Cloning and expression of the dog mast cell alpha-chymase gene.

Chymases are chymotrypsin-like serine proteinases secreted by mast cells. Alpha- and beta-chymases differ in structure, function, and mast cell subset- and species-specific expression. Seeking genetic regulatory elements shared by alpha-chymases, we sequenced the dog alpha-gene. Extensive homology was found in intronic and flanking sequences of the dog, human, and mouse alpha-chymase genes, but little in corresponding beta-chymase sequences. Repetitive elements probably derived from retroposons are unique features of the dog flank. DNA blots suggest that the dog alpha-gene, like its human counterpart, may be the genome's sole chymase, unlike in rodents, in which beta-chymases predominate. Nuclear runoff studies predict that transcriptional mechanisms explain differences in steady state chymase and tryptase mRNA levels between mastocytoma and non-mast cells. In dog BR mastocytoma cells incubated with phorbol ester, high steady state levels of alpha-chymase mRNA drop dramatically with little change in tryptase mRNA, whereas dexamethasone decreases expression of both mRNAs. Portions of the dog or human gene 5' flank transfected into BR cells drive expression of a reporter gene and define regions with active promoters. Thus, BR cells express high levels of alpha-chymase mRNA regulated independently of tryptase and support transcription using dog or human promoters. These studies reinforce the alphabeta-chymase dichotomy and suggest the utility of BR cells in probing regulation of alpha-chymase expression.

Long-term persistence of AZT-resistance mutations in the plasma HIV-1 of patients removed from AZT therapy.

Zidovudine (3'-azido-2',3'-dideoxythymidine) resistant isolates of human immunodeficiency virus type I (HIV-1) were previously demonstrated in zidovudine-treated AIDS patients. The genetic linkage of multiple mutations characteristic of zidovudine-resistance as well as dideoxyinosine-resistance were demonstrated by examining clones of viral reverse transcriptase after polymerase chain reaction (PCR) amplification of plasma culture DNA. The zidovudine-resistance mutations persisted in seven timepoints from four patients for 5 to 22 months despite cessation of zidovudine therapy (and while patients underwent ddI therapy). One patient's plasma virus isolate at 14 months possessed a genotype doubly resistant to ZDV and ddI. Virus recovered from four timepoints showed Intermediate to high levels of zidovudine-resistance. As these genotypes were mainly derived from plasma culture, the zidovudine resistant virus appears to persist and replicate well in vivo after cessation of zidovudine therapy.

Long-term persistence of zidovudine resistance mutations in plasma isolates of human immunodeficiency virus type 1 of dideoxyinosine-treated patients removed from zidovudine therapy.

Zidovudine (3'-azido-2',3'-dideoxythymidine)-resistant isolates of human immunodeficiency virus type 1 (HIV-1) were previously demonstrated in zidovudine-treated AIDS patients. The genetic linkage of multiple mutations characteristic of zidovudine resistance as well as dideoxyinosine resistance were demonstrated by examining clones of viral reverse transcriptase after polymerase chain reaction amplification of plasma culture DNA. The zidovudine resistance mutations persisted at seven time points from 4 patients for 5-22 months despite cessation of zidovudine therapy (and while patients underwent dideoxyinosine therapy). One patient's plasma virus isolate at 14 months possessed a genotype doubly resistant to zidovudine and dideoxyinosine. Virus recovered from four time points showed intermediate to high levels of zidovudine resistance. As these genotypes were mainly derived from plasma culture, the zidovudine-resistant virus appears to persist and replicate well in vivo after cessation of zidovudine therapy.

Zidovudine-resistant human immunodeficiency virus type 1 genomes detected in plasma distinct from viral genomes in peripheral blood mononuclear cells.

The emergence of zidovudine (3'-azido-2',3'-deoxythymidine)-resistant strains of human immunodeficiency virus type 1 (HIV-1) from AIDS patients treated with zidovudine has been linked to six amino acid substitutions localized within the viral polymerase gene. Here, in 2 patients, three resistance mutations were detected by polymerase chain reaction amplification of HIV-1 polymerase (reverse transcriptase) sequences from cultures of patient plasma only and not from the same patients' uncultured leukocytes. The differences in distribution of the mutant genotypes from the two sources were highly significant. Both plasma- and peripheral blood mononuclear cell (PBMC)-derived virus (and/or RNA genomes) should be studied in additional subjects to confirm the hypothesis raised by these data that zidovudine-resistant virus may be more frequent in plasma than in uncultured PBMC.

Serum concentrations of insulin-like growth factor II are not changed by short-term fasting and refeeding.

To determine the factors that regulate insulin-like growth factor II (IGF-II), we raised polyclonal antibodies to this peptide and developed a RIA that measures IGF-II in serum or plasma samples after extraction of IGF-binding proteins by C18 cartridge chromatography. The IGF-II antiserum was highly specific, exhibiting no cross-reactivity with IGF-I or insulin at the highest concentrations tested (10(-6) mol/L). As little as 0.43 micrograms/L IGF-II was detectable, and 50% displacement of tracer occurred at 1.7 microgram/L. The serum IGF-II concentrations of normal adults [mean, 634 +/- 170 (+/- SD) micrograms/L], patients with acromegaly (570 +/- 146 micrograms/L), and patients with hypopituitarism (156 +/- 58 micrograms/L) were similar to those reported by others. In eight obese subjects injected with GH (0.1 mg/kg ideal BW, im, every 48 h for 16 days), serum IGF-II concentrations did not rise significantly, whereas IGF-I concentrations increased 67%. Sixteen normal subjects, within 15% of ideal body weight, were fasted for 5 days on two to four occasions and refed diets of differing protein and calorie contents. Their mean serum IGF-II concentration before fasting (691 +/- 26 micrograms/L) was not significantly different from that after fasting (674 +/- 21 micrograms/L) or after refeeding (641 +/- 20 micrograms/L). In contrast, their mean IGF-I concentration decreased 42% with fasting and rose with refeeding. Unlike IGF-I, serum IGF-II concentrations do not appear to be regulated by short term changes in nutritional status. It is clear from this study and others that IGF-II and IGF-I are regulated differently despite their structural homology and the similarity of their actions in vitro.