Prostate specific antigen density: a method for differentiating prostate carcinoma from benign prostatic hypertrophy demonstrates ethnic variation.
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Aged↗
Biomedical subjects
S Clejan
Publications and source records attributed to S Clejan.
Rapid preparation of tissue DNA from paraffin-embedded blocks and analysis by polymerase chain reaction.
We have developed a new, easy, and more rapid method for DNA preparation, which avoids contamination. With this method, manual surgical blade scrapings from precisely targeted areas of paraffin block surfaces, without microtome cutting, were used to obtain tissues from 10 different neoplasms. Our results indicate the feasibility of DNA extraction from the scraped paraffin tissue for molecular genetic analysis. We applied this technique successfully to screen for the presence of human papillomavirus using the polymerase chain reaction (PCR) procedure in cases of endocervical, esophageal, and nasopharyngeal carcinomas, and to examine the expression of p53 gene from prostate and gastric adenocarcinomas. We conclude that this procedure is also suitable for purification of PCR products in analysis of the mutation or loss of allelic genes by Bstu I endonuclease digestion.
Base Sequence↗
Interaction of cholesterol with conformationally restricted phospholipids in vesicles.
The interaction of cholesterol with conformationally restricted analogs of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) in the liquid-crystalline phase has been studied in vesicles. These analogs contain one of three cyclopentane triols in place of the glycerol moiety found in natural phospholipids and make possible an analysis of whether a limitation of the conformational mobility in the glycerol backbone region affects the interaction with cholesterol. When cholesterol was incorporated into vesicles from cyclopentanoid phospholipids in which the acyl group vicinal to the head group is trans, the first-order rate constant for Cl- efflux is decreased similarly to that in vesicles from 'natural' DPPC or DPPG (about 50%). However, when the head group is in the unnatural 2 position, cholesterol has a much smaller effect on the rate of Cl- efflux (a decrease of about 20%). Cholesterol decreased the rate constants for valinomycin-mediated 86Rb+ efflux from vesicles of the cyclopentanoid PC analogs and of DPPC to a similar extent. The half-time values for spontaneous intervesicle cholesterol exchange were not markedly different using vesicles prepared with the natural glycerophospholipids and with the cyclopentano-phospholipids, suggesting that the geometrical orientation of the acyl chains or the head group has little influence on cholesterol desorption from the lipid/water interface.
1,2-Dipalmitoylphosphatidylcholine↗
Prostate specific antigen and prostatitis. I. Effect of prostatitis on serum PSA in the human and nonhuman primate.
Prostate specific antigen (PSA) has become a mainstay in the diagnosis and management of patients with prostate cancer. We have found, as have others, that it may be elevated in patients with prostatic inflammation. Ten patients had clinical evidence of prostatitis and elevated PSA levels. Six of these had persistently elevated levels after antibiotic treatment. After transrectal ultrasonography and biopsy, two had findings of adenocarcinoma, and the rest had a pathologic diagnosis of acute or chronic prostatitis. We studied this process in an experimental model of prostatitis using a nonhuman primate. We infected six cynomolgus monkeys and followed their PSA levels until resolution of the infection. The PSA peaked between 5 and 7 days after inoculation and gradually returned to baseline in 8 weeks. The dramatically elevated serum PSA levels in bacterial prostatitis can cause confusion in the diagnosis of prostatic carcinoma.
Adult↗
Prostate specific antigen and prostatitis. II. PSA production and release kinetics in vitro.
Elevations in serum prostate specific antigen (PSA) levels in patients with prostatitis are well known, but the pathophysiologic mechanisms involved with this phenomenon are poorly understood. We have recently evaluated the effect of prostatitis on PSA levels in primates. This data demonstrated a rapid rise in PSA, which subsequently fell along the biological decay curve. This suggested a release of sequestered PSA. We evaluated the effect of infection upon PSA production for the prostatic adenocarcinoma cell line LNCaP. The cell line was determined to produce 9.6 +/- 2.7 fg/cell/hr PSA with essentially linear kinetics. No effect was seen with dead bacteria, bacterial supernatant or complement upon the PSA production. Live E. coli had no effect for 4-8 hours at which time the cells sloughed and PSA production ceased. No release of stored PSA was seen. These data do not elucidate the reasons for increased serum PSA levels found with acute bacterial prostatitis, but indicate that it is not a storage phenomenon.
