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Biomedical subjects

S Kaufman

Publications and source records attributed to S Kaufman.

At least 145 records · Page 8Linked to original sources
PubMed1993

Induction of pterin synthesis is not required for cytokine-stimulated tryptophan metabolism.

Activation of the immune system which occurs in inflammatory disease leads to parallel increases in pterin synthesis and increased production of neuroactive L-tryptophan metabolites. Several model systems were studied to determine whether pterins, which are cofactors for hydroxylation reactions, could be required in the oxidative kynurenine pathway of L-tryptophan degradation. Treatment of mice with interferon-gamma increased L-tryptophan metabolism without any corresponding change in tissue biopterin concentrations. Cytokine-treated human fibroblasts, macrophages and glioblastoma cells all showed increases in kynurenine production, which were completely independent of pterin synthesis. When pterin synthesis de novo was blocked, either by an inhibitor of GTP cyclohydrolase or because of a genetic deficiency of one of the enzymes of the pathway of pterin biosynthesis, cytokine-stimulated increases in tryptophan metabolism were unaffected. Furthermore, increasing intracellular tetrahydrobiopterin concentrations by treating cells with sepia-pterin also had no effect on markers of tryptophan metabolism. Therefore, both normal and cytokine-stimulated L-tryptophan metabolism appears to be completely independent of pterin biosynthesis.

Animals↗
PubMed1993

Products of the tyrosine-dependent oxidation of tetrahydrobiopterin by rat liver phenylalanine hydroxylase.

Phenylalanine hydroxylase, a tetrahydrobiopterin (BH4)-dependent oxygenase, catalyzes the conversion of phenylalanine to tyrosine. During this physiological reaction, the oxidation of BH4 is tightly coupled to the hydroxylation of the amino acid substrate with a stoichiometry of 1 mol of BH4 oxidized for every mole of tyrosine formed. In the presence of an activator and certain analogues of either the amino acid or the pterin coenzyme, the enzyme also catalyzes a partially uncoupled reaction in which the amount of tetrahydropterin oxidized exceeds the amount of amino acid hydroxylated. With tyrosine as the amino acid analogue, the reaction is completely uncoupled, i.e., the enzyme functions as a tetrahydropterin oxidase with no net hydroxylation of the amino acid. We have investigated whether the normal pterin intermediate, a 4a-hydroxytetrahydropterin, is formed during the completely uncoupled reaction and whether oxygen is reduced exclusively to the level of water, as in the tightly coupled reaction, or whether some reduction to hydrogen peroxide occurs. We present direct evidence that both the normal hydroxytetrahydropterin intermediate and hydrogen peroxide are formed. The amount of hydrogen peroxide formed, however, is not stoichiometric with the amount of oxygen reduced, an indication that there are at least two pathways for this reduction, one leading to the formation of water and one to hydrogen peroxide. We also postulate that hydrogen peroxide and the hydroxytetrahydropterin are both derived from alternate routes of breakdown of a common precursor, the corresponding 4a-hydroperoxytetrahydropterin. By contrast, in the normal tightly coupled reaction, we suggest that this peroxy compound participates in the hydroxylation of the amino acid substrate.

Animals↗
PubMed1993

Tetrahydrobiopterin biosynthesis defects examined in cytokine-stimulated fibroblasts.

Incubation of primary skin fibroblast cultures with the cytokines interferon-gamma and tumour necrosis factor-alpha stimulates the de novo pathway of tetrahydrobiopterin biosynthesis. Fibroblasts from patients with the two most common types of genetic defects of tetrahydrobiopterin metabolism that cause hyperphenylalaninaemia show characteristic pterin responses predicted by the nature of the defect. Cells from a child with 6-pyruvoyl-tetrahydropterin synthase deficiency produce higher than normal levels of neopterin and no biopterin. Fibroblasts from dihydropteridine reductase-deficient children produce normal levels of tetrahydrobiopterin, which gradually becomes partially oxidized, and higher than normal levels of neopterin. As a model for cells with the rarest form of tetrahydrobiopterin deficiency, lack of GTP cyclohydrolase activity, normal fibroblasts were treated with 2,4-diamino-6-hydroxypyrimidine to inhibit GTP cyclohydrolase activity by > 90%, the level expected in patients with a GTP cyclohydrolase deficiency. Neopterin and biopterin synthesis rates of < 10% of normal levels were readily detectable. Therefore, analysis of the patterns of the pterins synthesized by fibroblasts can aid in the diagnosis of the hyperphenylalaninaemias caused by disorders of tetrahydrobiopterin metabolism.

Adult↗
PubMed1993

Deletion mutagenesis of rat PC12 tyrosine hydroxylase regulatory and catalytic domains.

