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Biomedical subjects

M Renz

Publications and source records attributed to M Renz.

At least 19 recordsLinked to original sources
PubMed2000

Quantitative expression analysis of genes regulated by both obesity and leptin reveals a regulatory loop between leptin and pituitary-derived ACTH.

Absence of the hormone leptin leads to dramatic increases in appetite, food intake, and adiposity. The primary site of action, at least with respect to appetite, is the hypothalamus. Leptin also has significant effects on the function(s) of peripheral organs involved in maintaining body composition. Some of these effects are mediated through direct interaction of leptin with its receptor on the target tissue, and some effects are indirectly mediated through secondary hormonal and neural pathways. Few of the genes that are responsible for regulating body composition and the peripheral effects of leptin are known. We have used a new gene profiling technology to characterize gene expression changes that occur in the pituitary, hypothalamus, fat, muscle, and liver in response to both obesity and treatment with exogenous leptin. These differences were then overlaid to allow the identification of genes that are regulated by obesity and at least partially normalized by leptin treatment. By using this process we have identified five genes (POMC, PC2, prolactin, HSGP25L2G, and one novel) that are both abnormally expressed in the pituitaries of obese mice and are sensitive to the effects of leptin. We also show that adrenocorticotropic hormone appears to be involved in a regulatory loop involving leptin.

Adipose Tissue↗
PubMed2000

Automated solid-phase extraction and two-step derivatisation for simultaneous analysis of basic illicit drugs in serum by GC/MS.

A combination of automated solid-phase extraction (SPE) and subsequent two-step derivatisation has been developed for the simultaneous analysis of basic drugs of abuse and cocaine metabolites in serum samples. Substances included in this procedure are morphine, codeine, methadone, cocaine, benzoylecgonine, methylecgonine, amphetamine, methamphetamine, MDMA, MDEA and MDA. SPE with mixed-mode cartridges (RP-C8 and cation-exchange) was fully automated with a Zymark RapidTrace SPE robot. GC/MS analysis was performed after derivatisation with a new two-step reaction by trifluoroacetic anhydride and 2,2,3,3,3-pentafluoropropanol. High recoveries (> 85%) with high reproducibility (CV 1.1-3.8%) were found for all drugs. High correlation coefficients (r > 0.998) were obtained due to the addition of deuterated standards prior to extraction. Experience obtained over 2 years of applying this method to drug analysis in serum is discussed.

Automation↗
PubMed1999

Screening for drugs in serum by electrospray ionization/collision-induced dissociation and library searching.

Factors influencing in-source collision-induced dissociation (ESI/CID) of organic molecules in a Perkin-Elmer/SCIEX ionspray source have been investigated. Breakdown curves of four drugs and organic compounds were acquired by monitoring the intensities of MH+ and specific fragment ions while ramping the orifice voltage. Haloperidol, diazepam, 1,4-acetamido-acetoxybenzene and diacetamido-1,2-benzene were found to be substances with characteristic breakdown curves, with maximums and points of intersection at orifice voltages between 20 and 70 V. The breakdown curves of haloperidol were used for comparison of ESI/CID with ionspray and turboionspray sources on three PE/SCIEX-API instruments. Using standardized source parameters and mass resolution, very similar fragmentation graphs were obtained for haloperidol with all instruments. Infusion of varying concentrations of haloperidol (0.1 to 10 micrograms/mL with ionspray, and 0.01 to 1 microgram/mL with turboionspray) yielded comparable breakdown curves. With turboionspray, a preconcentration of the aerosol occurred, yielding higher ion abundances. Solvent pH and the ratio of aqueous ammonium formate/acetonitrile had minor effects on the degree of fragmentation of haloperidol in a wide range. With these preconditions, a currently expanding mass spectral library of 400 drugs was set up by liquid chromatography/mass spectrometry with alternating orifice voltages (20, 50, and 80 V, respectively) in a looped experiment. An example of drug identification in a patient's serum with library search is shown.

Chromatography, High Pressure Liquid↗
PubMed1998

TcDJ1, a putative mitochondrial DnaJ protein in Trypanosoma cruzi.

A full length cDNA encoding a novel Trypanosoma cruzi DnaJ protein was cloned and characterized. The 324 amino acid protein encoded by the cDNA (TcDJ1) displays a characteristics J-domain, but lacks the Gly-Phe and zinc finger regions present in some other DnaJ proteins. Relative to four other T. cruzi DnaJ proteins, TcDJ1 has an amino terminal extension containing basic and hydroxylated resides characteristic of mitochondrial import peptides. A T. cruzi transfectant expressing epitope-tagged TcDJ1 was generated and subcellular fractions were produced. Western blot analysis revealed that the protein has a molecular mass of 29 kDa and is found in the mitochondrial fraction. The expression of TcDJ1 is developmentally regulated since the levels of both mRNA and protein are much higher in epimastigotes (replicative form) than in metacyclic trypomastigotes (infective form). Thus it may participate in mitochondrial biosynthetic processes in this organism.

