Estradiol effects on the growth hormone/insulin-like growth factor-1 axis in amenorrheic athletes.

Disruption of the growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis has been reported and studied in menopause, hypothalamic amenorrhea, and anorexia nervosa, but not in weight-stable amenorrheic athletes. We investigated the effects of short-term transdermal estradiol on basal and exercise-stimulated serum GH, IGF-1, and associated binding proteins (IGFBP-1 and IGFBP-3) in seven weight-stable female amenorrheic athletes with percentage body fats greater that 12%. Each subject received a 72 h placebo patch followed by 144 h of transdermal estradiol. Serum samples for GH, IGF-1, IGFBP-1, and IGFBP-3 were obtained at baseline (t1), 72 hr (t2), 144 hr (t3), and during three 90-minute trials of aerobic exercise. Basal, and exercise GH, IGF-1, and IGFBP-1 were not different between trials. Baseline IGFBP-3 decreased from t1 to t2 (p = 0.04) and serum free fatty acids increased from t1 to t2, and t1 to t3 (p = 0.04, and 0.02 respectively). These findings differ from postmenopausal women, and women having weightloss-associated amenorrhea, suggesting that estrogen, exercise, and nutritional deficiencies may have independent effects on the GH/IGF-1 axis.     

Sex determination in platypus and echidna: autosomal location of <Emphasis Type="Italic">SOX3</Emphasis> confirms the absence of <Emphasis Type="Italic">SRY</Emphasis> from monotremes

In eutherian ('placental') mammals, sex is determined by the presence or absence of the Y chromosome-borne gene SRY, which triggers testis determination. Marsupials also have a Y-borne SRY gene, implying that this mechanism is ancestral to therians, the SRY gene having diverged from its X-borne homologue SOX3 at least 180 million years ago. The rare exceptions have clearly lost and replaced the SRY mechanism recently. Other vertebrate classes have a variety of sex-determining mechanisms, but none shares the therian SRY-driven XX female:XY male system. In monotreme mammals (platypus and echidna), which branched from the therian lineage 210 million years ago, no orthologue of SRY has been found. In this study we show that its partner SOX3 is autosomal in platypus and echidna, mapping among human X chromosome orthologues to platypus chromosome 6, and to the homologous chromosome 16 in echidna. The autosomal localization of SOX3 in monotreme mammals, as well as non-mammal vertebrates, implies that SRY is absent in Prototheria and evolved later in the therian lineage 210-180 million years ago. Sex determination in platypus and echidna must therefore depend on another male-determining gene(s) on the Y chromosomes, or on the different dosage of a gene(s) on the X chromosomes.     

Dithiothreitol prevents age-associated decrease in oocyte/conceptus viability in vitro

The present study was designed to ascertain whether the negative effects on reproductive potential of post-ovulatory ageing in vitro of oocytes can be prevented by antioxidant therapy. Mouse metaphase II (MII) oocytes were aged in vitro for 12 h prior to insemination in the presence of varying concentrations of L-ascorbic acid, 6-methoxy- 2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), L-cystine dihydrochloride, ethylenediaminetetraacetic acid (EDTA), beta- mercaptoethanol and DL-dithiothreitol (DTT). In-vitro ageing of oocytes was associated with lower fertilization rate, higher proportion of concepti exhibiting cellular fragmentation at 24 h post-insemination and lower percentage of concepti reaching the blastocyst stage. Ascorbic acid, Trolox and EDTA had no effect on cellular fragmentation or potential of oocytes for development. However, the probability of an oocyte reaching the blastocyst stage was decreased (P < or = or = 0.05) in oocytes incubated in the presence of L-cystine (50 and 500 microM) and beta-mercaptoethanol (5, 50 and 500 microM) when compared to control aged oocytes. Age-associated cellular fragmentation at 24 h post-insemination was partially prevented (P < or = 0.05) by incubating oocytes in the presence of beta-mercaptoethanol (500 microM). DTT (50 and 500 microM) increased (P < or = 0.05) fertilization rate and number of cells at 81 h post-insemination to levels similar to those exhibited by control oocytes. Furthermore, both age-associated fragmentation at 24 h post-insemination (P < or = 0.05) and decreased potential of oocytes for development to the blastocyst stage (P < or = 0.05) were prevented, at least in part, by culturing oocytes in the presence of DTT (50 microM). Although the mechanism by which DTT exerts its beneficial effects on aged oocytes remains to be elucidated, it may protect oocytes by preventing oxidation of free thiol groups and/or altering a redox-independent signalling pathway that mediates cellular fragmentation and death.      

Characterization of 19 disease-associated missense mutations in the regulatory domain of the cystic fibrosis transmembrane conductance regulator

In order to gain a better insight into the structure and function of the regulatory domain (RD) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, 19 RD missense mutations that had been identified in patients were functionally characterized. Nine of these (I601F, L610S, A613T, D614G, I618T, L619S, H620P, G628R and L633P) resulted in aberrant processing. No or a very small number of functional CFTR proteins will therefore appear at the cell membrane in cells expressing these mutants. These mutations were clustered in the N- terminal part of the RD, suggesting that this subdomain has a folding pattern that is very sensitive to amino acid changes. Mutations that caused no aberrant processing were further characterized at the electrophysiological level. First, they were studied at the whole cell level in Xenopus laevis oocytes. Mutants that induced a whole cell current that was significantly different from wild-type CFTR were subsequently analysed at the single channel level in COS1 cells transiently expressing the different mutant and wild-type proteins. Three mutant chloride channels, G622D, R792G and E822K CFTR, were characterized by significantly lower intrinsic chloride channel activities compared with wild-type CFTR. Two mutations, H620Q and A800G, resulted in increased intrinsic chloride transport activities. Finally, T665S and E826K CFTR had single channel properties not significantly different from wild-type CFTR.      

