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PubMed1982

Purification and physicochemical characterization of human alpha 2-HS-glycoprotein.

A large-scale purification method for alpha 2 HS-glycoprotein from normal human pooled serum is presented. 130 mg of alpha 2 HS was obtained from 21 of normal serum and the yield was 13.6%. Charge heterogeneity on isoelectrofocusing of this protein is mainly due to sialic acid. By the measurement of the circular dichroism spectrum, the alpha-helix content was calculated as 11% and the beta-structure content was calculated as 21 to 33%. alpha 2HS consists of a single polypeptide chain (Mr 49,000) of which the N-terminal amino acid is alanine. The N-terminal sequence of 31 amino acids contains 19 hydrophobic residues.

Amino Acid Sequence↗
PubMed1992

Comparison of N-glycosides of fetuins from different species and human alpha 2-HS-glycoprotein.

Complex type N-glycosides of commercial bovine fetuin preparations from pooled fetal calf serum have been shown to contain comparable amounts of Gal4,4,4TRI (see structure A below) and Gal4,4,3TRI (structure B) as major asialo-structures. To investigate whether there is a clear genetic specificity for synthesis of these oligosaccharides, N-glycosides from two preparations of bovine fetuin, each from a single calf, were examined. Both of these structures were present in each calf, and there was only a subtle quantitative difference in the ratio of these two structures between the calves. Thus, a specific galactosyltransferase, presumably required for the biosynthesis of the Gal4,4,3TRI structure, may exist in both of these individual calves. Comparison of fetuin N-glycosides was also extended to sheep, pig, and human alpha 2-HS-glycoprotein, the human counterpart of bovine fetuin, using high-pH anion-exchange chromatography of the reducing oligosaccharides as well as HPLC of their pyridinylamino derivatives. The N-glycosides of ovine fetuin also have both Gal4,4,4TRI and Gal4,4,3TRI structures in a ratio similar to that of bovine fetuin. However, the major N-glycoside of porcine fetuin is of a fucosyl biantennary complex type structure (structure C below) and human alpha 2-HS-glycoprotein has an N-glycoside which is almost exclusively a nonfucosylated biantennary structure (structure D). This species-specific presence of N-glycosides of fetuins and comparison with N-glycosides of other glycoproteins suggest that the polypeptide sequence of a glycoprotein may affect its N-glycan structure by regulating the activity of specific glycosyltransferases. [formula: see text]

Amidohydrolases↗
PubMed1991

Rat tyrosine kinase inhibitor shows sequence similarity to human alpha 2-HS glycoprotein and bovine fetuin.

Human alpha 2-HS glycoprotein and bovine fetuin, abundant proteins of fetal plasma, are structural members of the fetuin family within the cystatin superfamily. They are characterized by the presence of two N-terminally located cystatin-like units and a unique C-terminal sequence segment not present in the other members of the cystatin superfamily. Search for related sequences revealed that the natural inhibitor of the insulin receptor tyrosine kinase [Auberger, Falquerho, Contreres, Pages, Le Cam, Rossi & Le Cam (1989) Cell (Cambridge, Mass.) 58, 631-640] shows sequence similarity to the mammalian fetuins. The sequence identity between rat tyrosine kinase inhibitor, human alpha 2-HS glycoprotein and bovine fetuin is 56 and 60% respectively (percentage of residues in identical positions). The sequence similarity extends over the entire protein structures, except the extreme C-terminal portions. In particular, the number and relative positions of the cysteine residues are invariant among the proteins, suggesting that the characteristic array of linearly arranged and tandemly repeated disulphide loops of the cystatin superfamily is also present in rat tyrosine kinase inhibitor. We conclude that rat tyrosine kinase inhibitor may be classified as a novel member of the mammalian fetuin family.

Amino Acid Sequence↗
PubMed2001

Calcium phosphate crystal-associated proteins: alpha-2-HS-glycoprotein, prothrombin fragment 1 and osteopontin.

To study the inhibitory effects of calcium phosphate-associated proteins on calcium oxalate crystallization and urinary concentrations of proteins in people who form stones and healthy controls. From 60 L of urine from healthy men, calcium phosphate-associated proteins (alpha-2-HS-glycoprotein, prothrombin fragment 1 and osteopontin) were obtained. The effects of the proteins on calcium oxalate (CaOx) crystallization were studied with a mixed suspension mixed product removal system. To examine urinary concentrations of the proteins, urine samples were collected from 17 healthy subjects and 15 stone formers and analyzed using anion-exchange chromatography and an enzyme immunoassay. Prothrombin fragment 1 (PTF1) and osteopontin (OPN) had strong inhibitory effects on CaOx crystallization, while alpha-2-HS-glycoprotein had a mild inhibitory effect. Urinary concentrations of PTF1 and OPN were lower in stone formers than in healthy controls. Low urinary concentrations of PTF1 and OPN might be one of the reasons for stone formation.

