Astaxanthin improves the developmental competence of in vitro-grown oocytes and modifies the steroidogenesis of granulosa cells derived from bovine early antral follicles.

In this study we investigated the effect of astaxanthin (Ax), which exhibits strong antioxidant activity, during invitro growth (IVG) on the developmental competence of oocytes and steroidogenesis of granulosa cells derived from early antral follicles. Bovine oocyte-cumulus-granulosa complexes collected from early antral follicles were cultured for 12 days in the presence or absence (control) of 500µM Ax. The viability of oocytes and antrum formation in the granulosa cell layer during IVG culture were greater in the presence than absence of Ax (P<0.05). Regardless of Ax treatment, 17ß-oestradiol production increased during IVG culture; however, progesterone production was significantly lower in the presence than absence of Ax (P<0.05). Reactive oxygen species levels were lower in Ax-treated oocytes than in controls after IVG (P<0.05). Although nuclear maturation and cleavage rates did not differ between the Ax-treated and control groups, Ax treatment led to weaker cathepsin B activity in oocytes and better blastocyst rates than in controls (P<0.05). Accordingly, Ax treatment during IVG increased the total number of cells in blastocysts (P<0.05). These results indicate that Ax supplementation of IVG medium improves the quality of bovine oocytes due to its antioxidative effects on growing oocytes and its suppression of the luteinisation of granulosa cells.

Detection of norovirus in food samples collected during suspected food-handler-involved foodborne outbreaks in Tokyo.

Although norovirus (NoV) is the major cause of gastroenteritis, with the largest number of NoV food poisoning cases in Japan, limited information is available regarding NoV detection in food. This study aimed to detect NoV in food samples during the 2015-2016 suspected foodborne outbreaks in Tokyo; 352 food samples from 64 NoV food poisoning outbreaks were collected. Bacterial culturing was performed for sample pretreatment and real-time reverse transcription polymerase chain reaction was conducted for NoV screening. The NoV detection rate was 1·7% (6/352). NoV-positive food samples included leftover boxed lunch, mackerel fillet (foodstuff), aburi salmon slice (partially seared salmon slice), raw tuna as a chirashizushi ingredient, raw amberjack as a sushi topping and ice for drinks. Since fresh fish as sushi toppings or ingredients and ice were consumed without heating, they may present a higher risk of viral infection. NoV-positive food samples were obtained from five outbreaks, wherein food handlers were NoV-positive in four. Each partial VP1 sequence from food samples matched completely with those in NoV-positive individuals and food handlers. Hence, food handlers play a potentially important role in food-based NoV transmission in all five outbreaks; therefore, hygiene education among them is essential to prevent NoV foodborne outbreaks. SIGNIFICANCE AND IMPACT OF THE STUDY: Significance and Impact of the Study: Norovirus (NoV) is a leading cause of foodborne outbreak in Japan. The most frequent route of transmission in NoV foodborne outbreaks is secondary contamination via infected food handlers. However, limited information is available regarding NoV contamination in food samples. This study reports the detection of NoV in food samples to elucidate the source and route of NoV infection leading to outbreaks for 2 years in Tokyo. Our data potentially contribute to education and the development of safe food-handling strategies among food handlers and employees in the food industry through elucidation of risk factors associated with NoV contamination.

Observation of the spatial distribution of gravitationally bound quantum states of ultracold neutrons and its derivation using the Wigner function.

Ultracold neutrons (UCNs) can be bound by the potential of terrestrial gravity and a reflecting mirror. The wave function of the bound state has characteristic modulations. We carried out an experiment to observe the vertical distribution of the UCNs above such a mirror at the Institut Laue-Langevin in 2011. The observed modulation is in good agreement with that prediction by quantum mechanics using the Wigner function. The spatial resolution of the detector system is estimated to be 0.7 µm. This is the first observation of gravitationally bound states of UCNs with submicron spatial resolution.

Haploinsufficiency of the paternal-effect gene Dnmt3L results in transient DNA hypomethylation in progenitor cells of the male germline.