The functional organization of rat tyrosine hydroxylase was investigated by deletion mutagenesis of the regulatory and catalytic domains. A series of tyrosine hydroxylase cDNA deletion mutants were amplified by PCR, cloned into the pET3C prokaryotic expression vector, and the mutant proteins were partially purified from E. coli. The results show that the deletion of up to 157 N-terminal amino acids activated the enzyme, but further deletion to position 184 completely destroyed catalytic activity. On the carboxyl end, the removal of 43 amino acids decreased but did not eliminate activity, suggesting that this region may play a different role in the regulation of the enzyme. These findings place the amino end of the catalytic domain between residues 158 and 184 and the carboxyl end at or prior to position 455. Deletions within the first 157 amino acids in the N-terminus caused an increase in hydroxylating activity, a decrease in the apparent Km for tyrosine and phenylalanine substrates, and a substantial increase in the Ki for dopamine inhibition. The results define this region of the N-terminus as the regulatory domain of tyrosine hydroxylase, whose primary functions are to restrict the binding of amino acid substrates and to facilitate catecholamine inhibition. The results also suggest that the well-established role of the regulatory domain in restricting cofactor binding may be secondary to an increase in catecholamine binding, which in turn lowers the affinity for the cofactor. These findings provide new insight into the functional organization and mechanisms of regulation of tyrosine hydroxylase.

Animals↗
PubMed1993

The influence of reproductive hormones on ANF release by rat atria.

The effects of subcutaneous (sc) administration of estradiol (50 micrograms/100 microliters/day x 10 day) or testosterone (15 mg/100 microliters twice a week x 2 weeks) on ANF release were examined in isolated perfused rat atria. The concentration of ANF in the perfusate was measured by radioimmunoassay, under basal conditions (atrial wall unstressed), and during atrial distention (intraluminal pressure raised to 4, 6, and 10 cm water). In both male and female control (vehicle-injected) groups, increased atrial pressure resulting in distention of the atrium caused a significant increase in ANF release. Estradiol increased basal secretion of ANF but did not influence stretch-induced stimulation of ANF secretion. By contrast, although testosterone did not affect basal secretion, it completely abolished the stretch-induced increase in ANF secretion. Neither estradiol nor testosterone affected atrial compliance.

Animals↗
PubMed1993

Splenic control of intravascular volume in the rat.

1. We tested the hypothesis that hypervolaemia causes an increase in intrasplenic filtration of cell-free fluid out of the vasculature. To this end we developed a preparation in the anaesthetized rat whereby the splenic vein could be non-occlusively cannulated. 2. Haematocrit and plasma protein concentrations were measured in the splenic afferent and efferent blood supplies. 3. In response to volume loading with saline (1% body weight), there was a sustained increase in the arterial-venous differential of haematocrit, i.e. there was a relative increase in the haematocrit of the blood draining from the spleen. There was no such change in plasma protein concentration. By contrast, this degree of volume loading had no effect on the haematocrit of blood passing through the hindquarters of the animal. 4. Following volume expansion, there was no significant difference in the protein concentration of the plasma and the lymph fluid collected from the splenic lymphatic duct. 5. Distension of the superior vena caval-right atrial junction by means of a small inflatable balloon, caused a similar increase in the splenic venous haematocrit, and again, no change in plasma protein concentration. 6. We interpret these results to mean that, in response to expansion of the intravascular space, there is increased intrasplenic filtration of plasma out of the blood and into the lymphatic system.

Animals↗
PubMed1993

Renal response to atrial stretch during pregnancy in conscious rats.

Chronic indwelling balloons were placed at the junction of the right atrium and the right superior vena cava of virgin female rats. The renal response to discrete standardized atrial stretch was tested in the conscious animals. The rats were then mated, and the renal response was retested at days 7, 14, and 20 of pregnancy. On day 21, the rats were killed, and the pressure-volume relationship of the right atria was measured. In response to atrial stretch, there was an increase in urine volume, urine sodium output, and urine potassium output and a decrease in free water clearance in the virgin rats. Subsequent to mating, these responses were completely abolished at all stages of pregnancy. The renal responses to atrial stretch were also abolished during pseudopregnancy. Central venous pressure did not change when the intracardiac balloons were inflated; there were also no changes in resting central venous pressure during pregnancy. There were no significant changes in unstressed atrial volume or in atrial compliance during pregnancy.

Animals↗
PubMed1993

Alpha 7 beta 1 integrin is a component of the myotendinous junction on skeletal muscle.

