Transfer and Expression of the Catabolic Plasmid pBRC60 in Wild Bacterial Recipients in a Freshwater Ecosystem

3-Chlorobenzoate (3Cba)-degrading bacteria were isolated from the waters and sediments of flowthrough mesocosms dosed with various concentrations of 3Cba and inoculated with a 3Cba-degrading Alcaligenes sp., strain BR60. Bacteria capable of 3Cba degradation which were distinct from BR60 were isolated. They carried pBRC60, a plasmid introduced with Alcaligenes sp. strain BR60 that carries a transposable element (Tn5271) encoding 3Cba degradation. The isolates expressed these genes in different ways. The majority of pBRC60 recipients were motile, yellow-pigmented, gram-negative rods related to the group III pseudomonads and to BR60 by substrate utilization pattern. They were capable of complete 3Cba degradation at both millimolar and micromolar concentrations. Two isolates, Pseudomonas fluorescens PR24B(pBRC60) and Pseudomonas sp. strain PR120(pBRC60), are more distantly related to BR60 and both produced chlorocatechol when exposed to 3Cba at millimolar concentrations in the presence of yeast extract. These species showed poor growth in liquid 3Cba minimal medium but could degrade 3Cba in continuous cultures dosed with micromolar levels of the chemical. Laboratory matings confirm that pBRC60 can transfer from BR60 to species in both the beta and gamma subgroups of the proteobacteria and that 3Cba gene expression is variable between species. Selection pressures acting on pBRC60 recipients are discussed.     

The alpha subunit of type II Ca2+/calmodulin-dependent protein kinase is highly conserved in Drosophila

A monoclonal antibody against rat brain type II Ca2+/calmodulin-dependent protein kinase (CaM kinase) precipitates three proteins from Drosophila heads with apparent molecular weights similar to those of the subunits of the rat brain kinase. Fly heads also contain a CaM kinase activity that becomes partially independent of Ca2+ after autophosphorylation, as does the rat brain kinase. We have isolated a Drosophila cDNA encoding an amino acid sequence that is 77% identical to the sequence of the rat alpha subunit. All known autophosphorylation sites are conserved, including the site that controls Ca(2+)-independent activity. The gene encoding the cDNA is located between 102E and F on the fourth chromosome. The protein product of this gene is expressed at much higher levels in the fly head than in the body. Thus, both the amino acid sequence and the tissue specificity of the mammalian kinase are highly conserved in Drosophila.     

The functional morphology of the respiratory system of the Holothyrida (= Tetrastigmata) Acari: Anactinotrichida

The respiratory system of Holothyrus coccinella Gervais (Holothyridae) and Allothyrus australasiae (Womersley) (Allothyridae) were examined. The stigma-peritreme complex is connected to tracheae and ventilated by indirect muscles. The peritreme provides an alternative route for the entry of air into the tracheal system, should a stigma be occluded by debris and retards water vapour transpiration, the mechanisms of which are compared in the two species.     

Production of antiserum to CRF associated with adrenal atrophy in a rabbit

Corticotropin releasing factor (CRF) was recently isolated from ovine hypothalami by its ability to stimulate adrenocorticotropin (ACTH) and β-endorphin release from dispersed rat pituitary cells. Intramuscular injection of synthetic ovine CRF conugated to bovine thyroglobulin with 1-ethyl-3(3-dimethylaminopropyl) carbodiimide and emulsified with Freund's complete adjuvant into a random bred New Zealand white rabbit resulted in antiserum production to CRF associated with adrenal atrophy. A decrease in the level of plasma coticosteroids was associated with an increase in mean total binding of ¹²⁵I-N-Tyr-CRF. Upon sacrifice, a decrease in pituitary content of ACTH and a decrease in adrenal weight and content of corticosteroids was observed in the rabbit producing antiserum to CRF. Adrenal atrophy was histologically verified with an observed decrease in the adrenal cortical zone not reflected in the zona glomerulosa. Individual cells were relatively larger either with more abundant pale cytoplasm or with distinctly vacuolated cytoplasm. The results presented here are consistent with a physiologically necessary role for this CRF or peptides with similar structures in the hypothalamic-pituitary-adrenal axis.     

