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Biomedical subjects

S Kaufman

Publications and source records attributed to S Kaufman.

At least 127 records · Page 7Linked to original sources
PubMed1994

Induction of tissue factor-like activity in monocytes by anti-cardiolipin antibodies.

Anti-cardiolipin Abs (ACLA) are present in the sera of patients with antiphospholipid syndrome (APLS) and are associated with high incidence of thromboembolic phenomena, fetal loss, thrombocytopenia, and prolongation of the phospholipid-dependent coagulation assays (lupus anticoagulant). Recently, it has been shown that APLS can be induced experimentally by using ACLA. However, the pathophysiology of thrombus formation in this syndrome is unknown. Monocytes generate a potent procoagulant activity (PCA) after stimulation with various substances. Increased PCA has been found in monocytes from patients with diseases that are associated with high incidence of thromboembolic phenomena. In the present study, we report that the monoclonal ACLA that were shown previously by us to induce APLS stimulate mononuclear cells to generate a potent PCA. The PCA resembled tissue factor (TF) in that it accelerated clotting through the extrinsic coagulation pathway, was abolished by phospholipase C, and was inhibited by anti-TF mAbs. The induction of TF-like activity by ACLA in monocytes was dose- and time-dependent. It was induced in monocytes and monocytic cell lines, but not in lymphoid or myeloid cells, and did not require T lymphocytes for expression. The generation of PCA was dependent on protein synthesis inasmuch as it was prevented by adding puromycin to the system and was not affected by cytarabine. The TF-like activity that is induced by ACLA in monocytes may activate coagulation and thereby play a major role in the pathogenesis of thrombus formation in APLS.

Adult↗
PubMed1994

High-level expression and deletion mutagenesis of human tryptophan hydroxylase.

Human tryptophan hydroxylase has been expressed as a soluble and active form in Escherichia coli by fusion with an affinity tag, maltose-binding protein. The fusion protein has been purified to near homogeneity by affinity chromatography on crosslinked amylose resin. The purified fusion protein has a specific activity of 86 nmol of 5-hydroxytryptophan per min per mg of fusion protein. A series of truncation mutants have also been made to explore the domain organization of tryptophan hydroxylase. All deletion mutants were subject to affinity purification and kinetic characterization. While removal of the N-terminal 164 amino acids completely inactivates the enzyme, deletion of the first 91 residues results in a 7-fold reduction in specific activity. From the C terminus, deletion of 36, 55, or 112 amino acids abolishes the activity, whereas deletion of 19 residues decreases the specific activity by approximately 11-fold. These results are consistent with a model for tryptophan hydroxylase in which the enzyme consists of an N-terminal regulatory domain, a catalytic core, and a small C-terminal region of uncertain but important function.

ATP-Binding Cassette Transporters↗
PubMed1994

Electrostatic activation of rat phenylalanine hydroxylase.

The conversion of phenylalanine to tyrosine is accelerated approximately five fold by phosphorylation of the enzyme which catalyzes this step, phenylalanine hydroxylase. To gain a clearer understanding of the mechanism of this activation, we have applied site-directed mutagenesis to specifically modify a clone of the hydroxylase at the phosphorylation site, the serine at position 16. We converted this serine residue to alanine and to glutamic acid. The wild-type and mutant proteins were purified and the activation states of the enzymes were examined with respect to the single phosphorylation site at position 16. Substitution of Ser16 with a negatively charged Glu residue resulted in activation of the enzyme, whereas substitution with an uncharged Ala residue did not. These results indicate that activation of the native enzyme by phosphorylation is due to the introduction of a negative charge, and suggest involvement of electrostatic interactions.

Alanine↗
PubMed1994

Evaluation of hemostatic activity of desamino-D-arginine vasopressin (DDAVP) in uremic rats.

DDAVP, an analog of vasopressin, has been shown to have hemostatic activity. Although it has been used clinically to control bleeding, there have been very few attempts to characterize this agent in experimental animals. We describe a new animal model which is fast, reliable and inexpensive, and which may be used to screen novel analogs of the peptide. Under sodium pentobarbital anesthesia, rats were bilaterally nephrectomized and implanted with indwelling intravenous cannulae. The next day they were re-anesthetized, a standardized cut was made in the tail and the tail was allowed to bleed into warm isotonic saline (25 ml). After 10 min., the tail was removed from the saline and the blood loss was measured either by laser nephelometry or by colorimetric analysis. DDAVP was then injected intravenously and the rat was allowed to rest quietly for 30 min., after which time a second incision was made in the tail and blood loss again measured for 10 min. Unlike bleeding time which was highly variable, blood loss proved to be a reliable index of the hemostatic activity. Thus we were able to demonstrate that DDAVP reduced blood loss in the uremic rats, whereas it was without effect in intact rats.

Animals↗
PubMed1994

Immunological profile changes following perioperative autologous vs. homologous blood transfusion in oncologic patients.

