Regression of mouse mammary gland anlagen in recombinants of Tfm and wild-type tissues: testosterone acts via the mesenchyme

In the male mouse, regression of the mammary gland anlagen is induced by testosterone during embryonic life. In the androgen-insensitive Tfm mouse, the gland anlagen are resistant to the testosterone action. To analyze cellular interactions in this process, we isolated the mammary gland anlagen from Tfm- and wild-type embryos. The epithelial buds were separated from the mesenchyme by trypsin-pancreatin treatment. From the epithelial and mesenchymal components, reciprocal recombinations were prepared and cultivated on millipore filter in the presence of testosterone. In combination with androgen-insensitive Tfm- mesenchyme, the wild-type buds survived the action of testosterone. On the other hand, in combination with wild-type mesenchyme, the androgen-insensitive Tfm epithelial buds were destroyed. The results show that testosterone induces detachment and degeneration of the buds via the mesenchyme.     

Production of specific antibodies to contractile proteins,and their use in immunofluorescence microscopy

Summary The injection of rabbits with insoluble or soluble G-actin from chicken smooth or striated muscle will produce antibodies that are equally reactive, and species and tissue non-specific in immunoprecipitation, immunofluorescence and actin-activated Mg²⁺-ATPase^** inhibition test. These antibodies have been used for the identification of actin-containing fibrils in a variety of tissues. When G-actins from chicken smooth or striated muscle are immobilized by chemical linkage to Affi-Gel 702 microbeads, their immunogenicity is increased, but the antibodies obtained against them are species-specific and will only react with actin and actin-containing structures from chicken and are therefore limited in use. It is concluded from this work that insoluble G-actin is the preferable immunogen to obtain precipitating antibodies for wide use.Abbreviations ATPase Adenosinetriphosphatase - FITC Fluoresceinisothiocyanate - SDS Sodiumdodecylsulfate This paper is dedicated to Dr. Dorothy M. Needham, University of Cambridge, England, in honour of her eightieth birthday     

Inhibitory effects of mersalyl and of antibodies directed against smooth-muscle myosin on a calcium adenosine triphosphatase of the plasma membrane from mouse liver cells.

The effect of mersalyl and of antibodies, directed against smooth-muscle myosin and skeletal muscle myosin, on the (Ca²⁺ + Mg²⁺)-activated adenosine triphosphatase (Ca,Mg)ATPase) system of mouse liver plasma membranes has been studied. Antismooth-muscle myosin inhibited by 38.6% at optimum substrate concentration the (Ca,Mg)ATPase with a K_m of 0.88 × 10^?3m. Mersalyl (0.5 mm) also inhibited this enzyme, the percentage inhibition being 44.6% at optimal substrate concentration. These results suggest the presence of a smooth-muscle myosin-like protein in the plasma membrane of mouse liver cells which has an associated (Ca,Mg)ATPase activity.     

Comparison of vascular smooth muscle cells from adult human,monkey and rabbit in primary culture and in subculture

Summary A method is presented for growing large numbers of pure isolated smooth muscle cells from adult human, monkey, and rabbit blood vessels in primary culture.In the first few days in culture these cells closely resembled those in vivo and could be induced to contract with angiotensin II, noradrenaline and mechanical stimulation. They stained intensely with antibodies against smooth muscle actin and myosin. Fibroblasts and endothelial cells did not stain with these antibodies thereby allowing the purity of each batch of cultures to be monitored. This was consistently found to be better than 99%. The smooth muscle cells modified or dedifferentiated after about 9 days in culture to morphologically resemble fibroblasts. At this stage cells could no longer be induced to contract and did not stain with the myosin antibodies. Intense proliferation of these cells soon resulted in a confluent monolayer being formed at which stage some differentiated characteristics returned. The modification or dedifferentiation process could be inhibited by the presence of a feeder layer of fibroblasts or endothelial cells, or the addition of cAMP to the culture medium.Smooth muscle cells which had migrated from explants in primary culture, and cells in subculture, had morphological and functional properties of dedifferentiated cells at all times.The advantages of differentiated rather than dedifferentiated smooth muscle cells in culture for the study of mitogenic agents in atherosclerosis is discussed.The authors wish to thank Professor H.H. Bentall of the Royal Postgraduate Medical School, Hammersmith Hospital, London, for making available human material, and Dr. S. Zeki of Department of Anatomy, University College London for material from monkeys. We are also extremely grateful to Professor G. Burnstock for the use of his laboratory facilitiesHolder of a John Halliday Travelling Fellowship from the Life Insurance Medical Research Fund of Australia and New ZealandResearch Fellow with the National Heart Foundation of AustraliaSupported by the Deutsche Forschungsgemeinschaft     

Proteomics of corynebacteria: From biotechnology workhorses to pathogens

Corynebacteria belong to the high G+C Gram-positive bacteria (Actinobacteria) and are closely related to Mycobacterium and Nocardia species. The best investigated member of this group of almost seventy species is Corynebacterium glutamicum, a soil bacterium isolated in 1957, which is used for the industrial production of more than two million tons of amino acids per year. This review focuses on the technical advances made in proteomics approaches during the last years and summarizes applications of these techniques with respect to C. glutamicum metabolic pathways and stress response. Additionally, selected proteome applications for other biotechnologically important or pathogenic corynebacteria are described.     

A novel Phytochrome B allele in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> exhibits partial mutant phenotype: a short deletion in N-terminal extension reduces Phytochrome B activity

During analysis of an Arabidopsis thaliana line possessing a Phytochrome A epiallele (phyA’), a partial Phytochrome B-deficient phenotype was observed, consisting of elongated hypocotyls in seedlings grown under constant white light or red light (660 nm). The observed hypocotyls were twice the length (8 mm) of wild-type (4 mm), but approximately half the length of a null phyB-9 mutant (14 mm). Several analyses were performed to characterize this apparent partial phyB mutant. Sequencing of the entire exonic region revealed three point mutations that altered codon usage, and one in-frame 12 base pair deletion. Each of the point mutations has been described in other lines that display wild-type phenotype, and therefore their effect is thought to be minimal, if any. The N-terminal deletion of amino acids 9 through 12 (GGGR) is a unique mutation found in this line. This deletion most likely contributes to the phyB mutant phenotype by lowering the binding affinity of the active form of Phytochrome B (Pfr) with Phytochrome Interacting Factor 3 (PIF3).     

Evolutionary origin of the medaka Y chromosome

Genetic sex determination in an XX-XY chromosome system can be realized through a locus on the Y chromosome that makes the undifferentiated gonad develop into a testis. Although this mechanism is widespread, only in two cases so far have the corresponding master male sex-determining genes been identified. One is Sry, which initiates testes determination in most mammals. The other is dmrt1bY (syn. dmy), from the fish medaka, Oryzias latipes. The mammalian Y is roughly estimated to be over 200 million years old. The medaka Y may be considerably younger. A comparative analysis of the genus Oryzias revealed that one sister species of the medaka has dmrt1bY on a homologous Y chromosome, whereas in another closely related species only a non-sex-linked pseudogene is present. In all other species, dmrt1bY was not detected. The divergence time for the different species was determined with mitochondrial DNA sequences. The timing was confirmed by independent calculations based on dmrt1 sequences. We show that the medaka sex-determining gene originated approximately 10 million years ago. This makes dmrt1bY and the corresponding Y chromosome the youngest male sex-determining system, at least in vertebrates, known so far.