STUDY QUESTION: How does haploinsufficiency of the paternal-effect gene Dnmt3L affect DNA methylation establishment and stability in the male germline? SUMMARY ANSWER: Reduced expression of DNMT3L in male germ cells, associated with haploinsufficiency of the paternal-effect gene Dnmt3L, results in abnormal hypomethylation of prenatal germline progenitor cells. WHAT IS KNOWN ALREADY: The DNA methyltransferase regulator Dnmt3-Like (Dnmt3L) is a paternal-effect gene required for DNA methylation acquisition in male germline stem cells and their precursors. In males, DNMT3L deficiency causes meiotic abnormalities and infertility. While Dnmt3L heterozygous males are fertile, they have abnormalities in X chromosome compaction and postmeiotic gene expression and sire offspring with sex chromosome aneuploidy. It has been proposed that the paternal effects of Dnmt3L haploinsufficiency are due to epigenetic defects in early male germ cells. DNA methylation is an essential epigenetic modification essential for normal germ cell development. Since patterns of DNA methylation across the genome are initially acquired in prenatal male germ cells, perturbations in methylation could contribute to the epigenetic basis of the paternal effects in Dnmt3L(+/-) males. STUDY DESIGN, SIZE, DURATION: This is a cross-sectional study of DNA methylation in Dnmt3L(+/+) versus Dnmt3L(+/-) male germ cells collected from mice at 16.5 days post-coitum (dpc), Day 6 and Day 70 (n = 3 per genotype, each n represents a pool of 2-20 animals). Additionally, DNA methylation was compared in enriched populations of spermatogonial stem cells (SSC)/progenitor cells from Dnmt3L(+/+) and Dnmt3L(+/-) males following ≈ 2 months in culture. MATERIALS, SETTING, METHODS: DNA methylation at intergenic loci along chromosomes 9 and X was examined by quantitative analysis of DNA methylation by real-time polymerase chain reaction at the time of initial acquisition of epigenetic patterns in the prenatal male germline (16.5 dpc) and compared with patterns in early post-natal spermatogonia (Day 6) and in spermatozoa in mice. DNA methylation status at CpG-rich sites across the genome was assessed in spermatogonial precursors from Day 4 male mice using restriction landmark genomic scanning. MAIN RESULTS AND THE ROLE OF CHANCE: At 16.5 dpc, 42% of intergenic loci examined along chromosome 9 and 10% of those along chromosome X were hypomethylated in Dnmt3L heterozygotes. By Day 6 and in spermatozoa, germ cell DNA methylation was similar in heterozygous and wild-type mice. DNA methylation stability of acquired patterns in wild-type and Dnmt3L(+/-) SSC/progenitor cell culture was analyzed at numerous loci across the genome in cells cultured in vitro and collected at passages 6-28. While the methylation of most loci was stable in culture over time, differences at ≈ 1% of sites were found between Dnmt3L(+/-) and Dnmt3L(+/+) cultures. LIMITATIONS, REASONS FOR CAUTION: Evaluation of DNA methylation in SSCs can only be performed after a period of culture limiting the investigation to changes observed during culture when compared with DNA methylation differences between genotypes that could be present at the beginning of culture establishment. WIDER IMPLICATIONS OF THE FINDINGS: The DNA methylation defects described here in prenatal male germline progenitor cells and SSC culture are the earliest epigenetic perturbations yet identified for a mammalian paternal-effect gene and may influence downstream epigenetic events in germ cells at later stages of development. Together, the results provide evidence of a 'window' of susceptibility in prenatal male germ cell precursors for the induction of epimutations due to genetic perturbations and, potentially, in utero environmental exposures.

The efficiency of male fertility restoration is dependent on the recovery kinetics of spermatogonial stem cells after cytotoxic treatment with busulfan in mice.

BACKGROUND: Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis and represent a crucial resource for male fertility restoration. It has not been well documented, however, whether the recovery of SSC population size after cytotoxic damage associates with the kinetics of male fertility restoration. We addressed this issue using the mouse as a model. METHODS: Following single injections of busulfan at 15, 30 or 45 mg/kg into male mice, we examined their ability to sire offspring at different times by natural mating and determined SSC numbers using spermatogonial transplantation. We measured testis physiological parameters (testis weights, sperm counts, serum and intratesticular testosterone levels, and histological assessments of spermatogenic recovery) and quantified the expression of glial-cell-line-derived neurotrophic factor (GDNF) transcripts. RESULTS: Regardless of busulfan doses, fertility was lost within 4 weeks after treatment, while more than 95% of SSCs were lost within 3 days. Fertility and SSC numbers gradually recovered with time, but the recoveries were delayed at higher busulfan doses. Interestingly, SSC numbers reached ∼30% of before-treatment levels by 4 weeks prior to the time of fertility restoration, across the dose groups. Sperm counts were ∼20% of before-treatment levels at the onset of fertility restoration, regardless of busulfan doses. We detected a significant increase in total GDNF mRNA per testis immediately after busulfan treatment. CONCLUSIONS: The loss and restoration of fertility after busulfan treatment are direct consequences of SSC loss and expansion. Our data suggest that there is a threshold in SSC numbers that allows for male fertility restoration and that the testicular somatic environment responds rapidly and temporarily to the loss of spermatogonia, including SSCs, by altering GDNF mRNA levels. This study provides fundamental information to clinically apply SSCs for male fertility restoration in the future.

