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Biomedical subjects

I Becker

Publications and source records attributed to I Becker.

At least 55 records · Page 3Linked to original sources
PubMed1997

Cut out or poke in--the key to the world of single genes: laser micromanipulation as a valuable tool on the look-out for the origin of disease.

The optical micromanipulation systems UV(ultraviolet)-Laser Microbeam and Optical Tweezers Trap, already proven to be powerful tools for 'non-contact' micro-manipulation of gametes, cells and organelles, have now made their way into the nanocosmos of genes and molecules. Force measurements of DNA transcription have been performed and selective DNA molecule micromanipulation gives insight into single molecule behaviour. Retrievement of selected single cells without contamination is an import prerequisite for further processing with modern methods of molecular biology. Laser micro-dissection allows to precisely eliminate any unwanted material or to isolate pieces of chromosomes or single cells of interest with high accuracy and efficiency. This enables the cell or chromosome specific molecular analysis of genes and genetic defects underlying disease, such as cancer or infection. This review article gives an overview of current topics of laser microbeam application in biological or medical research and advanced molecular diagnosis.

Animals↗
PubMed1997

Efficiency of single-cell polymerase chain reaction from stained histologic slides and integrity of DNA in archival tissue.

Molecular analysis of isolated single cells is a powerful tool for studying heterogeneity within a population of cells and for clarifying issues of cell origin and clonality. Here, we investigate the applicability of molecular techniques at a single-cell level by using routinely processed archival tissue. An ultraviolet laser in conjunction with a computer-controlled micromanipulator and a microscope were used for the contamination-free isolation of single tumor cells from stained sections of diffuse-type gastric cancer. A total of 1,328 single cells and 654 clusters of 10-30 cells each, taken from specimens of 14 patients, were analyzed for parts of the E-cadherin gene by the polymerase chain reaction (PCR). With increasing length in base pairs (bp) of the amplified fragments, the efficiency of single-cell PCR as measured by the rate of detectable amplification products declined from approximately 25% (156, 213, and 228 bp) to 14% (246 bp) and 11% (264 and 296 bp). For groups of 10-30 cells, a similar effect was seen at a higher level at 33% (246 bp), 31% (264 bp), and 26% (296 bp), respectively. To our knowledge, this is the first report that has studied the outcome of single-cell PCR on a large systematic scale. The average degree of DNA disintegration in paraffin-embedded, stained tissues was estimated to be approximately 100 bp when the aforementioned data were used in a mathematical model. This study provides evidence that in order to obtain reasonable sensitivity with single-cell PCR, short fragments, preferably < 200 bp long, should be used. Furthermore, whenever applicable, pooling of cells of interest may be another favorable option.

DNA↗
PubMed1996

Entamoeba histolytica: localization of a 30-kDa cysteine proteinase using a monoclonal antibody.

We produced a monoclonal antibody against a major cysteine proteinase of 30kDa from trophozoites of Entamoeba histolytica strain HM1:IMSS. The specificity of the monoclonal antibody was confirmed by specific inhibition of azocasein digestion and by electrophoretic analysis, in the presence of sodium dodecyl sulfate or on a substrate gel, of the antigen precipitated by the antibody. Immunofluorescent staining of trophozoites with the monoclonal antibody revealed heterogeneity in the intensity of whole cell fluorescence and subcellular localization of the stain. The latter was also observed in trophozoites, which were stained by conventional immunohistochemical methods, from experimental liver abscesses in hamsters. Ultrastructural analysis showed antigen distributed mainly in clear amorphous zones in the cytoplasm, which were not limited by a visible membrane. Proteinases are translocated from these compartments to phagocytic vacuoles after trophozoites ingest erythrocytes, suggesting that these regions might be a lysosomal equivalent of this primitive eukaryotic cell.

Animals↗
PubMed1996

Effects of altered phosphoenolpyruvate carboxylase activities on transgenic C3 plant Solanum tuberosum.

