Anx7 is required for nutritional control of gene expression in mouse pancreatic islets of Langerhans

BACKGROUND: Gene expression in islets of Langerhans is profoundly sensitive to glucose and other nutrients. Islets of Langerhans in the Anx7(+/-) knockout mouse exhibit a profound reduction in ITPR3 protein expression, defective intracellular calcium signaling, and defective insulin secretion. Additional data presented here also show that mRNA for ITPR3 is virtually undetectable in isolated Anx7(+/-) islets. IP3Receptor type 3 (ITPR3) expression in islets of Langerhans is closely regulated by secretory stimuli, and it has been suggested that the level of the ITPR3 expression controls the ability of the islets to respond to nutritional signals. We report that although control islets respond to glucose in vitro by a transient increment in ITPR3 mRNA, the islets from the Anx7(+/-) mouse remain low. We therefore hypothesized that the Anx7/IP3 Receptor(3)/Ca(2+) signaling pathway plays a role in beta cell responses to glucose, and that in the absence of the Anx7/ITPR3 signaling system, the islets would be unable to discriminate between fed or fasted states in vivo. MATERIALS AND METHODS: To test this hypothesis, we subjected Anx7(+/-) and control mice to either food and water ad libidum or to an overnight fast with access to water only. We then isolated the respective islets and compared nutrient-dependent changes in global gene expression under the four conditions using genome-based microarray technology. RESULTS: Anx7 protein expression in these islets is only about 50% of control levels in normal littermate controls, and IPTR3 message and protein are virtually zero. cDNA microarray analyses show that in control animals gene expression is significantly affected by the fasting state. Many of the affected genes have historical relevance to development and differentiation of islets. These include preproglucagon, APOJ, cadherin2, phosphoglucoisomerase, oncostatin M, PAX6, HGF, and cytokeratin 18. However, there are also many other nutritionally sensitive genes in control islets that are principally associated with cell division and DNA repair. The latter genes have not specifically been associated with islet physiology in the past. By contrast, Anx7(+/-) mouse islets exhibit a greatly reduced ability to discriminate genomically between fed and fasted states for all classes of identified genes. Many of the validated genes are specific to islets in comparison to liver tissue examined. Real-time quantitative RT-PCR analysis of islets from Anx7 heterozygous mice and littermate controls revealed remarkable down-regulation in PTEN, Glut-2, PDX-1, IGF-1, and Neuro D1 expression, but not in liver. CONCLUSIONS: We conclude that reduced gene dosage in the Anx7(+/-) islet, with concomitant loss of ITPR3 expression and consequent defects in Ca(2+) signaling, may substantially contribute to the mechanism of the loss of genomic discrimination, in vivo, between the fed and fasted states. We believe that the requirement for complete Anx7 gene dosage and IPTR3 expression in islets of Langerhans will prove to be of fundamental importance for understanding the mechanism of nutritional sensing in health and disease.     

Biochemical studies of H-2K antigens from a group of related mutants. II. Identification of a shared mutation in B6-H-2 bm6 , B6.C-H-2 bm7 , and B6.C-H-2 bm9

In an earlier paper, we presented evidence that two independent mutants of the bg series, B6-H-2 ^bm5 (bm5) and B6-H-2 ^bm16 (bm16) carry identical mutations such that tyrosine at residue number 116 of the H-2K^b molecule from the parent strain C57BL/6Kh is replaced by a phenylalanine in each of the two mutant molecules. In this paper, we demonstrate, using similar techniques, that the independent bg series mutants B6-H-2 ^bm6 (bm6), B6.C-H-2 ^bm7 (bm7), and B6.C-H-2 ^bm9 (bm9), which share biological properties with bm5 and bm16, can be grouped together because they share two identical mutations, one of which is common to bm5 and bm16, a Tyr to Phe interchange at residue number 116. In addition, a second mutation is at residue number 121, where a Cys in the H-2K molecule from 136 is substituted with an Arg in the mutant. Since all of the bg series mutants arose independently and share biological and biochemical characteristics, it is anticipated that study of these mutants could lead to some understanding of the high mutation rate in the K^b molecule.     

