Characterization of sheep hemopexin glycovariants

The hemopexin phenotype HpxB1 isolated from sheep serum, yields three major bands when subjected to starch gel and/or polyacrylamide gel electrophoresis which are here designated as subcomponents HpxB1-I, HpxB1-II and HpxB1-III. Electrospray mass spectrometric analysis of samples of the isolated subcomponents prepared by ion exchange chromatography showed that each was composed of three glycoproteins and that the major difference between the subcomponents was due to their constituent glycoproteins possessing different numbers of sialic acid residues. Combined analysis of the ESI-MS data and of the overall carbohydrate compositional data obtained by colorimetric procedures, leads to the composition of the glycan of each glycoprotein, and a combined methylation and 400 MHz H-NMR analysis of the alkaline cleaved glycans identified them as being of only the biantennaryN-acetyllactosamine type. Taking into account the molecular mass, the carbohydrate content and structure it may be concluded that each of the constituent glycoproteins contain fiveN-glycosidically linked glycans.Abbreviations HpxB1 hemopexin phenotype B1 - Man mannose - Gal galactose - GlcNAc N-acetylglucosamine - NeuAc N-acetylneuraminic acid - GlcNAc-ol N-acetylglucosaminitol     

Structural and immunochemical identification of Leb glycolipids in the plasma of a group O Le(a-b-) secretor

Total non-acid glycosphingolipids were isolated from the plasma of a healthy red blood cell group O Le(a-b-) salivary ABH secretor individual. Glycolipids were fractionated by HPLC and combined into eight fractions based on chromatographic and immunoreactive properties. These glycolipid fractions were analysed by thin-layer chromatography and tested for Lewis activity with antibodies reactive to the type 1 precursor (Le^c), H type 1 (Le^d), Le^a and Le^b epitopes. Fractions were structurally characterized by mass spectrometry (EI-MS and LSIMS) and proton NMR spectroscopy. Expected blood group glycolipids, such as H type 1, (Fuc1-2Gal1-3GlcNAc1-3Gal1-4Glc1-1Cer) were immunochemically and structurally identified. Inconsistent with the red cell phenotype and for the first time, small quantities of Le^b blood group glycolipids (Fuc1-2Gal1-3(Fuc1-4)GlcNAc1-3Gal1-4Glc1-1Cer) were immunochemically and structurally identified in the plasma of a Lewis-negative individual. These findings confirm recent immunological evidence suggesting the production of small amounts of Lewis antigens by Lewis negative individuals. Abbreviations: HPLC, high performance liquid chromatography; TLC, (high performance) thin layer chromatography; EI-MS, electron impact ionisation mass spectrometry; LSIMS, liquid secondary ion mass spectrometry; NMR, nuclear magnetic resonance spectroscopy. The sugar types are abbreviated to Hex for hexose, HexNAc forN-acetylhexosamine and dHex for deoxyhexose (fucose). The ceramide types are abbreviated to d for dihydroxy and t for trihydroxy base, n for non-hydroxy and h for hydroxy fatty acids; LCB, long chain base.     

Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification

S-2-(3 aminopropylamino) ethylphosphorothioic acid (WR-2721) shown to surpass radical scavenging thiols in their radioprotective efficacy in cancer-type diseases has been tested for its protective potential in the reperfused heart. We investigated the radical scavenger properties of the compound in a radical generating systemin vitro as well as in isolated rat hearts subjected to 30 min ischaemia and 30 min reperfusion with the electron-paramagnetic resonance spin trap technique. The action on high-energy phosphates is observed by means of phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy while its influence on left ventricular systolic segmental length change (SSLC) during 60 min reperfusion following 60 min regional ischaemia was assessed with a fibreoptic system in anaesthetized open-chest rats. WR-2721 (0.1 mM) reduced the vascular concentration of radical adduct in isolated hearts by up to 78% (275±84% of pre-ischaemic baseline values vs 1260±413%, p<0.05) between 5 and 12.5 min reperfusion. This was accompanied by a reduction of the left ventricular end diastolic pressure to pre-ischaemic values at 30 min of reperfusion (9±6 mmHg vs 42±8 mmHg in the absence of WR-2721, p<0.02). An accelerated recovery of creatine phosphate levels (78±5% of pre-ischaemia values vs 41±5% within 60 min reperfusion; p<0.05) was observed under similar conditions with NMR-spectroscopy, although the post-ischaemic tissue content of adenosine triphosphate was not affected. The administration of WR-2721 (0.5 mmol/kg body weight) ledin situ to an accelerated restoration of contractile activity in the post-ligated left ventricular area reflected by the post-ischaemic recovery of SSLC (64±10% of pre-ischaemic values compared with 27±6% in control animals 60 min following reperfusion; p<0.02). The present data confirm an effective reduction in the exposure of the reperfused heart to endogenously released free radicals by WR-2721, a partial preservation of high-energy phosphates and an improvement in post-ischaemic contractility and encourage further investigation of such favourable action in injured myocardium.     

