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PubMed1988

Morphology, yield and functional integrity of islet-like cell clusters in tissue culture of human fetal pancreata obtained after different means of abortion.

Morphology, yield and function were studied in cultured islet-like cell clusters (ICC) from 140 human fetal pancreata obtained after abortions of different types performed at 11-23 weeks of gestation (12 by hysterotomy, 75 by mechanical dilation and extraction, and 53 induced with prostaglandin). After collagenase digestion and culture in medium supplemented with 10% human serum, up to 2000 free-floating ICC were formed from a single pancreas. Randomly scattered insulin- and glucagon-immunoreactive cells were found in the medullary part of the ICC. More than 100 ICC developed in 100% of the hysterotomies and 87% of the mechanical abortions, but in only 53% of the prostaglandin-induced abortions. Insulin and glucagon levels in the culture medium decreased rapidly during the first 7 days of culture, but then remained stable for at least 31 days. The hysterotomy-derived ICC responded to 10 mmol/l theophylline plus 20 mmol/l glucose by a 12.2 +/- 3.1 (SEM, N = 7) fold increase in insulin release, as compared with a 5.4 +/- 0.9 fold response of the prostaglandin ICC (N = 16; P less than 0.02). Despite the low proportion of B-cells, (pro)insulin biosynthesis accounted for 10% of the total protein biosynthesis in low (2 mmol/l) glucose. In conclusion, the yield and viability of the ICC were clearly better, if prostaglandin had not been used for the induction of the abortion.

Abortion, Induced↗
PubMed1997

Regulation, replication, and integration functions of the Vibrio cholerae CTXphi are encoded by region RS2.

CTXphi is a filamentous phage that encodes cholera toxin, one of the principal virulence factors of Vibrio cholerae. CTXphi is unusual among filamentous phages because it can either replicate as a plasmid or integrate into the V. cholerae chromosome at a specific site. The CTXphi genome has two regions, the 'core' and RS2. Integrated CTXphi is frequently flanked by an element known as RS1 which is related to RS2. The nucleotide sequences of RS2 and RS1 were determined. These related elements contain three nearly identical open reading frames (ORFs), which in RS2 were designated rstR, rstA2 and rstB2. RS1 contains an additional ORF designated rstC. Functional analyses indicate that rstA2 is required for CTXphi replication and rstB2 is required for CTXphi integration. The amino terminus of RstR is similar to the amino termini of other phage-encoded repressors, and RstR represses the expression of rstA2. Although genes with related functions are clustered in the genome of CTXphi in a way similar to those for other filamentous phages, the CTXphi RS2-encoded gene products mediating replication, integration and repression appear to be novel.

Amino Acid Sequence↗
PubMed2002

An integrated, functionally annotated gene map of the DXS8026-ELK1 interval on human Xp11.3-Xp11.23: potential hotspot for neurogenetic disorders.

Human chromosome Xp11.3-Xp11.23 encompasses the map location for a growing number of diseases with a genetic basis or genetic component. These include several eye disorders, syndromic and nonsyndromic forms of X-linked mental retardation (XLMR), X-linked neuromuscular diseases and susceptibility loci for schizophrenia, type 1 diabetes, and Graves' disease. We have constructed an approximately 2.7-Mb high-resolution physical map extending from DXS8026 to ELK1, corresponding to a genetic distance of approximately 5.5 cM. A combination of chromosome walking and sequence-tagged site (STS)-content mapping resulted in an integrated framework and transcript map, precisely positioning 10 polymorphic microsatellites (one of which is novel), 16 ESTs, and 12 known genes (RP2, PCTK1, UHX1, UBE1, RBM10, ZNF157, SYN1, ARAF1, TIMP1, PFC, ELK1, UXT). The composite map is currently anchored with 89 STSs to give an average resolution of approximately 1 STS every 30 kb. By a combination of EST database searches and in silico detection of UniGene clusters within genomic sequence generated from this template map, we have mapped several novel genes within this interval: a Na+/H+ exchanger (SLC9A7), at least two zincfinger transcription factors (KIAA0215 and Hs.68318), carbohydrate sulfotransferase-7 (CHST7), regucalcin (RGN), inactivation-escape-1 (INE1), the human ortholog of mouse neuronal protein 15.6, and four putative novel genes. Further genomic analysis enabled annotation of the sequence interval with 20 predicted pseudogenes and 21 UniGene clusters of unknown function. The combined PAC/BAC transcript map and YAC scaffold presented here clarifies previously conflicting data for markers and genes within the Xp11.3-Xp11.23 interval and provides a powerful integrated resource for functional characterization of this clonally unstable, yet gene-rich and clinically significant region of proximal Xp.

