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PubMed2001

Long-term follicular dynamics and biochemical characteristics of dominant follicles in dairy cows subjected to acute heat stress.

The objective of this study was to examine the quality of successive dominant follicles (DFs) after induced heat stress. Non-lactating dairy cows expressing estrus at normal intervals were allocated randomly to heat stress (HS; n=8) and control (C; n=8) groups. Cows received GnRH (100 microg, i.m.) on Day 0, a progesterone CIDR-B device on Day 4 and prostaglandin (PGF(2alpha); 25mg, i.m.) on Day 7 upon removal of the CIDR device. The DF and follicles >5mm were aspirated on Day 8, and GnRH (100 microg) injected following aspiration, to initiate a new follicular wave. In this manner, a DF was aspirated every 8 days (one "follicular cycle") for 10 cycles. After the first follicular cycle, HS cows were placed in environmental chambers for 7 days during the second follicular cycle (8h per day at 43.3 degrees C set point and 16h per day at 24 degrees C for 4 days, and 8h per day at 43.3 degrees C set point and 16h per day at 32.2 degrees C set point for 3 days; relative humidity, 40%) and thereafter maintained outdoors with control cows at a mean ambient temperature (18.5 degrees C; range 12.7-26 degrees C). Rectal temperature increased (P<0.001) in HS as compared with C cows (39.28+/-0.01 degrees C versus 38.78+/-0.01 degrees C). Concentrations of estradiol (E(2); 1662+/-189 versus 1493+/-188ng/ml) and progesterone (P(4); 44.7+/-5 versus 54.1+/-5.1ng/ml) in follicular fluid (FF) of DF did not differ between C and HS treatments, respectively. Total FF protein concentration was greater (P<0.05) in HS (99.7+/-2.3mg/ml) than in C (92.7+/-2.3mg/ml). Heat shock protein 90 (Hsp 90) in FF was not altered by heat stress. IGF-II ligand blots were conducted with FF samples (n=79) from four HS and four C cows. There was a predominance of IGFBP-3 in 76 of 79 FF samples, indicating healthy follicular status, and only three FF samples had the lower molecular weight IGFBP-2 indicative of a poor quality follicle. Plasma P(4) and E(2) concentrations did not differ between C and HS groups. The number of class 1 and 3 follicles increased during and just after heat stress, but the number of class 2 follicles did not differ between C and HS cows. Heat stress appeared to induce a decrease in follicular dominance, but GnRH-induced follicular cycles resulted in development of healthy preovulatory follicles in both groups.

Animals↗
PubMed2000

Embryonic and postnatal accumulation of homogeneous substance in the endolymphatic sac in the guinea pig.

OBJECTIVE: The endolymphatic sac (ES) of vertebrates contains varying amounts of a homogeneous substance (HS) that stains deeply with basic aniline dyes. Histochemically, HS is characterized as a carbohydrate-protein complex, being both neutral and acidic in nature. In the present study, deposition of HS in the ES was studied in the guinea pig from the 3rd week of gestation to 104 weeks postnatally in order to find out if HS accumulates with age, at which point during embryonic development this substance appears, if its presence is correlated to the sense of hearing and if the amount of substance in the left versus right ear of one and the same animal is correlated to any degree. METHODS: Sixty-nine endolymphatic sacs were evaluated in 38 guinea pigs. The ES specimens were sectioned for light and transmission electron microscopy and the amount of HS filling was categorized in four groups: none, low, medium and a high level of substance. RESULTS: The substance was not discerned until after 7 weeks of gestation, when it filled only a minor part of the distal ES lumen. At 9 weeks gestation the nature of the substance altered, becoming homogeneous, as visualized by osmium-toluidine blue staining and approximately filling the distal half of the luminal space. In the postnatal period, 65% of ES specimens were filled with HS to the intermediate or proximal ES, whereas only 6.5% of the ES specimens were devoid of the substance. The extent of filling of the ES in the prenatal temporal bones was significantly less than postnatally (P < 0.0001, chi2-test). The extent of postnatal filling was not correlated with age. Left and right ears were closely correlated in one and the same animal. Phagocytic cells were often found at the border between clear endolymph and stainable substance. CONCLUSION: The appearance of HS seemed to coincide temporally with the onset of hearing during the prenatal period indicating that it could play a part in normal inner ear functioning in the guinea pig. The close correlation regarding the level of the HS in the left and right ear, both pre- and postnatally could reflect a general symmetry in endolymph pressure-volume conditions within the inner ear fluid systems, as well as in the environmental hydrostatic pressure in the posterior cranial fossa.