Stereospecific assignment of the NH2 resonances from the primary amides of asparagine and glutamine side chains in isotopically labeled proteins

An HMQC-based pulse scheme is presented for the stereospecific assignment of asparagineand glutamine side-chain amide protons. The approach makes use of the recently developedquantitative-J correlation spectroscopy [Bax, A. et al. (1994) Methods Enzymol., 239,79–105] to distinguish the E and Z primary amide protons and, as such, eliminates theneed for assignments derived from more time-consuming and potentially ambiguous NOEmethods. An application of this method to a uniformly 15N,13C-labeled cellulose-bindingdomain is presented. When used in combination with a NOESY-HSQC experiment, thepredominant 2 dihedral angles of two asparagine side chains in this protein can also bedefined.     

Characterization of electron transfer reactions of microperoxidase assembled at short-chain thiol-monolayers on gold

In this study thiol-monolayers were used in order to modify gold and provide suitable chemical functionalities for the immobilization of the small redox-active haem-containing peptide, microperoxidase (MP-11). Initially, we assembled a variety of thiol-containing species on the gold electrodes and measured a series of electron transfer reactions, each characteristic of the surface-modifier. Using suitable immobilization strategies, we subsequently covalently bound MP-11 to appropriate monolayers and found two characteristic electrochemical responses (i.e. using MP-11 bound to mercaptopropionic acid, redox peaks were seen at E^0′ = −315 mV and at +179 mV versus Ag|AgCl, with the former being attributed to the haem and the latter with the thiol monolayer). Exposure of the peptide-thiol surface to UV irradiation resulted in cleavage of the Au---S bond, leading to a decrease in the magnitude of both responses. Our work is supported by corroborative evidence showing the immobilization of the peptide, obtained using both X-ray photoelectron and reflectance Fourier transform infra-red spectroscopies. We hypothesize that differences in the ionic charges on the protein backbone account for the shift in E^0′ for MP-11, as observed in our system, when compared to that found for MP-11 immobilized by different strategies.     

Intrachain disulfide bond in the core hinge region of human IgG4.

IgG is a tetrameric protein composed of two copies each of the light and heavy chains. The four-chain structure is maintained by strong noncovalent interactions between the amino-terminal half of pairs of heavy-light chains and between the carboxyl-terminal regions of the two heavy chains. In addition, interchain disulfide bonds link each heavy-light chain and also link the paired heavy chains. An engineered human IgG4 specific for human tumor necrosis factor-alpha (CDP571) is similar to human myeloma IgG4 in that it is secreted as both disulfide bonded tetramers (approximately 75% of the total amount of IgG) and as tetramers composed of nondisulfide bonded half-IgG4 (heavy chain disulfide bonded to light chain) molecules. However, when CDP571 was genetically engineered with a proline at residue 229 of the core hinge region rather than serine, CDP571 (S229P), or with an IgG1 rather than IgG4 hinge region, CDP571(gamma 1), only trace amounts of nondisulfide bonded half-IgG tetramers were observed. Trypsin digest reversephase HPLC peptide mapping studies of CDP571 and CDP571(gamma 1) with on-line electrospray ionization mass spectroscopy supplemented with Edman sequencing identified the chemical factor preventing inter-heavy chain disulfide bond formation between half-IgG molecules: the two cysteines in the IgG4 and IgG1 core hinge region (CPSCP and CPPCP, respectively) are capable of forming an intrachain disulfide bond. Conformational modeling studies on cyclic disulfide bonded CPSCP and CPPCP peptides yielded energy ranges for the low-energy conformations of 31-33 kcal/mol and 40-42 kcal/mol, respectively. In addition, higher torsion and angle bending energies were observed for the CPPCP peptide due to backbone constraints caused by the extra proline. These modeling results suggest a reason why a larger fraction of intrachain bonds are observed in IgG4 rather than IgG1 molecules: the serine in the core hinge region of IgG4 allows more hinge region flexibility than the proline of IgG1 and thus may permit formation of a stable intrachain disulfide bond more readily.     

