Both avian and mammalian embryonic myoblasts are intrinsically heterogeneous
Adult skeletal muscles are composed of different fibre types. What initiates the distinctive muscle fibre type-specific specialization in a developing embryo is still controversial. In vitro studies of avian muscles have shown the expression of one of the slow myosin heavy chains, SM2, in only some myotubes. In this report we demonstrate the expression of another slow myosin heavy chain, SM1, restricted to only some chicken myotubes (presumptive slow) in vitro. We also demonstrate that as is the case for avian species, distinct fast and slow myogenic cells are detectable in mammalian species, human and rat, during in vitro development in the absence of innervation. While antibodies to fast myosin heavy chains stained all myotubes dark in these muscle cell cultures, antibodies to slow myosin heavy chains stained only a proportion of the myotubes (presumptive slow). The other myotubes were either unstained or only weakly stained with slow myosin heavy c hain antibodies. The muscle cell cultures prepared from different developmental stages of rat skeletal muscles showed a reduction in the number of slow myosin heavy chain-positive myotubes with advancing foetal growth. It is concluded that embryonic myogenic cells that are likely to form distinct fast or slow muscle fibre types are intrinsically heterogeneous, not only in avian but also in mammalian species, although extrinsic factors reinforce and modify such commitment throughout subsequent development.

title: Regenerative capability of upper and lower jaws in the newt.
abstract: The regenerating amphibian jaw represents an important model for studying pattern formation and the mechanisms underlying regeneration of facial structures. We have studied regeneration of upper and lower jaws in the urodele amphibian, Notophthalmus viridescens, using whole mount preparations stained for bone and cartilage, scanning electron microscopy and immunocytochemistry to further characterize these regenerating systems. In addition, we have investigated whether lower jaws of adults and larvae display similar regenerative ability. Although in adult animals the original shape of both the lower and upper jaws is rather faithfully reproduced following amputation, and the teeth and oral mucosa with its specialized sensory organs fully regenerate, significant differences in the regenerative ability of the various skeletal elements are observed. In fact, only tooth-bearing skeletal elements ossify, while the other elements of the regenerated skeleton remain cartilaginous for as long as 5 months after amputation. In contrast, a regenerated lower jaw in the larva is indistinguishable from an unamputated one at the same stage of development. Interestingly, regenerating adult jaws form directly bicuspid teeth, which are the type of teeth normally found in the adult, rather than the monocuspid teeth characteristic of larval jaws, indicating that jaw regeneration is not a recapitulation of development, in that an adult jaw blastema directly regenerates an adult jaw. Finally, we have studied the expression of tissue specific markers in normal and regenerating upper and lower jaws to establish whether the blastemal cells, which will form the missing part of the jaw, express any of these markers of the differentiated state, or are undifferentiated as suggested by their morphological appearance. Under our experimental conditions, no expression of markers of the differentiated state, such as those for muscle, cartilage and glands is detectable in early regenerates. On the contrary, the mesenchymal marker 22/31, whose expression in normal jaws is restricted to dermal fibroblasts and the dental pulp, is expressed in at least a half of the blastemal cells. The significance of these observations in relation to the origin of blastemal cells in the jaw will be discussed.

title: Regeneration of lower and upper jaws in urodeles is differentially affected by retinoic acid.
abstract: The vitamin A derivative retinoic acid (RA) is a powerful teratogen which can induce severe craniofacial and limb malformations if administered at certain stages of gestation. In addition this compound has been shown to affect patterning in regenerating systems. A classical example is the induction of supernumerary structures along the proximodistal axis of the regenerating amphibian limb. We have investigated the effect of RA on other regenerating systems, the amphibian lower and upper jaws, both in developing and adult animals. We report here that RA does not induce formation of extra structures either in the lower or in the upper jaw of adult newts under experimental conditions where duplications of the regenerating limb occur. However, RA selectively induces severe malformations in the upper jaw regenerate that resemble those induced in avian and mammalian embryos. Analysis of the expression of the newt retinoic acid receptors RAR alpha and delta in upper and lower jaws showed that RAR alpha was expressed at a significant level in the wound epidermis, but not in blastemal cells, whereas no RAR delta could be detected in the regenerate either by in situ hybridization or by using an anti-RAR delta antibody. Therefore, unlike in the limb, in jaws RAR delta is not up-regulated following amputation, and this difference in expression may be causally related to the different effects induced by RA on jaws and limbs. In order to establish whether retinoids affected regeneration of developing jaws in a similar fashion, their effects were studied in animals whose jaws had been amputated at different developmental stages. Under the experimental conditions used overall growth retardation and head defects were observed in the majority of embryos which had been amputated and treated with retinol palmitate (RP) between stages 26-28 and 38-39. In contrast, patterning of upper jaw regenerates in larvae amputated at stage 26-28 and 38-39. In contrast, patterning of upper jaw regenerates in larvae amputated at stage 45 was not significantly affected by the treatment, although the early phase of regeneration was slower than in controls. The different responses to retinoids of regenerating facial structures in embryos, larvae and adults will be discussed.