Evaluation of follicular development and oocyte quality in pre-pubertal cats.

Our study was conducted to assess the follicular development and availability of sound ovarian oocytes for in vitro production (IVP) of embryos in pre-pubertal cats. The relationship between body and ovarian weight was examined in 93 cats. The results revealed that ovarian weight rapidly increased until 100 days of estimated age. By histological evaluation of ovaries obtained from 11 pre-pubertal cats with estimated age of <20, 20-40 and 100-120 days, it was clarified that the increase in ovarian weight during kitten growth accompanied the increase in the number and size of antral follicles. The follicular diameter and percentage of normal oocytes in secondary/antral follicles also increased as estimated age (body weight) increased. The oocytes obtained from pre-pubertal cats with 100-120 days of estimated age were used for IVP of embryos. The results showed that the success rates of in vitro maturation, in vitro fertilization and development to blastocysts after in vitro culture in pre-pubertal cats were lower than in sexually mature cats. However, the percentage of blastocysts based on the cleaved embryos and cell number of blastocysts in pre-pubertal cats were comparable to those in mature cats. In conclusion, these results suggest that the ovaries of pre-pubertal cats with ≥100 days of age contain oocytes with in vitro developmental competence to blastocysts.

Effect of duration of in vitro maturation on nuclear maturation and fertilizability of feline oocytes.

This study was conducted to improve in vitro production of embryos from domestic cats using TCM-199 as an IVM medium. The time sequence of nuclear maturation and the optimal timing of in vitro insemination were examined. Most oocytes were at the germinal vesicle stage immediately after collection; however, 8.3% had already resumed meiosis before IVM culture. After 30 h of IVM culture, the percentage of oocytes at metaphase II (MII) reached a peak (75.5%) and did not change (P>0.05) from 30 to 48 h after IVM culture. The percentage of oocytes with two pronuclei was higher (P<0.05) for oocytes matured for 30 and 36 h (38.2 and 33.0%, respectively) than for those after IVM culture for only 24 h (18.5%). Total sperm penetration rate was highest (P<0.05) for oocytes that had been matured for 30 h (46.1%). After 30 h of IVM and 18 h of IVF culture, 66.3 and 24.8% of inseminated oocytes had cleaved and developed to the blastocyst stage, respectively. We concluded that IVM of feline oocytes for 30 h in TCM-199 resulted in optimal nuclear maturation and sperm penetration.

Increased catabolic rate of low density lipoproteins in humans with cholesteryl ester transfer protein deficiency.

The cholesteryl ester transfer protein (CETP) transfers lipids among lipoprotein particles and plays a central role in lipoprotein metabolism. Humans with genetic deficiency of CETP have both elevated HDL cholesterol and apolipoprotein A-I concentrations as well as decreased LDL cholesterol and apolipoprotein B levels. The present study was undertaken to elucidate the metabolic basis for the decreased LDL cholesterol and apo B levels in CETP deficiency. We conducted a series of in vivo apo B kinetic studies in tow unrelated homozygotes with CETP deficiency and in control subjects. A primed constant infusion of stable isotopically labeled phenylalanine was administered to the two CETP deficient subjects and control subjects and apo B kinetic parameters in VLDL, intermediate density lipoproteins, and LDL were obtained by using a multicompartmental model. The fractional catabolic rates (FCR) of LDL apo B were significantly increased in the CETP-deficient subjects (0.56 and 0.75/d) compared with the controls (mean FCR of 0.39/d). Furthermore, the production rates of apo B in VLDL and intermediate density lipoprotein were decreased by 55% and 81%, respectively, in CETP deficiency compared with the controls. In conclusion, CETP-deficient subjects were demonstrated to have substantially increased catabolic rates of LDL apo B as the primary metabolic basis for the low plasma levels of LDL apo B. This result indicates that the LDL receptor pathway may be up-regulated in CETP deficiency.