Antigens, Neoplasm↗
Digital rectal examination-associated alterations in serum prostate-specific antigen.
The effect of digital rectal examination (DRE) on measurement of serum prostate-specific antigen was investigated during a prostate cancer screening program of 2,736 ambulatory men. Serum samples were collected before and after DRE and values compared using the nonparametric Wilcoxon signed rank test. Small, yet statistically significant, increases were found associated with DRE. The magnitude of these increases, however, was of minor clinical importance. Patients who exhibited the largest increases with initial values greater than the upper limit of normal (4 micrograms/L) were found to have either benign prostatic hyperplasia, prostatitis, or prostatic carcinoma. The benign prostatic hyperplasia patients showed relatively low initial prostate-specific antigen values with similarly small increases related to DRE, whereas the prostatitis and cancer patients exhibited both higher initial prostate-specific antigen values and larger increases associated with DRE. Finally, patients with increases in prostate-specific antigen from less than 4 micrograms/L to greater than or equal to 4 micrograms/L comprised less than 2% of the reference range population, the majority of whom had post-DRE measurements of less than 5 micrograms/L. Thus, DRE does not appear to be a significant factor in falsely elevating prostate-specific antigen levels and should be of limited concern to the clinician obtaining serum samples after DRE.
Adult↗
Signal transduction by the erythropoietin receptor: evidence for the activation of phospholipases A2 and C.
Erythropoietin (Ep) is the peptide growth factor whose actions on the erythroid progenitor cell induce terminal differentiation. However, the intracellular signaling system that is activated by Ep is poorly understood. Our previous studies have implicated the lipoxygenase metabolites of arachidonic acid in the actions of Ep. In this study, we report an early (30 s to 5 min) increase in levels of two lipoxygenase metabolites: leukotriene B4 (LTB4; 3- to 5-fold) and 12-hydroxyeicosatetraenoic acid (12-HETE; 2-fold). These responses were blocked by an antibody to Ep, by lipoxygenase inhibitors, or by 1,6-di[O-(carbamoyl)cyclohexanone oxime]hexane (RHC80267), an inhibitor of diacylglycerol (DAG) lipase. RHC 80267 also significantly inhibited Ep-mediated proliferation. Ep induced the release of [3H]arachidonic acid from cellular phospholipids at 5 min and also increased DAG accumulation at 1 min with a maximum increase of 68.2% over control seen at 30 min. No increase in levels of inositol trisphosphate or phosphatidic acid was observed in response to Ep. Taken together, these data suggest that the signal transduction pathway of the Ep receptor includes the activation of phospholipases A2 and C, resulting in the liberation of DAG and arachidonate and the subsequent formation of LTB4 and 12-HETE.
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
Protonophore-resistance and cytochrome expression in mutant strains of the facultative alkaliphile Bacillus firmus OF4.
