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Biomedical subjects

C Holm

Publications and source records attributed to C Holm.

At least 19 recordsLinked to original sources
PubMed1992

Ovarian cancer staging: does it require a gynecologic oncologist?

Forty-seven patients with presumed Stages I-II invasive ovarian epithelial carcinoma were treated with intravenous 50 mg/m2 cis-platinum, for 2-18 cycles (median, 9), 50 mg/m2 doxorubicin for 2-14 cycles (median, 9), and/or 600 mg/m2 cyclophosphamide for 2-14 cycles (median, 6) after surgical staging by a gynecologic oncologist or a nononcologic surgeon. Mean follow-up is 6.8 years. Cumulative 5-year actuarial survival is 73 +/- 6%; 75 +/- 12% for Stage I and 71 +/- 8% for Stage II disease. When screened for poor prognosticators, only the specialty of the operating surgeon was identified (P < 0.05). Five-year actuarial survival and disease-free survival, respectively, for Stages I-II patients surgically staged by a gynecologic oncologist were 83 +/- 7% and 76 +/- 8%, compared to 59 +/- 11% (P < 0.05) and 39 +/- 11% (P < 0.03) for the group operated upon by a nononcologist.

Actuarial Analysis↗
PubMed1991

Expression of biologically active hormone-sensitive lipase in mammalian (COS) cells.

cDNAs encoding rat adipose tissue hormone-sensitive lipase were expressed in COS cells, under the control of the SV40 promoter to half the level in rat adipocytes, the richest native source of the enzyme. A cDNA lacking most of the long 5'-untranslated region of the full-length rat hormone-sensitive lipase cDNA was, with regard to the lipase activity, on the average 70% more efficiently expressed that the full-length cDNA. The recombinant protein was almost identical to hormone-sensitive lipase of rat adipose tissue with respect to specific activity, susceptibility to inhibitors, molecular size, phosphorylation and activation by cyclic AMP-dependent protein kinase. The described eukaryotic expression system will allow analysis of effects of amino acid substitutions introduced into the lipase molecule by site-directed mutagenesis.

Adenosine Monophosphate↗
PubMed1991

Genetic analysis of the gyrase A-like domain of DNA topoisomerase II of Saccharomyces cerevisiae.

We have undertaken a genetic analysis of heat-sensitive and cold-sensitive mutations in TOP2, the gene encoding yeast DNA topoisomerase II. Deletion mapping was used to localize 14 heat-sensitive and four cold-sensitive top2 mutations created by a method biased toward mutations in the 3' two-thirds of the gene. The mutations all appear to be located in the region of DNA topoisomerase II that shows homology to the "A" subunit of bacterial DNA gyrase. The heat-sensitive mutations and one cold-sensitive mutation lie in the center of the gene near the sequence that encodes the active site tyrosine. The three other cold-sensitive mutations map farther toward the 3' end of the gene. The cold-sensitive mutations exhibit intragenic complementation, and the complementation groups correspond to the physical map. We sequenced nine top2 mutations and found that the mutations are usually single missense mutations, frequently involve proline, and affect conserved regions of the protein. Suppressor analysis yielded two intragenic suppressors and seven independent isolates of an allele-specific extragenic suppressor we named tos1; tos1 is not allelic to any genes predicted to encode type I topoisomerase-related proteins. The two intragenic suppressors were tested for allele-specificity; the results revealed a complex pattern of suppression between heat-sensitive and cold-sensitive top2 alleles. These top2 mutations may have compensatory effects on the general stability of the protein.

Base Sequence↗
PubMed1991

The metabolism of ethyl esters of fatty acids in adipose tissue of rats chronically exposed to ethanol.

The concentration of ethyl esters of fatty acids as well as the activity of the enzyme synthesizing these esters (fatty acid ethyl ester synthase) were determined in adipose tissue of rats ingesting ethanol (9-16 g/kg body weight/day) for different periods of time. After 10 and 17 weeks of ethanol exposure about 300 nmol of ethyl esters of oleic, palmitic, stearic, and linoleic acids were found per gram adipose tissue. The ethyl esters disappeared after 1 week of abstinence. Closer analyses, using radioactive ethanol, revealed a half-life of the esters of less than 24 hr. The bulk of the esters was found in a membrane preparation of isolated adipocytes. Hormone-sensitive lipase hydrolyzed emulsified ethyl oleate as efficiently as that of trioleoylglycerol, but in mixed ethyl oleate/trioleoyl glycerol particles the hydrolysis of ethyl oleate was slower, suggesting a decreased accessibility. Synthase activity was found in adipose tissue from rats not exposed to ethanol. It doubled after 10 and 17 weeks of ethanol and decreased with a half-life of at least a week after abstinence. It was concluded that ethyl esters of fatty acids are formed in rat adipose tissue as previously shown in other tissues. They seem to be stored mainly in membranous parts of the adipocytes. Synthase activity is induced by ethanol. The elevated activity has a longer half-life, and may be useful as an indicator of alcohol abuse.

