PubMed Health⌕ Search

Biomedical subjects

S Michaelis

Publications and source records attributed to S Michaelis.

At least 19 recordsLinked to original sources
PubMed2004

Subcutaneous infection with Mycobacterium abscessus in a renal transplant recipient.

Transplant recipients show a high incidence of infections, often with atypical manifestations mainly because of drug-related immunosuppression. Mycobacterial infections can be life-threatening in immunosuppressed patients due to the risk of dissemination. There have been only few reports of cutaneous infections with Mycobacterium abscessus among immunosuppressed patients. We present a rare case with a sporotrichoid skin infection with M. abscessus in a renal recipient. The value of immunohistochemical detection of mycobacteria using an anti-BCG antibody in the diagnostic work-up skin biopsies is demonstrated. Long-term antibiotic treatment resulted in complete remission, although recurrences with atypical presentation were observed.

Aged↗
PubMed2003

Unusual manifestation of specific cutaneous involvement by B-cell chronic lymphocytic leukemia: spontaneous regression with scar formation.

We report a patient with specific cutaneous involvement by B-cell chronic lymphocytic leukemia, who demonstrated unusual clinical features during the course of the disease, namely several spontaneous regressions of skin lesions with the formation of scars. In addition, histologically proven keratoacanthoma was found. During the follow-up period of approximately 1.5 years, the patient experienced several recurrences of skin lesions and their partial spontaneous regression. The scars persisted and remained unchanged. We hypothesize that vascular injury combined with edema could have accounted for dermal ischemia and the subsequent development of the scarring lesions.

Aged↗
PubMed2003

Evaluation of living conditions of free-ranging animals by automated chronobiological analysis of behavior.

A biorhythmical method has been developed to assess behavior patterns and to evaluate the living conditions of animals that can be applied also to humans. All kinds of continuous and equidistant long-term recordings of behavior are suitable for this method. Time functions from an automatic telemetry system (ETHOSYS II) were analyzed macroscopically (comparison of daily levels) and microscopically (autocorrelation function and power spectral analysis). Degrees of functional coupling (DFCs) were calculated to identify and evaluate disturbances in behavior. Hierarchic frequency tuning of complex rhythmic functions of behaviors, leads primarily to period lengths that are synchronized with the 24-h period. DFCs, a measure of harmony between internal rhythms and the external 24-h period, were found to be high in well-adapted, healthy, and undisturbed individuals but were lowered during periods of adaptation, sickness, or social interaction. Specific stress conditions could be identified and evaluated in several species, under various conditions, using these biorhythmic analyses.

Animal Migration↗
PubMed2002

T-zone lymphoma with cutaneous involvement: a case report and review of the literature.

T-zone lymphoma (TZL) is a rare subtype of nodal peripheral T-cell lymphoma characterized by a clonal expansion of T-zone lymphocytes accompanied by a proliferation of other T-zone constituents. Non-specific cutaneous alterations are seen in about one-third of all cases, but specific cutaneous involvement is extremely rare. We present a case of TZL with secondary skin infiltration, review the literature on cutaneous manifestations of TZL and discuss the differential diagnosis of TZL.

Adult↗
PubMed2001

Yeast as a model system for studying endocytosis.

Endocytosis is the membrane trafficking process by which plasma membrane components and extracellular material are internalized into cytoplasmic vesicles and delivered to early and late endosomes, eventually either recycling back to the plasma membrane or arriving at the lysosome/vacuole. The budding yeast Saccharomyces cerevisiae has proven to be an invaluable system for identifying proteins involved in endocytosis and elucidating the mechanisms underlying internalization and postinternalization events. Through genetic studies in yeast and biochemical studies in mammalian cells, it has become apparent that multiple cellular processes are linked to endocytosis, including actin cytoskeletal dynamics, ubiquitylation, lipid modification, and signal transduction. In this review, we will highlight the most exciting recent findings in the field of yeast endocytosis. Specifically, we will address the involvement of the actin cytoskeleton in internalization, the role of ubiquitylation as a regulator of multiple steps of endocytosis in yeast, and the sorting of endocytosed proteins into the recycling and vacuolar pathways.

Actins↗
PubMed2001

The multispanning membrane protein Ste24p catalyzes CAAX proteolysis and NH2-terminal processing of the yeast a-factor precursor.

