The in vitro pharmacological profile of KR31080, a nonpeptide AT1 receptor antagonist

Summary— KR31080 (2-butyl-5-methyl-6-(1-oxopyridin-2-yl)-3-[[2'-(1H-tetrazol-5-yl) biphenyl-4-yl]methyl]-3H-imidazo[4,5-b] pyridine) is a potent inhibitor of angiotensin type 1 (AT₁) receptors in rabbit aorta and human recombinant AT₁ receptors. In the isolated rabbit thoracic aorta, KR31080 caused a nonparallel shift to the right of the concentration-response curves to angiotensin II (All) with decreased maximal response (pD'₂ = 10.1 ± 0.1), but had no effect on the contractile response induced by norepinephrine. KR31080 inhibited specific [¹²⁵I]AII binding to rabbit aortic membranes (AT, receptors) and [¹²⁵I][Sar¹, Ile⁸]AII binding to human recombinant AT₁ receptors in a concentration-dependent manner with IC₅₀ values of 0.84 ± 0.08 nM and 1.92 ± 0.15 nM, respectively, but did not inhibit specific [¹²⁵I)AII binding to bovine cerebellum membranes (ÀT₂ receptors). In the Scatchard analysis, KR31080 interacted with rabbit aortic AT₁ receptors in a competitive manner, similar to losartan. These results demonstrate that KR31080 is a potent and AT₁ selective angiotensin receptor antagonist which exerts a competitive antagonism in the [¹²⁵I]AII binding assay and insurmountable AT₁ receptor antagonism in the functional study.     

Release of cortical catecholamines by visual stimulation requires activity in thalamocortical afferents of monkey and cat

Catecholamine (CA) release was measured in vivo in the monkey and cat visual cortices electrochemically. Stereate-modified, graphite-paste electrodes were used to monitor changes in norepinephrine and dopamine release. Micromolar changes in CA concentration were obtained by stimulation of the eye with nonspecific (strobe) or specific (oriented bars, radial gratings) stimuli. CA release depended on which eye was illuminated. Electrodes passed tangentially through the striate area recorded release following visual stimulation of one eye or the other in succession, and the shift in eye dominance occurred at about 500 microns intervals. The magnitude of CA release was highly correlated with the ocular dominance of neuronal activity measured with tungsten microelectrodes. Light-stimulated release was not recorded in monkey area V2, V4, or somatosensory area 1, but was recorded in cat V2, suggesting that the presence of LGN afferents is associated with CA release. Results are discussed in terms of the role of geniculate activity and the specific role of CAs in cortical information processing.     

Obesity of Czech children and adolescents: relation to parental obesity and socioeconomic factors

The purpose of this study was to estimate the change in overweight and obesity prevalence and the influence of socioeconomic status and parental obesity on overweight and obesity of children and adolescents in the Czech Republic. The roles of family history of obesity, dietary factors and physical activity were evaluated. The quota sample of 1,417 children and adolescents aged 6.00–17.99 years was examined in November 2005 as a part of the survey Lifestyle and Obesity. The quota sample was nationally representative, and subjects were selected according to the gender, age, region, size of residential location and education of parents. In the subjects weight, height and waist circumferences were measured and BMI was calculated. Food intake and physical activity were estimated by food frequency and physical activity questionnaires. The results were compared with the data from the 6th National Anthropological Survey of Children and Adolescents 2001, the Czech Republic. The data was evaluated by Pearson’s chi-square test and by linear regression analysis with backward factor reduction. Enhancing prevalence of obesity was found in younger age categories in comparison with the year 2001. Parental overweight and obesity significantly increased the risk of overweight and obesity. BMI in children (6–12.99 years) was associated with the intake of sweetened carbonated drinks and potatoes including fried ones (positive association) and low-sugar carbonated drinks and physical activity (negative association). In adolescents (13–17.99 years) BMI was associated with parental obesity, fat meat intake, time spent on the computer (positive association) and fish intake and physical activity (negative association). The results show an increase in obesity prevalence in younger children in the Czech Republic. Parental obesity significantly enhanced the risk ratio of obesity predominantly in adolescents.     

LIPOPROTEIN(α) IN HEALTH AND DISEASE

SUMMARY Elevated plasma levels of Lp(a) do seem to influence the progression of atherosclerosis. Evidence is emerging that certain apo(a) isoforms may be more atherogenic than others, and in transgenic mice free apo(a) has been shown to be associated with accelerated atherosclerosis. Currently it is not known whether treating elevated Lp(a) levels will reduce progression of atherosclerosis and, as therapeutic options are limited, mass screening of Lp(a) levels in populations is not indicated. The presence of raised Lp(a) levels, however, warrants aggressive treatment to reduce other cardiovascular risk factors. Continuing research to investigate the relationship of the apo(a) gene to other genes, including the plasminogen gene and apo(a)-related genes, will add further information pertaining to the evolution, function, regulation and clinical implications of Lp(a).     

