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Biomedical subjects

M Pech

Publications and source records attributed to M Pech.

53 records · Page 3Linked to original sources
PubMed1986

Selective gene amplification in mammalian cells after exposure to 60Co gamma rays, 241Am alpha particles, or uv light.

Simian Virus 40 wild type (SV40)-transformed Chinese hamster embryo cells (Co631) contain about five viral copies integrated per cell genome. These SV40 sequences were used as endogenous indicator genes to study the response of mammalian cells to radiation at the gene level. An increase in copy number was detected by dispersed cell blotting and Southern analysis in combination with specific DNA hybridization. All types of radiation tested induce a 15- to 25-fold amplification of SV40 sequences without producing intact virus. The amplification is dose dependent and increases with time after irradiation: a maximum effect is observed at Day 3 after alpha particle or uv exposure and at Day 6 after gamma-ray exposure. A RBE of 6 can be calculated for alpha particles if amplification rates at Day 3 are compared. However, when the maximum effect is considered independent of time, no difference between different types of radiation is observed. Southern blots of genomic DNA show that not all integrated SV40 sequences are amplified upon radiation. Amplified sequences are found either in restriction fragments of relatively high molecular weight or in unit size fragments. SV40 amplification is selective in that the amplification of other genes, e.g., of alpha-actin, dhfr (dihydrofolate reductase), and of two oncogenes of the ras family (Kirsten ras and Harvey ras), was below detection level.

Alpha Particles↗
PubMed1985

A large section of the gene locus encoding human immunoglobulin variable regions of the kappa type is duplicated.

The structure of a new segment of the gene locus encoding the variable regions of human immunoglobulins of the Kappa type (VK) has been elucidated. This segment (cluster B) encompasses six VK sequences, which belong to three different subgroups and which are arranged in the same transcriptional orientation. Part of cluster B was found to be very similar to another region of the VK gene locus, which was cloned previously (cluster A). Sequence differences between the homologous region of clusters A and B range from 0.2% to 3.7% depending on the position of the VK sequences. The divergence is in the same range for genes and pseudogenes. Hybridization experiments with DNAs from different individuals clearly demonstrate that the two segments are located at different positions within the VK locus and do not represent allelic variants. The sequence homology between clusters A and B is higher than the homology of both clusters to an allelic variant, which is represented by a DNA segment that had been isolated from another individual. These results, together with a report in the literature of two other homologous regions in the VK locus, make it very likely that a major part of even the whole locus is duplicated. In this case, VK gene numbers would be higher than previously estimated on the basis of hybridization studies. An inverse orientation of VK gene clusters would explain published data on rearrangement products in B-cells if an inversion-deletion mechanism is assumed.

Alleles↗
PubMed1984

Immunoglobulin genes of different subgroups are interdigitated within the VK locus.

The variable regions of immunoglobulins are encoded by multigene families which are rearranged during B-cell differentiation. These families were classified in groups and subgroups based on their amino acid sequences. Genes belonging to a distinct subgroup are believed to occur in the genome within clusters. We are investigating the organization of human variable region genes of the kappa type (VK genes, ref. 1) in the germline and found now for the first time that VK sequences of three of the four different subgroups are interdigitated within the VK locus. We present evidence for the interspersion of two VKIII genes and a VKII pseudogene within an array of five VKI genes. All eight VK sequences are arranged in the same orientation. An evolutionary model for the generation of this 'mixed cluster' is discussed.

Biological Evolution↗
PubMed1984

Composite human VK genes and a model of their evolution.

A phage library and two cosmid libraries were screened for human VK genes. Two recombinant phage and four cosmid clones were analysed in detail by restriction mapping and sequencing. Each one contained a single VKI sequence. Two of these six sequences are potentially functional VK genes and four are pseudogenes. Two pseudogenes derived from different genomic DNAs are highly homologous and are therefore either allelic variants or the products of a recent duplication event. Comparisons of our sequences with all fully determined human VKI amino acid and DNA sequences reveal identical segments which at first sight appear like minigenes. But these segments do not coincide with the subregions and some of the segments include both, framework and complementarity determining regions (FR, CDR, ref. 2). The findings may be explained by an evolutionary model generating composite genes by gene conversion and selection.

