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Biomedical subjects

G Schlaf

Publications and source records attributed to G Schlaf.

12 recordsLinked to original sources
PubMed2000

Induction of functional anaphylatoxin C5a receptors on hepatocytes by in vivo treatment of rats with IL-6.

In normal rat liver, anaphylatoxin C5a receptors (C5aR) are only expressed by nonparenchymal cells, mainly Kupffer cells and hepatic stellate cells, but not by parenchymal cells, i.e., hepatocytes (HC). Nevertheless, C5a stimulates glucose output by HC. This HC-specific defense reaction is induced indirectly via prostanoids secreted by the C5aR-expressing Kupffer cells and hepatic stellate cells. It is shown here that under inflammatory conditions simulated by in vivo treatment of rats with IL-6 C5aR mRNA and protein were induced in HC in a time-dependent manner. Maximal mRNA and protein expression were observed at 4-8 h and 8-10 h, respectively, after IL-6 injection. The newly expressed receptors were functional, because recombinant rat C5a significantly activated glycogen phosphorylase in HC isolated from IL-6-treated but not in HC from control rats. In perfused livers of IL-6-treated animals in contrast to control animals, recombinant rat C5a-induced glucose output was not impaired by inhibition of prostanoid synthesis and function with the cyclooxygenase inhibitor indomethacin and the thromboxane receptor antagonist daltroban. These results indicate that HC-specific defense reactions might be differently regulated under normal and inflammatory conditions as shown here for the indirect prostanoid-dependent or direct C5a-induced activation of hepatocellular glycogen phyosphorylase and glucose output in control or IL-6-treated rats, respectively.

Animals↗
PubMed1999

Rat complement factor I: molecular cloning, sequencing and expression in tissues and isolated cells.

Factor I (FI) is a regulatory serine protease of the complement system which cleaves three peptide bonds in the alpha-chain of C3b and two bonds in the alpha-chain of C4b thereby inactivating these proteins. The human protein and the recently characterized mouse factor I are heterodimers of about 88,000 MW which consist of a non-catalytic heavy chain of 50,000 MW which is linked to a catalytic light chain of 38,000 MW by a disulphide bond. For the screening of a rat liver cDNA library we used a hybridization probe produced by polymerase chain reaction (PCR) using degenerated primers which corresponded to conserved parts of the human and the murine factor I nucleotide sequences. One of the identified sequences, which had a length of 2243 base pairs (bp), contained the complete coding region and the whole 3' untranslated region. The length of the coding region in rat consisted of 1812 bp followed by a 3' untranslated region of 207 bp including the polyadenylation signal and the beginning of the poly A tail. Comparison of the rat cDNA-derived coding sequence revealed identities of 87% to the mouse and of 78% to the human FI nucleotide sequence. The translation product of rat FI mRNA was 604 amino acid residues (aa) in length with an identity of 85% to the mouse (603 aa) and 69% to the human protein (583 aa). The comparison of the molecular mass predicted by the primary structure and derived from rat FI isolated from rat serum as detected in immunoblot analyses suggested a glycosylation of more than 20% of the total mass of the FI protein. Expression studies using reverse transcription (RT)-PCR assays indicated that FI-specific mRNA could neither be identified in B cells, nor in T cells, monocytes or granulocytes from rat and human peripheral blood nor in rat peritoneal macrophages. These data were in agreement with the results of RT-PCR obtained with several human lymphoma cell lines (Jurkat, MOLT-4, HUT102, Wil 2-NS, Ramos, Raji, U937) all of which were devoid of FI-specific mRNA. In accord with our data from two rat hepatoma cell lines (FAO and H4IIE) and one from man (HepG2) only isolated rat hepatocytes (HC) but neither Kupffer cells (KC), hepatic stellate cells (HSC; Ito cells) nor sinusoidal endothelial cells (SEC) expressed FI-specific mRNA. FI mRNA was also detected in human umbilical vein endothelial cells (HUVEC) and in the uterus and small intestine of the rat. Spleen and lymph nodes did not contain any detectable FI-specific mRNA.

