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Biomedical subjects

P Straub

Publications and source records attributed to P Straub.

At least 19 recordsLinked to original sources
PubMed2006

The translocation motif of hepatitis B virus improves protein vaccination.

Cell-penetrating peptides (CPPs) have been shown to improve antigen loading of dendritic cell vaccines. Here we asked whether fusion of a CPP to a protein improves its immunogenicity when this fusion protein is directly applied as vaccine. We used the cell-penetrating translocation motif (TLM) derived from the hepatitis B virus, because no size limitation of cargos has been observed. Increased immunogenicity was observed when TLM was fused to ovalbumin (TLM-ova). TLM-ova was found to be superior to ova in inducing proliferation and cytotoxicity of ova-specific CD8+ T cells in vitro and in vivo. Using ovalbumin-expressing thymoma cells (EG7-ova), an improved anti-tumor immune response was observed for TLM-ova vaccination versus vaccination with ova. Moreover, TLM-ova vaccination induced a higher titer of anti-ovalbumin IgG2a antibodies compared to ova. These data demonstrate that CPP-protein vaccines can improve cellular as well as humoral immune responses.

Amino Acid Motifs↗
PubMed2006

Impact of cytopathologist expert on diagnosis and treatment of pancreatic lesions in current clinical practice. A series of 106 endoscopic ultrasound-guided fine needle aspirations.

OBJECTIVES: To evaluate the role of a cytopathologist expert in interpreting difficult pancreatic endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) in terms of impact on diagnostic yield and therapy in current clinical practice. METHODS: Retrospective study of 106 EUS-FNA performed between January 2002 and September 2004. Forty-seven cases of difficult interpretation by the local cytopathologist were mailed to an expert cytopathologist. The final diagnosis was established by histology obtained by either surgery (n=30), non-equivocal FNA (n=40) or core (n=8) biopsies, or by a mean follow-up of 14 months (n=28). RESULTS: EUS-FNA involved 68 solid lesions (SL). Specimens collected were adequate in 63%. The overall sensitivity of EUS-FNA for the diagnosis of malignancy of SL was 88% versus 72% for local cytopathologist and 89% for expert. EUS-FNA with the reevaluation by expert of 39 SL gave a therapeutic impact in 47% of SL. Thirty-eight cystic lesions (CL) were evaluated. Specimens collected were adequate for cytology in 84% of cases and for biochemistry in 79%. The sensitivity of imaging+EUS-FNA+/-biochemistry for the diagnosis of a surgical CL was 58% with a therapeutic impact in 39.5%. CONCLUSION: Pancreatic EUS-FNA is an easy technique for experienced endosonographers, which can be safely performed on an outpatient basis. The possibility of mailing difficult cases to an expert cytopathologist increases the diagnostic yield. In these circumstances, EUS-FNA has a therapeutic impact in current practice in nearly one out of two pancreatic lesions.

Adenocarcinoma↗
PubMed1997

[Mercury erythema after accidental exposure to mercury vapor].

Mercury exanthem can be considered as a systemic contact dermatitis following exposure to mercury vapor in patients with a prior sensitization to mercurials. It is characterized by a symmetrically distributed erythematous eruption appearing predominantly in the major flexural areas, in the neck, the lower portion of the abdomen and the upper anteromedial part of the thighs. In some cases, small pustules develop over the erythematous surfaces. We report our observations of two patients with mercury exanthem after exposure to mercury vapor caused by a broken thermometer. One of these patients presented with an unusual bullous form of mercury exanthem. Although diagnosis of mercury exanthem can be essentially based on clinical features, confirmation of the patient's exposure to mercury should be obtained. Cutaneous patch tests often prove the sensitization to mercurials.

Accidents, Home↗
PubMed1997

[Spironolactone-induced pemphigoid].

BACKGROUND: Sporadic observations would suggest that certain drugs play a role in the development of pemphigoid. A recent case-control study on long-term drug use associated with pemphigoid was unable to confirm the suspected role of these drugs, but did demonstrate a significant association between the development of pemphigoid and use of spironolactone. CASE REPORT: An 82-year-old patient had bullous erythematous lesions on the left thigh suggestive of pemphigoid. Histology and direct cutaneous immunofluorescence confirmed the diagnosis. The patient had been taking alimemazine, adrafinil, flunarizine, spironolactone and furosemide as long-term treatment. Spironolactone alone was withdrawn. The cutaneous lesions regressed and have not recurred during the 18-month follow-up. DISCUSSION: The causal effect of spironolactone in this case of pemphigoid is quite plausible. This hypothesis is favored by the agreement with epidemiological data. Other observations are however required before the causal role of spironolactone in development of pemphigoid can be confirmed.

