A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

Multiple effects of genetic background on variegated transgene expression in mice

BLG/7 transgenic mice express an ovine beta-lactoglobulin transgene during lactation. Unusually, transgene expression levels in milk differ between siblings. This variable expression is due to variegated transgene expression in the mammary gland and is reminiscent of position-effect variegation. The BLG/7 line was created and maintained on a mixed CBA x C57BL/6 background. We have investigated the effect on transgene expression of backcrossing for 13 generations into these backgrounds. Variable transgene expression was observed in all populations examined, confirming that it is an inherent property of the transgene array at its site of integration. There were also strain-specific effects on transgene expression that appear to be independent of the inherent variegation. The transgene, compared to endogenous milk protein genes, is specifically susceptible to inbreeding depression. Outcrossing restored transgene expression levels to that of the parental population; thus suppression was not inherited. Finally, no generation-dependent decrease in mean expression levels was observed in the parental population. Thus, although the BLG/7 transgene is expressed in a variegated manner, there was no generation-associated accumulated silencing of transgene expression.     

Culture and Function of Electrofusion-Derived Clonal Insulin-Secreting Cells Immobilized on Solid and Macroporous Microcarrier Beads

In view of the advantages of the bulk production of clonal pancreaticbeta cells, an investigation was made of the growth and insulin secretoryfunctions of an electrofusion-derived cell line (BRIN-BD11) immobilizedon a solid microcarrier, cytodex-1 or a macroporous microcarrier,cultispher-G. For comparison, similar tests were performed usingBRIN-BD11 cells present in single cell suspensions or allowed toform pseudoislets. Similar growth profiles were recorded for eachmicrocarrier with densities of 4.4×10⁵±0.3 cells/ml and4.2×10⁵±0.2 cells/ml achieved using cytodex-1 andcultispher-G, respectively. Cell viability began to decline on day 5 ofculture. Insulin concentration in the culture medium reached a peak of26±2.0 ng/ml and 24±2.2 ng/ml for cells grown oncytodex-1 and cultispher-G, respectively. Cells grown on both types ofmicrocarrier showed a significant 1.5–1.8-fold acuteinsulin-secretory response to 16.7 mmol/l glucose. L-alanine (10 mmol/l) andL-arginine (10 mmol/l) also induced significant 3–4 fold increasesof insulin release. BRIN-BD11 cells immobilized on cytodex-1 or cultispher-Gout-performed single cell suspensions and pseudoislets in terms ofinsulin-secretory responses to glucose and amino acids. A 1.3-fold,2.2-fold and 1.7-fold stimulation of insulin secretion was observed forglucose, L-alanine and L-arginine respectively in single cellsuspensions. Corresponding increases for pseudoislets were1.6–1.8-fold for L-alanine and L-arginine, with no significantresponse to glucose alone. These data indicate the utility ofmicro-carriers for the production of functioning clonal beta cells.     

Discrimination of grasshopper (Orthoptera: Acrididae) diet and niche overlap using next‐generation sequencing of gut contents

Species of grasshopper have been divided into three diet classifications based on mandible morphology: forbivorous (specialist on forbs), graminivorous (specialist on grasses), and mixed feeding (broad‐scale generalists). For example, Melanoplus bivittatus and Dissosteira carolina are presumed to be broad‐scale generalists, Chortophaga viridifasciata is a specialist on grasses, and Melanoplus femurrubrum is a specialist on forbs. These classifications, however, have not been verified in the wild. Multiple specimens of these four species were collected, and diet analysis was performed using DNA metabarcoding of the gut contents. The rbcLa gene region was amplified and sequenced using Illumina MiSeq sequencing. Levins' measure and the Shannon–Wiener measure of niche breadth were calculated using family‐level identifications and Morisita's measure of niche overlap was calculated using operational taxonomic units (OTUs). Gut contents confirm both D. carolina and M. bivittatus as generalists and C. viridifasciata as a specialist on grasses. For M. femurrubrum, a high niche breadth was observed and species of grasses were identified in the gut as well as forbs. Niche overlap values did not follow predicted patterns, however, the low values suggest low competition between these species.     

Cell-to-cell communication and cellular environment alter the somatostatin status of delta cells

Introduction

Somatostatin, released from pancreatic delta cells, is a potent paracrine inhibitor of insulin and glucagon secretion. Islet cellular interactions and glucose homeostasis are essential to maintain normal patterns of insulin secretion. However, the importance of cell-to-cell communication and cellular environment in the regulation of somatostatin release remains unclear.

Methods

This study employed the somatostatin-secreting TGP52 cell line maintained in DMEM:F12 (17.5 mM glucose) or DMEM (25 mM glucose) culture media. The effect of pseudoislet formation and culture medium on somatostatin content and release in response to a variety of stimuli was measured by somatostatin EIA. In addition, the effect of pseudoislet formation on cellular viability (MTT and LDH assays) and proliferation (BrdU ELISA) was determined.

Results

TGP52 cells readily formed pseudoislets and showed enhanced functionality in three-dimensional form with increased E-cadherin expression irrespective of the culture environment used. However, culture in DMEM decreased cellular somatostatin content (P < 0.01) and increased somatostatin secretion in response to a variety of stimuli including arginine, calcium and PMA (P < 0.001) when compared with cells grown in DMEM:F12. Configuration of TGP52 cells as pseudoislets reduced the proliferative rate and increased cellular cytotoxicity irrespective of culture medium used.

Conclusions

Somatostatin secretion is greatly facilitated by cell-to-cell interactions and E-cadherin expression. Cellular environment and extracellular glucose also significantly influence the function of delta cells.     

