Latency-associated sense transcripts are expressed during in vitro human cytomegalovirus productive infection.

Published studies have identified novel sense transcripts in latent human cytomegalovirus (HCMV) infection. These cytomegalovirus latency-associated transcripts (CLTs) have start sites upstream from the MIE gene productive start site (PSS). We evaluated the expression of the sense CLTs in an in vitro HCMV productive infection. Transcripts with initiation sites upstream from the PSS were detected in infected human fibroblasts using reverse transcription-polymerase chain reaction and 5' rapid amplification of cDNA ends (RACE) analysis. DNA sequencing of 5' RACE PCR products confirmed that the start sites were consistent with sense CLT expression. Furthermore, ribonuclease protection assay analysis showed that transcripts initiating at the latent start site-1 were most abundant at late times postinfection after transcription from the PSS had decreased. In addition, transcription consistent with sense CLT expression was identified in HCMV-infected dTHP-1 cells, monocyte-derived macrophages, and endothelial cells, as well as in clinical isolate-infected human fibroblasts. These findings clearly demonstrate the expression of sense CLTs during in vitro HCMV infection.

Racial differences in clinically localized prostate cancers of black and white men.

PURPOSE: Tumor grade, deoxyribonucleic acid (DNA) ploidy, proliferation, p53 and bcl-2 expression were examined in clinically localized prostate cancers of black and white American men to learn whether these features showed racial differences. MATERIALS AND METHODS: A total of 117 prostate cancers (43 black and 74 white patients) obtained at radical prostatectomy for clinically localized disease were assigned Gleason scores by a single pathologist. Enzymatically dissociated nuclei from archival prostate cancers were examined by DNA flow cytometry using propidium iodide staining and the multicycle program to remove debris and sliced nuclei and to perform cell cycle analysis. For immunostaining after microwave antigen retrieval we used a DO-1/DO-7 monoclonal antibody cocktail for p53 and the clone 124 antibody for bcl-2. RESULTS: Significantly more black than white men had Gleason score 7 tumors. The DNA ploidy distribution of Gleason 6 or less tumors was similar for both races. As anticipated, the ploidy distribution of higher grade prostate cancer in white men was more abnormal but, unexpectedly, this was not found for higher grade prostate cancer in black men. No significant racial differences were found in S phase fractions, p53 or bcl-2 immunopositivity. However, for prostate cancer in black men there was a significant association between bcl-2 immunopositivity and higher S-phase fractions. CONCLUSIONS: The aggressive prostate cancers of black men may be characterized by the 2 features of high proliferation and a block to programmed cell death.

Behavioral and neurochemical changes in folate-deficient mice.

Weanling mice were fed an amino acid-based diet supplemented with 0 or 11.3 mumol folic acid/kg diet for approximately 38 days to study behavior and neurochemistry in folate deficiency. After approximately 5 wk, mice fed the unsupplemented diet weighted approximately 70% as much those fed the supplemented diet. After 2 wk, mice fed the unsupplemented diet consistently discarded (spilled) more food, and after approximately 5 wk, they had spilled 3 times more than mice fed the supplemented diet. Serum folate, brain folate and brain S-adenosylmethionine of mice fed the unsupplemented diet were 4, 53, and 60% as high, respectively, as those of mice fed the supplemented diet. Pathologic changes were not evident in brain, spinal cord, or skeletal muscle of folate-deficient mice. The hypothalamic 5-hydroxyindole acetic acid/serotonin ratio and caudate dopamine, homovanillic acid, and 3,4-dihydroxyphenylacetic acid concentrations were lower in deficient than control mice. Folate-deficient mice develop a behavioral activity, food spilling, which may have a neurochemical basis in the serotonin and dopamine systems.

Solid-phase extraction protocol for isolating retinol-d4 and retinol from plasma for parallel processing for epidemiological studies.