Aged↗
PubMed1996

[Cystic lymphangioma of the spermatic cord. Report of a new case].

We report a new case of spermatic cord lymphangioma in a young patient. It is a rare tumor of the lymphatic system. Only eleven cases have been reported in literature. This lesion tends to recur despite surgical ablation. The pathogenesis of this lesion is still controversial.

Adult↗
PubMed1995

Identification by in vitro mutagenesis of the interaction of two segments of C2MstC1, a chimera of cytochromes P450 2C2 and P450 2C1.

A hybrid cytochrome P450, C2MstC1, with 306 N-terminal amino acids derived from cytochrome P450 2C2 sequence and 184 C-terminal amino acids from cytochrome P450 2C1 acquires a novel progesterone 21-hydroxylase activity which is absent in the parent enzymes. Extension of the cytochrome P450 2C2 sequence to residue 382 reduced progesterone hydroxylase activity to 5% of that of C2MstC1, while further extension to residue 411 or 462 increased activity back to about 30 or 40%, respectively. In the chimera with cytochrome P450 2C2 sequence to residue 382, substitution of cytochrome P450 2C1 amino acids at positions 368, 369, and 374 increased progesterone hydroxylase activity to a level equivalent to that of C2MstC1. In the chimera with cytochrome P450 2C2 sequence extending to residue 411, substitutions of P450 2C1 amino acids at positions 386 and 388, in addition those at 368, 369, and 374, were required to obtain activities equivalent to that of C2MstC1, which suggests an interaction between these two regions. The lauric acid hydroxylase activities of all chimeras and mutant cytochromes P450 differed by 2-fold or less, demonstrating that the changes in progesterone hydroxylase activity reflected altered interactions with the substrate. Alignment of cytochrome P450 2C1 sequence with cytochromes P450cam, P450BM-3, and P450terp predicts that residues 368/369 and 386/388 are in adjacent antiparallel strands of the same beta-sheet, in agreement with the experimental data suggesting an interaction between these two regions.

Amino Acid Sequence↗
PubMed1995

Molecular analysis of autoantigens in hepatitis D.

In contrast to hepatitis B virus infection, autoimmunity is common in chronic hepatitis D. In 1983, microsomal autoantibodies were described that differ from the previously described liver-kidney microsomal (LKM)-1 and LKM-2 antibodies. Therefore, these were named LKM-3. In addition, autoantibodies against basal layer cells of rat forestomach (basal cell layer antibodies (BCLA)) and thymic stellate epithelial cells (stellate epithelial cell antibodies (SECA)), as well as thymic reticular (thymic reticular antibodies) and perithymocytic cells (perithymocytic cell antibodies), were reported to be specifically associated with hepatitis D. Antinuclear antibodies against nuclear membrane lamin C were also found to be specifically associated with chronic hepatitis D. A molecular identification of the nonnuclear antigens has not been achieved apart from the observation that BCLA and SECA both recognize an antigen of 46 kDa and that these antibodies are immunologically cross-reactive. After the identification of cytochrome P450 2D6 as the major LKM-1 antigen and cytochrome P450 2C9 as the LKM-2 antigen, family 1 uridine diphosphate-glucuronosyltransferases have been identified as the molecules expressing the major LKM-3 autoepitope. Future studies will have to assess the possible pathogenetic and diagnostic relevance of these autoantibodies. However, chronic hepatitis D seems to be a good clinical model for the study of virus-induced autoimmunity in man.

