Atherosclerosis: cell biology and lipoproteins.
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Arteriosclerosis↗
Biomedical subjects
K J Williams
Publications and source records attributed to K J Williams.
Development of a PCR-based allele-specific assay from an RFLP probe linked to resistance to cereal cyst nematode in wheat.
The RFLP locus Xglk605 identified by the probe Tag605 maps to a proximal position on the long arm of wheat chromosome 2B about 7 cM away from a gene conditioning resistance to cereal cyst nematode in the wheat line AUS10894. The clone Tag605 was partially sequenced and the PCR primer set AWP1 was designed. The 292-bp product, which showed no polymorphism between varieties, was cloned and sequenced. A single base difference was found in the sequence of the AWP1 products amplified and cloned from the wheat lines AUS10894 and "Spear'. PCR primers were designed with 3' termini that corresponded to the two alleles. A dual-PCR system was developed in which the primer sets AWP2 and AWP3 produced allele-specific amplification. The concentration of the oligonucleotide primers and the sequence of the primer-template mismatches were critical to the success of discriminatory allele amplification.
Alleles↗
Mammalian assay for site-specific DNA damage processing using the human H-ras proto-oncogene.
The human genomic H-ras proto-oncogene was inserted into an Epstein-Barr virus (EBV) vector (p220.2) that replicates synchronously with the cell cycle. Unique restriction enzyme sites, 30 bp apart, were created on either side of codon 12 to enable the construction of gapped heteroduplex (GHD) DNA. Depending upon experimental protocol, the gap could be located either on the coding (non-transcribed) strand or the non-coding (transcribed) strand. GHD DNA was created using a 1.8 kb segment of H-ras DNA containing exon 1, into which a synthetic 30 nucleotide oligomer containing a strand- and site-specific mismatched nucleotide was annealed. The 1.8 kb segment of H-ras DNA containing a codon 12; middle G:T, A:C or T:C mismatch has been religated with high efficiency into the EBV vector and transfected into NIH 3T3 cells using a mild liposome-mediated protocol. Subsequent hygromycin resistant NIH 3T3 colonies have been PCR amplified and sequenced. In this study, codon 12; middle nucleotide mismatch correction rates to wild-type G:C during replication in NIH 3T3 cells were 96.4% of G:T mismatches, 87.5% of A:C mismatches and 67% of T:C mismatches.
3T3 Cells↗
A randomised comparison of meropenem with cefotaxime or ceftriaxone for the treatment of bacterial meningitis in adults. Meropenem Meningitis Study Group.
Third-generation cephalosporins are presently the agents of choice for the empirical antimicrobial therapy of bacterial meningitis. However, a number of factors associated with these agents, namely the development of resistance by pneumococci, limited activity against some Enterobacteriaceae and Pseudomonas spp., and the possible adverse effects of their bacteriolytic mode of action, indicate that newer classes of antimicrobial agents be evaluated for the treatment of bacterial meningitis. Meropenem is a carbapenem antibiotic which is highly active against the major bacterial pathogens causing meningitis, and penetrates well into the cerebrospinal fluid. Two prospective randomised studies in 56 adult bacterial meningitis patients have compared meropenem 40 mg/kg 8-hourly, up to a maximum of 6 g/day (n = 28) with cephalosporin treatment, i.e. cefotaxime (n = 17) or ceftriaxone (n = 11). Patients were assessed by neurological examination, Glasgow Coma Score and Herson-Todd score. Clinical cure was observed in all 23 evaluable patients treated with meropenem (100%) and with 17 of the 22 evaluable cephalosporin-treated patients (77%). All pre-treatment isolates were eradicated except one isolate of Staphylococcus aureus in a cefotaxime-treated patient. Neurological sequelae were noted in three meropenem and four cephalosporin-treated patients. No patients in either treatment group experienced seizures after the start of therapy. This was despite the fact that a patient in each group had experienced seizures before therapy, several had underlying CNS disorders, and that doses of 6 g/day of meropenem were given. Hearing impairment was recorded in 11 meropenem and nine cephalosporin treated patients. Three patients in the meropenem group and one in the cephalosporin group died during treatment for reasons unrelated to study therapy. Overall, the results of this study indicate that meropenem is an effective and well-tolerated antibiotic for the treatment of bacterial meningitis in adults.
Adult↗
A reexamination of the NRMP matching algorithm. National Resident Matching Program.
