Biomedical subjects

G J Smith

Publications and source records attributed to G J Smith.

At least 91 records · Page 5Linked to original sources

PubMed1993

Confocal laser scanning immunofluorescence microscopy of the pulmonary surfactant system. Association of surfactant protein A with the nucleus of the alveolar type II cell.

BACKGROUND: Localization of surfactant protein A (SP-A) to nucleus of type II cells isolated from the lungs of rats has been reported. Data suggested that most SP-A was located within lamellar bodies of the type II cell; however, some SP-A was found in other cytoplasmic regions of the cell and in particular in the nucleus. EXPERIMENTAL DESIGN: Type II cells and type II cell nuclei isolated from the lungs of rats were reacted with affinity-purified antibodies against SP-A. Location of SP-A was determined by using fluorescein isothiocyanate-labeled secondary antibodies and the distribution of fluorescence examined by using a laser scanning microscope fitted with a confocal aperture. Two-dimensional electrophoresis followed by Western blotting was used to separate and identify type II cell nuclear proteins. RESULTS: Nuclei were isolated from type II cells obtained from elastase-digested rat lungs and examined for the presence of SP-A. The nuclei contained both focal and diffuse deposits of SP-A. Some regions within the nuclear matrix (in particular the nucleolus) appeared to be relatively devoid of SP-A. The perinuclear membrane stained intensely for SP-A where optical sectioning showed its presence as a patchwork of punctate deposits. Analysis of the SP-A associated with the nucleus by two-dimensional electrophoresis revealed that it consisted of a family of proteins with molecular masses of 26, 32, and 36 kDa and pI 5.1. Biosynthesis of nuclear SP-A in primary cultures of type II cells was sensitive to inhibitors of glycosylation resulting in the presence of only the lowest molecular weight unprocessed form. CONCLUSIONS: These data indicate that three basic forms of SP-A are associated with the nucleus of the type II cell and are especially concentrated on the perinuclear membrane.

Animals↗

PubMed1993

Improved ultrastructural lung preservation with prostaglandin E1 as donor pretreatment in a primate model of heart-lung transplantation.

Donor pretreatment with prostaglandin E1 as a pulmonary vasodilator has developed as a simple, effective means to provide excellent preservation in heart-lung transplantation. This study was undertaken to investigate the degree of ultrastructural preservation of the lung with prostaglandin E1 and other pulmonary vasodilators in a primate heart-lung transplantation model. Heart-lung transplantation was performed in 14 African green monkeys. Donor cardiac preservation was achieved with cold crystalloid cardioplegic solution (10 ml/kg). Lung preservation was achieved with cold, modified Euro-Collins solution delivered into the main pulmonary artery (60 ml/kg total). Vasodilator agents were administered intravenously 15 minutes before aortic crossclamping. The heart-lung grafts were stored at 4 degrees C for 6 hours. Three groups of animals were studied: five donors with prostaglandin E1 (0.1 to 4.0 micrograms/kg per minute), five donors with prostacyclin (0.1 to 0.35 micrograms/kg per minute), and four donors with nitroprusside (0.8 to 5.0 micrograms/kg per minute). After transplantation, arterial blood gas measurements and lung biopsies were performed at 1- and 3-hour intervals. Five formalin blocks per specimen were sectioned for hematoxylin and eosin staining. Cellular preservation and endothelial cell swelling were evaluated with electron microscopy. The specimens were graded for alveolar hemorrhage, endothelial cell swelling, and cellular preservation (grade 0, minimal, to grade 3, severe) and a mean score was obtained for each preservative agent. Prostaglandin E1-treated specimens demonstrated the least amount of endothelial swelling (mean score of 1.0) compared with prostacyclin- and nitroprusside-treated specimens (mean scores of 1.4 and 2.7, respectively). All nitroprusside-treated specimens demonstrated moderate to severe endothelial cell swelling. Interstitial and alveolar hemorrhage was noted in poorly preserved specimens, but there were no significant differences between groups. We conclude that prostaglandin E1 provides improved cellular preservation by decreasing the extent of endothelial cell swelling as observed on electron microscopy.

Alprostadil↗

PubMed1992

A simple technique for the determination of kappa and lambda immunoglobulin light chain expression by B cells in whole blood.