Amino Acid Sequence↗
PubMed1999

Differential expression of the C5a receptor on the main cell types of rat liver as demonstrated with a novel monoclonal antibody and by C5a anaphylatoxin-induced Ca2+ release.

The C5-anaphylatoxin (C5a) is a protein of 74 (human) or 77 (rat) amino acid residues, respectively, which is generated by limited proteolysis upon activation of the fifth component of complement. Its generation may be induced by both the classical and alternative pathways. C5a has been shown to indirectly increase glucose output from hepatocytes (HC) in perfused rat liver by inducing prostanoid release from Kupffer cells (KC) and hepatic stellate cells (HSC). A direct action of C5a on hepatocytes would require their expression of the specific C5a receptor (C5aR). In former studies using quantitative reverse transcription polymerase chain reaction (RT-PCR) it was shown that HC lack this receptor in contrast to KC, HSC and, probably, sinusoidal endothelial cells (SEC), all of which contained mRNA for the C5aR in decreasing amounts. Using a novel monoclonal antibody (mAb R63) against the rat receptor, expression of the rat receptor on the four cell types was investigated by FACS analysis, immunohistochemistry, and immunocytochemistry. The data obtained were confirmed by functional studies in which the Ca2+ response after stimulation of the isolated cells with recombinant rat C5a (rrC5a), the ligand for the receptor was recorded. The FACS and the immunocytochemical data presented here clearly indicate that rat HC do not express the C5aR, whereas KC have the highest expression level followed by HSC. SEC expressed the receptor only weakly. In line with these findings, a strong Ca2+ response was observed after stimulation of KC and HSC, and a weak one with SEC. However, no signal was obtained upon stimulation of HC. The results of this study support the indirect stimulation of glucose output from HC via prostanoid release from nonparenchymal liver cells and contradict the formerly proposed hypothesis of a direct action of C5 anaphylatoxin on hepatocytes.

Anaphylatoxins↗
PubMed1998

Determination of synapsin I and synaptophysin in body fluids by two-site enzyme-linked immunosorbent assays.

Two-site enzyme-linked immunosorbent assays (ELISA) have been established for the specific and sensitive determination of two membrane proteins of the small synaptic vesicles (SSV), namely: peripheral synapsin I and integral synaptophysin. The ELISA used highly specific capture monoclonal antibodies (mAB) and polyclonal antibodies (pAB) as detectors. For synapsin I, the mAB were newly generated, whereas for synaptophysin, the commercially available mAB SY38 was applied. In order to calibrate the ELISA and to raise pAB, both proteins were purified in the mg-range. Synapsin I was purified by conventional means from human and porcine brain and synaptophysin was purified by immunoaffinity chromatography from porcine brain. Using the ELISA, neither synapsin I nor synaptophysin could be determined in serum or cerebrospinal fluid (CSF) from healthy donors or patients suffering various neurological disorders or pheochromocytomas. For this reason, the degradation of both proteins in serum and CSF was investigated. With the exception of synaptophysin measured in serum, both proteins exhibited fast rates of degradation. Despite the negative results in human body fluids, the two ELISA are appropriate for the quantification of these membrane proteins in neuronal or neuroendocrine cell extracts or preparations of SSV.

Amino Acid Sequence↗
PubMed1996

A novel enzyme-linked immunosorbent assay for determination of synaptophysin as compared with other quantification procedures.

A two-sided enzyme-linked immunosorbent assay (ELISA) has been established for reliable, specific and sensitive determination of synaptophysin (SYN), an intrinsic membrane protein of the small synaptic vesicles. This ELISA used a highly specific monoclonal antibody (SY 38) as capture reagent and a specific SYN antiserum in combination with a secondary peroxidase-conjugated antibody for detection. Calibration was carried out with immunoaffinity-purified SYN from porcine cortex. The sensitivity was found to be improved substantially when the ELISA was compared with previously used dot-immunobinding assays. This ELISA allowed rapid and reliable determination of SYN from detergent lysed homogenates, partially and highly purified preparations of rat, porcine and human brain. SYN was determined in highly purified small synaptic vesicles, and it was calculated to be 5.8% of total detergent solubilized protein.