Two protonophore-resistant mutants, designated strains CC1 and CC2, of the facultative alkaliphile Bacillus firmus OF4 811M were isolated. The ability of carbonyl cyanide m-chlorophenylhydrazone (CCCP) to collapse the protonmotive force (delta mu H+) was unimpaired in both mutants. Both resistant strains possessed elevated respiratory rates when grown at pH 7.5, in either the presence or absence of CCCP. Membrane cytochromes were also elevated: cytochrome o in particular in strain CC1, and cytochromes aa3, b, c and o in strain CC2. Strain CC2 also maintained a higher delta mu H+ than the others when grown in the absence of CCCP. When grown in the presence of low concentrations of CCCP, strains CC1 and CC2 both maintained higher values of delta mu H+ than the wild-type parent and correspondingly higher capacities for ATP synthesis. In large-scale batch culture at pH 10.5, both mutant strains grew more slowly than the parent and contained significantly reduced levels of cytochrome o. Cells of stran CC1 also displayed a markedly altered membrane lipid composition when grown at pH 10.5. Unlike previously characterized protonophore-resistant strains of B. subtilis and B. megaterium, neither B. firmus mutant possessed any ability above that of the parent strain to synthesize ATP at given suboptimal values of delta mu H+. Instead, both resistant alkaliphile strains maintained a higher delta mu H+ and a correspondingly higher delta Gp than the parent strain when growing in sublethal concentrations of CCCP, apparently as a result of mutational changes affecting respiratory chain composition. Also of note in both the mutant and the wild-type strains was a marked elevation in the level of one of the multiple terminal oxidases, an aa3-type cytochrome, during growth at pH 7.5 in the presence of CCCP or during growth at pH 10.5, i.e. two conditions that reduce the bulk delta mu H+.
Adenosine Triphosphate↗
Prostate cancer screening in younger men: prostate-specific antigen and public awareness.
The American Cancer Society recommends annual digital rectal examination for men over forty years of age. We evaluated 414 men between forty and fifty-nine years of age with a questionnaire, digital rectal examination (DRE), and prostate-specific antigen (PSA) determination. One hundred ninety were forty to forty-nine years old, and 224 were fifty to fifty-nine years old. Four patients in the forty to forty-nine age group had elevated PSA determinations, and 7 had abnormal findings on DRE. Using prostate ultrasound and biopsy, no cases of prostate cancer were diagnosed. Ten patients in the fifty to fifty-nine age group had elevated PSA determinations, and 5 were diagnosed to have prostate cancer. These data suggest that PSA may have utility in detecting cancer in younger men.
Adenocarcinoma↗
Mutants of Bacillus species isolated on the basis of protonophore resistance are deficient in fatty acid desaturase activity.
The fatty acid desaturase activity in cell extracts of Bacillus subtilis was characterized and found to be O2 dependent, NADH dependent, and cyanide sensitive. In cell fractionation studies, only 10% of the desaturase activity was recovered in the membrane fraction; the addition of cytosolic factors, which by themselves were devoid of activity, restored membrane activity to the level found in the unfractionated cell extracts. NADH was preferred over NADPH as an electron donor, and palmitoyl-coenzyme A was used preferentially over stearoyl-coenzyme A as the straight-chain fatty acid substrate. An increase in desaturase activity was observed when either the growth or the assay temperature was lowered from 37 to 20 degrees C, although the assay temperature appeared to be the more important parameter. Three protonophore-resistant mutants of B. subtilis and a comparable mutant of Bacillus megaterium had been found to possess reduced levels of unsaturated fatty acids in their membrane phospholipids; their protonophore resistance was abolished when grown in the presence of an unsaturated fatty acid supplement. All of these strains were found to be either significantly deficient in or totally lacking desaturase activity in comparison with their wild-type parent strains. Full, protonophore-sensitive revertants of the mutants had levels of desaturase activity comparable to those of the wild-type. Temperature-sensitive revertants of two of the mutants, which grew at 32 degrees C but not at 26 degrees C in the presence of protonophore, exhibited desaturase activity comparable to that of the wild-type at 26 degrees C but lacked activity at 32 degrees C. These results indicate that the biochemical basis for protonophore resistance in these Bacillus mutants is a fatty acid desaturase deficiency.
Bacillus subtilis↗
Facultative alkaliphiles lack fatty acid desaturase activity and lose the ability to grow at near-neutral pH when supplemented with an unsaturated fatty acid.
