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Biomedical subjects

R W Clark

Publications and source records attributed to R W Clark.

At least 19 recordsLinked to original sources
PubMed1995

The Pope's confessor: a metaphor relating to illness in the analyst.

This paper examines some of the internal and external eventualities in the situation of illness in the analyst. The current emphasis on the use of the self as part of the analyzing instrument makes impairments in the analyst's physical well-being potentially disabling to the analytic work. A recommendation is made for analysts, both individually and as a professional group, to always consider this aspect of a personal medical problem.

Adult↗
PubMed1995

Low level quantification of cholesteryl ester transfer protein in plasma subfractions and cell culture media by monoclonal antibody-based immunoassay.

Sensitive immunoradiometric (IRMA) and ELISA assays for cholesteryl ester transfer protein (CETP) have been developed using two different monoclonal antibodies (MAbs). The MAbs were prepared against human plasma CETP and demonstrated specificity by their inhibition of cholesteryl ester transfer activity and by immunoblots of crude plasma fractions and whole media from transfected CHO cells. For these sandwich-type assays, one MAb, 2F8, is used for capture, and the second MAb, 2E7, is iodinated (IRMA) or conjugated with alkaline phosphatase (ELISA) and used for detection. Both assays are linear and provide sensitivities much greater than previously reported. The IRMA allows for the accurate quantification of CETP in the range of 0.5-20 ng/assay (5-200 ng/ml), the ELISA 0.05-5 ng/assay (0.5-50 ng/ml). Using the IRMA, the mean plasma CETP concentration in 44 normolipidemic individuals was determined to be 2.10 +/- 0.36 micrograms/ml; 2.05 +/- 0.33 for males (n = 25) and 2.16 +/- 0.40 for females (n = 19). Values ranged from 1.28 to 2.97 micrograms/ml and CETP mass correlated well with cholesteryl ester transfer activity (r = 0.913, n = 23). The distribution of CETP in human plasma was examined both by gel permeation fast protein liquid chromatography (FPLC) and by native gel electrophoresis. For FPLC using agarose resins, a low ionic strength, isotonic buffer system resulted in near total recoveries of CETP, and demonstrated a peak for CETP mass centered at molecular masses of 150 to 180 kilodaltons, larger than that for free monomeric CETP. Native acrylamide gel electrophoresis of plasma from six individuals, followed by 2F8/2E7 sandwich immunoblotting, showed CETP migrating within a size range of 170-220 kilodaltons. This result is consistent with suggestions that plasma CETP is associated with small-sized HDL. Agarose gel electrophoresis showed plasma CETP, as well as purified recombinant CETP, to be prebeta migrating. For determining the concentration of CETP in the media of cultured HepG2 cells, advantage was taken of the high sensitivity of the ELISA. CETP levels were found to increase linearly over the 100-h culture period, reaching 8.0 +/- 0.4 ng/ml (18.0 +/- 1.3 ng/mg cell protein). These sensitive, direct immunoassays for CETP mass should be valuable aids for examining the behavior of CETP in plasma and other complex systems, as well as for studying the synthesis and secretion of CETP by different cells and tissues.

Animals↗
PubMed1994

Inhibition of cholesteryl ester transfer protein activity in hamsters alters HDL lipid composition.

We investigated the role of cholesteryl ester transfer protein (CETP) in hamsters by using a monoclonal antibody (MAb) that inhibited hamster CETP activity. MAbs were prepared against partially purified human CETP and screened for inhibiton of 3H-cholesteryl oleate (CE) transfer from LDL to HDL in the presence of human plasma bottom fraction (d > 1.21 g/ml). Antibody 1C4 inhibited CE transfer activity in both human plasma bottom fraction (IC50 = approximately 4 micrograms/ml) and in whole plasma from male Golden Syrian hamsters (IC50 = approximately 30 micrograms/ml). Purified MAb 1C4 was injected into chow- and cholesterol-fed hamsters, and blood was collected for analysis of plasma CETP activity and HDL lipid composition. Plasma CETP activity was inhibited by 70%-80% at all and HDL lipid composition. Plasma CETP activity was inhibited by 70%-80% at all times up to 24 h following injection of 500 micrograms MAb 1C4 (approximately 3.7 mg/kg). The amount of antibody required for 50% inhibition at 24 h post-injection was 200 micrograms (approximately 1.5 mg/kg). Inhibition of hamster CETP activity in vivo increased hamster HDL cholesterol by 33% (P < 0.0001), increased HDL-CE by 31% (P < 0.0001) and decreased HDL-triglyceride by 42% (P < 0.0001) (n = 36) as determined following isolation of HDL by ultracentrifugation. An increase in HDL cholesterol and a redistribution of cholesterol to a larger HDL particle were also observed following fast protein liquid chromatography (FPLC) gel filtration of plasma lipoproteins.(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↗
PubMed1990

Selective inhibition of cholesterol synthesis in liver versus extrahepatic tissues by HMG-CoA reductase inhibitors.

