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H Haupt

Publications and source records attributed to H Haupt.

At least 19 recordsLinked to original sources
PubMed1992

The mapping by high-pH anion-exchange chromatography with pulsed amperometric detection and capillary electrophoresis of the carbohydrate moieties of human plasma alpha 1-acid glycoprotein.

The reducing oligosaccharides released from alpha 1-acid glycoprotein (AGP) by conventional hydrazinolysis have been analyzed by two different mapping techniques, using high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) and capillary electrophoresis (CE) with uv detection at 190 nm. The CE measurements proved about 4000 times more sensitive than the measurements by HPAE-PAD. The N-glycan pool was fractionated by Mono Q anion-exchange chromatography, and individual fractions so obtained were desialylated using Vibrio cholerae neuraminidase. The resulting asialo-N-glycans were further analyzed by HPAE-PAD, revealing 2 major, 4 intermediate, and 4 small peaks and at least 3 spikes, which counted for at least 13 different asialo-N-glycans. The carbohydrate structures were tentatively assigned by comparison of the Mono Q-separated N-glycans with the known AGP carbohydrate structures and known structures contained in a mapping database that allows structural assignment of N-glycans by mere comparison of retention times. In addition to the hitherto known AGP carbohydrate structures, we have tentatively identified a number of sulfated N-glycans that are currently being analyzed in more detail. We have also compared the glycan pools recovered from AGP using hydrazinolysis and glycopeptidase F (PNGase F). Approximately 40 distinct peaks could be detected in the hydrazinolysis-derived N-glycan pool by either technique (HPAE-PAD and CE), while about 30 distinct peaks were detected in the N-glycan pool derived by PNGase F digestion of the tryptic AGP digest of the same batch of AGP. These differences were attributed to an increased desialylation (approximately 3 mol%) during hydrazinolysis, based on the detection by HPAE-PAD and CE of free sialic acid and monosialylated oligosaccharides in the glycan pool derived by conventional hydrazinolysis. The integrity of the N-glycans' chitobiose core was examined by 500-MHz 1H NMR spectoscopy. The hydrazinolysis procedure could be optimized such that the hydrazinolysis-derived N-glycan pool was chromatographically essentially identical to the PNGase F-derived N-glycan pool. Hydrazinolysis proved best, with practically no loss of N-acetlylneuraminic acid and the closest resemblance to the PNGase F-derived N-glycan pool, using an automated apparatus. Notably, it was recognized that, in our hands, PNGase F digestion in the presence of sodium dodecyl sulfate resulted in partial desialylation of the liberated N-glycans.

Capillary Action↗
PubMed1992

Amino acid sequence of human plasma galactoglycoprotein: identity with the extracellular region of CD43 (sialophorin).

The amino acid sequence of galactoglycoprotein purified from human plasma was elucidated to 75% completeness by using chemical degradation of peptides and glycopeptides derived from digests of the protein with seven specific proteases. This sequence represents a polypeptide chain of approximately 220 amino acid residues including a high content of serine, threonine, alanine, and proline with one N-linked and multiple O-linked glycans. Comparison of peptide sequences from the native galactoglycoprotein and the deglycosylated derivative demonstrated the locations of 25 sites of O-glycosylation and three serine sites that are not glycosylated. The homogeneous N terminus was established as serine. C-terminal analysis revealed multiple C-terminal residues, suggesting that galactoglycoprotein molecules are of varying lengths. A search of a protein data base revealed that the galactoglycoprotein polypeptide is identical to the N-terminal (extracellular) polypeptide region of the blood-cell surface molecule CD43 (sialophorin, leukosialin). Further support of the relatedness of these molecules was obtained by immunoprecipitation of 125I-labeled galactoglycoprotein by monoclonal anti-CD43 antibodies. The composition and properties of the molecules together with the known structure of the gene encoding CD43 suggest that galactoprotein is derived by proteolytic cleavage from transmembrane "hexasaccharide CD43," known to be expressed on neutrophils, activated T lymphocytes, and platelets.

Amino Acid Sequence↗
PubMed1992

Intensity-dependent changes in oxygenation of cochlear perilymph during acoustic exposure.

This study examined the effects of acoustic exposure at different intensities on local oxygenation of the cochlea. The oxygen partial pressure (pO2) of perilymph in the basal scala tympani was measured polarographically in anesthetized guinea pigs exposed to either wide-band noise at 85 dB SPL or a 10 kHz pure tone at 90, 105, or 125 dB SPL for 1 h. Cochlear temperature, heart rate, arterial blood pressure and acid-base status were monitored. The cochlear microphonics (CM) and compound action potentials (CAP) were recorded before and after exposure. There were clear intensity-dependent differences in the effect of acoustic exposure on perilymphatic oxygenation. Moderate exposure intensities (85-90 dB SPL) were found to increase the pO2 by an average of about 20% of the initial level. In contrast, high intensity acoustic exposure (125 dB SPL) resulted in a mean decrease of about 20%. These changes persisted within a subsequent 30-min post-exposure period. There was no significant change in cochlear temperature and cardiorespiratory variables during and after any of the exposures as compared to the controls. CM and CAP amplitudes showed an extensive loss after acoustic overstimulation (125 dB SPL), but no permanent change with lower exposure intensities. These findings suggest that intracochlear oxygenation plays an important role in inner ear physiology during acoustic stimulation.

