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Biomedical subjects

D A Gordon

Publications and source records attributed to D A Gordon.

At least 37 records · Page 2Linked to original sources
PubMed1996

A novel abetalipoproteinemia genotype. Identification of a missense mutation in the 97-kDa subunit of the microsomal triglyceride transfer protein that prevents complex formation with protein disulfide isomerase.

The microsomal triglyceride transfer protein (MTP) is a heterodimer composed of the ubiquitous multifunctional protein, protein disulfide isomerase, and a unique 97-kDa subunit. Mutations that lead to the absence of a functional 97-kDa subunit cause abetalipoproteinemia, an autosomal recessive disease characterized by a defect in the assembly and secretion of apolipoprotein B (apoB) containing lipoproteins. Previous studies of abetalipoproteinemic patient, C.L., showed that the 97-kDa subunit was undetectable. In this report, [35S]methionine labeling showed that this tissue was capable of synthesizing the 97-kDa MTP subunit. Electrophoretic analysis showed two bands, one with a molecular mass of the wild type 97-kDa subunit and the other with a slightly lower molecular weight. Sequence analysis of cDNAs from additional intestinal biopsies showed this patient to be a compound heterozygote. One allele contained a perfect in-frame deletion of exon 10, explaining the lower molecular weight band. cDNAs of the second allele were found to contain 3 missense mutations: His297 --> Gln, Asp384 --> Ala, and Arg540 --> His. Transient expression of each mutant showed that only the Arg540 --> His mutant was non-functional based upon its inability to reconstitute apoB secretion in a cell culture system. The other amino acid changes are silent polymorphisms. High level coexpression in a baculovirus system of the wild type 97-kDa subunit or the Arg540 --> His mutant along with human protein disulfide isomerase showed that the wild type was capable of forming an active MTP complex while the mutant was not. Biochemical analysis of lysates from these cells showed that the Arg to His conversion interrupted the interaction between the 97-kDa subunit and protein disulfide isomerase. Replacement of Arg540 with a lysine residue maintained the ability of the 97-kDa subunit to complex with protein disulfide isomerase and form the active MTP holoprotein. These results indicate that a positively charged amino acid at position 540 in the 97-kDa subunit is critical for the productive association with protein disulfide isomerase. Of the 13 mutant MTP 97-kDa subunit alleles described to date, this is the first encoding a missense mutation.

Apolipoproteins B↗
PubMed1996

An inhibitor of the microsomal triglyceride transfer protein inhibits apoB secretion from HepG2 cells.

The microsomal triglyceride (TG) transfer protein (MTP) is a heterodimeric lipid transfer protein that catalyzes the transport of triglyceride, cholesteryl ester, and phosphatidylcholine between membranes. Previous studies showing that the proximal cause of abetalipoproteinemia is an absence of MTP indicate that MTP function is required for the assembly of the apolipoprotein B (apoB) containing plasma lipoproteins, i.e., very low density lipoproteins and chylomicrons. However, the precise role of MTP in lipoprotein assembly is not known. In this study, the role of MTP in lipoprotein assembly is investigated using an inhibitor of MTP-mediated lipid transport, 2-[1-(3, 3-diphenylpropyl)-4-piperidinyl]-2,3-dihydro-1H-isoindol-1-o ne (BMS-200150). The similarity of the IC50 for inhibition of bovine MTP-mediated TG transfer (0.6 microM) to the Kd for binding of BMS-200150 to bovine MTP (1.3 microM) strongly supports that the inhibition of TG transfer is the result of a direct effect of the compound on MTP. BMS-200150 also inhibits the transfer of phosphatidylcholine, however to a lesser extent (30% at a concentration that almost completely inhibits TG and cholesteryl ester transfer). When BMS-200150 is added to cultured HepG2 cells, a human liver-derived cell line that secretes apoB containing lipoproteins, it inhibits apoB secretion in a concentration dependent manner. These results support the hypothesis that transport of lipid, and in particular, the transport of neutral lipid by MTP, plays a critical role in the assembly of apoB containing lipoproteins.