Effects of temperature on papilloma growth in the newt, Cynops pyrrhogaster.

The effects of temperature on skin papillomas of newts (Cynops pyrrhogaster) were studied. Newts bearing these tumors were maintained for 30 weeks at 4 +/- 1 (S.E.), 10 +/- 1, 13 +/- 2, 25 +/- 1, and 30 +/- 1 degree. The diameters of the papillomas were measured externally. They decreased at 4, 25, and 30 degrees, and increased at 10 and 13 degrees. The effect of temperature on the tumor growth or regression began to appear from around the 10th week. Furthermore, when the temperatures were changed, it was possible to reverse the growth or regression of the tumors. Histologically, cell layers of the tumors became thin at 4, 25, and 30 degrees, but there were some differences between 4 and 25 or 30 degrees. The pigment layers became thick, and epidermal cells invaded dermal layer at 4 degrees, and clumps of cells separated from the surface of the tumors at 25 and 30 degrees. Mitotic indices were lower at 4, 25, and 30 degrees than in normal epidermis, but were about 3 times as high as normal tissue at 10 or 13 degrees. The relation between these results in the laboratory and seasonal fluctuations of these tumors in nature is discussed.

Mutagenic analysis of Vav reveals that an intact SH3 domain is required for transformation.

The vav proto-oncogene encodes a protein with multiple modulae domains that enable it to function as a mediator, linking tyrosine signaling to downstream events in hematopoietic cells. Circumstantial evidence suggests that protein-protein interactions exerted by two of these domains, the Src homology 2 (SH2) and the Src homology 3 (SH3), play an important role in the regulation of Vav activity. To study the relevance of the SH3 domain for the function of vav as a transforming gene, we have created several mutations in the SH3 domain located at its carboxy region. Substitution of the non-conserved aspartic acid 797 (to asparagine, D797N) retained the transforming potential of the vav oncogene, whereas substitutions of five highly conserved amino-acids: alanine 789 (to asparagine, A789N), leucine 801 (to arginine, L801R), tryptophan 821 (to arginine, W821R), glycine 830 (to valine, G830V) and valine 837 (to glutamic acid, V837E) greatly reduced its transforming potential. The mutant proteins resemble Vav in many biochemical properties; however, while the transforming mutant protein (D797N) associates with several unidentified proteins in a manner similar to that of Vav, the non-transforming mutant Vav proteins react very poorly with these proteins. Among the known Vav-interacting proteins, hnRNP-K associates with all mutant proteins except A789N and V837E whereas binding of Zyxin to any of the mutant proteins is not affected. Taken together, our results clearly demonstrate that the SH3 domain has a positive effect on vav activity and is needed for vav transformation. The vavSH3C associating protein(s) that are crucial for its activity as a transforming gene have probably not yet been identified.

Purification and immunological determination of alpha 2-macroglobulin in serum from injured rats.

Rat alpha 2-macroglobulin was isolated and purified from the pooled sera of turpentine-injected rats by sequential use of dextran sulphate, DEAE-cellulose and gel filtration chromatography. The final protein product obtained by this procedure proved to be alpha 2-macroglobulin of a high degree of purity based on electrophoretic, immunologic and centrifugal analysis. The alpha 2-macroglobulin preparation also binds stoichiometrically to trypsin preventing subsequent inhibition by protein trypsin inhibitors. SDS-polyacrylamide gel electrophoresis of rat alpha 2-macroglobulin after incubation with trypsin suggested that there are at least two susceptible peptide bonds in the 170,000-dalton alpha 2-macroglobulin subunit. The concentration of alpha 2-macroglobulin in the sera of rats was measured by electroimmuno assay a monospecific antiserum against alpha 2-macroglobulin. Purified alpha 2-macroglobulin was used as a standard. Sera from normal male rats contained 32 +/- 4 micrograms of alpha 2-macroglobulin per ml. To determine the time course of response of alpha 2-macroglobulin to inflammation, rats were subjected to either laparotomy or subcutaneous injection of turpentine. After either type of injury, the concentration of alpha 2-macroglobulin increased rapidly, reaching a maximum value of 110-140 times that of the control value by 24 h. Little difference was noted in responsiveness between the two sexes.