Two obligate alkaliphiles were found to have high levels of fatty acid desaturase, whereas two facultative alkaliphiles had no detectable activity. Supplementation of the growth medium of one facultative strain with palmitoleic acid, but not palmitic acid, at pH 7.5 inhibited growth. The obligate strain outgrows the facultative strain in a chemostat at a very high pH, whereas the converse is true at a pH of 7.5, and the two strains grow equally well at pH 9.0. Thus, the obligate strain is compromised at a near-neutral pH but is better adapted than a related facultative alkaliphile to an extremely alkaline pH.
Bacillus↗
Increased rates of lipid exchange between Mycoplasma capricolum membranes and vesicles in relation to the propensity of forming nonbilayer lipid structures.
We have studied the effects of modification of the endogenous phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG) content of the plasma membrane of Mycoplasma capricolum on the kinetics of spontaneous [14C]cholesterol and 14C-labeled phospholipid exchange between M. capricolum membranes and lipid vesicles. The PG/DPG molar ratio of M. capricolum membranes changed when cells were grown in media supplemented with 0.5 mM CaCl2 and/or egg phosphatidylcholine (PC) (10-20 micrograms/ml), increasing from 3.9 to 6.3 on supplementation with Ca2+; this ratio decreased to 1.1 in media supplemented with PC and to 1.8 in media containing both PC and Ca2+. The ratio of palmitate to oleate in both PG and DPG decreased when cells were grown with PC or with PC and Ca2+. Bilayer disruptions were seen in freeze-fracture electron micrographs of trypsin-treated M. capricolum membranes from cells grown with both Ca2+ and PC, and numerous lipidic particles and other bilayer disruptions were observed in trypsin-treated M. capricolum membranes and their lipid extracts. The rates of spontaneous exchange of 14C-labeled cholesterol and PC from membranes isolated from cells grown with PC and Ca2+ to acceptor lipid vesicles were exchanged by approximately 30%, and the rate of the rapidly exchangeable cholesterol pool in intact cells was enhanced by 64%. The enhancements in cholesterol and PC exchange rates are considered to result from structural defects expected in the M. capricolum membranes obtained from cells grown with Ca2+ supplementation. Our findings parallel previous examples of functional modifications of membranes induced by bilayer instability arising from a pretransitional state leading to the onset of a nonlamellar phase.
Calcium↗
Evidence of GTP-binding protein regulation of phospholipase A2 activity in isolated human platelet membranes.
G protein regulation of human platelet membrane phospholipase A2 activity was investigated at pH 8.0 and 9.0 by studying the effects of the nonhydrolyzable GTP analogue, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), and of F-/Al3+ ions on arachidonic acid (AA) release. The membrane acted as the source of the enzyme, the substrate, and the G protein. At pH 8.0, 10 and 100 microM GTP gamma S stimulated AA mobilization at least 6-fold. Optimum AA release conditions required 1 mM Ca2+ and 5 mM Mg2+. Nonspecific nucleotide effect was excluded since similar stimulatory effects on AA release were not observed by ATP, GTP, ADP, and NADP. Although at pH 9.0 the GTP gamma S-stimulated AA release was greater than at pH 8.0, it constituted only 26% of the total. At both pH values the effect of F- (10 mM) in the presence of Al3+ (2 microM) was similar to that of GTP gamma S. The G protein inhibitor, guanosine 5'-O-(2-thiodiphosphate), inhibited the GTP gamma S-stimulated AA release by about 80% at pH 8.0 and by 100% at pH 9.0. To determine a possible contribution to AA mobilization by the phospholipase C and diacylglycerol lipase pathway, the effects of neomycin, a phospholipase C inhibitor, were investigated. 100 microM neomycin did not inhibit the GTP gamma S-stimulated AA release at pH 8.0 and only slightly so (17%) at pH 9.0. At pH 8.0 in the presence of Ca2+ the released fatty acids consisted mainly of arachidonic and docosahexaenoic acids (80 and 8%, respectively). GTP gamma S had no effect on the fatty acid profile but only on their quantity. These results provide evidence of G protein regulation of phospholipase A2 activity in isolated platelet membranes.