Animals↗
PubMed1994

Cloning and characterization of cDNAs encoding mouse Ugt1.6 and rabbit UGT1.6: differential induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

In this report, cDNAs for mouse liver Ugt1.6 and rabbit liver UGT1.6 have been cloned and characterized. The predicted amino acid sequence of mouse Ugt1.6 is 93% and 78% similar to the rat and human UGT1.6 sequences, respectively, while the rabbit UGT1.6 is 79% and 83% similar to the rat and human UGT1.6 sequences, respectively. To examine the substrate specificities of the proteins encoded by the mouse Ugt1.6 and rabbit UGT1.6 cDNAs, the recombinants were expressed in monkey kidney COS-1 cells. Transfection of the mouse and rabbit recombinants allowed for the expression of the UGT1.6 proteins as determined by immunoprecipitation of newly synthesized protein. The expressed UGTs conjugated small planar phenolic molecules such as 4-nitrophenol, 1-naphthol, and 4-methylumbelliferone. While the bulky phenol 4-hydroxybiphenyl was not a substrate for the enzymes, 2-hydroxybiphenyl was an excellent substrate. Androgens and estrogens were not conjugated by either mouse Ugt1.6 or rabbit UGT1.6. In rodents, UGT1.6 mRNA is expressed constitutively and induced when the animals are treated with the Ah receptor ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Using wild-type mouse hepatoma cells and the Ah receptor deficient class II cells, it was demonstrated that induction of mouse Ugt1.6 was dependent upon a functional Ah receptor complex. However, when New Zealand white rabbits were treated with TCDD and liver mRNA was examined by Northern blot analysis, it was shown that TCDD had no effect on the induction of UGT1.6 mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Recognition of uridine diphosphate glucuronosyl transferases by LKM-3 antibodies in chronic hepatitis D.

Patients with chronic hepatitis D often have liver-kidney microsomal antibodies type 3 (LKM-3). These antibodies react with several microsomal antigens that have a molecular weight of 55 KDa and an isoelectric point of about 8. We studied the molecular nature of the antigen and, by immunoscreening a human liver cDNA expression library with KM-3 sera, found that uridine diphosphate glucuronosyl transferases (UGT) appeared as candidate antigens. We confirmed the identity of UGT as an antigen by reacting the sera with recombinant rabbit liver UGT proteins. Some sera reacted with rabbit UGT-2 proteins, but UGT-1 proteins were more sensitive and specific in detecting LKM-3 autoantibodies in patient sera. Anti-UGT-1 antibodies were detected in all LKM-3 positive sera from patients with hepatitis D and 1 out of 11 patients with autoimmune hepatitis type 2. Sera from patients who had hepatitis B only did not react with UGT proteins. The UGT proteins are part of the phase II enzymes of drug metabolism and are the first such enzymes to be identified as human autoantigens.

Antibody Specificity↗
PubMed1994

Differential effects of mutations in substrate recognition site 1 of cytochrome P450 2C2 on lauric acid and progesterone hydroxylation.

Mutations at amino acid positions 107-120, which are part of a predicted substrate recognition site [Gotoh, O. (1992) J. Biol. Chem. 267, 83-90], were analyzed in C2MstC1, a chimera of P450 2C2 and P450 2C1. This hybrid protein has a new activity for progesterone C21-hydroxylation in addition to the lauric acid (omega-1)hydroxylase activity present in both parent proteins. Various substitutions for highly conserved glycines at positions 111 and 117 and tryptophan at position 120 strongly decreased the lauric acid hydroxylase activity of P450 2C2 and C2MstC1 and the progesterone hydroxylase activity of C2MstC1. Activities of mutant proteins with substitutions at 107, 108, and 112-115 were also strongly reduced. Modest or no decreases in activity were observed for substitutions at 109, 110, 116, 118, and 119. Lauric acid hydroxylase activity decreased more in most C2MstC1 mutants than in those of P450 2C2, particularly at positions 107 and 108. A substitution of phenylalanine for valine-112 reduced progesterone hydroxylation by 30-fold while only moderately reducing lauric acid hydroxylase by 40%. This differential effect on two dissimilar substrates demonstrates the importance of residue 112 for substrate interactions. The results are consistent with a model in which residues 107-110 align with the B'-helix of the bacterial proteins P450cam and P450BM-3. This helix is followed by a substrate-contacting loop from 111 to 116, and residues 117-120 align with the C-helices of the bacterial proteins In this alignment, Trp-120 is positioned behind the heme such that it could participate in electron transfer from reductase.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Preference for aromatic substitutions at tryptophan-120, which is highly conserved and a potential mediator of electron transfer in cytochrome P450 2C2.

