Pre- and post-puberty expression of genes and proteins in the uterus of Bos indicus heifers: the luteal phase effect post-puberty.

Progesterone signaling and uterine function are crucial in terms of pregnancy establishment. To investigate how the uterine tissue and its secretion changes in relation to puberty, we sampled tissue and uterine fluid from six pre- and six post-pubertal Brahman heifers. Post-pubertal heifers were sampled in the luteal phase. Gene expression of the uterine tissue was investigated with RNA-sequencing, whereas the uterine fluid was used for protein profiling with mass spectrometry. A total of 4034 genes were differentially expressed (DE) at a nominal P-value of 0.05, and 26 genes were significantly DE after Bonferroni correction (P < 3.1 × 10-6 ). We also identified 79 proteins (out of 230 proteins) that were DE (P < 1 × 10-5 ) in the uterine fluid. When we compared proteomics and transcriptome results, four DE proteins were identified as being encoded by DE genes: OVGP1, GRP, CAP1 and HBA. Except for CAP1, the other three had lower expression post-puberty. The function of these four genes hypothetically related to preparation of the uterus for a potential pregnancy is discussed in the context of puberty. All DE genes and proteins were also used in pathway and ontology enrichment analyses to investigate overall function. The DE genes were enriched for terms related to ribosomal activity. Transcription factors that were deemed key regulators of DE genes are also reported. Transcription factors ZNF567, ZNF775, RELA, PIAS2, LHX4, SOX2, MEF2C, ZNF354C, HMG20A, TCF7L2, ZNF420, HIC1, GTF3A and two novel genes had the highest regulatory impact factor scores. These data can help to understand how puberty influences uterine function.

Association between KRAS mutation and lung metastasis in advanced colorectal cancer.

BACKGROUND: KRAS mutations have been associated with lung metastases at diagnosis of metastatic colorectal cancer (mCRC), but the impact of this mutation on subsequent development of lung metastasis is unknown. We investigated KRAS mutation as a predictor of lung metastasis development. METHODS: We retrospectively evaluated data from patients with mCRC whose tumour was tested for KRAS mutation from 2008 to 2010. The relationships of KRAS mutational status with time-to-lung metastasis (TTLM) and overall survival (OS) were analysed. RESULTS: Of the 494 patients identified, 202 (41%) had tumours with KRAS mutation. KRAS mutations were associated with a shorter TTLM (median 15.2 vs 22.4 months; hazard ratio=1.40; P=0.002) and a two-fold greater odds of developing lung metastases during the disease course in patients with liver-limited mCRC at diagnosis (72 vs 56%, P=0.007). Overall survival did not differ by KRAS status. CONCLUSIONS: Lung metastasis was more likely to develop during the disease course in patients whose tumour had a KRAS mutation than in those whose tumour did not have a KRAS mutation. This finding may have an impact on decision making for surgical resection of metastatic disease.

Novel orally available salvinorin A analog PR-38 inhibits gastrointestinal motility and reduces abdominal pain in mouse models mimicking irritable bowel syndrome.

The opioid and cannabinoid systems play a crucial role in multiple physiological processes in the central nervous system and in the periphery. Selective opioid as well as cannabinoid (CB) receptor agonists exert a potent inhibitory action on gastrointestinal (GI) motility and pain. In this study, we examined (in vitro and in vivo) whether PR-38 (2-O-cinnamoylsalvinorin B), a novel analog of salvinorin A, can interact with both systems and demonstrate therapeutic effects. We used mouse models of hypermotility, diarrhea, and abdominal pain. We also assessed the influence of PR-38 on the central nervous system by measurement of motoric parameters and exploratory behaviors in mice. Subsequently, we investigated the pharmacokinetics of PR-38 in mouse blood samples after intraperitoneal and oral administration. PR-38 significantly inhibited mouse colonic motility in vitro and in vivo. Administration of PR-38 significantly prolonged the whole GI transit time, and this effect was mediated by µ- and κ-opioid receptors and the CB1 receptor. PR-38 reversed hypermotility and reduced pain in mouse models mimicking functional GI disorders. These data expand our understanding of the interactions between opioid and cannabinoid systems and their functions in the GI tract. We also provide a novel framework for the development of future potential treatments of functional GI disorders.

Parameters of the reproductive tract, spermatogenesis, daily sperm production and major seminal plasma proteins of tropically adapted morada nova rams.