Adult↗
PubMed2000

Inhibitory effect of calcium phosphate-associated proteins on calcium oxalate crystallization: alpha2-HS-glycoprotein, prothrombin-F1 and osteopontin.

OBJECTIVE: To analyse urinary calcium phosphate- associated proteins and assess their inhibitory effects on calcium oxalate crystallization. Materials and methods Urine samples were collected over 24 h from five healthy men and calcium phosphate crystallization induced with NaOH solution. The bound proteins were separated on a cellulose column. To examine the effect of urinary calcium phosphate-associated proteins on calcium oxalate crystallization, 60 L of urine was collected from the healthy men. The effect of the separated fractions was studied in a mixed suspension/mixed product removal system. RESULTS: The separated proteins were identified as alpha2-HS-glycoprotein, prothrombin fragment 1 and osteopontin. Prothrombin fragment 1 and osteopontin strongly inhibited the growth of calcium oxalate crystals in artificial urine. CONCLUSION: alpha2-HS-glycoprotein, prothrombin fragment 1 and osteopontin selectively bound with calcium phosphate crystals in urine. Prothrombin fragment 1 and osteopontin in urine may strongly influence stone formation.

Adult↗
PubMed2000

Calcium phosphate crystal-associated proteins: alpha2-HS-glycoprotein, prothrombin F1, and osteopontin.

PURPOSE: To elucidate the process of urinary stone formation, many urinary constituents have been examined as inhibitors or promoters. MATERIALS AND METHODS: We analyzed specific binding proteins of calcium phosphate (CaP) crystals generated in human urine and found that the separated proteins were alpha2-HS-glycoprotein, prothrombin fragment 1 (PTF1), and osteopontin (OPN). We also studied their inhibitory effects on calcium oxalate crystallization and the quantitative differences in urinary excretion of the proteins in healthy individuals and stone formers. RESULTS: Both PTF1 and OPN had strong inhibitory effects on growth of calcium oxalate crystals. No differences in alpha2-HS-glycoprotein were found between healthy subjects and stone formers. Urinary concentrations of PTF1 and OPN were lower in stone formers than in healthy controls. No differences in these proteins were seen between single and recurrent stone formers. CONCLUSION: Low urinary concentrations of PTF1 and OPN might be one reason for stone formation.

Adolescent↗
PubMed1991

The effect of the alpha 2-HS-glycoprotein on the mitogen-induced lymphoblastic transformation and IL-2 production.

Authors investigated the in vitro effect of alpha 2-HS-glycoprotein on the lymphoblastic transformation and interleukin-2 production. The observations were made on the peripheral blood lymphocytes of healthy persons, on those of women taking oral contraceptives and on those of patients with systemic lupus erythematosus. The alpha 2-HS-glycoprotein inhibited the mitogen responses both in physiologic and in the halved concentrations. The inhibition of the interleukin-2 production seemed less remarkable.

Adolescent↗
PubMed1996

Two mutations in the locus control region hypersensitivity site-2 (5' HS-2) of haplotype 19 beta s chromosomes alter binding of trans-acting factors.

There are five major haplotypes associated with sickle cell anemia (SS). Individuals homozygous for haplotypes 3 (Senegal) and 31 (Saudi Arabian) have high fetal hemoglobin (HbF) levels (15 to 30% of total hemoglobin) whereas individuals homozygous for haplotypes 17 (Cameroon), 19 (Benin), and 20 (Bantu) have low HbF levels (1 to 10%). We previously identified several point mutations in the LCR 5'HS-2 that were specific for haplotype 19 beta s chromosomes (compared to the GenBank HUMHBB reference sequence, T-->G at position 8580, A-->G at position 8598, and A-->T at position 9114). We postulated that one or more of these mutations may alter the binding of specific trans-acting factors and ultimately affect the expression of HbF in these sickle cell patients. We performed gel mobility shift assays using 32P-end-labeled double-stranded 19mers corresponding to each of the LCR 5'HS-2 normal (GenBank) and mutant sequences. Nuclear extracts prepared from HeLa and HEL cells were used in our experiments and neither the normal nor mutant sequence at position 8580 bound trans-acting factors in either nuclear extract. The 8598 mutant increased binding of Sp1; using purified protein and both nuclear extracts. HEL extracts were used to quantify the increase in Sp1 binding to the 8598 mutation and we found an increase in binding of 66 and 47%, respectively, in two shifted bands. The 9114 mutation sharply decreased binding of an unknown trans-acting factor by 74%. This factor was present in both HeLa and HEL nuclear extracts.

