Identification of a small Na/H exchanger-like message in the rabbit myocardium

We examined the Na⁺/H⁺ exchanger message in isolated perfused rabbit hearts using Northern blot analysis with cDNA encoding for the rabbit cardiac Na⁺/H⁺ exchanger. A cDNA probe from the coding region of the rabbit myocardial Na⁺/H⁺ exchanger hybridized to mRNA of 5 kb under high stringency, and to a second 3.8 kb mRNA species under low stringency. When Northern blots were re-probed with a section of the 3′-untranslated region of the cDNA, the 5 kb message was apparent while the smaller 3.8 kb message was not. If isolated working rabbit hearts were subjected to ischemia we observed increases in the 3.8 kb message. Overall, the results show that a 3.8 kb mRNA product, which is homologous to the amiloride sensitive Na⁺/H⁺ exchanger, exists in the myocardium and increases during ischemia in the myocardium.     

Improvements in immunostaining samples embedded in methacrylate: localization of microtubules and other antigens throughout developing organs in plants of diverse taxa

Microtubules are important in plant growth and development. Localizing microtubules in sectioned material is advantageous because it allows any tissue of interest to be studied and it permits the positional relations of the cells within the organ to be known. We describe here a method that uses semi-thin (0.5–2 m) sections of material embedded in butyl-methylmethacrylate, to which 10 mM dithiothreitol was added. After removing the embedding material and using indirect immunofluorescence staining, we obtain clear images of microtubules, actin microfilaments, callose and pulse-fed bromodeoxyuridine. This method works on the root tissues of Arabidopsis thaliana(L.) Heynh, Pinus radiataD. Don, Zamia furfuraceaAit., Azolla pinnataR. Br. and on sporophytic tissues of Funaria hygrometricaHedw. In general, most of the cells in the organs studied are successfully stained. Using this method, we find that interphase meristematic cells in all of these species have microtubules not only in the usual cortical array but also throughout their cytoplasm. The presence of the calcium chelator ethylene glycol-bis(-aminoethyl ether)N,N,N,N-tetraacetic acid EGTA in fixation buffers led to some tissue damage, and did not enhance the preservation of microtubules. The common assumption that EGTA-containing buffers stabilize plant microtubules during fixation appears unwarranted.Abbreviations BrdU 5-bromodeoxyuridine - DTT dithiothreitol - EGTA ethylene glycol-bis(-aminoethyl ether) - N,N,N,N tetraacetic acid We thank Ann Cork for technical assistance, Professor B.E.S. Gunning (Australian National University) and Drs. A.R. Hardham (A.N.U.) and R.E. Williamson (A.N.U.) for intellectual and material support, Dr D. McCurdy (A.N.U.) for the purified anti-actin antibody, and Professor B. Stone (La Trobe University, Melbourne, Australia) for generously providing the anti-callose antibody. We also thank the Electron Microscopy Unit of A.N.U. for the use of facilities. L.C.F. gratefully acknowledges financial support from the National Sciences and Engineering Research Council of Canada.     

Growth of Octopine-Catabolizing Pseudomonas spp. under Octopine Limitation in Chemostats and Their Potential To Compete with Agrobacterium tumefaciens

The growth characteristics of five octopine-catabolizing pseudomonads have been determined in batch and continuous cultures. All five strains belonged to rRNA homology group I and showed a more psychrotrophic growth pattern than did Agrobacterium tumefaciens B6 and ATCC 15955. In chemostats limited by octopine, either as the source of carbon and nitrogen or the sole source of nitrogen, maximum specific growth rates and substrate affinities were lower than those in chemostats limited by glutamate. These growth dynamics were similar to those observed for Agrobacterium strains B6 and ATCC 15955 even though the catabolic genes and pathways are believed to be different in the two genera. An analysis of the yields in octopine-limited chemostats indicated that the use of octopine as the sole source of carbon and nitrogen was grossly inefficient. Octopine and presumably lysopine and octopinic acid provided a better source of nitrogen than of carbon. One of the Pseudomonas fluorescens strains, E175D, was able to produce its highest yield on octopine as a nitrogen source. Competition models formulated on pure culture parameters indicated that two of the Pseudomonas spp. would dominate A. tumefaciens B6 and ATCC 15955 when in simple competition for octopine as a limiting substrate.     

Ecophysiology of Bacteriophage S5100 Infecting Halobacterium cutirubrum

Increasing salinity reduces burst size and increases the latent period of infection of Halobacterium cutirubrum by lytic bacteriophage S5100. Cells become reversibly and persistently infected at saturation-level concentrations of NaCl. We propose that high salinity provides a natural refuge for sensitive host bacteria and that phage S5100 acts as a scavenger, proliferating when host viability is threatened by dilution of the environment.     

