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Biomedical subjects

S Diaz

Publications and source records attributed to S Diaz.

At least 73 records · Page 4Linked to original sources
PubMed1991

Pulmonary alveolar proteinosis: prolonged spontaneous remission in two patients.

Pulmonary alveolar proteinosis is a rare idiopathic diffuse airspace disease characterized by intra-alveolar accumulation of large quantities of lipoproteinaceous material, with preservation of the lung interstitium. The clinical course of pulmonary alveolar proteinosis is variable. Spontaneous resolution is known to occur in up to a quarter of the cases. We describe two patients with untreated pulmonary alveolar proteinosis who experienced complete clinical, functional and radiographic resolution. In follow-up periods of fourteen and six years, both patients have remained asymptomatic.

Adult↗
PubMed1991

[A new levodopa benserazide preparation for Parkinson's disease with motor fluctuations refractory to standard L-dopa].

As Parkinson's disease worsens, many patients develop motor fluctuations which usually correlate directly or indirectly with L-dopa plasma levels. A new L-dopa-benserazide HBS preparation (Madopar) a control release pharmaceutical formulation which is activated when it contacts gastric fluid thus providing more stable L-dopa plasma levels, was assayed. Ten patients with a diagnosis of idiopathic Parkinson's disease and motor fluctuations otherwise unresponsive to conventional therapy were selected. The average age was 62 years and the duration of the disease 9 years. The motor status was evaluated on an hourly basis with the King's College Parkinson's disease rating scale; in addition, a nocturnal disability scale (Lees) was used. Out of the 10 patients, 2 dropped out within the first month due to worsening of parkinsonian signs, while 7 of the remainders preferred HBS preparation to the previous treatment. The number of off hours in this group was reduced by 58% and motor fluctuation became less severe. In only 3 cases was it possible to use HBS as monotherapy while in the rest standard L-dopa had to be added, specially as morning doses. The average L-dopa daily dose was increased by 36%. Unwanted effects included psychiatric disturbances in two (in one L-dopa dose had to be reduced) and epigastralgia in one. Our findings suggest that this L-dopa-benserazide control release may be considered an able therapeutic formulation in the control of motor fluctuations in Parkinson's disease.

Adult↗
PubMed1990

Biosynthesis and turnover of O-acetyl and N-acetyl groups in the gangliosides of human melanoma cells.

We and others previously described the melanoma-associated oncofetal glycosphingolipid antigen 9-O-acetyl-GD3, a disialoganglioside O-acetylated at the 9-position of the outer sialic acid residue. We have now developed methods to examine the biosynthesis and turnover of disialogangliosides in cultured melanoma cells and in Golgi-enriched vesicles from these cells. O-Acetylation was selectively expressed on di- and trisialogangliosides, but not on monosialogangliosides, nor on glycoprotein-bound sialic acids. Double-labeling of cells with [3H]acetate and [14C]glucosamine introduced easily detectable labels into each of the components of the ganglioside molecules. Pulse-chase studies of such doubly labeled molecules indicated that the O-acetyl groups turn over faster than the parent molecule. When Golgi-enriched vesicles from these cells were incubated with [acetyl-3H]acetyl-coenzyme A, the major labeled products were disialogangliosides. [Acetyl-3H]O-acetyl groups were found at both the 7- and the 9-positions, indicating that both 7-O-acetyl GD3 and 9-O-acetyl GD3 were synthesized by the action of O-acetyltransferase(s) on endogenous GD3. Analysis of the metabolically labeled molecules confirmed the existence of both 7- and 9-O-acetylated GD3 in the intact cells. Surprisingly, the major 3H-labeled product of the in vitro labeling reaction was not O-acetyl-GD3, but GD3, with the label exclusively in the sialic acid residues. Fragmentation of the labeled sialic acids by enzymatic and chemical methods showed that the 3H-label was exclusively in [3H]N-acetyl groups. Analyses of the double-labeled sialic acids from intact cells also showed that the 3H-label from [3H]acetate was exclusively in the form of [3H]N-acetyl groups, whereas the 14C-label was at the 4-position. Pulse-chase analysis of the 3H/14C ratio showed that the N-acetyl groups of both GD3 and of the monosialoganglioside GM3 were turning over faster than the parent molecules. Selective periodate oxidation showed that both the inner and outer sialic acid residues of GD3 incorporated 3H-label in the in vitro reaction, and showed similar turnover of N-acetylation in the pulse-chase study. Taken together, these results indicate that both the O- and N-acetyl groups of the sialic acid residues of gangliosides turn over faster than the parent molecules. They also demonstrate a novel re-N-acetylation reaction that predicts the existence of de-N-acetyl gangliosides in melanoma cells.

