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M Volkenandt

Publications and source records attributed to M Volkenandt.

At least 73 records · Page 4Linked to original sources
PubMed1994

Transfection with a cDNA encoding a Ser31 or Ser34 mutant human dihydrofolate reductase into Chinese hamster ovary and mouse marrow progenitor cells confers methotrexate resistance.

Chinese hamster ovary (CHO) DHFR- cells were converted into the DHFR+ phenotype when they were transfected with a mammalian expression vector carrying human dihydrofolate reductase-encoding cDNAs (DHFR) containing a Ser31 or a Ser34 mutation. Furthermore, transfection of these mutants into wild-type CHO cells resulted in resistance to high levels of methotrexate (MTX), indicating that these human variants can act as dominant selectable markers. Southern blot analysis and polymerase chain reaction amplifications confirmed that the transfected plasmids were integrated into the CHO DNA. Gene copy number analysis revealed that both the Ser3 1 and the Ser3.4 mutants amplifiable when grown in increasing concentrations of MTX. Retrovirus-mediated gene transfer of the Ser31 mutant into mouse marrow progenitor cells also resulted in MTX-resistant CFU-GM (colony-forming unit-granulocyte macrophage) cells.

Animals↗
PubMed1994

Detection of Chlamydia trachomatis in semen by antibody-enzyme immunoassay compared with polymerase chain reaction, antigen-enzyme immunoassay, and urethral cell culture.

OBJECTIVE: To compare the results obtained by four different techniques for the detection of Chlamydia trachomatis in the male genital tract. DESIGN: Prospective study. SETTING: Andrology unit of a university hospital. PATIENTS: Male infertility patients. INTERVENTIONS: Analysis of semen samples and urethral swabs for the presence of C. trachomatis by recombinant antibody-enzyme-linked immunosorbent assay (rELISA), polymerase chain reaction (PCR), antigen-enzyme immunoassay (EIA) and McCoy cell culture. MAIN OUTCOME MEASURE: Detection of C. trachomatis. RESULTS: In 57 of 205 semen samples (27.8%) immunoglobulin A-antibodies against C. trachomatis were found. In contrast, only 1 of 56 semen samples (1.8%) was positive for C. trachomatis-DNA by PCR, only 1 of 139 semen samples (0.7%) was positive by antigen-EIA, and only 4 of 173 urethral swabs (2.3%) grew C. trachomatis in cell culture. CONCLUSIONS: The discrepancy of positive results found by the antibody-rELISA and direct methods for the detection of C. trachomatis indicates successful eradication of the microorganism in > 90% of antibody-positive men. Therefore, detection of antibodies against C. trachomatis in seminal plasma appears to be of limited diagnostic value.

Base Sequence↗
PubMed1994

p53 protein in benign and malignant sweat gland tumors.

Mutation of the p53 gene and increased levels of p53 protein are among the most frequent alterations in human cancers. To date, very little is known about the mechanisms underlying the development of sweat gland carcinomas. In this study, we analyzed 43 benign and 39 malignant sweat gland tumors for p53 protein level using the antibody PAB1801. Nine (23%) of 39 sweat gland carcinomas were positive for p53 protein. Among these carcinomas, six of 12 cases of extramammary Paget's disease were positive using immunohistochemistry. No other correlation between tumor subtype and p53 reactivity was detected. Among 43 benign sweat gland tumors, only one case displayed staining for p53. We conclude that p53 protein plays a role in a subset of sweat gland tumors, especially in extramammary Paget's disease.

Adenocarcinoma↗
PubMed1994

bcl-2 protein expression and correlation with the interchromosomal 14;18 translocation in cutaneous lymphomas and pseudolymphomas.