Alleles↗
PubMed1984

Molecular footprints of human immunoglobulin gene evolution: a new sequence family.

Analysis of the human VK (ref. 2) gene locus led to the detection of a new sequence family (L sequences). Its copy number is in the range of 10(2). The L sequences, which are about 500 bp long, are found as part of the 3' flanking regions of a clustered set of human VKI genes but they occur also separate from the genes. Models are discussed in which L sequences are viewed as molecular footprints of amplification and transposition processes of VK genes.

Amino Acid Sequence↗
PubMed1984

Organization and evolution of a gene cluster for human immunoglobulin variable regions of the kappa type.

An 80,000 base-pair region from the gene locus encoding the variable regions of the human immunoglobulins of the kappa type (V kappa genes) was cloned and analysed. The region comprises five V kappa sequences of subgroup I and one interspersed V kappa pseudogene of subgroup II. The six genes and pseudogenes are arranged at different distances but in the same orientation. The organization of the cluster can be explained by a series of amplification steps; the existence of a V kappa II pseudogene in a V kappa I gene cluster may have been the result of a transposition event; a final duplication step led to a second closely related copy of the cluster. From sequence data for altogether 16,000 base-pairs it appears that gene conversion-like events and subsequent selection contribute to both homogeneity and diversity of the V kappa repertoire.

Amino Acid Sequence↗
PubMed1983

Identification of the messenger (m) RNA coding for the constant fragment (c mu) of the heavy chain with cloned DNA in single cells by in situ hybridization.

Cells from a normal donor, a B-CLL and a T-ALL were labeled with an FITC conjugated rabbit antihuman IgM and hybridized with a rhodamine-conjugated cloned DNA coding for the c mu segment. The cells were measured simultaneously by immunofluorimetry for their surface content of IgM and their amount of m-RNA hybridizing with the cloned DNA. Thus it was possible to compare the transcription of the gene coding for the immunoglobulin mu chain and the expression of IgM in individual cells. It could be shown that besides the immunoglobulin mu containing B-cells, which were expected to contain the respective m-RNA, cells were found which did not express IgM but which were still positive for the m-RNA.

B-Lymphocytes↗
PubMed1982

Comparative study of G2 delay and survival after 241 Americium-alpha and 60cobalt-gamma irradiation.

Survival and G2 dalay following exposure to either 60Cobalt-gamma-rays or 241Americium-alpha-particles were studied in eight mammalian cell lines of human and animal origin including human fibroblasts from normal individuals and from patients with Ataxia telangiectasia or Fanconi's anemia. For both endpoints the effectiveness of alpha particles was greater as compared to gamma-rays. RBE values for G2 delay (4.6-9.2) were in general comparable to RBE values derived from initial slopes of survival curves (RBE alpha) but higher compared to the ratio of mean inactivation doses (RB-DML). Ataxia cells were particularly sensitive to cell killing by gamma-irradiation (D37 = 0.57 Gy), however, showed average sensitivity to alpha-particles of high LET (D37 = 0.30 Gy). With the exception of Ataxia cells, cell killing and G2 delay seem to be related processes if individual cell cycle parameters are taken into account.

Alpha Particles↗
PubMed1979

Nucleotide sequence of a highly repetitive component of rat DNA.

