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Biomedical subjects

F Lin

Publications and source records attributed to F Lin.

At least 127 records · Page 7Linked to original sources
PubMed1996

Reverse transcription/polymerase chain reaction (RT/PCR) amplification of very small numbers of transcripts: the risk in misinterpreting negative results.

Technical modifications of the reverse-transcription/polymerase chain reaction (RT/PCR) amplification method now permit its use to detect amplified products from as few as one abnormal cell, either isolated or mixed with a larger number of normal cells. We studied the reproducibility of such results using as targets low numbers of cells from chronic myeloid leukaemia (CML) patients and CML cell lines in quintuplicate two-step RT/PCR designed to amplify BCR-ABL sequences. When one K562 or KYO1 cell was diluted in 10(3) non-CML HL60 cells, an amplification product was obtained in each test; at greater dilutions BCR-ABL transcripts were detected erratically. Titration of cDNA synthesised from 5 x 10(7) cells from four CML patients showed that whereas positive BCR-ABL sequences could be amplified in some tests starting with as little as a 1 in 10(7) dilution of cDNA template (corresponding to 5-10 cells), the dilution threshold for reproducible amplification was around 1 to 5 in 10(5) (100-500 cells). Quantitative PCR analysis revealed that reactions from 1 in 10(7) diluted cDNA contained less than 10 BCR-ABL transcripts as the starting template. The stochastic nature of the amplification from such small numbers of transcripts was illustrated by results of 10 replicate PCR tests on cDNA from a patient expressing both b3a2 and b2a2 transcripts: dilutions of cDNA up to 1 in 10(5) yielded dual transcript amplification in all 10 tests, but the 1 in 10(7) cDNA dilution resulted in b3a2 and b2a2 products in three tests, b3a2 only in three, b2a2 only in one and no amplification in three tests. We conclude that this 'sampling effect' may yield false-negative results and thus misinterpretation of data regarding assessment of gene expression when the quantity of target material available for study is very small.

False Negative Reactions↗
PubMed1996

Magnetic resonance imaging of primary osteosarcoma of the lumbar vertebra with metastasis to the perirenal fat.

Primary osteosarcoma of the spine not associated with any predisposing factor is rare. The authors describe a case of osteosarcoma of the lumbar spine in a 44-year-old woman with biopsy-proven ulcerative colitis. The patient underwent bone scanning, computed tomography (CT) and magnetic resonance imaging (MRI), and the authors present the first record of the MRI appearance of this disease in a vertebral body. Relative to classical osteosarcoma of long bone, this case involved a higher degree of soft-tissue ossification, which was readily visible with both CT and MRI. The patient had extensive metastasis, including metastasis to the perirenal fat, and died 10 months after presentation.

Adipose Tissue↗
PubMed1996

Reconstitution with Philadelphia chromosome-negative recipient haematopoiesis early after allogeneic BMT for CML.

We report a 33-year-old man with Ph chromosome-positive CML who underwent an allogeneic BMT from an unrelated donor. DNA microsatellite studies showed complete donor chimaerism immediately after BMT followed by mixed chimaerism; by day + 45 haematopoiesis was exclusively of recipient origin. Throughout the first year post-transplant all marrow metaphases were Ph negative but with non-clonal rearrangements consistent with autologous recovery. Cytogenetic relapse of leukaemia was first detected 15 months post-transplant. This case is unusual in that non-malignant stem cells of recipient origin survived the transplant and reconstituted haematopoiesis very early after BMT. Later the leukaemic cells reasserted their 'proliferative' advantage.

Adult↗
PubMed1996

Quantitative molecular methods to monitor the response of CML patients to interferon-alpha.