Phosphoenolpyruvate carboxylase (PEPC) genes from Corynebacterium glutamicum (cppc), Escherichia coli (eppc) or Flaveria trinervia (fppc) were transferred to Solanum tuberosum. Plant regenerants producing foreign PEPC were identified by Western blot analysis. Maximum PEPC activities measured in eppc and fppc plants grown in the greenhouse were doubled compared to control plants. For cppc a transgenic plant line could be selected which exhibited a fourfold increase in PEPC activity. In the presence of acetyl-CoA, a known activator of the procaryotic PEPC, a sixfold higher activity level was observed. In cppc plants grown in axenic culture PEPC activities were even higher. There was a 6-fold or 12-fold increase in the PEPC activities compared to the controls measured in the absence or presence of acetyl-CoA, respectively. Comparable results were obtained by transient expression in Nicotiana tabacum protoplasts. PEPC of C. glutamicum (PEPC C.g.) in S. tuberosum leaf extracts displays its characteristic K(m) (PEP) value. Plant growth was examined with plants showing high expression of PEPC and, moreover, with a plant cell line expressing an antisense S. tuberosum (anti-sppc) gene. In axenic culture the growth rate of a cppc plant cell line was appreciably diminished, whereas growth rates of an anti-sppc line were similar or slightly higher than in controls. Malate levels were increased in cppc plants and decreased in antisense plants. There were no significant differences in photosynthetic electron transport or steady state CO2 assimilation between control plants and transformants overexpressing PEPC C.g. or anti-sppc plants. However, a prolonged dark treatment resulted in a delayed induction of photosynthetic electron transport in plants with less PEPC. Rates of CO2 release in the dark determined after a 45 min illumination period at a high proton flux density were considerably enhanced in cppc plants and slightly diminished in anti-sppc plants. When CO2 assimilation rates were corrected for estimated rates of mitochondrial respiration in the light, the electron requirement for CO2 assimilation determined in low CO2 was slightly lower in transformants with higher PEPC, whereas transformants with decreased PEPC exhibited an appreciably elevated electron requirement. The CO2 compensation point remained unchanged in plants (cppc) with high PEPC activity, but might be increased in an antisense plant cell line. Stomatal opening was delayed in antisense plants, but was accelerated in plants overexpressing PEPC C.g. compared to the controls.

Amino Acids↗
PubMed1996

Differential expression of chemokines in patients with localized and diffuse cutaneous American leishmaniasis.

The abundance of macrophages in localized cutaneous leishmaniasis (LCL) and diffuse cutaneous leishmaniasis (DCL) lesions and differences in the composition of T cell subsets indicate involvement of cell-specific chemotaxis processes. The expression of macrophage chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1 alpha and -1 beta, RANTES (regulated on activation, normal T cell expressed and secreted), I-309, and interleukin-8 were investigated in lesions of patients with LCL or DCL. In LCL, high levels of MCP-1 and moderate levels of MIP-1 alpha were detected. In DCL, MCP-1 expression was significantly lower and MIP-1 alpha expression was predominant. All other chemokines investigated were minimally expressed or absent. These findings suggest that MCP-1 and MIP-alpha are responsible for the recruitment of macrophages and T cells in cutaneous leishmaniasis. The results show that self-healing LCL is associated with higher levels of MCP-1, which may stimulate macrophage microbicidal mechanisms, and nonhealing DCL is associated with higher levels of MIP-alpha.

Animals↗
PubMed1996

Single-cell mutation analysis of tumors from stained histologic slides.

