[Plant lipoxygenases--their structures and functions].
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Amino Acid Sequence↗
Biomedical subjects
D Shibata
Publications and source records attributed to D Shibata.
Low temperature cultivation of Escherichia coli carrying a rice lipoxygenase L-2 cDNA produces a soluble and active enzyme at a high level.
Using a T7 RNA polymerase promoter system, rice lipoxygenase L-2 cDNA was expressed in E. coli as a fusion protein with 18 amino acid residues at the amino terminal end of the original enzyme. Incubation at 37 degrees C for 3 h in the presence of the inducer resulted in the production of inactive lipoxygenase. However, when induction was carried out at 15 degrees C for 16 h, active lipoxygenase, amounting to 3% of the total soluble protein, was produced. The enzyme was purified by ammonium sulfate precipitation and Mono-Q column chromatography to homogeneity at a yield of 80%. Expression of this protein should permit future site-directed mutagenesis of the gene and crystallization of the enzyme.
Amino Acid Sequence↗
Detection of c-K-ras mutations in fine needle aspirates from human pancreatic adenocarcinomas.
Formalin-fixed paraffin-embedded tissue specimens obtained by fine needle aspiration of pancreatic masses from 47 patients were examined retrospectively for cytology and the presence of mutant c-K-ras oncogenes. Point mutations of c-K-ras in codon 12 were detected by RNA-DNA RNAse A mismatch cleavage after in vitro DNA amplification of the cellular c-K-ras sequences by the polymerase chain reaction. Of the 36 patients with pancreatic adenocarcinoma, mutant c-K-ras oncogenes were detected in 18 of 25 (72%) with malignant cytologies, 2 of 8 (25%) with atypical cytologies, and 0 of 3 with benign aspiration cytologies. The remaining 11 patients without pancreatic adenocarcinomas did not have mutant c-K-ras genes detectable by the assay. The diagnosis of pancreatic adenocarcinoma was based upon clinical follow-up. The presence of mutant c-K-ras oncogenes did not significantly affect survival in the patients studied. Mutant c-K-ras genes were found at the time of initial clinical presentation in the majority of pancreatic adenocarcinomas, suggesting an important role of the mutation in oncogenesis. In conjunction with cytology, our approach represents an application for cancer diagnosis at the molecular genetic level.
Adenocarcinoma↗
Phytochrome-mediated activation of the gene for cytosolic glutamine-synthetase (GS1) during imbibition of photosensitive lettuce seeds.
A full-length cDNA encoding glutamine synthetase was isolated from a lambda gt11 library constructed from the poly(A)+ RNA isolated from lettuce seeds incubated under red light. The nucleotide sequence of the cDNA and the deduced sequence of amino acids showed a high degree of homology to those of the cytosol-type glutamine synthetase from other plants. Northern and dot-blot analyses of poly(A)+ RNA extracted from the seeds incubated under various light conditions showed that the activation of the gene for cytosolic glutamine-synthetase during imbibition of lettuce seeds is directly or indirectly regulated by phytochrome.
Amino Acid Sequence↗
Detection of specific t(14;18) chromosomal translocations in fixed tissues.
The present study was undertaken to establish the incidence of t(14;18) (q32:q21) chromosomal translocations detectable by a polymerase chain reaction (PCR) assay on fixed lymphoma biopsies. DNA samples from 113 formalin-fixed, paraffin-embedded tissue biopsies (non-Hodgkin's lymphomas, 96 cases; Hodgkin's disease, six cases; reactive, 11 cases) were amplified by the PCR. Of the 96 non-Hodgkin's lymphoma cases, 56 had a follicular pattern and 40 had a diffuse pattern. Polymerase chain reaction-amplifiable t(14;18) chromosomal translocations were detected in 23 of 43 follicular low-grade lymphomas, one of eight follicular intermediate grade lymphomas, one of five follicular high-grade lymphomas, and one of 10 diffuse large-cell lymphomas. The remaining 30 diffuse lymphomas represented the spectrum of the Working Formulation classification. There were six biopsy specimens of Hodgkin's disease and 11 biopsy specimens of follicular hyperplasia; all were negative. The translocation was not detected in 16 biopsies (non-Hodgkin's lymphomas, seven cases; follicular hyperplasia, nine cases) from patients infected with the human immunodeficiency virus. Since this procedure uses the widely available fixed paraffin-embedded material, correlative studies between histology and genetic aberrations can be readily undertaken.
Chromosomes, Human, Pair 14↗
Mutational activation of the c-K-ras gene in human pancreatic carcinoma.