Saccharomyces cerevisiae Ste24p is a multispanning membrane protein implicated in the CAAX proteolysis step that occurs during biogenesis of the prenylated a-factor mating pheromone. Whether Ste24p acts directly as a CAAX protease or indirectly to activate a downstream protease has not yet been established. In this study, we demonstrate that purified, detergent-solubilized Ste24p directly mediates CAAX proteolysis in a zinc-dependent manner. We also show that Ste24p mediates a separate proteolytic step, the first NH(2)-terminal cleavage in a-factor maturation. These results establish that Ste24p functions both as a bona fide COOH-terminal CAAX protease and as an a-factor NH(2)-terminal protease. Importantly, this study is the first to directly demonstrate that a eukaryotic multispanning membrane protein can possess intrinsic proteolytic activity.

Amino Acid Sequence↗
PubMed2001

Biochemical studies of Zmpste24-deficient mice.

Genetic studies in Saccharomyces cerevisiae identified two genes, STE24 and RCE1, involved in cleaving the three carboxyl-terminal amino acids from isoprenylated proteins that terminate with a CAAX sequence motif. Ste24p cleaves the carboxyl-terminal "-AAX" from the yeast mating pheromone a-factor, whereas Rce1p cleaves the -AAX from both a-factor and Ras2p. Ste24p also cleaves the amino terminus of a-factor. The mouse genome contains orthologues for both yeast RCE1 and STE24. We previously demonstrated, with a gene-knockout experiment, that mouse Rce1 is essential for development and that Rce1 is entirely responsible for the carboxyl-terminal proteolytic processing of the mouse Ras proteins. In this study, we cloned mouse Zmpste24, the orthologue for yeast STE24 and showed that it could promote a-factor production when expressed in yeast. Then, to assess the importance of Zmpste24 in development, we generated Zmpste24-deficient mice. Unlike the Rce1 knockout mice, Zmpste24-deficient mice survived development and were fertile. Since no natural substrates for mammalian Zmpste24 have been identified, yeast a-factor was used as a surrogate substrate to investigate the biochemical activities in membranes from the cells and tissues of Zmpste24-deficient mice. We demonstrate that Zmpste24-deficient mouse membranes, like Ste24p-deficient yeast membranes, have diminished CAAX proteolytic activity and lack the ability to cleave the amino terminus of the a-factor precursor. Thus, both enzymatic activities of yeast Ste24p are conserved in mouse Zmpste24, but these enzymatic activities are not essential for mouse development or for fertility.

Amino Acid Sequence↗
PubMed2001

The yeast a-factor transporter Ste6p, a member of the ABC superfamily, couples ATP hydrolysis to pheromone export.

ATP-binding cassette (ABC) proteins transport a diverse collection of substrates. It is presumed that these proteins couple ATP hydrolysis to substrate transport, yet ATPase activity has been demonstrated for only a few. To provide direct evidence for such activity in Ste6p, the yeast ABC protein required for the export of a-factor mating pheromone, we established conditions for purification of Ste6p in biochemical quantities from both yeast and Sf9 insect cells. The basal ATPase activity of purified and reconstituted Ste6p (V(max) = 18 nmol/mg/min; K(m) for MgATP = 0.2 mm) compares favorably with several other ABC proteins and was inhibited by orthovanadate in a profile diagnostic of ABC transporters (apparent K(I) = 12 microm). Modest stimulation (approximately 40%) was observed upon the addition of a-factor either synthetic or in native form. We also used an 8-azido-[alpha-(32)P]ATP binding and vanadate-trapping assay to examine the behavior of wild-type Ste6p and two different double mutants (G392V/G1087V and G509D/G1193D) shown previously to be mating-deficient in vivo. Both mutants displayed a diminished ability to hydrolyze ATP, with the latter uncoupled from pheromone transport. We conclude that Ste6p catalyzes ATP hydrolysis coupled to a-factor transport, which in turn promotes mating.

ATP-Binding Cassette Transporters↗
PubMed2001

[Surgical palliation of pancreatic carcinoma. Results of a 7 year period].