Chromosome Mapping↗
PubMed2006

Purdue ionomics information management system. An integrated functional genomics platform.

The advent of high-throughput phenotyping technologies has created a deluge of information that is difficult to deal with without the appropriate data management tools. These data management tools should integrate defined workflow controls for genomic-scale data acquisition and validation, data storage and retrieval, and data analysis, indexed around the genomic information of the organism of interest. To maximize the impact of these large datasets, it is critical that they are rapidly disseminated to the broader research community, allowing open access for data mining and discovery. We describe here a system that incorporates such functionalities developed around the Purdue University high-throughput ionomics phenotyping platform. The Purdue Ionomics Information Management System (PiiMS) provides integrated workflow control, data storage, and analysis to facilitate high-throughput data acquisition, along with integrated tools for data search, retrieval, and visualization for hypothesis development. PiiMS is deployed as a World Wide Web-enabled system, allowing for integration of distributed workflow processes and open access to raw data for analysis by numerous laboratories. PiiMS currently contains data on shoot concentrations of P, Ca, K, Mg, Cu, Fe, Zn, Mn, Co, Ni, B, Se, Mo, Na, As, and Cd in over 60,000 shoot tissue samples of Arabidopsis (Arabidopsis thaliana), including ethyl methanesulfonate, fast-neutron and defined T-DNA mutants, and natural accession and populations of recombinant inbred lines from over 800 separate experiments, representing over 1,000,000 fully quantitative elemental concentrations. PiiMS is accessible at www.purdue.edu/dp/ionomics.

Arabidopsis↗
PubMed1990

Analysis of mutations in the integration function of Moloney murine leukemia virus: effects on DNA binding and cutting.

The 3' terminus of the pol gene of Moloney murine leukemia virus encodes the integration (IN) protein, required for the establishment of the integrated provirus. A series of six linker insertion mutations and two single-base substitutions were generated within the region encoding the IN protein. Mutations were initially generated within an Escherichia coli plasmid expressing the IN protein, and the resulting variants were assayed for DNA-binding activity. Mutations which altered conserved cysteine residues within a potential DNA finger-binding motif resulted in lower or variable DNA binding, which appeared to be the result of variable protein folding. Upon renaturation, these proteins were able to nonspecifically bind DNA in a manner similar to that of the other mutant IN proteins and the parent. When reconstructed back into full-length virus, seven of the eight mutations were lethal. All mutants produced a stable IN protein in virions and mediated normal conversion of the retroviral RNA to its three DNA forms. Fine-structure analysis of the linear double-stranded viral DNA indicated that all seven lethal alterations within the IN protein blocked the formation of the 3' recessed termini that normally precedes integration.

Amino Acid Sequence↗
PubMed1989

Functional integrity of sympathetic efferent postganglionic axons in a region of stunned myocardium.