Animals↗
PubMed1996

The microsomal triglyceride transfer protein facilitates assembly and secretion of apolipoprotein B-containing lipoproteins and decreases cotranslational degradation of apolipoprotein B in transfected COS-7 cells.

We studied the role of microsomal triglyceride transfer protein (MTP) in the synthesis, secretion, and cotranslational degradation of apolipoprotein (apo) B using nonhepatic COS-7 cells that expressed C-terminally truncated forms of apoB (from apoB15 to apoB94) with or without the large subunit of human MTP. With the exception of apoB15 and apoB18, secretion of all of the apoB forms was stimulated by expression of MTP, even though a small amount of short apoB forms (</=apoB48) could be secreted by cells transfected with apoB alone. The majority of the apoB protein, including apoB72 and apoB94, was secreted as high density lipoprotein (1.08-1.17 g/ml). Pulse-chase experiments revealed that the secretion efficiency of apoB94 and apoB72 was low (ranging from 2 to 12%). The failure to secrete buoyant lipoproteins and the low secretion efficiency were associated with insufficient lipid synthesis by the cells. The incorporation of [3H]oleate into cellular triglyceride and phosphatidylcholine by COS cells over a 2-h period was 28 and 38%, respectively, of that by rat hepatoma (McA-RH7777) cells. In addition to the desired full-length apoB, cells transfected with large constructs (>/=apoB60) also produced smaller species with a size of approximately220 kDa (designated B48-like protein). Coexpression with MTP decreased formation of the B48-like proteins by 40-60%. The reduction in B48-like protein formation was specific to MTP expression; coexpression with other proteins (e.g. apoA-I or apoB15) did not alter B48-like protein production. Kinetic analysis suggested that B48-like proteins were produced concurrently (cotranslational) with the full-length apoB94 and apoB72 and were not products of post-translational degradation. Although some of the B48-like proteins might be derived from truncated species (approximately 7 kb in size) of apoB mRNA that were found in cells transfected with large apoB constructs, MTP coexpression did not affect the relative levels of the aberrant 7-kb RNA with respect to the full-length mRNA. However, coexpression of MTP decreased the accessibility of apoB to exogenous trypsin by 2-fold for apoB72 and by 10-fold for apoB94 in isolated microsomes. Thus, the reduced B48-like protein formation by MTP may be a consequence of attenuated cotranslational degradation during apoB translocation across the ER membrane. Formation of B48-like proteins was insensitive to N-acetyl-leucyl-leucyl-norleucinal, a cysteine protease inhibitor known to block post-translational degradation of apoB. These results indicate that MTP facilitates the assembly and secretion of lipoproteins containing apoB and also attenuates the formation of B48-like proteins, probably by assisting apoB translocation across the ER membrane.

Animals↗
PubMed1996

Silica and progressive systemic sclerosis (scleroderma): evidence for workers' compensation policy.

The occurrence of several confirmed cases of progressive systemic sclerosis (scleroderma) among male miners prompted a request by a member of the provincial parliament (MPP) of Ontario that the Industrial Disease Standards Panel (IDSP) evaluate the evidence for an occupational connection. A number of publications in reputable peer-reviewed medical journals offer case-control evidence gathered over four decades on three continents showing a rather clear-cut relationship between occupational exposure to crystalline silica and scleroderma. This article summarizes the evidence for a causal relationship and describes the process by which the members of the panel, using the criteria developed by Sir Austin Bradford Hill as a guide, made a finding of probable connection, the term mandated by the Workers' Compensation Act of Ontario. It provides insight into the difficulties encountered by those setting occupational disease policy when scientific certainty is unobtainable.

Canada↗
PubMed1996

Detection of stratified microbial populations related to Chlorobium and Fibrobacter species in the Atlantic and Pacific oceans.