Animals↗
PubMed2003

Capillary electrophoresis: a tool for studying interactions of glycans/proteoglycans with growth factors.

Heparan sulfate (HS) and heparin bind to various growth factors and modulate their activities. Interactions of heparin and HS with members of the fibroblast growth factor (FGF) family are prerequisites for binding of FGFs to their high affinity cell receptors. The sulfation patterns of distinct oligosaccharide domains within heparin and HS chains determine their high affinity binding with basic FGF (bFGF). In order to study the structural basis of interactions of HS with bFGF, we developed a capillary electrophoresis (CE) method in order to monitor the ability of HS-derived oligosaccharides to bind this growth factor. HS was degraded to Delta-di- and Delta-oligosaccharides with digestion with heparitinase and the obtained Delta-saccharides were analyzed by capillary zone electrophoresis (CZE), using 50 mM phosphate, pH 3.5, as operating buffer, reversed polarity (30 kV) and detection at 232 nm. Under these conditions all differently sulfated HS Delta-disaccharides and the various Delta-oligosaccharide groups were resolved. Following incubation of the digest with bFGF and re-electrophoresis of the mixture, the bFGF interacting oligosaccharide groups were easily detected and identified. In view of the obtained results, CE is a multipotent analytical tool for determining disaccharide composition in HS, separating the various oligosaccharide groups produced by the action of heparitinase and identifying those interacting with bFGF.

Animals↗
PubMed2003

C-reactive protein, clinical presentation, and ischemic activity in patients with chest pain and normal coronary angiograms.

OBJECTIVES: We sought to investigate the relationship among C-reactive protein (hs-CRP), clinical characteristics, exercise stress test responses, and ST-segment changes during daily life in patients with typical chest pain and normal coronary angiograms (CPNCA). BACKGROUND: Patients with CPNCA have coronary microvascular endothelial dysfunction and myocardial ischemia. Elevated hs-CRP levels have been related to atherogenesis and endothelial dysfunction. The relationship between hs-CRP and disease activity has not been previously investigated in CPNCA patients. METHODS: We studied 137 consecutive CPNCA patients (mean age, 57 +/- 9; 33 men). All completed standardized angina questionnaires, underwent exercise stress testing, 24-h ambulatory electrocardiogram (ECG) monitoring (Holter), and hs-CRP measurements at study entry. RESULTS: C-reactive protein levels (mg/l) were higher in patients with frequent (2.9 +/- 3.3) and prolonged (3.9 +/- 4.1) chest pain episodes, and in those with ST-segment depression on exercise testing (2.6 +/- 2.8) and Holter monitoring (3.4 +/- 3.1) compared with patients with occasional (1.3 +/- 1.2; p = 0.002) or shorter chest pain (1.5 +/- 1.3; p < 0.001) episodes, negative exercise stress testing (1.1 +/- 1.1; p < 0.001), and no ST-segment shifts on Holter monitoring (0.9 +/- 0.7; p < 0.001). Moreover, we found a correlation between hs-CRP concentration and number of ischemic episodes during Holter monitoring (r = 0.65; p < 0.001) and with the magnitude of ST-segment depression on exercise testing (r = -0.43; p < 0.001). The hs-CRP was the only independent variable (multivariate logistic regression) capable of predicting positive findings on Holter monitoring (odds ratio [OR], 3.8; confidence interval [CI], 2.3 to 6.2) and exercise testing (OR, 1.7; CI, 1.2 to 2.2). CONCLUSIONS: The hs-CRP correlates with symptoms and ECG markers of myocardial ischemia in CPNCA patients. Whether hs-CRP is related to the pathogenesis of angina in these patients deserves further investigation.

Adult↗
PubMed1996

C-type natriuretic peptide-mediated coronary vasodilation: role of the coronary nitric oxide and particulate guanylate cyclase systems.

