Biomedical subjects

L Fink

Publications and source records attributed to L Fink.

At least 37 records · Page 2Linked to original sources

PubMed1999

Rat porphobilinogen deaminase gene: a pseudogene-free internal standard for laser-assisted cell picking.

Analysis of gene expression and its transcriptional regulation requires a reliable access to target mRNA. However, mRNA extractions from homogenized tissue are limited because only average data are obtained, and cell-specific expression may not be addressed. Here, we describe a new method that combines the microscopic selection of oligocellular clusters or a few isotypic cell profiles from complex tissues by UV-laser-assisted cell picking with a simplified and highly efficient protocol for mRNA amplification. For positive control and quantitation reference, a reliable housekeeping gene is needed. For this purpose, we designed primers of the rat porphobilinogen deaminase (PBGD) gene. In contrast to many commonly used housekeeping primer pairs that co-amplify processed pseudogenes, this sequence allowed detection of a pseudogene-free rat cDNA sequence, thus eliminating the need for a DNase-digestion step. PBGD mRNA was consistently expressed in all complex tissues investigated and in 21 specific cell types harvested by laser-assisted cell picking. PBGD is suggested as a reliable new rat housekeeping gene, particularly suitable for analysis of oligocellular samples such as those obtained by laser-assisted cell picking in complex tissues.

Animals↗

PubMed1999

Angiotensin-converting enzyme (CD143) in neoplastic germ cells.

Angiotensin I-converting enzyme (ACE, CD143, Kininase II, EC 3.4.15.1) occurs in two isoforms; whereas the somatic isoform (sACE) appears in certain endothelial cells and some other cell types, the testicular isoform (tACE) was found in humans and various mammals only during spermiogenesis. An expression of ACE was reported formerly in some human seminomas, but its isoform type, cellular distribution, and pathogenetic meaning are not known. Therefore we analyzed normal human testes, 22 different testicular tumors, and 23 fetal and postnatal tissues of different stages of testicular development. By reverse transcriptase-polymerase chain reaction, ACE mRNA isoforms were assessed in homogenized tissue sections and in germ cells selectively isolated by laser-assisted cell picking. Immunohistochemistry was performed on consecutive sections using monoclonal antibodies specific to the human somatic isoform or both, sACE and tACE. In adult men, tACE was detectable in spermatids and spermatozoa, but normal spermatogonia and spermatocytes were not found to express ACE in any isoform. By contrast, both mRNA and protein of sACE were detectable in the cells of intratubular germ cell neoplasm, seminomas, and other testicular tumor types. Because sACE was also found in fetal germ cells, our findings point to profound differences in the regulation of ACE expression in fetal, mature adult, and neoplastic germ cells. They are in agreement with the concept that neoplastic germ cells phenotypically reflect an embryonic stage of cellular differentiation. Laser-assisted cell picking proved to be a reliable method to investigate differently regulated mRNA of cells which reside in close neighborhood within complex tissues.

Adult↗

PubMed1998

Dual cytoplasmic and nuclear distribution of the novel arsenite-stimulated human ATPase (hASNA-I).

The arsenite-stimulated human ATPase (hASNA-I) protein is a distinct human ATPase whose cDNA was cloned by sequence homology to the Escherichia coli ATPase arsA. Its subcellular localization in human malignant melanoma T289 cells was examined to gain insight into the role of hASNA-I in the physiology of human cells. Immunocytochemical staining using the specific anti-hASNA-I monoclonal antibody 5G8 showed a cytoplasmic, perinuclear, and nucleolar distribution. Subcellular fractionation indicated that the cytoplasmic hASNA-I was soluble and that the perinuclear distribution was due to association with the nuclear membrane rather than with the endoplasmic reticulum. Its presence in the nucleolus was confirmed by showing colocalization with an antibody of known nucleolar specificity. Further immunocytochemical analysis showed that the hASNA-I at the nuclear membrane was associated with invaginations into the nucleus in interphase cells. These results indicate that hASNA-I is a paralogue of the bacterial ArsA protein and suggest that it plays a role in the nucleocytoplasmic transport of a nucleolar component.

Adenosine Triphosphatases↗

PubMed1998

Rat cardiac neurons express the non-coding R-exon (exon 1) of the cholinergic gene locus.

Choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) are both encoded by the cholinergic gene locus from which, in the rat, five different species of ChAT mRNA and three different species of VAChT mRNA are produced. So far, discrimination between mRNA subtypes has been possible only in CNS homogenates or in cell cultures. In this study, cardiac neurons were microdissected from frozen sections of rat heart using a u.v. laser and harvested using a micromanipulator. RT-PCR demonstrated the expression of the non-coding R-exon and splicing to R1-type mRNA in the majority of cardiac neurons. The technique presented here is the first to allow subtype analysis of cholinergic locus mRNA species in neurons in situ.

