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Biomedical subjects

M Simon

Publications and source records attributed to M Simon.

At least 235 records · Page 13Linked to original sources
PubMed1995

Allelic losses on chromosomes 14, 10, and 1 in atypical and malignant meningiomas: a genetic model of meningioma progression.

To investigate chromosomal events that underlie formation and progression of meningiomas, we have examined a set of 18 benign (WHO grade I), 15 atypical (grade II), and 13 anaplastic/malignant (grade III) meningiomas for loss of heterozygosity (LOH) on chromosomes 1p, 6p, 9q, 10q, and 14q. Frequent loss of loci on these chromosomes was seen in grade II and grade III tumors, specifically, 14q (II and III, 47 and 55%), 1p (40 and 70%), and 10q (27 and 40%). In contrast, LOH for these loci was infrequent in benign meningiomas, specifically, 14q (0%), 1p (11%), and 10q (12%). The smallest common regions of deletion that could be defined were 14q24-q32, 1p32-pter, and 10q24-qter. These observations indicate the likely presence of tumor suppressor genes in these regions that are involved in the development of WHO grade II and grade III meningiomas. Because LOH for loci on chromosomes 1p and 10q was found in tumors of all grades and because the frequency of LOH in all three regions increased with tumor grade, these results would support a model for the formation of aggressive meningiomas through tumor progression.

Adult↗
PubMed1995

Structural requirements of peptide and MHC for DR(alpha, beta 1*0401)-restricted T cell antigen recognition.

We identified functionally important regions of the DR(alpha, beta 1*0401) peptide binding site and present a model of bound peptide. DR(alpha, beta 1*0401)-restricted T cell recognition and peptide binding of Mycobacterium leprae (ML) peptide 38-50 and overlapping peptides from the 18-kDa heat-shock protein were analyzed. ML38-50 is unusual in its restricted binding pattern, binding to only one of five DR4 subtypes and no other DR molecules tested. Amino acid substitutions were introduced into ML38-50 and the DR(alpha, beta 1*0401) peptide binding site at positions likely to influence peptide-MHC or peptide- or MHC-TCR interactions. Peptide binding, T cell proliferation, and computer modeling studies suggest that residues 39F, 42E, and 44D of ML38-50 interact with pockets 1, 4, and 6, respectively, of the peptide binding site. Only DR(alpha, beta 1*0401) substitutions at residues in pockets 4 or 7 prevented binding of ML38-50, while multiple substitutions at other positions negatively affected its T cell recognition. In contrast, T cell recognition of some high affinity ML peptides that overlapped ML38-50, and contained N-terminal extensions, was only abolished with pocket 4 substitutions. An inverse correlation of peptide affinity for DR(alpha, beta 1*0401) with negative effects of MHC substitutions on T cell recognition of the overlapping ML peptides was observed. Thus, some regions, such as pocket 4, dominantly influence T cell recognition of multiple DR(alpha, beta 1*0401)-binding peptides. However, each DR(alpha, beta 1*0401)-binding peptide appears to have unique properties that determine the outcome of its MHC-peptide interactions and the relative importance of other polymorphic pockets.

Amino Acid Sequence↗
PubMed1995

Conservative surgery and adjuvant radiation therapy in the management of adult soft tissue sarcoma of the extremities: clinical and radiobiological results.

PURPOSE: The outcome of adult patients with soft tissue sarcoma of the extremities treated with conservative surgery and adjuvant irradiation was evaluated to (a) determine the appropriate treatment volume and radiation dosage in the postoperative setting, and (b) correlate in vitro radiobiological parameters obtained prior to therapy with clinical outcome. METHODS AND MATERIALS: Sixty-four consecutive adult patients with soft tissue sarcoma of the extremities (40 lower, 24 upper) who underwent conservative surgery and adjuvant irradiation 7 preoperative, 50 postoperative, 7 perioperative) between 1978 and 1991 were reviewed. The initial radiation field margin surrounding the tumor bed/scar was retrospectively analyzed in all postoperative patients. Initial field margins were < 5 cm in 12 patients, 5-9.9 cm in 32 and > or = 10 cm in 6. Patients with negative pathological margins were initially treated with traditional postoperative doses (64-66 Gy); however, in later years the postoperative dose was reduced to 60 Gy. Thirteen cell lines were established prior to definite therapy, and radiobiological parameters (multitarget and linear-quadratic) were obtained and correlated with outcome. RESULTS: Postoperative patients treated with an initial field margin of < 5 cm had a 5-year local control of 30.4% vs. 93.2% in patients treated with an initial margin of > or = 5 cm (p = 0.0003). Five-year local control rates were similar in patients treated with initial field margins of 5-9.9 cm (91.6%) compared with those treated with > or = 10 cm margins (100%) (p = 0.49). While postoperative patients receiving < 60 Gy had a worse local control than those receiving > or = 60 Gy (p = 0.08), no difference was seen in local control between patients receiving less than traditional postoperative doses (60-63.9 Gy) (74.4% vs. those receiving 64-66 Gy (87.0%) (p = 0.5). The local control of patients treated in the later years of the study, with strict attention to surgical and radiotherapeutic technique, was 87.6%. Severe late sequelae were more frequent in patients treated with doses > or = 63 Gy compared to patients treated with lower doses (23.1% vs. 0%) (p < 0.05). Mean values for Do, alpha, beta, D, n and SF2 obtained from the 13 cell lines were 115.7, 0.66, 0.029, 2.15, 0.262, respectively. Four of the 13 cell lines established prior to therapy ultimately failed locally. The radiobiological parameters of these cell lines were similar to the other nine cell lines in terms of radiosensitivity. CONCLUSIONS: Our data confirm the importance of maintaining an initial field margin of at least 5 cm around the tumor bed/scar in the postoperative setting. No benefit was seen with the use of margins > or = 10 cm. In addition, patients undergoing wide local excision with negative margins can be treated with lower than traditional postoperative doses (60 Gy) without compromising local control and with fewer chronic sequelae. Finally, it does not appear that inherent tumor cell sensitivity is a major determinant of local failure following radiation therapy and conservative surgery in soft tissue sarcoma.