The interchromosomal 14;18 translocation occurs in approximately 70-80% of follicular lymphomas and in a lower proportion of high-grade non-Hodgkin lymphomas of the lymph nodes. This translocation results in the fusion of the bcl-2 oncogene on chromosome 18 with immunoglobulin heavy chain genes on chromosome 14, and in the expression of higher amounts of normal bcl-2 protein. We studied bcl-2 expression in biopsies of 108 patients with benign and malignant cutaneous lymphoproliferative diseases (B-cell lymphoma, primary cutaneous, 42; secondary cutaneous, 21; primary cutaneous T-cell lymphoma, 21; B-cell pseudolymphoma, 24), using a monoclonal anti-bcl-2 antibody on paraffin-embedded tissue sections, bcl-2 protein was detected immunohistochemically in 16 of 63 cases of cutaneous B-cell lymphoma, whereas cutaneous T-cell lymphomas and B-cell pseudolymphomas were negative. The proportion of bcl-2 protein expression was significantly higher in secondary (11/21) than in primary cutaneous B-cell lymphomas (5/42; chi 2 test, p < 0.001). Biopsies from 25 of these patients (B-cell lymphoma, 22; B-cell pseudolymphoma, three) were analyzed previously on the molecular level for the t(14;18), using polymerase chain reaction amplification of DNA obtained from paraffin-embedded sections. In four of 11 cases of bcl-2 protein-positive B-cell lymphoma (primary, one; secondary, three) the t(14;18) was detected by polymerase chain reaction. All other cases of B-cell lymphoma, including seven cases where bcl-2 protein was detected by immunohistology, and B-cell pseudolymphoma were negative. These results demonstrate: 1) bcl-2 protein is expressed in a small portion of cutaneous B-cell lymphomas; 2) bcl-2 protein expression is significantly more frequent in secondary than in primary cutaneous B-cell lymphoma; 3) only approximately one-third of cases expressing the bcl-2 protein are characterized also by the t(14;18). bcl-2 protein expression might indicate that the cutaneous manifestation of the lymphoma represents a secondary spread from a node-based lymphoma.

Blotting, Southern↗
PubMed1994

Molecular subtyping of Borrelia burgdorferi in erythema migrans and acrodermatitis chronica atrophicans.

Recently, three subtypes of Borrelia burgdorferi have been identified: Borrelia burgdorferi sensu stricto, Borrelia garinii, and the VS 461 group of Borrelia burgdorferi. These subtypes differ by nucleotide sequence variations within several Borrelia burgdorferi specific genes and most likely by their pathogenetic potential. To assess whether different subtypes of Borrelia burgdorferi might be associated with different cutaneous manifestations and clinical courses of Lyme disease, lesional skin biopsies from 35 patients with erythema migrans and 18 patients with acrodermatitis chronica atrophicans were analyzed. A Borrelia burgdorferi specific gene segment encoding a 26-kD protein with subtype specific nucleotide sequence variations was amplified by a nested polymerase chain reaction technique. For molecular subtyping, the products were transcribed into complementary RNA. Upon polyacrylamide gel electrophoresis, complementary RNA molecules separate into several metastable conformational forms resulting in patterns of bands highly specific for the nucleotide sequence of the transcribed molecules. In biopsy specimens of erythema migrans, the VS 461 subtype was detected in 28 of 35 and the Borrelia garinii subtype in six of 35 cases. In one of 35 cases of erythema migrans Borrelia burgdorferi sensu stricto as well as Borrelia garinii was detected. In contrast, in all 18 biopsies of acrodermatitis chronica atrophicans, only the VS 461 subtype was identified. This subtype is rarely found in the USA, where acrodermatitis chronica atrophicans is almost unknown. These data indicate that acrodermatitis chronica atrophicans might be closely associated with the VS 461 group of Borrelia burgdorferi.

Acrodermatitis↗
PubMed1994

Detection of clonal T-cell populations in gastrointestinal lymphomas by analysis of cRNA conformational polymorphisms of rearranged T-cell-receptor-gamma genes.

Analysis of complementary RNA molecules of junctional regions of rearranged T-cell-receptor-gamma genes show a pattern of conformational polymorphisms which is specific for an individual lymphocytic clone. In a blinded study we analysed formalin-fixed, paraffin-embedded histological specimens from gastrointestinal lymphomas and control tissues (lymphomas: pleomorphic T-cell 10, anaplastic large cell [Ki1+] 9, centroblastic 5, immunocytoma 1, B-CLL 2, Hodgkin's 2, centroblastic-centrocytic 1, MALT [mucosa associated lymphoid tissue] 1, T-cell acute lymphoblastic leukaemia 1, non-lymphoid or polyclonal lymphoid tissues 5). Junctional regions of rearranged TCR-gamma genes were amplified by the polymerase chain reaction and the products were transcribed into cRNA. Conformational patterns of cRNA molecules were analysed by polyacrylamide gel electrophoresis. 13/20 T-lineage lymphomas and the T-cell acute lymphocytic leukaemia displayed a distinct cRNA band pattern, all B-lineage lymphomas and the non-lymphoid control tissues were negative. Only one case of nasopharyngeal (lymphoepithelial, Schmincke-Regaud) carcinoma showed a faint cRNA banding pattern. This novel and non-radioactive assay allows for the rapid detection and molecular characterization of clonal lymphoid populations in minute histological biopsy specimens.