A highly repetitive component of rat DNA which could not yet be enriched by density gradient centrifugation was isolated with the help of the restriction nuclease Sau3AI. This nuclease converted the bulk of the DNA to small fragments and left a repetitive DNA component as large fragments which were subsequently purified by gel filtration and electrophoresis. This DNA component which was termed rat satellite DNA I is composed of tandemly repeated 370 bp blocks. According to sequence analysis the 370 bp repeats consist of alternating 92 and 93 bp units with homologous but not identical sequences. Methylation of CpG residues was correlated to the rate of cleavage by restriction nucleases. Significant homologies exist between the sequences of rat satellite DNA I and satellite DNAs of several other organisms. The divergence of the sequence of rat satellite DNA I was discussed with respect to evolutionary considerations.

Animals↗
PubMed1979

Patchwork structure of a bovine satellite DNA.

According to a previous restriction nuclease analysis, bovine 1.706 satellite DNA (density 1.706 g/cm3 in CsCl) is organized in an unusual structure of superimposed long- and short-range repeats (Streeck and Zachau, 1978). We have now determined the nucleotide sequence of this satellite DNA in both cloned fragments and fragments from the total satellite DNA. Each long-range repeat unit (about 2350 bp) is divided into four segments. Each segment consists of different variants of a basic 23 bp sequence which is itself composed of a dodecanucleotide and a related undecanucleotide. A total of 2400 nucleotides have been sequenced. Detailed analysis of the sequence divergence reveals that both the overall extent of divergence and the frequency of base changes at individual positions of the 23 bp repeats are characteristically different in the various segments. Preferentially methylated sites and a high incidence of symmetry elements are found. In two of the four segments, 22 of 23 bp of the prototype sequence are included in six overlapping elements of dyad symmetry and in a palindrome. A scheme for the evolution of the satellite DNA from a basic dodecanucleotide is proposed which is based on the different degrees of divergence for the various repeats superimposed in this satellite DNA.

Animals↗
PubMedDate not supplied

Thermoablation of liver metastases: efficacy of temporary celiac plexus block.

PURPOSE: To determine the efficacy of celiac plexus block during thermoablation of liver metastases. METHODS: Fifty-five consecutive patients underwent thermoablation therapy of liver tumors by laser-induced thermotherapy. Twenty-nine patients received a temporary celiac plexus block, 26 patients acted as control group. In both groups fentanyl and midazolam were administered intravenously upon request of the patient. The duration of the intervention, consumption of opiates, and individual pain sensations were documented. RESULTS: No complications resulting from the celiac plexus block were recorded. Celiac plexus block significantly reduced the amount of pain medication used during thermoablation therapy of liver tumors (with block, 2.45 mug fentanyl per kg body weight; without block, 3.58 mug fentanyl per kg body weight, p < 0.05; midazolam consumption was not reduced) in patients with metastases < or = 5 mm from the liver capsule. For metastases farther away from the capsule no significant differences in opiate consumption were seen. Celiac plexus block reduced the time for thermoablation significantly (178 min versus 147 min, p < 0.05) no matter how far the metastases were from the liver capsule. Average time needed to set the block was 12 min (range 9-15 min); additional costs for the block were marginal. As expected (as pain medications were given according to individual patients' needs) pain indices did not differ significantly between the two groups. CONCLUSION: In patients with liver metastases < or = 5 mm from the liver capsule, celiac plexus block reduces the amount of opiates necessary, simplifying patient monitoring. In addition celiac plexus block reduces intervention time, with positive effects on overall workflow for all patients.

Analgesics, Opioid↗
PubMedDate not supplied

Percutaneous transjugular direct porto-caval shunt in patients with Budd-Chiari syndrome.