Animals↗
PubMed1996

Large-scale purification of synaptophysin and quantification with a newly established enzyme-linked immunosorbent assay.

Synaptophysin (SYP I), an integral membrane protein, was purified on a large scale (0.55 - 2.7 mg) from isolated small synaptic vesicles (SSV) of porcine cortex. In order to achieve this, a conventional purification procedure which consists of size exlusion chromatography, hydrophobic interaction chromatography and chromatofocusing has been developed. This procedure was compared with purification of SYP I by immunoaffinity chromatography. The elution patterns of both procedures were monitored using sodium dodecylsulfate gel electrophoresis (SDS-PAGE) with subsequent Coomassie blue staining of proteins and simultaneous immunoblotting with SYP I-specific antibody. Contaminating proteins with relative molecular masses (M(r)) very similar to SYP I could be removed during the process of purification, demonstrating that the 38 kDa protein found after Triton X-100 lysis of enriched SSV does not exclusively represent SYP I. A specific antiserum was raised in rabbits using a highly purified preparation of SYP I. This antiserum was used in combination with a monoclonal antibody to establish a specific and sensitive enzyme-linked immunosorbent assay (ELISA) which allowed rapid and reliable quantification of this hydrophobic membrane protein in all purification steps, starting with Triton X-100-lysed brain homogenates. Using this ELISA, the concentration of SYP I in highly purified SSV was determined to be 5.8% of solubilized protein.

Animals↗
PubMed1996

[Sleep complaints and hypnotic use by the elderly--results of a representative survey in West Germany].

For the first time, a country-wide group of West-Germans were interviewed regarding their sleep complaints and hypnotic intake. Therefore 344 persons who were older than 65 years and 1653 subjects between the age of 14 and 65 years were representatively selected by a random route system. Around the age of 45 the prevalence of sleep-disturbances starts rising continuously and dramatically. Over the age of 65 nearly half of the West-Germans (43%) suffer from difficulties in initiating and/or maintaining sleep (DIMS) at least sometimes, 15% suffer frequently or always from these complaints. 79% of the sleep disturbed elderly (over the age of 65) are chronically ill, having sleep-complaints for more than 2 years or since childhood. The hypnotika intake increases with age as well. Around 45 years of age the irregular hypnotika intake (several times the year or month), and after 55 years of age, regular hypnotic intake (daily or several times the week) rapidly rises. After 65 years of age hypnotic intake increases much more than the sleep complaints do. So, every third elderly person (34%) takes hypnotics at least irregularly. The results of this study indicate that sleep complaints in the elderly are three times more common and more severe than in younger persons, and that they tend to chronify while treated with hypnotics.

Adolescent↗
PubMed1995

Origin and immunoregulation of autoantibodies against intestinal alkaline phosphatase.

In the sera of patients with acute bacterial infections specific autoantibodies (sIAPa) of the immunoglobulin class G (IgG) were found which bind to intestinal alkaline phosphatase (IAP) through the Fab portion. This was demonstrated using immunoaffinity (IA) isolation of sIAPa from patients' sera (particularly bacterial meningitis and ventriculitis) digestion with pepsin, purification of F(ab')2 fragments on protein A and subsequently binding on IAP coupled to CNBr (cyanogen bromide)-activated Sepharose. Immunoblots using specific anti-Fc and anti-Fab antibodies showed that the bulk of F(ab')2 fragments had bound. Additionally, binding of native IAP to the F(ab')2 fragments was observed after separation of F(ab')2 fragments using isoelectric focusing (IEF), blotting onto nitrocellulose and incubation with IAP. Moreover, we have demonstrated the occurrence of natural anti-IAP autoantibodies (nIAPa) which were isolated from sera of healthy individuals using IA chromatography. Investigation of isotype distribution revealed that IgG but not IgM or IgA were predominant even among nIAPa. The nIAPa fraction exhibited lower binding efficiencies on IEF blots than the sIAPa fraction, however, in contrast to sIAPa, cross-reactions with other autoantigens were observed for nIAPa. NIAPa and sIAPa did not show subclass restriction. As revealed by IEF the spectrotypes of sIAPa were found to be patient-specific, poly- to oligoclonal and stable during longer periods.

