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S Kaufman

Publications and source records attributed to S Kaufman.

At least 19 recordsLinked to original sources
PubMed1995

A novel natriuretic, hypotensive factor derived from the spleen.

Previous experiments have shown that the reduction in plasma volume observed after administration of atrial natriuretic factor is abolished by splenectomy. In order to determine whether the spleen contains a factor that influences cardiorenal homeostasis, rat spleens were homogenized in phosphate-buffered saline, centrifuged, subjected to ultrafiltration (molecular weight cut-off 10,000), extracted on C18 affinity columns and dried. After reconstitution in isotonic saline, the extract was injected i.v. into conscious rats. In response to this extract, there was a dose-dependent diuresis, natriuresis, kaliuresis and chloruresis which plateaued during the second and third hours following administration. Solute-free water clearance was inversely related to urine output. Blood pressure fell significantly from 109 +/- 3 mm Hg to 103 +/- 3 mm Hg during the first 10 min. after injection of the extract, and tended to remain depressed thereafter. There was no accompanying increase in heart rate. We conclude that the mammalian spleen contains a natriuretic, hypotensive factor that may play a role in cardiorenal homeostasis.

Animals↗
PubMed1995

Improved survival with preoperative chemotherapy followed by resection uncompromised by tumor response for advanced squamous cell carcinoma of the head and neck.

BACKGROUND: A total of 93 patients were treated with one of two preoperative chemotherapy regimens over a 15-year period. The study supports the importance of strict adherence to guidelines for ablative surgery. METHODS: A single surgeon performed the surgery and evaluated each patient prior to treatment. The extent of the planned operation was documented. RESULTS: The 5-year absolute survival of 88 patients who completed the protocol was estimated at 55%. The 40 cisplatin/5-fluorouracil-treated patients exhibited a significantly better outcome than the 48 cisplatin/bleomycin-treated patients (76% versus 43%, respectively). Comparison of a subset of 37 patients with a matched group from the standard control arm of the Head and Neck Contracts Program demonstrated a statistically significant improvement in overall survival over standard treatment. CONCLUSIONS: These data suggest that strict adherence to preoperative chemotherapy planning of ablative uncompromised surgery contribute to improved survival. Selective rather than routine postoperative radiotherapy may be advantageous.

Antibiotics, Antineoplastic↗
PubMed1992

Identity of 4a-carbinolamine dehydratase, a component of the phenylalanine hydroxylation system, and DCoH, a transregulator of homeodomain proteins.

The principal pathway for the metabolism of phenylalanine in mammals is via conversion to tyrosine in a tetrahydrobiopterin-dependent hydroxylation reaction occurring predominantly in the liver. Recently, the proposal that certain hyperphenylalaninemic children may have a deficiency of carbinolamine dehydratase, a component of the phenylalanine hydroxylation system, has widened the interest in this area of metabolism. Upon cloning and sequencing the dehydratase, we discovered that this protein is identical to DCoH, the cofactor which regulates the dimerization of hepatic nuclear factor 1 alpha, a homeodomain transcription factor. The identity of the nuclear and cytoplasmic proteins is demonstrated by size, immunoblotting, stimulation of phenylalanine hydroxylase, and dehydratase activity. The evolution of the dual functions of regulation of phenylalanine hydroxylation activity and transcription activation in a single polypeptide is unprecedented.

Amino Acid Sequence↗
PubMed1992

"7-tetrahydrobiopterin," a naturally occurring analogue of tetrahydrobiopterin, is a cofactor for and a potential inhibitor of the aromatic amino acid hydroxylases.