Interferon-alpha (IFN-alpha) induces cytogenetic responses of variable degree in patients with CML. We sought to establish the relationship between BCR-ABL transcript numbers measured by competitive two-step reverse transcription polymerase chain reaction (RT-PCR) and cytogenetic status in CML patients treated with IFN-alpha. All 305 samples from 133 patients investigated by RT-PCR were positive for BCR-ABL transcripts. In order to standardize results for variability in RNA and cDNA quality, we quantified total ABL transcripts in each sample as an internal control. The BCR-ABL : ABL ratios correlated well with the cytogenetic results. Quantitative nested PCR allowed the detection of residual BCR-ABL transcripts in all complete cytogenetic responders on IFN-alpha. We conclude that competitive PCR with internal controls is a reliable method for monitoring patients on IFN-alpha and reduces the need for repeated marrow investigations.

Antineoplastic Agents↗
PubMed1996

Molecular analysis of transient cytogenetic relapse after allogeneic bone marrow transplantation for chronic myeloid leukaemia.

Serial quantification of residual disease in CML patients after allogeneic BMT is useful for early detection of relapse. However, the fact that some cytogenetic relapses appear to be transient may complicate protocols for early therapeutic intervention based on molecular analysis and could result in the unnecessary treatment of some patients. To determine the frequency and significance of transient cytogenetic relapse, we have studied serial samples from 98 CML patients after allogeneic BMT by conventional cytogenetics and competitive RT-PCR for BCR-ABL mRNA. During the period of study, 26 patients had cytogenetic or haematologic evidence or relapse. In four cases (15% of those who relapsed; 4% of all patients) relapse appeared to be transient; i.e., subsequent marrow samples were completely Ph chromosome-negative despite the fact that there had been no change in treatment, including the level of immunosuppression. BCR-ABL mRNA levels broadly paralleled the cytogenetic findings. Of these four patients, two subsequently progressed to frank haematologic relapse and two remained strongly positive for BCR-ABL transcripts and are therefore presumably still at risk of relapse. Analysis of B cell-enriched, T cell-enriched and lymphoid-depleted fractions for three patients demonstrated that transient relapse was not due to the proliferations of BCR-ABL-positive lymphoid cells. In contrast, BCR-ABL-positive myeloid precursor cells were detected in two of three patients tested. We conclude that transient cytogenetic relapse followed by sustained remission is a relatively infrequent occurrence after current allogeneic transplant regimens.

Adult↗
PubMed1996

Synthesis and characterization of human gene 1 relaxin peptides.

The peptide encoded for by one of the two relaxin genes found in the human genome, designated H1, has been synthesized by the Boc-polystyrene solid phase method. The two chains which constitute relaxin, A- and B-, were assembled separately and, after cleavage, deprotection and purification, combined in solution at high pH to form the one intra- and two intermolecular disulfide bonds. Comprehensive chemical characterization including ion spray mass spectrometry of the peptide confirmed both its correct identity and high purity. The synthetic H1 relaxin was analyzed by circular dichroism spectroscopy and shown to possess a greater alpha-helical conformation in water than the corresponding H2 relaxin. The peptide had powerful direct chronotropic and inotropic effects in the isolated rat heart assay as did an analogue of the peptide in which the C-terminus of the B-chain was extended by four residues.

Amino Acid Sequence↗
PubMed1996

New marker for blood vessels in human ovarian and endometrial cancers.

Angiogenesis plays a critical role in tumor biology and may someday be a target for novel therapeutic interventions. To date, however, relatively few markers have been identified that can specifically distinguish between microvessels in benign versus malignant lesions. Here we report that the cationic heme-protein eosinophil peroxidase (EPO) was localized by in situ immunohistochemistry on the vascular endothelial cells and/or connective tissue stroma in 16 of 16 cases of human endometrial carcinoma and in 12 of 15 cases of ovarian carcinoma. Similar deposits of EPO were not detected in normal endometrial tissues or ovaries from five healthy subjects, in adjacent uninvolved tissues from four tumor-bearing subjects, or in any normal organs from five other subjects. These findings imply that eosinophil degranulation is a significant and previously unappreciated component of the interaction between ovarian and endometrial cancers and the host. Moreover, the abundant and highly specific nature of the EPO deposition near and within the microvessels of these cancers suggests that eosinophil degranulation is a new marker for tumor blood vessels that potentially could be exploited to treat these important types of cancers that currently lack highly effective therapies.