Conjugated Dopamine in Superfusates of Slices of Rat Striatum

Abstract: An acid-hydrolyzable conjugate of 3,4-dihydroxyphenylethylamine (dopamine, DA) was detected in superfusates from slices from rat striatum. The concentrations of endogenous free and conjugated DA, and of the acid metabolites (3,4-dihydroxyphenylacetic acid [DOPAC] and homovanillic acid [HVA]) in superfusates were measured using HPLC with electrochemical detection. Conjugated DA in superfusates represented 10–20% of the free DA under basal conditions and during release evoked by p -tyramine (5 × 10⁻⁶ M to 5 × 10⁻⁴ M ); much smaller amounts of conjugated DA overflowed into superfusate when DA was released by equimolar concentrations of β-phenylethyl-amine. Surprisingly, inhibition of monoamine oxidase by the inhibitors N -methyl- N -propargyl-3-(2,4-dichlorophenoxy)propylamine hydrochlo-ride (clorgyline) or N -methyl- N -2-propynylbenylamine (pargyline) had little effect on the amounts of conjugated DA present in superfusate. Under basal conditions, the amounts of conjugated DA in superfusate were always less than the amounts of DOPAC but quite similar to the amounts of HVA. However, during release of DA evoked by p -tyramine the concentrations of conjugated DA in superfusate showed much more pronounced increases than those of the acidic metabolites.     

Fucosylated protein of retinal cone photoreceptor outer segments: morphological and biochemical analyses

Cone outer segments (OS) of the goldfish retina are diffusely labeled after intravitreal injection of [(3)H]fucose while rod OS remain unlabeled. By electron microscopic radioautography, the OS of red- and blue-sensitive cones are heavily labeled while green- sensitive cone OS are lightly labeled. The time-course and pattern of OS labeling in all cone types from 30 min to 24 h resemble that of incorporation of other sugars into rhodopsin in rod OS. The nature of the cone OS-specific fucosylated component(s) was examined using biochemical techniques. Cone OS were prelabeled by intravitreal injection of [(3)H]fucose 24 h before sacrifice. Photoreceptor OS were isolated using a discontinuous sucrose density gradient and it was verified by electron microscopic radioautography that the only source of radioactivity in the preparations was cone OS. The different cone types could be recognized by the heaviness of labeling, characteristic membrane spacing, and 'staining' of green cone OS in vitro with horseradish peroxidase. After acid hydrolysis of prelabeled photoreceptor membranes, 90 percent of the counts were in the neutral sugar fraction which was analyzed by thin-layer chromatography. Approximately 70 percent of the radioactivity co-chromatographed with authentic fucose. SDS-PAGE/fluorography of prelabeled photoreceptor membranes revealed a single radioactive component that was lightly stained with coomassie blue and showed an apparent molecular weight of 33,000. This cone-derived band was separated from unlabeled rod opsin which was well stained and showed an apparent mol wt of 38,000. Isoelectric focusing under denaturing conditions produced two major and one minor band of radioactivity with isoelectric points of 8.2, 8.6, and 8.8 respectively. No radioactivity was found in association with a stained band corresponding in isoelectric point to that of bovine opsin (pl, 6.2). The fucosylated component was readily digested by pronase, indicating its protein nature. Washing of the isolated OS with isotonic and hypotonic buffers failed to extract major amounts of the radioactivity, suggesting that the fucosylated component is an integral membrane protein. The presence of a fucosylated protein thus represents a major difference between cone and rod OS in the goldfish and has enabled us to identify cone OS in preparations of isolated photoreceptor membranes and to demonstrate the separation of a cone-derived glycoprotein from rod opsin.     

Measurement of free and bound 5-hydroxytryptophan in plasma by liquid chromatography with electrochemical detection

A new method for the measurement of concentrations of 5-hydroxytryptophan (5HTP) in plasma is described. This method, which uses high-performance liquid chromatography with electrochemical detection, was used to measure 5HTP in plasma of patients with the carcinoid syndrome, and also in the plasma of rats after injection of 5HTP (30 mg/kg). A significant proportion of 5HTP was bound to macromolecules in plasma both in the rats and in the patients.     