This study describes the reproductive parameters of Morada Nova rams, a breed of hair sheep from Brazil and with unique adaption to tropical environments. At 42 weeks of age, 15 rams were subjected to semen collection and, 1 week later, animals were slaughtered for collection of testes, epididymis and accessory sex glands. We conducted 2-D electrophoresis of seminal plasma proteins and major spots of stained gels were identified by LC-MS/MS. Total RNA was isolated from testis, epididymis and vesicular glands and subjected to qPCR. At slaughter, scrotal circumference and testicular weight were 27.5 ± 0.5 cm and 109.5 ± 6.0 g, respectively. Seminiferous tubule (ST) diameter was 188.3 ± 4.0 µm and each testis contained 1.9 ± 0.1 Sertoli cells (×10(9) ). Each Sertoli cell supported 0.1 ± 0.01 A spermatogonia, 3.0 ± 0.2 pachytene spermatocytes and 7.7 ± 0.5 round spermatids/tubule cross section. Daily sperm production reached 5.6 × 10(6)  cells/g of testis parenchyma. Testis size appeared as indicative of ST diameter and associated with epididymal measurements, as well as with the population of round spermatids and Sertoli cells/testis. Rams with heavier testes had greater daily sperm production and more Sertoli cells/testis. We detected 90.9 ± 9.6 spots per 2-D gel of seminal plasma. Major seminal proteins were identified as ram seminal vesicle proteins at 14 and 22 kDa, representing 16.2% and 12.8% of the total intensity of valid spots in the gels, respectively. Expression of both genes was greater in the vesicular glands as compared to testis and epididymis. Pixel intensity for those proteins in the 2-D gels was significantly correlated with seminal vesicle weight. This is the first description of the basic reproductive aspects of Morada Nova rams, including protein profiles of their seminal plasma. These findings will allow a better understanding of their reproductive physiology.

The invasive bumblebee Bombus terrestris (Linnaeus, 1758) disrupts the adaptive function of heteranthery by indiscriminately visiting the pollinating and feeding anthers of Senna arnottiana flowers.

Heteranthery, the presence of different types of anthers on the same flower, is a floral adaptation that aims to balance the need for pollinators to collect pollen as a food resource while ensuring sufficient pollen for pollination. We investigate the role of heteranthery in the pollination of Senna arnottiana flowers and how it is affected by the behaviour of visiting bee species, with a particular focus on the impact of the invasive bumblebee Bombus terrestris. In three populations of S. arnottiana we measured the size of three sets of anthers and style, stigma-anther separation, pollen quantity and fruit set, and contrasted it with the body size, behaviour, and pollination effectiveness of all floral visitors. Different bee species visited S. arnottiana flowers, and their foraging behaviour varied. Large-bodied native bees, including Centris cineraria, Caupolicana sp. and Cadeguala occidentalis, preferentially visited short anthers, whereas B. terrestris, an exotic bumblebee, foraged from both short and long anthers without distinction. In addition, B. terrestris contacted the stigma at a lower rate than large-bodied native bees. Instead of concentrating its pollen-gathering efforts on the feeding anthers, as predicted by the "division of labor" hypothesis, B. terrestris indiscriminately visited both types of anthers similarly. This behaviour of B. terrestris may disrupt the adaptive significance of heteranthery by mixing the roles of pollination and feeding anthers of S. arnottiana. Therefore, our results highlight the potential disruption of this relationship by exotic pollinators and the need to consider it in conservation efforts.

Electrochemical Impedance Analysis for Corrosion Rate Monitoring of Sol-Gel Protective Coatings in Contact with Nitrate Molten Salts for CSP Applications.

The protective behaviour of ZrO2-3%molY2O3 sol-gel coatings, deposited with an immersion coating technique on 9Cr-1Mo P91 steel, was evaluated with corrosion monitoring sensors using the electrochemical impedance spectroscopy technique. The tests were carried out in contact with solar salt at 500 °C for a maximum of 2000 h. The results showed the highly protective behaviour of the coating, with the corrosion process in the coated system being controlled by the diffusion of charged particles through the protective layer. The coating acts by limiting the transport of ions and slowing down the corrosive process. The system allowed a reduction in the corrosion rate of uncoated P91 steel. The estimated corrosion rate of 22.62 µm·year-1 is lower than that accepted for in-service operations. The proposed ZrO2-3%molY2O3 sol-gel coatings are an option to mitigate the corrosion processes caused by the molten salts in concentrated solar power plants.