This article describes the development of a simple technique by which the numbers of surface immunoglobulin expressing cells can be analysed by dual fluorescence flow cytometry in samples of whole blood. We have compared the results obtained using this procedure with those obtained using samples prepared by traditional density gradient centrifugation, and demonstrate an excellent correlation between the two techniques. The method is applicable both to blood samples from normal individuals and from patients with B cell malignancies such as B-CLL and B-NHL. We have also confirmed previous findings that density gradient centrifugation may preferentially deplete certain lymphocyte subsets. This technique offers the following advantages: (i) it is rapid, (ii) it is accurate, (iii) it is reliable, (iv) it is useful in cases of lymphopenia, and (v) it requires only a small volume of blood. It is likely to be applicable to other situations in which the presence of serum factors interfere with antibody staining in whole blood.

Antibodies, Monoclonal↗

PubMed1992

Partial characterization of nuclear matrix attachment regions from human fibroblast DNA using Alu-polymerase chain reaction.

The proteinaceous nuclear matrix of mammalian cell nuclei has been suggested to be involved in the regulation of chromatin structure, DNA replication, and gene expression. Interaction between cellular DNA and the nuclear matrix is mediated by putative DNA binding sequences, matrix attachment regions (MARs), which may become altered during early events in cellular transformation. Among the cellular changes occurring during the development of neoplasia, all of which may potentially involve the nuclear matrix, are alterations in nuclear structure, loss of control of DNA replication, and significant modifications of cellular gene expression. Therefore, a better understanding of the interaction between DNA and the nuclear matrix is needed. Isolated matrix associated DNA from pulse labeled SV40 transformed human fibroblasts was shown to be enriched in newly replicated DNA, confirming the association of DNA replication with the nuclear matrix as observed by others. Subgenomic fractions of matrix associated DNA enriched in putative MARs sites were prepared from quiescent and logarithmically growing normal human fibroblasts and SV40 transformed human fibroblasts. These fractions of DNA were analyzed by Alu-polymerase chain reaction and agarose gel electrophoresis, revealing complex and unique patterns of DNA products for each cell type investigated. A number of prominent DNA fragments with similar molecular size were found to be present in the amplified DNA products of each DNA source, suggesting that these DNA fragments may represent common DNA sequences which contain MARs sites or which are associated with MARs sites. The application of Alu-polymerase chain reaction to the molecular analysis of nuclear matrix associated DNA may facilitate the isolation and characterization of potentially new human MARs sequences.

DNA↗

PubMed1992

Cancer cell motility-inhibitory protein in the Dunning adenocarcinoma model.

Cell motility has been associated with metastatic ability in the Dunning R3327 rat prostatic adenocarcinoma model. Cancer cell motility promoters but not inhibitors have been described by many investigators. Serum-containing and serum-free media conditioned by the nonmotile, nonmetastatic G Dunning subline inhibited the motility of the highly motile, highly metastatic MAT-LyLu subline. Motility inhibition by the G subline-conditioned serum-free media was lost upon heating to 100 degrees C and by treatment with trypsin. The motility inhibitory protein(s) had a molecular weight exceeding 50,000 as determined by diafiltration. G subline-conditioned RPMI 1640 contained several proteins with molecular weights of between approximately 53,000 and 116,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One of these bands may represent the first inhibitor of cancer cell motility identified.

Adenocarcinoma↗

PubMed1992

Hard metal interstitial pulmonary disease associated with a form of welding in a metal parts coating plant.

We describe two cases of hard metal pulmonary disease (one fatal) in workers employed in the same area of a metal coating plant using the detonation gun process for applying a durable metal surface to metal parts. In this form of welding, a mixture of powdered metals, including tungsten carbide and cobalt, is heated by ignition of a flammable gas and propelled from the end of the "gun" at high temperature and velocity to form a welded metal coating. This process is done in an enclosed chamber and with each application, large volumes of fine aerosols are created. Inhalation exposure to hard metal may occur during the mounting and removal of the metal parts between applications, in spite of engineering controls and industrial hygiene surveillance. One of the cases presented with minimal chest x-ray abnormalities and an obstructive pattern on pulmonary function testing, although subsequent open lung biopsy showed diffuse interstitial pulmonary fibrosis. The fact that two cases of hard metal pulmonary interstitial disease occurred where thorough exposure control procedures and a surveillance program for cobalt were in place may indicate the need for revisions of the current technology used when hard metal is applied in the detonation gun process.