INTRODUCTION: High perioperative complication rates in the 1980s led to preferred use of endoscopic therapy for surgical palliation of pancreatic cancer. This encouraged us to analyse our own patients retrospectively. MATERIAL AND METHODS: In the period from 1 January 1992 to 31 December 1998, 253 patients with an exocrine carcinoma of the pancreas were operated on at the St. Elisabeth Hospital Cologne-Hohenlind: 73 patients (28.9%) underwent curative resection (R0) while 180 patients (71.1%) had palliative operative treatment (R1/R2). Palliative resection was performed in 22 patients (8.7%). Intestinal bypass surgery was done in 113 patients (44.7%) as a gastrojejunostomy and in 16 patients (6.3%) as a duodenojejunostomy. A biliodigestive anastomosis was performed in 85 patients (33.6%). This procedure was combined with a gastroenterostomy in 78 patients (30.8%). In 18 patients (7.1%) no surgical palliation was possible and the operation finished as a diagnostic laparotomy. RESULTS: The overall mortality rate within the first 30 (60) days was 5.5% (12.7%). Patients whose carcinoma had been resected curatively had a 30 (60)-day mortality rate of 2.7% (4.1%), compared to a rate in palliatively treated patients (resection/bypass/probatoria) of 6.7% (16.1%). Patients with palliatively resected tumor had perioperative mortality of 4.5% (4.5%), whereas patients who did not undergo resection had 6.9% (17.7%). The survival rate for curatively resected patients after Kaplan-Meier extrapolation was 64.7% after 1 year and 31.2% and 26.2% after 3 and 5 years, with a median survival time of 552 days. Palliatively operated patients had a survival rate of 19.4%, 2.5% and 0% for 1, 3 and 5 years. Median survival time was 171 days in this situation. Compared to patients without resection (17.4% and 2.0%), patients with palliative resection had survival rates for 1 and 3 years of 40% und 5.9%. After 5 years none of these patients were alive. CONCLUSIONS: Our data show a high success of surgical palliation in pancreatic cancer in centers with a high frequency of pancreatic surgery. Patients that could not be cured (R1/R2), although undergoing extensive procedures, had better survival rates than patients treated with bypass surgery. Perioperative mortality rate was comparatively low. This justifies aggressive surgical management of pancreatic carcinoma.

Aged↗
PubMed2001

Hsp70 molecular chaperone facilitates endoplasmic reticulum-associated protein degradation of cystic fibrosis transmembrane conductance regulator in yeast.

Membrane and secretory proteins fold in the endoplasmic reticulum (ER), and misfolded proteins may be retained and targeted for ER-associated protein degradation (ERAD). To elucidate the mechanism by which an integral membrane protein in the ER is degraded, we studied the fate of the cystic fibrosis transmembrane conductance regulator (CFTR) in the yeast Saccharomyces cerevisiae. Our data indicate that CFTR resides in the ER and is stabilized in strains defective for proteasome activity or deleted for the ubiquitin-conjugating enzymes Ubc6p and Ubc7p, thus demonstrating that CFTR is a bona fide ERAD substrate in yeast. We also found that heat shock protein 70 (Hsp70), although not required for the degradation of soluble lumenal ERAD substrates, is required to facilitate CFTR turnover. Conversely, calnexin and binding protein (BiP), which are required for the proteolysis of ER lumenal proteins in both yeast and mammals, are dispensable for the degradation of CFTR, suggesting unique mechanisms for the disposal of at least some soluble and integral membrane ERAD substrates in yeast.

Calcium-Binding Proteins↗
PubMed2001

Topological and mutational analysis of Saccharomyces cerevisiae Ste14p, founding member of the isoprenylcysteine carboxyl methyltransferase family.

Eukaryotic proteins that terminate in a CaaX motif undergo three processing events: isoprenylation, C-terminal proteolytic cleavage, and carboxyl methylation. In Saccharomyces cerevisiae, the latter step is mediated by Ste14p, an integral endoplasmic reticulum membrane protein. Ste14p is the founding member of the isoprenylcysteine carboxyl methyltransferase (ICMT) family, whose members share significant sequence homology. Because the physiological substrates of Ste14p, such as Ras and the yeast a-factor precursor, are isoprenylated and reside on the cytosolic side of membranes, the Ste14p residues involved in enzymatic activity are predicted to be cytosolically disposed. In this study, we have investigated the topology of Ste14p by analyzing the protease protection of epitope-tagged versions of Ste14p and the glycosylation status of Ste14p-Suc2p fusions. Our data lead to a topology model in which Ste14p contains six membrane spans, two of which form a helical hairpin. According to this model most of the Ste14p hydrophilic regions are located in the cytosol. We have also generated ste14 mutants by random and site-directed mutagenesis to identify residues of Ste14p that are important for activity. Notably, four of the five loss-of-function mutations arising from random mutagenesis alter residues that are highly conserved among the ICMT family. Finally, we have identified a novel tripartite consensus motif in the C-terminal region of Ste14p. This region is similar among all ICMT family members, two phospholipid methyltransferases, several ergosterol biosynthetic enzymes, and a group of bacterial open reading frames of unknown function. Site-directed and random mutations demonstrate that residues in this region play a critical role in the function of Ste14p.