In order to investigate whether brief, repeated episodes of acute myocardial ischemia impair the function of the intrinsic sympathetic efferent post ganglionic cardiac nerves, 12 cycles of left anterior descending coronary artery occlusions (5 mins) and reperfusions (10 mins) were successfully performed in 10 dogs. Following each brief occlusion percentage systolic shortening in the ventral myocardium was reduced, consistent with myocardial stunning. Stellate ganglion stimulation increased intramyocardial pressures in the ventral and lateral regions of the left ventricle before and after each of the artery occlusions in which stimulations were performed. Isoproterenol and tyramine also augmented intramyocardial pressures in those regions. When the coronary artery was occluded permanently in seven of the dogs tested, stellate ganglion stimulation continued to augment intramyocardial pressure in the ventral region of the left ventricle for up to 35 mins. These results indicate that following repetitive episodes of brief occlusion of a coronary artery that are reported to 'stun' the involved myocardium in a transmural distribution, sympathetic efferent post ganglionic axons can augment inotropism in the affected zone. Furthermore, when a coronary artery is totally occluded thereafter for up to 35 mins these efferent sympathetic post ganglionic nerves continue to function. It is concluded that sympathetic efferent post ganglionic neurons innervating the ventricle can augment cardiac inotropism in a region of stunned myocardium.

Animals↗
PubMedDate not supplied

Eye position signals in the vestibular nuclei: consequences for models of integrator function.

Recordings from neurons in the vestibular nuclei indicate that the cells that carry eye position signals encode the position of a single eye (either ipsilateral or contralateral) during both conjugate and vergence eye movements. The fact that the vestibular nuclei are aware of the positions of each eye is not surprising as the otolith-based linear vestibulo-ocular reflex is known to change its behaviour as a function of uniocular eye position. This result suggests that the signal coming from the oculomotor velocity-to-position integrator specifies the position of each eye during vergence movements and thus must receive a vergence velocity input along with its conjugate velocity inputs. As there is no vergence system in laterally eyed animals, we have proposed two possible models of integrator arrangement that could have developed from conjugate directional (rather than uniocular) integrators in lower animals without frontally mounted eyes. Both of these models explain the existence of near-response cells and produce the required bidirectional gaze paretic nystagmus following unilateral lesions of one integrator. The models also make specific and different predictions concerning the effects of unilateral integrator lesions on the behaviour of the vergence system and thus make suggestions for further experiments.

Animals↗
PubMed1996

Differential effect of Ca2+ on oxidant-induced lethal cell injury and alterations of membrane functional integrity in renal cortical slices.

This study was undertaken to examine if modulations of intracellular and extracellular Ca2+ affect the lethal cell injury and impairment of membrane transport function induced by oxidants in rabbit renal cortical slices. The oxidant t-butylhydroperoxide (t-BHP) and H2O2 increased lactate dehydrogenase (LDH) release and inhibited PAH uptake in a dose-dependent manner, but the potency of H2O2 was 100 times lower than that of t-BHP. Catalase prevented the effect of H2O2 but not that of t-BHP, suggesting that lower potency of H2O2 is attributed to the endogenous catalase activity. t-BHP induced lipid peroxidation and inhibited microsomal (Na+)-(K+)-ATPase activity. Omission of Ca2+ from the medium or addition of Ca2+ channel blockers (verapamil, diltiazem, and nifedipine) prevented the oxidant-induced LDH release. Similar effect was observed by addition of La3+. Buffering intracellular Ca2+ with BAPTA/AM decreased the oxidant-induced LDH release. However, the oxidant-induced impairment in PAH uptake was not altered under the same conditions. Also, the inhibition of microsomal (Na+)-(K+)-ATPase activity by t-BHP was not affected by verapamil, La3+, and BAPTA/AM. Dithiothreitol and glutathione prevented the oxidant-induced LDH release and reduction of PAH uptake and impeded the oxidant-induced inhibition of (Na+)-(K+)-ATPase activity and lipid peroxidation. Effects of t-BHP on TEA uptake were similar to those on PAH uptake. Modulations of intracellular or extracellular Ca2+ had little effect on the oxidant-induced lipid peroxidation. Glycine did not exert protective effect against the oxidant-induced cell injury. These results suggest strongly that Ca2+ plays an important role in the oxidant-induced LDH release but not in the oxidant-induced alterations of membrane transport function in rabbit renal cortical slices. The role of Ca2+ in oxidant-induced LDH release is not apparently associated with peroxidation of membrane lipid.

