Glucose-stimulated insulin release by individual pancreatic beta cells: potentiation by glyburide.

To investigate the effect of glyburide on insulin secretion by individual beta cells from normal rats, we employed a reverse hemolytic plaque assay. Pancreata were harvested from female Wistar-Furth rats, the pancreatic islets isolated, and the latter dispersed into single cells. These cells were mixed with protein A-coated ox erythrocytes, the mixture was placed in a Cunningham chamber in the presence of insulin antiserum, and the cells were exposed to the various test substances. Having developed hemolytic plaques around the insulin-secreting cells with complement, the percentage of plaque-forming cells was determined and the plaque areas (reflecting the amount of insulin secreted) were quantitated. For the purpose of validation, we demonstrated that (i) plaque-forming (but not nonplaque-forming) cells could be identified as insulin secreting by an independent immunofluorescent technique, (ii), plaques did not form if insulin antiserum was deleted from the preparation, (iii) plaques failed to develop if insulin antiserum was preabsorbed with insulin, and (iv) incubation with non-protein A-coated RBC or omission of complement resulted in no plaque formation. In addition, both the percentage of plaque-forming cells and the mean plaque are increased upon exposure to glucose (0.75-20 mM) in a concentration-dependent manner at 5- and 60-min incubation times. Moreover, somatostatin suppressed the percentage of plaque-forming cells and diminished the mean plaque area of cells which continued to secrete insulin in response to glucose. Exposure of cells to 100 nM glyburide in the presence of 5 mM or 20 mM glucose had no effect on the percentage of plaque-forming cells present at 5 min or 60 min. Similarly, glyburide did not alter mean plaque area at 5 or 60 min when cells were co-incubated with 5 mM glucose. However, mean plaque area was markedly enhanced at 5 and 60 min in response to glyburide and 20 mM glucose. These results demonstrate that glyburide (i) does appear to enhance insulin secretion by an effect directly on the pancreatic beta cell; (ii) does not act by recruiting previously noninsulin-secreting cells into a secretory pool; (iii) does not potentiate the effect of glucose, at fed concentrations, on insulin secretion by individual cells; but (iv) does augment insulin secretion by beta cells stimulated with supraphysiologic concentrations of glucose.     

Culture of Amelanchier x grandiflora in a programmable micropropagation apparatus

A micropropagation system was developed to test concepts for automation and microenvironment alteration. Amelanchier x grandiflora Rehd. Princess Diana (serviceberry) shoot cultures were grown in seven-liter polycarbonate containers. Through computer control, the apparatus intermittently applied culture medium to the plant material according to a selected schedule. Shoot growth in the programmable system was compared with four micropropagation treatments: gelled and liquid medium in baby food jars and gelled and non-cycling liquid medium in a seven-liter vessel. Plants cultured in continuous contact with liquid medium became increasingly vitrified. Significantly greater shoot number, shoot length, shoot weight, and culture weight occurred in the programmable system than in jars with gelled medium. The combination of liquid medium, 7-liter vessel, and intermittent contact with medium caused a significantly higher proliferation rate than any combination of jar/vessel and gelled/liquid medium tested.     

Zinc deficiency and the formation of white structures in immature embryo cultures of wheat (Triticum aestivum L.)

The effect of microelements on the induction of embryogenic callus from epiblast and scutellum of wheat (Triticum aestivum L.) embryos was studied by the sequential omission of each of the microelements from Murashige & Skoog medium. Omission of iron caused a marked decrease in yield and poor shoot formation from embryogenic callus. The yield of embryogenic callus on medium without added manganese was also reduced. Omission of boron, copper-cobalt, iodine, and molybdenum had little effect on the induction of embryogenic epiblast callus. By contrast there was a marked increase in the formation of white structures on the medium without any microelements or, specifically without addition of zinc. Since the formation of typical embryoids of wheat is associated with the formation of white structures, our result highlights the importance of certain microelements on somatic embryogenesis of wheat.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS medium Murashige & Skoog medium     

Parasitism: A cryptic determinant of animal community structure

Traditionally, the major biotic determinants of animal community structure were assumed to be competition and predation. Recent theoretical, experimental and field studies indicate that parasitism is also important. The spectrum of effects that parasites have on host individuals is as broad as the range of parasite-host interactions. This review considers evidence for direct effects of parasites on fecundity and survival that influence host demography, and for indirect effects on host physiology and behavior that interface with competition and predation.     

Construction and expression of nonsense suppressor tRNAs which function in plant cells

An Arabidopsis thaliana L. DNA containing the tRNA(TrpUGG) gene was isolated and altered to encode the amber suppressor tRNA(TrpUAG) or the ochre suppressor tRNA(TrpUAA). These DNAs were electroporated into carrot protoplasts and tRNA expression was demonstrated by the translational suppression of amber and ochre nonsense mutations in the chloramphenicol acetyltransferase (CAT) reporter gene. DNAs encoding tRNA(TrpUAG) and tRNA(TrpUAA) nonsense suppressor tRNAs caused suppression of their cognate nonsense codons in CAT mRNAs, with the tRNA(TrpUAG) gene exhibiting the greater suppression under optimal conditions for expression of CAT. The development of these translational suppressors which function in plant cells facilitates the study of plant tRNA gene expression and will make possible the manipulation of plant protein structure and function.     