Acoustic Stimulation↗
PubMed1991

The effect of the carbohydrate moiety upon the size and conformation of human plasma galactoglycoprotein as judged by electron microscopy and circular dichroism. Structural studies of a glycoprotein after stepwise enzymic carbohydrate removal.

Galactoglycoprotein is a unique human plasma protein [76% carbohydrate (23% N-acetylneuraminic acid, 20% galactose, 3% mannose, 1% fucose and 29% N-acetylgalactosamine plus N-acetylglucosamine) and 24% polypeptide, a single polypeptide chain of about 200 amino acid residues that is high in serine and threonine content] [Schmid, Mao, Kimura, Hayashi & Binette (1980) J. Biol. Chem. 255, 3221-3226]. Highly purified exoglycosidases with well-defined specificities were used to prepare five derivatives of galactoglycoprotein in which sequential residues of N-acetylneuraminic acid, galactose, N-acetylglucosamine, a second galactose and N-acetylgalactosamine were removed with 83% of the total carbohydrate cleaved. C.d. shows that native galactoglycoprotein and all derivatives in aqueous buffer are predominantly random coil, suggesting that removal of a large number of electrostatic net charges, as well as the major portion of the carbohydrate moiety, does not alter the secondary structure of the polypeptide chain. Examination of the size and conformation of tungsten-shadowed galactoglycoprotein and asialo and agalacto derivatives by electron microscopy shows the size and conformation of all three preparations to be similar, with only minor differences in particle length and width.

Antigens, CD↗
PubMed1991

Measurements of perilymphatic oxygen tension in guinea pigs exposed to loud sound.

Using different types of custom-made oxygen-sensitive microelectrodes, the perilymphatic oxygen partial pressure (PO2) was determined in anesthetized guinea pigs. Cochlear temperature, heart rate, and arterial blood pressure and acid-base status were monitored. The PO2 in the basal scala tympani perilymph (200 microns below the round window membrane) was found to be 53 +/- 17 mmHg (mean +/- SD) in 33 normal animals. In 11 guinea pigs exposed to loud sound for 15 min (10 kHz pure tone, 125 dB SPL) there was on average a continuous decline in the perilymphatic PO2, which was significant only 30 min post-exposure. A considerable variation in response was found in individual animals. Mean arterial blood pressures showed a slightly increasing time course, while heart rates did not change significantly during the whole period of the experiment. Arterial acid-base status and PO2 values remained within normal limits and did not change significantly. Cochlear microphonics and compound action potentials were substantially decreased after acoustic overstimulation. The results are discussed with due consideration of sources of error.

Acoustic Stimulation↗
PubMed1990

Cochlear blood flow following temporary occlusion of the cerebellar arteries.

In order to induce acute transient local blood flow impairment in the guinea pig cochlea, terminal vessels arising from the proximal and middle third of the basilar artery and from the rostral part of the collateral vertebral artery near their junction were occluded by temporary (1-2 min) compression. Clear identification of the anterior inferior cerebellar artery was impeded due to great variation in branching of the cerebellar arteries. To quantify the effect of the vascular occlusion on the cochlear microcirculation, laser Doppler flowmetry was used. Successive compression of the cerebellar arteries resulted in individually differentiated effects on the Doppler signal from the cochlea. In about half of the experiments there was a decrease in the laser signal to 25-55% of the initial level, while in the other cases no clear decrease occurred. Proximal obstruction of the basilar artery was found to reduce the cochlear blood flow up to 10-25% of its initial level, indicating that the Doppler signal primarily reflects microcirculation within the membranous cochlea. The findings are discussed with regard to their significance as an animal model for acute transient cochlear hypoxia.

Animals↗
PubMed1990

Laser Doppler measurements of cochlear blood flow during loud sound presentation.

The laser Doppler flowmeter may give responses to loud sound that reflect the vibration of cochlear structures rather than changes in cochlear blood flow. The present study demonstrates that the positive artifactual response (i.e., increased flow reading) to sound at frequencies above approximately 5 kHz can be eliminated by using flowmeters which have electronic filters at 4 and 12 kHz, limiting the bandwidth of the optical Doppler shifted frequency range to lower frequencies. However, when using the 4 kHz filter a "residual" immediate negative response to loud high-frequency sound (10 kHz tone at 125 dB SPL) is evident at the beginning of the exposure. These findings are discussed with regard to the suitability of the method for investigating the effect of sound/noise on cochlear blood flow.

Animals↗
PubMed1990

Laser light transmission and laser Doppler blood flow measurements on the human, rat and guinea pig cochlea.

