Elemental distribution in striated muscle and the effects of hypertonicity: Electron probe analysis of cryo sections

A method of rapid freezing in supercooled Freon 22 (monochlorodifluoromethane) followed by cryoultramicrotomy is described and shown to yield ultrathin sections in which both the cellular ultrastructure and the distribution of diffusible ions across the cell membrane are preserved and intracellular compartmentalization of diffusabler ions can be quantitated. Quantitative electron probe analysis (Shuman, H., A.V. Somlyo, and A.P. Somlyo. 1976. Ultramicros. 1:317-339.) of freeze-dried ultrathin cryto sections was found to provide a valid measure of the composition of cells and cellular organelles and was used to determine the ionic composition of the in situ terminal cisternae of the sarcoplasmic reticulum (SR), the distribution of CI in skeletal muscle, and the effects of hypertonic solutions on the subcellular composition if striated muscle. There was no evidence of sequestered CI in the terminal cisternae of resting muscles, although calcium (66mmol/kg dry wt +/- 4.6 SE) was detected. The values of [C1](i) determined with small (50-100 nm) diameter probes over cytoplasm excluding organelles over nuclei or terminal cisternae were not significantly different. Mitochondria partially excluded C1, with a cytoplasmic/ mitochondrial Ci ratio of 2.4 +/- 0.88 SD. The elemental concentrations (mmol/kg dry wt +/- SD) of muscle fibers measured with 0.5-9-μm diameter electron probes in normal frog striated muscle were: P, 302 +/- 4.3; S, 189 +/- 2.9;C1, 24 +/- 1.1;K, 404 +/- 4.3, and Mg, 39 +/- 2.1. It is concluded that: (a) in normal muscle the "excess CI" measured with previous bulk chemical analyses and flux studies is not compartmentalized in the SR or in other cellular organelles, and (b) the cytoplasmic C1 in low [K](0) solutions exceeds that predicted by a passive electrochemical distribution. Hypertonic 2.2 X NaCl, 2.5 X sucrose, or 2.2 X Na isethionate produced: (a) swollen vacuoles, frequently paired, adjacent to the Z lines and containing significantly higher than cytoplasmic concentrations of Na and Cl or S (isethionate), but no detectable Ca, and (b) granules of Ca, Mg, and P = approximately (6 Ca + 1 Mg)/6P in the longitudinal SR. It is concluded that hypertonicity produces compartmentalized domains of extracellular solutes within the muscle fibers and translocates Ca into the longitudinal tubules.     

QTL for body weight,morphometric traits and stress response in European sea bass Dicentrarchus labrax

Natural mating and mass spawning in the European sea bass (Dicentrarchus labrax L., Moronidae, Teleostei) complicate genetic studies and the implementation of selective breeding schemes. We utilized a two‐step experimental design for detecting QTL in mass‐spawning species: 2122 offspring from natural mating between 57 parents (22 males, 34 females and one missing) phenotyped for body weight, eight morphometric traits and cortisol levels, had been previously assigned to parents based on genotypes of 31 DNA microsatellite markers. Five large full‐sib families (five sires and two dams) were selected from the offspring (570 animals), which were genotyped with 67 additional markers. A new genetic map was compiled, specific to our population, but based on the previously published map. QTL mapping was performed with two methods: half‐sib regression analysis (paternal and maternal) and variance component analysis accounting for all family relationships. Two significant QTL were found for body weight on linkage group 4 and 6, six significant QTL for morphometric traits on linkage groups 1B, 4, 6, 7, 15 and 23 and three suggestive QTL for stress response on linkage groups 3, 14 and 23. The QTL explained between 8% and 38% of phenotypic variance. The results are the first step towards identifying genes involved in economically important traits like body weight and stress response in European sea bass.     

Plant virus infection development as affected by heavy metal stress

The work was focused on the investigation of possible dependencies between the development of viral infection in plants and the presence of high heavy metal concentrations in soil. Field experiments have been conducted in order to study the development of systemic tobacco mosaic virus (TMV) infection in Lycopersicon esculentum L. cv. Miliana plants under effect of separate salts of heavy metals Cu, Zn and Pb deposited in soil. As it is shown, simultaneous effect of viral infection and heavy metals in tenfold maximum permissible concentration leads to decrease of total chlorophyll content in experiment plants mainly due to the degradation of chlorophyll a. The reduction of chlorophyll concentration under the combined influence of both stress factors was more serious comparing to the separate effect of every single factor. Plants' treatment with toxic concentrations of lead and zinc leaded to slight delay in the development of systemic TMV infection together with more than twofold increase of virus content in plants that may be an evidence of synergism between these heavy metal's and virus' effects. Contrary, copper although decreased total chlorophyll content but showed protective properties and significantly reduced amount of virus in plants.     

