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Mature human aorta contains a 70-kDa versican fragment, which reacts with a neoepitope antiserum to the C-terminal peptide sequence DPEAAE. This protein therefore appears to represent the G1 domain of versican V1 (G1-DPEAAE(441)), which has been generated in vivo by proteolytic cleavage at the Glu(441)-Ala(442) bond, within the sequence DPEAAE(441)-A(442)RRGQ. Because the equivalent aggrecan product (G1-NITEGE(341)) and brevican product (G1-EAVESE(395)) are generated by ADAMTS-mediated cleavage of the respective proteoglycans, we tested the capacity of recombinant ADAMTS-1 and ADAMTS-4 to cleave versican at Glu(441)-Ala(442). Both enzymes cleaved a recombinant versican substrate and native human versican at the Glu(441)-Ala(442) bond and the mature form of ADAMTS-4 was detected by Western analysis of extracts of aortic intima. We conclude that versican V1 proteolysis in vivo can be catalyzed by one or more members of the ADAMTS family of metalloproteinases.

Antibodies to repressible nonspecific acid phosphatase [APase; orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2] purified from Saccharomyces cerevisiae were used to detect the in vitro products of APase mRNA. Immunoprecipitation of cell-free synthesized protein and of in vivo enzyme from cell extracts has shown that derepression of enzyme synthesis in situ is the result of de novo appearance of functional mRNA followed by de novo protein synthesis. At least three unique APase polypeptides are synthesized in vitro from separate mRNAs and appear to be glycosylated in vivo to form secreted enzyme.

The combined and separate hemodynamic effects of dobutamine and IV nitroglycerin were compared in 12 patients with chronic congestive heart failure (nine with ischemic heart disease, two with idiopathic congestive cardiomyopathy, and one with valvular heart disease). Dobutamine (7.1 µg/kg/min) increased cardiac index from 2.4 ± 0.4 to 3.4 ± 0.9 L/min/m2(P < 0.001) and decreased pulmonary wedge pressure from 28 ± 5 to 16 ± 4 mm Hg (P < 0.001) while nitroglycerin (127 µg/min) alone increased cardia index to 2.8 L/min/m2(P < 0.001) and decreased wedge pressure to 14.3 mm Hg (P < 0.001). With both drugs, cardiac index increased to 3.5 ± 0.6 L/min/m2; (NS compared to dobutamine alone) wedge pressure decreased to 11 ± 4L/min/m2(P < 0.05 compared to dobutamine alone). Those beneficial hemodynamic effects occurred without a significant change in the double product of heart rate and blood pressure, and were associated with an improvement in the transmyocardial gradient. Thus, the greatly enhanced ventricular performance with dobutamine + nitroglycerin was associated with a better relationship between myocardial oxygen demand and supply.

We examined the genetic system responsible for transcriptional regulation of repressible acid phosphatase (APase; orthophosphoric-monoester phosphohydrolase [acid optimum, EC 3.1.3.2]) in Saccharomyces cerevisiae at the molecular level by analysis of previously isolated and genetically well-defined regulatory gene mutants known to affect APase expression. These mutants identify numerous positive- (PHO4, PHO2, PHO81) and negative-acting (PHO80, PHO85) regulatory loci dispersed throughout the yeast genome. We showed that the interplay of these positive and negative regulatory genes occurs before or during APase gene transcription and that their functions are all indispensible for normal regulation of mRNA synthesis. Biochemical evidence suggests that the regulatory gene products they encode are expressed constitutively. More detailed investigation of APase synthesis is a conditional PHO80(Ts) mutant indicated that neither PHO4 nor any other protein factor necessary for APase mRNA synthesis is transcriptionally regulated by PHO80. Moreover, in the absence of PHO80, the corepressor, presumed to be a metabolite of Pi, did not inhibit their function in the transcriptional activation of APase.