Formalin-fixed and paraffin-embedded tissues are a valuable resource for diagnosis and research. PCR is one of the most powerful methods of retrospective analysis of the DNA present in fixed tissues. One major problem with the molecular analysis of tissue samples, however, is cellular heterogeneity, ie, the large variety of cell types usually present in these specimens can mask cell-specific genetic alterations associated with disease. Herein we describe a procedure for obtaining and analyzing single cells recovered from stained histologic tissue sections without risking contamination from neighboring cells. An ultraviolet laser microbeam was used to physically destroy the tissue surrounding the single cells of interest. These cells, now freed from adjacent cells, were then easily retrieved with a motorized, computer-controlled micromanipulator and molecularly characterized through the use of PCR-based microanalysis. This accurate microdissection technique, followed by DNA amplification and direct sequencing, revealed a novel mutation in the gene coding for the cell adhesion molecule E-cadherin in single tumor cells that was absent in the adjacent single epithelial cells of a patient with early gastric cancer of the diffuse type. In this form of malignancy, tumor cells lose homophilic cell-to-cell interactions and invade the connective tissue as single cells. E-cadherin gene mutations have previously been detected in advanced diffuse-type gastric cancer and gastric carcinoma cell lines. The present study suggests that E-cadherin gene mutations may be an early event in gastric tumorigenesis. The laser-based isolation and subsequent molecular characterization of individual cells, as described herein, allows for micrometer-sized precision and should prove useful in detecting the nucleic acid abnormalities that underlie cancer, infection, and genetic disease.

Base Sequence↗
PubMed1995

Using evolutionary changes to achieve species-specific inhibition of enzyme action--studies with triosephosphate isomerase.

BACKGROUND: Many studies that attempt to design species-specific drugs focus on differences in the three-dimensional structures of homologous enzymes. The structures of homologous enzymes are generally well conserved especially at the active site, but the amino-acid sequences are often very different. We reasoned that if a non-conserved amino acid is fundamental to the function or stability of an enzyme from one particular species, one should be able to inhibit only the enzyme from that species by using an inhibitor targeted to that residue. We set out to test this hypothesis in a model system. RESULTS: We first identified a non-conserved amino acid (Cys14) whose integrity is important for catalysis in triosephosphate isomerase (TIM) from Trypanosoma brucei. The equivalent residues in rabbit and yeast TIM are Met and Leu, respectively. A Cys14Leu mutant of trypanosomal TIM had a tendency to aggregate, reduced stability and altered kinetics. To model the effects of a molecule targeted to Cys14, we used methyl methanethiosulfonate (MMTS) to derivatize Cys14 to a methyl sulfide. This treatment dramatically inhibited TIMs with a Cys residue at a position equivalent to Cys14, but not rabbit TIM (20% inhibition) or yeast TIM (negligible inhibition), which lack this residue. CONCLUSIONS: Cys14 of trypanosomal TIM is a non-conserved amino acid whose alteration leads to loss of enzyme structure and function. TIMs that have a cysteine residue at position 14 could be selectively inhibited by MMTS. This approach may offer an alternative route to species-specific enzyme inhibition.

Animals↗
PubMed1995

Expression of somatostatin receptor subtypes in cultured astrocytes and gliomas.

Expression of receptors for the neuropeptide somatostatin was investigated in vitro in rat and human astrocytes, glioma cell lines, and solid human glial tumors that were all immunopositive for the astrocytic marker glial fibrillary acidic protein. After affinity labelling with a peptide-gold conjugate of known biological activity, somatostatin-binding sites could be visualized at the light-and electron-microscopic level on the surface of glial cells. Glioma cells were generally labeled more strongly than were normal astrocytes and preferentially bound the ligand at their processes and not at their somata as were normal cells. Somatostatin transmembrane receptor (SSTR) subtype expression was probed by reverse transcription-polymerase chain reaction: In rat and human cortical astrocytes and in one glioma cell line (U 118), a pattern of three subtypes (SSTR-1, SSTR-2, and SSTR-4) was detected, whereas, in all other glioma cell lines and in six solid glial tumors investigated, the SSTR-2 subtype was relatively stronger, expressed either alone or in combination with SSTR-1; sometimes SSTR-3 or SSTR-4 was demonstrated in clearly reduced amounts. In astrocytes and gliomas, somatostatin reduced the levels of cyclic AMP elicited by the adenylate cyclase activator forskolin indicating that at least one of the receptor subtypes is negatively linked to adenylate cyclase. In contrast to other cell types, somatostatin did not inhibit the basal or the fetal calf serum-stimulated proliferation of astrocytes, glioma cell lines, or glial tumors in culture. Thus, strong SSTR-2 subtype expression characterizes glial tumors, but somatostatin is ineffective in inhibiting their growth.