We have reported the presence of c-K-ras oncogenes activated by single point mutations at codon 12 in a vast majority of human pancreatic carcinomas. Formalin-fixed, paraffin-embedded specimens from surgical resections, autopsies and biopsies were used as well as snap frozen surgical specimens. The high oncogene incidence has been confirmed in other studies and indicate that somatic mutational activation of the c-K-ras gene is an important event in the development, maintenance or progression of cancer of the exocrine pancreas. While the role that these point mutations play in any or all of these processes remains to be determined, their presence is useful clinically for the diagnosis of pancreatic carcinoma at the molecular genetic level. The detection of mutated c-K-ras oncogenes in fine needle aspirates of pancreatic masses, that by cytomorphology may be suspicious but not diagnostic of malignant disease, increases the sensitivity of the diagnosis for this cancer. The identification of codon 12 mutations in the c-K-ras gene in pancreatic adenocarcinomas has been possible by advances in recombinant DNA techniques, especially by the development of in vitro gene amplification by the polymerase chain reaction (PCR). The possibility of analysing formalin-fixed, paraffin-embedded tissue for the presence of genetic alterations as small as single point mutations by PCR in concert with other mutation detection techniques, should facilitate the molecular genetic analysis of pancreatic carcinoma. Retrospective studies using stored specimens are now feasible with the technology described and should yield important information on the molecular epidemiology and aetiology of this and other diseases.
Adenocarcinoma↗
Identification of a mislabeled fixed specimen by DNA analysis.
Formalin-fixed, paraffin embedded surgical specimens can be identified by genotypic analysis. DNA was extracted from single sections of fixed tissue and amplified by the polymerase chain reaction for the human leukocyte antigen (HLA) DQ alpha and low density lipoprotein receptor genes. These two loci are polymorphic and fixed specimens can be distinguished based on differences between these alleles. The examination can be performed in 2 days and the HLA DQ alpha test utilized a nonisotopic color detection method. In this case report, a mislabeled specimen was identified as a section from a mastectomy because they had identical genotypes. Molecular genetic analysis of the DNA present in fixed specimens can yield information not otherwise evident from the histology.
Breast↗
Molecular Cloning and Ethylene Induction of mRNA Encoding a Phytoalexin Elicitor-Releasing Factor, beta-1,3-Endoglucanase, in Soybean.
Soybean (Glycine max) beta-1,3-endoglucanase (EC 3.2. 1.39) is involved in one of the earliest plant-pathogen interactions that may lead to active disease resistance by releasing elicitor-active carbohydrates from the cell walls of fungal pathogens. Ethylene induced beta-1,3-endoglucanase activity to 2- to 3-fold higher levels in cotyledons of soybean seedlings. A specific polyclonal antiserum raised against purified soybean beta-1,3-endoglucanase was used to immunoprecipitate in vitro translation products, demonstrating that ethylene induction increased translatable beta-1,3-endoglucanase mRNA. Several cDNA clones for the endoglucanase gene were obtained by antibody screening of a lambda-gt11 expression library prepared from soybean cotyledons. Hybrid-select translation experiments indicated that the cloned cDNA encoded a 36-kilodalton precursor protein product that was specifically immunoprecipitated with beta-1,3-endoglucanase antiserum. Escherichia coli cells expressing the cloned cDNA also synthesized an immunologically positive protein. Nucleotide sequence of three independent clones revealed a single uninterrupted open reading frame of 1041 nucleotides, corresponding to a polypeptide of 347 residue long. The primary amino acid sequence of beta-1,3-endoglucanase as deduced from the nucleotide sequence was confirmed by direct amino acid sequencing of trypsin digests of the glucanase. The soybean beta-1,3-endoglucanase exhibited 53% amino acid homology to a beta-1,3-glucanase cloned from cultured tobacco cells and 48% homology to a beta-(1,3-1,4)-glucanase from barley. Utilizing the largest cloned cDNA (pEG488) as a hybridization probe, it was found that the increase in translatable beta-1,3-endoglucanase mRNA seen upon ethylene treatment of soybean seedlings was due to 50- to 100-fold increase in steady state mRNA levels, indicating that ethylene regulates gene expression of this enzyme important in disease resistance at the level of gene transcription.
Journal Article↗
Detection of occult CNS involvement of follicular small cleaved lymphoma by the polymerase chain reaction.