Hepatic specificity of inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase may be achieved by efficient first-pass liver extraction resulting in low circulating drug levels, as with lovastatin, or by lower cellular uptake in peripheral tissues, seen with pravastatin. BMY-21950 and its lactone form BMY-22089, new synthetic inhibitors of HMG-CoA reductase, were compared with the major reference agent lovastatin and with the synthetic inhibitor fluindostatin in several in vitro and in vivo models of potency and tissue selectivity. The kinetic mechanism and the potency of BMY-21950 as a competitive inhibitor of isolated HMG-CoA reductase were comparable to the reference agents. The inhibitory potency (cholesterol synthesis assayed by 3H2O or [14C]acetate incorporation) of BMY-21950 in rat hepatocytes (IC50 = 21 nM) and dog liver slices (IC50 = 23 nM) equalled or exceeded the potencies of the reference agents. Hepatic cholesterol synthesis in vivo in rats was effectively inhibited by BMY-21950 and its lactone form BMY-22089 (ED50 = 0.1 mg/kg p.o.), but oral doses (20 mg/kg) that suppressed liver synthesis by 83-95% inhibited sterol synthesis by only 17-24% in the ileum. In contrast, equivalent doses of lovastatin markedly inhibited cholesterol synthesis in both organs. In tissue slices from rat ileum, cell dispersions from testes, adrenal, and spleen, and in bovine ocular lens epithelial cells, BMY-21950 inhibited sterol synthesis weakly in vitro with IC50 values 76- and 188-times higher than in hepatocytes; similar effects were seen for BMY-22089. However, the IC50 ratios (tissue/hepatocyte) for lovastatin and fluindostatin were near unity in these models. Thus, BMY-21950 and BMY-22089 are the first potent synthetic HMG-CoA reductase inhibitors that possess a very high degree of liver selectivity based upon differential inhibition sensitivities in tissues. This cellular uptake-based property of hepatic specificity of BMY-21950 and BMY-22089, also manifest in pravastatin, is biochemically distinct from the pharmacodynamic-based disposition of lovastatin, which along with fluindostatin exhibited potent inhibition in all tissues that were exposed to it.

Animals↗
PubMed1988

Carbamate formation on tubulin: CO2/bicarbonate buffers protect tubulin from inactivation by reductive methylation and carbamoylation and promote microtubule assembly at alkaline pH.

Carbamoylation and reductive methylation of tubulin have been shown previously to inhibit microtubule assembly, probably by attack on essential internal lysine residues [Mellado, W., Slebe, J., & Maccioni, R.B. (1982) Biochem. J. 203, 675-681; Szasz, J., Burns, R., & Sternlicht, H. (1982) J. Biol. Chem. 257, 3697-3704]. We show first that this inhibition is blocked by the presence of HCO3-/CO2 buffer at physiological concentrations during the carbamoylation or reductive methylation. Under conditions that block assembly, the amount of radiolabeled cyanate or formaldehyde incorporated by these reactions in the absence of HCO3-/CO2 was approximately four carbamoyl or five methyl groups in a ratio of approximately 1.7 alpha chain/beta chain. In the presence of HCO3-/CO2, the formaldehyde incorporation is decreased roughly 0.5 mol in each of the alpha and beta chains, and cyanate incorporation, roughly 1.0 mol/mol of alpha or beta monomer. These results are consistent with the hypothesis that CO2 competed with formaldehyde or cyanate for uncharged amino groups and led to the reversible formation of carbamates. The complete antagonism of the inhibition of microtubule assembly by reductive methylation by CO2, even though the number of methyl groups incorporated was reduced by only 0.5 mol/tubulin monomer, was consistent with the possibility that reductive methylation opened up additional residues for attack. Indeed, using an adaptation of the method of Gros et al. for measurement of carbamates [Gros, G., Forster, R.E., & Lin, L. (1976) J. Biol. Chem. 251, 4398-4407], we found that reductive methylation with 2 mM formaldehyde (assembly blocked) did not decrease carbamate formation (carbamate formation was inhibited at higher formaldehyde concentrations).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Natural size classes in DNA from Chinese hamster metaphase chromosomes.

DNA from isolated Chinese hamster ovary (CHO) metaphase chromosomes can be obtained in three different molecular weight classes. The two largest forms have sedimentation coefficients of 80 and 120 S at 7,500 rpm. Based on sedimentation and speed dependence analysis these have molecular weights of 220 million and above 5,000 million, and are thought to be analogs of DNA classes observed in a prior study of human metaphase chromosomes. An extract can be converted to primarily the 80 S form through alkaline pH treatment of metaphase DNA. The third class (45 S DNA) is formed as a result of metaphase chromosome exposure to the nuclease Bal31, and has a mass distribution analogous to the CHO replicon.

