450 2E1.

High-level expression of human and rat cytochrome P450 2E1 (CYP2E1) was achieved using a baculovirus expression system. A full length cDNA encoding human CYP2E1 was cloned from a human liver cDNA library and sequenced using the dideoxy sequencing method. Insect cells were infected with the homologous recombinant baculoviruses containing the human and rat CYP2E1 cDNAs, respectively. The infected cells were harvested at a time when 450-nm peak intensities were at a maximal level and there was no 420-nm peak observed in the reduced CO difference spectrum. Both human and rat CYP2E1 were then purified to electrophoretic homogeneity by a relatively rapid and efficient procedure. The specific contents of the purified human and rat CYP2E1 were 13.8 and 17.0 nmol/mg protein, respectively. The lambda(max) of the reduced CO difference spectra of both purified rat and human CYP2E1 was found to be 451.5 nm. When the purified rat and human CYP2E1 were reconstituted with purified rat NADPH-P450 reductase and human cytochrome b5, they were able to metabolize several known CYP2E1 substrates: chlorzoxazone, p-nitrophenol, acetaminophen, and carbon tetrachloride. Interestingly, cytochrome b5 markedly stimulated the CYP2E1-mediated two-electron oxidation of the first three substrates, while it had almost no effect on the presumed one-electron reduction of carbon tetrachloride.

Effects of some K(+)-channel inhibitors on the electrical behaviour of guinea-pig isolated trachealis and on its responses to spasmogenic drugs.

1. A study has been made of the effects of inhibitors selective among plasmalemmal K(+)-channels on the sensitivity and responsiveness of guinea-pig trachealis muscle to carbachol, histamine and KCl. The effects of the K(+)-channel inhibitors on the resting membrane potential and spontaneous electrical activity of the trachealis cells have also been examined. 2. In indomethacin (2.8 microM)-treated trachealis muscle, dofetilide (1 microM) and glibenclamide (10 microM) were each devoid of spasmogenic activity. In contrast, 4-aminopyridine (4-AP, 62.5 microM--8 mM), charybdotoxin (ChTX, 100 nM) and iberiotoxin (IbTX, 100 nM) were each spasmogenic. Spasm evoked by 4-AP, IbTX or ChTX was reduced, though not abolished, by atropine (1 microM). Spasm evoked by 4-AP (1 mM), ChTX (100 nM) or IbTX (100 nM) was unaffected by tetrodotoxin (TTX; 3.1 microM) or by tissue pretreatment with capsaicin (1 microM for 30 min). Spasm evoked by IbTX or ChTX was abolished by nifedipine (1 microM). 3. Dofetilide (1 microM) and glibenclamide (10 microM) were each without effect on the tracheal sensitivity or responsiveness to carbachol, histamine or KCl. 4-AP (1 mM) antagonized carbachol, potentiated histamine but did not affect tissue sensitivity to KCl. When the effects of 4-AP were examined in the presence of atropine (1 microM), it potentiated all the spasmogens including carbachol. IbTX and ChTX (each 100 nM) potentiated all three spasmogens. Potentiation of histamine induced by 4-AP (1 mM) or IbTX (100 nM) was also observed in tissues treated with a combination of atropine (1 microM) and TTX (3.1 microM). 4. Dofetilide (1 and 10 microM) was without effect on the resting membrane potential or spontaneous electrical activity of the trachealis cells. 4-AP (1 mM) evoked depolarization and caused a small increase in the frequency of slow wave discharge. The depolarization evoked by 4-AP was abolished by atropine (1 microM). IbTX (100 nM) and ChTX (100 nM) each evoked little or no change in resting membrane potential but converted the spontaneous slow waves into spike-like, regenerative action potentials. These electrophysiological effects of IbTX and ChTX were unaffected by atropine (1 microM). 5. It is concluded that the dofetilide-sensitive, cardiac, delayed rectifier K(+)-channel is either not expressed in trachealis muscle or is of no functional importance in that tissue. The ATP-sensitive K(+)-channel (KATP) does not moderate tracheal sensitivity to spasmogens such as carbachol, histamine and KCl. The 4-AP-sensitive delayed rectifier K(+)-channel (Kdr) and the large Ca(2+)-dependent K(+)-channel (BKCa) each moderate trachealis muscle sensitivity to spasmogens. Neither Kdr nor BKCa plays an important role in determining the resting membrane potential of guinea-pig trachealis cells. However, the BKCa channel is responsible for limiting the effects of the increase in membrane Ca2+ conductance associated with the depolarizing phase of slow waves. It is BKCa channel opening that prevents the development of a slow wave into a spike-like regenerative action potential.