Adenocarcinoma↗
PubMed1995

CD and NMR determination of the solution structure of a peptide corresponding to T4 lysozyme residues 38-51.

Solid phase methods have been used to synthesise a peptide corresponding to residues 38-51 of T4 lysozyme. The peptide, LYS(38-51), encompasses helix B in the crystal structure of T4 lysozyme. CD and 1H-NMR analysis showed that the peptide was unstructured in aqueous solution but adopted a helical conformation in the more hydrophobic environment provided by 50% TFE and SDS micelles. The solution structure derived from the NMR data was similar to that of the helix in the X-ray structure, although there was some fraying at the N-terminus.

Bacteriophage T4↗
PubMed1995

Selenoperoxidase-dependent glutathione cycle activity in peroxide-challenged leukemia cells.

Murine leukemia L1210 cells rendered deficient in glutathione peroxidase (GPX) and phospholipid hydroperoxide glutathione peroxidase (PHGPX) by Se deprivation (L.Se(-) cells) were found to be more sensitive to tert-butyl hydroperoxide (t-BuOOH) cytotoxicity than Se-replete controls (L.Se(+) cells). Human K562 cells, which express PHGPX, but not GPX, were also more sensitive to t-BuOOH in the Se-deficient (K.Se(-)) than Se-satisfied (K.Se(+)) condition. In examining the metabolic basis for selenoperoxidase-dependent resistance, we found that glucose-replete Se(-) cells reduce t-BuOOH to t-butanol far more slowly than Se(+) cells, the ratio of the first-order rate constants approximating that of the GPX activities (L1210 cells) or PHGPX activities (K562 cells). Monitoring peroxide-induced changes in GSH and GSSG gave consistent results; e.g., glucose-depleted L.Se(+) cells exhibited a first order loss of GSH that was substantially faster than that of glucose-depleted L.Se(-) cells. Under the conditions used, peroxide-induced conversion of GSH to GSSG could be stoichiometrically reversed by resupplying D-glucose, indicating that no significant lysis or GSSG efflux and/or interchange had taken place. The apparent first-order rate constant for GSH decay increased progressively for L1210 cells expressing a range of GPX activities from approximately 5% to 100%, demonstrating that peroxide detoxification is strictly dependent on enzyme content. The initial rate of 14CO2 release from D-[1-14C]glucose supplied in the medium was much greater for L.Se(+) or K.Se(+) cells than for their respective Se(-) counterparts, consistent with greater hexose monophosphate shunt activity in the former. These results highlight the importance of selenoperoxidase action in the glutathione cycle as a means by which tumor cells cope with hydroperoxide stress.

Animals↗
PubMed1995

Structural organization of the human gene (LMNB1) encoding nuclear lamin B1.

We have determined the structural organization of the human gene (LMNB1) that encodes nuclear lamin B1, an intermediate filament protein of the nuclear envelope. The transcription unit spans more than 45 kb and the transcription start site is 348 nucleotides upstream from the translation initiation codon. Lamin B1 is encoded by 11 exons. Exon 1 codes for the amino-terminal head domain and the first portion of the central rod domain, exons 2 through 6 the central rod domain, and exons 7 through 11 the carboxyl-terminal tail domain of this intermediate filament protein. Intron positions are conserved in other lamin genes from frogs, mice, and humans but different in lamin genes from Drosophila melanogaster and Caenorhabditis elegans. In the region encoding the central rod domain, intron positions are also similar to those in the gene for an invertebrate nonneuronal cytoplasmic intermediate filament protein and the genes for most vertebrate cytoplasmic intermediate filament proteins except neurofilaments and nestin.

Animals↗
PubMed1995

Amplification and hyperexpression of the catalase gene in selenoperoxidase-deficient leukemia cells.