Base Sequence↗
PubMed2004

In vitro investigation on the impact of the surface-active excipients Cremophor EL, Tween 80 and Solutol HS 15 on the metabolism of midazolam.

The impact of the surface-active formulation ingredients Cremophor EL, Tween 80 and Solutol HS 15 on the intrinsic clearance (Clint) of midazolam (MDZ) was investigated in rat hepatocytes and microsomes. In rat hepatocytes with 0.003%, 0.03% and 0.3% (w/v) Solutol HS 15 already present in the incubation medium, the Clint was significantly reduced in a dose-dependent manner by about 25%, 30% and 50%, respectively. In the presence of Cremophor EL and Tween 80 a significant reduction in Clint by about 30% and 25%, respectively, was observed at 0.03% surfactant concentration. At 0.3% of Cremophor EL and Tween 80, Clint was reduced by about 50% and 20%, respectively. A reduction in Clint was also observed in experiments with rat liver microsomes. At surfactant concentrations up to 0.03%, cytotoxicity assays (lactate dehydrogenase release, adenosine triphosphate content) as well as light microscope investigations did not reveal any cytotoxic impact of the surfactants on the hepatocyte monolayer. A potential interaction of the surfactants with biological membranes was determined using phosphatidylcholine-cholesterol liposomes loaded with self-quenching concentrations of carboxyfluorescein. No marked release of carboxyfluorescein from the liposomes (that would be an indication for a surfactant-dependent disruption of membrane integrity) was observed up to concentrations of 0.03% of the different surfactants. It is concluded that cytochrome P450 3A mediated metabolism of MDZ seems to be prevented by all surfactants at concentrations above 0.03%. In our experiments the surfactants did not show toxic effects at concentrations that resulted in a decreased Clint of MDZ. Thus, a direct inhibition of the metabolizing enzymes, a molecular interaction with the microsomes as well as an alteration of membrane properties that did not yet result in a release of LDH have to be taken into consideration as reasons for the observed changes in the metabolism of MDZ.

Animals↗
PubMed2004

Application of headspace solid-phase microextraction (HS-SPME) and comprehensive two-dimensional gas chromatography (GC x GC) for the chemical profiling of volatile oils in complex herbal mixtures.

The coupling of headspace solid-phase microextraction (HS-SPME) with comprehensive two-dimensional gas chromatography (GC x GC) was shown to be a powerful technique for the rapid sampling and analysis of volatile oils in complex herbal materials. When compared to one-dimensional (1-D) GC, the improved analytical capabilities of GC x GC in terms of increased detection sensitivity and separation power were demonstrated by using HS-SPME/GC x GC for the chemical profiling (fingerprinting) of essential/volatile oils contained in herbal materials of increasing analytical complexity. More than 20 marker compounds belonging to Panax quinquefolius (American ginseng) can be observed within the 2-D contour plots of ginseng itself, a mixture of ginseng and another important herb (P. quinquefolius/Radix angelicae sinensis), as well as a mixture of ginseng and three other herbs (P. quinquefolius /R. angelicae sinensis/R. astragali/R. rehmanniae preparata). Such analytical capabilities should be important towards the authentication and quality control of herbal products, which are receiving increasing attention as alternative medicines worldwide. In particular, the presence of Panax in the herb formulation could be readily identified through its specific peak pattern in the 2-D GC x GC plot.

Chromatography, Gas↗
PubMed2003

Fast screening method for determining 2,4,6-trichloroanisole in wines using a headspace-mass spectrometry (HS-MS) system and multivariate calibration.

The system based on coupling a headspace sampler to a mass spectrometer (HS-MS), which is considered one kind of electronic nose, is an emergent technique for ensuring and controlling quality in industry. It involves injecting the headspace of the sample into the ionization chamber of the mass spectrometer where the analytes are fragmented. The result is a complex mass spectrum for each sample analyzed. When several samples are analyzed the data matrix generated is processed with chemometric techniques to compare and classify the substances from their volatile composition, in other words, to compare and classify their flavor. So far, information from electronic nose applications has mainly been qualitative. In this paper we present a quantitative study that uses a multivariate calibration. We analyzed several white wines using HS-MS to determine 2,4,6-tricholoranisole (TCA). This is an off-flavor that is a serious problem for the wine industry. The method is simple because it does not require sample preparation, only addition of sodium chloride being necessary for sample conditioning. Also, it provides a fast screening (10 min/sample) of the quantity of TCA in wines at ultratrace (sub microg L(-1)) levels.