Endocrine correlates of intra-specific variation in the mating system of the St. Peter's fish (Sarotherodon galilaeus)

The Challenge Hypothesis postulates that androgen levels are a function of the social environment in which the individual is living. Thus, it is predicted that in polygynous males that engage in social interactions, androgen levels should be higher than in monogamous animals that engage in parental care. In this study, we tested this hypothesis at the intra-specific level using a teleost species, Sarotherodon galilaeus, which exhibits a wide variation in its mating system. Experimental groups of individually marked fish were formed in large ponds with different operational sex-ratios (OSR) to study the effects of partner availability on blood plasma levels of sex steroids [11-ketotestosterone (11-KT), testosterone (T), and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P)] and gonadosomatic index (GSI). Polygyny mostly occurred in the female biased OSR groups. 17,20beta-P and gonadosomatic index did not differ among OSR groups. However, 11-KT was high in male biased OSR and positively correlated with aggressive challenges, thereby supporting the central postulate of the Challenge Hypothesis. The results of T were the inverse of those of 11-KT, probably because 11-KT is metabolized from T. 11-KT levels of polygynous males did not differ neither from those of monogamous males, nor from those of males that participated in parental care. These results do not support the expected relationships between polygyny, parental care, and androgen levels. The differences from expectations for 11-KT may be related to the fact that in S. galilaeus, the mating and the parenting phase are not clearly separated and thus, males may still fight and court while they are brooding.     

Genomic structure and expression of parathyroid hormone-related protein gene (PTHrP) in a teleost, Fugu rubripes

In this study we describe the isolation and characterisation of the parathyroid hormone-related protein (PTHrP) gene from the teleost Fugu rubripes. The gene has a relatively simple structure, compared with tetrapod PTHrP genes, composed of three exons and two introns, encompassing 2.25 kb of genomic DNA. The gene encodes a protein of 163 amino acids, with a putative signal peptide of 37 amino acids and a mature peptide of 126 amino acids. The overall homology with known tetrapod PTHrP proteins is low (36%), with a novel sequence inserted between positions 38 and 65, the absence of the conserved pentapeptide (TRSAW) and shortened C-terminal domain. The N-terminus shows greater conservation (62%), suggesting that it may have a hypercalcaemic function similar to that of tetrapod PTHrP. In situ localisation and RT–PCR have demonstrated the presence of PTHrP in a wide range of tissues with varying levels of expression. Sequence scanning of overlapping cosmids has identified three additional genes, TMPO, LDHB and KCNA1, which map to human chromosome 12, with the latter two mapping to 12p12-11.2. PTHrP in human also maps to this chromosome 12 sub-region, thus demonstrating conservation of synteny between human and Fugu.     

The parathyroid hormone family of peptides: structure, tissue distribution, regulation, and potential functional roles in calcium and phosphate balance in fish

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are two factors that share amino acid sequence homology and act via a common receptor. In tetrapods, PTH is the main endocrine factor acting in bone and kidney to regulate calcium and phosphate. PTHrP is an essential paracrine developmental factor present in many tissues and is involved in the regulation of ossification, mammary gland development, muscle relaxation, and other functions. Fish apparently lack an equivalent of the parathyroid gland and were long thought to be devoid of PTH. Only in recent years has the existence of PTH-like peptides and their receptors in fish been firmly established. Two forms of PTH, two of PTHrP, and a protein with intermediate characteristics designated PTH-L are encoded by separate genes in teleost fish. Three receptors encoded by separate genes in fish mediate PTH/PTHrP actions, whereas only two receptors have so far been found in terrestrial vertebrates. PTHrP has been more intensively studied than PTH, from lampreys to advanced teleosts. It is expressed in many tissues and is present in high concentration in fish blood. Administration of this peptide alters calcium metabolism and has marked effects on associated gene expression and enzyme activity in vivo and in vitro. This review provides a comprehensive overview of the physiological roles, distribution, and molecular relationships of the piscine PTH-like peptides.     

L1 (LINE-1) retrotransposable elements provide a "fossil" record of the phylogenetic history of murid rodents

The single most difficult problem in phylogenetic analysis is deciding whether a shared taxonomic character is due to common ancestry or one that appeared independently due to convergence, parallelism, or reversion to an ancestral state. Mammalian L1 retrotransposons undergo periodic amplifications in which multiple copies of the elements are interspersed in the genome. Because these elements apparently are transmitted only by inheritance and are retained in the genome, a shared L1 amplification event can only be an inherited ancestral character. We propose that L1 amplification events can be an excellent tool for analyzing mammalian evolution and demonstrate here how we addressed several refractory problems in rodent systematics using L1 DNA as a taxonomic character.      

Effects of 11-ketotestosterone on genital papilla morphology in the sex changing fish Lythrypnus dalli

Five days after implantation with 11-ketotestosterone (KT) female Lythrypnus dalli exhibited elevated urinary KT levels and male-like genital papilla morphology. Papilla length increased, width decreased, and length : width ratio increased. Control fish exhibited increases in papilla length, and length : width ratio on day 3, but not on day 5. Changes in the total area of the papilla were not statistically significant. It is suggested that KT mediates genital papilla morphology rapidly and thus may be a useful indicator of circulating KT in L. dalli.