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     

The geometries of interacting arginine-carboxyls in proteins

The geometries are reported for interacting arginine-carboxyl pairs obtained from 37 high resolution protein structures solved to a resolution of 2.0 A or better. The closest interatomic distance between the guanidinium and carboxyl is less than 4.2 A for 74 arginine and carboxyl groups, with the majority of these lying within hydrogen-bonding distance (2.6-3.0 A). Interacting pairs have been transformed into a common orientation, and arginine-carboxyl, and carboxyl-arginine geometries have been calculated. This has been defined in terms of the spherical polar angles T theta, T phi, and the angle P, between the guanidinium and carboxyl planes. Results show a clear preference for the guanidinium and carboxyl groups to be approximately coplanar, and for the carboxyl oxygens to hydrogen bond with the guanidinium nitrogens. Single nitrogen-single oxygen is the most common type of interaction, however twin nitrogen-twin oxygen interactions also occur frequently. The majority of these occur between the carboxyl oxygens and the NH1 and NE atoms of the arginine, and are only rarely observed for NH1 and NH2. The information presented may be of use in the modelling of arginine-carboxyl interactions within proteins.     

Glutamine synthetase isozymes in elasmobranch brain and liver tissues

Glutamine synthetase is present as isozymic forms in the elasmobranchs Squalus acanthias (dogfish shark) and Dasyatis sabina (stingray). Subcellular fractionation of elasmobranch brain and liver tissue shows the enzyme to be predominantly cytosolic in the former tissue and mitochondrial in the latter. For the cytosolic brain enzyme, the subunit Mr equals 42,000 in the stingray and 45,000 in the shark, as determined by sodium dodecyl sulfate-gel electrophoresis/Western blotting. The subunit Mr = 45,000 and 47,000, respectively, for stingray and dogfish mitochondrial liver enzymes. Translation of total brain RNA from both species gives immunoprecipitable nascent peptides of the same size as their respective mature enzymes. However, in liver tissue, translation of glutamine synthetase mRNA yields peptides of higher Mr than that of the mature enzymes. In dogfish liver, Mr = 50,000 for the translation product and, in stingray liver, Mr = 48,000. This suggests that the translocation of the enzyme into liver mitochondria may be via a signal or leader sequence mechanism. The larger liver isozyme of elasmobranch glutamine synthetase is found in kidney where it is also known to be mitochondrial. The smaller cytosolic isozyme occurs in retina, heart, gill, and rectal gland tissue as well as in brain.     

Assay and properties of N-acetylglucosamine-6-phosphate deacetylase from rat liver

A single-vial assay has been developed for N-acetylglucosamine-6-phosphate deacetylase, in which [3H]acetate released from 3H-acetyl-labeled substrate is measured in a biphasic liquid scintillation counting system after acidification of the reaction mixture. The deacetylase was partially purified from rat liver, and some of its properties were determined. Chromatography on a calibrated Sepharose CL-6B column indicated a molecular weight of 345,000. The Km for the substrate at pH 8.0 was 0.3 mM. Glucosamine 6-phosphate and glucose 6-phosphate inhibited the enzyme, whereas N-acetylgalactosamine, N-acetylglucosamine, N-acetylglucosamine 1-phosphate, and glucosamine 1-phosphate were without effect. The effects of several divalent cations were also examined. Under the conditions tested, Ca2+, Mg2+, and Ba2+ had essentially no effect, whereas Mn2+, Ni2+, and Cu2+ were inhibitory and Co2+ stimulated activity at low concentrations but inhibited above 5 mM. An increase in the ionic strength of the reaction mixture to 0.3 M decreased the activity by 40%.     

The effect of retinoic acid on proteoglycan biosynthesis in bovine articular cartilage cultures

The addition of retinoic acid to adult bovine articular cartilage cultures produces a concentration-dependent decrease in both proteoglycan synthesis and the proteoglycan content of the tissue. Total protein synthesis was not affected by the presence of retinoic acid, indicating that the inhibition of proteoglycan synthesis was not due to cytotoxicity. The proteoglycans synthesized in the presence of retinoic acid were similar in hydrodynamic size, ability to form aggregates with hyaluronate, and glycosaminoglycan composition to those of control cultures. However, the presence of larger glycosaminoglycan chains suggests that the core protein was substituted with fewer but longer glycosaminoglycan chains. In cultures maintained with retinoic acid, a decreased ratio of the large proteoglycan was synthesized relative to the small proteoglycan compared to that measured in control cultures. In cultures maintained with retinoic acid for 1 day and then switched to medium with 20% (v/v) fetal calf serum, the rate of proteoglycan synthesis and hexuronate contents increased within 5 days to levels near those of control cultures. Within 2 days of switching to medium with 20% (v/v) fetal calf serum, the relative proportions of the proteoglycan species were similar to those produced in cultures maintained in medium with 20% (v/v) fetal calf serum throughout. The rate of proteoglycan synthesis by bovine articular cartilage cultures exhibited an exponential decay following exposure to retinoic acid, with estimated half-lives of 11.5 and 5.3 h for tissue previously maintained in medium alone or containing 20% (v/v) fetal calf serum, respectively. The addition of 1 mM benzyl beta-D-xyloside only partially reversed the retinoic acid-mediated inhibition of proteoglycan synthesis. This indicates that the inhibition of proteoglycan synthesis by retinoic acid was due to both a decreased availability of xylosylated core protein and a decreased capacity of the chondrocytes to synthesize chondroitin sulfate chains.     