Acetates↗
PubMed1990

High-pressure liquid chromatography of sialic acids on a pellicular resin anion-exchange column with pulsed amperometric detection: a comparison with six other systems.

A wide variety of different sialic acids have been reported in nature. Following their release and purification, detection and quantitation of these molecules is now possible by a number of techniques. We and others have previously reported high-pressure liquid chromatography separation of sialic acids with several different columns, elution methods, and detection techniques. We report here a new method for the separation of sialic acids at neutral pH on a Carbopac PA-1 anion-exchange column of pellicular resin, with pulsed amperometric detection following postcolumn addition of alkali. The major advantages of this system are the separation of a variety of sialic acids, sensitive detection (into the picomole range), and the relative ease of use for preparative purposes. Using a set of defined sialic acid standards, this method is compared and contrasted with six other HPLC methods previously described by us and by others. The advantages and disadvantages of each system are also addressed. In the final analysis, no single method is adequate to completely separate and quantitate all of the known sialic acids. However, used in appropriate combinations, these methods allow exploration of the biology of sialic acids in a manner heretofore not possible.

Acetates↗
PubMed1990

A randomized trial of the Gyne T 380 and Gyne T 380 Slimline Intrauterine Copper devices.

To facilitate manufacture and insertion of the Gyne T 380 IUD, design changes were instituted. Copper collars were seated flush at the ends of the horizontal crossbar of the device. A randomized study of the Gyne T 380 Slimline, the new design, was undertaken in comparison with the standard Gyne T 380. A total of 996 women were enrolled, with 698 Slimline insertions and 298 of the standard Gyne T. No statistically significant difference in ease of insertion or in performance was detected between the models. At one year, the pregnancy rate of each model was below 0.5 per 100 and the continuation rate was 79-80 per 100. Pelvic inflammatory disease or endometritis was found in one percent of subjects in the first year. This is the seventh multicenter randomized study of a collared T IUD with 380 mm2 of copper surface. In all seven, the one-year gross pregnancy rate has been 1.2 per 100 or lower.

Adult↗
PubMed1990

Milk and plasma concentrations of the progestin ST-1435 in women treated parenterally with ST-1435.

A subcutaneous contraceptive capsule releasing progestin ST-1435 was used by 6 breast-feeding women. One to three paired milk and plasma samples were collected over a one-month period and the concentrations of ST-1435 were determined by radioimmunoassay. An improved and sensitive method for determination of ST-1435 in milk was developed. A column chromatographic purification of milk prior to radioimmunoassay decreased the blank and improved sensitivity. The average plasma concentration of ST-1435 was 62 +/- 20 pg/ml (mean +/- SD). The average milk concentration of ST-1435 was 38 pg/ml (ranging from 7 to 73 pg/ml), while the average milk to plasma ratio was 0.60 (ranging from 0.25 to 0.91). There was a significant correlation between ST-1435 concentrations in breast milk and plasma, indicating that the concentration in plasma is the major determinant for the amount of ST-1435 excreted into milk. Since studies with this drug have shown good contraceptive efficacy and low bioavailability after oral intake, ST-1435 is a good candidate for lactational contraception.

Contraceptive Agents, Female↗
PubMed1990

Mapping study of noradrenergic stimulation of vasopressin release.

