Exercise, Dietary Protein, and Combined Effect on IGF-1.

Insulin-like growth factor 1 (IGF-1) is a dichotomous hormone. While beneficial for growth/repair, and regulating muscle hypertrophy, high concentrations of IGF-1 are associated with increased risk of cancer and mortality. Factors thought to mediate IGF-1 include dietary protein and exercise. The purpose of this study was to analyze acute effects of dietary protein and/or exercise on plasma free IGF-1 and the time-course thereof to inform individuals who may benefit from increased IGF-1 (muscle growth/repair) or reduced IGF-1 (risk/diagnosis of cancer). Twenty-four participants (11 females, 24.9±4.6y) completed the three-way crossover study consisting of: (1)a high protein (42g) meal; (2)exercise (20min with four 30sec sprints); and (3)exercise followed by a high protein meal. Blood samples were collected fasted at rest, immediately after rest (or 5min after exercise), and at regular intervals throughout a 5h recovery. An additional fasted venipuncture was performed the morning following each condition (24h after baseline). Free IGF-1 was higher at immediately after exercise in the exercise condition (p=0.04). In the protein condition the 24h IGF-1 was 17.5% higher (p=0.02) than baseline. IGF-1 did not change over time in response to exercise with protein. The data gleaned from this study can enhance the knowledge of the time-course effects from protein and/or exercise on IGF-1. This study can provide a foundation for future research to investigate optimal timing and dosage to enhance muscle protein synthesis for athletes, as well as investigate whether consistent high protein meals may chronically elevate IGF-1 and increase the risk of deleterious health outcomes.

Molecular characterization of the zebrafish PEA3 ETS-domain transcription factor.

The PEA3 subfamily of ETS-domain proteins play important roles in regulating transcriptional activation and have been implicated in several tumorigenic processes. Here we describe the identification of a further member of this family from zebrafish which most likely represents a homologue of PEA3. A high degree of sequence conservation is observed in the ETS DNA-binding domain and acidic transcriptional activation domain. The DNA binding specificity of zebrafish PEA3 is virtually identical to that exhibited by mammalian family members and is autoregulated by cisacting inhibitory domains. Transcriptional activation by zebrafish PEA3 is potentiated by the ERK MAP kinase and protein kinase A pathways. During embryogenesis, PEA3 is expressed in complex spatial and temporal patterns in both mesodermal somites and ectodermal tissues including the brain, dorsal spinal chord and neural crest. Our characterisation of zebrafish PEA3 furthers our understanding of its molecular function and its expression profile suggests a novel role in cell patterning in the early vertebrate embryo.

Exogenous retinoic acid causes specific alterations in the development of the midbrain and hindbrain of the zebrafish embryo including positional respecification of the Mauthner neuron.

Exogenously applied retinoic acid given at the early stages of gastrulation causes abnormal development of the caudal midbrain and anterior hindbrain in vertebrate embryos. We describe the limits of the brain regions that are affected using neuroanatomical criteria in the zebrafish embryo. Analysis of the reticulospinal complex shows that the Mauthner cell, which normally differentiates in rhombomere 4, is duplicated either in this rhombomere or in rhombomere 2. Using probes for zebrafish krx20 and pax2, it is demonstrated that retinoic acid affects the expression domains of these regulatory genes in a manner that is consistent with the neuroanatomical data. Expression of the goosecoid gene, which expressed in the prospective anterior mesoderm from the onset of gastrulation, is unaffected by the doses of retinoic acid used in this study, reflecting the normal development of the anterior end of the embryo.

Insights into early vasculogenesis revealed by expression of the ETS-domain transcription factor Fli-1 in wild-type and mutant zebrafish embryos.

Fli-1 is an ETS-domain transcription factor whose locus is disrupted in Ewing's Sarcoma and F-MuLV induced erythroleukaemia. To gain a better understanding of its normal function, we have isolated the zebrafish homologue. Similarities with other vertebrates, in the amino acid sequence and DNA binding properties of Fli-1 from zebrafish, suggest that its function has been conserved during vertebrate evolution. The initial expression of zebrafish fli-1 in the posterior lateral mesoderm overlaps with that of gata2 in a potential haemangioblast population which likely contains precursors of blood and endothelium. Subsequently, fli-1 and gata2 expression patterns diverge, with separate fli-1 and gata2 expression domains arising in the developing vasculature and in sites of blood formation respectively. Elsewhere in the embryo, fli-1 is expressed in sites of vasculogenesis. The expression of fli-1 was investigated in a number of zebrafish mutants, which affect the circulatory system. In cloche, endothelium is absent and blood is drastically reduced. In contrast to the blood and endothelial markers that have been studied previously, fli-1 expression was initiated normally in cloche embryos, indicating that induction of fli-1 is one of the earliest indicators of haemangioblast formation. Furthermore, although fli-1 expression in the trunk was not maintained, the normal expression pattern in the anterior half of the embryo was retained. These anterior cells did not, however, condense to form blood vessels. These data indicate that cloche has previously unsuspected roles at multiple stages in the formation of the vasculature. Analysis of fli-1 expression in midline patterning mutants floating head and squint, confirms a requirement for the notochord in the formation of the dorsal-aorta. The formation of endothelium in one-eyed pinhead, cyclops and squint embryos indicates a novel role for the endoderm in the formation of the axial vein. The phenotype of sonic-you mutants implies a likely role for Sonic Hedgehog in mediating these processes.