Recombinant tissue-type plasminogen activator ameliorates ischemic derangements induced by thrombotic occlusion in closed chest anesthetized dogs.

Effects of thrombotic coronary occlusion followed by thrombolytic reperfusion with recombinant tissue-type plasminogen activator (rt-PA) on infarct size and left ventricular function were studied in anesthetized closed chest dogs. After thrombotic occlusion of the left anterior descending coronary artery was produced by a copper coil technique, 74 dogs were randomly alloted to three groups; dogs treated with rt-PA at 90 min (n = 23) (group I) and at 180 min (n = 25) (group II) of the thrombotic occlusion, and 26 dogs treated with saline solution (permanent thrombotic occlusion, group III). The loading dose of intravenous rt-PA was 8,160 IU/kg body weight per min at the initial 60 min and the maintenance dose was 2,450 IU/kg per min continuously infused for 24 h. Thrombolytic recanalization was achieved at 15 +/- 4 and 18 +/- 6 min after rt-PA infusion in groups I and II, respectively. Infarct size and area at risk were determined by triphenyltetrazolium chloride staining and postmortem angiography; infarct size/area at risk ratio was 10 +/- 3% (n = 10), 33 +/- 7% (n = 9) and 63 +/- 3% (n = 10) in groups I, II and III, respectively (difference significant among groups). To examine whether infarct size and left ventricular function after thrombolytic reperfusion differ from those after mechanical reperfusion, 39 other dogs (group IV) underwent mechanical coronary occlusion for 106 +/- 1 min (occlusion period comparable with that of group I) and reperfusion using a balloon catheter.(ABSTRACT TRUNCATED AT 250 WORDS)

Point mutation (-69 G-->A) in the promoter region of cholesteryl ester transfer protein gene in Japanese hyperalphalipoproteinemic subjects.

Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester (CE) from HDL to apolipoprotein (apo) B-containing lipoproteins and plays a crucial role in reverse cholesterol transport, which is a major protective system against atherosclerosis. Genetic CETP deficiency is the most common cause of a marked hyperalphalipoproteinemia (HALP) in the Japanese, and various mutations have been identified in the coding region as well as in the exon/intron boundaries in the CETP gene. In the present study, we identified a novel mutation in the promoter region of the CETP gene. This mutation was a G-to-A substitution at the -69 nucleotide of the promoter region (-69 G-->A), corresponding to the second nucleotide of the PEA3/ETS binding site (CGGAA) located upstream of the putative TATA box. Four (2.0%) of 196 unrelated subjects with a marked HALP (HDL cholesterol >/=2.59 mmol/L=100 mg/dL) were revealed to be heterozygous for the -69 G-->A mutation, and the allelic frequency of the mutant was 0.0102 in the subjects with a marked HALP. The subjects with the -69 G-->A mutation had low plasma CETP levels. Reporter gene assay showed that this mutation markedly reduced the transcriptional activities in HepG2 cells (8% of wild type). These results suggested that this mutation would be dominant negative. In conclusion, a novel -69 G-->A mutation in the CETP gene causes the decreased transcriptional activity leading to HALP.

Differential promoter usage in prolactin receptor gene expression: hepatocyte nuclear factor 4 binds to and activates the promoter preferentially active in the liver.