Most graduating medical students in the United States find their first professional appointments through the National Resident Matching Program (NRMP). This service receives rank-order lists of preferences from students and from hospitals, and then generates final assignments of students to hospitals through the use of a specific computerized matching algorithm. The author uses recent findings from the mathematics and economics literatures to demonstrate three difficulties with the NRMP's matching algorithm and the official descriptions thereof. First, the algorithm favors hospitals over students, a feature known to the NRMP since at least 1976, but, in the author's opinion, not made clear in NRMP literature for students. Second, the author argues that the NRMP's justification that its algorithm mimics orderly, noncentralized admission processes is not correct. Institutions operating under non-centralized procedures must typically make more initial offers than there are positions, in the realization that some fraction of their offers will be declined. This arrangement enlarges the choices available to many applicants, and thereby benefits them, whereas the NRMP's algorithm unrealistically assumes that no institution would ever send out any extra offers. Third, the NRMP's algorithm contains incentives for students to misrepresent their true preferences when constructing their rank-order lists. This feature is a substantial disadvantage of the current algorithm and is incorrectly described in literature distributed to students and in published articles from the NRMP.(ABSTRACT TRUNCATED AT 250 WORDS)
Algorithms↗
The response-to-retention hypothesis of early atherogenesis.
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Animals↗
Interaction of intravenous atrial natriuretic factor with furosemide in patients with heart failure.
Furosemide is frequently administered intravenously to patients with chronic heart failure. However, use of diuretics may cause neuroendocrine activation and by itself may not consistently afford diuresis. Atrial natriuretic factor (ANF) in pharmacologic doses is a vasodilator and has favorable neuroendocrinologic effects in patients with congestive heart failure. To examine whether exogenous ANF might enhance the effects of acute furosemide injection, we studied 14 patients with chronic stable heart failure and measured the effects of the combination of ANF and furosemide on hemodynamics, neuroendocrine activation, and urine output. Eight patients were randomly assigned to receive placebo plus furosemide (1.3 mg/kg intravenously). Six patients received ANF (2 micrograms/kg intravenously) plus furosemide at the same dose in a double-blind manner. The group receiving placebo plus furosemide exhibited a slight increase in mean arterial pressure (92 to 96 mm Hg; p < 0.03), systemic vascular resistance (1989 to 2271 dynes.sec.cm-5; p = 0.0007), and pulmonary capillary wedge pressure (22 to 24 mm Hg; p < 0.04) from baseline to 10 minutes. The group receiving ANF plus furosemide exhibited no change in mean arterial pressure and systemic vascular resistance from baseline to 10 minutes. Pulmonary capillary wedge pressure and mean pulmonary pressure were unchanged. In the group receiving placebo plus furosemide.(ABSTRACT TRUNCATED AT 250 WORDS)
Aged↗
Lipoprotein lipase and sphingomyelinase synergistically enhance the association of atherogenic lipoproteins with smooth muscle cells and extracellular matrix. A possible mechanism for low density lipoprotein and lipoprotein(a) retention and macrophage foam cell formation.
Prominent features of atheromata include smooth muscle cells, cholesteryl ester-loaded macrophage foam cells, extracellular matrix, extracellularly trapped and aggregated lipoproteins, and various enzymes including lipoprotein lipase (LpL) and sphingomyelinase (SMase). The interplay of these factors was investigated in cell culture. Incubation of bovine aortic smooth muscle cells for 18 h at 37 degrees C with low density lipoprotein (LDL) in the presence of LpL and SMase led to massive aggregation of LDL on the surface of the cells as viewed by phase, fluorescence (using 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate-LDL), and electron microscopy. This aggregation required both enzymes. Studies with 125I-LDL confirmed these observations: 125I-LDL cell association in the presence of LpL plus SMase was 50-100-fold greater than in the absence of the two enzymes and was 10-fold greater than in the presence of either enzyme alone. A similar effect (68-fold enhancement) was seen with 125I-labeled lipoprotein(a) (Lp(a)), another atherogenic lipoprotein. In all cases, 125I-lipoprotein degradation was relatively low (< 5% of cell-associated material). LpL/SMase-mediated association of 125I-LDL with smooth muscle cells was still observed when enzymatically inactive LpL was used. The effect was markedly diminished when the smooth muscle cells were treated with a combination of chondroitin ABC lyase and heparitinase or when mutant Chinese hamster ovary cells that lack cell-surface proteoglycans were used, indicating a specific role for cellular proteoglycans. When smooth muscle cells with 125I-LDL or 125I-Lp(a) aggregates were rinsed and then coincubated with mouse peritoneal macrophages for a further 24 h, visible aggregates disappeared, and there was marked 125I-lipoprotein degradation. Electron micrographs after 24 h of co-culture showed lipid-laden, foamy macrophages situated on top of smooth muscle cells, suggesting that the macrophages phagocytosed and metabolized the smooth muscle cell-associated LDL aggregates. Last, 125I-LDL association with smooth muscle cell extracellular matrix was also synergistically enhanced by LpL and SMase, to a level that was 19-fold greater than in the absence of the two enzymes. Thus, the interaction of LDL and Lp(a) with four atheroma components, namely, smooth muscle cells, extracellular matrix, LpL, and SMase, represents a physiologically plausible mechanism for massive, focal retention and aggregation of atherogenic lipoproteins in the arterial wall with subsequent macrophage foam cell formation.