The precise role of hypothalamic norepinephrine (NE) in the control of vasopressin (AVP) release has remained unclear, due to reports of both inhibitory and excitatory effects of NE and only a few studies with direct hypothalamic manipulations. The present study utilized a chronically implanted swivel brain cannula to investigate, in undisturbed and freely behaving rats, the impact of acute hypothalamic infusions of monoamines on circulating AVP levels. The first study examined and compared the responsiveness of six hypothalamic sites to NE infusion through the swivel cannula. Results indicated that the excitatory effect of central noradrenergic stimulation on serum AVP is highly site specific, localized to the paraventricular (PVN) and supraoptic (SON) nuclei. These two nuclei appeared to be equally responsive to NE infusion, yielding a threefold rise in serum AVP over baseline levels. In contrast, NE in the dorsomedial nucleus produced a significantly smaller increase in AVP, and no response was observed in the ventromedial nucleus, posterior hypothalamus, or perifornical lateral hypothalamus. Further tests conducted in the PVN showed this nucleus to respond in a dose-dependent manner to NE infusion. In contrast, under similar test conditions, dopamine caused only a small increase in AVP at a relatively high dose, while a PVN injection of serotonin produced no response. These results support the existence of an excitatory noradrenergic system controlling AVP release and specifically demonstrate that this function of NE is localized to the PVN and SON, in contrast to other hypothalamic areas, and is mimicked to some extent by dopamine but not by serotonin.

Animals↗
PubMed1989

Biosynthesis of N-glycolyneuraminic acid. The primary site of hydroxylation of N-acetylneuraminic acid is the cytosolic sugar nucleotide pool.

N-Glycolylneuraminic acid (Neu5Gc) is an oncofetal antigen in humans and is developmentally regulated in rodents. We have explored the biology of N-acetylneuraminic acid hydroxylase, the enzyme responsible for conversion of the parent sialic acid, N-acetylneuraminic acid (Neu5Ac) to Neu5Gc. We show that the major sialic acid in all compartments of murine myeloma cell lines is Neu5Gc. Pulse-chase analysis in these cells with the sialic acid precursor [6-3H]N-acetylmannosamine demonstrates that most of the newly synthesized Neu5Gc appears initially in the cytosolic low-molecular weight pool bound to CMP. The percentage of Neu5Gc on membrane-bound sialic acids closely parallels that in the CMP-bound pool at various times of chase, whereas that in the free sialic acid pool is very low initially, and rises only later during the chase. This implies that conversion from Neu5Ac to Neu5Gc occurs primarily while Neu5Ac is in its sugar nucleotide form. In support of this, the hydroxylase enzyme from a variety of tissues and cells converted CMP-Neu5Ac to CMP-Neu5Gc, but showed no activity towards free or alpha-glycosidically bound Neu5Ac. Furthermore, the majority of the enzyme activity is found in the cytosol. Studies with isolated intact Golgi vesicles indicate that CMP-Neu5Gc can be transported and utilized for transfer of Neu5Gc to glycoconjugates. The general properties of the enzyme have also been investigated. The Km for CMP-Neu5Ac is in the range of 0.6-2.5 microM. No activity can be detected against the beta-methylglycoside of Neu5Ac. On the other hand, inhibition studies suggest that the enzyme recognizes both the 5'-phosphate group and the pyrimidine base of the substrate. Taken together, the data allow us to propose pathways for the biosynthesis and reutilization of Neu5Gc, with initial conversion from Neu5Ac occurring primarily at the level of the sugar nucleotide. Subsequent release and reutilization of Neu5Gc could then account for the higher steady-state level of Neu5Gc found in all of the sialic acid pools of the cell.

Animals↗
PubMed1989

O-acetylation and de-O-acetylation of sialic acids. O-acetylation of sialic acids in the rat liver Golgi apparatus involves an acetyl intermediate and essential histidine and lysine residues--a transmembrane reaction?