Murine L1210 and human HL-60 leukemia cells grown for 5-7 days in medium containing 1% serum without selenium supplementation [Se(-) cells] were severely depressed in selenoperoxidase (SePX) activity relative to selenium-supplemented controls [Se(+) cells]. Catalase (CAT) activity in Se(-) cells was unaffected up to this point, but thereafter began to increase. Two manifestations of this increase have been differentiated for both cell lines: (a) short-term induction of CAT (up to approx. twofold) after 2-3 weeks, followed by (b) long-term selection for cells that irreversibly express much higher levels of CAT, e.g., > 100 times (L1210) and > 10 times (HL-60) the levels observed in Se(+) controls after approximately 20 weeks. Although superoxide dismutase, glutathione S-transferase, and glucose-6-P dehydrogenase activities were unchanged in Se(-) cells, GSH levels were elevated by 50-100%; like short-term CAT elevation, this could be reversed by supplying Se. Short-term Se(-) cells were more sensitive to H2O2-induced killing than Se(+) cells, evidently because SePX activity was important for peroxide detoxification. However, long-term Se(-) cells were markedly more resistant to H2O2 than Se(+) counterparts, consistent with the much higher levels of CAT in the former. Southern blot analysis revealed that the copy number of CAT DNA in a clone of long-term Se(-) L1210 cells was four- to fivefold greater than that in an Se(+) clone. Northern blot analysis of RNA from the same Se(-) clone showed a CAT mRNA level that was at least 40 times higher than that of the Se(+) control. Similar trends were observed for HL-60 cells. These results suggest that elevated CAT during long-term Se deprivation is a reflection of amplification and greater transcription of the CAT gene.

Animals↗
PubMed1995

Correlation between the proportion of Philadelphia chromosome-positive metaphase cells and levels of BCR-ABL mRNA in chronic myeloid leukaemia.

We have sought to define the relationship between the proportion of marrow metaphases showing the Philadelphia chromosome (Ph) and levels of BCR-ABL mRNA assessed by quantitative polymerase chain reaction (PCR) in patients with chronic myeloid leukaemia (CML). From a total of 141 patients, 164 PCR assays were performed on peripheral blood samples taken within 2 weeks of a bone marrow specimen analysed by cytogenetics. BCR-ABL mRNA was quantified in all 106 PCR-positive samples by competitive PCR; results ranged from < 10 to 3.4 x 10(6) transcripts/micrograms RNA. Twenty-one chronic-phase patients had a median of 5.0 x 10(5) BCR-ABL transcripts/micrograms RNA; no difference in levels of the fusion mRNA was found between 15 Ph-positive and six Ph-negative, BCR-ABL-positive patients. Ph-positive metaphases were not detected in any individual who was PCR negative (n = 58) and in only a single patient who was PCR positive with < 10(3) BCR-ABL transcripts/micrograms RNA (n = 44). Conversely, of 41 samples from patients in haematological remission who had > 10(3) BCR-ABL transcripts/micrograms RNA, 30 had at least one Ph-positive metaphase. The highest level of BCR-ABL transcripts at which Ph-positive metaphases were not detected was 1.5 x 10(4). For the 46 patients who had at least one Ph-positive metaphase, a good correlation (Spearman coefficient = 0.83, P < 0.0001) was found between the percentage of Ph-positive metaphases and BCR-ABL transcript levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone Marrow↗
PubMed1995

Immunolocalization of interleukin-1 alpha in rat mandibular molars and its enhancement after in vivo injection of epidermal growth factor.

Immunolocalization of interleukin-1 alpha in the first mandibular molars of rats from day 0-12 postnatally showed that the protein was localized in the epithelial stellate reticulum adjacent to the dental follicle. Staining of the stellate reticulum was most prominent in the early days postnatally and was absent by postnatal day 11. Injection of epidermal growth factor into rats at day 0 greatly increased the intensity of the staining for interleukin-1 alpha in the stellate reticulum. Epidermal growth factor (EGF) enhanced the gene expression of interleukin-1 alpha in stellate reticulum cells in vitro, and this study suggests there is enhanced translation of interleukin-1 alpha messenger RNA in the stellate reticulum following EGF injection. In turn, the interleukin-1 alpha may exert its effect on the dental follicle cells adjacent to the stellate reticulum because EGF also enhanced expression of the interleukin-1 receptor type I messenger RNA in cultured dental follicle cells as well as enhancing its expression in vivo. In view of the fact that injection of EGF will stimulate precocious eruption of teeth, its stimulus of interleukin-1 alpha synthesis in the stellate reticulum may be the mechanism by which EGF initiates a cascade of molecular events to signal the onset of tooth eruption.

