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.     

Genetic mapping of ossification of the posterior longitudinal ligament of the spine.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is recognized as a common disorder among Japanese and throughout Asia. Estimates of its prevalence are in the range of 1. 9%-4.3%. Although its etiology is thought to involve a multiplicity of factors, epidemiological and family studies strongly implicate genetic susceptibility in the pathogenesis of OPLL. In this study we report an identification of a predisposing locus for OPLL, on chromosome 6p, close to the HLA complex. The evidence for this localization is provided by a genetic-linkage study of 91 affected sib pairs from 53 Japanese families. In this sib-pair study, D6S276, a marker lying close to the HLA complex, gives evidence for strongly significant linkage (P = .000006) to the OPLL locus. A candidate gene in the region, that for collagen 11A2, was analyzed for the presence of molecular variants in affected probands. Of 19 distinct variants identified, 4 showed strong statistical associations with OPLL (highest P = .0004). These observations of linkage and association, taken together, show that a genetic locus for OPLL lies close to the HLA region, on chromosome 6p.     

Identification of the sex pheromone of Ostrinia palustralis

By means of gas chromatography with electroantennographic detection, gas chromatography-mass spectrometry and a series of bioassays, (E)-11-tetradecenyl acetate (E11-14:OAc) and (Z)-11-tetradecenyl acetate (Z11-14:OAc) at a ratio of 99:1 were identified as female sex pheromone components of Ostrinia palustralis. The average amounts of E11- 14:OAc and Z11-14:OAc in a single sex pheromone gland were 37.2±24.4 ng and 0.3±0.2 ng, respectively. In a wind-tunnel bioassay, the binary blend of E11- and Z11-14:OAc elicited the same male behavioral responses as did virgin females.     

In Vitro Evaluation of 11C-Labeled (S)-Nicotine, (S)-3-Methyl-5-(1-Methyl-2-Pyrrolidinyl)isoxazole, and (R,S)-1-Methyl-2-(3-Pyridyl)azetidine as Nicotinic Receptor Ligands for Positron Emission Tomography Studies

Abstract: The binding characteristics of the novel ¹¹C-labeled nicotinic ligands (R,S)-1-methyl-2-(3-pyridyl) azetidine (MPA) and (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole (ABT-418) were investigated in comparison with those of (S)-[¹¹C]nicotine in vitro in the rat brain to be able to predict the binding properties of the new ligands for positron emission tomography studies in vivo. The data from time-resolved experiments for all ligands indicated fast binding kinetics, with the exception of a slower dissociation of [¹¹C]MPA in comparison with (S)-[¹¹C]nicotine and [¹¹C]ABT-418. Saturation experiments revealed for all ligands two nicotinic receptor binding sites with affinity constants (K_D values) of 2.4 and 560 nM and binding site densities (B_max values) of 65.5 and 223 fmol/mg of protein for (S)-[¹¹C]nicotine, K_D values of 0.011 and 2.2 nM and B_max values of 4.4 and 70.7 fmol/mg of protein for [¹¹C]MPA, and K_D values of 1.3 and 33.4 nM and B_max values of 8.8 and 69.2 fmol/mg of protein for [¹¹C]ABT-418. In competing with the ¹¹C-ligands, epibatidine was most potent, followed by cytisine. A different rank order of potencies was found for (?)-nicotine, (+)-nicotine, MPA, and ABT-418 displacing each of the ¹¹C-ligands. Autoradiograms displayed a similar pattern of receptor binding for all ligands, whereby [¹¹C]MPA showed the most distinct binding pattern and the lowest nonspecific binding. We conclude that the three ¹¹C-labeled nicotinic ligands were suitable for characterizing nicotinic receptors in vitro. The very high affinity of [¹¹C]MPA to nicotinic acetylcholine receptors, its low nonspecific binding, and especially the slower dissociation kinetics of the [¹¹C]MPA from the putative high-affinity nicotinic acetylcholine receptor binding site compared with (S)-[¹¹C]nicotine and [¹¹C]ABT-418 raise the level of interest in [¹¹C]MPA for application in positron emission tomography.     