A gene lineage (SAR406) related to Chlorobium and Fibrobacter species was found in 16S rRNA gene clone libraries prepared from samples from two oceans. The clone libraries were constructed from total picoplankton genomic DNA to assess bacterial diversity in the lower surface layer. The samples were collected by filtration from a depth of 80 m at a site in the western Sargasso Sea and from a depth of 120 m at a site in the Pacific Ocean, approximately 70 km from the Oregon coast. The PCR and primers which amplified nearly full-length 16S rRNA genes were used to prepare the clone libraries. Among the diverse gene clones in these libraries were two related clones (SAR406 and OCS307) which could not be assigned to any of the major bacterial phyla. Phylogenetic analyses demonstrated that these genes were distant relatives of the genus Fibrobacter and the green sulfur bacterial phylum, which includes the genus Chlorobium. The inclusion of SAR406 in phylogenetic trees inferred by several methods resulted in support from bootstrap replicates for the conclusion that Fibrobacter and Chlorobium species and SAR406 are a monophyletic group. An oligonucleotide probe that selectively hybridized to clone SAR406 was used to examine the distribution of this gene lineage in vertical profiles from the Atlantic and Pacific Oceans and in monthly time series at 0 and 200 m in the Atlantic Ocean. During stratified periods, the genes were most abundant slightly below the deep chlorophyll layer. Seasonal changes in the surface abundance of SAR406 rDNA were highly correlated with chlorophyll a levels (r = 0.75).

Bacteria↗
PubMed1996

Cryocrystalglobulinemia mimicking rheumatoid arthritis and vasculitis.

We describe a patient with longstanding arthropathy resembling rheumatoid arthritis (RA) and cold induced purpura for many years, concomitant with IgG kappa paraproteinemia. At autopsy, the patient was found to have evidence of widespread vascular occlusions due to precipitated crystals as well as tissue deposition of crystals. Neither vasculitis nor synovitis was observed despite the clinical picture of both. Blood and synovial fluid obtained antemortem were found to spontaneously form crystals when stored at 4 degrees C, and the washed crystals were composed of the same monoclonal IgG kappa previously found in serum. Cryocrystal formation occurred at 33 degrees C and neutral pH. This patient thus had cryocrystalglobulinemia mimicking clinical manifestations of RA and vasculitis.

Arthritis, Rheumatoid↗
PubMed1995

A cell-specific and selective effect on transactivation by the androgen receptor.

The androgen (AR) and glucocorticoid receptors (GR) are related ligand-activated transcriptional regulators which bind the same cis-acting element and are coexpressed in a variety of cell types. Despite a shared DNA binding site, these receptors mediate diverse cellular responses. To explain this paradox, the existence of cell-specific factors that interact with, and modulate the function of, distinct receptors has been proposed. Prostate epithelial cell growth is sensitive to androgens, but is not affected by glucocorticoids, even though both AR and GR are expressed in these cells. We have recently isolated a unique panel of prostate epithelial cell lines from normal rats and have used these cell lines to examine cell-specific steroid responses. In this study, we compared the abilities of AR and GR to enhance transcription of several different reporter genes regulated by simple (i.e., noncompsite) hormone response elements (HREs) in prostate and nonprostate cell lines. The cell-specific effect occurred independently of the AR hormone binding domain and could be observed with a GAL4 fusion protein containing only the AR N-terminal regulatory domain. Gel shift analyses showed that the relative DNA binding affinity of AR for a probe containing a simple HRE was similar in prostate and nonprostate cell extracts. Presently, the only factors known to mediate steroid receptor-specific gene regulation are cJun and cFos, but there were no cell-specific differences in the functional levels of these proteins which could account for a preferential effect on AR-dependent transcription. Taken together, these results suggest that cell-specific activities exist which can preferentially modulate transcriptional transactivation by AR.

Animals↗
PubMed1995

Dynamic digital traction for unstable comminuted intra-articular fracture-dislocations of the proximal interphalangeal joint.

