Characterization of phycoerythrin-containing Synechococcus spp. populations by immunofluorescence

Using an immunofluorescence assay developed to identify serogroups(i.e. clusters of strains labelled by one antiserum), the compositionof natural populations of phycoerythrin-containing Synechococcusspp. was examined. The 7803 (open ocean clone)-serogroup wasfound in most oceanic regions, but was most prevalent (up to85%) in tropical and subtropical waters during spring and summer.At coastal Long Island stations it was most abundant (up to65%) when water temperatures were >22°C. The seasonaland geographic distribution of the 7803-serogroup appeared tobe limited by water temperature. No consistent pattern was observedin the per cent composition with depth in the Sargasso Sea orat coastal to offshore stations in the North-west Atlantic Oceanor eastern tropical North Pacific Ocean. The 8016 (coastal clone)-serogroupwas abundant at coastal and estuarine stations off Long Island(up to 95 %) and its appearance was also correlated with warmwater temperature (> 15°C). However, this serogroup remaineda constant proportion of the population at the Long Island Soundstation during early winter months (through January) when abundanceof the 7803-serogroup was negligible. Owing to limited data,the oceanic distribution of the 8016-serogroup is not yet discernible.Lastly, antisera to the phycocyanin-dominant Synechococcus spp.clones failed to label any cells in samples collected from severaloceanic stations. Thus, these strains appear to be limited tocoastal and estuarine regions, which is consistent with predictionsfrom experiments comparing the photosynthetic performance ofthe phycoerythrin-dominant and phycocyanin-dominant clones. ¹Present address: Department of Oceanography, University ofHawaii, Honolulu, HI 96822, USA     

Thermal adaptations in lizard muscle function

Summary This study was undertaken to investigate thermal adaptations in muscle contractile properties in closely-related lizards with different preferred body temperatures (PBT). The species examined all belong to theSphenomorphus group of Australian skinks (Scincidae: Lygosominae). Preferred body temperatures are conservative at the generic level as follows:Ctenotus, 35°C;Sphenomorphus, 30°C;Eremiascincus, 25°C. Contractile properties of the fast glycolytic portion of the iliofibularis muscle were measured. Translational adaptations are evident in several isometric factors, including tetanic tension (Po), twitch tension (Pt), twitch time to peak tension (TPT), and twitch half-relaxation time (1/2 RT). Capacity adaptations are not evident in rates of tetanic tension development (dPo/dt) or in maximal velocities of isotonic shortening (V _max). Rotational adaptations are not evident in any contractile properties. Thermal limits on upper response temperatures are about 5°C warmer inCtenotus than in the more cryophilic species, indicative of resistance adaptation in muscle performance. Despite these adaptive shifts, there is little indication that muscle functional capacities are optimized or equalized at PBT in these lizards.Abbreviations FG fast glycolytic - IF iliofibularis muscle - PBT preferred body temperature - Po tetanic tension - Pt twitch tension - 1/2RT twitch half relaxation time - TPT twitch time to peak tension     

Biochemical Characterization of Molybdenum-Cofactor in a Cyanobacterium, Nostoc muscorum

Cell-free extracts of nitrate-grown Nostoc muscorum containnitrate reductase and molybdenum-cofactor activities. Whilenitrate reductase activity is associated with the paniculatefraction, cofactor activity is found predominantly in the solublefraction. This activity was distributed between two pools. Inone pool, the molybdenumcofactor is associated with a carrier(protein) of approximately 30,000 Da with an S_{20, w} between2.3 and 2.5. The carrier-bound cofactor is non-dialyzable andis found along with the major proteins during filtration inSephadex G-25 and G-100. The second pool contains free or unboundcofactor. It is separated from soluble proteins by dialysiswith a membrane with a pore-size of 10 to 15 kDa. However, itis retained with a membrane with a pore size of 1 kDa. It isin the included volume during chromatography through SephadexG-25. Its molecular mass is estimated to be between 1,000 and5,000 Da. The molybdenum content was proportional to cofactoractivity in both pools. Reducing agents increased cofactor activity.However, activity in both pools was sensitive to heat, acid,and oxidative treatments. The carrier protein appears to givesome protection. ¹Fulbright Scholar from Department of Biological Sciences, R.D. University, Jabalpur-482001, India. To whom reprint requestsshould be addressed. (Received June 22, 1987; Accepted August 21, 1987)     

Transport Properties of the Tomato Fruit Tonoplast : I. Identification and Characterization of an Anion-Sensitive H-ATPase