Isolated intact rat liver Golgi vesicles utilize [acetyl-3H]coenzyme A to add 3H-O-acetyl esters to sialic acids of internally facing endogenous glycoproteins. During this reaction, [3H]acetate also accumulates in the vesicles, even though the vesicles are impermeant to free acetate. On the other hand, entry of intact AcCoA into the lumen of the vesicles could not be demonstrated, and permeabilization of the vesicles did not alter the reaction substantially (Diaz, S., Higa, H. H., Hayes, B. K., and Varki, A. (1989) J. Biol. Chem. 264, 19416-19426). When vesicles prelabeled with [acetyl-3H] coenzyme A are permeabilized with saponin, we can demonstrate a [3H]acetyl intermediate in the membrane that can transfer label to the 7- and 9-positions of exogenously added free N-acetylneuraminic acid but not to glucuronic acid or CMP-N-acetylneuraminic acid. This labeled acetyl intermediate represents a significant portion of the radioactivity incorporated into the membranes during the initial incubation and cannot be accounted for by nonspecifically "trapped" acetyl-CoA in the permeabilized vesicles. There was no evidence for involvement of acetylcarnitine or acetyl phosphate as an intermediate. The overall acetylation reaction appears to involve two steps. The first step (utilization of exogenous acetyl-CoA to form the acetyl intermediate) is inhibited by coenzyme A-SH (apparent Ki = 24-29 microM), whereas the second (transfer from the acetyl intermediate to sialic acid) is not affected by millimolar concentrations of the nucleotide. Studies with amino acid-modifying reagents indicate that 1 or more histidine residues are involved in the first step of the acetylation reaction. Diethylpyrocarbonate (which can react with both nonsubstituted and singly acetylated histidine residues) also blocks the second reaction, indicating that the acetyl intermediate on both sides of the membrane involves histidine residue(s). Taken together with data presented in the preceding paper, these results indicate that the acetylation of sialic acids in Golgi vesicles may occur by a transmembrane reaction, similar to that described for the acetylation of glucosamine in lysosomes (Bame, K. J., and Rome, L. H. (1985) J. Biol. Chem. 260, 11293-11299). However, several features of this Golgi reaction distinguish it from the lysosomal one, including the nature and kinetics of the reaction and the additional involvement of an essential lysine residue. The accumulation of free acetate in the lumen of the vesicles during the reaction may occur by abortive acetylation (viz. transfer of label from the acetyl intermediate to water). It is not clear if this is an artifact that occurs only in the in vitro reaction.

Acetates↗
PubMed1989

O-acetylation and de-O-acetylation of sialic acids. 7- and 9-o-acetylation of alpha 2,6-linked sialic acids on endogenous N-linked glycans in rat liver Golgi vesicles.

We have previously shown that radioactivity from [acetyl-3H]AcCoA is concentrated into isolated intact rat liver Golgi vesicles. The incorporated radioactivity occurred in acid-soluble and acid-insoluble components, and the acid-insoluble fraction included O-acetylated sialic acids (Varki, A., and Diaz, S. (1985) J. Biol. Chem. 260, 6600-6608). Nearly all of the protein-associated radioactivity was found to be in sialic acids alpha 2-6-linked to N-linked oligosaccharides on endogenous glycoproteins. Incubation of the vesicles with CMP-[3H]sialic acid resulted in labeling of a very similar group of glycoproteins. The 3H-O-acetyl groups were found at both the 7- and the 9-positions of N-acetylneuraminic acid residues at the end of the labeling reaction. Although 7-O-acetyl groups can undergo migration to the 9-position under physiological conditions, kinetic studies using O-acetyl-14C-labeled internal and O-acetyl-3H-labeled external standards indicate that during the labeling, release, and purification, negligible migration occurred. Studies with mild periodate oxidation provided further confirmation that O-acetyl esters are added directly to both the 7- and the 9-positions of the sialic acids in this system. The acid-soluble, low molecular weight component is released from the vesicles by increasing concentrations of saponin, and its exit parallels that of CMP-[14C]sialic acid taken up during the incubation. The vesicles themselves are impermeant to free acetate. However, even after short incubations, this saponin-releasable radioactivity was almost exclusively in [3H] acetate and not in [3H]acetyl-CoA. The apparent Km for accumulation of the [3H]acetate is almost identical with that for the generation of the acid-insoluble O-acetylated sialic acids. Most of this accumulation of free acetate is also blocked by coenzyme A-SH. Only a small portion arises from the action of an endogenous esterase on the 3H-O-acetylated sialic acids. Taken together, the results indicate that accumulation of free [3H]acetate occurs within the lumen of the vesicles in parallel with O-acetylation of sialic acids and is probably a product of abortive acetylation. It is not known if this reaction occurs in vivo. Permeabilization of Golgi vesicles to low molecular weight molecules with saponin does not alter the rate of acetylation substantially. Furthermore, double label studies suggest that the intact acetyl-CoA molecule does not gain access to the lumen of the vesicles. These results indicate that the acetylation reaction may have a different mechanism from previously described Golgi glycosylation reactions, wherein specific transporters concentrate sugar nucleotides for use by luminally oriented transferases.