Reduction of vanadate to vanadyl by a strain of Saccharomyces cerevisiae

Three strains of Saccharomyces cerevisiae, SC-1, DBVPG 6173 and DBVPG 6037, were studied for vanadate resistance in complex Sabouraud medium since they did not thrive in different minimal media (yeast nitrogen base with and without amino acids). The strain SC-1 was resistant up to 16 mm of vanadate, whereas the strains DBVPG 6173 and DBVPG 6037 were inhibited by 8 mm and 4 mm vanadate, respectively. The vanadate resistance in strain SC-1 was constitutive and due to the reduction of this oxyanion to vanadyl, which was detected by EPR spectroscopy and visible spectroscopy. The transformation of vanadate to vanadyl took place during the exponential growth phase; 10 mm of vanadate was reduced to vanadyl outside the cells since the oxyanion was not detected in the cell biomass and only a negligible concentration of vanadyl (25 nmoles mg cells dry weight) was found in the biomass. The other two vanadate-sensitive yeast strains only accumulated vanadate and did not reduce the oxyanion to vanadyl.     

SINGLE-CELL PIGMENTATION OF PORPHYRA LINEARIS ANALYZED BY FOURIER TRANSFORM MULTI-PIXEL SPECTROSCOPY AND IMAGE ANALYSIS1

The novel method of Fourier transform multi-pixel spectroscopy was used for the nondestructive analysis of and comparison of pigmentation in different regions of live thalli of the red alga Porphyra linearis. Because the thallus in this alga consists of a monolayer of nonoverlapping cells, we were able to analyze the pigmentation of single cells by combining light absorbance with natural fluorescence data. From the image of each cell in the vegetative male and female reproductive and holdfast regions, more than 4 ± 10⁴ fluorescence and absorbance spectra were obtained. Specific pigments in the different regions were localized by the use of a software program of similarity mapping followed by image construction. The reconstructed images revealed subcellular localization of each pigment according to specific spectroscopic fingerprints. The results showed that the vegetative and female reproductive cell types had a significantly higher content of phycoerythrin than of phycocyanin, and quite similar chlorophyll a levels. Most of the holdfast cells were poorly pigmented, but had more chlorophyll a than phycoerythrin or phycocyanin. The male reproductive cells contained only traces of pigments. Thus, by using Fourier transform multipixel spectroscopy, we were able to characterize the pigmentation of different regions of the thallus and follow the distribution patterns of the different pigments on the subcellular level along the differentiation gradient of the alga.     

Interaction of Co,Mn, Mg and Al with d(GCGTACGC): a spectroscopic study

Spectroscopic study on the interactions of trace elements Co, Mn, Mg and Al with d(GCGTACGC) indicated the following: Al and Mg did not alter T_m values. Mn enhanced T_m at lower concentration and decreased it at higher concentrations. Interestingly Co at higher concentration elevated the T_m. These studies also showed lower concentrations of Mn displaced EtBr, whereas Al could displace it at higher ionic strength. Mg and Co displaced EtBr fluorescence at moderate concentrations. The binding constant values and CD spectra clearly indicated strong binding of these elements to DNA.     

Control of the rate of phosphocreatine resynthesis after exercise in trained and untrained human quadriceps muscles

We examined the effect of differences in exercise intensity on the time constant (t _c) of phosphocreatine (PCr) resynthesis after exercise and the relationships betweent _c and maximal oxygen uptake (VO_2max) in endurance-trained runners (n = 5) and untrained controls (n = 7) (average VO_2max = 66.2 and 52.0 ml · min^–1 · kg^–1, respectively). To measure the metabolism of the quadriceps muscle using phosphorus nuclear magnetic resonance spectroscopy, we developed a device which allowed knee extension exercise inside a magnet. All the subjects performed four types of exercise: light, moderate, severe and exhausting. The end-exercise PCr: [PCr + inorganic phosphate (P_i)] ratio decreased significantly with the increase in the exercise intensity (P < 0.01). Although there was little difference in the end-exercise pH, adenosine diphosphate concentration ([ADP]) and the lowest intracellular pH during recovery between light and moderate exercise, significant changes were found at the two higher intensities (P < 0.01). These changes for runners were smaller than those for the controls (P < 0.05). The _c remained constant after light and moderate exercise and then lengthened in proportion to the increase in intensity (P < 0.05). The runners had a lowert _c at the same PCr and pH than the controls, particularly at the higher intensity (P < 0.05). There was a significant correlation betweent _c and [ADP] in light exercise and betweent _c and both end-exercise PCr and pH in severe and exhausting exercise (P < 0.05). The threshold of changes in pH andt _c was a PCr: (PCr + P_i) ratio of 0.5. There was a significant negative correlation between the VO_2max andt _c after all levels of exercise (P<0.05).However, in the controls a significant correlation was found in only light and moderate exercise (P < 0.05). These findings suggest the validity of the use oft _c at an end-exercise PCr:(PCr + P_i) ratio of more than 0.5 as a stable index of muscle oxidative capacity and the correlation between local and general aerobic capacity. Moreover, endurance-trained runners are characterized by the faster PCr resynthesis at the same PCr and intracellular pH.