Adolescent↗
PubMed1995

The KIN28 gene is required both for RNA polymerase II mediated transcription and phosphorylation of the Rpb1p CTD.

Kin28p, associated with cyclin Ccl1p, is a putative cyclin-dependent kinase (CDK) of the p34cdc2 family in Saccharomyces cerevisiae. Search for mutations co-lethal (syn mutations) with a kin28 thermosensitive mutation (kin28-ts3) has uncovered genetic interactions between gene KIN28 and genes RAD3, SIN4, STI1 and CDC37. The genetic interaction between KIN28 and the CDC37 cell division cycle gene suggests that a connection exists between the activity of CDK-Kin28p and cell-cycle progression. Both RAD3 and SIN4 gene products are implicated in the RNA polymerase II transcription process. Here we show that RNA polymerase II transcription is drastically reduced in a kin28-ts mutant, at restrictive temperature. This impairment correlates with a markedly decreased phosphorylation of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Rpb1p). Thus, the Kin28 gene product is required in vivo for RNA polymerase II phosphorylation and transcriptional activity as recently suggested by experiments using an in vitro reconstituted system.

Cyclin-Dependent Kinases↗
PubMed1995

Metabolic regulation of active retinoid concentrations in cultured human epidermal keratinocytes by exogenous fatty acids.

Retinol metabolism was examined in cultured human epidermal keratinocytes incubated with medium containing physiological concentrations of bovine serum albumin-free fatty acid complexes and [3H]retinol. Unsaturated 16- and 18-carbon fatty acids: (1) promoted up to a 50% increase in the steady state total cell retinoid mass which was accounted for as retinyl ester corresponding to the added fatty acid, (2) decreased the rate of endogenous retinyl ester utilization up to 80%, (3) decreased the steady state cellular concentrations of retinol, 3,4-didehydroretinol and their respective carboxylic acids up to 80%, and (4) did not alter the rate of retinoic acid metabolism. Increasing the medium retinol concentration augmented the fatty acid-stimulated increase in esters and blunted the extent of decrease in alcohols and acids. Saturated fatty acids with 14, 16, 17, and 18 carbons also reduced the rate of retinyl ester utilization and the cellular concentrations of the retinoid alcohols and acids but had little to no effect on retinyl ester synthesis. The data demonstrate that exogenous fatty acids possess the capacity to regulate the cellular concentrations of substrate retinoid alcohols, active retinoid acids, and the overall rate of retinol metabolism via substrate-mediated stimulation of retinol esterification and decreasing retinyl ester utilization. Fatty acids thus possess the potential to play a physiological role in modulating retinoid signaling in keratinocytes by regulating cellular concentrations of active retinoids.

Cells, Cultured↗
PubMed1995

A second nitrogen permease regulator in Saccharomyces cerevisiae.

We describe a Saccharomyces cerevisiae mutant affected in its urea and proline transport capacities, and a gene coding for a protein complementing this mutation. This protein is not membrane-embedded and contains two PEST sequences, often found in regulatory factors. The mRNA is not down-regulated under nitrogen catabolite repression, and is induced by urea and proline. In the mutant, the PUT4 mRNA encoding the proline permease is not affected, whereas the DUR3 mRNA, involved in urea active transport, is strongly increased. Our data suggest that this protein is a post-transcriptional regulator of nitrogen permeases.

Amino Acid Sequence↗
PubMed1995

Suppressors of thermosensitive mutations in the DNA polymerase delta gene of Saccharomyces cerevisiae.