Acyltransferases↗
PubMed1990

Segregation of recombined chromosomes in meiosis I requires DNA topoisomerase II.

To understand better the similarities and differences between meiosis and mitosis, we examined the meiotic role of DNA topoisomerase II, an enzyme that is required mitotically to disentangle sister chromatids at the time of chromosome segregation. In meiosis, we found that topoisomerase II is required only at the time of nuclear division. When cold-sensitive top2 mutants are induced to sporulate at the restrictive temperature, they undergo premeiotic DNA synthesis and commitment to meiotic levels of recombination but fail to complete the first meiotic nuclear division. The introduction of a mutation blocking recombination relieves the requirement for topoisomerase II in meiosis I. These results suggest that topoisomerase II is required at the time of chromosome segregation in meiosis I for the resolution of recombined chromosomes.

Cell Nucleus↗
PubMed1990

Incorporation of hormone-sensitive lipase into phosphatidylcholine vesicles.

Enzymatically active, detergent-solubilized purified hormone-sensitive lipase (HSL) was incorporated into phosphatidylcholine (PC) vesicles, using a detergent-dialysis procedure with small PC vesicles, obtained by sonication, as phospholipid source and CHAPS, a zwitterionic bile-salt derivative, as detergent. Association of enzyme protein with the PC vesicles was verified by floatation in a discontinuous dextran gradient and by gel chromatography. An average of 35% of added HSL was incorporated into the vesicles. The vesicles were shown, by quasi-elastic light scattering and electron microscopy, to have a diameter of approximately 160 nm. The vesicle-associated HSL could be phosphorylated by cyclic AMP-dependent protein kinase. The vesicles were stable, both with regard to enzyme activity and size, for at least 4 days when stored at 4 degrees C. The preparation of detergent-free, vesicle-associated and stable HSL provides new possibilities to study some of its properties, and supports and extends the previous report (Holm, C., Fredrikson, G., and Belfrage, P., J. Biol. Chem. 261, 15659-15661, 1986) which demonstrated the amphiphilic character of HSL.

Animals↗
PubMed1990

Sequelae of lateral ovarian transposition in unirradiated cervical cancer patients.

The sequelae of lateral ovarian transposition (LOT) in cervical cancer patients has been examined only in the light of the effect of pelvic radiation therapy on ovarian preservation. Preservation of ovarian function has not been examined in the absence of radiation therapy, and symptomatic ovarian cyst formation in transposed ovaries with the need for subsequent surgery has not been addressed in either radiated or unirradiated cervical cancer patients. We studied 84 premenopausal FIGO stage IA or IB cervical cancer patients treated by primary radical hysterectomy between the years 1978 and 1988. None of these patients received adjuvant radiation therapy. Fifty-nine of eight-four patients had radical hysterectomy (RH) without LOT. These patients were compared to 25 of 84 patients who had LOT in addition to RH. The incidence of symptomatic ovarian cysts, the majority requiring operative intervention, was 24% in the ovarian transposition patients as compared to 7.4% in those who had RH alone. This threefold increase in symptomatic benign ovarian cyst formation in the translocated ovary was significant (P = .048). On the other hand, LOT in these RH patients does not appear to increase the incidence of early menopause (P greater than 0.05). On follow-up of those patients who did not incur additional surgery or radiation, 4.3% became menopausal, as compared to 4.1% of those patients undergoing RH alone, with the mean ages of the two groups being comparable.

Adult↗
PubMed1990

Cloning by function: an alternative approach for identifying yeast homologs of genes from other organisms.

Studies of cell physiology and structure have identified many intriguing proteins that could be analyzed for function by using the power of yeast genetics. Unfortunately, identifying the homologous yeast gene with the two most commonly used approaches--DNA hybridization and antibody cross-reaction--is often difficult. We describe a strategy to identify yeast homologs based on protein function itself. This cloning-by-function strategy involves first identifying a yeast mutant that depends on a plasmid expressing a cloned foreign gene. The corresponding yeast gene is then cloned by complementation of the mutant defect. To detect plasmid dependence, the colony color assay of Koshland et al. [Koshland, D., Kent, J. C. & Hartwell, L. H. (1985) Cell 40, 393-403] is used. In this paper, we test the feasibility of this approach using the well-characterized system of DNA topoisomerase II in yeast. We show that (i) plasmid dependence is easily recognized; (ii) the screen efficiently isolates mutations in the desired gene; and (iii) the wild-type yeast homolog of the gene can be cloned by screening for reversal of the plasmid-dependent phenotype. We conclude that cloning by function can be used to isolate the yeast homologs of essential genes identified in other organisms.