A patient with follicular small cleaved lymphoma presented with an unusual clinical relapse in the central nervous system (CNS) without morphologic evidence of lymphoma cells in the cerebral spinal fluid (CSF). Molecular genetic analysis of the small number of cells in the CSF after in vitro DNA amplification by the polymerase chain reaction demonstrated the presence of an abnormal translocation sequence between chromosomes 14 and 18. A similar translocation could be detected from the original fixed archival lymph node biopsy and from a small proportion of circulating mononuclear cells. These results indicated that occult lymphoma cells were present in the CSF and peripheral blood. Secondary CNS lymphoma involvement was identified at autopsy. This case demonstrates the enhanced sensitivity of lymphoma diagnosis from poorly cellular specimens after in vitro DNA amplification.
Aged↗
Human T-cell lymphotropic virus type I (HTLV-I)-associated adult T-cell leukemia-lymphoma in a patient infected with human immunodeficiency virus type 1 (HIV-1).
A patient had adult T-cell leukemia-lymphoma in the unusual setting of coinfection with human immunodeficiency virus type 1 (HIV-1) and human T-cell lymphotropic virus type I (HTLV-I). The leukemic cells were CD4 positive and showed clonal genetic rearrangement of the T-cell receptor complex. Cytogenetic analysis showed three clonal karyotypic abnormalities: trisomy 3 and two translocations [t(1;15), (X;1)]. The patient was seropositive for HIV and HTLV-I; HTLV-I and HIV-1 DNA sequences were detected in peripheral blood leukocytes by the polymerase chain reaction. The HTLV-I sequences were detected in a relatively high proportion of mononuclear cells (at least 1 in 30 cells), whereas HIV-1 sequences were detected in a smaller proportion of cells (at least 1 in 3000 cells). Clinical remission was achieved after chemotherapy. There was a decrease in the proportion of HTLV-I positive mononuclear cells (at least 1 in 1000 cells), whereas the proportion of HIV-1 positive cells was relatively unchanged (at least 1 in 1000 cells). Adult T-cell leukemia-lymphoma in the setting of HIV coinfection may become increasingly common because asymptomatic retroviral coinfections are frequent.
Adult↗
Detection of human papillomavirus DNA in fine-needle aspirations of metastatic squamous-cell carcinoma of the uterine cervix using the polymerase chain reaction.
Routinely processed fine-needle aspirations of metastatic squamous-cell carcinoma (SCC) were analyzed for human papillomavirus type 16 (HPV-16) using the polymerase chain reaction (PCR), an in vitro DNA amplification method. HPV-16 DNA was detected in five of seven (71%) metastases from SCC of the uterine cervix. In two cases in which the primary tumor was available for comparison, the HPV-16 DNA content of the primary tumor and of the metastasis was identical. HPV-16 was not found in a metastatic SCC from the lung or in a metastatic nasopharyngeal SCC. These findings demonstrate that HPV-16 DNA sequences can be readily detected in routinely processed fine-needle aspirations using the polymerase chain reaction. The finding of HPV-16 DNA in a metastasis may serve to direct a search for a primary site of origin.
Adolescent↗
Three cDNA sequences coding for glutamine synthetase polypeptides in Oryza sativa L.
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Amino Acid Sequence↗
Detection of human papillomavirus in formalin-fixed, invasive squamous carcinomas using the polymerase chain reaction.
We analyzed 88 formalin-fixed, paraffin-embedded invasive squamous carcinomas for human papillomavirus-related DNA sequences (HPV types 16 and 18) following in vitro gene amplification using the polymerase chain reaction. HPV DNA sequences were found in 35 of 50 (70%) carcinomas of the anogenital region, including four of four (100%) anal, six of eight (75%) vulvar, nine of 14 (64%) vaginal, two of five (40%) penile, and 14 of 19 (74%) cervical tumors. Nine of 25 (36%) oropharyngeal squamous carcinomas contained HPV DNA sequences, including four of 10 (40%) laryngeal, three of eight (38%) buccal, and two of seven (29%) glossal tumors. HPV DNA sequences were not found in 13 esophageal carcinomas. Of the 44 cases that contained viral DNA, HPV-16 was detected in 41 cases (93%) and HPV-18 in five cases (11%), while both types were found in two cases (one anal and one vulvar). HPV DNA sequences were found in 43 of 83 (52%) nonverrucous and in one of five (20%) verrucous carcinomas, but this difference was not significant. These findings demonstrate that HPV DNA sequences are more frequently associated with anogenital than oropharyngeal squamous carcinomas and can be readily detected in routinely processed tissues using the polymerase chain reaction.
Blotting, Southern↗
Analysis of human immunodeficiency virus and cytomegalovirus infection by polymerase chain reaction in the acquired immunodeficiency syndrome. An autopsy study.
