Two-dimensional 1H NMR of three spin-labeled derivatives of bovine pancreatic trypsin inhibitor

Three nitroxide spin-labeled monoderivatives of bovine pancreatic trypsin inhibitor were prepared with the amino-specific reagent succinimidyl 1-oxy-2,2,5,5-tetramethyl-3-pyrroline-3-carboxylate. The monoderivatives were purified by ion-exchange and affinity chromatography. Thin-layer maps of tryptic peptides of the monoderivatives showed that the spin-label was incorporated at either the alpha-amino group, Lys-15, or Lys-26. Two-dimensional J-correlated 1H NMR spectra of the monoderivatives were recorded. Spectra were also recorded after reduction by ascorbic acid of the nitroxide label to hydroxylamine. With the nitroxide label present, significant line-broadening effects on many of the cross peaks in the spectra were observed. The extent of line broadening for the C alpha H-NH cross peaks was qualitatively correlated with the distance between the labeled amino group and the average C alpha H-NH position in the crystal structure. The spin-label affects cross peaks of protons within approximately 15 A. This study suggests that it is feasible to accumulate sufficient intramolecular distances in order to determine protein solution structures with the aid of distance geometry algorithms.     

A myb gene required for leaf trichome differentiation in Arabidopsis is expressed in stipules

The GL1 gene is required for the initiation of differentiation of hair cells (trichomes) on the crucifer, Arabidopsis thaliana. This gene has been localized to a 4.5 kb DNA fragment by molecular complementation of gl1 mutants. DNA sequence analysis has shown that the protein encoded by GL1 contains a Myb DNA-binding motif. Southern analysis and subsequence analysis of isolated lambda clones has established that GL1 is a member of an extensive myb gene family in Arabidopsis. The putative GL1 promoter directs the expression of the GUS reporter gene in non-trichome-bearing structures that appear to be stipules. This pattern of expression suggests that GL1 may control the synthesis of a diffusible signal that activates the developmental pathway for trichome differentiation.     

Identification of rat S14 protein and comparison of its regulation with that of mRNA S14 employing synthetic peptide antisera

The rat S14 gene encodes a protein of unknown function and has an amino acid sequence unrelated to any published sequences. Expression of mRNA S14 and lipogenesis in liver, fat, and mammary gland are regulated coordinately by dietary and hormonal stimuli, suggesting that the S14 protein may be associated with lipogenesis. Antisera to synthetic peptides corresponding to portions of the deduced amino acid sequence of the protein were used to identify the protein and to compare its regulation with that of mRNA S14. Antisera specifically recognized the in vitro translation product of mRNA S14 as defined by its migration on two-dimensional gel electrophoresis. A product of identical Mr was identified on Western blots of liver homogenates from hyperthyroid, carbohydrate-fed rats. Subcellular fractionation showed that S14 protein is primarily cytosolic. The protein was detectable in tissues with abundant S14 gene expression, including hyperthyroid liver and epididymal fat and hypothyroid brown adipose tissue, whereas it was undetectable in hypothyroid liver and euthyroid kidney, testis, and spleen. Diurnal variation in hepatic mRNA S14 correlated with comparable changes in levels of the protein. Surprisingly, no S14 protein was observed in the livers of chronically (3 week) hypothyroid rats treated with triiodothyronine (T3) until 12 h had elapsed, despite attainment of maximal levels of mRNA S14 within 4 h. Rapid appearance of protein after T3 treatment was observed in both euthyroid and short term (4 day) hypothyroid rats, suggesting that long-term hypothyroidism is associated with a defect in the translational efficiency of mRNA S14.     