Characterization of the rat PRL receptor (PRLR) gene has revealed three separate untranslated exon 1 sequences, each associated with a different transcription start site and 5'-flanking sequence. We show by RT-PCR that exon 1A is expressed primarily in liver but is also detectable in ovary and mammary gland. Exon 1B expression is observed exclusively in the ovary, whereas exon 1C is expressed in all three tissues. Transient transfection of luciferase reporter constructs containing parts of the 5'-flanking regions (0.3-1.1 kb) of exon 1A, 1B, and 1C, respectively, showed activity of the 1A promoter in Chinese hamster ovary (CHO) cells, the human hepatoma cell line, HepG2, and the rat hepatoma cell line, H4II, which was 10- to 14-fold increased compared with the activity of the promoter-less luciferase vector. No activity of the 1A promoter was detected in the human mammary cell line, T-47D. Relative to a vector containing the Simian virus 40 (SV40) promoter, the 1A promoter had 20% activity in H4II cells and 1-3% activity in CHO and HepG2 cells. The 1B promoter produced a 6.1-fold increase of luciferase activity in CHO cells (approximately 2% of the SV40 promoter), whereas no significant activity was detected in HepG2, H4II, and T-47D cells. The 1C promoter was strongly active in T-47D cells (approximately 64-fold over control) and moderately active in the other cell lines tested (9- to 13-fold over control). 5'-Deletion analysis of the 1A promoter revealed that a fragment containing -83/ +81 bp, relative to the transcription start site, was sufficient to drive transcription in hepatoma cells, whereas this construct was inactive in CHO cells. Cotransfection of CHO cells with the -83/+81 construct and an expression vector encoding the liver-enriched transcription factor, hepatocyte nuclear factor 4 (HNF4), revealed a dose-dependent transactivation of the proximal 1A promoter with a maximal stimulation of approximately 10-fold. Electrophoretic mobility shift assays showed binding of HNF4 to the sequence -14/+24 of the 1A promoter, and mutational analysis revealed that the sequence GGGCAAAGTCA at position +11/+21 is required for this binding. We conclude that the 1A, 1B, and 1C promoters of the PRLR gene are used in a cell type- dependent way that may play a role in differential hormonal regulation of the gene. In particular, we have shown that HNF4 operates on the proximal 1A promoter and may be responsible, in combination with other factors, for the increased activity of this promoter in adult female liver.

Production of chronic congestive heart failure by rapid ventricular pacing in the rabbit.

OBJECTIVE: The aim was to produce a model of low output congestive heart failure by rapid pacing in rabbits. METHODS: To perform rapid pacing in rabbits, a custom made pacemaker was developed which is light (about 80 g) and can pace at up to 400 beats.min-1 for more than two weeks. A thoracotomy was done and two electrodes were sutured onto the left ventricle. A central venous pressure line was chronically implanted. With the use of this pacemaker, rabbits were paced at 350-400 beats.min-1 for several weeks. RESULTS: Central venous pressure increased from 1.4(SEM 0.2) to 6.4(0.5) mm Hg (p < 0.01, n = 14). After pacing for 16.1(1.6) d, haemodynamic studies were performed under anaesthesia with thiamylal sodium. Left ventricular end diastolic pressure was higher in the paced rabbits (n = 10) than in the control rabbits which underwent sham operation but were not paced (n = 6), at -0.6(0.6) v 19.3(2.0) mm Hg (p < 0.01). Cardiac output [673(56) v 536(45) ml.min-1, p < 0.10] and +dP/dt [1433(97) v 722(51) mm Hg.s-1, p < 0.01] were lower in the paced rabbits (n = 7-8) than in the control rabbits (n = 6). The paced rabbits had more ascites [1.9(1.0) v 45.9(18.9) ml, p < 0.05] and pleural effusion [0.4(0.3) v 12.9(6.7) ml, p < 0.10] than control rabbits. Plasma noradrenaline was higher in the paced rabbits (n = 11) than in the control rabbits (n = 7), at 1.59(0.43) v 0.60(0.05) ng.ml-1 (p < 0.05). The ratio of wet heart weight or lung weight to body weight was higher (p < 0.01) in the paced rabbits than in the control rabbits. CONCLUSIONS: Chronic biventricular congestive heart failure can be produced in rabbits by rapid pacing.

Effects of long term treatment with an alpha 1 adrenoceptor blocker on cardiac hypertrophy, contractility, and myosin isoenzymes in spontaneously hypertensive rats.