Arachidonic Acid↗
Mutation of Bacillus firmus OF4 to duramycin resistance results in substantial replacement of membrane lipid phosphatidylethanolamine by its plasmalogen form.
Mutant strains of alkalophilic Bacillus firmus OF4 that were selected for resistance to duramycin had greatly reduced levels of membrane diacylphosphatidylethanolamine, as had been found in studies of such mutants of Bacillus subtilis. In the B. firmus strains, however, substantial levels of plasmenylethanolamine were found. This is an unusual membrane component for an aerobic eubacterium, but the presence of trace amounts even in the wild type was confirmed in experiments with 32Pi-labeled growth medium. The membrane lipid composition of the duramycin-resistant strains had several other changes that also left alkalophilic growth unimpaired.
Anti-Bacterial Agents↗
Permeability studies of lipid vesicles from alkalophilic Bacillus firmus showing opposing effects of membrane isoprenoid and diacylglycerol fractions and suggesting a possible basis for obligate alkalophily.
Previous studies of the membrane lipids of extremely alkalophilic bacilli had indicated that both facultative and obligate alkalophiles contained a substantial fraction of isoprenoid lipid as well as high concentrations of cardiolipin. Facultative alkalophiles differed from obligate strains in having a phospholipid fatty acid composition that would be expected to result in a more ordered membrane structure. Current studies of ion permeability in vesicles prepared from lipids from obligately alkalophilic Bacillus firmus RAB and its facultatively alkalophilic strain, OF4, support the suggestion that membranes of the latter strain form a tighter barrier structure, with the difference especially pronounced at near neutral pH values. The water permeability of whole cells and the reflection coefficients for acetamide in vesicles were also consistent with a tighter membrane in the facultatively alkalophilic strain than in the obligately alkalophilic strain. The permeability properties of vesicles prepared from phospholipids from these organisms were studied as a function of the addition of either homologous membrane isoprenoid or diacylglycerol. For each permeability parameter that was assayed, in lipids from both strains, the isoprenoid fraction decreased the permeability, whereas the diacylglycerol fraction increased the permeability of the vesicles to solute.
Bacillus↗
Large decreases in membrane phosphatidylethanolamine and diphosphatidylglycerol upon mutation to duramycin resistance do not change the protonophore resistance of Bacillus subtilis.
Duramycin-resistant mutant strains were selected from wild-type Bacillus subtilis (BD99) and its protonophore-resistant mutant derivative, strain AG1A3. Analyses of the membranes of the duramycin-resistant mutants showed that they had little or no phosphatidylethanolamine and diphosphatidylglycerol as determined by chemical detection after thin-layer chromatography. Small amounts of these phospholipids must remain in the mutant strains, however, because during studies of incorporation of exogenous, radioactive fatty acids, label associated with palmitoleic acid was found in chromatographic positions that corresponded to the expected positions of phosphatidylethanolamine and diphosphatidylglycerol. The duramycin-resistant strains both showed elevated levels of phosphatidylglycerol and aminoacyl(lysyl)phosphatidylglycerol. The duramycin-resistant derivative of protonophore-resistant AG1A3 (AG1A3-DR4), but not that of the wild type, also showed a decreased content of neutral relative to polar lipid in the membrane. The composition of neutral lipid in that strain was higher in free fatty acids and lower in 1,2-diacylglycerol than its parent strain. AG1A3-DR4 also contained appreciable levels of lysophosphatidylethanolamine and somewhat elevated diglycosyldiacylglycerol relative to the other strains in the study. The protonophore resistance of AG1A3 was unaltered by mutation to duramycin resistance. Nor was there any change in the efficacy of exogenous palmitoleic acid in diminishing the protonophore resistance of AG1A3-DR4. This phenomenon persists upon dramatic reduction in the content of phosphatidylethanolamine and diphosphatidylglycerol even though those phospholipids are normally the preferred sites of incorporation of the exogenous unsaturated fatty acids that mediate the effect.