The ability of 2-amino-4-hydroxy-7-[dihydroxylpropyl-(L-erythro)-5,6,7,8-tetrahyd ropterin] ("7-tetrahydrobiopterin" or 7-BH4) to substitute for the natural cofactor tetrahydrobiopterin (BH4) has been studied in vitro in the reactions of the three mammalian aromatic amino acid hydroxylases. With rat liver phenylalanine hydroxylase, the apparent Km for 7-BH4 is 160 microM, a value that is approximately 60-fold greater than that for the natural cofactor. In contrast, the hydroxylase reaction is severely inhibited by as little as 1 microM 7-BH4 when assayed in the presence of physiological concentrations of BH4. This inhibition can be overcome either by an increase in the concentration of BH4 or a decrease in the concentration of phenylalanine. With both rat brain tryptophan hydroxylase and rat pheochromocytoma tyrosine hydroxylase, the Km value for 7-BH4 is about one order of magnitude greater than the Km for BH4. Accordingly, 7-BH4 is a poor competitive inhibitor of both tryptophan and tyrosine hydroxylase. Thus, our results suggest that the observed hyperphenylalaninemia in patients who excrete 7-BH4 in their urine may arise directly from the inhibition of phenylalanine hydroxylase by low levels of this pterin. On the other hand, it is less likely that low levels of 7-BH4 would affect the activity of tyrosine or tryptophan hydroxylase in vivo.

Animals↗
PubMed1992

Regulation of recombinant rat tyrosine hydroxylase by dopamine.

Recombinant rat PC12 tyrosine hydroxylase, also called tyrosine 3-monooxygenase [L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2], purified from Escherichia coli is in an activated form with a low Km for the tetrahydrobiopterin cofactor and a pH optimum of 6.5. Pretreatment with low levels of the derived product, dopamine, inhibits catalytic activity, increases the Km for the cofactor, and shifts the pH curve towards a more acidic pH optimum. Labeled dopamine binds to tyrosine hydroxylase with high affinity (Kd = 1 microM) but low stoichiometry (r = 0.08 mol/mol of enzyme subunit). The binding of dopamine results in the appearance of a blue-green chromophore with lambda max at approximately 660 nm, which is consistent with the formation of a catecholamine-iron complex. In the absence of dopamine, the recombinant enzyme cannot be further activated by phosphorylation with cAMP-dependent protein kinase, although as much as 1 mol of phosphate is incorporated per mol of subunit. In contrast, the enzyme pretreated with dopamine is activated by phosphorylation in the same fashion and to the same extent as the native hydroxylase. The results suggest that the high-affinity binding of catecholamine products is a pivotal post-translational modification that determines the state of enzyme activation and the response to phosphorylation.

Animals↗
PubMed1992

Distribution of 4a-hydroxytetrahydropterin dehydratase in rat tissues. Comparison with the aromatic amino acid hydroxylases.

A 4a-carbinolamine intermediate is generated stoichiometrically during the tetrahydrobiopterin-dependent phenylalanine hydroxylation reaction catalyzed by phenylalanine hydroxylase. The dehydration of the carbinolamine is catalyzed by the enzyme, 4a-hydroxytetrahydropterin dehydratase. We have now examined the distribution of the dehydratase activity in various rat tissues by activity measurements and by immunoblot analysis to explore the possibility that the dehydratase may also play a role in tyrosine and tryptophan hydroxylation. The only two tissues that express relatively high dehydratase activity are liver and kidney, which are also the only two tissues that express phenylalanine hydroxylase activity. The dehydratase activity was generally very low in those tissues which contain high levels of tyrosine and tryptophan hydroxylase activity, except for the pineal gland. These results suggest that the dehydratase may not play an important role in the regulation of the synthesis of those neurotransmitters which are derived from the hydroxylated aromatic amino acids.

Animals↗
PubMed1992

Phenylalanine-induced phosphorylation and activation of rat hepatic phenylalanine hydroxylase in vivo.

Rats were given intraperitoneal injections of 2 mCi of carrier-free 32Pi and substances known to activate liver phenylalanine hydroxylase. After 30 min, these animals were anesthetized and their livers removed for analysis of enzyme activity, 32Pi incorporation into immunoprecipitated phenylalanine hydroxylase and [gamma-32P]ATP specific activity. Following glucagon treatment, rat liver phenylalanine hydroxylase activity was stimulated more than 6-fold when assayed in the presence of the natural cofactor, tetrahydrobiopterin (BH4). Glucagon injection also resulted in an incorporation of 0.41 mol of 32Pi/mol of hydroxylase subunit (approximately 50,000 Da). In vivo stimulation of phenylalanine hydroxylase activity and 32Pi incorporation by glucagon had been previously observed in this laboratory (Donlon, J., and Kaufman, S. (1978) J. Biol. Chem. 253, 6657-6659). However, we show for the first time in the present study that in vivo treatment with phenylalanine alone results in a 4-fold increase in the BH4-dependent activity of phenylalanine hydroxylase concomitant with a significant incorporation of phosphate into phenylalanine hydroxylase (0.51 mol of 32Pi/mol of hydroxylase subunit). It is further demonstrated in vivo that the combined treatment with phenylalanine and glucagon results in a greater than 10-fold stimulation of BH4-dependent activity and the greatest level of 32Pi incorporation (0.75 mol of 32Pi/mol of hydroxylase subunit). Phenylalanine did not produce an elevation in plasma glucagon in these animals. A model is, thereby, proposed with respect to the ligand binding effects of phenylalanine on the state of phosphorylation and activation of phenylalanine hydroxylase. The significance of these regulatory roles are considered in light of the probable physiological environment of the enzyme.