To determine the effect of perioperative blood transfusion on immunological parameters, T cells, T-cell subsets, and concanavalin A-induced suppression were measured in 25 patients with colorectal and breast cancer. During the operation, 15 patients received autologous blood and 10 patients had homologous transfusion. The immunological status was again determined after curative surgery. Before surgery, normal percentage of T lymphocytes, decreased ratios of helper/suppressor cells, and impaired con A-induced suppression were found. Following the operation, the helper and suppressor cell percentages reversed to normal, whereas the con A-induced suppression remained impaired. This change was significantly more pronounced in patients who received autologous blood transfusion than in the other group. Autotransfusion has an impact on immune parameters that might prove less detrimental to the clinical outcome in oncologic surgery than homologous transfusion.

Adult↗
PubMed1994

Cloning and expression of rabbit and human brain tryptophan hydroxylase cDNA in Escherichia coli.

Rabbit and human brain tryptophan hydroxylase were cloned and expressed in Escherichia coli. Each of the respective cDNAs, including the complete coding sequence of tryptophan hydroxylase, was obtained by reverse transcription of rabbit or human brain mRNA and subcloned into the expression vector pET-3C. The expressed rabbit brain tryptophan hydroxylase activity, measured in the presence of tetrahydrobiopterin, represents approximately a 50-fold enhancement in yield (units/g tissue (wet wt) over that of a rabbit brain extract. Likewise, the level of expressed human brain tryptophan hydroxylase is approximately 57 times the average yield previously reported for a human brain homogenate and approximately 10-times the activity of homogenates of human raphe nucleus. The rabbit brain and pineal-derived tryptophan hydroxylase sequences varied by disparities in six amino acid residues (99% identity). The human carcinoid and brain peptide sequences varied by disparities in 18 amino acid residues (96% identity). Several properties of both expressed enzymes were studied and compared with those of native tryptophan hydroxylases.

Amino Acid Sequence↗
PubMed1994

The effect of tetrahydrobiopterin on the in situ phosphorylation of tyrosine hydroxylase in rat striatal synaptosomes.

Tetrahydrobiopterin (BH4), the obligatory cofactor of the aromatic amino acid hydroxylases, decreased the in situ 32P-phosphorylation of tyrosine hydroxylase (TH) in rat striatal synaptosomes. Incubation of pre-32P-labeled synaptosomes with BH4 in the presence of a permeant analogue of cAMP decreased the cAMP-stimulated level of 32P label incorporation into TH by about 50%, as determined by immunoprecipitation and autoradiography of SDS-polyacrylamide gels. The extent of inhibition mirrored changes in intrasynaptosomal BH4 levels and varied both as a function of BH4 concentration and length of incubation. A similar decrease in the amount of TH 32P-labeling was observed with the precursor of BH4, sepiapterin. This effect, in turn, was reversed by the inhibitor of sepiapterin reductase, N-acetyl-serotonin. Finally, exposure of pre-32P-labeled synaptosomes to the inhibitor of protein phosphatase 2A, okadaic acid, blocked the response to BH4. Collectively, the data suggest that BH4 stimulates the dephosphorylation of TH in situ and thus may play a dual role both as a cofactor for catalysis and a regulator of hydroxylase activity.

Animals↗
PubMed1994

Perfluoroctyl bromide as a blood pool contrast agent for computed tomographic angiography.

RATIONALE AND OBJECTIVES: We determined whether perfluoroctyl bromide (perflubron) could be used as a computed tomography (CT) angiographic agent by studying vessel visibility (celiac artery, superior mesenteric artery [SMA], and renal arteries) with spiral CT and three-dimensional (3D) reconstructions. METHODS: Five rhesus monkeys were examined with a perflubron emulsion (90% [w/v] perfluorochemical; administered intravenously at a dose of 1.5 ml/kg and at a rate of 0.5 ml/sec. Spiral CT was performed immediately and at 5 hr after injection. Three dimensional images of the aorta at the level of the celiac artery, SMA, and renal arteries were reconstructed and blindly rated 0-4 (0 = not seen; 4 = excellent visualization) by two observers. RESULTS: All the vessels had the best ratings immediately after injection: celiac artery, 2.8 +/- 0.42; SMA, 2.7 +/- 0.48; left renal artery, 2.1 +/- 0.99; and right renal artery, 1.2 +/- 1.03. The ratings after the 5-hr delay were as follows: celiac artery, 1.3 +/- 1.34; SMA, 1.5 +/- 1.08; left renal artery, 1.5 +/- 0.97; and right renal artery, 1.2 +/- 0.79. CONCLUSIONS: Spiral CT angiography with a perflubron emulsion successfully demonstrated all vessels immediately and at 5 hr after contrast agent infusion. Further refinements of the dose, rate, and reconstruction technique are expected to increase vessel visibility over this wide imaging window.

Angiography↗
PubMed1994

Unusual bleeding manifestations of amyloidosis in patients with multiple myeloma.