Animals↗
PubMed1994

E-cadherin gene mutations provide clues to diffuse type gastric carcinomas.

The calcium-dependent homophilic cell adhesion molecule and candidate suppressor gene, E (epithelial)-cadherin, plays a major role in the organization and integrity of most epithelial tissues. Diffusely growing gastric carcinomas show markedly reduced homophilic cell-to-cell interactions. We speculated that mutations in the E-cadherin gene may be responsible for the scattered phenotype of this type of carcinoma. For that reason we have examined E-cadherin in 26 diffuse type, 20 intestinal type and 7 mixed gastric carcinomas (Laurén's classification) at the DNA, RNA, and protein levels. Reverse transcription polymerase chain reaction and direct sequencing of amplified E-cadherin complementary DNA fragments revealed inframe skipping of either exon 8 or exon 9 in 10 patients with diffuse tumors and an exon 9 deletion in one patient with a mixed carcinoma; both exons encode putative calcium binding domains. These alterations were not seen in nontumorous gastric tissues. Splice site mutations responsible for the exon deletions were identified in six of these patients, eliminating the possibility of alternative splicing mechanisms. Five of these splice site alterations were confirmed as somatic mutations. Non-splice site mutations were observed in three diffuse type tumors, namely a 69-base pair deletion of exon 10 and two point mutations, one of which destroys a putative calcium binding region. Immunohistochemical evaluation showed E-cadherin immunoreactivity in tumors and lymph node metastases of patients expressing abnormal mRNA. The allelic status of the E-cadherin gene was analyzed in one patient, revealing loss of heterozygosity with retention of a mutated E-cadherin allele. Overall, E-cadherin mutations were identified in 50% (13 of 26) of the diffuse type and in 14% (1 of 7) of the mixed carcinomas. In contrast, two silent E-cadherin mutations (not changing the amino acid sequence) were detected in two tumors of the intestinal type. Our study provides strong in vivo evidence that E-cadherin gene mutations may contribute to the development of diffusely growing gastric carcinomas and support a tumor/metastasis suppressor gene hypothesis.

Base Sequence↗
PubMed1994

Inhibition of viroid infection by antisense RNA expression in transgenic plants.

Complex formation between different antisense RNAs directed against either plus-strand or minus-strand sequences of the potato spindle tuber viroid (PSTVd) was studied using temperature-gradient gel electrophoresis and immunochemical detection with an antibody specific for double-stranded RNA. Short minus-strand sequences were directed against the upper central conserved region (UCCR) of plus-strand viroid replication intermediates, a plus-strand corresponding to the left half of the rod-like secondary structure (VL+) against minus-strand replication intermediates. It was shown that antisense RNA forms complexes with the corresponding target RNA only with low yield during incubation at low (physiological) temperatures but with high yield during in vitro transcription of the target RNA when the antisense RNA is already present in the solution. The antisense RNA sequences were integrated into Solanum tuberosum L. by Agrobacterium tumefaciens transformation. Antisense RNA expression in vivo was analyzed by Northern analysis. Infection tests were performed using the transgenic potato lines in order to evaluate their degree of resistance against PSTVd infection. Although some lines showed a significant inhibition of viroid accumulation, a high variability of viroid infection in different transgenic potato lines was obtained. Since strongly infected plants were observed in all transgenic lines 6 to 8 weeks post inoculation, a threshold concentration of viroid, overcoming the antisense effect has to be assumed. When the rate of viroid accumulation was tested using agroinfection assays on leaf discs, a stronger antisense effect could be achieved.