Adult↗

PubMed1992

Isolation by fluorescence-activated cell sorting of cells of a human lymphoblastoid cell strain containing mutations in the lambda immunoglobulin gene.

Fluorescence-activated cell sorting (FACS) was used to establish clonal populations of a human lymphoblastoid cell strain that contain spontaneously occurring and N-methyl-N'-nitro-N-nitrosoguanidine-induced mutations in the lambda immunoglobulin gene. Multiple rounds of FACS using a monoclonal antibody specific for the membrane-expressed human lambda immunoglobulin were used to enrich the population fraction of cells lacking a wild-type lambda immunoglobulin on the cell surface. Approximately 20% of the clonal populations established after five rounds of FACS-mediated enrichment did not express the lambda immunoglublin epitope recognized by the monoclonal antibody used for selection. However, evaluation of the FACS-selected mutant clonal populations with a polyclonal antilambda antibody, or a monoclonal antibody directed against a different epitope on the lambda immunoglobulin made by the T5-1 cells, indicated that the mutant clonal populations expressed lambda immunoglobulin on their cell surfaces. Additionally, the presence of lambda mRNA and of both secreted and cytoplasmic lambda protein confirmed the transcription and translation of the lambda immunoglobulin gene. These data suggest that FACS-mediated selection employing epitope-specific monoclonal antibodies provides a powerful technique for isolation of cell populations that express mutations within the coding region of the lambda immunoglobulin gene.

Blotting, Northern↗

PubMed1992

Space habitat contaminant growth models.

This paper outlines the need for and role of Contaminant Growth Models (CGM) in designing space habitats and space mission operations. The tremendous complexity of the contamination problem and the current lack of data suggests that a "layered" approach to CGM development and utilization be made in conjunction with medical/toxicological research and risk assessments. Two space habitat contaminant examples: thermodegradation and hydrazine, are utilized to show how such a model might be useful in analyzing the environment of a space habitat. The examples also depict how NASA's interdisciplinary Center for Space Environmental Health (CSEH) will combine medical/toxicological research results with engineering environmental system design tools to set standards and help in the design, prediction, monitoring and safe control of space habitats. Since the CSEH project is just beginning, the contents of the paper are descriptive of the goal and the approach being taken, rather than conclusive in nature.

Ammonia↗

PubMed1992

A long-lived transient resulting from flash photolysis of hematoporphyrin in aqueous solution.

A long-lived transient with a lifetime of several hundred microseconds was observed following the flash photolysis of aqueous solutions of hematoporphyrin buffered at pH 7.5. The transient-ground state difference absorption spectrum was determined 500 microseconds after flash photolysis. The yield of this species was found to increase with increasing hematoporphyrin concentration and it was also found to depend on the excitation wavelength. The lifetime of the species is not significantly affected by the presence of oxygen. Because the triplet state of hematoporphyrin is not the only long-lived species produced by flash photolysis of aqueous hematoporphyrin solutions, the observed triplet state extinction coefficients will be lower than the true value and hence the triplet state yields of hematoporphyrin determined by the flash photolysis, "complete conversion" technique, are only upper limits. The formation of the long-lived species is discussed in terms of electron transfer between the monomer partners in hematoporphyrin dimer and aggregates which are present in aqueous solutions of hematoporphyrin, particularly in concentrated solutions.

Hematoporphyrins↗

PubMed1992

Altered levels and protein kinase C-mediated phosphorylation of substrates in normal and transformed mouse lung epithelial cells.

Protein phosphorylation and protein kinase C (PKC) levels were analyzed in intact cultures of spontaneously transformed, chemically transformed, and untransformed mouse pulmonary epithelial cell lines. It was found that although the transformed cell lines contained about 80% less protein kinase C, measured as total enzyme activity or binding of [3H]phorbol ester, phosphorylation events after phorbol ester treatment could still be easily detected. A commonly described Mr 80-kDa protein kinase C substrate (p80, 80 K, MARKS) was identified using 2D-PAGE, following phosphorylation in intact cells, and found to have reduced availability for phosphorylation in the transformed cell lines C4SE9, C1SA5 and NULB5 in comparison to the untransformed C4E10 and C1C10. Available levels of p80 were further analyzed in heat-denatured extracts from all cell lines using partially purified bovine brain PKC and correlated well with changes seen in intact cells. It was also noted that all transformed cell lines contained large amounts of a family of phosphoproteins of Mr 55-65 kDa, that could not be detected in the untransformed cell lines and whose phosphorylation state was increased by protein kinase C activation. This protein was found to be located in the nucleus. Hence, spontaneously and chemically transformed mouse pulmonary epithelial cells exhibit reduced levels of PKC, along with an altered pattern of PKC-mediated phosphorylation.