Effects of process errors on the production of ethanol by Escherichia coli KO11

Escherichia coli KO11 was previously constructed for the production of ethanol from both hexose and pentose sugars in hemicellulose hydrolysates by inserting the Zymomonas mobilis genes encoding pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB). This biocatalyst appears relatively resistant to potential process errors during fermentation. Antibiotics were not required to maintain the maximum catabolic activity of KO11 even after deliberate contamination with up to 10% soil. Fermentations exposed to extremes of temperature (2 h at 5°C or 50°C) or pH (2 h at pH 3 or pH 10) recovered after re-adjustment to optimal fermentation conditions (35°C, pH6) although longer times were required for completion in most cases. Ethanol yields were not altered by exposure to extremes in temperature but were reduced by exposure to extremes in pH. Re-inoculation with 5% (by volume) from control fermentors reduced this delay after exposure to pH extremes. Received 24 July 1997/ Accepted in revised form 16 April 1998     

sn-1-Acylglycerol-3-phosphate acyltransferase of Escherichia coli causes insertion of cis-11 eicosenoic acid into the sn-2 position of transgenic rapeseed oil

The plsC gene of Escherichia coli encoding sn-1-acylglycerol-3-phosphate acyltransferase was modified by inserting an endoplasmic reticulum retrieval signal to its 3 end and introduced into rapeseed (Brassica napus L.) plants under the control of a napin promotor. In developing seeds from transgenic plants an sn-1-acylglycerol-3-phosphate acyltransferase activity was detectable which showed substrate specificities typical of the E. coli enzyme. Moreover, seed oil from the transformants unlike that from untransformed plants contained substantial amounts of triacylglycerol species esterified with very-long-chain fatty acids at each glycerol position. Analysis of fatty acids at the sn-2 position of triacylglycerol showed hardly any very-long-chain fatty acids in untransformed plants, but in certain transformants these fatty acids were present, namely about 4% erucic acid and 9% eicosenoic acid. These data demonstrate that the bacterial acyltransferase can function in developing rapeseed and alters the stereochemical composition of transgenic rape seed oil by directing very-long-chain fatty acids, especially cis-11 eicosenoic acid, to its sn-2 position.     

Two‐dimensional reference map for the basic proteome of the human dorsolateral prefrontal cortex (dlPFC) of the prefrontal lobe region of the brain

We describe a 2‐DE proteomic reference map containing 227 basic proteins in the dorsolateral prefrontal cortex region of the human brain. Proteins were separated in the first dimension on pH 6–11 IPG strips using paper‐bridge loading and on 12% SDS‐PAGE in the second dimension. Proteins were subsequently identified by MS and spectra were analyzed using an in‐house proteomics data analysis platform, Proline. The 2‐DE reference map is available via the UCD 2‐DE Proteome Database ( http://proteomics‐portal.ucd.ie:8082 ) and can also be accessed via the WORLD‐2DPAGE Portal ( http://www.expasy.ch/world‐2dpage/ ). The associated protein identification data have been submitted to the PRIDE database (accession numbers 10018–10033). Separation of proteins in the basic region resolves more membrane associated proteins relevant to the synaptic pathology central to many neurological disorders. The 2‐DE reference map will aid with further characterisation of neurological disorders such as bipolar and schizophrenia.     

A cytotoxic deoxocucurbitacin from Desfontainia spinosa

A new cytotoxic triterpene, 11-deoxocucurbitacin I was isolated from Desfontainia spinosa. The structure of this compound was elucidated by extensive analysis of the high field ¹H NMR and ¹³C NMR spectra and field desorption mass spectrometry. This is the first example of a cucurbitacin lacking an 11-keto functionality.