Assessment of acrosome and nuclear abnormalities in human spermatozoa with large vacuoles.

BACKGROUND: Spermatozoa with large vacuoles (SLV) may have a negative impact on embryo development. The origin of these vacuoles is unknown. We evaluated acrosome and nucleus alterations in isolated SLV, versus unselected spermatozoa. METHODS: We studied 20 patients with teratozoospermia. Spermatozoa from the native semen sample and spermatozoa presenting a vacuole occupying >13.0% total head area, isolated under high magnification (×6600), were assessed. Confocal and transmission electron microscope evaluations were performed on SLV and native sperm, respectively. Acrosome morphology and DNA fragmentation were analysed using proacrosin immunolabelling (monoclonal antibody 4D4) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay. Chromatin condensation was evaluated with aniline blue staining. Sperm aneuploidy was assessed using fluorescence in situ hybridization. RESULTS: SLV represented 38.0 ± 5.10% of motile spermatozoa obtained after gradient density centrifugation. Vacuoles were mainly in the anterior and median sperm head (45.7 ± 2.90 and 46.1 ± 3.00%, respectively). Abnormal acrosomes were increased in SLV compared with unselected spermatozoa (77.8 ± 2.49 versus 70.6 ± 2.62%; P = 0.014). Microscopic observations showed an exclusively nuclear localization of large vacuoles. Complete DNA fragmentation was higher in native spermatozoa (P < 0.0001) than SLV, while chromatin condensation was altered in SLV (P < 0.0001). Aneuploidy and diploidy rates were increased in SLV (P < 0.0001). CONCLUSIONS: Sperm vacuoles were exclusively nuclear. In our selected teratozoospermic population, aneuploidy and chromatin condensation defects were the main alterations observed in SLV. Based on results from this small sample of spermatozoa, we propose a global impairment of the spermatogenesis process as a common origin of the morphological alterations.

Differences in stationary-phase cells of a commercial Saccharomyces cerevisiae wine yeast grown in aerobic and microaerophilic batch cultures assessed by electric particle analysis, light diffraction and flow cytometry.

We applied electric particle analysis, light diffraction and flow cytometry to obtain information on the morphological changes during the stationary phase of Saccharomyces cerevisiae. The reported analyses of S. cerevisiae populations were obtained under two different conditions, aerobic and microaerophilic, at 27°C. The samples analysed were taken at between 20 and 50 h from the beginning of culture. To assist in the interpretation of the observed distributions a complexity index was used. The aerobically grown culture reached significantly greater cell density. Under these conditions, the cell density experienced a much lower reduction (3%) compared with the microaerophilic conditions (30%). Under aerobic conditions, the mean cell size determined by both electric particle analysis and light diffraction was lower and remained similar throughout the experiment. Under microaerophilic conditions, the mean cell size determined by electric particle analysis decreased slightly as the culture progressed through the stationary phase. Forward and side scatter distributions revealed two cell subpopulations under both growth conditions. However, in the aerobic growing culture the two subpopulations were more separated and hence easier to distinguish. The distributions obtained with the three experimental techniques were analysed using the complexity index. This analysis suggested that a complexity index is a good descriptor of the changes that take place in a yeast population in the stationary phase, and that it aids in the discussion and understanding of the implications of these distributions obtained by these experimental techniques.

Immunological diagnosis of tuberculosis based on recombinant antigens ESAT-6 and CFP-10 in children from an endemic area in northeast Brazil.

Diagnostic tests for tuberculosis (TB) using interferon gamma (IFN-γ) responses produced by T lymphocytes after stimulation by early secretory antigen target 6 (ESAT-6), culture filtrate protein 10 (CFP-10) or purified protein derivate (PPD) were carried out using ELISA (enzyme-linked immunosorbent assay) in whole blood culture supernatants from children with suspected TB disease (n=21), latent TB infection (LTBI; n=17) and negative controls (NC; n=21) from Recife, Pernambuco, Brazil. The results were analysed using the ROC (receiver operating characteristic) curves and the areas under the curve (AUC) generated varied from 0.5 to 1.0 with higher values indicating increased discriminatory ability. Comparisons of AUCs were made using non-parametric assumptions, and the differences were considered significant if P<0.05. The ROC curve showed a statistical difference (P = 0.015) between the LTBI and NC groups with an AUC of 0.731, TB disease and NC (AUC=0.780; P=0.002) and a group with TB (latent infection+disease, n=38) and NC (AUC=0.758; P = 0.001) when the antigen used was ESAT-6. No statistical difference was found between the groups when CFP-10 or PPD was used. In conclusion, the ESAT-6 test may be the most appropriate for diagnosis of childhood TB, both LTBI and TB disease, when associated with epidemiological and clinical data, especially in endemic areas such as Brazil.