Solid-phase extraction permits the parallel processing of samples in large numbers. We have applied this technique to the isolation of retinol isotopomers from plasma of humans participating in a study of vitamin A stable isotope dilution. The isotopomers were analyzed by gas chromatography/mass spectrometry. The extraction involves the separation of retinol from its aqueous matrix with a C18 silica-based sorbent followed by removal of lipid contaminants with an aminopropyl silica-based sorbent. Overall recovery of retinol from plasma was 47.2% +/- 1.8%. Purity of the retinol isolated from plasma is comparable with that obtained with a single HPLC method. This method permits the preparation of 32 samples per day by one analyst. Elimination of the need for HPLC permits sample preparation in the field with a minimum of equipment and technical skill.

The active sites of cytochromes P450 IA1, IIB1, IIB2, and IIE1. Topological analysis by in situ rearrangement of phenyl-iron complexes.

The reactions of cytochromes P450 IA1, IIB1, IIB2, and IIE1 with phenyldiazene yield complexes with absorption maxima at either 474 or 480 nm. Anaerobic extraction of the prosthetic group from the phenyldiazene-treated proteins followed by its exposure to oxygen and strong acid produces an equal mixture of the four possible N-phenylprotoporphyrin IX regioisomers. Exposure of the anaerobically extracted heme complexes to oxygen in the absence of strong acid results in their decomposition to heme and products other than N-phenylprotoporphyrin IX. These results show that the 474/480 nm absorption maxima are due to sigma phenyl-iron complexes. Treatment of the intact hepatic cytochrome P450 complexes with K3Fe(CN)6 results in disappearance of the 474/480 nm band. Extraction of the prosthetic group then yields only the two N-phenylprotoporphyrin IX regioisomers with the N-phenyl group on pyrrole rings A and D. The same regioisomer pattern is obtained if the cytochrome P450IA1 phenyl-iron complex is simply warmed to 37 degrees C, but this thermal rearrangement occurs much less readily, if at all, with the complexes of the other isozymes. The regioisomers with the N-phenyl on pyrrole rings A and D are obtained in a 2:1 ratio with isozyme IA1, 1:1 with IIB2, 1:1.7 with IIB1, and 1:2 with IIE1. These results establish that the active sites of these cytochrome P450 isozymes have a common architecture despite their gross differences in substrate specificity. In this architecture, the region directly above pyrrole rings A and D is relatively open whereas that above pyrrole rings B and C is occluded by protein residues.

Secobarbital-mediated inactivation of rat liver cytochrome P-450b: a mechanistic reappraisal.

Administration of the allylbarbiturate secobarbital (SB) to phenobarbital-pretreated rats is known to result in structural and functional loss of hepatic cytochrome P-450 and generation of N-alkylated prosthetic heme derivatives. Isozyme-selective functional markers have led us to confirm P-450b as the isozyme selectively inactivated by the drug. In contrast to its inactivation by allylisopropylacetamide, such SB-inactivated P-450b is not amenable to structural and functional repair by exogenous heme, for reasons that remain unclear. In an effort to gain some insight, we have explored various possible mechanisms. In the course of these studies with rat liver microsomes enriched in P-450b as well as isolated purified P-450b, we have found that, along with prosthetic heme alkylation, a significant fraction of the hemoprotein also undergoes drug-mediated alkylation of the apocytochrome, presumably at the active site. Accordingly, an equimolar ratio of irreversibly bound drug to functionally inactive residual P-450b chromophore is observed after incubation of the purified isozyme with SB and NADPH. Thus, P-450b-mediated oxidative metabolism of SB appears to partition not only between prosthetic heme alkylation and epoxidation but apoprotein alkylation as well.

Drug-induced modulations of hepatic heme metabolism. Neurological consequences.

Administration of the porphyrinogenic agent DDEP to PB-pretreated rats results in acute hepatic heme depletion, which is a characteristic of acute hepatic porphyria. Such acute heme depletion is associated with impaired hepatic tryptophan degradation and enhanced serotonergic tone in the CNS. We showed that intestinal motility in these rats is also significantly decreased, indicating that the serotonergic tone of the enteric nervous system may also be enhanced. In addition, the marked hepatic accumulation of glucogenic precursors, observed in parallel, indicates that the elevated tryptophan levels may also block hepatic glucogenesis. The clinical implications of these findings to acute heme-deficient states, such as the acute hepatic porphyrias, was discussed.