Immunization against a 70 kDa band that co-purifies with skeletal muscle integrins has resulted in an antibody directed against the avain alpha 7 integrin subunit. The specificity of the antibody was established by patterns of tissue staining and cross-reactivity with antibodies directed against the cytoplasmic domain of the rat alpha 7 cytoplasmic domain. On sections of adult skeletal muscle the alpha 7 integrin was enriched in the myotendinous junction (MTJ). This localization was unique as neither the alpha 1, alpha 3, alpha 5, alpha 6 and alpha v subunit localizes in the myotendinous junction. The distribution of the alpha 7 subunit in the MTJ was examined during embryonic development. alpha 7 expression in the junction is first apparent around embryo day 14 and is almost exclusively at the developing MTJ at this stage. alpha 3 is expressed with distinctive punctate staining around the junctional area in earlier embryos (11-day). The time of appearance of the alpha 7 subunit in the MTJ correlates with the insertion of myofibrils into subsarcolemmal densities and folding of the junctional membrane, suggesting a role of the alpha 7 integrin in this process. Vinculin is present throughout development of the myotendinous junction, suggesting that the alpha 7 integrin recognizes a preformed cytoskeletal structure. The presence of the alpha 7 subunit in the myotendinous junction and the alpha 5 subunit in the adhesion plaque demonstrates a molecular difference between these two adherens junctions. It also points to possible origins of junctional specificity on muscle. Differences between these two junctions were developed further using an antibody against phosphotyrosine (PY20). Phosphotyrosine is thought to participate in the organization and stabilization of adhesions. The focal adhesion and the neuromuscular junction, but not the MTJ, contained proteins phosphorylated on tyrosine.

Amino Acid Sequence↗
PubMed1993

Alkaline phosphatase in human semen: an investigation using enzyme inhibitors and gel electrophoresis.

Human seminal alkaline phosphatase was investigated with respect to its electrophoretic mobility, heat lability, and susceptibility to inhibition by phenylalanine, tartrate, and homoarginine. Total alkaline phosphatase activity in 30 samples of human semen was measured colorimetrically, using p-nitrophenylphosphate as substrate. Using linear regression analysis, no significant correlation was found between the enzyme activity and the sperm count, sperm motility, semen volume, and the concentrations of seminal inositol and fructose. The alkaline phosphatase activity was higher in the earlier portion of split ejaculate samples. Sodium DL-tartrate (42 mmol/l), which inhibits acid phosphatase, did not inhibit seminal alkaline phosphatase significantly. L-Homoarginine (10 mmol/l), an inhibitor of the liver and bone isoenzymes, inhibited the seminal enzyme (53%), whereas L-phenylalanine (12 mmol/l), a strong inhibitor of placental alkaline phosphate, decreased activity by about 10%. Electrophoresis of semen samples on agarose revealed a broad band which was not sharpened after treatment with neuraminidase. Semen total alkaline phosphatase was essentially totally inactivated by heating at 56 degrees C for 15 min or 10 min at 65 degrees C; similar behaviour has been reported for the liver and bone isoenzymes. Electrophoresis after heating did not reveal a residual band of heat-stable placental-like alkaline phosphatase. Semen alkaline phosphatase appears to contain more than one isoenzyme, but placental-like alkaline phosphatase cannot be more than a minor component.

Alkaline Phosphatase↗
PubMed1993

Diabetes in American Indians. An overview.

OBJECTIVE: To review the growth of diabetes as a major health problem for American Indians and Alaska Natives. RESEARCH DESIGN AND METHODS: Pertinent studies of diabetes in Indians previously published are reviewed and compared with current data. RESULTS: Although diabetes rates may vary among different Indian tribes, diabetes has become a widespread health problem. CONCLUSIONS: Our understanding of the natural history of diabetes among Indians has improved, but better strategies to prevent complications and ultimately to prevent diabetes are urgently needed in Indian communities.

Adolescent↗
PubMed1993

Prevalence of diagnosed diabetes among American Indians and Alaska Natives, 1987. Estimates from a national outpatient data base.

OBJECTIVE: To estimate the prevalence of diagnosed diabetes among American Indians and Alaska Natives served by the IHS. RESEARCH DESIGN AND METHODS: This was a cross-sectional study of the 1987 IHS national outpatient data base. RESULTS: Prevalence rates of diagnosed diabetes determined from the IHS outpatient data base were consistent with recent studies of diabetes in different IHS areas. IHS-wide, age-adjusted prevalence was 69/100,000, or 2.8 times the U.S. rate. There was considerable variation in prevalence rates of diabetes throughout the country, with rates ranging from 15.3/100,000 in Alaska to 119.2/100,000 in southern Arizona. CONCLUSIONS: This study documented the high prevalence of diabetes among American Indians and Alaska Natives and the wide variation in rates between different tribal groups. This study also demonstrated the feasibility of using an outpatient data base to estimate rates of disease that have uniform methods of diagnosis and result in frequent clinic visits.

Age Factors↗