Animals↗
PubMed1985

Degradation of functional integrity during long-term storage of a freeze-dried biological membrane.

Trehalose, and to some extent a few other carbohydrates, is capable of stabilizing the structure and function of isolated biological membranes during lyophilization. In this paper the results of investigations into the long-term stability of the lyophilized membrane-carbohydrate mixtures were reported. The effects of varying water content, oxygen level, and light on the rates of oxidation, browning, and degradation of biological activity were reported. The efficiency with which three carbohydrates stabilized membrane structure was also reported, with glucose shown to be less efficient than maltose or trehalose. Increased water content accelerated loss of biological activity, possibly because, under the same conditions, nonenzymatic browning and photooxidation were accelerated also. Glucose-containing samples were especially unstable at elevated humidities. Efficiency of preservation could be maximized by storage under conditions of low oxygen, low humidity, and dark, and by the inclusion of high levels of trehalose.

Animals↗
PubMed1993

Genetic organization and sequence of the region encoding integrative functions from Lactobacillus gasseri temperate bacteriophage phi adh.

A 2.0-kb fragment from the Lactobacillus gasseri temperate bacteriophage phi adh contained the essential genetic determinants for site-specific integration. The nucleotide sequence of this fragment was determined. An open reading frame (intG), which adjoined the phage attachment site (attP), encoded a deduced protein related to the integrase family. The organization of this region was comparable to other phage site-specific recombination systems.

Amino Acid Sequence↗
PubMed2004

Phenobarbital responsiveness as a uniquely sensitive indicator of hepatocyte differentiation status: requirement of dexamethasone and extracellular matrix in establishing the functional integrity of cultured primary rat hepatocytes.

We used a serum-free, highly defined primary hepatocyte culture model to investigate the mechanisms whereby dexamethasone (Dex) and extracellular matrix (ECM) coordinate cell differentiation and transcriptional responsiveness to the inducer, phenobarbital (PB). Low nanomolar levels of Dex and dilute concentrations of ECM overlay were essential in the maintenance of normal hepatocyte physiology, as assessed by cell morphology, LDH release, expression of the hepatic nuclear factors C/EBPalpha, -beta, -gamma, HNF-1alpha, -1beta, -4alpha, and RXRalpha, expression of prototypical hepatic marker genes, including albumin and transferrin, and ultimately, cellular capacity to respond to PB. The loss of hepatocyte integrity produced by deficiency of these components correlated with the activation of several stress signaling pathways including the MAPK, SAPK/JNK, and c-Jun signaling pathways, with resulting nuclear recruitment of the activated protein-1 (AP-1) complex. In Dex-deficient cultures, normal cellular function, including the PB induction response, was largely restored in a dose-dependent manner by reintroduction of nanomolar additions of the hormone, in the presence of ECM. Our results demonstrate critical and cooperative roles for Dex and ECM in establishing hepatocyte integrity and in the coordination of an array of liver-specific functions. These studies further establish the PB gene induction response as an exceptionally sensitive indicator of hepatocyte differentiation status.

Albumins↗
PubMed1985

Hyaluronidase does not prevent deterioration of vascular functional integrity during reperfusion after no-flow ischemia in isolated rabbit hearts.

Effects of hyaluronidase on myocardial water content and distribution, and on coronary vascular hemodynamics and endothelial cell transport function were assessed in isolated rabbit hearts during 3.5 hours of reperfusion after 30 minutes of global, no-flow ischemia. In nonischemic control hearts, perfusion pressure, left ventricular end-diastolic pressure, maximum +dP/dt, and intravascular clearance of radiolabeled albumin remained constant during 5 hours of continuous perfusion, while the mean-transit time and vascular into extravascular space clearance of radiolabeled albumin increased 1.5X and 2.5X baseline, respectively. During reperfusion after 30 minutes of no flow, perfusion pressure increased 53% and interstitial fluid volume increased 2-fold, while left ventricular end-diastolic pressure and maximum +dP/dt returned to control levels. The rate of intravascular clearance of radiolabeled albumin decreased 38%, and the mean-transit time and vascular-into-extravascular space clearance of albumin increased approximately 3X and 5X baseline, respectively. Hyaluronidase blocked the ischemia-reperfusion-induced increases in total water content and in interstitial fluid volume and reduced the increases in perfusion pressure and mean-transit time of radiolabeled albumin by 40% and 45%, respectively, but did not prevent the increase in albumin vascular-into-extravascular space clearance and the decrease in albumin clearance from the coronary vasculature. These findings indicate that hyaluronidase does not prevent ischemia-reperfusion-induced increases in albumin permeation of the coronary vasculature, and suggest that its protective effect on ischemic myocardium is mediated, instead, by reducing interstitial edema and vascular resistance.