Acute Disease↗
PubMed1995

[Chronification of sleep disorders. Results of a representative survey in West Germany].

In a representative selection of German citizens who were older than 13 years of age, 1,997 were asked about their sleep complaints. They were also asked how frequently a physician was consulted and how often sleeping pills were taken. According to our results sleep disturbances are an important health problem in Germany. Every fourth person suffers at least sometimes from difficulties in falling asleep and/or staying asleep, problems which are not due to external influences. Seven percent suffer frequently or always from these complaints; 15% report that they are frequently tired or that they are always tired during the day. Ten percent of all persons suffering from sleep complaints take sleeping pills daily or at least sometimes during the week. Forty-five percent of all persons who take hypnotics daily still frequently or always suffer from difficulties in falling asleep and/or staying asleep. Furthermore, sleep complaints tend to become chronic: 75% of the sleep-disturbed population are chronically ill, having had complaints for more than 2 years of since childhood. They suffer from sleep disturbances and from reduced performance during the following day. Nevertheless, it appears as though neither patients nor physicians take insomnia seriously. This might answer the question of why only 17% of the persons who do not suffer more than 2 years from sleep disturbances and only 49% of the chronically ill population consult a doctor because of their sleep disturbances. The results of this study indicate the importance of informing patients and physicians about insomnia and different ways of treating it.

Adolescent↗
PubMed1989

Survival in multiple sclerosis.

Survival from onset of symptoms was calculated for two series of MS patients, a population-based epidemiologic series from southern Lower Saxony of 224 cases, and a revised hospital series of 1429 cases from 11 neurologic centers throughout the Federal Republic of Germany. Median survival for the epidemiologic series was 35-42 years, with no finer estimate possible. Observed survival was 3/5 expected survival after 35 years of illness. Survival in this series was similar to that from two other population-based reports. No significant difference was found between the sexes, though patients with onset at age 35 + had a significantly shorter survival than those with onset at age under 35 years. The much larger hospital series, where median survival was 30 years, demonstrated significant differences separately favoring females and young onsets. However, the ratios of observed to expected survival from population life tables were strikingly similar among all four subgroups defined by sex and age at onset. While overall there is a substantial reduction in the expectation of life in MS, the differential survival by sex and age appears to be a reflection of the expected survival by sex and age per se, and not that of the disease.

Adult↗
PubMed1986

Prognostic indicators in multiple sclerosis.

The influence of sex, age at onset, course of the disease and initial symptomatology on the mortality of patients with multiple sclerosis is analysed. A sample of 1926 patients was followed up prospectively over 4.9 years. Both, the mortality ratio (number of observed to expected deaths) and the excess death rate are calculated. Whereas the mortality ratio as a parameter of overall mortality is influenced by a variety of factors, such as age and sex; the excess death rate represents the number of extra deaths per 1000 exposed to risk in an indicated year and is, therefore, a parameter of the mortality which is attributed to MS. The excess death rate was comparable for the sexes, it was slightly higher for patients with a higher age at onset and it was clearly higher for the progressive course. Patients with initial diplopia and sensory signs and symptoms had the lowest excess death rate, whereas patients with pareses, cerebral and sphincter disturbances at onset showed the highest excess death rate.

Adult↗