A Simple, Two-Color Fluorescence Detection Method for Membrane Blotting Analysis Using Alkaline Phosphatase and Horseradish Peroxidase

We have developed a one-step, two-color fluorescence detectionmethod using simultaneously two fluorogenic substrates for bothSouthern and Western blots on nylon membranes. For this enzyme-mediatedreporter system, a mixture of (i) 3-hydroxy-N-2'-biphenyl-2-naphthalenecarboxamidephosphate ester (HNPP), a substrate for alkaline phosphataseand (ii) N-(4-amino-5-methoxy-2-methylphenyl)benzamide (AMMB),a fluorogenic substrate for horseradish peroxidase was used.The reaction with these substrates produces blue (HNPP) andyellow (AMMB) fluorescent signals under ultraviolet light (302nm). Therefore, this simple method allows the simultaneous visualizationof two different targets on a single nylon membrane, e.g. nucleicacids or proteins.     

Exogenous delta-crystallin gene expression as probe for differentiation of teratocarcinoma stem cells

We developed an experimental system in which differentiation of teratocarcinoma stem cell is probed by expression of stably introduced exogenous genes. We used chicken delta-crystallin gene (delta gene) and its derivative (Mo delta gene) driven by long terminal repeat (LTR) promoter of Moloney murine leukemia virus (Mo-MuLV). Neither of the genes was expressed in the undifferentiated condition. Differentiation to primitive endoderm induced by retinoic acid (RA) led to expression of delta but not Mo delta, while differentiation to more advanced endodermal cells by RA plus dibutyryl cAMP elicited Mo delta expression in addition to delta. These results are interpreted as a consequence of differential activation/suppression of gene expression through enhancer elements associated with the genes.     

Sequence analysis of the gene encoding a novel l-2,4-diaminobutyrate decarboxylase of Acinetobacter baumannii: similarity to the group II amino acid decarboxylases

The gene (ddc) encoding a novel enzyme, l-2,4-diaminobutyrate decarboxylase (DABA-DC; EC 4.1.1.-) in Acinetobacter baumannii was sequenced, and an open reading frame of 1,530 nucleotides was detected. The sequence of 20 N-terminal amino acids of purified DABA-DC and of its proteolytic peptide fragments coincided with those deduced from the nucleotide sequence determined. Comparison of the predicted amino acid sequence of the A. baumannii enzyme with those of other pyridoxal 5′-phosphate-dependent decarboxylases revealed significant similarity to the group II amino acid decarboxylases and conservation of the putative pyridoxal 5′-phosphate-binding domain. Received:20 February 1996 / Accepted 15 April 1996     

Genetic control of the immune response in lines of chickens selected for graft-versus-host competences

The immune response of chickens to goat erythrocytes has been examined. The H line selected for high competence of graft-versus-host reaction (GVHR) showed higher immune responses than the L line selected for low GVHR competence. It appeared also that immune responses were controlled by the B blood group locus, which is the major histocompatibility locus in chickens. The relative immune responsiveness of B genotypes were B ¹¹/¹¹> B ⁹/ B ¹¹> B ⁹/B⁹.
Treatment of antiserum with 2-mercaptoethanol (2-ME) proved that the difference in immune responses between lines was due mainly to the 2-ME resistant antibody and that the difference between the B genotypes was due to the 2-ME sensitive antibody.     

Lesion studies of neural mechanisms underlying the mating dance of the silkmoth,Bombyx mori

Lesion studies were carried out to reveal neural mechanisms controlling a characteristic mating behavior, referred to as ‘mating dance’ in a maleBombyx mori which appears in reaction to the sex pheromone of the conspecific female. The experiments revealed 2 essential neural mechanisms involved in the mating dance. One is a flight motor system in the thorax, which organizes the wing vibration representing the mating dance. The other, a dance command element, is in the head ganglia. Once activated by the female sex pheromone, the latter can maintain its excitation for several minutes without additional stimulation. This long-lasting neuronal excitation was suggested to descend via cervical connectives to the thoracic ganglia and activate the flight motor system into operation.     

Projection of statocyst sensory neurons associated with crescent hairs in the crayfish Procambarus clarkii Girard

Summary Both smooth muscle and striated muscle are present in the iris of the chick embryo. The two types of musculature form mixed clusters which include undifferentiated cells and many nerve fibres, but they are structurally quite distinct and have different origins. The smooth musculature originates around the 10th day from a laminar invagination (iridial lamella) of the posterior epithelium, and is therefore an ectodermal derivative. The striated musculature appears slightly later than the smooth musculature and originates from undifferentiated cells which are regarded as mesenchymal. After the 15th day in ovo the smooth musculature stops growing; its cells become confined to an area very near the pupillary margin and many develop pigment granules in the sarcoplasm. Many smooth muscle cells seem to undergo regressive changes; however, cells with the typical appearance of visceral muscle cells are still present in the iris of 3-month-old chickens. High density of innervation and vasculari/ation, wide range of striated muscle fibre diameters, presence of lipid vacuoles and of large clusters of mitochondria in the striated fibres, occurrence of peripheral couplings of the sarcoplasmic reticulum, and presence of numerous fibroblast processes in the interstices between fibres, characterize the sphincter pupillae of the mature iris.This work was supported by grants from the Medical Research Council and the Central Research Fund of the University of London     

A retinoic acid responsive gene, MK, produces a secreted protein with heparin binding activity

MK is a gene whose expression increases transiently during retinoic acid-induced differentiation of embryonal carcinoma cells. MK polypeptide was secreted by differentiating HM-1 embryonal carcinoma cells and by L-cells transfected with an MK cDNA under the control of the beta-actin promoter and Rous sarcoma virus enhancer. MK polypeptide was found to have heparin binding activity. Conditioned medium of the transfected L-cells promoted growth of PC-12 pheochromocytoma cells. These findings support the view that MK polypeptide is a secreted factor involved in regulation of growth and differentiation.