In order to test the applicability of laser-Doppler flowmetry in monitoring cochlear blood flow clinically, the thickness and the helium-neon laser light transmission of specimens of human, rat and guinea pig promontory bone and human skin were determined. Furthermore, comparative laser-Doppler measurements were taken from the promontory in patients, rats and guinea pigs. Due to the different thicknesses of the promontory bone in different species, the light transmission was found to be considerably higher for the animal cochlea (rat, 15%; guinea pig, 6.6%) than the human cochlea (1.7%). However, a clearly higher laser-Doppler signal was recorded from both the human and the rat cochleas as compared with the guinea pig. The relative laser light attenuation by the human skin specimens corresponded to that of the human promontory bone. The findings are discussed with regard to the suitability of the laser-Doppler method for blood flow measurements in the human cochlea.

Aged↗
PubMed1990

[Chemistry and clinical significance of human plasma proteins].

Enormous progress has been made in the course of the past few years in the various fields of plasma protein research. The primary and disulfide bridge structures are now known for almost all of the 120 proteins thus far isolated from human plasma, including trace and ultratrace proteins as well as a number of genetic variants. Genetic cloning and the derivation of the amino-acid sequence from the nucleotide sequence have played a decisive role here. However, we are only in possession of the exact three-dimensional structure for a small number of plasma proteins. The major problem in this respect is, at present, the lack of suitable protein crystals for X-ray structure analysis. We still do not know the physiological function of a large number of plasma proteins, despite the fact that they, in part, have been well characterised both physically and chemically and could be assigned to their respective protein families on the basis of their amino-acid sequence. The development of techniques for protein structure determination is relatively well advanced today, yet we lack methods of illuminating the structure-function relationship. There are more than 20 different highly purified protein preparations in virus-safe form available today for substitution therapy. To this effect new purification procedures have been developed which pay particular attention to virus elimination and inactivation. Should present indications be confirmed, one may assume that further plasma proteins (e. g. proteinase inhibitors, apolipoproteins, fibronectin) could be of significance in therapy and prophylaxis. Unlimited amounts of human blood are not available. Gene technology offers a promising alternative, at least for the production of plasma protein administered to patients in small amounts. Work is being done intensively on various blood coagulation factors and proteinase inhibitors at the moment, and factor VIII: C is already being successfully used for the treatment of patients with hemophilia A. However, it will no doubt take years before recombinant plasma proteins are in a position to extensively replace traditional preparations. In the field of diagnostic investigation with plasma proteins immunochemical methods of determination have assumed an increasing significance during the course of the last two decades. Particularly the development of automated techniques which allow serial quantification of individual proteins, has made protein profiling for diagnosis and monitoring in a number of diseases a routine procedure in many clinical laboratories.

Amino Acid Sequence↗
PubMed1989

The arrangement of disulfide loops in human alpha 2-HS glycoprotein. Similarity to the disulfide bridge structures of cystatins and kininogens.

The complete disulfide loop structure of human alpha 2-HS glycoprotein has been elucidated. alpha 2-HS glycoprotein isolated from human plasma was found to be a two-chain protein composed of a heavy and a light chain. The heavy chain comprises the A-chain of alpha 2-HS glycoprotein (Yoshioka, Y., Gejyo, F., Marti, T., Rickli, E. E., Bürgi, W., Offner, G. D., Troxler, R. F., and Schmid, K. (1986) J. Biol. Chem. 261, 1665-1676) and part of the connecting peptide which has been predicted from the corresponding cDNA sequence (Lee, C. C., Bowman, B. H., and Yang, F. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4403-4407), whereas the light chain corresponds to the beta-chain of alpha 2-HS glycoprotein (Gejyo, F., Chang, J. L., Bürgi, W., Schmid, K., Offner, G. D., Troxler, R. F., Van Halbeek, H., Dorland, L., Gerwig, G. J., Vliegenthart, J. F. G. (1983) J. Biol. Chem. 258, 4966-4971). Twelve half-cystine residues are present in the alpha 2-HS glycoprotein molecule, and 11 of them are positioned in the heavy chain and a single one in the light chain of the molecule; they form six disulfide bridges. The first and the last half-cystine residues of the amino acid sequence of alpha 2-HS glycoprotein are engaged in the formation of a loop spanning the extreme NH2- and COOH-terminal portions of the molecule, thereby connecting the heavy and light chains. The other 10 half-cystines residues are linked consecutively in the heavy chain and form five loops which span 4-19 amino acid residues. Among them are two pairs of loops which are characterized by mutual sequence homology. The particular arrangement of disulfide loops in alpha 2-HS glycoprotein is similar to the patterns of linearly arranged and tandemly repeated disulfide loops of cysteine proteinase inhibitors, i.e. the cystatins and the kininogens. It is concluded that alpha 2-HS glycoprotein represents a structural prototype of a novel family among the cystatin superfamily, characterized by the presence of two cystatin-like building blocks. Extensive similarity among the NH2-terminal sequences of alpha 2-HS glycoprotein and human histidine-rich glycoprotein suggest that the latter protein is another candidate protein of this new family.

Amino Acid Sequence↗