Cytochemical localization of 5'-nucleotidase in the frog (Rana pipiens) retina. A histochemical and cytochemical study

Localization of 5'-nucleotidase in the frog retina was investigated using histochemical and cytochemical techniques. Light-microscopic observations revealed the presence of this enzyme in the inner retinal layers (the nerve fiber layer, ganglion cell layer, and inner plexiform layer). Ultrastructural investigations revealed that the enzyme activity is associated with the plasma membranes of the Müller cell processes, whereas the Müller cell processes present in the outer retinal layers did not demonstrate any detectable enzyme activity. This observation would appear to confirm our previous findings, that 5'-nucleotidase is an ectoenzyme, but its distribution in frog retina differs from that in rodents and it is only present in the inner layers of the retina. The prominent localization of 5'-nucleotidase on the glial plasma membrane may be viewed in the context of the widely accepted interaction between neurones and glial cells. Since nucleotides do not penetrate the plasma membrane, a mechanism to produce membrane-permeable adenosine, important for neuronal function, is postulated. It is known that 5'-nucleotidase produces adenosine by hydrolyzing adenosine 5'-monophosphate (5'-AMP). Therefore one would expect that the glial membrane-bound enzyme can accomplish the final step in this mechanism by producing the adenosine in the extracellular spaces.     

Influence of Temperature of Incubation and Type of Growth Medium on Pigmentation in Serratia marcescens

Maximal amounts of prodigiosin were synthesized in either minimal or complete medium after incubation of cultures at 27 C for 7 days. Biosynthesis of prodigiosin began earlier and the range of temperature for formation was greater in complete medium. No prodigiosin was formed in either medium when cultures were incubated at 38 C; however, after a shift to 27 C, pigmentation ensued, provided the period of incubation at 38 C was not longer than 36 hr for minimal medium or 48 hr for complete medium. Washed, nonpigmented cells grown in either medium at 38 C for 72 hr could synthesize prodigiosin when suspended in saline at 27 C when casein hydrolysate was added. These suspensions produced less prodigiosin at a slower rate than did cultures growing in casein hydrolysate at 27 C without prior incubation at 38 C. Optimal concentration of casein hydrolysate for pigment formation by suspensions was 0.4%; optimal temperature was 27 C. Anaerobic incubation, shift back to 38 C, killing cells by heating, or chloramphenicol (25 mug/ml) inhibited pigmentation. Suspensions of washed cells forming pigment reached pH 8.0 to 8.3 rapidly and maintained this pH throughout incubation for 7 days. Measurements of viable count and of protein, plus other data, indicated that cellular multiplication did not occur in suspensions of washed cells during pigment formation. By this procedure utilizing a shift down in temperature, biosynthesis of prodigiosin by washed cells could be separated from multiplication of bacteria.     

Fatty acids. part 42 The preparation and properties of methyl monomercaptostearates. some related thiols, and some methyl epithiostearates

Some saturated and unsaturated mercapto C₁₈ esters have been obtained for the first time. Such compounds are prepared from hydroxy esters via their mesyloxy derivatives by reaction with sodium hydrogen sulphide or with potassium thioacetate (followed by deacetylation) or from alkene esters by radical addition of thioacetic acid. The mercapto esters are readily identified by infrared, nuclear magnetic resonance, and mass spectroscopic procedures, preferably after acetylation or trifluoroacetylation.γ-Mesyloxy alkenes furnish tetrahydrothiophen rather than mercapto alkenes and methyl 9,12-epithiostearate was synthesised by an independent route from thiophen.     

Rearing, reproductive behaviour and gamma sterilization of fruit fly, Dacus zonatus (Diptera: Tephritidae)

Investigations were made on rearing, reproductive behaviour and gamma sterilization of one-day old male adults of Dacus zonatus. The larvae were successfully reared on an artificial diet based on wheat shorts. Adult emergence ranged from 89–99% with a sex ratio of about 1:1. Mating occurred at dusk and its duration ranged from 8–13 hours. Males mated a second time with the same female but preferred mating if the already mated female was replaced with a sexually mature virgin female. The optimum dosage for inducing sterility amongst one-day old male adults was 12 kR.