High-resolution 1H NMR study of the solution structure of alamethicin

A 1H NMR study of the peptide alamethicin, which forms voltage-gated ion channels in membranes, is described. The molecule was studied in methanol as a function of temperature and pH. A complete assignment of the spectra is given, including several stereospecific assignments. Alamethicin was found to have a structure substantially similar to the crystal although, in solution, the C-terminal dipeptide adopts a somewhat extended conformation. The overall conformation was insensitive to the ionization of the side chain of the only ionizable group, Glu-18.     

Primary structure analysis proves that protein phosphatases 2C1 and 2C2 are isozymes

The two recently discovered forms of protein phosphatase 2C, termed 2C1 and 2C2, were digested with CNBr or trypsin, and several peptides corresponding to two regions of the protein were sequenced. These studies revealed close homology between the two enzymes with 49 identities over the 62 residues that could be compared directly. The results establish that protein phosphatases 2C1 and 2C2 are the products of different genes. The C-terminus of protein phosphatase 2C2 has also been identified.     

Doppler studies in the growth retarded fetus and prediction of neonatal necrotising enterocolitis,haemorrhage, and neonatal morbidity.

In 82 consecutive cases of intrauterine growth retardation managed by established criteria fetal Doppler studies identified 29 fetuses with absence of end diastolic frequencies in the fetal aorta. These same fetuses were significantly more growth retarded (p less than 0.001) and had an earlier gestational age at delivery (p less than 0.001) than those with end diastolic frequencies present. A subgroup of these cases was analysed in more detail to examine the prognostic value of this phenomenon for the neonate. Two groups of neonates of equivalent gestational age and with a birth weight below 2000 g were compared. There were 26 neonates with absent end diastolic frequencies (group 1) and 20 with end diastolic frequencies (group 2) in the fetal aorta. Those in group 1 were more likely to suffer perinatal death (p less than 0.05), necrotising enterocolitis (p less than 0.01), and haemorrhage (p less than 0.05). Only 4 (15%) of the babies in group 1 had an uncomplicated neonatal period compared with 15 (75%) in group 2 (p less than 0.001). The circulatory changes identified in these cases may provide a more sensitive measure of critical fetal compromise than current techniques and thus allow the clinician to deliver the fetus before irreversible tissue damage has occurred.     

cDNA Clones for Corn Leaf NADH:Nitrate Reductase and Chloroplast NAD(P):Glyceraldehyde-3-Phosphate Dehydrogenase : Characterization of the Clones and Analysis of the Expression of the Genes in Leaves as Influenced by Nitrate in the Light and Dark

cDNA clones were selected from a corn (Zea mays L.) leaf lambda gt11 expression library using polyclonal antibodies for corn leaf NADH:nitrate reductase. One clone, Zmnrl, had a 2.1 kilobase insert, which hybridized to a 3.2 kilobase mRNA. The deduced amino acid sequence of Zmnrl was nearly identical to peptide sequences of corn leaf NADH:nitrate reductase. Another clone, Zm6, had an insert of 1.4 kilobase, which hybridized to a 1.4 kilobase mRNA, and its sequence coded for chloroplastic NAD(P)⁺:glyceraldehyde-3-phosphate dehydrogenase based on comparisons to sequences of this enzyme from tobacco and corn. When nitrate was supplied to N-starved, etiolated corn plants, nitrate reductase, and glyceraldehyde-3-phosphate dehydrogenase mRNA levels in leaves increased in parallel. When green leaves were treated with nitrate, only nitrate reductase mRNA levels were increased. Nitrate is a specific inducer of nitrate reductase in green leaves, but appears to have a more general effect in etiolated leaves. In the dark, nitrate induced nitrate reductase expression in both etiolated and green leaves, indicating light and functional chloroplast were not required for enzyme expression.