Recruiting of somatotroph cells after combined somatostatin, GHRH and growth hormone (GH) secretagogue stimulation in a study of pituitary GH reserve in prepuberal female rats

Diagnostic confirmation of growth hormone (GH) deficiency in children and adults is based on stimulation tests designed to assess the pituitary reserve by measuring the amount of GH released into the bloodstream; however, the results obtained by this means cannot provide any direct indication of the amount of GH actually produced by pituitary somatotroph cells. The present paper sought to test the hypothesis that release of GH following administration of specific stimuli does not accurately reflect the somatotroph cell response, and that the amount of GH released into the bloodstream may often be greater or smaller than the amount synthesized. GH release and changes in the proportion of somatotroph cells were charted in prepuberal female Wistar rats, following administration of several different GH stimuli: GHRH (1 microg/kg), GHRP-6 (1 microg/kg), GHRELIN (1 microg/kg) and combined GHRH-based treatments, with or without SRIH pretreatment (1 microg/kg) 90 minutes earlier. Peak serum GH values were recorded 15 minutes after administration of GHRH+GHRELIN and GHRH+GHRP-6; maximum stimulation in terms of an increased proportion of somatotroph cells occurred 15 minutes after combined adminstration of GHRH + GHRELIN. SRIH pretreatment (- 90 min) inhibited GH release, with a subsequent "escape" and lack of response to stimulation which lasted at least 30 minutes except following administration of GHRH. However, combined administration of GHRH+GHRELIN maintained stimulation of the somatotroph cell population. In conclusion, the results suggest that the enhanced GH release prompted by stimulation tests used to diagnose GH deficiency in prepuberal female rats does not fully reflect somatroph cell dynamics, and that not all the GH produced and stored by somatotroph cells is released into the bloodstream.     

Improved gating of a chimeric alpha7-5HT3A receptor upon mutations at the M2-M3 extracellular loop

Acetylcholine-evoked currents of the receptor chimera alpha7-5HT3A V201 expressed in Xenopus oocytes are strikingly small when compared to the amount of alpha-bungarotoxin binding sites detected at the oocyte membrane. Since the chimeric receptor is made of the extracellular N-terminal region of the rat alpha7 nicotinic acetylcholine receptor and the C-terminal region of the mouse 5-HT3A receptor, which includes the ion channel, we hypothesized that communication between these two regions was not optimal. Here, we show that mutating to aspartate several adjacent positions in the M2-M3 extracellular linker increases current amplitudes to different extents, thus confirming the important role of this region on receptor gating.     

Role of energetic coenzyme pools in the production of L-carnitine by Escherichia coli

The aim of this work was to understand the steps controlling the biotransformation of trimethylammonium compounds into L(-)-carnitine by Escherichia coli. The high-cell density reactor steady-state levels of carbon source (glycerol), biotransformation substrate (crotonobetaine), acetate (anaerobiosis product) and fumarate (as an electron acceptor) were pulsed by increasing them fivefold. Following the pulse, the evolution of the enzyme activities involved in the biotransformation process of crotonobetaine into L(-)-carnitine (crotonobetaine hydration), in the synthesis of acetyl-CoA (ACS: acetyl-CoA synthetase and PTA: ATP: acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid (ICDH: isocitrate dehydrogenase) and glyoxylate (ICL: isocitrate lyase) cycles was monitored. In addition, the levels of carnitine, the cell ATP content and the NADH/NAD(+) ratio were measured in order to assess the importance and participation of these energetic coenzymes in the catabolic system. The results provided an experimental demonstration of the important role of the glyoxylate shunt during biotransformation and the need for high levels of ATP to maintain metabolite transport and biotransformation. Moreover, the results obtained for the NADH/NAD(+) pool indicated that it is correlated with the biotransformation process at the NAD(+) regeneration and ATP production level in anaerobiosis. More importantly, a linear correlation between the NADH/NAD(+) ratio and the levels of the ICDH and ICL (carbon and electron flows) and the PTA and ACS (acetate and ATP production and acetyl-CoA synthesis) activity levels was assessed. The main metabolic pathway operating during cell metabolic perturbation with a pulse of glycerol and acetate in the high-cell density membrane reactor was that related to ICDH and ICL, both regulating the carbon metabolism, together with PTA and ACS enzymes (regulating ATP production).     