Major metabolic homocysteine-derivative, homocysteine thiolactone, exerts changes in pancreatic beta-cell glucose-sensing, cellular signal transduction and integrity

Homocysteine can be converted to its reactive thioester, homocysteine thiolactone. Cytotoxic properties of these amino thiols have been attributed to protein homocysteinylation, increased oxidative stress, DNA damage and apoptosis. This study used pancreatic BRIN-BD11 beta-cells to examine functional defects caused by acute and long-term exposure to homocysteine thiolactone in comparison with homocysteine. Acute and long-term exposure to both agents caused concentration-dependent inhibitions of glucose-induced insulin secretion while impairing the insulin-secretory responses to alanine, KCl, elevated Ca(2+), forskolin and PMA. Acute exposures also caused significant reduction in the amplitude of KCl-induced membrane depolarisation but no effects on changes of intracellular Ca(2+) induced by alanine or KCl. Cellular insulin content and DNA damage were not altered following culture, however, there were early signs of apoptosis consistent with impaired cellular integrity. In conclusion, exposure to homocysteine thiolactone, like homocysteine, induced beta-cell dysfunction and demise by mechanisms independent of changes in membrane potential and [Ca(2+)](i).     

Differential Acute and Long Term Actions of Succinic Acid Monomethyl Ester Exposure on Insulin-Secreting BRIN-BD11 Cells

Esters of succinic acid are potent insulin secretagogues, and have been proposed as novel antidiabetic agents for type 2 diabetes. This study examines the effects of acute and chronic exposure to succinic acid monomethyl ester (SAM) on insulin secretion, glucose metabolism and pancreatic beta cell function using the BRIN-BD11 cell line. SAM stimulated insulin release in a dose-dependent manner at both non-stimulatory (1.1mM) and stimulatory (16.7mM) glucose. The depolarizing actions of arginine also stimulated a significant increase in SAM-induced insulin release but 2-ketoisocaproic acid (KIC) inhibited SAM induced insulin secretion indicating a possible competition between the preferential oxidative metabolism of these two agents. Prolonged (18hour) exposure to SAM revealed decreases in the insulin-secretory responses to glucose, KIC, glyceraldehyde and alanine. Furthermore, SAM diminished the effects of nonmetabolized secretagogues arginine and 3-isobutyl-1-methylxanthine (IBMX). While the ability of BRIN-BD11 cells to oxidise glucose was unaffected by SAM culture, glucose utilization was substantially reduced. Collectively, these data suggest that while SAM may enhance the secretory potential of non-metabolized secretagogues, it may also serve as a preferential metabolic fuel in preference to other important physiological nutrients and compromise pancreatic beta cell function following prolonged exposure.     

Functional examination of microencapsulated bioengineered insulin-secreting beta-cells.

Clonal insulin-secreting BRIN-BD11 cells engineered by electrofusion were encapsulated inside natrium alginate beads and cultured in RPMI 1640 culture media. Acute insulin secretory responses to glucose and amino acids were compared between microencapsulated cells and non-encapsulated cells maintained in monolayer culture. Encapsulated cells exhibited a 1.5-fold, 2.9-fold and 4.2-fold increase (P< 0.001) in insulin release in response to 16.7 mmol/l glucose, 10 mmol/l L-arginine and 10 mmol/l L-alanine respectively. Insulin output by non-encapsulated cells was approximately 30% greater but the relative magnitudes of responses were similar. This is the first study to demonstrate the stability of cellular engineered insulin-secreting cells encapsulated in alginate beads, illustrating the utility of this approach for cellular engineering and potential transplantation in diabetes.     

BIOCHEMICAL GENETICS OF MOSQUITOFISH. IV. CHANGES OF ALLELE FREQUENCIES THROUGH TIME AND SPACE

Gene frequency data from samples of Gambusia affinis populations at 76 localities across the Savannah River drainage were used to investigate temporal and spatial patterns in population genetic structure. Localities in the Par Pond system on the Savannah River Plant were sampled in 1971, 1977, and 1979. Allelic frequencies in these populations were generally stable through time, although significant temporal changes were observed among samples from Pond C, an impoundment receiving thermal effluent. Significant spatial heterogeneity in allele frequencies was observed on both microgeographic and regional scales. Populations within the Par Pond system were spatially subdivided at four of the five loci surveyed (mean F_ST = 0.051). Subdivision was even more pronounced when samples from across the Savannah River drainage were compared (mean F_ST = 0.196). A hierarchial analysis of gene diversity (G_ST) demonstrated that most of the genic diversity across the drainage exists as within-subdivision diversity. Even when populations from such contrasting habitats as rivers, creeks, ponds, and reservoirs are compared, an average of only 13% of the total gene diversity was attributed to between-group diversity. Greatest between-group gene diversity was observed when reservoirs were compared with one another. This general pattern of low between-habitat diversity suggests that differential selection pressures are not playing a major role in producing the observed levels of subdivision. In the Par Pond system, neither single locus nor multilocus genetic distances were significantly associated with geographic distance or with its reciprocal. For samples from over the Savannah River drainage, significant correlations between genetic and geographic distance were observed only for the Gpi-2 and Pgm-2 loci. Thus, there was a general lack of concordance between genetic and geographic distances. Spatial autocorrelation demonstrated patterns consistent with Wright's isolation by distance model. Significant positive correlations in allelic frequencies among neighboring populations were observed for five of six alleles; allelic frequencies in more distantly separated populations were typically not correlated.