OBJECTIVES: We tested the hypothesis that C-type natriuretic peptide (CNP) mediates coronary vasodilation through activation of cyclic guanosine monophosphate (cGMP) by way of particulate guanylate cyclase. BACKGROUND: CNP has known peripheral vasodilator properties, and preliminary data have suggested that it can function as a coronary vasodilator. METHODS: The actions of CNP were studied in instrumented dogs and in organ chamber rings in the presence and absence of a known antagonist to particulate guanylate cyclase, HS-142-1. Additionally, the actions of HS-142-1 were tested on acetylcholine-mediated coronary vasodilation, and immunohistochemical staining was utilized to localize the presence of CNP in the coronary endothelium. RESULTS: CNP relaxed isolated coronary arteries with (mean +/- SEM 45.9 +/- 7%*) and without (72.0 +/- 7%*) an endothelium (*p < 0.05 for CNP effect alone, p < 0.05 for endothelium vs. no endothelium with CNP). Intracoronary infusions increased coronary blood flow (baseline, 64.6 +/- 5.1 ml/min; CNP-5, 79.9 +/- 6.1*; CNP-20, 103.3 +/- 13.6* [*p < 0.05 vs. baseline value]) and reduced coronary vascular resistance (baseline, 1.6 +/- 0.3 mm Hg/ml per min; CNP-5, 1.4 +/- 0.3*; CNP-20, 1.2 +/- 0.3*). Intracoronary injections increased coronary blood flow (delta baseline coronary flow, 30 +/- 9* ml/min [*p < 0.05]). HS-142-1 significantly attenuated these increases (delta coronary flow, 30 +/- 9* ml/min [CNP] to 14 +/- 6 [CNP + HS-142-1] [p < 0.05 CNP vs. CNP + HS-142-1]) and the relaxation of organ chamber rings (56 +/- 7% [CNP] to 18 +/- 6% [HS-142-1 + CNP]). Finally, CNP was localized to the coronary endothelium and smooth muscle by immunohistochemical staining. CONCLUSIONS: CNP functions as a coronary vasodilator through activation of cGMP by way of particulate guanylate cyclase. CNP-mediated coronary vasodilation is attenuated by intracoronary HS-142-1. Intracoronary HS-142-1 does not affect acetylcholine-mediated coronary vasodilation. These observations support a role for exogenous CNP as a potent coronary vasodilator.

Animals↗
PubMed2002

Dopaminergic modulation of aldosterone secretions on changes of sodium intake in aldosterone-producing adenoma.

BACKGROUND: Impairment of renal dopamine excretion on high-salt diet (HS) may account for increased blood pressure (BP) in hypertensive subjects. Whether such impairment of dopaminergic activity exists in the adrenal gland is unknown. The purpose of this study was to differentiate degrees of dopaminergic inhibition of aldosterone secretion in patients with aldosterone-producing adenoma (APA). METHODS: A total of 15 patients with unilateral APA were fed a low-salt diet (LS) for 1 week, followed by another week on HS. At the end of each diet period, 24-h ambulatory BP recording, daily urine catecholamine measurement, and a metoclopramide test were performed. RESULTS: A high-salt diet increased both daytime and nighttime BP (P < .001), and urine dopamine excretion (P < .01). Intravenous injection of 10-mg metoclopramide increased plasma aldosterone concentrations (PAC) on both diets. The areas-under-the-curve for PAC between LS and HS were not different, but the area-under-the-curve for PAC increment was greater on HS (P < .05). Six patients with increment areas on HS greater than those on LS by 50% were termed "suppressible," and the remainders as "nonsuppressible." On HS, the so-called suppressible subjects had greater urine dopamine and less urine norepinephrine excretions (P < .05). The nonsuppressible subjects had greater percentage increase of nighttime BP by HS than the suppressible (for systolic BP, 13.1% v 4.5%, P < .01; for mean BP, 12.0% v 5.1%, P < .01, respectively), but no difference in daytime BP. CONCLUSIONS: Two subtypes of APA were defined according to their responses to metoclopramide during salt manipulation. On HS, the nonsuppressible subjects, with less dopaminergic inhibition of aldosterone secretion, had less urinary DA excretion and greater BP elevation. The renal and adrenal dopaminergic activities are regulated in a parallel fashion.

Adrenal Cortex Neoplasms↗
PubMed1997

Tissue-specific regulation of the sodium pump in DOCA-salt hypertension.