Cloning and expression of the gene involved in Sanfilippo B syndrome (mucopolysaccharidosis III B)

Sanfilippo B syndrome is caused by a deficiency of alpha-N- acetylglucosaminidase, a lysosomal enzyme involved in the degradation of heparan sulphate. Accumulation of the substrate in lysosomes results in degeneration of the central nervous system with progressive dementia often combined with hyperactivity and aggressive behaviour. In order to clone the deficient gene, we purified the enzyme from human placenta and obtained amino acid sequence information. Alignment of one of the CNBr generated internal peptides to sequence from the database revealed the chromosomal location of the gene in the 5' upstream flanking region of the gene for 17-beta-hydroxysteroid-dehydrogenase at 17q21.1. The available DNA sequence was used to clone the cDNA coding for alpha-N- acetylglucosaminidase and analyse its gene structure. The gene is fully contained in the 5' upstream flanking region of the gene for 17-beta- hydroxysteroid-dehydrogenase and interrupted by five introns. The cDNA clone has a length of 2575 bp and encodes a protein of 743 amino acids. Chinese hamster ovary cells transfected with the cDNA construct show alpha-N-acetylglucosaminidase activity about 17-fold over background. This will allow correction studies with NAG deficient Sanfilippo B cell lines and facilitate the development of enzyme replacement therapy for these patients.      

Tissue fixation effects on immunohistochemical staining of caspase-3 in brain tissue.

Fixation methods for tissue often vary amongst clinical and research laboratories. To evaluate the effects of fixation method on studies of brain tissue, we examined immunohistochemical outcomes amongst 2 fixatives, 4 caspase-3 antibodies, and 2 species (human infants and piglets). Fixatives were 10% neutral buffered formalin (NBF) or 10% NBF and glacial acetic acid (FAA). Antibodies for caspase-3 were commercially obtained and included 2 for active caspase-3, and 2 for procaspase-3 (CASP3 and CPP32). Immunohistochemical staining of caspase-3 varied with fixation method, with the greatest effect of fixation method observed for the active caspase-3 antibodies and this effect was present in both species. In NBF-fixed tissue, active caspase-3 immunoreactivity was only visible microscopically, and was specific to neuronal cell bodies. In FAA-fixed tissue, active caspase-3 immunoreactivity was visible macroscopically, and predominantly present in fiber tracts and fasciculi compared with neuronal bodies. Fixation and species differences were also identified for the procaspase-3 antibodies, CASP3 and CPP32, where FAA-fixed pig tissue showed abundant staining of blood vessels that were not observed in the NBF-fixed pig tissue or in the human tissue. This study characterizes differences in immunohistochemical outcomes using commercially available antibodies for caspase-3, according to tissue fixation method and species.     

Skeletal muscle mitochondrial function and lean body mass in healthy exercising elderly

BACKGROUND: The decline in muscle mass (sarcopenia) with aging may be related to a decline in mitochondrial function. However, investigators have yet to reach a consensus as to whether a decline in mitochondrial function can be attenuated by physical activity has yet to reach a consensus. METHODS: Using dynamic 31PMRS to measure mitochondrial function, we measured baseline Phosphocreatine (PCr), inorganic phosphate (Pi), phosphodiester (PDE), [ADP], pH and recovery times (t(1/2)) for PCr and [ADP] following exercise, in 45 older (73+/-4 years, SD), and 20 younger subjects (25+/-4 years, SD) who were matched for body mass across high and low activity levels and within age and sex groupings. RESULTS: Baseline PCr, and Pi, were lower, and PDE higher in the older subjects compared to younger subjects (all P<0.01). The t(1/2)(ADP) was longer in older subjects (P<0.001) controlling for age and sex in the low activity group (P=0.02). In the older low activity groups, t(1/2)(PCr) was longer than high activity groups. Higher PDE levels were positively correlated with longer t(1/2)(PCr) in the older low activity females (both P<0.05). CONCLUSIONS: Our data suggests that mitochondrial function declines with age in healthy, exercising elderly adults and that the decline appears to be influenced by the level of physical activity.     

Prognostic value of exercise testing soon after myocardial infarction.

The prognostic value of a limited treadmill exercises test performed one day before hospital discharge after acute myocardial infarction was studied in 210 consecutive patients who had no over heart failure and had been free of chest pain for at least four days. No complications occurred. During a one-year follow-up period 28 of 43 patients (65 per cent) who had chest pain during the test reported angina, as compared with 60 of 167 (36 per cent) who had no chest pain during test (P less than 0.001). The one-year mortality rates were 2.1 per cent (three of 146) in patients without changes in the S-T segment during exercise and 27 per cent (17 of 64) in those with depression of the S-T segment (P less than 0.001). Sudden death occurred in one of 146 (0.7 per cent) patients who showed no change in the S-T segment and in 10 of 64 (16 per cent) with depression of the segment (P less than 0.001). Thus, a limited treadmill exercise test performed before hospital discharge after acute myocardial infarction is safe and can predict mortality in the subsequent year.