Unstable intra-articular fracture-dislocations about the proximal interphalangeal joint present a formidable management dilemma. Fourteen patients with comminuted injuries of the proximal interphalangeal joint were treated with dynamic digital traction and followed for an average of 24 months. Dorsal fracture-dislocation and pilon-type injuries made up the majority of cases. Average proximal interphalangeal joint articular surface involvement was 80%. Immediate motion was instituted after traction application. The device was applied in the office with readily available materials. At final follow-up examination, active arc of motion averaged 89 degrees at the proximal interphalangeal joint and 95 degrees for patients sustaining isolated injuries. X-ray films revealed fracture union, joint remodeling, and preservation of joint space. Patient satisfaction was high, with return to previous levels of activity and excellent functional joint restoration. Dynamic digital traction is recommended for unstable proximal interphalangeal joint fractures with comminution, including those associated with subluxation or dislocation.

Adult↗
PubMed1995

Microsomal triglyceride transfer protein: a protein complex required for the assembly of lipoprotein particles.

The mechanism of assembly of lipoprotein particles in the lumen of the endoplasmic reticulum is an important but poorly understood biological problem. A knowledge of this process is of great practical importance because possession of elevated levels of lipoproteins is one of the major risk factors for the development of atherosclerosis. This review describes a major advance in the delineation of the mechanisms involved in the assembly and secretion of apolipoprotein-B-containing lipoproteins: the demonstration of a requirement for microsomal triglyceride transfer protein.

Journal Article↗
PubMed1994

Cloning and regulation of hamster microsomal triglyceride transfer protein. The regulation is independent from that of other hepatic and intestinal proteins which participate in the transport of fatty acids and triglycerides.

Microsomal triglyceride transfer protein (MTP) is a heterodimer consisting of protein disulfide isomerase and a unique large subunit. Recent studies showing that an absence of MTP is a cause of abetalipoproteinemia indicate that MTP is required for the assembly of very low density lipoproteins in the liver and chylomicrons in the intestine. In this study, complementary DNA encoding the large subunit of hamster MTP was cloned. The cDNA sequence was used to design a 50-base pair oligonucleotide probe for a solution hybridization assay to quantitate MTP large subunit mRNA levels in a study of MTP regulation in male Syrian Golden hamsters. In animals fed a low fat diet, MTP exhibited a proximal to distal gradient of expression in the intestine. MTP activity and large subunit mRNA levels in the liver were about 25 and 10% that found in the proximal intestine, respectively. To investigate the effect of diet on MTP, hamsters were maintained for 31 days on one of four diets: 1) control low fat, 2) high fat, 3) low fat, high sucrose, or 4) diet 1 followed by a 48-h fast. The high fat diet increased MTP large subunit mRNA levels in the liver and throughout the small and large intestine. A 55 and 126% increase was observed in the liver and intestine (duodenum and jejunum), respectively. A 40% increase of intestinal MTP protein mass was also observed. The high sucrose diet caused a significant 55% increase in hepatic MTP mRNA levels but did not significantly affect the intestinal mRNA levels. MTP mRNA levels were unchanged in response to fasting. A short term dietary study showed that intestinal MTP mRNA was up-regulated within 24 h after initiating a high fat diet. An acute hepatic response was not observed. The regulation of MTP mRNA levels by high fat diets was compared to that of the liver fatty acid binding protein (L-FABP) and apolipoprotein B (apoB). ApoB mRNA levels were not significantly affected by a high fat diet. Although L-FABP mRNA levels were increased in the liver and intestine, the onset of the changes did not parallel that of MTP. These results suggest that L-FABP, apoB, and MTP, three proteins which play important roles in the transport of fatty acids and triglyceride in the liver and intestine, are not coordinately regulated by diet in hamsters.

Amino Acid Sequence↗
PubMed1994

Secretion of apolipoprotein B-containing lipoproteins from HeLa cells is dependent on expression of the microsomal triglyceride transfer protein and is regulated by lipid availability.