DNA polymerases (Pol) alpha, delta and epsilon are necessary for replication of nuclear DNA. Pol delta interacts permanently or transiently with numerous accessory proteins whose identification may shed light on the function(s) of Pol delta. In vitro mutagenesis was used to induce thermosensitive (ts) mutations in the DNA polymerase delta gene (POL3). We have attempted to clone two recessive extragenic suppressors of such ts mutants (sdp1 for mutation pol3-14 and sdp5-1 for mutation pol3-11) by transforming thermoresistant haploid strains pol3-14 sdp1 and pol3-11 sdp5-1 with wild-type genomic libraries in singlecopy or multicopy vectors. None of the thermosensitive transformants so obtained was identified as being sdp1 or sdp5-1. Instead, three genes were cloned whose products interfere with the activity of suppressors. One of them is the type 1 protein phosphatase gene, DIS2. Another is a novel gene, ASM4, whose gene product is rich in asparagine and glutamine residues.

Amino Acid Sequence↗
PubMed1995

Expression of vascular endothelial growth factor in renal vascular disease and renal allografts.

Vascular endothelial growth factor (VEGF) is a dimeric glycoprotein that exerts a proliferative effect specifically on endothelial cells. VEGF can increase vascular permeability and collagenase activity, is chemotactic for monocytes, and may dilate blood vessels. It can be induced by phorbol ester and cAMP in both mesenchymal and epithelial cells. In vitro cell culture experiments suggest that VEGF is upregulated by oxygen deprivation. In this study we tested whether in vivo acute and/or chronic reduction of renal blood flow by vascular obstruction would result in increased expression of VEGF mRNA and protein. Three normal kidneys, five human kidneys with narrowing of preglomerular vessels by vascular rejection or by vasculitis, and eight kidneys with nephrosclerosis and/or diabetic nephropathy were examined. In situ hybridization with 35S-labelled riboprobes showed a pronounced expression of VEGF mRNA in acutely hypoxic proximal and distal tubules of both the cortex and medulla; VEGF protein was demonstrated in the epithelia of these tubules by immunohistochemistry. In kidneys with chronically reduced blood flow, the majority of atrophic tubules were negative for VEGF mRNA and protein, although interstitial cells expressed VEGF mRNA. In arcuate arteries showing intimal and adventitial fibrosis, some medial smooth muscle cells were positive for VEGF mRNA. In glomeruli with segmental sclerosis, viable podocytes showed a prominent signal for VEGF mRNA. Mesangial cells did not express VEGF in the cases studied. It is possible that hypoxia itself led to the upregulation of VEGF in tubular epithelia and vascular smooth muscle cells. The vasodilatory and permeability-promoting effects of the endothelial growth factor produced by damaged tubular epithelia may constitute a mechanism to alleviate a decrease in blood flow and substrate availability and to re-establish vascular integrity.

Acute Disease↗
PubMed1995

Stage-specific induction of terminal deoxynucleotidyl transferase in a T-lymphoid line upon coculture with a thymic stromal line.

We previously reported an in vitro T-cell differentiation system in which the L4 lymphoid clone was cocultured with the St3 stromal line derived from the same murine thymic tumor, 15#4T.L4 cells in L4-St3 cocultures sequentially express Thy-1 and CD4 in a manner typical of normal thymocytes. In contrast, L4 cells grown in medium alone retain their Thy-1-CD4- phenotype. We also isolated L4 subclones from the coculture with increasingly differentiated phenotypes with respect to Thy-1 and CD4. We now report induction of an additional thymocyte differentiation marker, terminal deoxynucleotidyl transferase (TdT) in 15#4T cells (and to a lesser extent subcloned L4 cells) upon coculture with St3 stroma. Coculture of 15#4T cells with St3 stroma resulted in expression of TdT as measured by ribonuclease protection for TdT RNA and Western immunoblotting for TdT protein. Cocultured L4 cells were induced for TdT expression to a lesser degree and for a shorter period of time. The magnitude of TdT RNA induction was maximal for cell lines with the least mature differentiation phenotype (15#4T and L4: Thy-1-CD4-) and decreased proportionally for subclones with increasingly mature phenotype, e.g., L4E cells (Thy-1+CD4+). TdT protein was undetectable by Western immunoblotting and immunofluorescent staining of the L4E subclone on or off stroma. Recombination-activating gene-1 (RAG-1), which is expressed in immature thymocytes during T-cell receptor rearrangement, but suppressed in mature thymocytes, was also examined using the ribonuclease protection assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Topology of the pore-region of a K+ channel revealed by the NMR-derived structures of scorpion toxins.