Detection of chromosomes and estimation of aneuploidy in human spermatozoa using fluorescence in-situ hybridization

The development and application of fluorescence in-situ hybridization (FISH) has opened the way for comprehensive studies on numerical chromosome abnormalities in human spermatozoa. FISH can be rapidly applied to large numbers of spermatozoa and thus overcomes the major limitation of karyotyping spermatozoa after penetration of zona-free hamster oocytes. The simultaneous hybridization of two or more chromosome-specific probes to spermatozoa and subsequent detection of the bound probes using different fluorescent detection systems enables two or more chromosomes to be localized simultaneously in the same spermatozoon and provides a technique for undertaking reasonable estimates of aneuploidy. The most commonly used probes are those which bind to the centromeric region of specific chromosomes. Most studies to date have concentrated on estimating aneuploidy in spermatozoa from normospermic men, although reports are beginning to appear on aneuploidy in spermatozoa from subfertile and infertile men. Multi- probe FISH studies have generally reported disomy (hyperhaploidy) estimates of 0.05-0.2% per chromosome. There is preliminary evidence that some chromosomes such as X, Y and 21 are predisposed towards higher rates of non-disjunction during spermatogenesis. There are also suggestions of inter-donor variability in aneuploidy frequencies for specific chromosomes, although this requires confirmation in larger studies. While FISH is clearly a powerful technique that has many applications in reproductive medicine, it must also be realized that it does have limitations and the technology itself is still evolving and has yet to be fully validated on spermatozoa.      

Effect of estradiol and progesterone on daily rhythm in food intake and feeding patterns in Fischer rats

The product of meal number x meal size, over time, is food intake. Because estrogens modulate feeding activity via their action on the hypothalamus, and because there is a diurnal rhythm in the expression of cytoplasmic estrogen receptors and in estrogen binding activity, the present study examined the effects of ovariectomy and later hormone therapy on acute changes in body weight, and on the meal number-to-meal size relationship as reflected by food intake in the dark/light feeding patterns, in adult female rats in the intact state and after ovariectomy. Twelve female Fischer rats were randomized into ovariectomy and sham operation groups. A rat eater meter measured the feeding indexes for 15 days before and 25 days after ovariectomy, and later for 35 days with hormone therapy. We report: (a) mean body weight gain was linear before and up to ovariectomy, while exponential after ovariectomy; (b) increase in daily food consumption is mainly via an increase in food intake during the light phase; (c) light phase meal number remains unchanged, meal size significantly increases, with the resultant increase in overall food intake; (d) during the dark phase, meal size also significantly increases, but is accompanied by a proportional decrease in meal number, resulting in unchanged dark-phase food intake; and (e) estrogen restoration with either estradiol valerate or estradiol-progesterone combination, reversed the above changes. Data show that in the female Fischer 344 rat: (a) changes in daily rhythm in food intake are brought about by differential effects of the hormones on both meal size and meal number in both the total daily levels as well as in the dark-to-light distribution; (b) estadiol appears to have a tonic inhibitory effect on the light phase meal size and a phasic effect on the dark phase meal size and number, but no significant effect on the light-phase meal number; and (c) in the Fischer rats, progesterone augments estradiol's effect on these indicies.     

Sequencing of the alpha-synuclein gene in a large series of cases of familial Parkinson's disease fails to reveal any further mutations. The European Consortium on Genetic Susceptibility in Parkinson's Disease (GSPD)

A mutation in exon 4 of the human alpha-synuclein gene was reported recently in four families with autosomal dominant Parkinson's disease (PD). In order to examine whether mutations in this exon or elsewhere in the gene are common in familial PD, all seven exons of the alpha- synuclein gene were amplified by PCR from index cases of 30 European and American Caucasian kindreds affected with autosomal dominant PD. Each product was sequenced directly and examined for mutations in the open reading frame. No mutations were found in any of the samples examined. We conclude that the A53T change described in the alpha- synuclein gene is a rare cause of PD or may even be a rare variant. Mutations in the regulatory or intronic regions of the gene were not excluded by this study.      

Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography

In this study, we have used time-lapse video cinematography to study fertilization in 50 human oocytes that had undergone intracytoplasmic sperm injection (ICSI). Time-lapse recording commenced shortly after ICSI and proceeded for 17-20 h. Oocytes were cultured in an environmental chamber which was maintained under standard culture conditions. Overall, 38 oocytes (76%) were fertilized normally, and the fertilization rate and embryo quality were not significantly different from 487 sibling oocytes cultured in a conventional incubator. Normal fertilization followed a defined course of events, although the timing of these events varied markedly between oocytes. In 35 of the 38 fertilized oocytes (92%), there were circular waves of granulation within the ooplasm which had a periodicity of 20-53 min. The sperm head decondensed during this granulation phase. The second polar body was then extruded, and this was followed by the central formation of the male pronucleus. The female pronucleus formed in the cytoplasm adjacent to the second polar body at the same time as, or slightly after, the male pronucleus, and was subsequently drawn towards the male pronucleus until the two abutted. Both pronuclei then increased in size, the nucleoli moved around within the pronuclei and some nucleoli coalesced. During pronuclear growth, the organelles contracted from the cortex towards the centre of the oocyte, leaving a clear cortical zone. The oocyte decreased in diameter from 112 to 106 microm (P < 0.0001) during the course of the observation period. The female pronucleus was significantly smaller in diameter than the male pronucleus (24.1 and 22.4 microm respectively, P = 0.008) and contained fewer nucleoli (4.2 and 7.0 respectively, P < 0.0001). After time-lapse recording, oocytes were cultured for 48 h prior to embryo transfer or cryopreservation. Embryo quality was related to fertilization events and periodicity of the cytoplasmic wave, and it was found that good quality embryos arose from oocytes that had more uniform timing from injection to pronuclear abuttal and tended to have a longer cytoplasmic wave. In conclusion, we have shown that time-lapse video cinematography is an excellent tool for studying fertilization and early embryo development, and have demonstrated that human fertilization comprises numerous complex dynamic events.