Liver tissue graft rejection in murine major histocompatibility complex mutants

Liver tissue grafts between seven H-2 mutants and their parental strains have been studied. Each of these mutants was originally identified by reciprocal mutant—parental strain skin graft rejection. However, liver grafts among mutants and parental standard strains are not uniformly rejected. Liver graft rejection also fails to correlate with mutant—parental stimulation in CML and MLC. In addition, the immune reaction pattern of female mutant animals against grafts of male liver differs from the reaction pattern found in parental standard strains. Several explanations for the differences between immune response to liver and skin grafts are proposed, including different T cell subsets involved in recognition, availability of antigenic sites to immunocompetent cells, and structural differences between mutant and parental H-2 antigens. Abbreviations used in this paper: bml, 2, 3, 4,14; dml; fm2=mutants of strains C57BL/6, B10.D2 and B10.M respectively; B6=C57BL/6     

Studies on the mechanism of action of hexamethylene bisacetamide, a potent inducer of erythroleukemic differentiation

Hexamethylene bisacetamide (diacetyldiamino hexane) is a potent inducer of erythroid differentiation in murine erythroleukemia cells. Hexamethylene bisacetamide and the closely related pentamethylene bisacetamide were synthesized with radioactive labels in various portions of the molecule and the uptake, metabolism, and intracellular distribution determined. Bisacetamides are taken up by the cell; an intracellular concentration equal to the extracellular concentration is achieved by 6–8 h. Commitment to differentiation is not detected until at least 10 h after equilibration. Both uptake and commitment to differentiate are concentration and temperature dependent. The majority of the compound is deacetylated upon cell entry and the acetate portion incorporated nonspecifically into lipid and protein. Acetate competes with the incorporation of hexamethylene bisacetamide into protein and lipid, but does not affect inducing activity. The diamine portion of the molecule is detected only in the cytoplasm, in a trichloroacetic acid-soluble and acetylated form, whereas the acetate moiety is detected in both cytoplasm and nucleus and in both a trichloroacetic acid-soluble and insoluble form. The cellular uptake of diamines and bisacetamides (acetylated diamines) are similar, but acetylation of the diamine greatly increases inducing activity.     

Development of a nuclear polyhedrosis virus in midgut cells and penetration of the virus into the hemocoel of the armyworm,Pseudaletia unipuncta

The development of a nuclear polyhedrosis virus (NPV) in larval midgut cells of the armyworm, Pseudaletia unipuncta, is similar to that of other NPV. In the nucleus, the envelopes around the nucleocapsids seem to be derived de novo or from the inner layer of the nuclear envelope wich forms cisternae, blebs, or infoldings. The nucleocapsids are also enveloped by synhymenosis during passage through the nuclear membrane, the cell membrane, or the endoplasmic reticulum membrane. Both enveloped and unenveloped nucleocapsids may enter the cytoplasm through the nuclear pore or budding through the nuclear membrane. From the cytoplasm the virions may enter the hemocoel through the basal cell and basement membranes or through the endoplasmic reticulum, intercellular space, and the basement membrane.     

Fitness of Karyotypes in DROSOPHILA PSEUDOOBSCURA

In the dynamics of the survival of chromosomal polymorphism selection may be operating at the genic level, at the chromosomal level or at the supergene level. Tests designed to distinguish between these levels were run on Drosophila pseudoobscura. There was no evidence for heterosis, a necessary requirement for gene-determined chromosomal polymorphism. A strong chromosmal selection was observed. No evidence was found for the presence within one locality of more than a single superallele for each supergene (= gene order). These results are compared to those found by others.     

Regulation of Rhodopsin-eGFP Distribution in Transgenic Xenopus Rod Outer Segments by Light

The rod outer segment (OS), comprised of tightly stacked disk membranes packed with rhodopsin, is in a dynamic equilibrium governed by a diurnal rhythm with newly synthesized membrane inserted at the OS base balancing membrane loss from the distal tip via disk shedding. Using transgenic Xenopus and live cell confocal imaging, we found OS axial variation of fluorescence intensity in cells expressing a fluorescently tagged rhodopsin transgene. There was a light synchronized fluctuation in intensity, with higher intensity in disks formed at night and lower intensity for those formed during the day. This fluctuation was absent in constant light or dark conditions. There was also a slow modulation of the overall expression level that was not synchronized with the lighting cycle or between cells in the same retina. The axial variations of other membrane-associated fluorescent proteins, eGFP-containing two geranylgeranyl acceptor sites and eGFP fused to the transmembrane domain of syntaxin, were greatly reduced or not detectable, respectively. In acutely light-adapted rods, an arrestin-eGFP fusion protein also exhibited axial variation. Both the light-sensitive Rho-eGFP and arrestin-eGFP banding were in phase with the previously characterized birefringence banding (Kaplan, Invest. Ophthalmol. Vis. Sci. 21, 395–402 1981). In contrast, endogenous rhodopsin did not exhibit such axial variation. Thus, there is an axial inhomogeneity in membrane composition or structure, detectable by the rhodopsin transgene density distribution and regulated by the light cycle, implying a light-regulated step for disk assembly in the OS. The impact of these results on the use of chimeric proteins with rhodopsin fused to fluorescent proteins at the carboxyl terminus is discussed.