Thyroid hormone and circadian regulation of the binding activity of a liver-specific protein associated with the 5'-flanking region of the S14 gene

Recent studies have described a DNase I hypersensitive site in the 5'-flanking region of the rat hepatic S14 gene that is closely associated with its expression. A 111-base pair subfragment (-389 to -279) of this region interacts specifically in a gel shift assay with a protein present in hepatic nuclear protein extracts. This protein, designated P1, was not present in extracts of other tissues, even those in which the gene is expressed and hormonally regulated. The binding activity of P1 is exceedingly low in extracts from hypothyroid rats and is markedly increased by administration of thyroid hormone. However, the slow accumulation of P1 after thyroid hormone administration indicates that increased levels of P1 are not necessary for the acute hormonal induction of S14 gene expression. The level of P1 binding activity increases in the evening, synchronous with circadian variation of hepatic mRNA S14. Since neither P1 binding activity nor circadian variation in mRNA-S14 levels are observed in the other tissues expressing the S14 gene, P1 may function to modulate the circadian rhythm observed in hepatic S14 gene expression. DNase I footprinting analysis revealed that P1 binds to a defined nucleotide sequence, 5'-AAAAGAGCTATTGATTGCCTGCA-3', located between -310 and -288 in the S14 gene.     

Growth hormone acts at a pretranslational level in hepatocyte cultures

We have examined the effects of ovine growth hormone and recombinant DNA synthesized human growth hormone on hepatocytes maintained in serum free cultures. Both growth hormone preparations augmented or attenuated 3 specific mRNA sequences as revealed by two-dimensional gel electrophoresis of [35S] methionine labeled products synthesized in vitro in an mRNA dependent rabbit reticulocyte lysate system. The results clearly indicate that growth hormone, free of potential pituitary contaminants, acts directly on hepatocytes at a pretranslational level.     

Thyroid hormone-carbohydrate interaction at the hepatic nuclear level

We have studied the interaction between triiodothyronine (T3) and carbohydrate (CHO) in the induction of hepatic lipogenic enzymes under both in vivo and in vitro conditions. Our studies demonstrate a synergistic relationship between T3 administration and CHO feeding in the induction of these enzymes. Likewise, in states characterized by CHO deprivation such as starvation and diabetes, the response to T3 is also inhibited. Studies in the aging animal have documented a diminished response both to CHO and to T3. Our studies suggest that T3 multiplies a primary CHO-generated signal by a constant factor, and that this signal declines with age. Additional studies with primary hepatocyte cultures provide evidence that glucose is the main factor responsible for the induction of hepatic malate dehydrogenase: decarboxylating (EC 1.1.1.40) (ME). Glucose induces ME in the absence of changes in extrahepatic hormones or metabolites and in the complete absence of T3. In the cultured hepatocyte system, T3 also acts as a constant multiplier of the primary glucose-derived signal. Our results provide further support for the thesis that the primary action of T3 at the molecular level is a multiplication of other nuclear signals. The complexity of response pattern to both T3 and CHO administration, however, is illustrated by recent studies in which we have analyzed the translated products of total poly(A+) RNA extracted from livers of rats subjected to various physiological stimuli.     

Colorimetric device for measurement of transvascular fluid flux in blood-perfused organs

The aim of this study was to develop a device capable of measuring transvascular fluid flux in blood-perfused organs. For any given blood flow through the organ (QT), transvascular flux (QF) can be considered as the fraction of QT exchange. Presumably, QF would change the background concentration of an impermeable tracer residing in the perfusate. Thus QF could be calculated from the relative changes in tracer concentration for any given QT. We have used Blue Dextran (1 g/l of blood) as the reference tracer. Because the minimum molecular weight of Blue Dextran is 2 X 10(6), we anticipated it to behave as an impermeable tracer in most organs. QF was simulated with continuous infusions of plasma, normal saline solution, and a 50% mixture of both. Changes in Blue Dextran concentration were continuously followed colorimetrically by changes in transmission of specific light at a wavelength of 632 nm. Because 632-nm light is affected by hematocrit and O2 saturation changes, two additional wavelengths were used: 815-nm, which is not affected by saturation or Blue Dextran concentration changes, was used to account for changes in hematocrit, and 887-nm specific light, which is not affected by Blue Dextran, served to correct for saturation changes. Red cells could not be used as the reference tracer because of the possibility of hematocrit changes independent of fluid flux (Fahraeus effect). The device so constructed proved capable of measuring rates of fluid infusion in the order of 0.1% of QT with a variability of 10% around the mean.     