Animals↗
Codons 12 and 13 of H-ras protooncogene interrupt the progression of DNA synthesis catalyzed by DNA polymerase alpha.
Mutagenesis of protooncogenes has been postulated to contribute to the initiation and progression of human cancer. Activating mutations in the H-ras gene are predominantly single-base substitutions and are most frequently identified at codons 12, 13, and 61. We have analyzed the effects of DNA sequence context at specific codons that are hot spots for ras mutation with respect to abnormalities in copying by purified DNA polymerase alpha, a major eucaryotic replication enzyme. Exon 1 of H-ras gene was inserted into M13 mp19, single-stranded DNA constructs were isolated, and the progression of synthesis by polymerase alpha was measured. Strong termination sites were found in codons 12 and 13. Pausing at these codons is abolished when the template is mutated at the middle base of codon 12, the same alteration that converts H-ras into an activated oncogene. Resistance of codon 12 in double-stranded constructs to digestion with restriction enzymes and computer investigation of the ras sequence suggest that these termination sites are in a region of secondary structure. The frequency of sequence alterations within DNA chains that have been extended past codons 12 and 13 was found to be < 0.01. We consider a variety of mechanisms by which the potential secondary structure involving codons 12 and 13 may contribute to the pausing of DNA polymerase alpha and to the generation of clustered mutations at this site.
Base Sequence↗
Origins of chemotherapy.
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Anti-Infective Agents↗
The readers' NIH.
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Financing, Government↗
Mechanisms by which lipoprotein lipase alters cellular metabolism of lipoprotein(a), low density lipoprotein, and nascent lipoproteins. Roles for low density lipoprotein receptors and heparan sulfate proteoglycans.
We sought to investigate effects of lipoprotein lipase (LpL) on cellular catabolism of lipoproteins rich in apolipoprotein B-100. LpL increased cellular degradation of lipoprotein(a) (Lp(a)) and low density lipoprotein (LDL) by 277% +/- 3.8% and 32.5% +/- 4.1%, respectively, and cell association by 509% +/- 8.7% and 83.9% +/- 4.0%. The enhanced degradation was entirely lysosomal. Enhanced degradation of Lp(a) had at least two components, one LDL receptor-dependent and unaffected by heparitinase digestion of the cells, and the other LDL receptor-independent and heparitinase-sensitive. The effect of LpL on LDL degradation was entirely LDL receptor-independent, heparitinase-sensitive, and essentially absent from mutant Chinese hamster ovary cells that lack cell surface heparan sulfate proteoglycans. Enhanced cell association of Lp(a) and LDL was largely LDL receptor-independent and heparitinase-sensitive. The ability of LpL to reduce net secretion of apolipoprotein B-100 by HepG2 cells by enhancing cellular reuptake of nascent lipoproteins was also LDL receptor-independent and heparitinase-sensitive. None of these effects on Lp(a), LDL, or nascent lipoproteins required LpL enzymatic activity. We conclude that LpL promotes binding of apolipoprotein B-100-rich lipoproteins to cell surface heparan sulfate proteoglycans. LpL also enhanced the otherwise weak binding of Lp(a) to LDL receptors. The heparan sulfate proteoglycan pathway represents a novel catabolic mechanism that may allow substantial cellular and interstitial accumulation of cholesteryl ester-rich lipoproteins, independent of feedback inhibition by cellular sterol content.
Animals↗
Retroviral reverse transcriptases: error frequencies and mutagenesis.
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Biological Evolution↗
Pancreatic pseudocyst: recommendations for operative and nonoperative management.