Das LängenverhÄltnis der Eu- und Heterochromatischen Abschnitte Riesenchromosomenartiger Bildungen Verglichen mit dem der Prophasechromosomen bei Bryonia Dioica

Zusammenfassung In den 128- und 256-ploiden Kernen der Klebstoffhaare von Bryonia dioica fanden sich im Jahre 1960 riesenchromosomenartige Gebilde, die eine Mittelstellung zwischen Riesenchromosomen und Endochromozentren einnehmen. Sie unterscheiden sich — vorwiegend durch das Vorhandensein von kompaktem Heterochromatin — von den im Jahre 1953 am gleichen Pflanzenmaterial aufgefundenen chromatischen Strukturen.Entsprechend der diploiden Chromosomenzahl sind 20 riesenchromosomenartige Bildungen vorhanden. Zwei Paare von ihnen stehen mit dem Nukleolus in Verbindung; sie werden als SAT I und SAT II bezeichnet, da sie den Nukleolenchromosomen der Mitose entsprechen.Das aus zahlreichen Messungen errechnete Längenverhältnis eu- und heterochromatischer Abschnitte der riesenchromosomenartigen Bildungen SAT I und SAT II ist gegenüber dem der entsprechenden Chromosomen eines mittleren Stadiums der Prophase aus dem Endosperm zugunsten der euchromatischen Abschnitte verschoben. Zu dem gleichen Ergebnis gelangt man, wenn man einige der übrigen, nicht näher bestimmbaren riesenchromosomenartigen Bildungen bzw. Mitosechromosomen vergleicht.Diese Tatsache wird mit der Vorstellung der Tendenz des kompakten Heterochromatins, seine starke Spiralisierung weitgehend unverändert beizubehalten, zu erklären versucht. Lockeres Heterochromatin und Euchromatin von Riesenchromosomen und riesenchromosomenartigen Bildungen strecken sich dagegen im Laufe der Polyploidisierung.Während des Überganges von 128-Ploidie zur 256-Ploidie verlängern sich die riesenchromosomenartigen Bildungen um das 1,26fache. Die Gesamtverlängerung gegenüber den mitotischen Chromosomen ist im Vergleich zu anderen Pflanzen sehr gering (12,54 bei 256-Ploidie).Im Verlauf der Prophase findet im Gegensatz zu Vicia und Rhoeo nur ein einmaliger Abbau des Heterochromatins im sog. Zerstäubungsstadium statt.In den endopolyploiden Kernen der Basalzelle der Haare und in den triploiden Kernen des Endosperms kommen Vakuolen vor, die aus dem Nukleolus in den Kernraum ausgestoßen werden.     

Primary production: Marine ecosystems

The seas occupy the greater part (70%) of the earth's surface, but their total net primary production is less than that of the land. Depletion of nutrients in the lighted surface waters is responsible for low productivities of most of the open ocean; higher productivities occur in coastal waters and areas of upwelling. A recent estimate of total production for the marine plankon is 50×10 ⁹ metric tons of dry matter per year; the total with benthic production may be 55×10⁹ tons/year. The value for the plankton may be too low; there are persistent problems in measuring productivity with the radiocarbon technique. Because harvestable fish populations are concentrated in a limited area of more productive waters, where they are subject to overharvest and pollution effects, major increase in food production from the seas is not likely.This paper was presented at the symposium, The Primary Production of the Biosphere, given at the Second Congress of the American Institute of Biological Sciences, Miami, Florida, October 24, 1971.     

Pyridoxal 5′-phosphate levels in brain after treatments which impair cerebral glucose metabolism

Pyridoxal 5-phosphate (PLP) concentrations were measured in brains of rats to determine whether a deficiency of this coenzyme was a common feature in hepatic coma, ethanol intoxication, and in animals treated withl-dopa or with 5-hydroxytryptophan (5-HTP) alone or with inhibitors of MAO or ofl-aromatic amino acid decarboxylase. These treatments have been shown previously to be associated with reduced conversion of glucose to amino acids in brain. Cerebral PLP concentrations were reduced after some of these treatments, notably injection of ethanol, orl-dopa alone or with -phenylisopropylhydrazine, an inhibitor of MAO, or of 5-HTP together withN-[-(chlorophenoxy)ethyl]cyclopropylamine hydrochloride, Lilly 51641, another MAO inhibitor. However, in other circumstances where inhibition of conversion of glucose to amino acids has been shown {treatment with 5-HTP, or with Lilly 51641 or with [N-(d,l-seryl)-N-2,3,4-trihydroxybenzyl]hydrazine, an inhibitor ofl-aromatic amino acid decarboxylase, together withl-dopa or with 5-HTP}, PLP levels in brain were unchanged, or were increased (in hepatectomized rats).