Anisoles↗
PubMed2005

Determination of organotin compounds by headspace solid-phase microextraction-gas chromatography-pulsed flame-photometric detection (HS-SPME-GC-PFPD).

A method based on Headspace solid-phase microextraction (HS-SPME, with a 100 mum PDMS-fiber) in combination with gas-chromatography and pulsed flame-photometric detection (GC-PFPD) has been investigated for simultaneous determination of eight organotin compounds. Monobutyltin (MBT), dibutyltin (DBT), tributyltin (TBT), monophenyltin (MPhT), and the semi-volatile diphenyltin (DPhT), triphenyltin (TPhT), monooctyltin (MOcT), and dioctyltin (DOcT) were determined after derivatization with sodium tetraethylborate. The conditions used for the extraction and preconcentration step were optimised by experimental design methodology. Tripropyltin (TPrT) and diheptyltin (DHepT) were used as internal standards for quantification of volatile and semi-volatile organotin compounds, respectively. The analytical precision (RSD) for ten successive injections of a standard mixture containing all the organic tin compounds ranged between 2 and 11%. The limits of detection for all the organotin compounds were sub ng (Sn) L(-1) in water and close to ng (Sn) kg(-1) in sediments. The accuracy of the method was evaluated by analysis of two certified reference material (CRM) sediment samples. The HS-SPME-GC-PFPD was then applied to the analysis of three harbour sediment samples. The results showed that headspace SPME is an attractive tool for analysis of organotin compounds in solid environmental matrices.

Chromatography, Gas↗
PubMed2005

Influence of the angiotensin converting enzyme inhibitor ramipril on high-sensitivity C-reactive protein (hs-CRP) in patients with documented atherosclerosis.

UNLABELLED: Some medications have been shown to produce reductions in hs-CRP levels after initiating therapy. Whereas the role of the renin-angiotensin system in the inflammatory process has been documented in more detail during the last few years, the impact of an ACE-inhibitor therapy on this process has not been fully understood so far. The aim of this study was to investigate the effect of a therapy with the angiotensin-converting enzyme (ACE) inhibitor ramipril on hs-CRP plasma concentrations in patients with atherosclerosis. METHODS AND RESULTS: A total of 24 patients were enrolled in this prospective, uncontrolled, open-label multicenter study. Inclusion criteria were documented atherosclerosis, baseline high-sensitivity C-reactive protein between 3 and 12 mg/l, LDL-Cholesterol < or =150 mg/dl and no previous treatment with ACE inhibitors or angiotensin receptor blockers. Ten patients, pretreated with statins, and 10 patients not previously treated with statins were eligible for statistical analysis. Baseline high-sensitivity C-reactive protein was significantly decreased from 3.99+/-1.61 mg/l (mean+/-SD) to 2.72+/-1.19 mg/l (-32%) after 3 months treatment with 10 mg ramipril daily (p=0.0002). The decrease was more pronounced in patients who had not been treated with statins previously (-1.50 mg/l+/-1.44 mg/l) compared to those who were pretreated (-0.90 mg/l+/-0.93 mg/l). CONCLUSIONS: The ACE inhibitor ramipril administered in a daily dose of 10 mg to patients with atherosclerosis reduces the high-sensitivity C-reactive protein concentration. This effect may contribute to cardiovascular risk reduction mediated by ramipril aside from the blood pressure lowering effect.

Adult↗
PubMed1992

Effects of HS-142-1, a novel non-peptide ANP antagonist, on diuresis and natriuresis induced by acute volume expansion in anesthetized rats.

In this study we used HS-142-1, a novel non-peptide antagonist for the atrial natriuretic peptide (ANP) receptor, to clarify the possible physiological significance of ANP in acute hypervolemia. Substantial volume expansion in anesthetized rats induced a strong diuresis and natriuresis. These renal responses were significantly blocked by HS-142-1 at a dose of 3.0 mgkg-1 i.v. This observation suggests that ANP and its guanylyl cyclase-coupled receptor are, under the present conditions, physiologically involved that appears to be responsible for the renal responses in the volume homeostasis.