We have examined the regulation of repressible acid phosphatase (APase; orthophosphoric-monoester phosphohydrolase [acid optimum], EC 3.1.3.2) in Saccharomyces cerevisiae at the physiological and molecular levels, through a series of repression and derepression experiments. We demonstrated that APase synthesis is tightly regulated throughout the growth phase and is influenced by exogenous and endogenous Pi pools. During growth in a nonlimiting Pi medium, APase is repressed. When external Pi becomes limiting, there is a biphasic appearance of APase mRNA and enzyme. Our data on APase mRNA half-lives and on the flux of intracellular Pi and polyphosphate during derepression are consistent with a mechanism of transcriptional autoregulation for the biphasic appearance of APase mRNA. Accordingly, preculture concentrations of Pi control the level of corepressor generated from intracellular polyphosphate degradation. When cells are fully derepressed, APase mRNA levels are constant, and the maximal linear accumulation rate of APase is observed. A scheme to integrate phosphorus metabolism and phosphatase regulation in S. cerevisiae is proposed.

The synthesis of repressible acid phosphatase in Saccharomyces cerevisiae was examined under conditions of blocked derepression as described by Toh-e et al. (Mol. Gen. Genet. 162:139-149, 1978). Based on a genetic and biochemical analysis of the phenomenon these authors proposed a new regulatory model for acid phosphatase expression involving a simultaneous interaction of regulatory factors in the control of structural gene transcription. We demonstrate here that under growth conditions that fail to produce acid phosphatase the enzyme is readily inactivated. Furthermore, we demonstrate under these conditions the production of acid phosphatase mRNA which is active both in vitro and in vivo in the synthesis of enzyme. This eliminates any step prior to translation of acid phosphatase polypeptide as an explanation for the phenomenon. We interpret our results for the block in appearance of acid phosphatase as a result of both deaccelerated growth and cellular biosynthesis during derepression, accompanied by an enhanced instability of the enzyme.

The immunological events mediated by, and antigen specificity of, allograft-bound lymphocytes (ABLs) are poorly understood. To further define the role of anti-body-mediated rejection, a rejected allograft from a patient with primary anti-TBM disease was sterilely minced and pressed through a microscreen. The ABLs were isolated by density gradient centrifugation. Using this technique, 8.5x106ABLs were isolated. Then 1x106washed ABLs/ml were suspended in RPMI 1640 with 20 fetal calf serum and cultured in microtiter plates with media only, or with pokeweed mitogen plates with media only, or with pokeweed mitogen (PWM) (100 $mUg/culture). The cells were incubated for 7 days and supernatants were collected and assayed for total IgG and IgM by a solid-phase enzyme immunoassay (EIA) and reactivity with normal human kidney targets by indirect immunofluorescence (IF) and immunoperoxidase (IP) techniques. Total IgG production was 500 ng/ml for both spontaneous and PWM stimulated cells. No IgM production was detected. IF and IP studies demonstrated IgG-anti-TBM antibodies in the spontaneous supernatants only. IgG antibodies reactive with peritubular capillaries (anti-PTC) were also noted. IgG-anti-TBM anti-bodies and antibodies reactive with arterioles were subsequently demonstrated by direct immunofluorescence techniques in the rejected allograft. Analysis of serum samples obtained at the time of allograft rejection showed no IF or IP reactivity with the Kidney targets. Subsequent analysis of anti-TBM production by the patient's peripheral blood mononuclear cells (PBMs) showed IgM-anti-TBM only. These studies suggest that the IgG-anti-TBM and IgG-anti-PTC antibodies reactive with the allograft resulted from in situ antibody production by ABLs; the role of anti-TBM antibodies in mediating the AR is unclear, but their presence suggests recurrence of the original disease in the allograft. Anti-PTC antibodies could be important in mediation of the vascular AR.

Two clones from a lambda phage collection containing yeast genes regulated by inorganic phosphate were shown by low-stringency hybridization to select three mRNAs that direct the in vitro synthesis of repressible acid phosphatase (EC 3.1.3.2) polypeptides p60, p58, and p56. By higher stringency hybridization one yeast fragment [8 kilobases (kb)] selects p60 mRNA and the other (5 kb) selects p56 mRNA. These EcoRI digestion fragments were subcloned in yeast transformation vectors and hybridization selection assignments were confirmed by measuring enzyme and mRNA levels in transformants. Enzyme and mRNA levels in (8-kb) high copy number transformants grown in high inorganic phosphate medium revealed a hitherto undetected acid phosphatase protein, P57, which is believed to correspond to the constitutive enzyme encoded by PHO3. The identify of the 8-kb fragment purported to contain the PHO5/PHO3 genes was confirmed by genetic mapping of an integrated copy of this fragment. The site of integration of the 5-kb fragment was demonstrated to be unlinked to the PHO5/PHO3 genes.