Animals↗
PubMed1995

Prognostic significance of human papillomavirus DNA in vulvar carcinoma.

OBJECTIVE: To determine the histopathologic, epidemiologic, and prognostic significance of human papillomavirus (HPV) DNA in primary invasive vulvar cancer. METHODS: From December 1981 through October 1992, primary tumor tissue from 55 newly diagnosed vulvar cancers was evaluated for the presence of HPV DNA. The DNA was extracted from tumor tissue and subjected to the polymerase chain reaction (PCR) using highly conserved consensus L1 primers that detect 25 different HPV genotypes and primers specific for HPV type 6/E6, type 16/E7, and type 18/E6 gene sequences. All PCR products were hybridized to type-specific radiolabeled probes. The association between the presence of HPV DNA and histologic, epidemiologic, and clinical characteristics was analyzed. RESULTS: Thirty-three (60%) tumors contained HPV DNA. Patients younger than 70 years of age or who smoked were more likely to have HPV-positive vulvar cancers. Twenty-one (95%) of 22 tumors classified as basaloid, warty, or verrucous contained HPV DNA, whereas 12 (39%) of 31 typical squamous tumors contained HPV (P < .001). Two adenocarcinomas were negative for HPV. Tumors with or without HPV DNA did not differ with respect to International Federation of Obstetricians and Gynecologists stage (size and nodal status), tumor grade, or therapy. Using life-table analysis, the absence of HPV DNA and the presence of regional nodal metastasis were predictive of recurrence and death from vulvar cancer. When controlling for lesion size, age, tumor grade, and nodal metastasis using the Cox proportional hazards model, only HPV status remained an independent prognostic factor. CONCLUSION: Human papillomavirus DNA is more common in vulvar cancers of young women who smoke than in older nonsmokers. Patients with HPV-negative tumors are at an increased risk of recurrence and death from vulvar cancer.

Adult↗
PubMed1995

Molecular methods for monitoring malignant disease after bone marrow transplantation.

Significant progress has been made over the past few years in the characterization of genetic defects responsible for the development of leukemia. In addition to contributing to our understanding of the biology of malignancy, defined genetic lesions may serve as markers of the abnormal cells. Such markers are useful for diagnostic purposes and for the detection of residual malignancy in patients after therapy or as contamination in peripheral blood or bone marrow harvests. In particular, detection of tumor-specific lesions or other tumor-associated markers by the techniques of cell and molecular biology has prompted a reevaluation of the definitions of remission and relapse.

Bone Marrow Transplantation↗
PubMed1995

Detection of the BCR-ABL gene by reverse transcription/polymerase chain reaction and fluorescence in situ hybridization in a patient with Philadelphia chromosome negative acute lymphoblastic leukaemia.

The Philadelphia (Ph) chromosome is detected in leukaemia cells in approximately 20% of adults with acute lymphoblastic leukaemia (ALL). When treated with chemotherapy alone, Ph-positive ALL has a poor prognosis, and patients may benefit from bone marrow transplantation in first remission. Here we report a patient with chromosomally normal bone marrow, in all 60 cells analysed, who was found to have the p210-type BCR-ABL chimaeric transcript by RT/PCR. Fluorescence in situ hybridization was labelled cosmid probes for BCR and ABL showed the presence of BCR-ABL juxtaposition on a normal chromosome 22 in leukaemia cell metaphases. We conclude that molecular and cytogenetic methods should be used in conjunction to detect the BCR-ABL gene rearrangement in ALL.

Adult↗