Amino Acid Sequence↗
PubMed2001

Chromosomal imbalances in gastric cancer. Correlation with histologic subtypes and tumor progression.

DNA copy number changes were analyzed by comparative genomic hybridization (CGH) in 38 gastric carcinomas and correlated with tumor histologic type and progression. Gains of copy numbers were observed in all tumors, affecting all chromosomes except chromosome 16. The average number of copy number gains was 7 (range, 1-13), most frequently located on chromosomes 11, 12, 15, 17, and 20 in 45% to 97% of tumors. High-level amplifications were found on chromosomes 12, 15, 17, and 20; the latter was affected most frequently (66%). Loss of DNA copy numbers was detected in 14 tumors affecting 7 chromosomes. No statistically significant differences in the frequency and pattern of chromosomal imbalances were observed in tumor histologic type (Lauren classification) and grade of differentiation, as well as the prognostic parameters depth of invasion (pT) and lymph node involvement (pN). Our results indicate that in gastric cancer there is no specific recurrent pattern of DNA aberrations to be correlated with tumor histologic type or stage. However, CGH analysis could reveal new, recurrent genetic changes in gastric cancer affecting chromosomes sites that harbor genes known to participate in tumorigenesis and progression of several human malignant neoplasms.

Carcinoma↗
PubMed2000

Reconstitution of the Ste24p-dependent N-terminal proteolytic step in yeast a-factor biogenesis.

The yeast mating pheromone a-factor precursor contains an N-terminal extension and a C-terminal CAAX motif within which multiple posttranslational processing events occur. A recently discovered component in a-factor processing is Ste24p/Afc1p, a multispanning endoplasmic reticulum membrane protein that contains an HEXXH metalloprotease motif. Our in vivo genetic characterization of this protein has demonstrated roles for Ste24p in both the N-terminal and C-terminal proteolytic processing of the a-factor precursor. Here, we present evidence that the N-terminal proteolysis of the a-factor precursor P1 can be accurately reconstituted in vitro using yeast membranes. We show that this activity is dependent on Ste24p and is abolished by mutation of the Ste24p HEXXH metalloprotease motif or by mutation of the a-factor P1 substrate at a residue adjacent to the N-terminal P1 cleavage site. We also demonstrate that N-terminal proteolysis of the P1 a-factor precursor requires Zn(2+) as a co-factor and can be inhibited by the addition of the metalloprotease inhibitor 1,10-orthophenanthroline. Our results are consistent with Ste24p itself being the P1-->P2 a-factor protease or a limiting activator of this activity. Interestingly, we also show that the human Ste24 homolog expressed in yeast can efficiently promote the N-terminal processing of a-factor in vivo and in vitro, thus establishing a-factor as a surrogate substrate in the absence of known human substrates. The results reported here, together with the previously reported in vitro reconstitution of Ste24p-dependent CAAX processing, provide a system for examining the potential bifunctional roles of yeast Ste24p and its homologs.

4-Butyrolactone↗
PubMed1998

Endoplasmic reticulum membrane localization of Rce1p and Ste24p, yeast proteases involved in carboxyl-terminal CAAX protein processing and amino-terminal a-factor cleavage.