A proposal that tryptophan-120 of P450 2C2 might mediate electron transfer has been tested by substitution of a series of aliphatic and aromatic residues at this position. Activity in transfected COS1 cells expressing enzymes substituted with the aromatic residues, tyrosine, phenylalanine, and histidine, was 25% to 60% of wild type. Activities in enzymes containing serine, arginine, or aliphatic amino acid substitutions were less than 10%, except for alanine (25%). Alanine is the only naturally occurring substitution for tryptophan at this position in mammalian cytochromes P450 and is present in the electron pathway proposed on the basis of the crystal structure of cytochrome c and cytochrome c peroxidase. The preference for aromatic residues or the small aliphatic amino acid, alanine, at position 120 is consistent with a role for this tryptophan in electron transfer.

Amino Acid Sequence↗
PubMed1993

Hydrophobic side chain requirements for lauric acid and progesterone hydroxylation at amino acid 113 in cytochrome P450 2C2, a potential determinant of substrate specificity.

To determine the requirements for hydrophobic amino acids at position 113 in cytochrome P450 2C2, a series of hydrophobic and uncharged polar amino acids was substituted for isoleucine in P450 2C2 and in C2MstC1, a chimera of P450 2C2 and P450 2C1. Lauric acid hydroxylase activity was determined in COS1 cells transfected with P450 2C2 mutants and both lauric acid and progesterone hydroxylase activities were determined for C2MstC1 variants. In P450 2C2, 40 to 120% of the wild type (omega-1) lauric acid hydroxylase activity was retained in all hydrophobic mutants, but activity was reduced to near background by substitutions of the polar amino acids, tyrosine and cysteine. Likewise, in C2MstC1 mutants, hydrophobic substitutions were 20 to 50% as active as wild type for lauric acid hydroxylation, and polar amino acids again resulted in strong reductions of activity. In contrast, a different pattern of activity for progesterone C21-hydroxylase was observed for C2MstC1 mutants. A valine substitution had a modest effect on activity but substitutions of leucine and alanine reduced progesterone C21-hydroxylase activity 5- to 7-fold, respectively, and the large hydrophobic amino acid, phenylalanine, reduced activity about 30-fold. No changes in the regiospecificity of progesterone hydroxylation were observed for any of the mutants. Similar steady-state levels of immunoprecipitated, radiolabeled protein were observed for each mutant except for the glycine substitution which resulted in degradation of the protein. The different patterns of the effects of the mutations on progesterone and lauric acid hydroxylase activity provide additional support for the critical role of residue 113 in substrate recognition. The low activities in mutant proteins with hydrophilic amino acid substitutions indicate that the hydrophobic nature of this residue is important. The hydrophobic requirements are more stringent for a larger, more rigid steroid substrate than for a saturated fatty acid with a flexible hydrocarbon tail.

Amino Acid Sequence↗
PubMed1993

Cassette mutagenesis of a potential substrate recognition region of cytochrome P450 2C2.

Cassette mutagenesis was used to analyze the effects of mutations at amino acid positions 107-120 in cytochrome P450 2C2 which are part of a predicted substrate recognition site, SRS-1 (Gotoh, O. (1992) J. Biol. Chem. 267, 83-90), and terminate with a highly conserved tryptophan. All the mutant enzymes were expressed in COS1 cells at approximately the same level as wild type as determined by immunoprecipitation of radiolabeled products. Substitutions of amino acids from 107 to 110 and from 116 to 119 resulted in mutant enzymes that retained substantial lauric acid hydroxylase activity suggesting that these residues are not critical determinants of substrate specificity. In contrast, amino acids 112-115 were characterized by strong decreases in activity in at least one mutation tested. A substitution of phenylalanine for valine 112 reduced activity about 3-fold. At position 113, substitution of leucine for isoleucine had little effect, but activity was reduced 15-fold by substitution with cysteine. A conservative mutation at position 114 reduced activity 5-fold, a nonconservative mutation at position 115 resulted in a 30-fold reduction of lauric acid hydroxylation. Substitution of glutamic acid for glycine at position 109 had little effect on activity, while more conservative substitutions of valine for glycines at positions 111 and 117 resulted in about 50- and 7-fold reductions of activity, respectively. Substitution of leucine for tryptophan 120 resulted in a 7-fold reduction of activity indicating that tryptophan at this position contributes to, but is not essential for, enzymatic activity in spite of its presence in nearly all eukaryotic cytochromes P450. The results are consistent with a model in which amino acids 112-114, and possibly 115, align with a loop of bacterial P450cam sequence containing substrate contacting amino acids, tyrosine 96 and phenylalanine 98. Glycines at either end of the loop in cytochrome P450 2C2 appear to be critical for activity.