From 1983 to 1990, 76 patients with pancreatic pseudocyst (PP) were managed. Computed tomographic scan (CT) was the primary diagnostic tool (88%). Ethanol accounted for 71 per cent and biliary disease 6 per cent of the occurrence of PP. Thirty-eight patients required surgery and 37 were managed nonoperatively. Forty-four PP complications occurred in 29 patients (4 bleeding, 1 ruptured, 13 gastric outlet obstruction, 10 infected, 6 hyperbilirubinemia, 5 pancreatic ascites, 5 pulmonary insufficiency) at a range of 1 day to 5 weeks from diagnosis; all but one occurred during initial hospitalization. Indications for surgery included complications,12 nonresolution or persistence of symptoms,18 and expansion.9 Internal drainage was accomplished in 40 per cent, (half within 4 weeks of diagnosis), 40 per cent underwent distal resection, and 15 per cent external drainage. There were two deaths in the series. Chronic pancreatitis, gallstone etiology, and gastric outlet obstruction significantly correlated with surgical management of the PP. Endoscopic retrograde cholangiopancreatography (ERCP) was helpful in planning the surgical procedure; 70 per cent of those undergoing ERCP had their operative plan altered. Percutaneous drainage failed in six of eight cases. The authors conclude that nonoperative management is safe and effective in 50 per cent of PP patients, if close radiographic follow-up is maintained until resolution.
Acute Disease↗
Leakage from vitrified radioactive waste.
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Glass↗
Multiple role juggling and daily mood states in working mothers: an experience sampling study.
The effects of multiple role juggling (i.e., simultaneously attending to demands of different roles) on daily mood states of employed mothers were examined. Ss completed activity and mood questionnaires 8 times a day for 8 days. Multiple role juggling had immediate negative effects on task enjoyment and mood. However, contrast effects and habituation to role juggling occurred when mood and satisfaction were examined over time. Furthermore, mood states tended to spill over from one episode to the next within a day, but contrast effects were found across days. These results reflect the complex nature of psychological adjustment to multiple role occupancy.
Adaptation, Psychological↗
Antibiotic treatment and associated prolonged prothrombin time.
The incidence and type of pathology causing a prolonged prothrombin time and clinical bleeding episodes were assessed in a multicentre study of 1109 patients receiving cefotetan, a N-methyl-thiotetrazole (NMTT), or equivalent antibiotics. There was no significant difference in the incidence of a prolonged prothrombin time (9.9% with cefotetan, 8.0% with comparable antibiotics) of clinical bleeding episodes. However, prothrombin time increases of greater than 12 seconds were significantly (p = 0.002) greater with cefotetan (3.8%) than with comparators (0.8%). In both antibiotic groups increases in prothrombin time were more likely following surgery and in patients who were older, with a high platelet count, low albumin, or higher urea and creatinine concentrations. All antibiotic treatment can be associated with prolonged prothrombin times and new agents should always be assessed in a large multicentre study before the practical, clinical importance of haemostatic defects can be defined.
Adolescent↗
Lipoprotein lipase modulates net secretory output of apolipoprotein B in vitro. A possible pathophysiologic explanation for familial combined hyperlipidemia.
We showed previously that net secretory output of apolipoprotein B (apo B) from cultured human hepatoma cells (HepG2) is regulated by rapid reuptake of nascent lipoproteins before they have diffused away from the vicinity of the cells. We now sought to determine if the nascent lipoproteins could be remodeled to enhance or impede reuptake. We found that lipoprotein lipase (LpL), an enzyme that hydrolyzes lipoprotein triglyceride, reduced HepG2 output of apo B to one-quarter to one-half of control. The reduction was apparent during co-incubations as short as 2 h and as long as 24 h. Heparin, which blocks receptor-mediated binding of lipoproteins, abolished the effect of LpL on apo B output, without causing enzyme inhibition. To assess uptake directly, we prepared labeled nascent lipoproteins. LpL tripled the cellular uptake of labeled nascent lipoproteins, from 15.2% +/- 0.7% to 48.7% +/- 0.3% of the total applied to the cells. Cellular uptake of 125I-labeled anti-LDL receptor IgG was unaffected by LpL; thus, LpL enhanced reuptake by altering lipoproteins, not receptors. Because LpL is present in the space of Disse in the liver, we conclude that LpL may act on newly secreted lipoproteins to enhance reuptake in vivo. LpL deficiency would reduce local reuptake of apo B, which would appear as overproduction, thereby providing a mechanistic link between partial LpL deficiency and familial combined hyperlipidemia.
Apolipoproteins B↗