Diminished thyroxine-binding globulin in pubertal diabetic children.

OBJECTIVE: To determine the effect of diabetes on thyroid hormone and thyroxine-binding globulin (TBG) concentrations during puberty. RESEARCH DESIGN AND METHODS: Total thyroxine (TT4), free thyroxine (FT4), and TBG levels of 171 thyroid microsomal antibody-negative subjects with normal thyroid-stimulating hormone (TSH) levels were measured and compared with those of nondiabetic adolescents. A random subset of 68 diabetic patients (40 boys and 28 girls) and 51 control subjects (24 boys and 27 girls) were analyzed for puberty-related changes. RESULTS: Most TT4 levels of diabetic subjects (80% of girls and 63% of boys) were below the 50th percentile for the normal range. TT4 increased with age in girls (r = 0.25, P < 0.04) but not in boys. FT4 was within normal limits in both sexes. TBG measurements were below the 50th percentile and 20% were below the 95% CI for both sexes; TT4 correlated with TBG in boys (r = 0.54, P < 0.001) and in girls (r = 0.58, P < 0.001). Duration of diabetes had no effect, whereas TT4 and FT4 levels were higher in girls with the lowest HbA1 levels (r = -0.29, P < 0.01 and r = -0.45, P < 0.01). Levels of TBG were reduced for all male pubertal stages (P < 0.01) and for early and late female pubertal stages (P < 0.01). There was no direct relationship between glucose control or the duration of diabetes and levels of TBG. CONCLUSIONS: Because TT4 levels are low and correlate with the low levels of TBG, it is important to measure free thyroid hormone and TSH levels in diabetic adolescents to establish euthyroidism.

Renin and aldosterone at high altitude in man.

Measurements have been made of hormonal changes relevant to salt and water balance during prolonged exposure to hypoxia to improve our understanding of the syndrome of acute mountain sickness. We have attempted to delineate the detailed inter-relationships between the renin-aldosterone and the vasopressin systems by a metabolically controlled study, involving an orthostatic stress (45 degrees head-up tilt) and an injection of a standard dose of ACTH to test adrenal responsiveness. Three Caucasian medical students underwent a 7-day equilibration at 150 m (Lima, Peru), followed by a 6-day sojourn at 4350 m (Cerro de Pasco, Peru) and a final 7 days at 150 m. Measurements were made of sodium and potassium balance, body weight and the 24-h renal excretion of vasopressin, cortisol and aldosterone 18-glucuronide. These variables showed little change, except for that of aldosterone 18-glucuronide, which fell sharply at altitude and rebounded even more sharply on return to sea level. At altitude, basal plasma levels of renin activity and aldosterone fell, and the response to orthostasis was attenuated, but the fall of plasma renin activity, as compared to plasma aldosterone, was delayed; on return to sea level this dissociation was exacerbated with the return of normal renin responsiveness lagging behind that of aldosterone. We suggest that unknown factors which dissociate the orthodox renin-aldosterone relationship, other than the activity of the angiotensin I-converting enzyme, are operative on exposure to hypoxia.

Specific binding sites for 125I-labelled human luteinizing hormone (hLH) in microsomal fractions from Drosophila.

Microsomal fractions prepared from Drosophila flies and larvae bound 125I-labelled human luteinizing hormone (hLH) in a displaceable manner. Binding increased linearly with increasing microsome concentration, and was dependent on the pH and metal ion concentration of the medium, and on the temperature and duration of incubation. Scatchard plots of 125I-hLH binding demonstrated the presence of two distinct binding sites, one with high affinity and low capacity, the other with low affinity and high capacity. 125I-hLH binding was inhibited in a dose-dependent fashion by partially purified human gonadotropin preparations, but not by other proteins, hormones and peptides. Highly purified human chorionic gonadotropin (hCG) or hLH preparations were much less effective at inhibiting 125I-hLH binding to Drosophila microsomes than partially purified gonadotropins. This was due to the presence of a contaminant present in partially purified commercial hCG preparations. Chromatography of crude hCG preparations clearly resolved the LH-binding inhibitor from hCG. Moreover, whereas hCG (and epidermal growth factor-like factors present in crude hCG preparations) were not bound to CM-Sepharose, the Drosophila LH-binding inhibitor was adsorbed strongly. Fractionation of 125I-hLH tracer on concanavalin A-Sepharose suggested that Drosophila binding sites did not preferentially bind a subfraction of the hormone tracer. These data suggest that high affinity binding sites for hLH/hCG-like molecules are present in Drosophila microsomes.