Remote nitrogen-15 isotope effects on addition of cyanide to NAD

The reversible reaction NAD + CN(-)----NAD-CN was examined for remote secondary 15N isotope effects caused by isotopic substitution at the ring nitrogen of the nicotinamide group. These were compared with analogous effects for dehydrogenase-catalyzed reactions, since both cyanide and the hydride ion add at the N-4 position of the nicotinamide ring. The 15N effects on the rate constants for the forward and reverse processes were examined directly by conducting both the normal and isotopic reactions simultaneously under carefully controlled conditions in the sample and reference cells of a dual-beam spectrophotometer. In both cases, the 15N kinetic isotope effect differed from 1.00 by considerably less than 0.01. The 15N equilibrium isotope effect, 15K, was obtained as the ratio of equilibrium constants measured separately with natural-abundance and labeled NAD by using a concentration jump procedure [1.004 +/- 0.002 (cyanide addition)]. A similar value for 15K of 1.010 +/- 0.008 was obtained in an analogous manner for the reaction catalyzed by lactate dehydrogenase: NAD + lactate----pyruvate + NADH + H+. The latter value is significantly smaller than a previously reported value obtained from kinetic studies [1.044 +/- 0.012; Cook, P. F., Oppenheimer, N. J., & Cleland, W. W. (1981) Biochemistry 20, 1817]. The present value also is smaller than might be expected for a change in bond order from 4 to 3 [Cleland, W. W. (1980) Methods Enzymol. 64, 104-125] on the basis of the canonical resonance structures for NAD and NADH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sand,swamp and weed vegetation at the estuary of the Rubin River (Palestine)

Zusammenfassung Der vorliegende Artikel gibt in grossen Zügen die Vegetation eines Platzes von einigen Quadratkilometern Ausdehnung nahe der Mittelmeerküste zwischen Jaffa und Askalon, wo bis vor kurzem ein mit Tamarisken und Schilfrohr bestandener Sumpf und eine aus leicht zerfallendem Kalksandstein aufgebaute Höhe noch ursprüngliche Pflanzengesellschaften getragen haben, während in einer Uebergangszone Ackerkultur auf sandigem Boden getrieben worden ist. Die ursprüngliche Vegetation ist durch Pflugkultur und Drainage des schweren Bodens, Aufforstung und bauliche Anlagen auf der Höhe zwecks Anlage einer Versuchsfarm verdrängt worden.Auf den Abhängen der Höhe fanden sich kniehohe Bestände von Perennen, in denen Andropogon hirtus und Ononis stenophylla vorherrschten. Die sandige Uebergangszone trug eine Assoziation, die von Eig als Ormenetum mixtae beschrieben worden ist und die er als segetale Variante seines (auf roten Sandböden der Küstenebene Palästinas sehr verbreiteten) Eragrostetum bipinnatae auffasste.Auf dem schwarzen, alluvialen Boden des Sumpfgebietes wurden noch Reste eines Phragmitetum typhetosum festgestellt, an anderer Stelle djungelartig dichte Phragmitesbestände ohne Begleitart in der Oberschicht des Röhrichts, aber mit einer Krautschicht, in der Alopecurus myosuroides vorherrschte. Die mit sinkendem Grundwasserspiegel auftretenden Veränderungen der ursprünglichen Vegetation werden skizziert. Nahe dem Grundwasser breitet sich Panicum repens in dichten Rasen aus. Höher über dem Grund-wasser findet man Bestände von Rubus sanctus mit Festuca arundinacea und Stachys viticina vergesellschaftet. Diesen nahe verwandt sind Altiherbosa, in denen Arten von Rumex, Lotus, Ononis, Mentha, Verbena, Inula, Pulicaria etc. ausser den genannten auftreten. Mehr segetalen Charakter haben Bestände von Ammi Visnaga und Cichorium pumilum, die nach mehrmaligem Pflügen beobachtet wurden, sowie die Prosopis Stephaniana-Cynara syriaca-Assoziation Eigs. Eine exakte Analyse der Abhängigkeit dieser artenreichen Pflanzenbestände vom Grundwasser kann leider nicht gegeben werden.Es wird an einigen Beispielen gezeigt, wie die besprochenen Folge-Gesellschaften schliesslich mit fortschreitender landwirtschaftlicher Nutzung von der auf schweren Böden der Küstenebene üblichen Unkrautvegetation der bewässerten Kulturen abgelöst werden.Auf leicht salzigen Böden fand sich ein Bestand von Juncus acutus-Plantago crassifolia, während Spergularia media-Hordeum marinum f. microstachys stärkeren Salzgehalt auf begrenzten Stellen mit weissem Boden anzuzeigen schien.Die Aufführung von ca. 250 Arten beansprucht ein gewisses pflanzengeographisches Interesse, da (1) über die Vegetation der Küstenregion zwischen Jaffa und Askalon recht wenig Angaben in der Literatur vorliegen und (2) weiter südlich permanente Sümpfe sich wegen des schnell trockener werdenden Klimas nicht mehr ausbilden können.