Animals↗
PubMed1987

A nuclease-derived fragment of metaphase DNA and its relationship to the replicon.

When isolated Chinese hamster cells (CHO) metaphase chromosomes are treated with nuclease Bal-31, the DNA is reduced to a size class that is resistant to further degradation. This size class resembles the distribution of replicon sizes in this particular cell line in both average size and size range. Tests based on molecular weight (MW) analysis were devised to locate the origin of replication within the Bal-31 segments. The evidence indicates that replication origins are positioned at or near the center of these segments. The tests were made possible by the additional discovery that BrdU-substituted DNA is highly susceptible to Bal-31 nuclease attack while still contained in the isolated metaphase chromosome.

Animals↗
PubMed1986

Characterization of alterations in plasma lipoprotein lipid and apoprotein profiles accompanying hepatoma-induced hyperlipidemia in rats.

Alterations in plasma lipoprotein lipid and apoprotein accompanying the hyperlipidemia of rats bearing Morris hepatoma 7288C were characterized. In tumor-bearing animals all plasma lipid classes except cholesterol ester (CE) were elevated, particularly free cholesterol (FC) and triglyceride (TG), which increased by 57 and 63%, respectively. Fasting only partially reduced the tumor-induced hyperlipidemia and had no effect on the ratios of FC/CE and TG/CE. Analysis of plasma lipoproteins revealed an elevation of VLDL, IDL, and LDL in host rats, with more than a 2-fold increase in both lipid and protein of VLDL. In contrast, the three high density fractions, HDL2, HDL3, and d greater than 1.21 g/ml, were reduced. The inverse changes in concentration of host lipoproteins of lower versus higher density indicate a defective catabolism of TG-rich lipoprotein. This possibility is supported by the analysis of apolipoprotein. The percentage of total apoprotein contributed by apo C-I and C-II was reduced in all host fractions except HDL2, while the C-IIIs remained unchanged except for a small decrease in C-III-3 of host VLDL and a slight increase in the combined C-IIIs of HDL2. These changes were reflected in the decreased C-I+C-II/C-III ratios of all host lipoprotein fractions. Apo E levels remained similar to control values except for a significant decrease in HDL2. Host VLDL showed increased apo A-IV and A-I content, while A-IV was decreased in HDL2. Changes in apo B profiles were also observed.

Animals↗
PubMed1985

Secretion of a nonspecific lipid transfer protein by hepatoma cells in culture.

Nonspecific lipid transfer protein (sterol carrier protein2) has previously been proposed to function as (i) a catalyst for intracellular movement of newly synthesized phospholipid, (ii) a cofactor in the biosynthesis and metabolism of cholesterol, and (iii) a cofactor in the feedback inhibition of cholesterol synthesis. Each of these functions is based upon the premise that nonspecific lipid transfer protein (nsLTP) is cytosolic. However, evidence presented in this report suggests that, at least in the case of cultured hepatoma cells, nsLTP is secreted. This conclusion is supported by three observations. First, after culture of hepatoma cells for 10 h, 88% of the nsLTP (as judged by its phosphatidylethanolamine transfer activity) appears in the medium, whereas the cytosolic level of transfer activity remains unchanged. Furthermore, this is accompanied by the appearance in the medium of a polypeptide of Mr 12,200-12,500, which corresponds to the known molecular weight of nsLTP. Finally, it was observed that the appearance of both the activity and the polypeptide in the medium are inhibited by monensin, an inhibitor of secretion. Thus their appearance seems to represent secretion and not simply leakage from the cells. Further evidence that nsLTP does not play an important role in the cytosolic transport of phospholipid and sterol is provided by our observation that hepatoma cells containing a level of nsLTP only 10-15% of that found in liver nevertheless possess near-normal membrane phospholipid compositions and retain the ability to feedback-inhibit cholesterol biosynthesis.

Animals↗
PubMed1985

Mass characteristics of DNA obtained from chromosomes of a human carcinoma cell line.

Sedimentation studies of DNA from chromosomes extracted from human mitotic cells showed that high-molecular-weight DNA can be obtained if cell hypotonic treatments and prolonged metaphase blocks are avoided. Two types of large double-stranded DNA were observed. One of these (Mr = 2.5 X 10(8)) appeared as a size class with characteristics reminiscent of the chromosomal DNA subunit hypothesis. However, this DNA is the decay product of larger molecules, whose minimum molecular weight is 6 X 10(8).

Antineoplastic Agents↗
PubMed1984

Videoendoscopic determination of the mechanism of obstruction in obstructive sleep apnea.