Proteins terminating in the CAAX motif, for example Ras and the yeast a-factor mating pheromone, are prenylated, trimmed of their last three amino acids, and carboxyl-methylated. The enzymes that mediate these activities, collectively referred to as CAAX processing components, have been identified genetically in Saccharomyces cerevisiae. Whereas the Ram1p/Ram2p prenyltransferase is a cytosolic soluble enzyme, sequence analysis predicts that the other CAAX processing components, the Rce1p and Ste24p proteases and the Ste14p methyltransferase, contain multiple membrane spans. To determine the intracellular site(s) at which CAAX processing occurs, we have examined the localization of the CAAX proteases Rce1p and Ste24p by subcellular fractionation and indirect immunofluorescence. We find that both of these proteases are associated with the endoplasmic reticulum (ER) membrane. In addition to having a role in CAAX processing, the Ste24p protease catalyzes the first of two cleavage steps that remove the amino-terminal extension from the a-factor precursor, suggesting that the first amino-terminal processing step of a-factor maturation also occurs at the ER membrane. The ER localization of Ste24p is consistent with the presence of a carboxyl-terminal dilysine ER retrieval motif, although we find that mutation of this motif does not result in mislocalization of Ste24p. Because the ER is not the ultimate destination for a-factor or most CAAX proteins, our results imply that a mechanism must exist for the intracellular routing of CAAX proteins from the ER membrane to other cellular sites.

Cell Fractionation↗
PubMed1998

Dual roles for Ste24p in yeast a-factor maturation: NH2-terminal proteolysis and COOH-terminal CAAX processing.

Maturation of the Saccharomyces cerevisiae a-factor precursor involves COOH-terminal CAAX processing (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) followed by cleavage of an NH2-terminal extension (two sequential proteolytic processing steps). The aim of this study is to clarify the precise role of Ste24p, a membrane-spanning zinc metalloprotease, in the proteolytic processing of the a-factor precursor. We demonstrated previously that Ste24p is necessary for the first NH2-terminal processing step by analysis of radiolabeled a-factor intermediates in vivo (Fujimura-Kamada, K., F.J. Nouvet, and S. Michaelis. 1997. J. Cell Biol. 136:271-285). In contrast, using an in vitro protease assay, others showed that Ste24p (Afc1p) and another gene product, Rce1p, share partial overlapping function as COOH-terminal CAAX proteases (Boyartchuk, V.L., M.N. Ashby, and J. Rine. 1997. Science. 275:1796-1800). Here we resolve these apparently conflicting results and provide compelling in vivo evidence that Ste24p indeed functions at two steps of a-factor maturation using two methods. First, direct analysis of a-factor biosynthetic intermediates in the double mutant (ste24Delta rce1Delta) reveals a previously undetected species (P0*) that fails to be COOH terminally processed, consistent with redundant roles for Ste24p and Rce1p in COOH-terminal CAAX processing. Whereas a-factor maturation appears relatively normal in the rce1Delta single mutant, the ste24Delta single mutant accumulates an intermediate that is correctly COOH terminally processed but is defective in cleavage of the NH2-terminal extension, demonstrating that Ste24p is also involved in NH2-terminal processing. Together, these data indicate dual roles for Ste24p and a single role for Rce1p in a-factor processing. Second, by using a novel set of ubiquitin-a-factor fusions to separate the NH2- and COOH-terminal processing events of a-factor maturation, we provide independent evidence for the dual roles of Ste24p. We also report here the isolation of the human (Hs) Ste24p homologue, representing the first human CAAX protease to be cloned. We show that Hs Ste24p complements the mating defect of the yeast double mutant (ste24Delta rce1Delta) strain, implying that like yeast Ste24p, Hs Ste24p can mediate multiple types of proteolytic events.

Amino Acid Sequence↗
PubMed1998

Mammalian prenylcysteine carboxyl methyltransferase is in the endoplasmic reticulum.

Prenylcysteine carboxyl methyltransferase (pcCMT) is the third of three enzymes that posttranslationally modify C-terminal CAAX motifs and thereby target CAAX proteins to the plasma membrane. Here we report the molecular characterization and subcellular localization of the first mammalian (human myeloid) pcCMT. The deduced amino acid sequence of mammalian pcCMT predicts a multiple membrane-spanning protein with homologies to the yeast pcCMT, STE14, and the mammalian band 3 anion transporter. The human gene complemented a ste14 mutant. pcCMT mRNAs were ubiquitously expressed in human tissues. An anti-pcCMT antiserum detected a 33-kDa protein in myeloid cell membranes. Ectopically expressed recombinant pcCMT had enzymatic activity identical to that observed in neutrophil membranes. Mammalian pcCMT was not expressed at the plasma membrane but rather restricted to the endoplasmic reticulum. Thus, the final enzyme in the sequence that modifies CAAX motifs is located in membranes topologically removed from the CAAX protein target membrane.

Amino Acid Sequence↗