Anti-Bacterial Agents↗
The protonophore resistance of Bacillus megaterium is correlated with elevated ratios of saturated to unsaturated fatty acids in membrane phospholipids.
Growth of the protonophore-resistant strain of Bacillus megaterium, strain C8, in the presence of oleic acid markedly reduced its resistance to low concentrations of carbonylcyanide m-chlorophenylhydrazone (CCCP). Growth of the CCCP-sensitive wild-type strain in the presence of stearic acid increased the resistance of that strain to growth inhibition by protonophore. Studies of the membrane lipids indicated that in the absence of additions to the medium, membranes from C8 contained greatly reduced levels of monounsaturated fatty acids relative to the wild type; wild-type levels were restored by growth of C8 in the presence of oleic acid, concomitant with the loss of resistance. Conversely, growth of the wild type on stearic acid increased the ratio of saturated/unsaturated fatty acids in the membrane, concomitant with a modest increase in the resistance of the wild-type strain to CCCP. The exogenous oleic acid was preferentially incorporated into phosphatidylethanolamine, diphosphatidylglycerol, and 1,2-diacylglycerol, whereas stearic acid was incorporated preferentially into phosphatidylglycerol, and into the small component of free fatty acids. Depending upon the growth conditions, changes in membrane lipid-to-membrane protein ratio and in the ratios of polar lipid components were observed, but none of those changes correlated as did the changes in saturated fatty-acid-to-unsaturated fatty-acid ratio with protonophore resistance. This latter correlation was further suggested by experiments in which the protonophore resistance of wild type B. megaterium was shown to increase with increasing growth temperature without any temperature-dependent loss of protonophore efficacy. The experiments here support the hypothesis developed from work with Bacillus subtilis that changes in the fatty acid composition of the membrane phospholipids affect energy coupling, and make it clear that simple increases or decreases in the hydrolytic activity of ATPase in the uncoupler-resistant mutants of bacilli are not correlated with resistance in some direct way.
Bacillus megaterium↗
Isolation and characterization of uncoupler-resistant mutants of Bacillus subtilis.
Three mutant strains of Bacillus subtilis were isolated on the basis of their ability to grow in the presence of 5 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP). The mutants (AG2A, AG1A3, and AG3A) were also resistant to 2,4-dinitrophenol, and AG2A exhibited resistance to tributyltin and neomycin. The mutants all exhibited (i) elevated levels of membrane ATPase activity relative to the wild type; (ii) slightly elevated respiratory rates, with the cytochrome contents of the membranes being the same as or slightly lower than those of the wild type; (3) a passive membrane permeability to protons that was indistinguishable from that of the wild type in the absence of CCCP and that was increased by addition of CCCP to the same extent as observed with the wild type; and (4) an enhanced sensitivity to valinomycin with respect to the ability of the ionophore to reduce the transmembrane electrical potential. Finally and importantly, starved whole cells of all the mutants synthesized more ATP than the wild type did upon energization in the presence of any one of several agents that lowered the proton motive force. Studies of revertants indicated that the phenotype resulted from a single mutation. Since a mutation in the coupling membrane might produce such pleiotropic effects, an analysis of the membrane lipids was undertaken with preparations made from cells grown in the absence of CCCP. The membrane lipids of the uncoupler-resistant strains differed from those of the wild type in having reduced amounts of monounsaturated C16 fatty acids and increased ratios of iso/anteiso branches on the C15 fatty acids. Correlations between protonophore resistance and the membrane lipid compositions of the wild type, mutants, and revertants were most consistent with the hypothesis that a reduction in the content of monounsaturated C16 fatty acids in the membrane phospholipids is related, perhaps casually, to the ability to synthesize ATP at low bulk transmembrane electrochemical gradients of protons.
Adenosine Triphosphatases↗