Animals↗

PubMed1992

An ultrastructural study of hydrogen peroxide production by cultured fetal and neonatal rat lung cells exposed to hyperoxia.

Hydrogen peroxide production in organotypic cultures of fetal and newborn rat lung cells has been investigated using an ultrastructural histochemical method, in which the quantity and location of an electron-dense reaction product derived from the interaction of cerium chloride and hydrogen peroxide was detected. Hydrogen peroxide was present in fetal cell cultures exposed to hyperoxia (50% oxygen) compared to controls maintained in 10% oxygen. This increase could be limited by incubation of cultures with ascorbic acid and preincubation with dexamethasone. On the other hand, in newborn rat lung cell cultures, less hydrogen peroxide was identified in cultures including those exposed to hyperoxia (50% oxygen).

Animals↗

PubMed1992

Bile acids influence the growth, oestrogen receptor and oestrogen-regulated proteins of MCF-7 human breast cancer cells.

The effects of the major human serum bile acid, glycochenodeoxycholic acid (GCDC), as well as unconjugated chenodeoxycholic acid (CDC), on the MCF-7 human breast cancer cell line have been studied in vitro under oestrogen and bile acid deprived culture conditions. GCDC increased the growth of the breast cancer cells over the range 10-300 microM. At concentrations in excess of the bile acid binding capacity of the medium cell growth was prevented. In contrast 10 microM CDC tended to reduce cell growth. Oestrogen (ER) and progesterone (PgR) receptors, pS2 and total cathepsin D were quantified by monoclonal antibody based immunoassays. Ten to 100 microM GCDC and 10 microM CDC down-regulated ER protein and this was accompanied by induction of the oestrogen-regulated proteins PgR, pS2 and possibly cathepsin D, including increased secretion of the latter two proteins into the culture medium. All these changes were quantitatively similar to those observed with 10 nM oestradiol. The bile acid effects on ER and PgR were not due to interference with the assay procedures. Cells incubated with 50 microM GCDC or 10 microM CDC had higher pmolar concentrations of the bile acids than controls. This study suggests that naturally occurring bile acids influence the growth and steroid receptor function of human breast cancer cells.

Bile Acids and Salts↗

PubMed1992

Microprocesses in perception and personality.

This paper utilizes an ontogenetically validated personality test, the Meta-Contrast Technique (MCT). The instrument focuses on the microdevelopment of perception (percept genesis) reflecting the process of individual adaptation to and construction of reality. Empirical evidence for the adequacy of the MCT paradigm in neuropsychological personality research is presented. In all, 45 patients were tested; thirty-three patients with various forms of supratentorial brain tumors were examined and compared with a group of 12 cerebrovascular disease (CVD) patients. Without the examiner's knowledge of degree of tumor malignancy, the MCT results allowed differentiation among tumors of various histological types. Uninformed of the final diagnosis, patients with highly malignant gliomas showed panic-related anxiety and schizoid-like regressions, in contrast both to patients with low and nonmalignant brain tumors and to the group of CVD patients.

Adult↗

PubMed1992

Ex vivo perfusion of intestinal allografts with anti-T cell monoclonal antibody/ricin A chain conjugates for the suppression of graft-versus-host disease.