Efficacy of a two-component compression system for the therapy of venous leg ulcers.

OBJECTIVE: The performance of a two-component compression system in daily practice was assessed in an outpatient clinic where patients with a wide variety of comorbidities and wound characteristics are treated. METHODS: In the single-centred observational study, patients with venous leg ulcers (VLUs) on one or both legs were treated with a two-component compression system for up to 12 months or until the ulcer healed. The only exclusion criteria were an ABPI <0.8, immobilisation and limited ankle joint flexibility. Study assessment parameters included ulcer size on entry into the study, the presence of skin irritations, occurrence of adverse events and ulcer recurrence. The primary outcome measure was the mean time to healing. RESULTS: In total, 136 patients with VLUs were included. Baseline median ulcer duration was 7.5 months and the baseline median size was 4.3cm(2). The average reduction in total ulcer surface was 2.9 +/- 5.5cm(2) per month. Of the wounds, 90.4% healed after 12 months; the mean healing time was three months (Kaplan-Meier, CI 95%: 3-4). The baseline wound size had a significant influence on mean healing time (Cox proportional hazards model; p<0.0001), wounds >4.3cm(2) healed within a mean of five months, while wounds <4.3cm(2) healed within a mean of two months (log rank test: p<0.00001). CONCLUSION: Healing results were encouraging and suggest a good applicability in the daily practice of VLU therapy. Further studies will include systematic assessment of patient concordance. DECLARATION OF INTEREST: None.

Apolipoprotein E, periodontal disease and the risk for atherosclerosis: a review.

The association between cardiovascular and periodontal diseases is characterized by chronic inflammatory processes, with a high prevalence worldwide and complex genetic-environment interactions. Although apolipoprotein E4 (ApoE4), one of the isoforms coded by a polymorphic APOE gene, has been widely recognized as a risk factor for cardiovascular diseases and as an immunoinflammatory factor, less is known regarding how ApoE4 affects atherosclerosis in periodontitis patients. The aim of this review was to investigate the potential underlying mechanisms related to APOE4 that could increase the risk of periodontal disease and, ultimately, of atherosclerosis. There have only been a few studies addressing apoE polymorphisms in patients with chronic periodontitis. To date, no studies have been performed that have assessed how ApoE4 affects atherosclerotic disease in chronic periodontitis patients. Although clinical studies are warranted, experimental studies have consistently documented the presence of periodontal pathogens, which are usually found in the oral cavity and saliva, in the atherosclerotic plaques of ApoE-deficient mice. In addition, in this review, the potential role of the APOE4 allele as an example of antagonistic pleiotropy during human evolution and its relation to oral health is discussed.

Major seminal plasma proteome of rabbits and associations with sperm quality.

The present study was conducted to describe the major seminal plasma proteome of rabbits and potential associations between seminal proteins and semen criteria. Semen samples were collected from 18 New Zealand adult rabbits, and seminal plasma proteins were analyzed by 2-D SDS-PAGE and tandem mass spectrometry. Sperm motility, vigor, concentration, morphology and membrane sperm viability were evaluated. Rabbits ejaculated 364 ±â€¯70 million sperm/ml, with 81 ±â€¯6.1% motile cells, 3.8 ±â€¯0.2 vigor and 66.7 ±â€¯2.5% sperm with normal morphology. Based on the viability and acrosome integrity assay, there were 65.8 ±â€¯2.5% live sperm with intact acrosome and most spermatozoa had both intact acrosome and functional membrane. On average, 2-D gels of rabbit seminal plasma had 232 ±â€¯69.5 spots, as determined by PDQuest software (Bio Rad, USA). Mass spectrometry allowed the identification of 137 different proteins. The most abundant proteins in rabbit seminal plasma were hemoglobin subunit zeta-like, annexins, lipocalin, FAM115 protein and albumin. The intensity of the spots associated with these five proteins represented 71.5% of the intensity of all spots detected in the master gel. Multiple regression models were estimated using sperm traits as dependent variables and seminal plasma proteins as independent ones. Also, sperm motility had positive association with beta-nerve growth factor and cysteine-rich secretory protein 1-like and a negative one with galectin-1. The percentage of rabbit sperm with intact membrane was related to seminal plasma protein FAM115 complex and tropomyosin. Then, the population of morphologically normal sperm in rabbit semen was positively linked to carcinoembryonic antigen-related cell adhesion molecule 6-like and down regulated by seminal plasma isocitrate dehydrogenase. Based on another regression model, the variation in the percentage of live sperm with intact acrosome was partially explained by the amount of leukocyte elastase inhibitor and the peptidyl-prolyl cis-trans isomerase A in the rabbit seminal fluid. The current study reports the identification of 137 proteins of rabbit seminal plasma. Major proteins of seminal secretion relate primarily to prevention of damages caused by lipid peroxide radicals and oxidative stress, membrane functionality, transport of lipids to the sperm membrane and temperature regulation. Moreover, finding seminal plasma proteins as indicators of semen parameters will improve assisted reproductive technologies.