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Zusammenfassung Zucht und Fortpflanzungsverhalten von Dacus zonatus (Saunders) wurde untersucht. Die Larven wurden vier Generationen lang an einer Diät aus Weizenkleie, Bierhefe, granuliertem Zucker, Agar, Nipagin, Salzsäure und Wasser gehalten. Die Arbeit gibt Daten über Verpuppungsprozentsatz (69,3%), Puppengewicht, Dauer der Ei + Larvenperiode, Schlüpfen der Adulten, Präovipositionszeit, Fruchtbarkeit, Fertilität und Lebensdauer der Adulten. Die Schlüpfrate der Adulten betrug 89–99%, das Geschlechtsverhältnis lag etwa bei 1:1. Die Kopulation findet während der Abenddämmerung statt, sie dauert 8–13 Stunden. Maximum der Kopulationen zwischen dem 10. und 15. Tag nach dem Schlüpfen. Männchen paarten sich ein zweites Mal mit dem gleichen Weibchen, bevorzugten jedoch geschlechtsreife jung-fräuliche Weibchen. Die Eiablage begann am 2.–7. Tag nach der Paarung, die Eizahl betrug bei gepaarten Weibchen 91–564.Die optimale Dosis zur Erzeugung von 99,3% Sterilität bei Bestrahlung von einem Tag alten Männchen war 12 kR. Die Lebensdauer der Bestrahlten war vermindert.

     

Formic Hydrogenlyase and the Photoassimilation of Formate by a Strain of Rhodopseudomonas palustris

A photosynthetic bacterium isolated by enrichment on media containing formate as major source of cell carbon was identified as a strain of Rhodopseudomonas palustris. It grew on a wide range of simple organic compounds including alcohols, fatty acids, and hydroxyacids, on a chemically defined medium with biotin and p-aminobenzoic acid as essential growth factors. The organism grew on formate or photoautotrophically with molecular hydrogen or thiosulfate only in the presence of yeast extract. Ability to photoassimilate formate could be shown only in organisms grown in the presence of formate. The organism contained an inducible formic hydrogenlyase consisting of a soluble formic dehydrogenase, a particulate hydrogenase, and one or more intermediate, but as yet unidentified, electron carriers. The formic hydrogenlyase could be reconstituted from a particulate hydrogenase and a partially purified soluble formic dehydrogenase. Some properties of the formic dehydrogenase and hydrogenase have been compared with that of the formic hydrogenlyase system.     

Inhibition of prolyl hydroxylase by poly(ADP-ribose) and phosphoribosyl-AMP. Possible role of ADP-ribosylation in intracellular prolyl hydroxylase regulation

Poly(ADP-ribose) prepared by incubating NAD+ with rat liver nuclei inhibited the hydroxylation reaction catalyzed by purified prolyl hydroxylase (proline,2-oxoglutarate dioxygenase, EC 1.14.11.2) in vitro. Near complete inhibition of the enzyme was seen in the presence of 6 nM (ADP-Rib)18 with a Ki(app) of 1.5 nM. The monomer unit of poly(ADP-ribose), adenosine diphosphoribose (ADP-Rib), was found to be a weak inhibitor. On the other hand, poly(ADP-ribose)-derived phosphoribosyl-AMP (PRib-AMP) and its dephosphorylated product, ribosyl-ribosyl-adenine (Rib-RibA), inhibited the enzyme in nanomolar concentrations (Ki(app) 16.25 nM). The order of inhibition was (ADP-Rib)18 greater than PRib-AMP, Rib-RibA much greater than ADP-Rib. These results suggested that the 1"----2' ribosyl-ribosyl moiety in these compounds was involved in the inhibition of the enzyme. The possibility that intracellular prolyl hydroxylase is regulated by the involvement of ADP-ribosylation reactions was examined in confluent cultures of skin fibroblast treated with 20 mM lactate. The activity of prolyl hydroxylase was stimulated by 145% over that of untreated cultures. In the lactate-treated cells, the level of NAD+ was lowered and the total ADP-ribosylation of cellular proteins reduced by 40%. These observations imply that the lactate-induced activation of cellular prolyl hydroxylase is mediated by a reduction in ADP-ribosylation and that the synthesis and degradation of ADP-ribose moiety(ies) may possibly regulate prolyl hydroxylase activity in vivo.     

