Mercury decreases the frequency of induced but not spontaneous clustering of acetylcholine receptors

Studies with environmental levels of various metals typically focus on observable neurological symptoms in newborns and adults. Use of the C2C12 skeletal muscle cell line as a developmental model enabled us to test whether environmental insults prevented myotube formation or the assembly of the postsynaptic component of the neuromuscular synapse. Specifically, we asked whether the inorganic metal mercury interfered with the fusion of myoblasts into myotubes, acetylcholine receptor (AChR) clustering, or the agrin signaling events that precede AChR clustering. C2C12 myotubes grown in culture medium containing 10 M mercuric chloride were morphologically indistinguishable from control myotubes at the light-microscopic level, and myoblasts fused into myotubes normally. However, myotubes pretreated with mercury demonstrated a decreased frequency of AChR clustering induced by agrin and other experimental manipulations. Furthermore, mercury pretreatment decreased the agrin-induced tyrosine phosphorylation of the AChR subunit, thus inhibiting the agrin signal transduction pathway. In contrast, mercury failed to decrease the frequency of spontaneous AChR clustering, suggesting that spontaneous AChR clustering differs from agrin-induced AChR clustering in some significant way.This work was supported in part by Midwestern University     

Neuroendocrinology of nutritional infertility

Natural selection has linked the physiological controls of energy balance and fertility such that reproduction is deferred during lean times, particularly in female mammals. In this way, an energetically costly process is confined to periods when sufficient food is available to support pregnancy and lactation. Even in the face of abundance, nutritional infertility ensues if energy intake fails to keep pace with expenditure. A working hypothesis is proposed in which any activity or condition that limits the availability of oxidizable fuels (e.g., undereating, excessive energy expenditure, diabetes mellitus) can inhibit both gonadotropin-releasing hormone (GnRH)/luteinizing hormone secretion and female copulatory behaviors. Decreases in metabolic fuel availability appear to be detected by cells in the caudal hindbrain. Hindbrain neurons producing neuropeptide Y (NPY) and catecholamines (CA) then project to the forebrain where they contact GnRH neurons both directly and also indirectly via corticotropin-releasing hormone (CRH) neurons to inhibit GnRH secretion. In the case of estrous behavior, the best available evidence suggests that the inhibitory NPY/CA system acts primarily via CRH or urocortin projections to various forebrain loci that control sexual receptivity. Disruption of these signaling processes allows normal reproduction to proceed in the face of energetic deficits, indicating that the circuitry responds to energy deficits and that no signal is necessary to indicate that there is an adequate energy supply. While there is a large body of evidence to support this hypothesis, the data do not exclude nutritional inhibition of reproduction by other pathways and processes, and the full story will undoubtedly be more complex than this.     

Autoclave method for rapid preparation of bacterial PCR-template DNA

An autoclave method for preparing bacterial DNA for PCR template is presented, it eliminates the use of detergents, organic solvents, and mechanical cellular disruption approaches, thereby significantly reducing processing time and costs while increasing reproducibility. Bacteria are lysed by rapid heating and depressurization in an autoclave. The lysate, cleared by microcentrifugation, was either used directly in the PCR reaction, or concentrated by ultrafiltration. This approach was compared with seven established methods of DNA template preparation from four bacterial sources which included boiling Triton X-100 and SDS, bead beating, lysozyme/proteinase K, and CTAB lysis method components. Bacteria examined were Enterococcus and Escherichia coli, a natural marine bacterial community and an Antarctic cyanobacterial-mat. DNAs were tested for their suitability as PCR templates by repetitive element random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE) analysis. The autoclave method produced PCR amplifiable template comparable or superior to the other methods, with greater reproducibility, much shorter processing time, and at a significantly lower cost.     

Real-time quantitative polymerase chain reaction (QPCR) to identify Myxobolus cerebralis in rainbow trout Oncorhynchus mykiss

This study describes the development of a TaqMan real-time quantitative polymerase chain reaction (QPCR) technique using the heat-shock protein 70 (Hsp 70) and 18S ribosomal DNA (18S rDNA) sequences to identify Myxobolus cerebralis and attempt to quantify infection severity within rainbow trout fry Oncorhynchus mykiss. Rainbow trout for this study were exposed to M. cerebralis under natural river conditions and examined for infection by histology, polymerase chain reaction (PCR) and QPCR analysis at 900 Celsius temperature units (CTUs) following exposure. Detection sensitivity by QPCR was shown to be equal to traditional PCR but greater than histopathology. Primer/probe combinations developed for this study were capable of specifically detecting M. cerebralis DNA in infected fish tissue and single triactinomyxon (TAM) spores with a sensitivity of 12.5 and 6.3 pg microl(-1) of DNA for the Hsp 70 and 18S rDNA sequences, respectively. A strong relationship between QPCR and infection severity was found for the Hsp 70 probe when parasite copy number and histology scores of 0-4 were compared (R2 = 0.96, p = 0.003). However, a reduction in copy number was observed at higher histology scores for the 18S probe (scores of 4 and 5) and the Hsp 70 probe (score of 5). The results of this study demonstrate that QPCR analysis is an effective tool for detecting M. cerebralis in fish tissue and may provide a relative indication of infection severity.     