Effects of acute or 3-day treatments of Hypericum caprifoliatum Cham. & Schltdt. (Guttiferae) extract or of two established antidepressants on basal and stress-induced increase in serum and brain corticosterone levels.

Since depressive patients present alterations in the hypothalamo-pituitary-adrenal (HPA) axis that are normalised by antidepressants, this HPA axis has been considered as a target of their actions. We have investigated the mechanism of action of a cyclohexane extract of Hypericum caprifoliatum (HCP), which displays antidepressant like activity, by studying, in mice, the influence of HCP and of two established antidepressant drugs, imipramine and bupropion, administered either acutely or semi-chronically (once a day, three consecutive days), on serum and brain cortex corticosterone levels, either in basal conditions or shortly after a forced-swimming session (FSS). Administered acutely, imipramine (20 mg/kg, per os (p.o.)), bupropion (30 mg/kg, p.o.) and HCP (360 mg/kg, p.o.) significantly reduced the immobility time and had no effects on FSS-induced increase of serum and cortical corticosterone levels. Conversely, 3 days repeated treatment with imipramine or bupropion resulted in a significant reduction of immobility time and FSS-induced increase of serum and cortical corticosterone levels. In a different way, repeated treatment with HCP significantly reduced the immobility time and only cortical corticosterone levels in stressed mice. These results indicate that short-term treatments with antidepressants are sufficient to induce modifications in the HPA axis reactivity to stress; and that apparently HCP has an influence on corticosterone levels by a mechanism diverse from the other tested antidepressants.

Delayed gastric emptying and altered antrum protein metabolism during activity-based anorexia.

BACKGROUND: Anorexia nervosa, a restrictive eating disorder, is often associated with gastrointestinal disorders, particularly a delayed gastric emptying. However, the mechanisms remained poorly documented. Thus, we aimed to evaluate gastric emptying and antrum protein metabolism in the Activity-Based Anorexia model (ABA). METHODS: Females C57Bl/6 mice were randomized into 3 groups: Control, ABA, and Limited Food Access (LFA). Food access has been progressively limited from 6 h/day at day 6 to 3 h/day at day 9 and until day 17. ABA mice had free access to an activity wheel. Gastric emptying was assessed. On gastric extracts, a proteomic analysis was performed, as well as an evaluation of protein synthesis and protein oxidation. KEY RESULTS: Both LFA and ABA mice exhibited a delayed gastric emptying compared with Controls (P < .05). Proteomic approach revealed 15 proteins that were differentially expressed. Among these proteins, we identified 2 clusters of interest contributing to (i) the organization of muscle fiber with ACTA2, VCL, KRT19, KRT8, and DES proteins and (ii) "heat shock proteins" with STIP1, HSPD1, and HSPA8 proteins. ABA mice specifically exhibited an increased rate of gastric oxidized proteins. CONCLUSIONS AND INFERENCES: Delayed gastric emptying observed in anorectic conditions appears to be secondary to malnutrition. However, an oxidative stress is specifically present in the stomach of ABA mice. Its role remains to be further studied.

Expression and activity profiling of the steroidogenic enzymes of glucocorticoid biosynthesis and the fdx1 co-factors in zebrafish.