Base Sequence↗
PubMed1994

Detection of monoclonal lymphoid cell populations by polymerase chain reaction technology.

Molecular studies have substantially broadened our understanding of cutaneous lymphoproliferative disorders and have been introduced in the routine assessment of lesions suggestive of a lymphoma. As compared with traditional Southern blot analyses, PCR-based assays for the detection of monoclonal lymphoid populations in clinical specimens offer several advantages. In general, they are faster to perform and allow for the detailed molecular analysis of very small tissue specimens, including sections from formalin-fixed, paraffin-embedded biopsy specimens. The PCR-based assays for the analysis of CTCL involve the amplification of highly variable junctional sequences of rearranged TCR-gamma genes, which represent a unique molecular marker for an individual lymphoid cell and a clonal disease developing from this cell. Using various strategies and gel electrophoretic systems, the PCR products can be separated to detect subsets of identical amplification products representing identical gene rearrangements in a clonal lymphoid population in the analyzed specimen. Although the molecular evidence for a monoclonal cell population as provided by PCR technology is an important factor in the diagnosis of a cutaneous lymphoma, only the synopsis of all available clinical, histomorphologic, immunohistochemical, and molecular data will lead to the adequate assessment of a cutaneous lesion.

Clone Cells↗
PubMed1994

Molecular detection and characterization of clonal cell populations in acute lymphocytic leukemia by analysis of conformational polymorphisms of cRNA molecules of rearranged T-cell-receptor-gamma and immunoglobulin heavy-chain genes.

Junctional regions of rearranged T-cell-receptor-gamma (TCR) and immunoglobulin heavy-chain (IgH) genes represent an idiotypic DNA sequence for an individual lymphocytic cell, and any clone or clonal disease developing from this cell. In this study, a novel methodology for detection and characterization of clone specific DNA sequences in patients with acute lymphocytic leukemia (ALL) was developed. Junctional regions of rearranged TCR-gamma IgH genes in specimens of bone marrow aspirates of patients with ALL (precursor-B-ALL ten, T-ALL two, null-ALL one; ALL not classified one), of a patient with lymphoid blast crisis of chronic myeloid leukemia, of a B-cell chronic lymphocytic leukemia, and in DNA from peripheral blood mononuclear cells of ten healthy volunteers were amplified by polymerase chain reaction (PCR). The PCR products were transcribed into complementary RNA (cRNA). Conformational polymorphisms of cRNA molecules were analyzed by non-denaturing polyacrylamide gel electrophoresis. A specific cRNA banding pattern for rearranged TCR-gamma or IgH genes was observed in all patients with lymphocytic leukemia. In contrast, analysis of DNA from healthy volunteers yielded a smear of confluent polymorphisms representing multiple different cRNA molecules. In two patients with precursor-B-ALL, cRNA banding patterns of junctional regions of rearranged TCR-gamma genes were analyzed in sequential bone marrow aspirates. The banding patterns disappeared after chemotherapy and achievement of blast clearance. This novel and rapid molecular assay offers several advantages as compared to Southern blot analyses and previous PCR based methodologies for the detection of clonal lymphocytic populations. With this methodology, studies on the clonal evolution of lymphoproliferative disorders (e.g. the prognostic significance of the emergence of additional clones) can be performed more easily than with any other traditional molecular method.

B-Lymphocytes↗
PubMed1994

Abnormalities of p53 protein expression in cutaneous disorders.