The architecture of the pore-region of a voltage-gated K+ channel, Kv1.3, was probed using four high affinity scorpion toxins as molecular calipers. We established the structural relatedness of these toxins by solving the structures of kaliotoxin and margatoxin and comparing them with the published structure of charybdotoxin; a homology model of noxiustoxin was then developed. Complementary mutagenesis of Kv1.3 and these toxins, combined with electrostatic compliance and thermodynamic mutant cycle analyses, allowed us to identify multiple toxin-channel interactions. Our analyses reveal the existence of a shallow vestibule at the external entrance to the pore. This vestibule is approximately 28-32 A wide at its outer margin, approximately 28-34 A wide at its base, and approximately 4-8 A deep. The pore is 9-14 A wide at its external entrance and tapers to a width of 4-5 A at a depth of approximately 5-7 A from the vestibule. This structural information should directly aid in developing topological models of the pores of related ion channels and facilitate therapeutic drug design.

Amino Acid Sequence↗
PubMed1995

Expression of N-cadherin and alpha-catenin in astrocytomas and glioblastomas.

We examined levels of mRNA and protein for N-cadherin, the predominant cadherin in neural tissues, and mRNA levels for the cadherin-associated protein, alpha-catenin, in a series of gliomas and in glioblastoma cell lines. mRNA levels for N-cadherin and alpha-catenin were significantly higher in glioblastomas than in low-grade astrocytomas or normal brain, while the levels of intact N-cadherin protein were similar in glioblastomas, low-grade astrocytomas and brain. In addition, there was no consistent relationship between invasiveness and expression of N-cadherin and alpha-catenin in highly invasive vs minimally invasive tumours within the same histopathological grade. To assess further the relationship between cadherin expression and neural tumour invasion, we measured N-cadherin expression, calcium-dependent cell adhesion and motility of several glioblastoma cell lines. While all N-cadherin-expressing lines were adhesive, no correlation was seen between the level of N-cadherin expression and cell motility. Together, these findings imply that, in contrast to the role played by E-cadherin in carcinomas, N-cadherin does not restrict the invasion of glioblastomas.

Astrocytoma↗
PubMed1995

Monoclonal antibodies to human epidermal filaggrin, some not recognizing profilaggrin.

To improve understanding of human profilaggrin processing to filaggrin, we produced seven monoclonal antibodies against epidermal filaggrin (AHF1-7). They were characterized on human epidermis by indirect immunofluorescence, immunogold labeling, and immunoblotting and found to be directed against seven different epitopes of (pro)filaggrin. AHF1-5 labeled the keratohyalin granules and the fibrous matrix of the lower corneocytes, and recognized filaggrin and profilaggrin. AHF6 also labeled the keratohyalin granules and the corneocyte matrix, but only recognized filaggrin. In addition to this reactivity within the upper epidermis, AHF4-6 stained the cytoplasm of the basal cells, and cross-reactivity of AHF5 and AHF6 with cytokeratin K14 was revealed on immunoblots. It is interesting that AHF7 recognized filaggrin, but not profilaggrin, and labeled only the corneocyte matrix and not the keratohyalin granules. This indicates that filaggrin and cytokeratins share several antigenic determinants and that filaggrin bears at least one epitope absent from its precursor. The original series of monoclonal antibodies described here appears to be a powerful tool for studying human profilaggrin processing in normal conditions and in the keratinization disorders in which processing is altered.

Antibodies, Monoclonal↗
PubMed1995

The rheumatoid arthritis-associated autoantibodies to filaggrin label the fibrous matrix of the cornified cells but not the profilaggrin-containing keratohyalin granules in human epidermis.

Since they were first described, serum IgG antibodies to the stratum corneum of rat oesophagus epithelium, highly specific for rheumatoid arthritis (RA), have been consensually called antikeratin antibodies (AKA). However, we recently demonstrated that they actually recognize three new proteins of rat oesophagus epithelium distinct from cytokeratins, and also human epidermal filaggrin. In this work we provided further evidence that AKA and RA-associated anti-filaggrin autoantibodies are the same antibodies. Moreover, analysing by indirect immunofluorescence on human skin a large series of 212 well characterized RA sera and anti-filaggrin autoantibodies purified from RA sera by affinity chromatography, we demonstrated the specific binding of AKA to the stratum corneum of human epidermis and the absence of any staining of the granular keratinocytes. This binding was confirmed and the AKA antigen precisely localized in human epidermis by immunoelectron microscopy. The antigen was found to be restricted to the filaggrin-containing intracellular fibrous matrix of the corneocytes, up to the desquamating cells. In contrast, MoAbs directed to human filaggrin and to profilaggrin, its precursor, not only stained the intracellular matrix of the lower corneocytes but also the keratohyalin granules of the granular cells, where profilaggrin is stored. These results reinforced by the absence of immunoblotting reactivity of RA sera to profilaggrin suggest that the epitopes recognized by AKA are absent from profilaggrin. Their identification may provide more insight into the pathogenesis of RA.

Adult↗