Agrobacterium tumefaciens↗
PubMed1993

Leukotriene B4 and C4 production in isolated rat gastric mucosal cells.

Dispersed rat gastric mucosal cells (F0) were separated into five fractions (F1-F5) by counterflow elutriation, with F1 representing the smallest and F5 the largest cell diameters. In F0-F5, leukotriene B4 (LTB4) release in response to 10(-5) M calcium ionophore A-23187 was 7.68 +/- 1.26, 51.6 +/- 10.8, 72.4 +/- 10.4, 7.1 +/- 0.7, 5.7 +/- 0.6, and 11.6 +/- 3.4 pg.10(6) cells-1 x 30 min-1. In the identical fractions, sulfidopeptide release in response to A-23187 was 200.6 +/- 20.5, 1,116.0 +/- 166.6, 1,309.4 +/- 163.2, 189.8 +/- 25.8, 108.0 +/- 18.0, and 158.4 +/- 54.0 pg.10(6) cells-1 x 30 min-1. High-pressure liquid chromatography verified the radioimmunologically determined LTB4 and identified LTC4 as the only sulfidopeptide LT released. LT release from F2 cells in response to A-23187 was time and dose dependent, reaching maximal stimulation at 10(-5) M A-23187. This response was blocked by the dual inhibitor of cyclooxygenase and lipoxygenases, BW755C (2 x 10(-5)-2 x 10(-4) M), by the selective 5-lipoxygenase inhibitor L-651,392 (10(-7)-10(-5) M), and by MK-886 (10(-9)-10(-7) M), which blocks translocation of 5-lipoxygenase. The postreceptor stimuli dibutyryl adenosine 3',5'-cyclic monophosphate, forskolin, 12-O-tetradecanoyl-phorbol-13-acetate, and oleyl-acetyl-glycerol failed to induce LT release. However, 10(-4) M arachidonic acid increased basal LT release up to eightfold and increased A-23187-stimulated LT release by an additional 30%.(ABSTRACT TRUNCATED AT 250 WORDS)

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
PubMed1992

Deletions in the short arm of chromosome 8 are present in up to 90% of human colorectal cancer cell lines.

Cytogenetic analyses of human colon cancer cells have revealed non-random deletions in chromosome arm 8p, among other chromosomal changes. By using 8p-specific DNA probes we could identify allelic loss in 87% of colon cancer cell lines. Corresponding analyses in direct preparations of colon tumor tissues revealed a minimal value of 40% of allelic loss but were obstructed in many instances by contaminating normal tissue. These findings add to the number of non-random genetic alterations occurring during colon carcinogenesis.

Alleles↗
PubMed1991

The multidrug-resistance gene MDR1 is expressed in human glial tumors.

The most consistantly reported alteration of multidrug-resistant carcinoma cells is the overexpression of a membrane glycoprotein, termed P-glycoprotein. In this study we examined whether the strong intrinsic chemotherapy resistance of glial tumors might be related to the expression of the MDR1 gene which codes for P-glycoprotein. Fourteen glial tumors were examined immunohistochemically using the monoclonal antibody C219. In addition, RNA samples of 11 of these tumors were analysed using a sensitive Northern blot assay. P-glycoprotein is expressed in all 14 glial tumors; the number of stained tumor cells, however, varied considerably ranging from 0.3% to 15%. There was no correlation between the number of MDR1-positive cells and the histological malignancy. Varying amounts of MDR1 mRNA were detectable in 7 from 11 examined tumors. The results of our study show that the MDR1 gene is expressed in human glial tumors and suggest that the multidrug transporter may contribute to the clinical non-responsiveness of these tumors to chemotherapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Role of leukocytes and amebic proteinases in experimental rat testicular necrosis produced by Entamoeba histolytica.