title: Study of properties of cholinephosphotransferase from fetal guinea pig lung mitochondria and microsomes
abstract: SummaryWe have reported earlier that cholinephosphotransferase (EC 2.7.8.2) is present in both mitochondria and microsomes of fetal guinea pig lung. This study was designed to compare the properties of mitochondrial and microsomal cholinephosphotransferase in fetal guinea pig lung. Various parameters, such as substrate specificity, Km values, sensitivity to N-ethylmaleimide, dithiothreitol and trypsin were measured. Both showed significant preference for unsaturated diacylglycerols over saturated diacylglycerols. Data on Km and Vmax indicate that the affinity of this enzyme for different diacylglycerols varies between the two forms. The ID50 values for N-ethylmaleimide were 20 mM and 12.5 mM for the mitochondrial and microsomal form of the enzyme, respectively. Dithiothreitol showed an inhibitory effect on both; however, the mitochondrial form was inhibited less than the microsomal form. The effects of N-ethylmaleimide and dithiothreitol on both forms of enzyme indicated that the microsomal cholinephosphotransferase requires a higher concentration of -SH for its activity than the mitochondrial enzyme does. The enzyme was inhibited by trypsin in both mitochondria and microsome under isotonic condition suggesting that this enzyme is on the outside of the membrane in both endoplasmic reticulum and mitochondria.

title: Glycogen content & structure & some enzymes of glycogen metabolism in human foetal organs.
abstract: The glycogen content, and its structure and the enzymes involved in glycogenolysis in human foetal organs were studied at different periods of gestation. Of all the tissues studied glycogen content was found to be the highest in cardiac muscle. Very little glycogen was present in the foetal liver at 9-12 wk of gestation, this increased progressively to nearly 2 per cent at 24 wk. Glycogen content of placenta was lower than that of skeletal muscle and liver. The level of glycogen in adipose tissue, placenta and cerebrum was not high enough to play any role in glucose homeostasis of the foetus. Human foetal liver and skeletal muscle glycogen showed the normal branched structure while the liver glycogen was found to be unusually stable. Glycogen phosphorylase activity in the foetal liver and muscle was found to be low, i.e., about a fifth and a fourth of adult liver and muscle activity respectively. The stability of foetal liver glycogen and phosphorolytic activity in the liver and muscle indicate negligible glycogenolysis during foetal development. Glucose-6-phosphatase activity in foetal liver was undetectable below 12 wk of gestation, the activity increasing progressively up to 24 wk.

title: Identifying rare variants from exome scans: the GAW17 experience
abstract: Genetic Analysis Workshop 17 (GAW17) provided a platform for evaluating existing statistical genetic methods and for developing novel methods to analyze rare variants that modulate complex traits. In this article, we present an overview of the 1000 Genomes Project exome data and simulated phenotype data that were distributed to GAW17 participants for analyses, the different issues addressed by the participants, and the process of preparation of manuscripts resulting from the discussions during the workshop.

title: Cell density-dependent transcriptional activation of endocrine-related genes in human adipose tissue-derived stem cells.
abstract: Adipose tissue is recognized as an endocrine organ that plays an important role in human diseases such as type II diabetes and cancer. Human adipose tissue-derived stem cells (ASCs), a distinct cell population in adipose tissue, are capable of differentiating into multiple lineages including adipogenesis. When cultured in vitro under a confluent condition, ASCs reach a commitment stage for adipogenesis, which can be further induced into terminally differentiated adipocytes by a cocktail of adipogenic factors. Here we report that the confluent state of ASCs triggers transcriptional activation cascades for genes that are responsible for the endocrine function of adipose tissue. These include insulin-like growth factor 1 (IGF-1) and aromatase (Cyp19), a key enzyme in estrogen biosynthesis. Despite similar adipogenic potentials, ASCs from different individuals display huge variations in activation of these endocrine-related genes. Bioinformatics and experimental data suggest that transcription factor Foxo1 controls a large number of "early" confluency-response genes, which subsequently induce "late" response genes. Furthermore, siRNA-mediated knockdown of Foxo1 substantially compromises the ability of committed ASCs to stimulate tumor cell migration in vitro. Thus, our work suggests that cell density is an important determinant of the endocrine potential of ASCs.