We describe two patients who developed severe haemorrhagic complications. One manifested massive vaginal bleeding and the second haemopericardium with cardiac tamponade. In both cases histopathological examination showed subendothelial deposits of amyloid. Since other local and systemic causes of the haemorrhagic complications were excluded the bleeding was most probably due to amyloid angiopathy. Therefore, amyloid angiopathy should be considered in the differential diagnosis of bleeding in patients with amyloidosis associated with multiple myeloma.

Aged↗
PubMed1994

Influence of pregnancy on ANF release from isolated atria.

Isolated perfused right atria were prepared from virgin cycling rats and from rats at 7, 14, and 21 days of pregnancy. Intraluminal pressure was raised from the basal control level (atmospheric) to 4, 6, and 10 cmH2O. Basal and stretch-induced release of atrial natriuretic factor (ANF) into the perfusate was measured by radioimmunoassay. When atria derived from unmated and from 7-day pregnant rats were distended, mean ANF secretion into the perfusion medium increased by 64 +/- 16 and 89 +/- 31 pg/ml at the highest distending pressure of 10 mmHg. (Mean basal secretion for the two groups was 196 +/- 104 and 181 +/- 63 pg/ml, respectively.) However, at 14 and 21 days, distension failed to elicit any significant increase in ANF release. There was no significant difference between the groups with respect to basal secretion of ANF, nor did pregnancy influence atrial compliance or volume. The slopes of the pressure-volume curves for atria derived from virgin, 7-, 14-, and 21-day-pregnant rats were 0.76 +/- 0.17, 0.623 +/- 0.178, 0.811 +/- 0.177, and 1.050 +/- 0.173, respectively. These results are in agreement with our findings in vivo that plasma ANF levels are elevated at 7 days of pregnancy but that, despite the progressive increase in intravascular volume, they decrease to control levels at 14 and 21 days. Our latest data suggest that this fall is a result of changes in the secretory characteristics of the atrial tissue itself and not necessarily of pregnancy-induced changes in atrial filling.

Analysis of Variance↗
PubMed1994

Regulation of nitric oxide synthesis by proinflammatory cytokines in human umbilical vein endothelial cells. Elevations in tetrahydrobiopterin levels enhance endothelial nitric oxide synthase specific activity.

We have examined cytokine regulation of nitric oxide synthase (NOS) in human umbilical vein endothelial cells (HUVEC). 24-h treatment with IFN-gamma (200 U/ml) plus TNF (200 U/ml) or IL-1 beta (5 U/ml) increased NOS activity in HUVEC lysates, measured as conversion of [14C]L-arginine to [14C]L-citrulline. Essentially, all NOS activity in these cells was calcium dependent and membrane associated. Histamine-induced nitric oxide release, measured by chemiluminescence, was greater in cytokine-treated cells than in control cells. Paradoxically, steady-state mRNA levels of endothelial NOS fell by 94 +/- 2.0% after cytokine treatment. Supplementation of HUVEC lysates with exogenous tetrahydrobiopterin (3 microM) greatly increased total NOS activity, and under these assay conditions, cytokine treatment decreased maximal NOS activity. IFN-gamma plus TNF or IL-1 beta increased endogenous tetrahydrobiopterin levels and GTP cyclohydrolase I activity, the rate-limiting enzyme of tetrahydrobiopterin synthesis. Intracellular tetrahydrobiopterin levels were higher in freshly isolated HUVEC than in cultured cells, but were still limiting. We conclude that inflammatory cytokines increase NOS activity in cultured human endothelial cells by increasing tetrahydrobiopterin levels in the face of falling total enzyme; similar regulation appears possible in vivo.

Amino Acid Oxidoreductases↗
PubMed1994

[Infective endocarditis and drug addiction].

A total of 103 patients (P) with infectious endocarditis (IE) were treated during the period 1982-1993; 72% of them were male. Mean age was 46.4 (SD 19.7). Eighteen patients were intravenous drug addicts (D) (17.5%). The first case of drug addiction in the IE group was seen in 1986; since then, there was a steady increase of this condition, reaching 50% of all cases. The average age of D patients was 28.17 +/- 8.15 years significantly lower than that corresponding to non-drug addicts (ND), 48.64 +/- 19.5 (p < 0.00001). The infection was located mainly in the left heart of the ND (93%), and in the right heart of the D (78%), the difference being statistically significant. All the cases of left IE occurred on previously damaged valves. Right location in ND was found in all cases in immunosuppressed individuals being treated for systemic diseases, except in one patient having an endocavitary pacemaker. No differences were found between ND and D with regard to the presence of fever, cutaneous manifestations, splenomegaly or cerebrovascular accident. Differences were noted, however, in the case of respiratory involvement, which showed a significantly higher frequency in D (38.9% versus 12.9%; p < 0.014). Blood cultures were positive in 81.6% of the total number of patients, no difference being found between the two groups. Staphylococcal infections were predominant in D (44%) and streptococcal infections in ND (48%), the difference being statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