Base Sequence↗
PubMed1990

Assignment of seven genes to distinct intervals on the midportion of human chromosome 19q surrounding the myotonic dystrophy gene region.

Hybridization studies using a panel of somatic cell hybrids with subchromosomal segments of 19q have localized the genes encoding hormone-sensitive lipase (LIPE), carcinoembryonic antigen (CEA), and small nuclear ribonucleoprotein polypeptide A (SNRPA) to various regions of 19q13.1; the cellular receptor for poliovirus sensitivity (PVS) to 19q13.2; and the genes coding for prostate-specific antigen (APS), human pancreatic kallikrein (KLK1), and small nuclear ribonucleoprotein 70-kD polypeptide (SNRP70) to 19q13.3----qter. Our results exclude several of these genes from being seriously considered as a candidate for the myotonic dystrophy gene on 19q.

Animals↗
PubMed1989

Human adipose tissue hormone-sensitive lipase: identification and comparison with other species.

The mRNA for human hormone-sensitive lipase (HSL) was identified using Northern blot analysis and a cDNA-probe for rat HSL. As in the rat, human adipose tissue expresses a single mRNA species of 3.3 kb. Using Western blotting with a polyclonal rabbit antibody towards rat adipose tissue HSL, the corresponding enzyme in human adipose tissue was identified with an apparent 88 kDa polypeptide, thus slightly larger than the rat and bovine 84 kDa, and the mouse and guinea-pig 82 kDa species. Additional evidence for the identification was provided by the inhibition of HSL diacylglycerol lipase activity by the anti-rat HSL antibody, and by NaF, DFP and Hg2+, known inhibitors of HSL. The concentration of the enzyme, as reflected by its activity per g tissue and the specific activity was about two thirds of that in the rat adipose tissue (200 g rats). The identification of the human enzyme protein made it possible to directly demonstrate its phosphorylation by cAMP-dependent protein kinase, thus extending the previous report regarding activation of the lipase with this kinase and ATP-Mg2+ in human adipose tissue extracts (Khoo, J.C., Aquino, A.A. and Steinberg, D. (1974) J. Clin. Invest. 53, 1124-1131).

Adipose Tissue↗
PubMed1989

Differential requirement of DNA replication for the cytotoxicity of DNA topoisomerase I and II inhibitors in Chinese hamster DC3F cells.

The cytotoxicity of topoisomerase inhibitors is thought to result from the induction of enzyme-mediated DNA breaks. The fact that these breaks reverse rapidly in cells programmed to die, led us to investigate further the cytotoxic mechanisms of topoisomerase I (camptothecin) and topoisomerase II inhibitors (VP-16 and amsacrine) in Chinese Hamster lung fibroblasts (DC3F). Exposures (30 min) to camptothecin produced limited cell killing with approximately 20% of the cells naturally resistant. This resistance was overcome by increasing the drug exposure time. Inhibition of DNA synthesis by 5-min pretreatments with aphidicolin or hydroxyurea abolished the cytotoxicity of camptothecin without changing the level of camptothecin-induced DNA breaks. A good correlation was found between the degree of DNA synthesis inhibition by aphidicolin and the reduction of camptothecin cytotoxicity. In similar experiments performed with topoisomerase II inhibitors, aphidicolin prevented only partially against VP-16- and amsacrine-induced cytotoxicities, yet had no effect upon drug-induced DNA breaks. These results indicate that the production of topoisomerase-mediated DNA breaks by antitumor drugs is not sufficient for cell killing. Instead, an interference of moving DNA replication forks with drug-stabilized topoisomerase-DNA complexes is critical for cell death. The cytotoxicity of camptothecin seemed to be completely related to this process, while that of topoisomerase II inhibitors seemed to involve additional mechanisms in DC3F cells.

Amsacrine↗
PubMed1989

Rapid purification of detergent-solubilized bovine hormone-sensitive lipase by high performance hydrophobic interaction chromatography.

Hormone-sensitive lipase (HSL), the enzyme controlling the rate of adipose tissue lipolysis and also possibly involved in the regulation of steroidogenesis, has been purified from bovine omental adipose tissue. Partially detergent-solubilized, delipidated and purified HSL was obtained through step-elution at conventional DEAE ion-exchange chromatography, followed by concentration on hydroxylapatite. High performance hydrophobic interaction chromatography (HPHIC) on phenylsilica then resulted in an increase of HSL protein purity from 2% to more than 70%. Final purification of the enzyme to apparent homogeneity (greater than 95% protein purity), concentration and removal of most of the detergent was obtained by high performance cation exchange chromatography on Mono S. At least 0.5 mg of highly stable HSL was obtained from 5 kg of bovine omental fat within four working days. The purified lipase had a lower specific activity than previously reported for the corresponding rat enzyme but the preparations have proved very useful for enzyme structure studies and as an antigen.