Animals↗
PubMed1992

Role of spleen in ANF-induced reduction in plasma volume.

Atrial natriuretic factor (ANF) causes an increase in hematocrit that cannot be accounted for by urinary losses. The mechanism behind this phenomenon was studied in intact and splenectomized rats. Rat ANF 99-126 was infused i.v. for 30 min into conscious rats at rates of 0 (saline control), 0.05, or 0.1 microgram/min. Plasma volume was then determined by dilution of the dye, Evan's Blue. In one group of rats, red cell volume was determined using 51Cr-labelled erythrocytes. ANF infusion was continued uninterrupted throughout the experiments. In the intact rats, ANF (0.10 microgram/min) caused hematocrit to increase from 38.9 +/- 0.5 to 41.2 +/- 0.4% (p < 0.005). Splenectomy so attenuated this response to ANF that it failed to reach significance. Similarly, ANF (0.10 microgram/min) caused plasma volume to fall from 5.1 +/- 0.1 to 4.5 +/- 0.1 mL/100 g body wt. (p < 0.005) in the intact rats, but did not affect plasma volume in the splenectomized rats. As a result, blood volume was significantly reduced by ANF in the intact rats, but remained unchanged in the splenectomized rats. Red cell volume did not change in response to infusion of ANF, nor did ANF affect the rate of clearance of Evan's Blue out of the plasma. It is concluded that the spleen is an important site of movement of protein-poor fluid out of the vasculature, and that this exchange is influenced by ANF.

Animals↗
PubMed1992

Purification and biochemical characterization of recombinant rat liver phenylalanine hydroxylase produced in Escherichia coli.

Phenylalanine hydroxylase, important in phenylalanine metabolism in mammals, is regulated through short-term (activation) and long-term (induction) mechanisms. To help elucidate the structure-function relationships involved in the activation of this enzyme, we have isolated and characterized full-length cDNA clones to rat phenylalanine hydroxylase. Recombinant rat phenylalanine hydroxylase was placed into an expression vector in Escherichia coli. The enzyme has been purified to homogeneity and its physical and catalytic properties have been characterized. The molecular weight and the fluorescence emission spectrum of the recombinant enzyme were identical to those of the native enzyme. The recombinant enzyme could be activated by incubation with phenylalanine or lysolecithin or by phosphorylation, as is the rat liver enzyme. The extent of activation is the same as that for the native enzyme in each case except for phenylalanine, which activates the recombinant enzyme only 5- to 10-fold rather than the 15- to 30-fold activation observed with the native enzyme. The kinetic constants determined for the recombinant enzyme are also essentially the same as those reported for the native enzyme. We conclude that this enzyme is essentially identical to the native enzyme and should be very useful in the future study of this important hydroxylase.

Animals↗
PubMed1991

High-level expression of rat PC12 tyrosine hydroxylase cDNA in Escherichia coli: purification and characterization of the cloned enzyme.

A rat cDNA containing the complete coding sequence for rat tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) was isolated from a rat PC12 cDNA library and subcloned in a bacterial expression plasmid, and large amounts of functional enzyme were produced in Escherichia coli. The recombinant enzyme was purified approximately 20-fold to a final specific activity of 1.8 mumol/min per mg of protein, with a yield of 30%. As much as 1 mg of pure protein could be obtained from 1 g of wet bacterial cells. The purified hydroxylase was shown to be homogeneous by denaturing polyacrylamide electrophoresis and isoelectric focusing. Amino acid analysis of the N terminus (25 residues) revealed 100% identity with rat PC12 tyrosine hydroxylase, as deduced from its cDNA sequence. Several of the kinetic properties of the recombinant enzyme resembled those of the native PC12 hydroxylase. However, in contrast to the native enzyme, the purified recombinant hydroxylase was shown to be in an activated form. Phosphorylation with cAMP-dependent protein kinase resulted in stoichiometric incorporation of phosphate, but the kinetic profile of the recombinant enzyme was unaffected. Several clues to these differences are considered that may provide insight into the structural features important to the regulation of tyrosine hydroxylase.

