Whole-mount in situ hybridization to mouse embryos

The mouse is well-established as the major animal model for the study of mammalian development. Rapid progress in large-scale cDNA and also genomic sequencing projects is identifying new mouse genes at an unprecedented rate. As a first step toward understanding the function of these novel genes, it is important to determine their developmental expression pattern. Here we provide a reliable, sensitive method for whole-mount in situ hybridization using the mouse embryo.     

Characterization of novel antimicrobial peptides from the skins of frogs of the Rana esculenta complex

Rana esculenta is a hybridogenetic hybrid between Rana ridibunda and Rana lessonae and so is best considered as a complex of interbreeding species rather than a discrete single species. In this study, antimicrobial peptides were isolated from a pooled extract of the skins of specimens of the R. esculenta complex collected in the wild. In addition to several peptides belonging to the brevinin and esculentin families that have been previously isolated from skin secretions of a single specimen of R. esculenta, three newly described members of the brevinin-2 family (brevinin-2Ei, brevinin-2Ej, and brevinin-2Ek) and one member of the temporin family (temporin-1Ec) were purified and characterized. In addition, three structurally related peptides with no sequence similarity with antimicrobial peptides isolated from other species of ranid frogs, that potently and selectively inhibit the growth of the Gram-positive bacterium Escherichia coli (minimal inhibitory concentration (MIC<5 microM)), were identified. These peptides show limited amino acid sequence similarity to the homologous exon gene products that encode the N-terminal flanking peptides of preprocaerulein, preproxenopsin, and preprolevitide and so have been termed caerulein precursor-related fragments (CPRF-Ea, CPRF-Eb, and CPRF-Ec). The data suggest that there may be considerable polymorphism among specimens from different populations of the R. esculenta complex. It is proposed that the distribution and amino acid sequences of skin antimicrobial peptides may be useful markers for taxonomic classification of particular sub-populations and for an understanding of phylogenetic interrelationships.     

Developmental expression of the <Emphasis Type="Italic">Xenopus laevis Tbx20</Emphasis> orthologue

We have isolated the Xenopus orthologue of the T-box gene, Tbx20, and characterized its developmental expression profile. We show that Tbx20 is one of the earliest markers of heart tissue in Xenopus, and is expressed throughout all cardiac tissue during later stages of development. In addition, we also observe expression in the cement gland, the jugular vein, the lung bud, the cloacal aperture, rhombomeres 2, 4, 6 and 8, and in a subset of motor neurons.     

Isolation of the opioid peptide Leu-Val-Val-hemorphin-7 from bronchoalveolar lavage fluid of a patient with non-small cell lung cancer

The decapeptide Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe was isolated in high yield (1.5 nmol/ml) from bronchoalveolar lavage (BAL) fluid from a patient with an adenocarcinoma of the lung. This peptide, termed LVV-hemorphin-7 represents residues 32-41 of the beta-chain of hemoglobin and has been shown to be an endogenous ligand for opioid receptors. The N-terminal flanking peptide of LVV-hemorphin-7 [residues (1-31) of hemoglobin beta-chain] was also isolated in high yield. Neither peptide was detected in BAL fluid from the tumor-free lung of the same patient or from patients with non-neoplastic inflammatory lung disease. LVV-hemorphin-7 was not identified in BAL fluid from seven additional patients with non-small cell lung cancer, indicating that the formation of the peptide is unlikely to be of any diagnostic significance. However, the ability of LVV-hemorphin-7 to inhibit angiotensin-converting enzyme suggests that its formation may be of pathophysiological significance in the regulation of tumor blood flow in certain patients.     

Structure, antihyperglycemic activity and cellular actions of a novel diglycated human insulin

Human insulin was glycated under hyperglycemic reducing conditions and a novel diglycated form (M(r) 6135.1 Da) was purified by RP-HPLC. Endoproteinase Glu-C digestion combined with mass spectrometry and automated Edman degradation localized glycation to Gly(1) and Phe(1) of the insulin A- and B-chains, respectively. Intraperitoneal (i.p.) administration of diglycated insulin to mice alone or in combination with glucose (7 nmol/kg) resulted in a 43-61% and 11-34% reduction in glucose lowering activity, respectively, compared with native insulin. Consistent with these findings, diglycated insulin (10(-9) to 10(-7) mol/liter) was 22-38% less effective (P < 0.001) than native insulin in stimulating glucose uptake, glucose oxidation and glycogen production in isolated mouse abdominal muscle.     

Surface ectoderm is necessary for the morphogenesis of somites

The paraxial mesoderm of the neck and trunk of mouse embryos undergoes extensive morphogenesis in forming somites. Paraxial mesoderm is divided into segments, it elongates along its anterior posterior axis, and its cells organize into epithelia. Experiments were performed to determine if these processes are autonomous to the mesoderm that gives rise to the somites. Presomitic mesoderm at the tailbud stage was cultured in the presence and absence of its adjacent tissues. Somite segmentation occurred in the absence of neural tube, notochord, gut and surface ectoderm, and occurred in posterior fragments in the absence of anterior presomitic mesoderm. Mesodermal expression of Dll1 and Notch1, genes with roles in segmentation, was largely independent of other tissues, consistent with autonomous segmentation. However, surface ectoderm was found to be necessary for elongation of the mesoderm along the anterior-posterior axis and for somite epithelialization. To determine if there is specificity in the interaction between ectoderm and mesoderm, ectoderm from different sources was recombined with presomitic mesoderm. Surface ectoderm from only certain parts of the embryo supported somite epithelialization and elongation. Somite epithelialization induced by ectoderm was correlated with expression of the basic-helix-loop-helix gene Paraxis in the mesoderm. This is consistent with the genetically defined requirement for Paraxis in somite epithelialization. However, trunk ectoderm was able to induce somite epithelialization in the absence of strong Paraxis expression. We conclude that somitogenesis consists of autonomous segmentation patterned by Notch signaling and nonautonomous induction of elongation and epithelialization by surface ectoderm.     

Effects of endothelin-1 and homologous trout endothelin on cardiovascular function in rainbow trout

The cardiovascular effects of endothelin (ET)-1 and the recently sequenced homologous trout ET were examined in unanesthetized trout, and vascular capacitance curves were constructed to evaluate the responsiveness of the venous system to ET-1. A bolus dose of 667 pmol/kg ET-1 doubled ventral aortic pressure; produced a triphasic pressor-depressor-pressor response in dorsal aortic pressure (P(DA)); increased central venous pressure, gill resistance, and systemic resistance; and decreased cardiac output, heart rate, and stroke volume. These responses were dose dependent. Bolus injection of trout ET (333 or 1,000 pmol/kg) produced essentially identical, dose-dependent cardiovascular responses as ET-1. Dorsal aortic infusion of 1 and 3 pmol. kg(-1). min(-1) ET-1 and central venous infusion into the ductus Cuvier of 0.3 and 1 pmol. kg(-1). min(-1) produced similar dose-dependent cardiovascular responses, although the increase in P(DA) became monophasic. The heightened sensitivity to central venous infusion was presumably due to the more immediate exposure of the branchial vasculature to the peptide. Infusion of 1 pmol. kg(-1). min(-1) ET-1 decreased vascular compliance but had no effect on unstressed blood volume. These results show that ETs affect a variety of cardiovascular functions in trout and that branchial vascular resistance and venous compliance are especially sensitive. The multiplicity of effectors stimulated by ET suggests that this peptide was extensively integrated into cardiovascular function early on in vertebrate phylogeny.