Fixed autopsy tissues from nine patients infected with the human immunodeficiency virus (HIV) were assayed for the presence of HIV provirus and/or cytomegalovirus by the polymerase chain reaction. The HIV provirus was detected in lymphoid tissues from all nine patients. With nonlymphoid tissue, HIV was detected 32% (9/28) of tissues with chronic inflammation and in 7% (4/57) of tissues without chronic inflammation. Cytomegalovirus was detected in tissues from six of the nine patients, often in the absence of inclusions, and was most often detected in the adrenal gland and lung. Widely disseminated cytomegalovirus infection was present in three patients with characteristic cytomegalovirus inclusions. A similar pattern of widely disseminated HIV was not identified. These studies correlate histologic features with the presence of specific viral sequences in HIV-infected patients.
Acquired Immunodeficiency Syndrome↗
Postmortem enzyme immunoassay for human immunodeficiency virus.
The reliability of postmortem enzyme immunoassay testing for antibody to human immunodeficiency virus (HIV) was assessed in blood and vitreous humor. Vitreous humor tested up to 34 hours post mortem and blood tested at least 58 days post mortem were consistently repeatedly positive for HIV antibody in patients with histopathologic criteria and premortem HIV test results that satisfied the Centers for Disease Control definition of acquired immunodeficiency syndrome (AIDS). There were no false-negative results in serum specimens, although one third of vitreous specimens that were tested 34 hours or more post mortem were negative in patients with AIDS. There were no false-positive results in control specimens from patients at autopsy without evidence of AIDS, despite prolonged postmortem intervals producing hemolysis and autolysis of the specimens. Postmortem enzyme immunoassay testing of blood and vitreous humor may be useful to screen for HIV infection in high-risk groups and for diagnosis of patients with histopathologic evidence of AIDS for medical, legal, and epidemiologic purposes.
Acquired Immunodeficiency Syndrome↗
Human immunodeficiency viral DNA is readily found in lymph node biopsies from seropositive individuals. Analysis of fixed tissue using the polymerase chain reaction.
Human immunodeficiency virus (HIV) DNA was detected in formalin-fixed, paraffin-embedded lymph node biopsies after in vitro DNA amplification by the polymerase chain reaction. Twenty-three of 25 biopsies from HIV seropositive individuals were positive for HIV DNA including 11 with follicular hyperplasia, six with follicular involution, two who were partially involved with Kaposi's sarcoma, one with granulomatous lymphadenitis, and three with non-Hodgkin's lymphoma. The remaining two biopsies from seropositive individuals lacking detectable HIV DNA also contained non-Hodgkin's lymphomas. An average of 0.0001 to 0.01 HIV DNA copies per cell was estimated to be present in biopsies with follicular hyperplasia or involution. The positive lymphoma biopsies contained approximately tenfold fewer HIV DNA. In contrast, 19 of 20 biopsies from seronegative or low risk individuals were negative for HIV DNA. The sole exception was a seronegative individual with chronic adenopathy from follicular hyperplasia and a history of prostitute contact. The studies demonstrated a high prevalence of HIV DNA in non-lymphomatous lymph node biopsies from HIV infected individuals.
Base Sequence↗
Analysis of DNA sequences in forty-year-old paraffin-embedded thin-tissue sections: a bridge between molecular biology and classical histology.
DNA sequences from human tissues paraffin embedded 40 yr ago were studied using the in vitro gene amplification technique known as the polymerase chain reaction. Although significant DNA degradation was observed, single copy genomic sequences and viral segments were readily detected from single 5- to 10-microns tissue sections. This demonstrates that the world-wide collection of archival paraffin-embedded tissues may be used to study the association of biological agents (viral, bacterial, or parasitic) or endogenous DNA lesions with disease over time and to carry out retrospective studies on material where the clinical outcome has already been established. This will be especially valuable in studying rare cancers and other rare diseases.
Base Sequence↗
Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes.
Using in vitro gene amplification by the polymerase chain reaction (PCR) and mutation detection by the RNAase A mismatch cleavage method, we have examined c-K-ras genes in human pancreatic carcinomas. We used frozen tumor specimens and single 5 micron sections from formalin-fixed, paraffin-embedded tumor tissue surgically removed or obtained at autopsy. Twenty-one out of 22 carcinomas of the exocrine pancreas contained c-K-ras genes with mutations at codon 12. In seven cases tested, the mutation was present in both primary tumors and their corresponding metastases. No mutations were detected in normal tissue from the same cancer patients or in five gall bladder carcinomas. We conclude from these results that c-K-ras somatic mutational activation is a critical event in the oncogenesis of most, if not all, human cancers of the exocrine pancreas.
Adenocarcinoma↗