Amino Acid Sequence↗
PubMed1997

Potent alpha 4 beta 1 peptide antagonists as potential anti-inflammatory agents.

The migration, adhesion, and subsequent extravasation of leukocytes into inflamed tissues contribute to the pathogenesis of a variety of inflammatory diseases including asthma, rheumatoid arthritis, inflammatory bowel disease, and multiple sclerosis. The integrin adhesion receptor alpha 4 beta 1 expressed on leukocytes binds to the extracellular matrix protein fibronectin and to the cytokine inducible vascular cell adhesion molecule-1 (VCAM-1) at inflamed sites. Binding of alpha 4 beta 1 to VCAM-1 initiates firm adhesion of the leukocyte to the vascular endothelium followed by extravasation into the tissue. Monoclonal antibodies generated against either alpha 4 beta 1 or VCAM-1 can moderate this inflammatory response in a variety of animal models. Recently peptides containing a consensus LDV sequence based on the connecting segment-1 (CS-1) of fibronectin and cyclic peptides containing an RCD motif have shown promise in modulating leukocyte migration and inflammation presumably by blocking the interaction of alpha 4 beta 1 with VCAM-1. Here we describe novel, highly potent, cyclic peptides that competitively inhibit alpha 4 beta 1 binding to VCAM-1 and fibronectin at sub nanomolar concentrations. The structure of a representative analog was determined via NMR spectroscopy and used to facilitate optimization of peptide leads. The peptides discussed here utilize similar functional groups as the binding epitope of VCAM-1, inhibit lymphocyte migration in vivo, and are highly selective for alpha 4 beta 1. Furthermore the structure--activity relationships described here have provided a template for the structure-based design of small molecule antagonists of alpha 4 beta 1-mediated cell adhesion processes.

Animals↗
PubMed1995

A C-terminally-anchored Golgi protein is inserted into the endoplasmic reticulum and then transported to the Golgi apparatus.

Unlike conventional membrane proteins of the secretory pathway, proteins anchored to the cytoplasmic surface of membranes by hydrophobic sequences near their C termini follow a posttranslational, signal recognition particle-independent insertion pathway. Many such C-terminally-anchored proteins have restricted intracellular locations, but it is not known whether these proteins are targeted directly to the membranes in which they will ultimately reside. Here we have analyzed the intracellular sorting of the Golgi protein giantin, which consists of a rod-shaped 376-kDa cytoplasmic domain followed by a hydrophobic C-terminal anchor sequence. Unexpectedly, we find that giantin behaves like a conventional secretory protein in that it inserts into the endoplasmic reticulum (ER) and then is transported to the Golgi. A deletion mutant lacking a portion of the cytoplasmic domain adjacent to the membrane anchor still inserts into the ER but fails to reach the Golgi, even though this mutant has a stable folded structure. These findings suggest that the localization of a C-terminally-anchored Golgi protein involves at least three steps: insertion into the ER membrane, controlled incorporation into transport vesicles, and retention within the Golgi.

Amino Acid Sequence↗
PubMed1995

The major dermatomyositis-specific Mi-2 autoantigen is a presumed helicase involved in transcriptional activation.

OBJECTIVE: To characterize the complementary DNA (cDNA) and protein sequences of autoantigens recognized by anti-Mi-2 antibodies, using recombinant Mi-2 proteins for improved autoantibody detection. METHODS: A cDNA expression library was immunoscreened, and cDNA isolation, alignment, and sequence analysis were performed. Northern blotting and in situ hybridization techniques were used. A recombinant protein (rMi-2) was synthesized. Immunoprecipitation of 35S-methionine-labeled HEp-2 cell proteins and immunoblotting of rMi-2 and natural nuclear proteins were performed. Immunofluorescence studies were done with anti-Mi-2 positive sera of dermatomyositis (DM) patients, and with human or rabbit antibodies specific for rMi-2. Antibody screening of systemic lupus erythematosus, rheumatoid arthritis, DM, and antinuclear antibody-positive human sera was performed using an rMi-2 protein enzyme-linked immunosorbent assay (ELISA). RESULTS: A major antigen recognized by anti-Mi-2 positive sera of DM patients was found to constitute a 218-kd nuclear protein (218-kd Mi-2) encoded on chromosome 12 and to belong to the SNF2/RAD 54 helicase family. Human and rabbit antibodies that were affinity purified using the recombinant protein reacted with and precipitated a nuclear protein of similar size, which was also recognized by anti-Mi-2 sera. Anti-218-kd Mi-2 antibodies detected by rMi-2 protein ELISA seemed to be mainly restricted to sera from patients with DM. CONCLUSION: The molecular characterization of the 218-kd Mi-2 antigen may contribute to our understanding of autoimmune phenomena in DM. The use of immunoreactive recombinant proteins allows structural and functional studies of the helicase and the development of sensitive and accurate antibody screening tests.