Alterations in sodium pump activity have been associated with volume-sensitive hypertension, but little is known regarding the molecular regulation of the catalytically active alpha-subunit of the sodium pump in these models. We examined changes in the mRNA abundance of the alpha-isoforms in tissues that might participate in sodium and volume regulation in the deoxycorticosterone acetate (DOCA)-high salt rat model. These tissues included kidney, heart, aorta, pituitary, and hypothalamus. This study assessed alterations arising from changes in dietary salt intake alone, from DOCA administration alone, and those requiring both DOCA and high salt with their attendant volume expansion and hypertension. Increased sodium intake produced no significant change in any isoform in the five tissues studied. DOCA administered with a low salt diet produced no significant change in any of the alpha-isoforms in any of the tissues studied. The combination of DOCA and high salt (HS), on the other hand, brought about a twofold increase in renal alpha1-mRNA abundance compared with control (alpha1, CTL: 101.1 +/- 9.3, DOCA-HS: 197.3 +/- 22.9, P < .0001). DOCA-HS also induced a marked increase in both alpha1- and alpha2-mRNA in the aorta (alpha1, CTL: 122.5 +/- 33.3 v DOCA-HS: 487.2 +/- 59.9, P = .001; alpha2, CTL: 126.6 +/- 40.0 v DOCA-HS: 559.8 +/- 271.7, P = .01). In contrast DOCA-HS animals showed a significant reduction in the alpha2-, but not alpha1-, mRNA abundance in heart (alpha2, CTL: 118.0 +/- 11.7 v DOCA-HS: 61.1 +/- 9.1, P = .006). No change was observed in pituitary or hypothalamus with DOCA-HS. Of factors known to modulate the mRNA abundance of the sodium pump, only the putative endogenous sodium pump inhibitor might account for the changes in the aorta and kidney. Reductions in aldosterone or hypertension might reduce alpha2 in the heart. Only the renal response would favor sodium reabsorption, which could contribute to the hypertensive process.

Animals↗
PubMed1990

Cross-reactivity of monoclonal anti-DNA antibodies with heparan sulfate is mediated via bound DNA/histone complexes.

To study in more detail the cross-reactive binding of anti-DNA antibodies to heparan sulfate (HS) and heparan sulfate proteoglycan (HSPG) purified from glomerular basement membranes (GBM), the binding pattern of 31 murine IgG anti-DNA MoAbs, derived from MRL/lpr, NZB/W and graft-versus-host diseased mice, was analysed. In ELISA we found binding of 10 anti-DNA MoAbs to HS. Seven of the 10 anti-HS positive clones bound to HSPG but not to the HSPG core protein in ELISA and/or on Western blots. However, DNase-I treatment partly reduced this binding, whereas after purification of MoAb by protein-A sepharose chromatography under dissociative conditions, all clones completely lost their binding capacity to HS and HSPG. Culturing of hybridoma cells in the presence of 3H-thymidine revealed DNA bound to the MoAb. Although the binding to HS and HSPG could be reconstituted by the addition of the protein-A column effluent, this was not possible by the addition of DNA alone. Therefore, we performed immunoprecipitation of the effluent with purified MoAb and subsequent SDS-PAGE which showed that the complex also contained histones. However, histones alone were also not able to reconstitute the binding to HS and HSPG. It is concluded that binding of anti-DNA MoAb to HS and GMB-HSPG is mediated via bound complexes containing both DNA and histones. A comparable reaction with polyclonal anti-DNA Ab might play a role in the pathogenesis of SLE nephritis, since histones have a very high affinity for HS, the major glycosaminoglycan of the GBM.

Animals↗
PubMed1999

Fibroblast growth factor receptor signalling is dictated by specific heparan sulphate saccharides.