Development of radioimmunoassays for the measurement of aldosterone in unprocessed plasma and simple plasma extracts.

Inexpensive and rapid radioimmunoassay techniques for the measurement of aldosterone in unprocessed plasma and simple plasma extracts are described. The use of low pH (pH 5.0) and merthiolate to minimise plasma protein binding and the use of aldosterone-free plasma in the standards allows the measurement of aldosterone in 50 microliter of unprocessed plasma, which has been found useful in the diagnostic screening and classification of hyperaldosteronism. Despite quantitative recovery of added (+)-aldosterone and high specificity, the aldosterone content of unprocessed plasma is overestimated, probably by the presence of a water-soluble compound which closely resembles aldosterone. The use of a simple preliminary dichloromethane extraction procedure gives an excellent correlation with values obtained after chromatography. Values are given for chosen normal people and people with benign essential hypertension, using both assay procedures in three different physiological contexts.

A radioimmunoassay for the measurement of tetrahydroaldosterone 3-glucosiduronic acid in human plasma.

A simple, reliable and specific radioimmunoassay procedure for the measurement of the principal aldosterone metabolite, tetrahydroaldosterone 3-glucosiduronic acid, in human plasma is described. The glucosiduronate is extracted from plasma (generally 200 microliter) by the use of Amberlite XAD-2 resin, eluted with acidic, aqueous ethanol, separated from unconjugated steroids by partition and estimated using a specific antibody. A disequilibrium system is used, but with a conventional charcoal separation; the advantages of such a system are discussed. Data obtained by incorporating chromatographic separations into the assay procedure is presented as evidence for the specificity of the method. Data concerning the long-term storage of plasma samples is given, together with plasma concentrations of the glucosiduronate in both normal people and in people with abnormalities of aldosterone secretion.

Activation or detoxification of mutagenic and carcinogenic compounds in transgenic Drosophila expressing human glutathione S-transferase.

Sensitivity of transgenic Drosophila melanogaster with expression of a human gene encoding the glutathione S-transferase alpha subunit (GSTA1-1) to 1,2:5,6-dibenzanthracene (DBA) and 1,2-dichloroethane (DCE) was investigated in the somatic mutation and recombination test (SMART). We performed the same assay in control transgenic flies expressing the bacterial lacZ gene. Three types of transgenic Drosophila strains carrying GSTA1-1 were used: two transgenic strains homozygous for the second chromosome with a single-copy transgene insertion and one strain with two transgene insertions. Larvae carrying the lacZ gene were significantly more sensitive to genotoxic effects of DBA than those carrying three copies of the GSTA1-1 gene. The larvae with lacZ expression showed significantly lower sensitivity to DCE compared with those expressing GSTA1-1. Finally, a pretreatment with buthionine-sulphoximine (BSO) in experiment with DCE significantly decreased the frequency of mutation events in larvae with three GSTA1-1 copies in comparison with others.

Genotoxicity of N-nitroso-N-methylurea and acetone oxime in the transgenic Drosophila carrying the human gene encoding a subunit of glutathione S-transferase.

The genotoxic effects of N-nitroso-N-methylurea (MNU) and acetone oxime (ACOX) were tested in the Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster. We have performed the same assay on transgenic flies expressing the human gene encoding a glutathione S-transferase alpha subunit (HGST). The SMART assay is used here to demonstrate genotoxicity and to determine the effect of human glutathione S-transferase on the genotoxic response. Three types of Drosophila strains were used: non-transgenic strains first described by Szabad (1986), transgenic strains derived from the Szabad strains but expressing the bacterial lacZ gene, and similarly derived transgenic strains expressing the HGST gene. MNU was highly genotoxic in both transgenic and non-transgenic flies. The non-transgenic lies were significantly more sensitive to the genotoxic effects of MNU compared to both types of transgenic flies. There were statistically significant differences between the transgenic HGST crosses and transgenic lacZ and non-transgenic control crosses but there was no significant difference between the genotoxic response to MNU in flies from the transgenic cross with lacZ and from the cross carrying three copies of HGST. ACOX also proved to be genotoxic to both non-transgenic and transgenic flies. However, flies carrying three copies of the gene were significantly more resistant to the genotoxic effect of ACOX than those transgenic flies with two or no copies of the human gene.