Analysis of the expression and function of Wnt-5a and Wnt-5b in developing and regenerating axolotl (Ambystoma mexicanum) limbs.
Urodele amphibians are unique adult vertebrates because they are able to regenerate body parts after amputation. Studies of urodele limb regeneration, the key model system for vertebrate regeneration, have led to an understanding of the origin of blastema cells and the importance of positional interactions between blastema cells in the control of growth and pattern formation. Progress is now being made in the identification of the signaling pathways that regulate dedifferentiation, blastema morphogenesis, growth and pattern formation. Members of the Wnt family of secreted proteins are expressed in developing and regenerating limbs, and have the potential to control growth, pattern formation and differentiation. We have studied the expression of two non-canonical Wnt genes, Wnt-5a and Wnt-5b. We report that they are expressed in equivalent patterns during limb development and limb regeneration in the axolotl (Ambystoma mexicanum), and during limb development in other tetrapods, implying conservation of function. Our analysis of the effects of ectopic Wnt-5a expression is consistent with the hypothesis that canonical Wnt signaling functions during the early stages of regeneration to control the dedifferentiation of stump cells giving rise to the regeneration-competent cells of the blastema.

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: Both avian and mammalian embryonic myoblasts are intrinsically heterogeneous
abstract: 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: 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: Mechanism of intestinal entry of infectious prion protein in the pathogenesis of variant Creutzfeldt-Jakob disease.
abstract: The pathogenesis of variant Creutzfeldt-Jakob disease (vCJD) is most likely to be dependent on intestinal entry of orally ingested infectious prion proteins, though tonsils or other oral portals of entry are possible. The exact route of entry of infectious prion proteins is uncertain but receptors for prion proteins such as laminin receptor precursor (LRP) may be expressed on intestinal brush border. Cellular prion protein (PrP(c)) is expressed on intestinal enteric nervous system and is separated by a single layer of epithelial cells from ingested infectious prion proteins. Intestinal M cells in the Peyer's patches may also transcytose prion proteins which may be transported to the lymphatic system by migrating dendritic cells underlying the M cells. The relative importance of the several potential portals of intestinal entry of infectious prion proteins is uncertain but may determine susceptibility of the population and also potential preventive strategies.

title: Diagnostic accuracy in retinoblastoma.
abstract: This study was done to ascertain the accuracy of diagnosis and possible risk of misdiagnosis in suspected retinoblastoma cases by different pre-operative diagnostic procedures like fundoscopy, ultrasonography, computerised tomography, and aqueous cytology study. Twenty-four eyes excised with diagnosis of suspected retinoblastoma were included in the study. Final diagnosis was reached by histopathological examination of excised eyes and compared with pre-operative diagnosis by different diagnostic procedures and accuracy of each procedure was calculated. Twenty-two cases were finally diagnosed as retinoblastoma and 2 cases (8.33%) as other than retinoblastoma. Computerised tomography and aqueous humour cytology were two most specific (100%) tests. Computerised tomography was the most sensitive (95.4%) test. Aqueous cytology was poorly sensitive (9.09%). Both ultrasonography and fundoscopy demonstrated sensitivity over 90% but with low specificity (50%). When test results of fundoscopy and ultrasonography were combined, specificity of this combination was 100% and sensitivity was 95.4%, which match exactly the values of computerised tomography. Combination of fundoscopy and ultrasonography has accuracy as high as that of computerised tomography. In patients with clear media this combination can be used as an effective alternative to computerised tomography to reach a diagnosis with comparable accuracy.

title: Mechanical phenotype is important for stromal aromatase expression
abstract: Evidence that aromatase expression in tumor-associated breast stroma is elevated, provides a rationale for use of aromatase inhibitors (AIs) in breast cancer treatment. However, regulation of local aromatase expression in cancer-free breast stroma is poorly understood. Recent clinical work indicates that stromal cells in dense breast tissue tend to express higher levels of aromatase than their counterpart from non-dense tissue. Consistent with the clinical observation, our cell culture-based study indicated that cell density, cell shape, and extracellular matrix (ECM) significantly induced stromal aromatase expression via a distinct signal transduction pathway. In addition, we identified a number of cell surface markers that are commonly associated with aromatase-expressing stromal cells. As mammographic density is one of the strongest and most prevalent risk factors for breast cancer, these findings provide a potential mechanistic link between alterations in tissue composition of dense breast tissue and increased stromal aromatase expression. Further exploration of the in vitro model system may advance understanding of an important problem in breast cancer biology.

title: A novel non-parametric regression reveals linkage on chromosome 4 for the number of externalizing symptoms in sib-pairs.
abstract: In this report, we present results of a genome-wide linkage scan using as a phenotype the number of externalizing symptoms associated with alcohol use disorders. Subjects were collected by the Collaborative Study on the Genetics of Alcoholism project from families in which at least three first degree relatives were affected by alcohol dependence. We use a novel non-parametric regression method based on kernel smoothing for our analysis. We report a statistically significant linkage close to the ADH gene cluster on Chromosome 4. We also obtain evidence for epistatic interaction between a region on Chromosome 1 and one on Chromosome 15. Although alcoholism as a covariate does not have any effect on the linkage scan, it has an effect on the epistatic interaction.

title: An IL-6 link between obesity and cancer.
abstract: Obesity is a growing epidemic all over the world that by virtue of inducation of a chronic, low-grade, and systemic inflammation leads to an increased risk of a number of diseases, including cancer. IL-6 an important cytokine in the increased risk to cancer in obese patients mainly because of its pro-inflammatory activity. Some data suggest that IL-6 might increase the risk of certain cancers such as those that originate from breast, liver, prostate, colon, and esophagus. A better understanding of the regulation and role of IL-6 in obesity-associated cancer is required to develop effective therapeutic approaches.