The spatial and temporal expression of steroidogenic genes in zebrafish has not been fully characterised. Because zebrafish are increasingly employed in endocrine and stress research, a better characterisation of steroidogenic pathways is required to target specific steps in the biosynthetic pathways. In the present study, we have systematically defined the temporal and spatial expression of steroidogenic enzymes involved in glucocorticoid biosynthesis (cyp21a2, cyp11c1, cyp11a1, cyp11a2, cyp17a1, cyp17a2, hsd3b1, hsd3b2), as well as the mitochondrial electron-providing ferredoxin co-factors (fdx1, fdx1b), during zebrafish development. Our studies showed an early expression of all these genes during embryogenesis. In larvae, expression of cyp11a2, cyp11c1, cyp17a2, cyp21a2, hsd3b1 and fdx1b can be detected in the interrenal gland, which is the zebrafish counterpart of the mammalian adrenal gland, whereas the fdx1 transcript is mainly found in the digestive system. Gene expression studies using quantitative reverse transcriptase-PCR and whole-mount in situ hybridisation in the adult zebrafish brain revealed a wide expression of these genes throughout the encephalon, including neurogenic regions. Using ultra-high-performance liquid chromatography tandem mass spectrometry, we were able to demonstrate the presence of the glucocorticoid cortisol in the adult zebrafish brain. Moreover, we demonstrate de novo biosynthesis of cortisol and the neurosteroid tetrahydrodeoxycorticosterone in the adult zebrafish brain from radiolabelled pregnenolone. Taken together, the present study comprises a comprehensive characterisation of the steroidogenic genes and the fdx co-factors facilitating glucocorticoid biosynthesis in zebrafish. Furthermore, we provide additional evidence of de novo neurosteroid biosynthesising in the brain of adult zebrafish facilitated by enzymes involved in glucocorticoid biosynthesis. Our study provides a valuable source for establishing the zebrafish as a translational model with respect to understanding the roles of the genes for glucocorticoid biosynthesis and fdx co-factors during embryonic development and stress, as well as in brain homeostasis and function.

Dominant T- and B-cell epitopes in an autoantigen linked to Chagas' disease.

In Chagas' disease caused by Trypanosoma cruzi, a paradigm of autoimmune disease, both autoantibodies and autoreactive T cells have been described. We have identified a novel dominant autoantigen, named Cha, recognized by the majority of sera from T. cruzi-infected humans and mice. We noted significant homologies between amino acids 120-129 of Cha, where the B-cell epitope maps, and an expressed sequence tag from T. cruzi, and also between amino acids 254-273 of Cha and a repeated amino acid sequence from the shed acute-phase antigen (SAPA) of T. cruzi. Moreover, T. cruzi-infected mice contain autoreactive T cells that can cross-react with Cha and the SAPA homologous peptides. Transfer of T cells from infected mice triggered anti-Cha (120-129) Ab production in naive recipients. Interestingly, heart tissue sections from those adoptive transferred mice showed cardiac pathology similar to T. cruzi-infected mice. Our results demonstrate the presence of both T- and B-cell cross-reactive epitopes in the Cha antigen. This dual mimicry may lead to T/B cell cooperation and give rise to a pathological immunodominant response against Cha in T. cruzi infected animals.

In vitro effects of crude extracts of Parkia biglobosa (Mimosaceae), Stereospermum kunthianum (Bignoniaceae) and Biophytum petersianum (Oxalidaceae) on corticosteroid secretion in rat.

Previous studies conducted in guinea pig, rat and rabbit have revealed that crude extracts from Parkia biglobosa, Stereospermum kunthianum and Biophytum petersianum exert hypotensive and/or hypoglycemic activities. Since corticosteroids are involved in the control of arterial blood pressure and glycemia, we have investigated the possible effects of these plant extracts on rat adrenal tissue in vitro. Short-term administration of crude semi-ethanolic extracts of P. biglobosa and S. kunthianum to perifused rat adrenal tissue did not induce any significant changes in corticosteroid output. Conversely, the B. petersianum extract caused a dose-dependent increase in corticosterone and aldosterone secretion. Repeated infusions or prolonged administration of B. petersianum extract did not produce any apparent attenuation of the steroid response. Altogether, these data indicate that a semi-ethanolic extract of B. petersianum dose-dependently stimulates corticosterone and aldosterone secretion in rat without any desensitization phenomenon.

Regression of hormone-dependent mammary tumors in Sprague-Dawley rats as a result of tamoxifen or pharmacologic doses of 17 beta-estradiol: cyclic adenosine 3',5'-monophosphate-mediated events.