Animals↗

PubMed1998

Real-time quantitative RT-PCR after laser-assisted cell picking.

The present study describes a technique for quantitation of mRNA in a few isotypic cells obtained from an intact organ structure by combining laser-assisted cell picking and real-time PCR. The microscopically controlled lasering of selected cells in stained tissue sections was applied to lung alveolar macrophages, which are unique in that they can alternatively be gathered as a pure cell population from intact lungs by bronchoalveolar lavage as a reference technique. TNF-alpha was chosen as the transcriptionally inducible target gene to be quantified in alveolar macrophages of control rat lung, as well as low- and high-challenge lungs stimulated by endotoxin and IFN-gamma nebulization. Online fluorescence detection for quantitation of the number of amplified copies was based on 5' nuclease activity of Taq polymerase cleaving a sequence-specific dual-labeled fluorogenic hybridization probe. A pseudogene-free sequence of PBGD served as an internal calibrator for comparative quantitation of target. A quick procedure and minimized loss of template were achieved by avoiding RNA extraction, DNase digestion and nested-PCR. Using this approach, we demonstrated dose-dependent manifold upregulation of the ratio of TNF-alpha mRNA copies per one copy of PBGD mRNA in alveolar macrophages of the challenged lungs. The quantitative data obtained from laser-picked alveolar macrophages were well matched with those of lavaged alveolar macrophages carried out in parallel. We suggest that this new combination of laser-assisted cell picking and real-time PCR has great promise for quantifying mRNA expression in a few single cells or oligocellular clusters in intact organs, allowing assessment of transcriptional regulation in defined cell populations.

Animals↗

PubMed1998

Properties of tumour suppressor p53 in murine hepatocyte lines transformed by hepatitis B virus X protein.

Persistent infection by hepatitis B virus (HBV) correlates with the prevalence of hepatocellular carcinoma. It has recently been demonstrated that the complete viral genome very efficiently transforms the immortalized murine hepatocyte line FMH202 in vitro. Here it is shown that the viral transactivating protein X (HBx) is sufficient to transform FMH202 cells, albeit with lower efficiency. Clonal cell lines expressing HBx mRNA in moderate or high amounts grew in soft agar and formed tumours in nude mice. Growth efficiency in soft agar of HBx transformed cell lines was much lower than that of cell lines transformed with the complete genome, and latency of tumour induction in nude mice was significantly longer after inoculation of HBx than of HBV transformed FMH202 cell lines. A marker of complete transformation, p53, was found to be phosphorylated more strongly in HBx transfected cell lines than in controls, and a cellular kinase was found to be associated with p53 complexes from HBx transformed cell lines. p53 was of wild-type conformation and was located in the nucleus of transformed cells.

Animals↗

PubMed1997

A laboratory approach to the evaluation of hereditary hypercoagulability.

The concept of hypercoagulability and especially its evaluation in the clinical laboratory has changed dramatically during the last few years. The genetic basis and the mechanisms of the various factors responsible for hypercoagulability are briefly reviewed with emphasis on the most common genetic deficiencies. The major thrust of this review centers on the cost-effective approach to examining patients with a personal or family history of venous thrombosis. Several new concepts dealing with thrombotic risk are presented with a focus on the theory that multiple factors cause thrombosis in affected patients. A proposal for a cost-effective sequential testing scheme for the accurate diagnosis of hereditary hypercoagulability is discussed. The knowledge of thrombotic risk factors is evolving rapidly, requiring the clinical laboratory to remain flexible. Ultimately, the clinical laboratory must take a leading role in the diagnosis of hereditary thrombotic disease by serving as the consultant to the primary caregiver by providing an up-to-date and cost-effective evaluation.

Antithrombin III↗

PubMed1997

Oxidative stress in diabetic macrovascular disease: does homocysteine play a role?