To investigate the role of amebic proteinases and host leukocytes, we studied amebiasis experimentally in the rat testis. The degree of inflammation and necrosis produced by different strains was correlated with proteinase activity and with zymograms. Intratesticular injection of axenically grown trophozoites of a pathogenic strain (HM-1 of Entamoeba histolytica) produced indistinguishable lesions in normal animals and leukopenic rats (less than 1000 leukocytes/mm3), suggesting that granulocytes do not contribute to the formation of lesions in this model. Testicular lesions produced by five different strains of E. histolytica ranging from highly virulent to almost nonpathogenic were proportional to the proteinase activity of each amebic strain. Inhibition of amebic proteinases in vitro and subsequent injection into the rat testis markedly reduced the inflammatory lesions resulting from highly virulent E. histolytica. The pathogenicity of three other amebae (E. laredo, E. moshkovskii, and E. invadens) was generally proportional to their proteinase activity; however, E. laredo showed high proteinase activity and caused minimal tissue damage. These results suggest that the pathogenic potential of Entamoeba spp. in the rat testis may be related to the type as well as the level of their proteinase activity.

Animals↗
PubMed1991

Animal experiments with a new dura graft (polytetrafluorethylene)--results.

A comparative animal experiment study on rabbits was performed to test a synthetic dura (expanded polytetrafluorethylene = ePTFE = Gore-Tex Surgical Membrane). For the purpose of comparison with the ePTFE implanted in the left hemisphere, lyophilized natural dura (Lyodura) was used and removed from the subjects after two, eight and twelve weeks. There were no rejection reactions. The lyophilized dura showed pronounced environmental reaction, with cellular permeation and indications of revitalization. The ePTFE manifested only slight reaction and remained inert in an enclosing sheath of connective tissue. The biocompatibility and other properties, such as simple handling and resteriliziability, of ePTFE can thus be assessed as positive.

Animals↗
PubMed1991

Pathogenesis of acute experimental amebic liver abscess in hamsters.

The hypothesis was tested that tissue necrosis in acute experimental amebic liver abscess in hamsters is not caused directly by the parasite but rather, indirectly, by the destruction of closely surrounding leukocytes that release their lysosomal enzymes and damage neighboring liver cells. Axenically grown trophozoites of Entamoeba histolytica strain HM-1 were injected intraportally into normal, hypocomplementemic, and leukopenic hamsters, and the lesions were studied histologically 2, 5, 10, and 24 hr after injection. Hypocomplementemia (less than 5% of normal CH50) and leukopenia (less than 1,000 leukocytes/ml) were achieved and sustained for up to 72 hr with repeated intraperitoneal injections of goat anti-hamster C3 and anti-hamster leukocyte antibodies, respectively. Decrease or absence of polymorphonuclear leukocytes in the vicinity of intrahepatic amebas effectively blocked deleterious effects of the parasites on surrounding hepatocytes. We conclude that in acute experimental amebic liver abscess the direct effect of the parasites on hepatocytes and their stroma is not responsible for tissue necrosis, but rather it is due to their indirect action through the destruction of inflammatory cells.

Acute Disease↗
PubMed1990

[Role of leukocytes and amebic proteases in experimental testicular necrosis produced in the rat by Entamoeba histolytica].

We examined the participation of polymorphonuclear leucocytes and amebic proteinases upon tissue damage by means of an experimental model of acute amebic lesions developed in rat's testicle. In leukopenic rats (less than 1,000 leucocytes/ml) intratesticular injection of axenic E. histolytica's trophozoites (HM-1) produced lesions undistinguishable from the normal controls. On the other hand, inhibition of 80% (average) of the proteinase activity by means of previous incubation of the trophozoites with human a2M gave way to minimal inflammatory lesions almost undistinguishable from the controls which were injected with PBS-A. Our data suggest that in this experimental model of acute amebiasis polymorphonuclear leukocytes do not participate in the tissue damage and that amebic proteinases are responsible for Entamoeba histolytica's virulence.

Animals↗