Signalling by fibroblast growth factors (FGFs) through FGF receptors (FGFRs) depends on the cell-surface polysaccharide heparan sulphate (HS) [1] [2]. HS has an ordered domain structure of highly diverse saccharide motifs that present unique displays of sulphate, carboxyl and hydroxyl groups [3]. These motifs interact with many proteins, particularly growth factors. HS binds both to FGFs [4] [5] [6] and FGFRs [7], and probably activates signalling by facilitating ligand-induced receptor dimerisation [8] [9]. Nevertheless, the extent to which specific HS saccharide sequences play a regulatory role has not been established. By screening a library of structurally diverse HS decasaccharides in bioassays of FGF signalling mediated by three different FGFR isoforms, we found that saccharides showed specificity for both ligands and receptors; some saccharides selectively activated FGF signalling through different FGFR isoforms, others acted as negative regulators. We conclude that HS saccharides play critical roles in dictating the specificity of ligand-receptor interactions in FGFR signalling. Controlled alterations in HS structures [10] would provide a mechanism for regulation of cellular responsiveness to growth factors that bind HS.

Animals↗
PubMed1999

Up-regulation by human recombinant transforming growth factor beta-1 of collagen production in cultured dermal fibroblasts is mediated by the inhibition of nitric oxide signaling.

BACKGROUND: Hypertrophic scarring remains the most disabling sequela for burn survivors. Little is known about its pathogenesis. Collagen accumulation, however, has been consistently observed in burn hypertrophic scars (HS). STUDY DESIGN: We have studied collagen production in the dermal fibroblasts derived from HS, which has developed for 9 months to 2 years. Reconstructive surgery was performed to remove HS from which the fibroblasts were cultured. Similarly, the normal cells were grown from the patient's donor site (DS), which provided autografting to the HS site. Collagen production in HS and DS fibroblasts was compared and analyzed in minimal essential amino acid medium containing 5% fetal bovine serum with inclusion of L-ascorbic acid (100 microg/mL) and beta-aminopropoinitrile (100 microg/mL) by monitoring a 20-h [3H]proline incorporation into bacterial collagenase III-digestible protein in the conditioned media. RESULTS: We failed to detect any significant difference in collagen production in vitro between HS and DS. Irrespective of the fibroblasts from HS or DS, collagen production was substantially stimulated by human recombinant transforming growth factor beta-1 (TGF-beta1) (20 ng/mL) by approximately 250% after a 3-day pretreatment. In contrast, sodium nitroprusside (SNP) at 100 microM exhibited significant suppression (68%), which was rescued by hemoglobin (10 microM). TGF-beta1 significantly decreased nitric oxide (NO) production by 55%. In contrast, NO level drastically increased by 350% following SNP treatment. Epidermal growth factor showed no effect on either collagen production or NO level. The linear regression analysis shows a significant inverse correlation (r = 0.72; p < 0.05) of NO level with collagen production, suggesting the involvement of NO signaling in the modulation of collagen production. Consistent with the notion, we further showed that N-nitro-L-arginine methyl ester (100 microM) caused a synergistic stimulation and an arrested inhibition of collagen production in the presence of TGFbeta-1 and SNP, respectively. 8-BrcGMP (300 microM) mimicked the NO inhibitory action, while methylene blue (50 microM) restored the collagen production which was inhibited by SNP. Moreover, 8-BrcGMP offset the stimulation of collagen production. CONCLUSIONS: The dermal fibroblasts derived from HS were not different from normals with respect to collagen production and their responses to regulations. The inhibition of collagen production was achieved by a cGMP-dependent NO action. TGFbeta-1 inhibited NO/cGMP signaling to ensure its stimulatory effect on collagen production in the dermal fibroblasts.

Burns↗
PubMed1999

Suppressed phenylalanine ammonia-lyase activity after heat shock in transgenic Nicotiana plumbaginifolia containing an Arabidopsis HSP18.2-parsley PAL2 chimera gene.

The activity of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) after heat shock (HS) in leaves and buds of transgenic Nicotiana plumbaginifolia containing an Arabidopsis HSP18.2 promoter-parsley phenylalanine ammonia-lyase 2 (HSP18.2-PAL2) chimera gene was examined. Immediately after HS treatment at 44 degrees C for 5 h, the PAL activity in both transgenic and normal (untransformed) plants was 35-38% lower than that before HS. At normal temperature (25-26 degrees C), the PAL activity recovered within 5 h of ending the HS treatment in normal plants, but not until 12-24 h in transgenic plants containing the HSP18.2-PAL2 gene. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed the presence of parsley PAL2 mRNA in transgenic plants, which remained for 8-12 h following 5-h HS at 44 degrees C; the mRNA was not observed before HS. The content of chlorogenic acid (CGA; 3-caffeoylquinic acid) decreased drastically 8-12 h after HS in transgenic plants, but only slightly in normal plants. Thus, the decrease in PAL activity accompanied by expression of the parsley PAL2 gene after HS treatment corresponded to the decrease in CGA synthesis. These results might be attributed to post-transcriptional degradation of endogenous PAL mRNA triggered by transcription of the transgene.