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Résumé L'article présent décrit la végétation d'un terrain s'étendant sur quelques kilométres carrés non loin du littoral méditerranéen entre Jaffa et Askalon. Tout récemment, un marais couvert de tamaris et de roseaux, et une hauteur composée d'un grès calcaire friable, y présentaient encore des groupements vegetaux primitifs, tandis que dans une zone intermédiaire on poussait la charrue sur un sol sablonneux. La végétation primitive a été détruite ou supplantée par le drainage du sol argileux, par l'afforestation, les constructions d'une ferme d'essais.Sur la pente de la colline se-trouvalent des groupes d'herbes perennantes parmi lesquelles prédominaient Andropogon hirtus et Ononis stenophylla. La zöne de transition sablonneuse portait une association qu' Eig a décrite comme Ormenetum mixtae. Il tenait cette association pour une variante ségétale de son Eragrostetum bipinnatae qui se trouve fort répandu sur les terrains sablonneux rouges de la plaine littorale palestinienne.Sur le sol noir, alluvial du terrain marécageux on constatait encore les restes d'un Phragmitetum typhetosum. En un autre endroit poussaient drus comme en pleine jungle des groupes de Phragmites sans espèces satellites dans la strate supérieure. Par contre on y trouvait une strate herbacée ou dominait Alopecurus myosuroides. Les modifications de la végétation primitive qui apparaissent avec l'abaissement de la nappe phréatique sont esquissés. Peu au dessus du fond de la nappe s'étend en touffes épaisses Panicum repens. Plus haut on trouve des peuplements de Rubus sanctus mêlés à Festuca arundinacea et Stachys viticina. Etroitement apparentés à ceux-là nous trouvons des Altiherbosa dans lesquels apparaissent en dehors de celles déjà mentionnèes des espèces de Rumex, Lotus, Mentha, Verbena, Inula, Pulicaria, etc.Un caractère plus ségétal portent les groupements d' Ammi Visnaga et et Cichorium pumilum qui furent constatés après un labourage répété, ainsi que l'association à Prosopis Stephaniana-Cynara syriaca d'Eig. Il est malheureusement impossible de donner une analyse exacte de la dépendance de la nappe d'eau de ces groupements végétaux si riches en espèces.Quelques exemples choisis montrent comment ces stades successifs de la dégradation de la végétation primitive sont définitivement supplantés par une végétation de mauvaises herbes qu'on trouve ordinairement sur les terrains lourds du littoral, dans les champs de labour ainsi que parmi les cultures irriguées.Sur des terres légèrement salines, on rencontre un groupement végétal à Juncus acutus-Plantago crassifolia tandis que Spergularia media et Hordeum marinum f. brachystachys semblent indiquer une teneur en sel élevée en des endroits limités à sol blanchâtre.L'énumération de quelques 250 espèces présente un certain intérêt phytogéographique, car (1°) la littérature offre bien peu de données exactes sur la flore du littoral entre Jaffa et Askalon; et (2°) plus loin vers le Sud le climat qui se dessèche rapidement ne permet plus la formation de marécages permanents.