Amino Acid Sequence↗
PubMed1993

Deletion of a conserved tetrapeptide, PPGP, in P450 2C2 results in loss of enzymatic activity without a change in its cellular location.

The sequence proline-proline-glycine-proline is highly conserved in cytochrome P450 families 1 and 2, and similar proline rich sequences are found in other cytochromes P450. Since this sequence immediately follows the NH2-terminal hydrophobic membrane insertion signal, it potentially could function as a signal either for retention of cytochrome P450 in the endoplasmic reticulum or for its correct orientation in the membrane. To test this possibility, DNA sequence coding for this tetrapeptide was deleted from cytochrome P450 2C2 cDNA. Translation of the mutated mRNA in a reticulocyte cell-free system containing canine microsomal membranes resulted in the insertion of the protein into the membrane with a topology indistinguishable from that of normal cytochrome P450 2C2. The mutated protein was expressed in COS1 cells and its distribution, assayed by immunofluorescence, was similar to that of cytochrome P450 2C2. Furthermore, if a short peptide containing a potential glycosylation site was fused to the N-terminus of the mutant protein, the new hybrid protein was glycosylated in COS1 cells and the carbohydrate moiety remained sensitive to cleavage by endoglycosidase H. These results indicate that the protein was inserted and retained in the endoplasmic reticulum membrane. Pulse-chase studies showed that the mutated protein was degraded about four times as fast as cytochrome P450 2C2. In contrast to cytochrome P450 2C2, no (omega-1) hydroxylase activity was detected in COS1 cells expressing the mutated protein at similar steady-state levels as the wild-type protein. These results indicate that, although the conserved PPGP tetrapeptide is not required for cellular localization of cytochrome P450 in the endoplasmic reticulum membrane, its deletion decreases the stability of the protein and abolishes enzymatic activity.

Amino Acid Sequence↗
PubMed1992

Biochemical characterization of binding sites for dihydropyridine and omega-conotoxin in brain of adult chicken.

Binding studies using the calcium channel blockers omega-conotoxin and dihydropyridine revealed a rather equal amount of binding sites in brain from adult chicken. The omega-conotoxin binding sites could be solubilized using digitonin, without substantial loss, whereas a great decrease in dihydropyridine binding sites was observed, indicating that both types of binding sites have different sensitivity to solubilization. In contrast to ion exchange chromatography where both binding sites comigrated, glycoprotein affinity chromatography led to a different partition of the binding sites in the flow through and eluate fractions. Our results indicate that both types of calcium channel blockers bind to different targets in adult chicken.

Animals↗
PubMed1989

[Superselective angiography of the circumflex arteries in preoperative evaluation of femoral neck fractures].

The study of arterial vascularity in the head and neck of the femur is performed as an emergency exploration of femoral neck fractures by means of selective catheterization. The systematization of vascular lesions thus obtained is compared with the anatomical types of fractures. The posteromedial circumflex artery is not injured definitively as a rule, so that conservative osteosynthesis can be preferred to arthroplasty.

Angiography↗
PubMed1988

Interferon treatment inhibits onset of herpes simplex virus immediate-early transcription.

Pretreatment of primary cultures of splenic mouse macrophages with murine IFN-alpha/beta leads to a stable inhibition of herpes simplex virus type 1. Analysis of viral DNA, RNA, and protein synthesis identifies expression of "immediate-early" genes as a major target of IFN-mediated inhibition. Determination of viral DNA in the nuclei early after infection, i.e., before onset of DNA replication, suggests that virus uptake, transport to the nucleus, and DNA stability are not decreased in IFN-pretreated macrophages. Nuclear runoff transcription analysis shows a significant reduction of immediate-early transcription rates following IFN treatment. End-specific probes for the ICP4 gene locate the inhibition to the onset of transcription. Northern blot analysis reveals a decrease in ICP4 transcripts in accordance with the observed inhibition of transcription. The observed inhibition of early gene transcription may be a consequence of decreased immediate-early gene expression.

Animals↗