Chylomicron-chylomicron remnant clearance by liver and bone marrow in rabbits. Factors that modify tissue-specific uptake

The metabolism of [14C]cholesterol- and [3H]retinol-labeled chylomicrons obtained from canine thoracic duct or rabbit mesenteric lymph was investigated in normal fasted rabbits. Typically, 70-80% of the chylomicrons injected into the rabbits were cleared from the plasma in 20 min, and their uptake was accounted for principally by the liver and the bone marrow. Surprisingly, the bone marrow was a major site of uptake; the uptake ranged from about half that of the liver to a nearly equal amount. The importance and specificity of chylomicron-chylomicron remnant uptake by the bone marrow were established by demonstrating that (a) bone marrow throughout the body accumulated these lipoproteins, (b) the level of uptake was consistent regardless of how the values were calculated or how the chylomicrons were prepared, (c) the uptake represented specific binding, and (d) radiolabeled intestinal lipoproteins induced in vivo delivered cholesterol and retinol to the marrow. Electron microscopic examination of the rabbit bone marrow established that perisinusoidal macrophages uniquely accounted for the uptake of the chylomicrons. Whereas liver cleared a variety of both triglyceride-rich lipoproteins (chylomicrons, chylomicron remnants, and very low density lipoproteins) and cholesterol-rich lipoproteins (beta-very low density lipoproteins and high density lipoproteins containing apolipoprotein E), bone marrow uptake appeared to be restricted to the triglyceride-rich lipoproteins. More chylomicron remnants (generated in a hepatectomized rabbit) were cleared by the liver than by the bone marrow, and the addition of excess apolipoprotein E to chylomicrons resulted in their preferential uptake by the liver. The role of chylomicron-chylomicron remnant delivery of lipids or lipid-soluble vitamins to rabbit bone marrow is open to speculation, and whether triglyceride-rich lipoprotein uptake occurs to a significant extent in the bone marrow of humans remains to be determined.     

Ricin B chain fragments expressed inEscherichia coli are able to bind free galactose in contrast to the full length polypeptide

Deleted forms of ricin B chain (RTB) containing only one of the two galactose binding sites were produced inE. coli and targeted to the periplasm by fusion to theompA orompF signal sequences. The proteins were then isolated from the periplasm and their sugar binding properties assessed. Previous studies investigating the properties of such proteins produced inXenopus laevis oocytes suggested that deleted forms of RTB, when not glycosylated, retain their ability to bind simple sugars, unlike the full-length unglycosylated proteins. When produced inE. coli however we found that only one, EB733, of a number of deleted forms of RTB closely related to those previously produced inXenopus laevis oocytes, bound to simple sugars. All of the deletion forms of RTB were found to bind in the asialofetuin binding assay; an assay which has been previously utilized to measure binding of lectins to the terminal galactose residues of glycoprotein oligosaccharides. However, in contrast to glycosylated RTB, binding of the deletion mutants could be competed to only a small degree or not at all with galactose. The only deletion mutant observed to bind to free galactose when produced inE. coli corresponded closely to the complete domain 2 of RTB. It is assumed that this mutant forms a stable structure similar to that of the C-terminal domain in the full-length protein. The structural integrity of EB733 was not only suggested by its sugar binding properties and solubility but also by its consistently higher level of expression and the absence of any apparent susceptibility toE. coli proteases.Abbreviations RTA ricin toxin A chain - RTB ricin toxin B chain - ER endoplasmic reticulum - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - IPTG isopropyl -d-thiogalactopyranoside     

Field evaluation of a model of photothermal flowering responses in a world lentil collection

A model to predict flowering time in diverse lentil genotypes grown under widely different photothermal conditions was developed in controlled environments. The present study evaluated that model with a world germ plasm collection of 369 accessions using two field environments in Syria and two in Pakistan. Photoperiod alone accounted for 69% of the variance in 1/f, the reciprocal of time (d) from sowing to flower. In contrast, temperature alone did not account for a significant proportion of variation in flowering time due to the exposure of plants to supra-optimal temperatures in the late-sown Syrian trial. With the model mean pre-flowering values of photoperiod and temperature combined additively to account for 90.3% of the variance of 1/f over accessions. The correlation of field-derived estimates of temperature sensitivity of accessions to glass-house-derived estimates was significant at P = 0.05, but the equivalent correlation for estimates of photoperiodic sensitivity was higher at P < 0.01. Flowering in the field was better measured as time from sowing to 50% plants in flower rather than time to first bloom or its node number. Dissemination of the lentil crop following domestication in West Asia to the lower latitudes such as Ethiopia and India has depended on selection for intrinsic earliness and reduced sensitivity to photoperiod. Movement from West Asia to the higher latitudes accompanied by spring sowing has resulted in a modest reduction in photoperiod sensitivity and an increase in temperature sensitivity.