Abnormalities in the p53 gene and in expression of its protein product are among the most frequent changes demonstrated in a variety of human cancers. p53 Is a nuclear phosphoprotein that in the natural form or "wild-type" can bind to DNA and prevent cells from entering into the S phase of the cell cycle. There is an increase in wild-type p53 after exposure of the skin to UV light, which allows for DNA repair before replication that would make DNA damage permanent. A loss of this protective influence destabilizes the genome. Mutation of the p53 gene commonly causes a defective protein that is degraded more slowly and accumulates in the cell to the extent that it becomes detectable by routine immunocytochemistry. These abnormalities precede the development of cancer in some examples. Studies of precursor lesions have used mainly immunohistochemical techniques that show p53 protein overexpression. The relationship between such overexpression and actual mutation of the p53 gene is controversial because overexpression of "wild-type" p53 protein also can occur. Mutations in the p53 gene have been observed in many actinic keratoses, basal cell carcinomas, and squamous cell carcinomas, and in a small proportion of malignant melanomas. Specific types of pyrimidine transitions have pointed to a role for UV light in these mutations. Molecular analysis is needed to determine whether or not immunocytochemical staining is truly reflective of mutation or is due to some other mechanism that causes an increased expression of wild-type p53.

Gene Expression↗
PubMed1993

Methotrexate resistance in an in vivo mouse tumor due to a non-active-site dihydrofolate reductase mutation.

A series of methotrexate (MTX)-resistant L1210 leukemia murine ascites tumors were developed in vivo and analyzed for drug resistance. Three of 20 tumors studied expressed an altered dihydrofolate reductase (DHFR) and each was identical, having a C to T base transition at nucleotide 46 in the DHFR gene as demonstrated by PCR and direct sequencing. This transition results in a Gly to Trp substitution at amino acid 15 of the enzyme. Purified altered enzyme displays significantly lower binding affinity for the antifolates MTX, trimetrexate, edatrexate, and trimethoprim with respective Ki values 165-, 76-, 30-, and 28-fold higher than values obtained for enzyme isolated from parental tumor (wild-type enzyme). Substrate (dihydrofolate) and cofactor (NADPH) binding is also diminished for the mutant enzyme, although to a lesser extent (17.3- and 3.6-fold higher Km, respectively). Gly-15 is highly conserved for all vertebrate species of DHFR but has no known interaction(s), either directly or indirectly, with bound cofactor, substrate, or inhibitor. Protein molecular modeling reveals that the affected residue is 9-12 A away from the enzyme active site and located in a region analogous to the mobile Met-20 loop domain characterized for Escherichia coli DHFR.

Amino Acid Sequence↗
PubMed1993

Molecular evidence for a clonal relationship between lymphomatoid papulosis and Ki-1 positive large cell anaplastic lymphoma.

Currently, considerable controversy surrounds questions about the clonal evolution of lymphomas in patients with lymphomatoid papulosis. In order to analyze a possible clonal relationship between lesions of lymphomatoid papulosis and a Ki 1+ large cell anaplastic lymphoma in the same patient, a highly specific molecular probe for the malignant lymphoid clone of the large cell anaplastic lymphoma was developed. As a clone specific molecular marker, highly variable junctional sequences of rearranged T-cell receptor-gamma genes were used. An oligonucleotide primer complementary to these sequences was synthesized and, using the polymerase chain reaction, clone specific DNA was detected in all lesions of lymphomatoid papulosis of the patient. These results provide evidence for a clonal relationship between lesions of lymphomatoid papulosis and large cell anaplastic lymphoma developing in the same patient.

Aged↗
PubMed1993

The polymerase chain reaction. Method and applications in dermatopathology.

Since it was first reported in 1985, the polymerase chain reaction (PCR) has revolutionized the way molecular studies are performed, and has developed into one of the most powerful tools in molecular pathology. By use of a cyclic change of temperature, a specific and exponential in vitro amplification of a target DNA sequence can be achieved within hours. As a template for PCR reactions, total genomic DNA is used; this can be readily extracted from clinical specimens. Very low quantities of DNA, as well as DNA degraded by fixation, can also be used as a template for PCR reactions, allowing formalin-fixed, paraffin-embedded tissue to become amenable to detailed molecular analysis. Sequences specific for certain viruses and other microorganisms, as well as molecular marker sequences associated with various types of human cancer, can be readily detected in paraffin-embedded tissue sections. The methodology of PCR, along with various applications in dermatopathology, are reviewed.

DNA↗