Adrenal Gland Neoplasms↗
PubMed1991

Decreased CD45RA T cells in B-cell chronic lymphatic leukemia patients: correlation with disease stage.

T-cell subsets CD4, CD8 and suppressor-inducers (CD45RA) were determined in 20 patients with B-cell chronic lymphatic leukemia (B-CLL). The proportion of CD4 and CD45RA was decreased when compared with T cells from normal subjects. CD8 was markedly increased. The activity of concanavalin A-induced suppressor cells was not significantly different from that of normal controls and was negatively correlated to the percentage of CD4 of B-CLL patients. The selective loss of CD45RA cells was more prominent in patients in advanced Rai stages of the disease (III to IV) than in early stages (0 to II). Six patients of the advanced stages group suffered from autoimmune hemolytic anemia, whereas no patient in the early stages of disease showed an autoimmune phenomenon. Our results may indicate a mechanism of autoimmunity in B-CLL similar to that of patients with autoimmune diseases.

Aged↗
PubMed1991

The hydroxylation of phenylalanine and tyrosine by tyrosine hydroxylase from cultured pheochromocytoma cells.

Pheochromocytoma tyrosine hydroxylase was reported to have unusual catalytic properties, which might be unique to the tumor enzyme (Dix, T. A., Kuhn, D. M., and Benkovic, S. J. (1987) Biochemistry 24, 3354-3361). Two such properties, namely the apparent inability to hydroxylate phenylalanine and an unprecedented reactivity with hydrogen peroxide were investigated further in the present study. Tyrosine hydroxylase was purified to apparent homogeneity from cultured pheochromocytoma PC12 cells. The purified tumor enzyme was entirely dependent on tetrahydrobiopterin (BH4) for the hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine and hydrogen peroxide could not substitute for the natural cofactor. Indeed, in the presence of BH4, increasing concentrations of hydrogen peroxide completely inhibited enzyme activity. The PC12 hydroxylase exhibited typical kinetics of tyrosine hydroxylation exhibited typical kinetics of tyrosine hydroxylation, both as a function of tyrosine (S0.5 Tyr = 15 microM) and BH4 (apparent Km BH4 = 210 microM). In addition, the enzyme catalyzed the hydroxylation of substantial amounts of phenylalanine to tyrosine and 3,4-dihydroxyphenylalanine (apparent Km Phe = 100 microM). Phenylalanine did not inhibit the enzyme in the concentrations tested, whereas tyrosine showed typical substrate inhibition at concentrations greater than or equal to 50 microM. At higher substrate concentrations, the rate of phenylalanine hydroxylation was equal to or exceeded that of tyrosine. Essentially identical results were obtained with purified tyrosine hydroxylase from pheochromocytoma PC18 cells. The data suggest that the tumor enzyme has the same substrate specificity and sensitivity to hydrogen peroxide as tyrosine hydroxylase from other tissues.

Animals↗
PubMed1991

Tetrahydrobiopterin, a cofactor for rat cerebellar nitric oxide synthase, does not function as a reactant in the oxygenation of arginine.