Six patients with obstructive sleep apnea were studied with our system of simultaneous video recording of the polysomnographic record and the endoscopic image from the pharyngeal airway. Preoperative and postoperative recordings were made during sleep in each patient. Tracheotomized patients recreated their preoperative laryngeal inlet obstruction and its immediate cessation by alternately opening and closing the tracheotomy tube. This demonstration coupled with the physical examination findings of "disproportionate anatomy," leads to the determination that the mechanism of obstructive sleep apnea is twofold: (1) an underlying CNS propensity to hypotonia of pharyngeal musculature during sleep and (2) either an isolated obstructive upper airway lesion or a combination of alterations in normal relationships within the upper airway that cause a passive narrowing of the upper airway. This combination of altered relationships is collectively referred to as "disproportionate anatomy."

Adult↗
PubMed1983

Acute mastoiditis in children: a review of 54 cases.

Fifty-four children with acute mastoiditis were managed at the Los Angeles County-University of Southern California Medical Center from 1972 through 1982. Our criteria for the diagnosis of acute mastoiditis are acute or subacute otitis media, postauricular swelling and erythema, protrusion of the auricle, and clouding of mastoid air cells on radiographs. Thirty-one (57%) recovered with conservative therapy consisting of early myringotomy and intravenous antibiotic, usually ampicillin. Twenty-three patients were managed surgically. The indication for surgery in each case was the clinical diagnosis of subperiosteal abscess; mastoid radiographs played no part in the decision to operate. Two of the 23 patients managed surgically had only incision and drainage of abscess; simple mastoidectomy was performed on 20 and radical mastoidectomy on one. Etiologic bacteria were cultured in 21 instances, S. pyogenes was cultured in 9, S. pneumoniae was cultured in 6, H. influenzae in 1, enterococci in 1, anaerobes in 2, and M. tuberculosis in 2.

Acute Disease↗
PubMed1983

Anthropometric characteristics, cardiac abnormalities and adrenergic activity in patients with primary disorders of sleep.

Sixty-one patients (pts) with sleep apnea (SA), 35 with narcolepsy (N) and 24 with idiopathic hypersomnolence (H) were studied in the Clinical Research Center. The height to body weight ratio was less than normal in SA pts (0.32 +/- 0.01 vs 0.45 +/- 0.01, p less than 0.01), but normal in N and H pts (0.45 +/- 0.02 and 0.45 +/- 0.01, respectively). Twenty-four hour urinary epinephrine (E) plus norepinephrine (NE) was greater than normal (p less than 0.01), but not different among SA, N and H pts. The incidence of mitral valve prolapse (MVP) was greater in N (49%) and H (58%) compared to SA (20%) (p less than 0.01). The ratio of the pre-ejection period to the left ventricular ejection time (PEP/LVET) was abnormal (greater than 0.42) in 36% of the pts with SA and only in one pt with N and H (p = 0.02). The % shortening of the echocardiographic internal diameter (% delta D) was abnormal (less than 28%) in 28% of pts with SA and normal in all pts with N and H (p = 0.02). The incidence of malignant ventricular dysrhythmias (24 hour Holter) was 26% in SA, 3% in N and 4% in H (p = 0.04). Thus, pts with impaired alertness have high adrenergic tone despite evidence for a role of catecholamines in wakefulness. Left ventricular dysfunction, malignant dysrhythmias and obesity are common findings in SA, while MVP is very common in N and H.

Adult↗
PubMed1983

Role of lipid transfer proteins in the abnormal lipid content of Morris hepatoma mitochondria and microsomes.

The level of nonspecific lipid transfer protein in three Morris hepatomas of varying degrees of differentiation has been found to be less than 10% of the level in either host or control livers, whereas phosphatidylcholine transfer activity is slightly reduced for hepatomas compared to host livers (20 to 40%), though nearly the same as levels present in livers of non-tumor-bearing rats. Two major differences have been observed in the lipid compositions of tumor mitochondrial and microsomal membranes compared to those of normal membranes. For all three hepatomas, the cholesterol content of mitochondria is markedly elevated, and the phospholipid content of microsomes is reduced (per mg of protein). As a consequence of the above alterations, the cholesterol: phospholipid ratio of both the microsomal and mitochondrial membranes of these tumors is increased, and the extent of this increase correlates well with the degree of differentiation of the hepatomas. If an important physiological role of lipid transfer proteins is to transfer newly synthesized lipids from microsomes to other cellular organelles, one would expect a 10-fold decline of nonspecific lipid transfer protein activity in hepatomas to result in phospholipid and cholesterol accumulation in microsomes and depletion in mitochondria. The observed decline of phospholipid in microsomes and accumulation of cholesterol in mitochondria from hepatomas suggest that this is not an important function of the nonspecific lipid transfer protein. Finally, preliminary studies of the possibility that the elevated cholesterol: phospholipid levels in hepatoma membranes affect the activities of enzymes of these membranes are reported.

Animals↗