Animals↗
PubMed2000

Integrating functional genomic information into the Saccharomyces genome database.

The Saccharomyces Genome Database (SGD) stores and organizes information about the nearly 6200 genes in the yeast genome. The information is organized around the 'locus page' and directs users to the detailed information they seek. SGD is endeavoring to integrate the existing information about yeast genes with the large volume of data generated by functional analyses that are beginning to appear in the literature and on web sites. New features will include searches of systematic analyses and Gene Summary Paragraphs that succinctly review the literature for each gene. In addition to current information, such as gene product and phenotype descriptions, the new locus page will also describe a gene product's cellular process, function and localization using a controlled vocabulary developed in collaboration with two other model organism databases. We describe these developments in SGD through the newly reorganized locus page. The SGD is accessible via the WWW at http://genome-www.stanford.edu/Saccharomyces/

Database Management Systems↗
PubMed2006

Structural and functional integrity of the coxsackievirus B3 oriR: spacing between coaxial RNA helices.

The enterovirus oriR is composed of two helices, X and Y, anchored by a kissing (K) interaction. For proper oriR function, certain areas of these helices should be specifically oriented towards each other. It was hypothesized that the single-stranded nucleotides bridging the coaxial helices (Y-X and K-Y linkers) are important to determine this orientation. Spatial changes were introduced by altering the linker length between the helices of the coxsackievirus B3 oriR. Changing the linker lengths resulted in defective RNA replication, probably because of an altered oriR geometry. The identity of the linker residues also played a role, possibly because of sequence-specific ligand recognition. Although each point mutation altering the primary sequence of the Y-X spacer resulted in defective growth at 36 degrees C, the mutations had a wild-type phenotype at 39 degrees C, indicating a cold-sensitive phenotype. The results show that the intrinsic connection between oriR structure and function is fine-tuned by the spacing between the coaxial RNA helices.

Base Sequence↗
PubMed1986

Sustained and transient temporal integration functions depend on spatial frequency, not grating area.

Contrast sensitivity for sinusoidal gratings was measured by a two-alternative forced-choice method and plotted as a function of duration. Gratings of large or small area and low or high spatial frequency (0.75 or 8.25 c/deg) were used. The temporal integration curve was much flatter in the low spatial frequency conditions than in the high frequency conditions, but its shape did not depend on the area of the grating. The results support the established view that the visual system exhibits transient temporal properties at low spatial frequencies and sustained properties at higher frequencies, but disconfirm the recent proposal that temporal integration depends on the area of the grating (expressed in square-cycles) rather than its spatial frequency.

Fovea Centralis↗
PubMed1999

Amino acid residues involved in the functional integrity of Escherichia coli methionine aminopeptidase.

Amino acid residues in the metal-binding and putative substrate-binding sites of Escherichia coli methionine aminopeptidase (MAP) were mutated, and their effects on the function of the enzyme were investigated. Substitution of any amino acid residue at the metal-binding site resulted in complete loss of the two cobalt ions bound to the protein and diminished the enzyme activity. However, only Cys70 and Trp221 at the putative substrate-binding site are involved in the catalytic activity of MAP. Changing either of them caused partial loss of enzyme activity, while mutations at both positions abolished MAP function. Both residues are found to be conserved in type I but not type II MAPs.

Amino Acid Substitution↗