Adenosine Triphosphatases↗
PubMed1994

Interferon-gamma-inducible protein p16. A new target of antinuclear antibodies in patients with systemic lupus erythematosus.

OBJECTIVE: To determine the cellular expression and localization of interferon-gamma (IFN gamma)-inducible protein p16, a new antigen specificity of antinuclear antibodies (ANA), and to evaluate the prevalence of anti-p16, particularly in SLE patients. METHODS: Serum levels of anti-p16 were determined by immunoblotting with recombinant p16 and cellular p16 messenger RNA (mRNA) by Northern blotting. We also utilized immunoprecipitation of 35S-methionine-labeled proteins, immunostaining of blotted proteins of subcellular fractions, and immunofluorescence studies with affinity-purified rabbit and human anti-recombinant p16. RESULTS: Protein p16 was localized within the nucleolus and nucleoplasm and constitutively expressed in Raji, HeLa, HEp-2, K562, and HL-60 cells. Synthesis of mRNA and protein was increased in the presence of IFN gamma. The prevalence of anti-p16 was 29% in 374 SLE patients (35% in those positive for anti-double-stranded DNA) and 0% in 188 healthy individuals. Anti-p16 was always accompanied by positive findings on indirect immunofluorescence for ANA. CONCLUSION: Anti-p16 represents a main ANA specificity. Anti-p16 may help to elucidate IFN gamma-dependent nuclear processes and to link autoantibody production to states of IFN gamma activation.

Animals↗
PubMed1994

Macrogolgin--a new 376 kD Golgi complex outer membrane protein as target of antibodies in patients with rheumatic diseases and HIV infections.

Antibodies against the Golgi complex (GC) were found by indirect immunofluorescence microscopy in the serum of two patients with sclerodermia and Sjögren syndrome. Serum from one patient was used to screen clones from an oligo (dT) primed HeLa cDNA expression library. Four overlapping cross hybridizing clones (G1, G12, G13, G14) were found. One additional 5' clone (G15) was retrieved from a random primed lambda gt11 human thyroid cDNA library by nucleic acid hybridization, exploiting sequence information of clone G12. Additional clones for both the 5' and 3' ends were generated by RF-PCR from HeLa cell mRNA. Alignment of the overlapping clones resulted in a consensus cDNA of 10,300 bp encoding a protein of 376 kD. A corresponding mRNA of about 10 kb was found in Northern blots of RNA from various cultured cells. The most distinct features of the protein were the extraordinarily high fraction of alpha-helical domains containing heptad repeats with the probability of forming coiled-coils and the structure similarities with the myosin family and the yeast intracellular transport protein USO1. Five overlapping cDNA fragments covering the entire open reading frame were used to synthesize recombinant proteins for affinity purification of the antibodies in the two patients' sera. By use of these affinity purified antibodies, staining of the GC of various cultured cell lines was reproduced. The antibody target was dissociated within 15 min after brefeldin A exposure of cultured cells, a phenomenon, which was fully reversible within 30 min after withdrawal of the drug. Sucrose step gradient separation of GC enriched microsomal fractions from rat liver showed a natural antigen of about 380 kD co-fractionating with the GC marker galactosyltransferase. KCl extraction, Triton X-114 partition, as well as trypsin digestion of microsomal fractions revealed that the hydrophilic protein has to be located on the cytoplasmatic surface of GC vesicles. Using the five blotted recombinant protein fragments, anti-GC antibodies were found in 18 of 164 (11%) HIV positive patients but in none of the 64 healthy controls. HIV patients as well as the two original patients showed a diverse antibody spectrum recognizing different epitopes of the recombinant proteins. The protein characterized herein, for which we propose the provisional name macrogolgin, constitutes the largest protein known so far associated with the GC.

Amino Acid Sequence↗
PubMed1994

HLA class II genes and antibodies against recombinant U1-nRNP proteins in patients with systemic lupus erythematosus. SLE Study Group.