STUDY OBJECTIVE: The aim was to investigate the effects of long-term treatment with an alpha 1 blocker, bunazosin hydrochloride, on blood pressure, heart weight, myocardial contractility, and ventricular myosin isoenzyme pattern in spontaneously hypertensive rats (SHR). DESIGN: Bunazosin hydrochloride was given orally in a dose of 10 mg.kg-1.d-1 for 8-9 weeks. Isometric tension development was measured in isolated left ventricular papillary muscles. The left ventricular myosin isoenzyme pattern was determined by pyrophosphate gel electrophoresis. SUBJECTS: 14 male SHR (seven treated and seven untreated rats) aged 23 to 24 weeks were studied. MEASUREMENTS AND MAIN RESULTS: The blood pressure of the bunazosin treated rats was approximately 14% lower than that of the untreated rats at the end of the treatment period. The ventricular weight of treated SHR was significantly lower (around 8%) than that of untreated rats, but there were no significant differences between the two groups in the mechanical data obtained from isolated left ventricular papillary muscles. The myosin isoenzyme pattern of the left ventricle was significantly shifted toward VM-1 in the bunazosin treated SHR, the VM-1 concentration in the treated group being 38% greater than in the untreated group. CONCLUSIONS: These results indicate that long term treatment with bunazosin hydrochloride reduces blood pressure and leads to the regression of cardiac hypertrophy in SHR. Myocardial energetics (as represented by the myosin isoenzyme pattern) were affected, but there was no influence on myocardial tension development.

Regulation of prolactin receptor mRNA expression in peripheral lymphocytes in rats in response to changes in serum concentrations of prolactin.

In the present study we have evaluated the absolute number of the two forms of prolactin (PRL) receptor mRNA in rat peripheral blood lymphocytes and the modulation of receptor mRNA induced by changes in serum levels of endogenous PRL or by administration of ovine PRL. Lymphocytes expressed low levels of both forms of PRL receptor transcripts. Repeated treatments with ovine PRL significantly reduced levels of mRNA encoding the long form PRL receptor, whereas expression was markedly increased by repeated doses of bromocriptine. In contrast, the mRNA level of short form PRL receptor was unchanged by both treatments. The expression of long form transcripts was also markedly decreased in lymphocytes from pituitary-grafted rats. Therefore it appears that in rat peripheral lymphocytes PRL has a negative effect on the expression of its own receptor.

Quantitative analysis by polymerase chain reaction of growth hormone receptor gene expression in human liver and muscle.

A single form of GH receptor (GHR) messenger RNA (mRNA) of 4.5 kilobases, encoding the full-length GHR, has been found in man. To measure the absolute number of mRNA molecules encoding the GHR in human tissues, we developed a quantitative polymerase chain reaction assay. An internal control RNA was constructed by inserting a 50-basepair fragment of the rat PRL receptor complementary DNA into a portion of the human GHR complementary DNA. The internal control RNA and the target mRNA were amplified together with the same set of primers. Twenty-four cycles of amplification were used to satisfy an exponential phase of amplification. It was possible to detect as few as 500 molecules of target mRNA/micrograms total RNA. In 3 liver samples obtained from normal donors at the time of transplant, the amount of GHR mRNA ranged from 0.5 +/- 0.1 to 1.4 +/- 0.4 x 10(6) molecules/micrograms total RNA. These results were confirmed by slot blot analysis of the same samples. The number of receptor transcripts did not appear to be correlated with the receptor-binding capacity found in the 3 liver samples. In 7 muscle biopsies, GH receptor mRNA varied between 4.0 +/- 0.4 and 34.6 +/- 1.4 x 10(4) molecules/micrograms total RNA. This technique allows direct measurement of GHR gene expression in human tissues and represents a valuable tool, particularly for tissues such as muscle, in which the receptor protein cannot be measured using conventional binding assays.

Role of cardiac angiotensin II in isoproterenol-induced left ventricular hypertrophy.

Angiotensin II (Ang II) has been shown to induce proliferation of cardiac myocytes. To examine the role of Ang II in left ventricular (LV) hypertrophy, isoproterenol was infused subcutaneously into 9-week-old male Wistar rats at 4.2 mg/kg/day for 7 days. Infusion of isoproterenol increased LV weight and Ang II concentrations in plasma and in LV tissue. In anephric rats, LV weight and tissue Ang II were increased similarly, but plasma Ang II was not changed by isoproterenol. Concomitant oral administration of trandolapril and isoproterenol prevented increases in both LV Ang II and LV weight. Treatment with hydralazine decreased blood pressure in a similar way as trandolapril but did not affect either LV weight or LV Ang II. Plasma Ang II was not decreased by either trandolapril or hydralazine when administered in combination with isoproterenol. These results suggest that cardiac tissue Ang II regulates myocyte growth in isoproterenol-induced LV hypertrophy, and the reduction of Ang II partly explains the prevention of cardiac hypertrophy by the converting enzyme inhibitor.