To elucidate the role of the microsomal triglyceride transfer protein (MTP) in lipoprotein assembly, MTP and apolipoprotein B-53 (apoB 53; the N-terminal 53% of apoB) were expressed in HeLa cells. The results showed that apoB-53 could be expressed in HeLa cells with or without expression of MTP. In contrast, efficient secretion of apoB-53 required expression of MTP. Ultracentrifugal density flotation analysis showed that apoB-53 was secreted predominantly as a particle with the density of high density lipoprotein. An essentially identical apoB-53 particle density distribution was obtained after transient expression of apoB-53 in McArdle RH-7777 rat hepatoma cells. The mass of apoB-53 secreted was greater, and the flotation density was lower, from cells fed lipid, suggesting that apoB secretion in HeLa cells was regulated by lipid availability, similar to what has been described for lipoprotein-producing cell lines. These results indicate that MTP is necessary and sufficient to direct the regulated secretion of apoB-53 in HeLa cells.

Animals↗
PubMed1994

Apolipoprotein (apo) B and apoII gene expression are both estrogen-responsive in chick embryo liver but only apoII is estrogen-responsive in kidney.

Estrogen regulates the hepatic synthesis of a variety of proteins required for egg yolk production in oviparous vertebrates. In chickens, two of these proteins, apolipoprotein (apo) B and apoII, comprise the major protein components of specialized very low density lipoprotein particles that transport triacylglycerols and cholesterol to the developing egg yolk. In the adult, apoB is synthesized constitutively in liver, small intestine, and kidney but is estrogen-responsive only in the liver. In this work we have examined the embryonic expression of the apoB and apoII genes in yolk sac, liver, kidney, and small intestine. The 14 kb apoB mRNA was first detected at day 3 of development in vascular yolk sac, a tissue involved in the transfer of yolk lipids into the embryonic circulation. Constitutive apoB mRNA expression was detectable in liver at day 6.5 and in kidney at day 7.5, but in intestine was barely detectable before hatching. The hepatic apoB gene acquired estrogen-responsiveness at day 6.5 and its hormone-dependent expression increased throughout development in concert with the estrogen-responsive expression of the apoII gene. In contrast, the constitutively expressed apoB gene in kidney remained unresponsive to estrogen. Surprisingly, the apoII gene was found to be responsive to estrogen in both the embryonic kidney and small intestine. ApoII mRNA induction by estrogen in kidney at day 11 was at 10% of the level in the liver but estrogen-responsiveness decreased later in development and was low in the adult.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Tocotrienols regulate cholesterol production in mammalian cells by post-transcriptional suppression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

Tocotrienols are natural farnesylated analogues of tocopherols which decrease hepatic cholesterol production and reduce plasma cholesterol levels in animals. For several cultured cell types, incubation with gamma-tocotrienol inhibited the rate of [14C]acetate but not [3H] mevalonate incorporation into cholesterol in a concentration- and time-dependent manner, with 50% inhibition at approximately 2 microM and maximum approximately 80% inhibition observed within 6 h in HepG2 cells. 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase total activity and protein levels assayed by Western blot were reduced concomitantly with the decrease in cholesterol synthesis. In HepG2 cells, gamma-tocotrienol suppressed reductase despite strong blockade by inhibitors at several steps in the pathway, suggesting that isoprenoid flux is not required for the regulatory effect. HMG-CoA reductase protein synthesis rate was moderately diminished (57% of control), while the degradation rate was increased 2.4-fold versus control (t1/2 declined from 3.73 to 1.59 h) as judged by [35S]methionine pulse-chase/immunoprecipitation analysis of HepG2 cells treated with 10 microM gamma-tocotrienol. Under these conditions, the decrease in reductase protein levels greatly exceeded the minor decrease in mRNA (23 versus 76% of control, respectively), and the low density lipoprotein receptor protein was augmented. In contrast, 25-hydroxycholesterol strongly cosuppressed HMG-CoA reductase protein and mRNA levels and the low density lipoprotein receptor protein. Thus, tocotrienols influence the mevalonate pathway in mammalian cells by post-transcriptional suppression of HMG-CoA reductase, and appear to specifically modulate the intracellular mechanism for controlled degradation of the reductase protein, an activity that mirrors the actions of the putative non-sterol isoprenoid regulators derived from mevalonate.

Acetates↗
PubMed1993

Effects of intracisternally injected serotonin on cerebral blood flow in cats during winter and summer, and after dark exposure.