Use of RNA isolated from feces as a promising tool for the early detection of colorectal cancer

Colorectal cancer is one of the most common forms of cancer worldwide. Early detection would allow patients to be treated surgically and halt the progression of the disease; however, the current methods of early detection are invasive (colonoscopy and sigmoidoscopy) or have low sensitivity (fecal occult blood test). The altered expression of genes in stool samples of patients with colorectal cancer can be determined by RT-PCR. This is a noninvasive and highly sensitive technique for colorectal cancer screening. According to information gathered in this review and our own experience, the use of fecal RNA to determine early alterations in gene expression due to malignancy appears to be a promising alternative to the current detection methods and owing to its low cost could be implemented in public health services.     

Population genetic structure of a specialist leafhopper on Zea: likely anthropogenic and ecological determinants of gene flow

Corn leafhopper, Dalbulus maidis DeLong & Wolcott (Hemiptera: Cicadellidae), is a specialist herbivore on the genus Zea (Poaceae). The genera Dalbulus and Zea evolved in central Mexico. We sought to determine whether population genetic structuring is prevalent in corn leafhoppers inhabiting three of its host plants: (1) the highland species perennial teosinte (Zea diploperennis Iltis, Doebley & Guzman), (2) the mid‐ to lowland‐species Balsas teosinte (Zea mays ssp. parviglumis Iltis & Doebley), and (3) the ubiquitous domesticated maize (Zea mays ssp. mays L.). We used amplified fragment length polymorphisms to detect population structuring and genetic differentiation among corn leafhoppers on the three host plants in western‐central and ‐northern Mexico. Our results showed that corn leafhopper in Mexico is composed of at least two genetically discrete populations: an ‘Itinerant’ population associated with the annual hosts maize and Balsas teosinte, which appears to be widely distributed in Mexico, and a ‘Las Joyas’ population restricted to perennial teosinte and confined to a small mountain range (Sierra de Manantlán) in western‐central Mexico. Our results further suggested that population structuring is not due to isolation by distance or landscape features: Las Joyas and Itinerant corn leafhopper populations are genetically distinct despite their geographic proximity (ca. 4 km), whereas Itinerant corn leafhoppers separated by hundreds of kilometers (>800 km), mountain ranges, and a maritime corridor (Sea of Cortez) are not genetically distinct. Based on our results and on published ethnohistorical and archaeological data, we propose pre‐Columbian and modern scenarios, including likely ecological and anthropogenic influences, in which the observed genetic population structuring of corn leafhopper could have originated and could be maintained. Also, we hypothesize that after evolving on the lowland Balsas teosinte, corn leafhopper expanded its host range to include maize and then the highland perennial teosinte, following the domestication and spread of maize within the last 9 000 years.     

The regulation of myosin phosphatase in pregnant human myometrium

Myometrial smooth muscle contractility is regulated predominantly through the reversible phosphorylation of MYLs (myosin light chains), catalysed by MYLK (MYL kinase) and MYLP (MYL phosphatase) activities. MYLK is activated by Ca2+-calmodulin, and most uterotonic agonists operate through myometrial receptors that increase [Ca2+]i (intracellular Ca2+ concentration). Moreover, there is substantial evidence for Ca2+-independent inhibition of MYLP in smooth muscle, leading to generation of increased MYL phosphorylation and force for a given [Ca2+]i, a phenomenon known as 'Ca2+-sensitization'. ROCK (Rho-associated kinase)-mediated phosphorylation and inhibition of MYLP has been proposed as a mechanism for Ca2+-sensitization in smooth muscle. However, it is unclear to date whether the mechanisms that sensitize the contractile machinery to Ca2+ are important in the myometrium, as they appear to be in vascular and respiratory smooth muscle. In the present paper, we discuss the signalling pathways regulating MYLP activity and the involvement of ROCK in myometrial contractility, and present recent data from our laboratory which support a role for Ca2+-sensitization in human myometrium.     

A chloroplast-derived C4V3 polypeptide from the human immunodeficiency virus (HIV) is orally immunogenic in mice

Although the human immunodeficiency virus (HIV) causes one of the most important infectious diseases worldwide, attempts to develop an effective vaccine remain elusive. Designing recombinant proteins capable of eliciting significant and protective mammalian immune responses remain a priority. Moreover, large-scale production of proteins of interest at affordable cost remains a challenge for modern biotechnology. In this study, a synthetic gene encoding a C4V3 recombinant protein, known to induce systemic and mucosal immune responses in mammalian systems, has been introduced into tobacco chloroplasts to yield high levels of expression. Integration of the transgene into the tobacco plastome has been verified by Southern blot hybridization. The recombinant C4V3 protein is also detected in tobacco chloroplasts by confocal microscopy. Reactivity of the heterologous protein with both an anti-C4V3 rabbit serum as well as sera from HIV positive patients have been assayed using Western blots. When administered by the oral route in a four-weekly dose immunization scheme, the plant-derived C4V3 has elicited both systemic and mucosal antibody responses in BALB/c mice, as well as CD4+ T cell proliferation responses. These findings support the viability of using plant chloroplasts as biofactories for HIV candidate vaccines, and could serve as important vehicles for the development of a plant-based candidate vaccine against HIV.     