Acetates↗
PubMed1989

Norepinephrine in the paraventricular nucleus stimulates corticosterone release.

Hypothalamic cells containing corticotropin-releasing factor are believed to be densely innervated by noradrenergic terminals. However, the role of norepinephrine (NE) in the control of the hypothalamo-pituitary-adrenal axis has remained undefined, with both excitatory and inhibitory effects suggested by the literature. The present experiments tested the effects of direct hypothalamic infusion of NE on the release of corticosterone (CORT) in awake and freely moving rats. Norepinephrine infusion into the paraventricular nucleus (PVN) produced a dose-dependent increase in circulating levels of CORT. In a mapping study, this stimulatory effect of NE was found to be anatomically localized. The strongest rise in CORT levels (up to 12 micrograms%) was observed after injection into the PVN, where NE acted in a dose-dependent fashion. A somewhat smaller effect was also detected with NE in the dorsomedial nucleus, while no response occurred after injection just dorsal to the PVN, into the ventromedial or supraoptic nuclei, or into the lateral or posterior hypothalamus. Serotonin infusion into the PVN produced a small but statistically reliable increase in circulating CORT levels. However, dopamine injection into this nucleus had no observable effect. These results agree with recent studies suggesting an excitatory function of PVN NE in the pituitary-adrenal axis.

Animals↗
PubMed1987

Biochemical and genetic evidence for distinct membrane-bound and cytosolic sialic acid O-acetyl-esterases: serine-active-site enzymes.

A cytosolic sialic acid-specific O-acetyl-esterase was previously described that can remove O-acetyl esters from the 9-position of sialic acids. We show that rat liver Golgi vesicles contain a distinct sialic acid-esterase located within the lumen of the same vesicles that add O-acetyl esters to sialic acids. Studies of a retinoblastoma cell line genetically deficient in the cytosolic enzyme also confirm the existence of distinct membrane-associated sialic acid esterase activity. We developed a sensitive, specific and facile assay, which measures release of [3H]acetyl groups from [3H-acetyl]9-O-acetyl-N-acetylneuraminic acid. Using this assay, we show that rat liver membranes may contain different sialic acid O-acetyl-esterases. The membrane-associated enzyme(s) bind to Concanavalin A Sepharose, whereas the cytosolic enzyme does not. Membrane-bound and cytosolic esterases are inactivated by di-isopropyl-fluorophosphate, showing they are serine-active-site enzymes.

Acetylesterase↗
PubMed1987

Purification of an acid protease from Mucor rouxii that inactivates chitin synthetase.

An acid protease was purified from the mycelial form of Mucor rouxii by a method which involved salt and acid precipitation, gel filtration and anion-exchange chromatography. The enzyme had a molecular mass of 16,000 Da. Its optimum pH was 4.0, maximal activity was obtained at 50 degrees C, and it was inactivated at 70 degrees C. It was not affected by leupeptin or N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) but diazoacetyl-DL-norleucine methyl ester (DNME) in the presence of Cu2+ and more noticeably pepstatin A, strongly inhibited the activity. This acid protease did not activate "zymogenic" chitin synthetase from the fungus, but brought about its inactivation even at low concentrations and after short periods of incubation time.

Aspartic Acid Endopeptidases↗