The estrogen and cyclic adenosine 3',5'-monophosphate (cAMP)-binding activities changed markedly when 7,12-dimethylbenz[a]anthracene-induced mammary tumors of Sprague-Dawley rats regressed following daily injections of either tamoxifen or pharmacologic doses of 17 beta-estradiol. cAMP binding increased eightfold to tenfold, whereas estrogen binding increased twofold to threefold in regressing tumor nuclei at 5 days after either treatment, which resulted in an inversion of the ratio of estrogen binding to cAMP binding found in growing tumor nuclei. Concomitantly, both binding activities were depleted from the cytosol. In the regressing tumors, the cAMP level increased twofold and nuclear cAMP-dependent protein kinase activity increased threefold to fourfold, with a 70-80% decrease in the cytoplasmic protein kinase activity. The rise in the nuclear protein kinase activity was abolished when cycloheximide was given with tamoxifen or with high doses of 17 beta-estradiol, which suggests that the increased activity is due to new protein synthesis. In the regressing tumor nuclei, the phosphorylation of the regression-associated proteins increased, whereas the phosphorylation of growth-associated proteins decreased. These data suggest that the mammary tumor regression induced by tamoxifen or high doses of estrogen proceed through a series of cAMP-mediated events.

Cyclic adenosine 3':5-monophosphate receptor proteins in hormone-dependent and -independent rat mammary tumors.

The molecular species of cyclic adenosine 3':5'-monophosphate (cAMP) receptor proteins (high-affinity-binding proteins) present in hormone-dependent and -independent rat mammary carcinomas were identified and characterized. Three major types of cAMP receptor proteins, with molecular weights of 39,000, 48,000, and 56,000, specifically incorporated the photoaffinity label, 8-azido-cyclic adenosine 3':5'-[32P]monophosphate and were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the tumor cytosols. The M.W. 48,000 and 56,000 receptor proteins appeared to be the regulatory subunits of cAMP-dependent protein kinase types I and II, respectively, and the M.W. 39,000 receptor protein was the proteolytic fragment of the M.W. 56,000 receptor protein. The relative amounts of these cAMP receptor proteins varied from one tumor type to another and showed no correlation with respect to the hormone dependency of tumors. Under two-dimensional gel electrophoresis, however, the M.W. 56,000 receptor protein from hormone-dependent tumors migrated as a doublet and shifted to either a more acidic or more basic charge than that of the receptor protein of hormone-dependent tumors. The alteration of the charge of the receptor did not affect the affinity for cAMP binding, because both hormone-dependent and hormone-independent tumor cytosols exhibited the dissociation constant for cAMP of approximately 10(-8) M. The M.W. 56,000 cAMP receptor protein from hormone-dependent tumors exhibited self-phosphorylation, but that from hormone-independent tumors did not. The diethylaminoethyl cellulose elution profiles of cAMP receptor proteins also differed between hormone-dependent and -independent tumors; cAMP binding activity from hormone-dependent tumors coeluted with cAMP-dependent protein kinase activity, whereas most of the cAMP binding activity from hormone-independent tumors eluted at a higher ionic strength than did cAMP-dependent protein kinase activity. These results suggest that the charge alteration of cAMP receptor proteins, which appears to occur at a site remote from that of cAMP binding, may be associated with the hormone independency of mammary tumors.

Current and future applications of flow cytometry in aquatic microbiology.

Flow cytometry has become a valuable tool in aquatic and environmental microbiology that combines direct and rapid assays to determine numbers, cell size distribution and additional biochemical and physiological characteristics of individual cells, revealing the heterogeneity present in a population or community. Flow cytometry exhibits three unique technical properties of high potential to study the microbiology of aquatic systems: (i) its tremendous velocity to obtain and process data; (ii) the sorting capacity of some cytometers, which allows the transfer of specific populations or even single cells to a determined location, thus allowing further physical, chemical, biological or molecular analysis; and (iii) high-speed multiparametric data acquisition and multivariate data analysis. Flow cytometry is now commonly used in aquatic microbiology, although the application of cell sorting to microbial ecology and quantification of heterotrophic nanoflagellates and viruses is still under development. The recent development of laser scanning cytometry also provides a new way to further analyse sorted cells or cells recovered on filter membranes or slides. The main infrastructure limitations of flow cytometry are: cost, need for skilled and well-trained operators, and adequate refrigeration systems for high-powered lasers and cell sorters. The selection and obtaining of the optimal fluorochromes, control microorganisms and validations for a specific application may sometimes be difficult to accomplish.