Hematological indicators of stress in longline-captured sharks

For many shark species, little information exists about the stress response to capture and release in commercial longline fisheries. Recent studies have used hematological profiling to assess the secondary stress response, but little is known about how, and to what degree, these indicators vary interspecifically. Moreover, there is little understanding of the extent to which the level of relative swimming activity (e.g., sluggish vs. active) or the general ecological classification (e.g., coastal vs. pelagic) correlates to the magnitude of the exercise-induced (capture-related) stress response. This study compared plasma electrolytes (Na(+), Cl(-), Mg(2+), Ca(2+), and K(+)), metabolites (glucose and lactate), blood hematocrit, and heat shock protein (Hsp70) levels between 11 species of longline-captured sharks (n=164). Statistical comparison of hematological parameters revealed species-specific differences in response to longline capture, as well as differences by ecological classification. Taken together, the blood properties of longline-captured sharks appear to be useful indicators of interspecific variation in the secondary stress response to capture, and may prove useful in the future for predicting survivorship of longline-captured sharks where new technologies (i.e., pop-up satellite tags) can verify post-release mortality.     

Living in the past: phylogeography and population histories of Indo-Pacific wrasses (genus Halichoeres) in shallow lagoons versus outer reef slopes

Sea level fluctuations during glacial cycles affect the distribution of shallow marine biota, exposing the continental shelf on a global scale, and displacing coral reef habitat to steep slopes on oceanic islands. In these circumstances we expect that species inhabiting lagoons should show shallow genetic architecture relative to species inhabiting more stable outer reefs. Here we test this expectation on an ocean-basin scale with four wrasses (genus Halichoeres): H. claudia (N?=?194, with ocean-wide distribution) and H. ornatissimus (N?=?346, a Hawaiian endemic) inhabit seaward reef slopes, whereas H. trimaculatus (N?=?239) and H. margaritaceus (N?=?118) inhabit lagoons and shallow habitats throughout the Pacific. Two mitochondrial markers (cytochrome oxidase I and control region) were sequenced to resolve population structure and history of each species. Haplotype and nucleotide diversity were similar among all four species. The outer reef species showed significantly less population structure, consistent with longer pelagic larval durations. Mismatch distributions and significant negative Fu's F values indicate Pleistocene population expansion for all species, and (contrary to expectations) shallower histories in the outer slope species. We conclude that lagoonal wrasses may persist through glacial habitat disruptions, but are restricted to refugia during lower sea level stands. In contrast, outer reef slope species have homogeneous and well-connected populations through their entire ranges regardless of sea level fluctuations. These findings contradict the hypothesis that shallow species are less genetically diverse as a consequence of glacial cycles.     

Genetic counseling in Usher syndrome: linkage and mutational analysis of 10 Colombian families

Usher Syndrome (US), an autosomal recessive disease, is characterized by retinitis pigmentosa (RP), vestibular dysfunction, and congenital sensorineural deafness. There are three recognized clinical types of the disorder. In order to improve genetic counseling for affected families, we conducted linkage analysis and DNA sequencing in 10 Colombian families with confirmed diagnosis of US (4 type I and 6 type II). Seventy-five percent of the US1 families showed linkage to locus USH1B, while the remaining 25% showed linkage to loci USH1B and USH1C. Among families showing linkage to USH1B we found two different mutations in the MYO7A gene: IVS42-26insTTGAG in exon 43 (heterozygous state) and R634X (CGA-TGA) in exon 16 (homozygous state). All six US2 families showed linkage to locus USH2A. Of them, 4 had c.2299delG mutation (1 homozygote state and 3 heterozygous); in the remaining 2 we did not identify any pathologic DNA variant. USH2A individuals with a 2299delG mutation presented a typical and homogeneous retinal phenotype with bilateral severe hearing loss, except for one individual with a heterozygous 2299delG mutation, whose hearing loss was asymmetric, but more profound than in the other cases. The study of these families adds to the genotype-phenotype characterization of the different types and subtypes of US and facilitates genetic counseling in these families. We would like to emphasize the need to perform DNA studies as a prerequisite for genetic counseling in affected families.