title: Volatilization of Mercury by Resting Mercury-Resistant Bacterial Cells

abstract: The mercuric ion reduction system encoded by the Hg2+ inducible mer operon confers bacterial resistance to mercuric ion. The mer A gene product which is a FAD-containing enzyme catalyzes the reduction of Hg2+ to volatile elemental mercury with the help of intracellular thiols and NADPH as a cofactor (Schottel 1974; Summers and Silver 1978; Fox and Walsh 1982; Misra 1992). Our earlier studies have shown that growing cells of different mercury-resistant bacteria reduce Hg2+ compounds to Hg(O) (Ray et al. 1989; Pahan et al. 1990a; Gachhui et al. 1989). We have also shown the effect of thiol compounds and flavins on mercury-degrading enzyme activities in mercury-resistant bacteria (Pahan et al. 1990b). Here we report that resting cells of mercury-resistant bacteria survive in a buffer system for several hours, synthesize inducible mercury-degrading enzymes and volatilize mercury from a mercury-containing buffer system. We know of no information regarding studies of mercury-degrading enzymes in resting mercury-resistant bacterial cells.

title: Cell density is a critical determinant of aromatase expression in adipose stromal cells
abstract: Obesity is associated with an increased risk of breast cancer among postmenopausal women. This is at least partly due to excessive estrogen production in adipose tissue of obese women. Aromatase, the key enzyme in estrogen biosynthesis, is an important target in endocrine therapy for estrogen receptor (ER)-positive postmenopausal breast cancer. In this study we show that high confluency of human adipose stromal cells (ASCs) cultured in vitro can significantly stimulate aromatase gene expression and reduce the expression of breast tumor suppressor BRCA1 and members of the NR4A orphan nuclear family. Furthermore, small interfering RNA (siRNA)-mediated knockdown of Nurr1, a member of the NR4A family, substantially increased aromatase expression. Lastly, we found that the cell density-triggered inducibility of aromatase expression varies in ASCs isolated from different disease-free individuals. Our finding highlights the impact of increased cell number on estrogen biosynthesis as in the case of excessive adiposity.

title: The role of sodium dodecyl sulfate (SDS) micelles in inducing skin barrier perturbation in the presence of glycerol.
abstract: The stratum corneum (SC) serves as the skin barrier between the body and the environment. When the skin is contacted with an aqueous solution of the surfactant sodium dodecyl sulfate (SDS), a well-known model skin irritant, SDS penetrates into the skin and disrupts this barrier. It is well established, both in vitro and in vivo, that the SDS skin penetration is dose-dependent, and that it increases with an increase in the total SDS concentration above the critical micelle concentration (CMC) of SDS. However, when we added the humectant glycerol at a concentration of 10 wt% to the aqueous SDS contacting solution, we observed, through in vitro quantitative skin radioactivity assays using (14)C-radiolabeled SDS, that the dose dependence in SDS skin penetration is almost completely eliminated. To rationalize this important observation, which may also be related to the well-known beneficial effects of glycerol on skin barrier perturbation in vivo, we hypothesize that the addition of 10 wt% glycerol may hinder the ability of the SDS micelles to penetrate into the skin barrier through aqueous pores that exist in the SC. To test this hypothesis, we conducted mannitol skin permeability as well as average skin electrical resistivity measurements in vitro upon exposure of the skin to an aqueous SDS contacting solution and to an aqueous SDS + 10 wt% glycerol contacting solution in the context of a hindered-transport aqueous porous pathway model of the SC. Our in vitro studies demonstrated that the addition of 10 wt% glycerol: (i) reduces the average aqueous pore radius resulting from exposure of the skin to the aqueous SDS contacting solution from 33 +/- 5 Angstrom to 20 +/- 5 Angstrom, such that a SDS micelle of radius 18.5 +/- 1 Angstrom (as determined using dynamic light-scattering measurements) experiences significant steric hindrance and cannot penetrate into the SC, and (ii) reduces the number density of aqueous pores in the SC by more than 50%, thereby further reducing the ability of the SDS micelles to penetrate into the SC and perturb the skin barrier.