Animals↗
PubMed1988

Cell cycle-associated change in the expression of the proliferation-sensitive and heat-shock protein hs x 70 (IEF14): increased synthesis during mitosis.

The major heat-inducible protein of transformed human amnion cells (AMA) has been identified as the proliferation-sensitive polypeptide IEF14 (Mr 66 kDa; HeLa protein catalogue). From its mobility in two-dimensional gels (Mr and pI) as well as from the fact that this protein is immunoprecipitated by mAb C92 F3-5 (W. J. Welch, and J. P. Suhan, (1986) J. Cell Biol. 103, 2035-2052), we concluded that this polypeptide is either closely related or identical to the 72 kDa inducible stress human protein hs X 70 (H. R. B. Pelham (1986) Cell 46, 959-961). It is further shown that in AMA cells the rate of synthesis of this protein increases preferentially during mitosis. These results provide further evidence suggesting that the levels of hs X 70 can be modulated by mechanisms independent of heat shock.

Amnion↗
PubMed1980

Detection of attograms of antigen by a high-sensitivity enzyme-linked immunoabsorbent assay (HS-ELISA) using a fluorogenic substrate.

In an attempt to increase the sensitivity of the ELISA technique three factors were examined: the use of a fluorogenic substrate (4MU-P), an increase in the antigen binding surface area, and the prolongation of the incubation time with the substrate. The use of the fluorogenic substrate and fluorometric detection method increased the sensitivity of the assay 16--39 times as compared to the colorigenic substrate (PNP-P). A 4-fold increase in the antigen binding surface area resulted in a 1000-fold increase in sensitivity, while prolonged incubation periods with the substrate at 10 degrees C yielded a further increase in sensitivity of 10--50-fold. Therefore, the present high sensitivity ELISA method (HS-ELISA) has the potential of increasing the sensitivity of the previously described method (Voller et al., 1974) by 1.6--19.5 X 10(5) times. HS-ELISA can detect as low as 3--10 attog/ml (3--10 X 10(-18) g/ml) antigen (Rb alpha M F(ab')2) or 24,000 molecules or purified mouse myeloma IgG. Our findings suggest that with simple modifications enzyme immunoassays can reach extreme levels of sensitivity.

Animals↗
PubMed1983

Subcellular distribution and kinetic parameters of HS mouse liver aldehyde dehydrogenase.

1. Aldehyde dehydrogenase subcellular distribution studies were performed in a heterogeneous stock (HS) of male and female mice (Mus musculus) with propionaldehyde (5 mM and 50 microM) and formaldehyde (1 mM) and NAD+ or NADP+. 2. The relative percents of distribution were: cytosolic 55-68%, mitochondrial 12-20%, microsomal 9-18% and lysosomal 3-15% for both propionaldehyde concentrations and NAD+. 3. Kinetic experiments using propionaldehyde and acetaldehyde with NAD+ revealed two separate enzymes, Enzyme I (low Km) and Enzyme II (high Km) in the cytosolic and mitochondrial fractions. 4. The kinetic data also indicated a spectrum of cytosolic low Km values that exhibited a bimodal distribution with one congruent to 40 microM and one congruent to 5 microM. 5. It was concluded that there was no significant difference in aldehyde-metabolizing capability between male and female HS mice, compared on a per gram of liver basis. The cytosolic low Km enzyme plays a major role in aldehyde oxidation at moderate to low aldehyde concentrations.

Aldehyde Dehydrogenase↗
PubMed2006

Femtosecond dynamics of proteoheparan sulfate (HS-PG) after UV excitation--a readout for arteriosclerotic nanoplaque formation?

Ultrashort UV laser pulses were used to excite tryptophan residues of heparan sulfate proteoglycan (HS-PG) in blood substitute Krebs solution. Tryptophan fluorescence is sensitive to the environment, so its shift and decay indicate the conformation and solvation state of the protein. We monitored stimulated emission and excited-state absorption by probing with delayed white-light femtosecond pulses. Comparison with bare tryptophan revealed transient absorption features which are characteristic for HS-PG. Furthermore, the effect of adding calcium salt was investigated. Differences in the spectra from solutions with and without calcium developed during several minutes, which points to changes in protein conformation, but could only be measured in the sub-ps regime. These results provide a first step to a better understanding of the molecular formation of nanoplaques in blood vessels. The goal of this work is to open a way towards biosensing of the initial stages in atherogenesis allowing for a risk assessment in cardiovascular disease.

Amino Acid Sequence↗