Ultracytochemistry of cholera-toxin binding sites in ciliary processes

Summary Cholera toxin reduces the rate of formation of aqueous humor in concentrations (10^–11 M) that do not disturb the morphology of the aqueoushumor forming epithelial cells of the ciliary processes of the rabbit eye. The search for an endogenous mediator of aqueous-humor formation comparable to cholera toxin in its mode of operation prompted us to map the distribution of cell surface receptors for cholera toxin in the ciliary processes of the eyes of rabbits. Cytochemical studies were carried out with the use of conjugates of cholera toxin to fluorescein isothiocyanate (CT-FITC) and to horseradish peroxidase (CT-HRP), and of the B subunit of cholera toxin to horseradish peroxidase (B-HRP). Multiple fluorescent CT-FITC binding sites were observed on the outer nonpigmented epithelial layer near the crests of the processes. Processes incubated with CT-HRP in vitro showed surface staining of 30–40% of the nonpigmented epithelial cells. A prominent reaction product was observed along the basal and lateral plasma membranes of these cells. In vivo studies carried out after arterial infusion of B-HRP showed a reproducible dense reaction product between the apical surfaces of the pigmented epithelium (PE) and of the nonpigmented epithelium (NPE) facing each other. Aggregations of reaction product were observed with the electron microscope in the extracellular space between the apices of PE and NPE. The apical plasma membrane of the endothelium of the blood vessels near the crests of the ciliary processes was stained after either in vivo or in vitro exposure to peroxidase conjugates. These findings indicate that the cell-surface receptors which mediate the action of cholera toxin on aqueous humor formation are very likely localized in the apical plasma membranes of the epithelium of the ciliary processes.Supported in part by USPHS grant # EY-00237, the Connecticut Lions Eye Research Foundation, Inc., and Research to Prevent Blindness, Inc.     

Enzyme-Linked Immunosorbent Assay of Substance P: A Study in the Eye

A solid phase enzyme-linked immunosorbent assay for quantitation of substance P is presented. The assay measures the capacity of soluble substance P to compete with the solid phase antigen for a limited quantity of specific substance P antibody. The solid-phase antigen consists of a synthetic substance P.poly-D-glutamic acid conjugate coated to polystyrene micro-ELISA plate wells. Soluble substance P and antibodies to substance P are first preincubated together and then added to the wells containing solid-phase antigen. Subsequently the wells are incubated with anti-antibodies conjugated to alkaline phosphatase. The wells are finally incubated with p-nitrophenyl phosphate an the absorbance is read in a spectrophotometer 16--24 hr after the start of the assay. The threshold for detection of substance P was 5--10 pg per well (0.25 ml). Substance P was extracted from rabbit eyes and the values obtained with the present method are compared with previously reported values based on radioimmunoassay.     

MORPHOLOGY AND DISTRIBUTION OF NUCLEI DURING DEVELOPMENT IN CYMOPOLIA BARBATA (CHLOROPHYCOPHYTA,DASYCLADALES)1

The morphology and distribution of a variety of types of nucleus in the apex, in young and mature gametangia, and in older regions of the Cymopolia cell were studied by light and electron microscopy. Spherical and convoluted nuclei 2–7μm in diameter were observed in apical regions of the vegetative siphon. Nuclei 4 μm in diameter were present in the young primary laterals and in developing gametangia. A single characteristic nucleus migrates from the siphon into the primary lateral of the second basipetal whorl. It is further transported into one of several possible secondary laterals and determines the development of gametangia which become multinucleate in the fourth or fifth whorls. Nuclei are characterized by size, shape, nucleolar morphology, nucleoplasmic inclusions and the ultrastructure of the perinuclear cytoplasm. Although no “primary nucleus” characteristic of the uninucleate genera, Acetabularia and Batophora was observed, some nuclei of Cymopolia have features in common with secondary nuclei of these genera.     

Analysis in vivo of translational mutants of the rIIB cistron of bacteriophage T4

We have mapped the mutants isolated by Nelson et al. (1981) that reduce the amount of rIIB protein synthesized during bacteriophage T4 infection of Escherichia coli B and characterized their rIIB expression in vivo. These mutants fall into four distinct groups in terms of mapping and phenotype. We have located the probable site of each mutation on the DNA sequence. We have also analyzed a number of other mutations near the initiating AUG of rIIB with respect to their rIIB expression. In some of these mutants, ribosomal recognition of the wild-type initiating AUG is precluded and so initiation occurs at a different AUG, which, in some instances, we have identified.     

TRACHELOMONAS HISPIDA VAR. CORONATA (EUGLENOPHYCEAE). I. ULTRASTRUCTURE OF CYTOSKELETAL AND FLAGELLAR SYSTEMS1,2

Trachelomonas hispida var. coronata Lemm. has a fibrous, mucilaginous, ovoid, mineralized envelope (lorica), the ornamentation and coloration of which are capricious in culture. Cells exhibit a radial distribution of most organelles: (i) A cortical endoplasmic reticulum, (ii) parietal chloroplasts, and (iii) a median vacuolar region surrounded by several Golgi bodies and diverse vesicles. Associated with the emergent flagellum is a “paraflagellar complex” that consists of dense globules, cross-striated ribbon-like structures, a paraflagellar body, and an array of parallel striated filaments. The stigma consists of a single layer of pigmented granules that partially surrounds the canal/reservoir transition zone where microtubular bands intersect. A microtubular cytoskeleton consists of pellicular microtubules, peri-canal microtubules, stigma-associated microtubules and para-reservoir microtubules. The thickenings on the posterior, concave margins of the pellicular strips suggest that this pellicle is of intermediate complexity between those of Euglena spirogyra (Ehrenb. and Trachelomonas volvocina (Ehrenb.).