To investigate a possible involvement of HLA-class II alleles in the genetic predisposition for the formation of anti-U1-nRNP antibody-in systemic lupus erythematosus (SLE), genomic DNA of 178 patients was typed for the DRB1, DQA1 and DQB1 alleles using a polymerase chain reaction (PCR) and non-radioactive-oligonucleotide typing. Antibodies against recombinant U1-nRNP proteins (U1-A-, U1-C- and 70K-protein) were determined by ELISA. Anti-U1-C antibody was found in 26 (14.7%), anti-U1-A in 34 (19.2%) and anti-70K in 17 (9.6%) patients. A joint occurrence was observed for these antibodies against the recombinant U1-nRNP proteins: anti-U1-C and anti-U1-A antibodies occurred together more frequently than alone and than together with anti-U1-70K antibodies. The frequency of DRB1*04 was slightly increased in the patients with anti-U1-C as compared to the patients without anti-U1-C (P < 0.05, Pcorr = n.s., RR = 2.4). The DQA1*0301 allele, which is in linkage disequilibrium with DRB1*04, is found more frequently in anti-U1-C-positive than in antibody-negative patients. The DQB1*0303 allele, detected in 12 of 176 SLE patients, was absent in the patients with any of the antibodies against the U1-nRNP proteins. All these deviations may be due to chance alone. We concluded that the presence of antibodies against recombinant U1-nRNP proteins was not significantly associated with any HLA DRB1, DQA1 and DQB1 allele in our group of SLE patients.

Alleles↗
PubMed1994

Molecular genetic analyses of a 376-kilodalton Golgi complex membrane protein (giantin)

Molecular genetic analyses of a 376-kDa Golgi complex (GC) membrane protein (giantin) are described. The immunoglobulin G fraction of a human serum containing antibodies against GC antigens as revealed by indirect immunofluorescence microscopy with Hep-2 cells was used to screen a HeLa cDNA expression library, yielding four overlapping cross-hybridizing clones. Additional cDNA clones were retrieved from a lambda gt11 human thyroid cDNA library or generated by reverse transcriptase-mediated PCR from HeLa cell mRNA. Alignment of the clones resulted in a consensus cDNA of 10,300 bp encoding a protein of 376 kDa. The corresponding mRNA with a size of about 10 kb was detected by Northern (RNA) blotting of HeLa, Hep-G2, and Jurkat cell RNA. Sequence analyses of the protein revealed an extraordinarily high content of heptad repeats with the probability of forming coiled coils similar to the proteins of the myosin family. Five overlapping recombinant proteins covering the entire sequence were synthesized and used for antibody production in rabbits and for affinity purification of human and rabbit antibodies. Indirect immunofluorescence experiments also done with brefeldin A-treated Hep-2 and Pt K1 cells revealed an identical GC staining of both the affinity-purified human and rabbit antibodies. Double labeling experiments with antibodies against the GC marker mannosidase II as well as immunoelectron microscopic studies confirmed the localization of the protein within the GC. A corresponding endogenous large-molecular-mass protein of about 390 kDa was found in [35S]methionine-labeled Hep-2 cell lysates as well as in GC-enriched subcellular fractions from rat liver. The protein as well as the recently described proteins golgin-95 and golgin-160 (M. J. Fritzler, J. C. Hamel, R. L. Ochs, and E. K. L. Chan, J. Exp. Med. 178:49-62, 1993) may belong to a new group of Golgi proteins with a high content of heptad repeats which may exert functions in scaffold formation or vesicle transport. As far as can be concluded from immunological and personally communicated partial cDNA sequence data, the protein seems to be identical with a 400-kDa Golgi protein (giantin) recently described (A. D. Linstedt and H. P. Hauri, Mol. Biol. Cell 4:679-693, 1993). Therefore, we agreed to adopt the name giantin.

Amino Acid Sequence↗
PubMed1994

Hepatitis C virus infections in dialysis units: prevalence of HCV-RNA and antibodies to HCV.

HCV infection causes serious complications in dialysis patients that lead to problems in management of patients in dialysis units. Determination of HCV-RNA is at present essential for monitoring the course of HCV infection. Reports concerning HCV-RNA in dialysis patients are mostly from Asian dialysis units; therefore, an analysis of dialysis patients in Europe was undertaken. From 1515 patients 2630 blood samples were screened for HCV-RNA and anti-HCV. Two-thirds of patients positive in an anti-HCV test containing a mixture of three antigens (EIA-II, Ortho) were further analysed for antibodies against these individual antigens. From 523 patients multiple samples were tested. Related on dialysis units from which all the attending patients were tested, mean prevalence of HCV-RNA was 8.4%, of anti-HCV 13.2%. Concerning all plasma samples from dialysis patients sent to our laboratory for investigation of HCV-RNA and anti-HCV the prevalence of HCV-RNA was 21.9%, of anti-HCV 23.1%, HCV-RNA was present in 76% of anti-HCV positive patients and in 4.1% of anti-HCV negative patients (1.3% of single and 6.8% of multiple tested patients). Acute and chronic infections with self-limited, persistent or intermittent viraemia were observed with changes and fluctuations of both HCV markers. With the exception of differences in onset of antibody production in some patients following acute infections, there were no major differences of dialysis patients compared to patients without dialysis treatment as far as antibody spectrum and detectability of HCV-RNA were concerned.

Base Sequence↗