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1.
J Am Soc Nephrol ; 9(3): 439-43, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9513906

ABSTRACT

Acute uremia (ARF) causes metabolic defects in glucose and protein metabolism that contribute to muscle wasting. To examine whether there are also defects in the metabolism of essential amino acids in ARF, we measured the activity of the rate-limiting enzyme for branched-chain amino acid catabolism, branched-chain ketoacid dehydrogenase (BCKAD), in rat muscles. Because chronic acidosis activates muscle BCKAD, we also evaluated the influence of acidosis by studying ARF rats given either NaCl (ARF-NaCl) or NaHCO3 (ARF-HCO3) to prevent acidosis, and sham-operated, control rats given NaHCO3. ARF-NaCl rats became progressively acidemic (serum [HCO3] = 21.3 +/- 0.7 mM within 18 h and 14.7 +/- 0.8 mM after 44 h; mean +/- SEM), but this was corrected with NaHCO3. Plasma valine was low in ARF-NaCl and ARF-HCO3 rats. Plasma isoleucine, but not leucine, was low in ARF-NaCl rats, and isoleucine tended to be lower in ARF-HCO3 rats. Basal BCKAD activity (a measure of active BCKAD in muscle) was increased more than 17-fold (P < 0.01) in ARF-NaCl rat muscles, and this response was partially suppressed by NaHCO3. Maximal BCKAD activity (an estimate of BCKAD content), subunit mRNA levels, and BCKAD protein content were not different in ARF and control rat muscles. Thus, ARF increases branched-chain amino acid catabolism by activating BCKAD by a mechanism that includes acidosis. Moreover, in a muscle-wasting condition such as ARF, there is a coordinated increase in protein and essential amino acid catabolism.


Subject(s)
Muscle, Skeletal/metabolism , Uremia/metabolism , Wasting Syndrome/metabolism , 3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide) , Acidosis/prevention & control , Acute Disease , Administration, Oral , Amino Acids/metabolism , Animals , Carbonates/administration & dosage , Carbonates/therapeutic use , Enzyme Activation/drug effects , Isoenzymes/analysis , Isoenzymes/drug effects , Isoenzymes/genetics , Isoleucine/blood , Isoleucine/drug effects , Ketone Oxidoreductases/drug effects , Ketone Oxidoreductases/genetics , Ketone Oxidoreductases/metabolism , Male , Mitochondria/enzymology , Multienzyme Complexes/drug effects , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Muscle, Skeletal/chemistry , Muscle, Skeletal/pathology , RNA, Messenger/analysis , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Transfer RNA Aminoacylation , Valine/blood , Valine/drug effects
2.
Int J Tissue React ; 20(3): 85-9, 1998.
Article in English | MEDLINE | ID: mdl-9894180

ABSTRACT

The nonsteroidal antiinflammatory drug sulindac (sulfoxide) is known to cause regression and prevent recurrence of adenomas in patients with familial adenomatous polyposis. The mechanism of action does not appear to require inhibition of prostaglandin synthesis since the sulfone metabolite of sulindac (FGN-1) retains the antineoplastic properties of sulindac but lacks inhibitory effects on cyclooxygenase, types 1 and 2. FGN-1 has been shown to induce apoptosis in a variety of tumor cell lines, and selective apoptosis of neoplastic cells has been proposed to account for its antineoplastic properties. Since angiogenesis is necessary for tumor progression and may be related to apoptosis, it is possible that inhibition of angiogenesis may also contribute to the antineoplastic properties of sulindac or FGN-1. In order to test this possibility, cells derived from several different types of human lung tumors were grafted intradermally in Balb/c mice. Sulindac sulfoxide and its sulfide and sulfone metabolites were administered for 3 days orally, in a daily dose of 0.025-0.5 mg, and angiogenesis was measured after 72 h using a previously described method. The results showed that sulindac sulfoxide and sulfone statistically inhibited angiogenesis.


Subject(s)
Adenocarcinoma/pathology , Antineoplastic Agents/pharmacology , Carcinoma, Squamous Cell/pathology , Lung Neoplasms/pathology , Neovascularization, Pathologic/pathology , Sulindac/analogs & derivatives , Adenocarcinoma/drug therapy , Adult , Aged , Animals , Carcinoma, Squamous Cell/drug therapy , Female , Humans , Lung Neoplasms/drug therapy , Male , Mice , Mice, Inbred BALB C , Middle Aged , Neoplasm Transplantation , Sulindac/pharmacology
3.
Circulation ; 94(6): 1412-22, 1996 Sep 15.
Article in English | MEDLINE | ID: mdl-8823001

ABSTRACT

BACKGROUND: Thrombolytic therapy reduces mortality in patients with acute myocardial infarction, but significant limitations exist with the use of currently available agents. In the present report, we describe the thrombolytic and antithrombotic potencies of a hybrid recombinant plasminogen activator consisting of an antifibrin antibody 59D8 (AFA) and low-molecular-weight single-chain urokinase-type plasminogen activator (scuPA). METHODS AND RESULTS: A thrombolysis model in which thrombi are preformed in vivo in juvenile baboons was developed to compare the potencies of AFA-scuPA, recombinant tissue plasminogen activator (rTPA), and recombinant scuPA (rscuPA) in lysing nonocclusive 111In-labeled platelet-rich arterial-type thrombi and 125I-labeled fibrin-rich venous-type thrombi. Systemic infusion of 1.89 nmol/kg AFA-scuPA produced thrombolysis that was comparable to that obtained with much higher doses of TPA (14.2 nmol/kg) and rscuPA (28.5 nmol/kg). When steady-state plasma concentrations are normalized, AFA-scuPA lyses thrombi sixfold more rapidly than scuPA and TPA (P < .001) and reduces the rate of formation more than comparable doses of rscuPA (P < .0001). At equivalent thrombolytic doses, AFA-scuPA produced fewer antihemostatic effects than either rTPA or rscuPA. Template bleeding time measurements were shorter (3.5 +/- 0.12 minutes for AFA-scuPA versus 5.3 +/- 0.36 and 5.2 +/- 0.04 minutes for rTPA and rscuPA, respectively; P < .05), alpha 2-antiplasmin consumption was less (P < .05), and D-dimer generation was lower (P < .05). CONCLUSIONS: We conclude that antibody targeting of scuPA to fibrin increases thrombolytic and antithrombotic potencies with less impairment of hemostasis compared with rTPA and rscuPA.