Journal Article↗
PubMed2006

Surface coverage and structure of mixed DNA/alkylthiol monolayers on gold: characterization by XPS, NEXAFS, and fluorescence intensity measurements.

Self-assembly of thiol-terminated single-stranded DNA (HS-ssDNA) on gold has served as an important model system for DNA immobilization at surfaces. Here, we report a detailed study of the surface composition and structure of mixed self-assembled DNA monolayers containing a short alkylthiol surface diluent [11-mercapto-1-undecanol (MCU)] on gold supports. These mixed DNA monolayers were studied with X-ray photoelectron spectroscopy (XPS), near-edge X-ray absorption fine structure spectroscopy (NEXAFS), and fluorescence intensity measurements. XPS results on sequentially adsorbed DNA/MCU monolayers on gold indicated that adsorbed MCU molecules first incorporate into the HS-ssDNA monolayer and, upon longer MCU exposures, displace adsorbed HS-ssDNA molecules from the surface. Thus, HS-ssDNA surface coverage steadily decreased with MCU exposure time. Polarization-dependent NEXAFS and fluorescence results both show changes in signals consistent with changes in DNA orientation after only 30 min of MCU exposure. NEXAFS polarization dependence (followed by monitoring the N 1s --> pi* transition) of the mixed DNA monolayers indicated that the DNA nucleotide base ring structures are oriented more parallel to the gold surface compared to DNA bases in pure HS-ssDNA monolayers. This indicates that HS-ssDNA oligomers reorient toward a more-upright position upon MCU incorporation. Fluorescence intensity results using end-labeled DNA probes on gold show little observable fluorescence on pure HS-ssDNA monolayers, likely due to substrate quenching effects between the fluorophore and the gold. MCU diluent incorporation into HS-ssDNA monolayers initially increases DNA fluorescence signal by densifying the chemisorbed monolayer, prompting an upright orientation of the DNA, and moving the terminal fluorophore away from the substrate. Immobilized DNA probe density and DNA target hybridization in these mixed DNA monolayers, as well as effects of MCU diluent on DNA hybridization in complex milieu (i.e., serum) were characterized by surface plasmon resonance (SPR) and (32)P-radiometric assays and reported in a related study (Gong, P.; Lee, C.-Y.; Gamble, L. J.; Castner, D. G.; Grainger, D. W. Anal. Chem. 2006, 78, 3326-3334.).

Alkanes↗
PubMed1995

Structure-function assessment of the role of the helical stack domain in the properties of human recombinant protein C and activated protein C.

The role of the helical stack (HS) in defining the properties of human recombinant (r) protein C (PC) and activated protein C (APC) was assessed. To do so, several mutations were made in this region of the molecule and their effects on the proteins examined. Substitution of the entire HS of PC (residues 38-46) by that of human coagulation factor (f) IX (residues 39-47), yielding r-[HSIX]PC, did not result in any substantial changes in the gamma-carboxyglutamic acid domain (GD)-related Ca(2+)-dependent properties of PC or APC, suggesting that the conformation of the HS may play a more dominant role in these Ca(2+)-dependent properties than do the specific amino acids that differ between these two HS regions. On the other hand, the catalytic efficiency of activation of r-[HSIX]PC by the thrombin/thrombomodulin complex was reduced to approximately one-third of that of wtr-PC, a result that demonstrates a specific role for the HS of PC in this activation process. Another mutation, [Ser42-->Pro], was generated in the HS region of r-PC, providing r-[S42P]PC, a change that according to the empirical algorithm based on the Chou-Fasman secondary structure rules, would disrupt the alpha-helical conformation of the HS. The anticoagulant activity of the corresponding r-[S42P]APC was found to be approximately 35% of that of wtr-APC. Because of the lack of any notable effects of this mutation on other GD-related Ca(2+)-dependent properties of r-PC and r-APC, the basis of this anticoagulant activity loss may be due to its nonmaximal alignment with substrate on the PL surface. The results of this study indicate that the role of the HS of r-PC and r-APC is to provide a region of the protein that is needed to assure optimal alignment on the PL or cell surface of the active site of the enzyme with that of the cleavage sites of the substrates, perhaps by functioning as a scaffold for separation of the active site of APC from the PL surface.