An anion-sensitive H⁺-translocating ATPase was identified in membrane vesicles isolated from mature green tomato (Lycopersicon esculentum) fruit. The H⁺-ATPase was associated with a low density membrane population having a peak density of 1.11 grams per cubic centimeter, and its activity was inhibited by NO₃⁻, N,N′-dicyclohexylcarbodiimide and diethylstilbestrol but not by vanadate, azide, molybdate, or oligomycin. This H⁺-ATPase has an unusual pH dependence indicating both a slightly acidic and a near neutral peak of activity. Chloride was found to be a potent stimulator of ATPase activity. The K_m for the H⁺-ATPase was approximately 0.8 millimolar ATP. The characteristics of this H⁺-ATPase are very similar to those described for a number of plant cell tonoplast H⁺-ATPases suggesting that the activity identified in tomato fruit membranes is tonoplast-associated. This report demonstrates the feasibility of isolating tonoplast vesicles from acidic fruit tissues for studies of transport activities associated with fruit development and maturation.     

The effect of the crossability loci Kr1 and Kr2 on fertilization frequency in hexaploid wheat x maize crosses

Summary Dominant alleles of the Kr1 and Kr2 genes reduce the crossability of hexaploid wheat with many alien species, including rye and Hordeum bulbosum, with Kr1 having the greater effect. However, a cytological study of wheat ovaries fixed 48 h after pollination showed that the wheat genotypes Highbury (kr1, Kr2) and Chinese Spring (Hope 5B) (kr1, kr2) were crossable with Seneca 60 maize, fertilization occurring in 14.4 and 30.7% of embryo sacs respectively. The latter figure was similar to the 29.7% fertilization found in Chinese Spring (kr1, kr2). Most embryo sacs in which fertilization occurred contained an embryo but lacked an endosperm and where an endosperm was formed it was usually highly aberrant. All three wheat x maize combinations were karyotypically unstable and rapidly eliminated maize chromosomes to produce haploid wheat embryos.     

Structure of the platelet membrane glycoprotein IIb. Homology to the alpha subunits of the vitronectin and fibronectin membrane receptors

The platelet membrane glycoprotein IIb X IIIa heterodimer complex (GPIIb X IIIa) is the platelet receptor for adhesive proteins, containing binding sites for fibrinogen, von Willebrand factor, and fibronectin on activated platelets. GPIIb X IIIa also appears to be a member of a family of membrane adhesive protein receptors that plays a major role in cell-cell and cell-matrix interactions. GPIb is the larger component of this platelet receptor and is composed of two disulfide-linked subunits. In this report we describe the analysis of cDNA clones for human GPIIb that were isolated from a lambda gt11 expression library prepared using RNA from HEL cells. A total of 3.3 kilobases of cDNA was sequence, revealing a continuous open reading frame encoding both GPIIb subunits. The cDNA encodes 1039 amino acids: 137 constituting the smaller subunit, 871 constituting the larger subunit, and 30 constituting an NH2-terminal signal peptide. No homology was found between the larger and smaller subunits. The smaller subunit contains a 26-residue hydrophobic sequence near its COOH terminus that represents a potential transmembrane domain. Four stretches of 12 amino acids present in the larger subunit are homologous to the calcium binding sites of calmodulin and troponin C. Northern blot analysis using HEL cell RNA indicated that the mature mRNA coding for GPIIb is 4.1 kilobases in size. A comparison of the GPIIb coding region with available cDNA sequences of the alpha-chains of the vitronectin and fibronectin receptors revealed 41% DNA homology and 74% and 63% amino acid homology, respectively. Our data establish the amino acid sequence for the human platelet glycoprotein IIb and provide additional evidence for the existence of a family of cellular adhesion protein receptors.     

Acquisition of antigens characteristic of adult pericentral hepatocytes by differentiating fetal hepatoblasts in vitro

Antigens specific to pericentral hepatocytes have been studied in adult mouse liver, during fetal development, and in cultured fetal hepatoblasts. Antibody reactive with glutamine synthetase stained all fetal liver cells but almost all cells lost this antigen after birth; only a single layer of pericentral cells retained it in adulthood. In contrast, monoclonal antibodies to major urinary protein (MUP) did not detect the antigen until approximately 3 wk after birth, after which time the cells within 6-10 cell diameters of the central veins were positive. Cultured fetal liver cells from embryos at 13 +/- 1 d of gestation were capable of differentiating in vitro to mimic events that would occur had the cells remained in the animal. About 10-20% of the explanted cells grew into clusters of hepatocyte-like cells, all of which stained with albumin antibodies. MUP monoclonals were reactive with one-half of the differentiated fetal hepatocytes. Glutamine synthetase was present in all hepatocytes after several days in culture and gradually decreased and remained in only occasional cells, all of which also contained the MUP antigen. These findings suggest that a sequence of gene controls characterizes expression of specific genes in developing liver, and that differentiating fetal hepatoblasts are capable of undergoing similar patterns of gene activity in culture.     