BACKGROUND: Non-insulin-dependent diabetes mellitus (NIDDM) and hyperhomocysteinemia are both associated with increased lipid peroxidation (oxidative stress). This may contribute to the accelerated vascular disease associated with these conditions. It is not known whether the coexistence of elevated homocysteine levels will stimulate oxidative stress further than that caused by diabetes alone. METHODS: Plasma concentrations of thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation, were measured in patients with NIDDM who had previously had a methionine load test; some of the patients had hyperhomocysteinemia. RESULTS: Plasma TBARS concentrations were elevated in diabetics with vascular disease. The additional presence of hyperhomocysteinemia was not associated with a further increase in plasma TBARS concentrations. CONCLUSIONS: Lipid peroxidation is increased in patients with diabetes mellitus and macrovascular disease and is not further elevated by the coexistence of elevated homocysteine levels. It is possible that diabetes maximally stimulates oxidative stress and any further acceleration of vascular disease in patients who have coexistent hyperhomocysteinemia is mediated through mechanisms other than lipid peroxidation.

Adult↗

PubMed1997

The Human Genome Project: view from the National Institutes of Health.

Because nearly all human disease is influenced by heritable alterations in the structure or function of genes, the issues and principles of medical genetics are coming to bear across medical disciplines. This "genetics revolution" is driven by the rapid pace in which scientists are identifying and isolating genes linked to human disease and is a direct outcome of the Human Genome Project. DNA tests are proving to be the most immediate commercial application of gene discovery, and the one seen most frequently by clinicians. To handle the growing populations who will seek genetic technologies as part of their health care, services must be expanded to help guide patients through the testing process so they make informed decisions about genetic testing. Care-givers will need to know not only about the medical benefits and risks of genetic technologies, but also about the psychosocial and legal implications of these technologies in the public arena. Until these issues are resolved, the technical ability to perform tests for DNA mutations should not be confused with a mandate to offer them. Safeguards must be in place to ensure that these tests are used wisely, maximizing their potential benefits to patients and minimizing their potential risks.

Attitude to Health↗

PubMed1996

Hyperhomocysteinemia following a methionine load in patients with non-insulin-dependent diabetes mellitus and macrovascular disease.

In the setting of an outpatient diabetic clinic, we determined whether macrovascular disease in patients with diabetes mellitus is associated with hyperhomocysteinemia (elevated plasma homocysteine [H(e)] concentrations) following a methionine load. Methionine-load tests were performed in 18 healthy controls, 11 diabetics without vascular disease (five insulin-dependent [IDDM] and six non-insulin-dependent [NIDDM]); and 17 diabetics with vascular disease (five IDDM and 12 NIDDM). All subjects were male, and there was no significant difference in mean age among the three groups. We measured plasma H(e) concentrations before and 2, 4, 6, 8, and 24 hours after an oral methionine load. Hyperhomocysteinemia (peak plasma H(e) concentration > control mean +/- 2 SD) occurred with significantly greater frequency (seven of 18, 39%) in patients with NIDDM as compared with age-matched controls (7%), being more common in those with macrovascular disease (five of 12, 41%). The area under the curve (AUC) over 24 hours, reflecting the total period of exposure to H(e), was also elevated with greater frequency in patients with NIDDM and macrovascular disease (33%) as compared with controls (0%). We conclude that hyperhomocysteinemia is associated with macrovascular disease in a significant proportion of patients with NIDDM. Further investigation of this association may determine whether hyperhomocysteinemia contributes to the increased frequency and accelerated clinical course of vascular disease in patients with diabetes mellitus.

Administration, Oral↗

PubMed1994

Recombinant soluble human thrombomodulin: a randomized, blinded assessment of prevention of venous thrombosis and effects on hemostatic parameters in a rat model.

UNLABELLED: Thrombomodulin is an endothelial surface receptor that binds thrombin and accelerates the activation of protein C. We compared the effects of a recombinant thrombomodulin analog (TME), recombinant hirudin (r-HIR), heparin sodium (HEP), and normal saline (Control) on thrombus formation, activated partial thromboplastin time (APTT), thrombin time (TT), platelet aggregation and tail transection bleeding time (BT) in a rat model of vena cava thrombosis. RESULTS: TME, r-HIR and HEP prevented venous thrombosis in this model in a dose-dependent manner. At the dose required to reduce vena cava thrombosis by 50% (ED50), TME did not prolong the APTT or TT as did HEP and r-HIR. Platelet aggregation in response to thrombin was not effected by TME but was inhibited by both r-HIR and HEP. BT did not differentiate the agents tested. CONCLUSION: TME inhibited venous thrombosis in a rat vena cava model with less effect on hemostatic variables than HEP or r-HIR.

Animals↗

PubMed1994

Initial reliability and validity of a new retrospective measure of child abuse and neglect.