Update on the recommended viewing protocol for FAXIL threshold contrast detail detectability test objects used in television fluoroscopy.

The significance of varying the viewing conditions that may affect the perceived threshold contrast of X-ray television fluoroscopy systems has been investigated. Factors investigated include the ambient room lighting and the viewing distance. The purpose of this study is to find the optimum viewing protocol with which to measure the threshold detection index. This is a particular problem when trying to compare the image quality of television fluoroscopy systems in different input field sizes. The results show that the viewing distance makes a significant difference to the perceived threshold contrast, whereas the ambient light conditions make no significant difference. Experienced observers were found to be capable of finding the optimum viewing distance for detecting details of each size, in effect using a flexible viewing distance. This allows the results from different field sizes to be normalized to account for both the magnification and the entrance air kerma rate differences, which in turn allow for a direct comparison of performance in different field sizes.

Cosegregation of the renin gene with an increase in mean arterial blood pressure in the F2 rats of SHR-WKY cross.

Using the restriction endonuclease, BgI I, Samani et al. found a restriction fragment length polymorphism (RFLP) for the renin gene in spontaneously hypertensive rats (SHR) and its normotensive control Wistar-Kyoto (WKY) rats. This RFLP was confirmed in our laboratory in SHR and WKY rats using a rat renin cDNA probe. The correlation of blood pressure and the renin RFLP was examined in 106 F2 rats produced from F1 rats, the offspring of a cross between SHR males and WKY females. Systolic blood pressure was measured by the tail cuff method at 12 weeks of age. Mean arterial blood pressure of anesthetized rats was measured by cannulation of the femoral artery prior to sacrifice. The frequency of renin genotype showed a typical 1:2:1 Mendelian ratio in F2 rats of SHR and WKY cross. The mean arterial blood pressure of F2 rats homozygous with the SHR allele was significantly higher than F2 rats that were heterozygous or homozygous for the WKY allele. No significant difference in systolic blood pressure was observed in these F2 rats. Thus, the renin gene RFLP cosegregates with an increase in mean arterial blood pressure in the F2 rats of SHR and WKY cross.

Sex influence on renal alpha 2-adrenergic receptor density in the spontaneously hypertensive rat.

Male spontaneously hypertensive rats (SHR) have higher blood pressure than females. We compared renal alpha 2-adrenergic receptor density among intact SHR and Wistar-Kyoto (WKY) rats of both sexes, male and female SHR gonadectomized at 4 weeks of age, and gonadectomized SHR supplemented with testosterone. Additional groups of SHR were treated with enalapril (30 mg/kg per day), an angiotensin-converting enzyme inhibitor, from 5 to 14 weeks of age. Renal alpha 2-adrenergic receptor density was higher in males than females in both SHR and WKY rats. Female SHR and WKY rats had identical low renal alpha 2-adrenergic receptor density. Castration of male SHR reduced the male-female differences in blood pressure and renal alpha 2-adrenergic receptor density by 60%. Treatment with testosterone raised blood pressure and renal alpha 2-adrenergic receptor density to the intact male levels in both gonadectomized males and females. Treatment with enalapril decreased blood pressure but not renal alpha 2-adrenergic receptor density in both male and female SHR. We conclude that (1) both renal alpha 2-adrenergic receptor density and blood pressure are influenced by sex in SHR and WKY, (2) renal alpha 2-adrenergic receptor density like blood pressure is regulated by androgens, and (3) increased renal alpha 2-adrenergic receptor density is not a consequence of high blood pressure in male SHR.