Subject(s)
Antibodies, Monoclonal/immunology , Fibrin/immunology , Plasminogen Activators/pharmacology , Thrombolytic Therapy , Thrombosis/prevention & control , Urokinase-Type Plasminogen Activator/pharmacology , Animals , Hemostasis/drug effects , Male , Recombinant Proteins , Tissue Plasminogen Activator/pharmacology
4.
J Biol Chem ; 270(13): 7431-6, 1995 Mar 31.
Article in English | MEDLINE | ID: mdl-7706289

ABSTRACT

The capacity of antisense sequences to the thrombin receptor to selectively inhibit thrombin receptor expression and limit mitogenic responses in vascular wall cells was investigated in vitro. Eight phosphorothioate oligodeoxynucleotides based on the sequences of the rat thrombin receptor (including sense, antisense, scrambled, and missense controls) were synthesized, characterized, and purified by high performance liquid chromatography. The antisense oligodeoxynucleotide (ODN 4) inhibitory effect was sequence-specific and both time-and concentration-dependent. A reduction in serum or alpha-thrombin-induced smooth muscle cell (SMC) proliferation was noted as early as 3 days at 30 microM (82%; 6.17 +/- 1.01 versus 34.08 +/- 3.89 x 10(4) cells/well; p < 0.05) and at a dose as low as 15 microM after 4 days in culture (19%; p < 0.05). Nonspecific effects were enhanced after prolonged exposure of SMC to the antisense oligodeoxynucleotide (> or = 6 days). A reduction of inositol phosphate generation greater than 50% (p < 0.05) was detected after exposure of SMC to antisense but not to sense or scrambled nucleotide sequences. This was observed after stimulation with both thrombin and SFFLRN (thrombin receptor peptide agonist). Northern blot analysis and enzyme-linked immunosorbent assays revealed 50 and 22% decreases, respectively, in thrombin receptor mRNA and protein (cell surface) levels in antisense oligonucleotide-treated (72 h) SMC as compared to untreated cells, suggesting that thrombin receptor down-regulation occurred at the pretranslational level. Thus, thrombin receptor-specific antisense sequences inhibit growth-related effects both of serum and thrombin on smooth muscle cells, potentially providing a new strategy for selective inhibition of receptor-mediated arterial injury responses.


Subject(s)
Aorta, Thoracic/cytology , Muscle, Smooth, Vascular/cytology , Oligonucleotides, Antisense/pharmacology , Receptors, Thrombin/biosynthesis , Amino Acid Sequence , Animals , Aorta, Thoracic/drug effects , Base Sequence , Blotting, Northern , Cell Division/drug effects , Cells, Cultured , Down-Regulation/drug effects , Male , Molecular Sequence Data , Muscle, Smooth, Vascular/drug effects , Oligodeoxyribonucleotides/chemical synthesis , Oligodeoxyribonucleotides/pharmacology , Oligonucleotides, Antisense/chemical synthesis , Oligopeptides/pharmacology , Organothiophosphates , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , Rats , Rats, Sprague-Dawley , Receptors, Thrombin/genetics , Thrombin/pharmacology
5.
J Biol Chem ; 268(8): 5550-6, 1993 Mar 15.
Article in English | MEDLINE | ID: mdl-8449917

ABSTRACT

To determine whether the fibrin-binding domains of tissue plasminogen activator (tPA) can confer enhanced catalytic activity to single-chain urokinase-type plasminogen activator (scuPA), we constructed, expressed, and characterized the kinetics of five recombinant tPA/scuPA hybrid molecules. The hybrid molecules are: 1) tPA3-50 (tPA finger)/scuPA138-411, 2) tPA177-256 (tPA kringle2)/scuPA140-411 (scuPA catalytic), 3) tPA3-50/tPA177-256/scuPA140-411, 4) scuPA1-47 (scuPA growth factor)/tPA177-256/scuPA140-411, and 5) scuPA1-138 (scuPA growth factor and kringle)tPA127-256/scuPA139-411. The amidolytic activity of all hybrids was comparable, as were the kinetics for conversion from single-chain to two-chain plasminogen activator. We found that 1) the lag time prior to achieving maximal velocity among these hybrids varied, 2) hybrids 2, 3, 4, and 5 were 2-134-fold more potent (by kcat/Km) than hybrid 1, and 3) those hybrid proteins containing the tPA finger domain (hybrids 1 and 3) gave a 2-fold increase in catalytic efficiency in the presence of DESAFIB (reptilase-digested fibrinogen). These kinetic differences are likely mediated by changes in the tertiary structure of the scuPA catalytic domain resulting from interactions between catalytic and noncatalytic domains in the presence of fibrin.


Subject(s)
Tissue Plasminogen Activator/metabolism , Urokinase-Type Plasminogen Activator/metabolism , Animals , Base Sequence , Catalysis , Cell Line , DNA , Fibrin/metabolism , Fibrinogen/metabolism , Kinetics , Molecular Sequence Data , Plasminogen/metabolism , Recombinant Fusion Proteins/metabolism , Structure-Activity Relationship
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