High-performance liquid chromatographic assay of lactic, pyruvic and acetic acids and lactic acid stereoisomers in calf feces, rumen fluid and urine

To facilitate clinical investigation of metabolic acidosis, a high-performance liquid chromatographic method was adapted and validated for the chiral separation of D-(-) and L-(+)-lactic acid in calf feces, rumen fluid and urine. A non-chiral method was also adapted and validated for the separation of pyruvic, acetic and DL-(+/-)-lactic acids in calf feces and DL-(+/-)-lactic and pyruvic acids in rumen fluid. Separation and quantification were achieved using a reversed phase sulphonated polystyrenedivinylbenzene analytical column for pyruvic, acetic and racemic lactic acids and by a 3 microm octadecylsilane (ODS) packed analytical column coated with N,N-dioctyl-L-alanine as the chiral selector for the separation of lactic acid enantiomers with Cu(II)-containing eluents by stereoselective ligand exchange chromatography. Endogenous analytes were present in validation samples over a range of concentrations (0.2-14.8 mmol/l). For the stereoselective assay, mean intra-day accuracy ranged from 90.6 to 108.4% and intra-day precision from 0.3 to 13.8%. For the non-stereoselective assay, mean intra-day accuracy ranged from 90.4 to 108.8% and intra-day precision from 1.5 to 11.1%. The limit of quantitation was 1.0 mmol/l for D- and L-lactic acid, 0.06125 mmol/l for pyruvic acid, 1.0 mmol/l for DL-lactic acid and 1 mmol/l for acetic acid. These assays can be used to study the role of the gastrointestinal tract and kidney in metabolic acidosis.     

Phylogeography of the false smooth snakes, Macroprotodon (Serpentes, Colubridae): mitochondrial DNA sequences show European populations arrived recently from Northwest Africa

Mitochondrial DNA (1075 bp: cytochrome b, 300 bp; 12S rRNA, 393 bp; and 16S rRNA, 382 bp) corroborates the monophyly of the genus Macroprotodon and of the species M. mauritanicus, M. abubakeri, and M. brevis. The subspecies M. brevis ibericus is also monophyletic. The mtDNA tree presented here indicates that M. cucullatus consists of at least two separate units and may possibly represent a primitive morphology rather than a species in its own right. However, this hypothesis is tentative since it is only reflects the history of a single evolutionary unit (mtDNA). A definitive understanding of the evolution of M. cucullatus will not be possible until informative nuclear markers are added to the mitochondrial data. Macroprotodon appears to have originated in the Maghreb region of NW Africa and speciated there around 4-5.5 million years ago around the end of the Miocene period, after which its three main lineages may each have expanded north into more mesic conditions. The group also spread eastwards into coastal areas of Libya quite recently and on to Egypt and Israel. Later still, M. b. ibericus from extreme north Morocco reached the Iberian Peninsula, and M. mauritanicus from Tunisia or Algeria colonised the Balearic Islands of Menorca and Mallorca. Both these range extensions may result from very recent natural colonisations or even from accidental human introduction. Recency of origin of Iberian and Balearic populations is indicated by uniformity of their mtDNA even across large distances, and its great similarity to that of populations in source regions. Isolated populations assigned to M. cucullatus in the Hoggar mountains (southern Algeria) and Western Sahara are probably relicts from quite recent periods of climatic amelioration in the North African desert.     

Association between gravitational force and tissue metabolism in periparturient rats

Recently, interest in mammalian reproduction and offspring survival in altered gravity has been growing. Because successful lactation is critical for mammalian neonate survival, we have been studying the effect of gravity metabolism. We have shown an exponential relationship between glucose metabolic rate in mammary tissue of periparturient rats and an increase in gravity load. In this study we showed that changes in mammary metabolic rate due to gravity force were accompanied by a decrease in glucose metabolism in adipose tissue and by a reduced size of adipocytes. We assume that these changes are likely due to changes in prolactin or leptin levels related to altered gravity load.     

Localization and interaction of NHERF isoforms in the renal proximal tubule of the mouse

In expression systems and in yeast, Na/H exchanger regulatory factor (NHERF)-1 and NHERF-2 have been demonstrated to interact with the renal brush border membrane proteins NHE3 and Npt2. In renal tissue of mice, however, NHERF-1 is required for cAMP regulation of NHE3 and for the apical targeting of Npt2 despite the presence of NHERF-2, suggesting another order of specificity. The present studies examine the subcellular location of NHERF-1 and NHERF-2 and their interactions with target proteins including NHE3, Npt2, and ezrin. The wild-type mouse proximal tubule expresses both NHERF-1 and NHERF-2 in a distinct pattern. NHERF-1 is strongly expressed in microvilli in association with NHE3, Npt2, and ezrin. Although NHERF-2 can be detected weakly in the microvilli, it is expressed predominantly at the base of the microvilli in the vesicle-rich domain. NHERF-2 appears to associate directly with ezrin and NHE3 but not Npt2. NHERF-1 is involved in the apical expression of Npt2 and the presence of other Npt2-binding proteins does not compensate totally for the absence of NHERF-1 in NHERF-1-null mice. Although NHERF-1 links NHE3 to the actin cytoskeleton through ezrin, the absence of NHERF-1 does not result in a generalized disruption of the architecture of the cell. Thus the mistargeting of Npt2 seen in NHERF-1-null mice likely represents a specific disruption of pathways mediated by NHERF-1 to achieve targeting of Npt2. These findings suggest that the organized subcellular distribution of the NHERF isoforms may play a role in the specific interactions mediating physiological control of transporter function.