Studies with purified nitric oxide synthase from rat cerebellum have confirmed previous reports that product formation is enhanced by tetrahydrobiopterin [H4B; 6-(L-erythro-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropterin]. The effect of the natural isomer, (6R)-H4B, is observed at extremely low (less than 0.1 microM) concentrations and is remarkably selective. At these concentrations, only the diastereoisomer (6S)-H4B, the structural isomer 7-(L-erythro-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropterin, and 7,8-dihydrobiopterin showed detectable effects. Our observations are inconsistent with a stoichiometric role for H4B in the oxygenation of arginine [e.g., Stuehr, D. J., Kwon, N. S., Nathan, C. F., Griffith, O. W., Feldman, P. L. & Wiseman, J. (1991) J. Biol. Chem. 266, 6259-6263]. Activity is initially independent of added H4B; enhanced product formation with H4B is observed only as incubation progresses. The effect of H4B is catalytic, with each mole of added H4B supporting the formation of greater than 15 mol of product. Recycling of H4B was excluded by direct measurement during nitric oxide synthesis and by the demonstration that nitric oxide synthase is not inhibited by methotrexate. These combined results exclude H4B as a stoichiometric reactant and suggest that H4B enhances product formation by protecting enzyme activity against progressive loss. Preliminary studies indicate that the decreased activity in the absence of added H4B does not depend on catalytic turnover of the enzyme. The role of H4B may be allosteric or it may function to maintain some group(s) on the enzyme in a reduced state required for activity.

Amino Acid Oxidoreductases↗
PubMed1991

7-Tetrahydrobiopterin is an uncoupled cofactor for rat hepatic phenylalanine hydroxylase.

Rat hepatic phenylalanine hydroxylase requires both a tetrahydropterin cofactor and molecular oxygen to convert phenylalanine to tyrosine. During the physiological hydroxylation, a single mol of the natural cofactor, tetrahydrobiopterin, is oxidized for each mol of phenylalanine converted to tyrosine. Artificial conditions have been devised in which the oxidation of the tetrahydropterin is uncoupled from the hydroxylation of the aromatic amino acid substrate. Recently, an isomer of tetrahydrobiopterin, 7-tetrahydrobiopterin, has been isolated from the urine of certain mildly hyperphenylalaninemic children. We report in this communication that 7-tetrahydrobiopterin may be an inefficient cofactor for phenylalanine hydroxylase because, in vitro, the phenylalanine-dependent oxidation of 7-tetrahydrobiopterin is accompanied by the hydroxylation of the aromatic amino acid substrate only about 15% of the time, i.e. the enzymatic oxidation of 7-tetrahydrobiopterin is about 85% uncoupled from the hydroxylation of the amino acid substrate.

Animals↗
PubMed1991

A staphylococcal plasmid that replicates and expresses ampicillin, gentamicin and amikacin resistance in Escherichia coli.

Plasmid pPG1 from Staphylococcus aureus coding for ampicillin (Apr), gentamicin (Gmr) and amikacin (Akr) resistance was transformed into Escherichia coli. Transformation efficiency was about 2 x 10(3) transformants/micrograms of plasmid DNA. The plasmids present in the E. coli transformants were identical to pPG1 according to their restriction patterns. The copy number of pPG1 was estimated to be at least 20-times less in E. coli than in S. aureus. The minimal inhibitory concentrations (MICs) for Ap and Gm were lower in E. coli than in S. aureus. However, the MIC for Ak was higher in E. coli transformants than in S. aureus. pPG1 was maintained in the E. coli transformants for at least 80 generations at 37 degrees C without antibiotic selection pressure.

Amikacin↗
PubMed1991

Leukocyte alkaline phosphatase and carcinoembryonic antigen in metastatic colorectal cancer patients.

Peripheral blood leukocyte alkaline phosphatase scores and plasma carcinoembryonic antigen levels in 26 patients with metastatic colorectal cancer were compared to those in 30 healthy controls. Patients had metastases to the liver and abdomen. The mean leukocyte alkaline phosphatase score in the metastatic colorectal cancer patients was significantly higher than in the control group (246 +/- 65 vs, 52 +/- 26, p less than 0.001); and the mean carcinoembryonic antigen level in the patients was also significantly higher than in the controls (110 +/- 100 vs, 4.9 +/- 3 ng/ml, p less than 0.001). One hundred percent of the metastatic cancer patients had elevated LAP scores and 73% of these patients had elevated CEA levels. There was a difference between the mean CEA levels in the patients with liver metastases and those with abdominal metastases (162 +/- 135 vs, 39 +/- 53 ng/ml, p less than 0.04). The results suggest that although both markers were elevated in metastatic colorectal cancer, the LAP score seems to be more useful in detecting metastatic disease, since we found 11% false negatives with the CEA level and 0% false negatives with the LAP score.

Abdominal Neoplasms↗