This investigation reports on two experiments: (a) The examination of the effects of intracisternally injected serotonin on cerebral blood flow and systemic pressure in cats during winter (1/10-3/7) and summer (7/6-9/3) at a latitude of 39' 40", and (b) the effects of similarly injected serotonin on the same parameters in 11 cats after exposure to 3 days of continuous darkness during the summer months. Serotonin (5 mg) injected intracisternally produced significant decreases in cerebral blood flow and systemic pressure that lasted from 60 to 180 min after injection. Blood flow decreases, expressed as percentage change from baseline, seen during winter (n = 24) periods were significantly greater [analysis of variance (ANOVA), p < 0.02] from decreases seen during summer (n = 25) periods. Following serotonin injection, systemic pressure decreases between the winter and summer cats were not significantly different. Eleven summer cats were subjected to 3 days of continuous darkness before being injected with serotonin. Blood flow decreases in the dark-exposed cats were significantly (p < 0.05) greater than those seen in normal light-exposed cats, whereas systemic pressure changes were not different. These studies suggest that the seasonal photoperiod may affect the sensitivity of cerebral vessels to intracisternally injected serotonin in cats.

Analysis of Variance↗
PubMed1992

Distribution of developmental myosin isoforms in isolated A-segments.

Immunogold labelling was used to determine the distribution of myosin isoforms within the A-bands of developing chicken pectoralis muscles. Previous localization studies led to the suggestion that neonatal myosin is preferentially located in the centre of heterogeneous thick filaments that contain either embryonic or adult myosin in addition to neonatal myosin. To further explore the possibility that neonatal myosin may serve to nucleate thick filament assembly, a method was developed to isolate A-segments (arrays of myosin filaments) from myofibrils in the presence of MgATP. A-bands usually dissociate into thick and thin filaments in a relaxing buffer, but the inclusion of an antibody against M-line protein prevented separation of the thick filament array. Well-ordered A-segments, approximately 1.5 microns in length, were prepared from muscles 12, 29, 40 days, and approximately 1 year after hatching. After reaction with monoclonal antibodies specific for neonatal and adult myosins, the A-segments were labelled with gold-conjugated secondary antibodies prior to negative staining. An antibody which cross-reacts with embryonic myosin was used to localize that epitope in A-bands of myofibrils from day 1 and day 3 posthatch muscles. At ages where expression of neonatal myosin was high, extensive gold labelling of A-segments was observed in the electron microscope. However, no preferential distribution of antibodies was observed at any age, independent of whether embryonic or adult myosin was coexpressed with the neonatal myosin, suggesting that neonatal myosin is not segregated to any particular region in the A-bands of developing muscles.

Age Factors↗
PubMed1992

Characterization of a panel of rat ventral prostate epithelial cell lines immortalized in the presence or absence of androgens.

We have transfected rat ventral prostate (RVP) epithelial cells with a plasmid containing the SV40 large T-antigen in an attempt to establish a panel of cell lines that will be useful in molecular genetic studies of prostate cell function. Since the distribution of cell types in the RVP is dramatically affected by androgen withdrawal and replacement, cells isolated from normal, castrated, or castrated rats that were given daily injections of testosterone were used in these experiments. Cell lines were established in media that were supplemented or depleted of androgens to accommodate the possible requirements of different prostate cell types. Numerous cell lines were isolated which retain characteristics of RVP epithelial cells and five of these cell lines were studied in detail. All five cell lines express the SV40 large T-antigen, supporting the role of this viral protein in immortalization. The RVP cell lines were shown to contain high levels of functional glucocorticoid receptors, but very low levels of androgen binding activity even though androgen receptor RNA could be detected. It was determined that the decreased androgen receptor activity in the RVP cells was apparently due to low receptor expression based on the results of transient transfection assays using androgen receptor cDNA. Taken together, the biochemical, cytological, and morphological characterizations of the RVP cell lines suggest that they may all have been derived from basal prostate epithelial cells despite the initial differences in androgen status of the animal and the level of androgens in the culture media.

Acid Phosphatase↗