Deregulation of arginine biosynthesis in Synechococcus sp. PCC7942

Summary In order to deregulate arginine biosynthesis in Synechococcus sp. PCC7942, d-arginine-resistant cell lines were selected following ethyl methanesulfonate mutagenesis of wild-type (WT) cells. Three of these arginine-producing mutant (APM) cell lines, APM1, APM31 and APM40, were putative regulatory mutants based upon secretion of l-arginine into their growth medium. HPLC of lyophilized post-harvest supernatants of APM 31 and 40 resolved two predominant amino acids, arginine and citrulline. In-vitro activity of N-acetylglutamate kinase (NAGK), the proposed regulatory enzyme of the arginine pathway, was about 100-fold less sensitive to l-arginine inhibition in extracts from APM 31 and 40 than the enzyme in WT extracts. The enzyme from APM 1 was 20-fold less sensitive to l-arginine inhibition than WT. The most likely site of mutation in each of the APM cell lines is in the gene for NAGK, rendering the enzymes insensitive to l-arginine feedback control. These strains can be utilized for the phototrophic production of arginine. Offprint requests to: S. E. Bingham     

Functional regulation of brain pyruvate dehydrogenase: Postnatal development, anesthesia and food-deprivation

The activity of brain pyruvate dehydrogenase complex (PDHC), is regulated by reversible phosphorylation of the alpha subunit of the E1 component (pyruvate dehydrogenase, EC 1.2.4.1) of PDHC. Using an in vitro back-titration assay, we have evaluated the postnatal development of E1 phosphorylation, as well as the effects of acute pentobarbital administration and food-deprivation on cerebral cortical E1 phosphorylation in synaptosomal and free mitochondrial compartments of the albino rat. Between birth and postnatal day 25, the back-titration phosphorylation increased ca 4-fold, with the largest increase occurring between days 15 and 20. The phosphorylation of E1 in the synaptosomal, but not free mitochondrial fraction, was decreased during pentobarbital anesthesia. Following 72 h of food-deprivation, E1 phosphorylation was decreased in both subcellular fractions.

The postnatal increase in E1 back-titration phosphorylation is consistent with and similar in magnitude to previously reported increases in the specific enzymatic activity of PDHC. These results also highlight the potential importance of localized subcellular alterations in mitochondrial metabolism and further validate the back-titration phosphorylation of E1 as a valuable tool for the study of central nervous system PDHC metabolism.     

Assumption validity in human optimal foraging: The Barí hunters of Venezuela as a test case

Optimal foraging theory is being used increasingly as a means of understanding human foraging behavior. One of the central assumptions of optimal foraging theory is that prey items or patches are encountered sequentially and as a Poisson process. Using empirical data gathered on the Barí hunters of Venezuela, we assess the validity of this central assumption. We compare our observed distribution of encounter frequencies with an expected Poisson distribution, utilizing chisquare tests and graphic representations. The results are strikingly consonant with the expected Poisson distribution and lend support to the applicability of optimal foraging models to human hunting behavior.     

Site-specific creation of uridine from cytidine in apolipoprotein B mRNA editing.

Human apolipoprotein (apo) B mRNA is edited in a tissue specific reaction, to convert glutamine codon 2153 (CAA) to a stop translation codon. The RNA editing product templates and hybridises as uridine, but the chemical nature of this reaction and the physical identity of the product are unknown. After editing in vitro of [32P] labelled RNA, we are able to demonstrate the production of uridine from cytidine; [alpha 32P] cytidine triphosphate incorporated into RNA gave rise to [32P] uridine monophosphate after editing in vitro, hydrolysis with nuclease P1 and thin layer chromatography using two separation systems. By cleaving the RNA into ribonuclease T1 fragments, we show that uridine is produced only at the authentic editing site and is produced in quantities that parallel an independent primer extension assay for editing. We conclude that apo B mRNA editing specifically creates a uridine from a cytidine. These observations are inconsistent with the incorporation of a uridine nucleotide by any polymerase, which would replace the alpha-phosphate and so rule out a model of endonucleolytic excision and repair as the mechanism for the production of uridine. Although transamination and transglycosylation remain to be formally excluded as reaction mechanisms our results argue strongly in favour of the apo B mRNA editing enzyme as a site-specific cytidine deaminase.     

Construction of synthetic genes using PCR after automated DNA synthesis of their entire top and bottom strands.

A new method is described for the direct construction of synthetic genes by applying a modified version of the polymerase chain reaction (PCR) to crude oligonucleotide mixtures made by automated solid phase DNA synthesis. Construction of the HIV-1 393 bp rev gene and the 655 bp nef gene by this method is illustrated. The sequences for the entire top and bottom strands of rev were each programmed into an automated DNA synthesizer. Following DNA synthesis, the two crude oligonucleotide solutions were mixed together, specific primers were added, and the target gene was amplified by a modified PCR technique. Although the longer (greater than 200 bases) strands comprise a very small percentage of the total DNA after solid phase synthesis, this method uses PCR to 'find' and amplify such strands to create the target gene. The rev gene constructed by this method was found to contain 4 sequence errors, which were subsequently corrected by site-directed mutagenesis. In order to evaluate the source of sequence errors, several nef genes were made from the top and bottom strand DNA synthesis solutions using independent PCR's. Results suggest that sequence errors arose from both DNA synthesis and PCR. The utility of this method in producing a functional gene is demonstrated by expression of rev in E.coli.