OBJECTIVE: This report presents initial findings on the reliability and validity of a new retrospective measure of child abuse and neglect, the Childhood Trauma Questionnaire. METHOD: Two hundred eighty-six drug- or alcohol-dependent patients were given the Childhood Trauma Questionnaire as part of a larger test battery, and 40 of these patients were given the questionnaire again after an interval of 2 to 6 months. Sixty-eight of the patients were also given a structured interview for child abuse and neglect, the Childhood Trauma Interview, that was developed by the authors. RESULTS: Principal-components analysis of responses on the Childhood Trauma Questionnaire yielded four rotated orthogonal factors: physical and emotional abuse, emotional neglect, sexual abuse, and physical neglect. Cronbach's alpha for the factors ranged from 0.79 to 0.94, indicating high internal consistency. The Childhood Trauma Questionnaire also demonstrated good test-retest reliability over a 2- to 6-month interval (intraclass correlation = 0.88), as well as convergence with the Childhood Trauma Interview, indicating that patients' reports of child abuse and neglect based on the Childhood Trauma Questionnaire were highly stable, both over time and across type of instruments. CONCLUSIONS: These findings provide strong initial support for the reliability and validity of the Childhood Trauma Questionnaire.

Adult↗

PubMed1994

An introductory orientation to clinical pathology core and on-call responsibilities.

An introductory 4-week orientation for clinical pathology is described. There were 76 hours of lectures, 74 hours of conferences, and 68 hours of laboratories for a total of 221 hours. During the orientation, all calls handled by the residents were evaluated as to resolution, patient outcome, and interaction required. Eighty calls were received during the orientation from 57 technologists (71%), 16 physicians (20%), and seven nurses (9%). The calls originated concerning the following: blood banking, 37 (46%); hematology, 21 (27%); chemistry, 14 (18%); microbiology, five (6%); and administration, three (4%). Sixty percent of the calls were consultative and 40% were supervisory. Ninety-nine percent were handled appropriately by the residents. Patient outcome was moderately or significantly affected in 44% of all calls, divided between 67% of all consultative calls and 9% of all supervisory calls. Significant pathologist interaction was required in 49% of all calls, divided between 71% of the consultative calls and 16% of the supervisory calls. Using this integrated, dynamic system of resident instruction, on-call experience, and evaluation, residents quickly gain confidence in handling call, didactic clinical consultation, and patient management. The orientation and on-call system described provides for a relevant and dynamic system for resident education.

Curriculum↗

PubMed1993

Coincidental polycythemia vera and multiple myeloma: case report and review.

Polycythemia vera (PCV) and multiple myeloma are both clonal disorders of hematopoietic stem cells. The simultaneous occurrence of these diseases in an individual patient is rare. A case of synchronous PCV and smoldering myeloma is presented and the literature is reviewed. The issues of clinical importance in this unusual case include the mechanisms of anemia in multiple myeloma, the difficulty in using anemia as a parameter on which to base the initiation of therapy for myeloma, and the risks of treatment-induced leukemia and myelodysplasia.

Aged↗

PubMed1993

Pathogenic significance of interleukin-6 in angioimmunoblastic lymphadenopathy-type T-cell lymphoma.

Patients with angioimmunoblastic lymphadenopathy (AILD)-type T-cell lymphoma may develop hypergammaglobulinemia. Among four cases of AILD-type T-cell lymphoma that we have studied, we detected a correlation between the number of plasma cells in tissue and the extent of interleukin-6 (IL-6) expression in lymphoma cells. We did not detect IL-6 in three patients who had no hypergammaglobulinemia and whose tissues showed only minimal plasma cell infiltration. In the fourth patient we observed an abundant IL-6 production by lymphoma cells, which accounted for a B-cell plasmacytic tissue response and for hypergammaglobulinemia. The pathogenic significance of IL-6 was substantiated by a concomitant decrease in the serum IL-6 level, measurable tumor mass, and immunoglobulin levels, as well as by a decline in the proportion of plasmacytoid cells in peripheral blood promptly on administration of chemotherapy. Plasmacytoid B cells could be maintained in culture in the presence of IL-6, but viability was lost on co-incubation with anti-IL-6. Interleukin-1 and tumor necrosis factor were not produced by T lymphoma cells and were incapable of sustaining plasmacytoid B-cell viability in vitro. Small amounts of IL-4 were noted in T lymphoma cells. Thus, in this case of AILD-type T-cell lymphoma, tumor cells with a T-cell phenotype produced IL-6 in large quantities, explaining the accompanying B-cell and plasmacytic histologic changes and humoral disease manifestations, including marked hypergammaglobulinemia. Although not all cases of AILD-type T-cell lymphoma have an accompanying plasma cell proliferation and hypergammaglobulinemia, and although the cytokine network in these patients may be more complex than has been recognized, this case with IL-6 expression serves to illustrate the utility of cytokine assays in the analysis of the histopathologic and clinical heterogeneities of peripheral T-cell lymphomas.