Amino Acid Sequence↗
PubMed2003

Molecular weight characteristics of humic substances from different environments as determined by size exclusion chromatography and their statistical evaluation.

Recorded molecular weights (MWs) for humic substances (HS) range from a few hundred to millions of daltons. For purposes of defining HS as a specific class of chemical compounds, it is of particular importance to ascertain if this broad range of MWs can be attributed to actual variability in molecular properties or is simply an artifact of the analytical techniques used to characterize HS. The main objectives of this study were (1)to establish if a preferential range of MWs exists for HS and (2) to determine any consistent MW properties of HS. To reach the goal, we have undertaken an approach to measure under standardized conditions the MW characteristics of a large set of HS from different natural environments. Seventy-seven humic materials were isolated from freshwater, soil, peat, and coal, such that each possessed a different fractional composition: humic acid (HA), fulvic acid (FA), and a nonfractionated mixture of HA and FA (HF). Size exclusion chromatography (SEC) was used as the analytical technique to determine molecular weight characteristics. The MW distributions were characterized by number (Mn) and weight (Mw) average MW, and by polydispersity. The complete range of Mw values varied within 4.7-30.4 kDa. The maximum Mw values were observed for peat HF and soil HA, whereas the smallest weights were measured for river water HF. Maximum values of polydispersity (3.5-4.4) were seen for peat HF and soil HA, while much lower values (1.6-3.1) were found for all preparations isolated with XAD-resins. Statistical evaluation showed consistent Mw and Mn variations with the HS source, while polydispersity was mostly a function of the isolation procedure used. A conclusion was made that HS have a preferential range of MW values that could characterize them as a specific class of chemical compounds.

Chromatography, Gel↗
PubMed2005

Humic substances are soft and permeable: evidence from their electrophoretic mobilities.

Due to the complexity of the humic substances (HS), mathematical models have often been employed to understand their roles in the environment. Since no consensus exists with respect to the structure and conformation of the HS, models have alternatively given them properties corresponding to impermeable hard spheres or fully permeable polyelectrolytes. In this study, the hydrodynamic permeability of standard HS (Suwannee River fulvic, humic, and peat humic acids) are evaluated as a function of pH and ionic strength. A detailed theoretical model is used to determine the softness parameter (lambda0), which characterizes the degree of flow penetration into the HS on the basis of measured values of electrophoretic mobilities, diffusion coefficients, and electric charge densities. Their motion in an electric field is evaluated by a rigorous numerical evaluation of the governing electrokinetic equations for soft particles. The hydrodynamic impact of the polyelectrolyte chains is accounted for by a distribution of Stokes resistance centers and partial dissociation of the hydrodynamically immobile ionogenic groups distributed throughout the polyelectrolyte. The results demonstrate thatthe studied HS are small (radius ca. 1 nm), highly charged (500-650 C g(-1) when all sites are dissociated), and very permeable (typical flow penetration length of 25-50% of the radius, depending on pH). The HS also coagulate slightly when lowering the pH of the solution. Modeling of the HS as hard spheres with a charge and slip plane located at the surface is thus physically inappropriate, as are a number of analytical theories for soft particles that hold for low to moderate electrostatic potentials and large colloids. The shortcomings of these simpler approaches, when interpreting the electrophoretic mobilities of HS, are highlighted by comparison with rigorous theoretical predictions.

Colloids↗
PubMed2004

Long-range order of organized oligonucleotide monolayers on Au(111) electrodes.