The purpose of the study was to evaluate the feasibility and effectiveness of direct porto-caval shunts in patients with Budd-Chiari syndrome (BCS) in whom there is no access to the hepatic veins during transjugular intrahepatic portosystemic shunt (TIPSS). We included six consecutive patients with fulminant/acute Budd-Chiari syndrome (mean age: 35 years) in whom a conventional TIPSS was not possible due to inaccessible hepatic veins. We performed a direct porto-caval shunt via a transhepatic approach. Patients were followed up by means of clinical examination, laboratory investigations, and Doppler ultrasound. TIPSS implantation from the inferior vena cava (IVC) was successful in all six patients (100%). The median transhepatic shunt length was 9 cm (8-10 cm). No procedure-related complications were observed in our patients. Early shunt occlusion occurred in three out of six patients (50%). In all three of these patients, the stent used to stabilize the shunt ended 1-2 cm before reaching the IVC. All occlusions were successfully recanalized. One of these patients developed recurrent early shunt as well as mesenteric and splenic vein occlusions. She died 7 days after TIPSS placement due to an unmanageable coagulation disorder. The remaining five patients were followed up by planned clinical examination and laboratory investigations (mean follow-up time was 15 months; patient 1 was followed up for 13 months, patient 2 for 14 months, patient 3 for 15 months, and patients 4 and 5 for 16 months) and all displayed a complete and durable resolution of liver failure and ascites without reintervention. In patients with acute liver failure originating from BCS and inaccessible hepatic veins, a direct transhepatic porto-caval shunt can be performed safely and effectively under ultrasound guidance. Future studies in larger patient groups should investigate if the patency of transcaval TIPSS with long transhepatic shunt segments is similar compared to conventional TIPSS via the hepatic vein.

Adult↗
PubMedDate not supplied

Volume changes of experimental carotid sidewall aneurysms due to embolization with liquid embolic agents: a multidetector CT angiography study.

Iodine-containing polyvinyl alcohol polymer (I-PVAL) is a novel precipitating liquid embolic that allows for artifact-free evaluation of CT angiography (CTA). As accurate aneurysm volumetry can be performed with multidetector CTA, we determined volumes of experimental aneurysms before, immediately after, and 4 weeks after embolization of 14 porcine experimental carotid sidewall aneurysms with this liquid embolic. An automated three-dimensional software measurement tool was used for volumetric analysis of volume-rendering CTA data. Furthermore, intra-aneurysmal pressure changes during liquid embolization were measured in four silicone aneurysms and potential polymer volume changes within 4 weeks were assessed in vitro. Liquid embolic injection was performed during temporary balloon occlusion of the aneurysm neck, resulting in a mean occlusion rate of 98.3%. Aneurysms enlarged significantly during embolization by 61.1 +/- 28.9%, whereas a significant shrinkage of 5.6 +/- 2.7% was observed within the follow-up period. Histologic analysis revealed an inflammatory foreign body reaction with partial polymer degradation. In silicone aneurysm models, intra-aneurysmal pressure remained unchanged during liquid embolic injection, whereas balloon inflation resulted in a mean pressure increase of 31.2 +/- 0.7%. No polymer shrinkage was observed in vitro. The aneurysm enlargement noted was presumably due to pressure elevation after balloon inflation, which resulted in dilatation of the weak venous wall of the newly constructed aneurysm--another shortcoming of this experimental aneurysm model. The volume decrease after 4 weeks expressed partial polymer degradation.

Angiography, Digital Subtraction↗
PubMedDate not supplied

Connective tissue growth factor in retrocorneal membranes and corneal scars.

We studied the localization and distribution of connective tissue growth factor (CTGF) in corneal scar tissue and membranes using in situ hybridization in 8 corneas from keratoplasty and 4 normal corneas. Identification of the cells was done with immunohistochemistry for SM-alpha-actin, vimentin, and Lu5. CTGF mRNA was found in activated corneal fibroblasts in 7 of 8 scars, 7 of 8 retrocorneal membranes and 2 subepithelial membranes, whereas the control corneas showed no CTGF mRNA expression. Vimentin was positive in all scars, retrocorneal and subepithelial membranes, SM-alpha-actin in 7 of 8 scars and 6 of 8 retrocorneal membranes. These results suggest that CTGF plays a crucial role in corneal wound healing and membrane formation.

Actins↗