Aged↗

PubMed1992

Prognosis of patients with sustained ventricular tachycardia and of survivors of cardiac arrest not inducible by programmed stimulation.

The aim of this study was to analyze the long-term clinical outcome of 60 prospectively studied patients with documented sustained ventricular tachyarrhythmia that was not inducible during baseline programmed ventricular stimulation: 39 with cardiac arrest due to noninfarction ventricular fibrillation (VF) and 21 with mild hemodynamically compromising sustained ventricular tachycardia (VT). Left ventricular ejection fraction was 55 +/- 14% in the VF group and 50 +/- 13% in the VT group (difference not significant). Patients were discharged without conventional antiarrhythmic drugs and received only empirical beta-blocker therapy. During a mean follow-up period of 21 +/- 16 months (mean +/- SD), 10 of 60 patients (17%) died suddenly. The actuarial incidence of sudden death at 1 and 4 years was similar in both groups (VF group, 10 and 20%; VT group, 16 and 16%) (p = 0.48). The actuarial incidence of sudden cardiac death was significantly higher in patients with left ventricular ejection fraction < or = 40% than in those with > 40% (1-year incidence in VF group, 40 vs 0%; VT group, 50 vs 0%) (p = 0.005 and p = 0.01, respectively). Multivariate regression analysis identified left ventricular ejection fraction < or = 40% and previous myocardial infarction as the only independent predictor of sudden cardiac death. The occurrence of frequent ventricular pairs during Holter monitoring was the only independent predictor of sustained VT recurrences. It is concluded that patients with sustained ventricular tachyarrhythmia in whom arrhythmia was non-inducible during baseline ventricular stimulation and not treated with antiarrhythmic therapy have a favorable outcome if left ventricular ejection fraction is high.(ABSTRACT TRUNCATED AT 250 WORDS)

Arrhythmias, Cardiac↗

PubMed1992

Faster rates of DNA unwinding under alkaline conditions in xrs-5 cells may reflect chromatin structure alterations.

The Chinese hamster ovary (CHO) cell line xrs-5 is a radiation-sensitive mutant isolated from CHO-K1 cells. The radiation sensitivity is associated with a defect in DNA double-strand break rejoining. The DNA alkaline unwinding technique was used to measure the DNA single-strand breakage caused by gamma-rays in xrs-5 and CHO-K1 cells. Greater rates of DNA unwinding were found in xrs-5 cells as compared to CHO-K1. Independent measurement of DNA strand breakage by DNA filter elution or pulsed-field gel electrophoresis failed to show any difference between the two cell lines. The greater rate of unwinding in xrs-5 cells may reflect an alteration in chromosome structure.

Animals↗

PubMed1992

Thrombomodulin expression in malignant pleural mesothelioma and pulmonary adenocarcinoma.

Thrombomodulin (TM) is a glycoprotein of molecular weight 75,000 kd that is normally present in restricted numbers of cells, including endothelial and mesothelial cells. In this study, the authors tested the possibility of using anti-TM to facilitate the diagnosis of mesothelioma. All of the 31 mesotheliomas and the two mesothelioma cell lines (MS-1 and MS-2) tested were stained positively with anti-TM. The specificity of anti-TM staining in mesothelioma cells was further confirmed by in situ hybridization of MS-1 cells with a TM-specific probe. The expression of TM in MS-1 cells was increased markedly when these cells were induced by 12-0-tetradecanyl phorbol 13-acetate (TPA) to differentiate. The expression of TM in mesothelioma cells, however, did not correlate with any particular phase of the cell cycle. In an attempt to differentiate pleural mesothelioma from pulmonary adenocarcinoma, the authors compared the expression of TM, carcinoembryonic antigen (CEA), and Leu M1 in these two types of tumors. Only four of 48 (8%) pulmonary adenocarcinomas were stained positively by antibodies to TM. Therefore, immunohistochemical staining with antibodies to TM yielded 100% sensitivity and 92% specificity for diagnosis of mesothelioma. All of the mesotheliomas stained negatively for CEA and Leu M1, except for one, which showed minimal focal positivity for Leu M1. In contrast, 79% and 60% of adenocarcinomas stained positively for CEA and Leu M1, respectively. These findings suggest that immunocytochemical staining with anti-TM should be added to the battery of tests to increase the diagnostic sensitivity and specificity for differentiating mesothelioma from pulmonary adenocarcinoma.

Adenocarcinoma↗