Oligonucleotides modified by a hexamethylene linker group adsorb on gold electrodes via Au-S bond formation. We have obtained novel data for adsorption of thiol-modified (HS) single-strand HS-10A and double-stranded HS-10AT oligonucleotides and for analogous thiol-free 10A (A = adenine) and 10T (T = thymine) nonspecifically adsorbed as reference molecules. Mercaptohexanol has served as a second reference molecule. The data are based on cyclic and differential pulse voltammetry, interfacial capacitance data, and in situ scanning tunneling microscopy (STM) directly in an aqueous buffer solution, with electrochemical potential control of both the sample electrode and the tip. All the data are based on single-crystal, atomically planar Au(111)-electrode surfaces. The high sensitivity of such surfaces provides accurate HS-10A and HS-10AT electrode coverages on the basis of the reductive desorption of the Au-S bond. The coverage is high and in keeping with dense monolayers of adsorbed HS-10A and HS-10AT in an upright or tilted orientation, with the oligonucleotide backbone repelled from the strongly negatively charged electrode surface. Adsorbed thiol-free 10A only gives a Au(111)-reconstruction peak, while 10T shows a subtle pattern involving pronounced voltammetric adsorption peaks indicative of both nonspecific adsorption via single thymine units and potential-dependent structural reorganization in the surface layer. In situ STM supports these findings at the molecular level. In situ STM of HS-10A discloses large, highly ordered domains at strongly negative sample potentials. Reversible domain formation and disordering could, moreover, be controlled by an electrochemical potential variation in the negative and positive directions, respectively. 10A and 10T did not form ordered adsorbate domains, substantiating that domain formation rests on adsorption of thiol-modified oligonucleotide adsorption in an upright or tilted orientation. The comprehensive, high-resolution information reported may hold prospects for single-molecule electronic conduction and molecular-scale mapping of oligonucleotide hybridization.

Biosensing Techniques↗
PubMedDate not supplied

Heparan sulfate 2-O-sulfotransferase (Hs2st) and mouse development.

Heparan sulphate 2-O-sulphotransferase (Hs2st) acts at an intermediate stage in the pathway of biosynthesis of heparan sulphate (HS), catalysing the transfer of sulphate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to the C2-position of selected hexuronic acid residues within the maturing HS chain. It is well established that 2-O-sulphation within HS, particularly of iduronate residues, is essential for HS to participate in a variety of high-affinity ligand-binding interactions. HS plays a central role in embryonic development and cellular function, modulating the activities of an extensive range of growth factors. Interestingly, in contrast to the early failure of embryos entirely lacking HS, Hs2st(-/-) mice survive until birth, but die perinatally due to a complete failure of kidney formation. The phenotype of Hs2st(-/-) mutant kidneys suggests that signalling between two tissues, ureteric bud and metanephric mesenchyme, is disrupted. We discuss candidate signalling molecules that may mediate this interaction. The HS generated by these mice lacks 2-O-sulphate groups but is extensively modified above wild type levels by O-sulphation at C-6 of glucosamine-N-sulfate (GlcNS) residues. We will discuss the potentially altered role of this atypical HS in growth factor signalling.

Animals↗
PubMed1996

Ankyrin-1 mutations are a major cause of dominant and recessive hereditary spherocytosis.

Hereditary spherocytosis (HS) is the most common inherited haemolytic anaemia in Northern Europeans. The primary molecular defects reside in the red blood cell (RBC) membrane, particularly in proteins that link the membrane skeleton to the overlying lipid bilayer and its integral membrane constituents. Ankyrin-1 is the predominant linker molecule. It attaches spectrin, the major skeletal protein, to the cytoplasmic domain of band 3, the RBC anion exchanger. Two-thirds of patients with HS have combined spectrin and ankyrin-1 deficiency; deficiency of band 3 occurs in about 15 to 20% (ref.1). These data suggest that ankyrin-1 or band 3 defects may be common in HS. To test this we screened all 42 coding exons plus the 5' untranslated/promoter region of ankyrin-1 and the 19 coding exons of band 3 in 46 HS families. Twelve ankyrin-1 mutations and five band 3 mutations were identified. Missense mutations and a mutation in the putative ankyrin-1 promoter were common in recessive HS. In contrast, ankyrin-1 and band 3 frameshift and nonsense null mutations prevailed in dominant HS. Increased accumulation of the normal protein product partially compensated for the ankyrin-1 or band 3 defects in some of these null mutations. Our findings indicate that ankyrin-1 mutations are a major cause of dominant and recessive HS (approximately 35 to 65%), that band 3 mutations are less common (approximately 15 to 25%), and that the severity of HS is modified by factors other than the primary gene defect.

Ankyrins↗