Expression of hemopoietic histocompatibility antigens on H-2-loss variants of F1 hybrid lymphoma cells: evidence consistent with trans gene regulation

H-2 heterozygous marrow stem cells, lymphoid progenitor cells, and leukemia/lymphoma cells do not express hemopoietic or hybrid histocompatibility (Hh) antigens, which are important transplantation antigens recognized during the rejection of normal or neoplastic hemopoietic cells. The Hh-1b determinant of the H-2b haplotype maps to the D region of H-2. We have tested the hypothesis that gene(s) at or near H-2D of the H-2d haplotype down-regulate the expression of Hh-1b in the trans configuration. We used Abelson leukemia virus-transformed pre-B lymphoma cells (ACCb) of BALB/c X BALB.B (H-2d X H-2b) origin, as well as variant lines of ACCb, which were selected for resistance to monoclonal anti-H-2 antibodies plus complement. B6D2F1 (H-2b X H-2d), C3B6F1 (H-2k X H-2b), or B6 (H-2b) mice were infused with inocula of 5 X 10(6) B6 bone marrow cells (BMC). Proliferation of donor-derived marrow cells was judged in terms of DNA synthesis by measuring the splenic incorporation of 5-iodo(125I)-2'-deoxyuridine (IUdR) 5 days after cell transfer. B6 BMC grew much better in B6 than in F1 hybrid host mice, an expression of "hybrid resistance". As observed previously, the injection of EL-4 (H-2b, Hh-1b) tumor cells prior to infusion of B6 (H-2b, Hh-1b) BMC enhanced the growth of B6 BMC in F1 hybrid mice. Therefore, this in vivo "cold target cell competition" type of assay can be used to detect the expression of Hh-1b antigens. Unlike EL-4 (H-2b) cells, hybrid resistance was not affected by prior infusion of (H-2b X H-2d) heterozygous ACCb cells. In contrast, three ACCb variant cell lines, H-2d-, Ld-Dd-, and Dd-, enhanced the growth of B6 BMC in F1 hosts. The ACCb H-2b- cell line did not affect hybrid resistance to B6 BMC. The loss of gene expression on the H-2d chromosome at or very near the H-2Dd locus is correlated with the appearance Hh-1b, as determined by the in vivo cold target competition assay. These results support the hypothesis that heterozygous cells possess trans-acting, dominant, down-regulatory genes mapping near H-2D that control the Hh-1 phenotype of lymphoid tumor cells.     

Calcium Dependence of Vasoactive Intestinal Peptide-Stimulated Cyclic AMP Accumulation in Rat Hippocampal Slices

Previous work has shown that incubation of hippocampal slices in medium without added calcium markedly attenuates the capacity of vasoactive intestinal peptide (VIP) to elevate cyclic AMP levels. The present studies examined the mechanism that confers calcium dependence on VIP stimulation of cyclic AMP accumulation in hippocampal slices. Calcium dependence was apparent immediately on slice preparation and was reversible only if calcium ions were added back very early during slice incubation (within 5 min). The cyclic AMP response to VIP was not abolished by preincubating slices in 100 microM adenosine, suggesting that calcium-dependent, VIP-induced release of adenosine does not mediate VIP elevation of cyclic AMP. VIP-stimulated cyclic AMP accumulation was not decreased by agents that block calcium influx (verapamil, nifedipine, magnesium ions), or by calmodulin antagonists (trifluoperazine, calmidozolium). In fact both verapamil (100 microM) and magnesium (14 mM) augmented VIP stimulation of cyclic AMP generation. Incubation of slices with the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX) did not affect VIP activation of cyclic AMP accumulation if slices were incubated without added calcium, but MIX did enhance VIP elevation of cyclic AMP content in slices incubated with calcium. Thus calcium dependence of the cyclic AMP response to VIP in hippocampal slices is unlikely to result from VIP-dependent calcium influx, from interactions with calmodulin, or from calcium-inhibited phosphodiesterase(s).(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of phospholipid peroxides in biological samples

Peroxidation of membrane lipids has been hypothesized to play a key role in various types of tissue degeneration and pathology. Lipid peroxides are formed when oxygen reacts with an unsaturated fatty acid chain. Virtually all of the unsaturated fatty acids in biological systems are bound by ester linkages in phospholipids or triglycerides. Phospholipid and triglyceride peroxides are primary products of lipid peroxidation and have rarely been measured. Most of the commonly used methods for detection of lipid peroxidation are based on detection of malondialdehyde or other chemical species that are derived from oxidized fatty acids. This review presents an overview of recently developed methods aimed at identifying and measuring oxidized phospholipids and triglycerides which are direct evidence of the occurrence of lipid peroxidation in vivo.