Adipose Tissue↗
PubMed1989

Hydroxyurea potentiation of the antineoplastic activity of cyclophosphamide and 4'-(9-acridinylamino)methanesulfon-M-anisidide (AMSA) in the brown Norway rat myelocytic leukemia model.

The activities of hydroxyurea (HU), 4'-(9-acridinylamino)methanesulfon-M-anisidide (AMSA) and cyclophosphamide (CY) were examined in the brown Norway rat myelocytic leukemia model in experiments designed to determine the synergy, optimal drug sequencing, and therapeutic index of combinations of these agents. A single dose of CY or four consecutive daily doses of AMSA produced increased survival in leukemic rats, with a positive-slope dose-response curve up to the maximum tolerated dose (MTD). HU at 1/2 MTD produced a minimal antileukemic effect but significantly potentiated the antineoplastic activity of 1/2 MTD of CY or AMSA with no significant toxic death rate. Drug-sequence experiments demonstrated that maximal synergy was achieved when HU was given immediately after CY but immediately before or during AMSA administration. No significant cure rate was seen with any CY/HU or HU/AMSA sequence. The three drugs given in the sequence of CY followed 3 days later by HU and AMSA simultaneously, however, was curative in the majority of rats with advanced leukemia, whereas other sequences were more toxic or less effective. Each of the drugs in these experiments was given at 1/2 of its single-agent MTD. HU significantly potentiates the antineoplastic effect of CY and AMSA in a drug-sequence-dependent manner in this model, apparently with an improved therapeutic index.

Amsacrine↗
PubMed1989

DNA topoisomerase II must act at mitosis to prevent nondisjunction and chromosome breakage.

The hypothesis that DNA topoisomerase II facilitates the separation of replicated sister chromatids was tested by examining the consequences of chromosome segregation in the absence of topoisomerase II activity. We observed a substantial elevation in the rate of nondisjunction in top2/top2 cells incubated at the restrictive temperature for one generation time. In contrast, only a minor increase in the amount of chromosome breakage was observed by either physical or genetic assays. These results suggest that aneuploidy is a major cause of the nonviability observed when top2 cells undergo mitosis at the restrictive temperature. In related experiments, we determined that topoisomerase II must act specifically during mitosis. This latter observation is consistent with the hypothesis that the mitotic spindle is necessary to allow topoisomerase II to complete the untangling of sister chromatids.

Aneuploidy↗
PubMed1988

Hormone-sensitive lipase: sequence, expression, and chromosomal localization to 19 cent-q13.3.

Hormone-sensitive lipase, a key enzyme in fatty acid mobilization, overall energy homeostasis, and possibly steroidogenesis, is acutely controlled through reversible phosphorylation by catecholamines and insulin. The 757-amino acid sequence predicted from a cloned rat adipocyte complementary DNA showed no homology with any other known lipase or protein. The activity-controlling phosphorylation site was localized to Ser563 in a markedly hydrophilic domain, and a lipid-binding consensus site was tentatively identified. One or several messenger RNA species (3.3, 3.5, or 3.9 kilobases) were expressed in adipose and steroidogenic tissues and heart and skeletal muscle. The human hormone-sensitive lipase gene mapped to chromosome 19 cent-q13.3.

Amino Acid Sequence↗
PubMed1988

Effects of dopamine on the portal circulation after therapeutic hepatic artery ligation.

The effects of exogenous dopamine (2, 4 and 6 micrograms.kg-1.min-1 i.v.) on the portal circulation were studied in six patients following therapeutic hepatic artery ligation. Portal blood flow (PBF) was measured by the continuous thermodilution technique. Portal venous pressure (PVP, n = 3) was monitored through the thermodilution catheter to allow derivation of preportal vascular resistance (PVR). Blood samples were taken through the portal venous catheter for measurement of dopamine. A significant increase in PBF and a decrease in PVR were observed during graded i.v. dopamine infusion. Thus, PBF was 961 +/- 119 ml.min-1 during control conditions and increased to 1446 +/- 221 ml.min-1 during the dopamine infusion at 6 micrograms.kg-1.min-1. No significant changes in mean arterial pressure or PVP were observed during dopamine administration. The pharmacokinetics of dopamine did not differ from that previously reported in patients with an intact arterial supply. In conclusion, our data indicate that exogenous dopamine consistently increases PBF by preportal vasodilation, also in patients with a surgically restricted hepatic arterial blood supply.

Adult↗