The removal of T lymphocytes from intestinal allografts prior to transplantation would prevent graft-versus-host disease and might also weaken the unusually severe rejection response mounted by graft recipients. Ex vivo perfusion by monoclonal antibody-toxin conjugates represents a potentially ideal approach to achieve this goal. Monoclonal antibody-toxin conjugates were prepared by coupling the mouse anti rat CD5 antibody MRC OX19 to the A chain of ricin. The resultant conjugate contained 1 or 2 ricin A chain molecules per molecule of immunoglobulin and had high selective toxicity for rat T lymphocytes. Following ex vivo perfusion of the small intestine, the mesenteric lymph nodes and Peyer's patches were removed and the level of penetration of the MRC OX19 antibody was assayed by immunohistological techniques. In lymph nodes, there was ready access of the antibody to the medulla, and to a lesser extent to the B cell areas of the cortex. However, only the T cells in the peripheral regions of the paracortex were stained, suggesting that the paracortex was resistant to penetration by blood-borne antibody, and was being stained only by diffusion from the lymph node medulla. Some penetration of the antibody also occurred in the immediate vicinity of the postcapillary venules. In the Peyer's patches, staining was seen in the germinal centers, but not at all in the T cell areas. The addition of agents such as histamine to the perfusate to increase vascular permeability did not alter this picture. These studies suggest the presence of a potentially interesting resistance to penetration of the paracortex of the lymph node by blood-borne substances. Nevertheless, sufficient penetration occurred to influence favorably the course of GVD disease following ex vivo perfusion prior to transplantation of the donor intestine with the MRC OX19-ricin A chain conjugate.

Animals↗

PubMed1992

Reduction of graft-versus-host reactivity after small bowel transplantation: ex vivo treatment of intestinal allografts with an anti-T cell immunotoxin.

A specific T lymphocyte immunotoxin was used to pre-treat small bowel grafts in an attempt to prevent graft-versus-host (GVH) reactivity and GVH disease in a rat transplant model. The immunotoxin used was a conjugate of the anti-CD5 MoAb MRC OX-19 with ricin A chain. The grafts were perfused ex vivo with a standard solution of immunotoxin followed by incubation at 4 degrees C for 1 h before transplantation. In a semi-allogeneic strain combination (parent to F1 hybrid offspring) graft treatment with immunotoxin led to a prolongation of recipient survival compared with groups receiving similar transplants without immunotoxin treatment. An additive effect on survival was observed when the host was treated with cyclosporin. The effect of immunotoxin was greater than that of mesenteric lymphadenectomy in increasing host survival. The effect of graft treatment with the immunotoxin on cellular migration from graft to host lymphoid tissues was assessed in fully allogeneic transplantation (PVG to DA). Host lymphoid tissues were subjected to immunohistochemical analysis using a MoAb specific for donor class I MHC antigens. Graft treatment with the immunotoxin led to a significant decrease in the number of graft cells found in host lymphoid tissues 7 days after transplantation. However, this effect was less marked than that achieved by graft mesenteric lymphadenectomy. With our current protocol graft treatment with a specific T cell immunotoxin can significantly reduce but not abolish GVH reactivity in rat small bowel transplantation.

Animals↗

PubMed1991

Application of confocal scanning laser microscopy in experimental pathology.

Confocal scanning laser microscopy (CSLM) represents an exciting new tool for scientific disciplines which focus on mechanistic studies such as experimental pathology. Enhanced resolution in the specimen plane and rejection of out-of-focus fluorescence flare allow analysis of specific nucleic acid sequences, enzymes, structural macromolecules, and cellular homeostasis utilizing fluorescent probes. Four different experimental applications are discussed which utilize CSLM to evaluate pathological processes at the subcellular, cellular, and tissue levels. Programmed cell death, or apoptosis, is a natural process of significance both during development and as a response to toxic stimuli. CSLM-imaging of nuclei of human B lymphoblastoid cells following exposure to a monofunctional alkylating agent suggests that the degradation of chromatin characteristic of apoptosis may occur in asymmetric patterns. Surfactant apoprotein-A is the major non-serum protein component of pulmonary surfactant and is essential for the extracellular function of surfactant. CSLM of alveolar type II cells suggests that apoprotein-A is present in both the cytoplasm, predominantly in lamellar bodies, and in the nucleus. The tumor promoter, phorbol myristate acetate, rapidly stimulated the formation of vacuoles in human neutrophils. CSLM using Lucifer Yellow as a probe suggests that cylindrical vacuoles are formed by fluid-phase pinocytosis. The blood-nerve barrier (BNB) in peripheral nerves may be an important target during toxin-induced neuropathies. Ricin-induced permeability of the BNB in the rat was rapidly visualized by CSLM as leakage of fluorescein isothiocynate (FITC)-dextran into the endoneurial compartment.

Animals↗

PubMed1991

Clonal analysis of neoplastic transformation in cultured diploid rat liver epithelial cells.

Expression of TGF-alpha mRNA, which correlates well with the ability of cells to condition medium with an EGF-like activity, clonally segregates best with tumorigenicity among the several single phenotypes considered in this study. The results of unreported studies in which we have analyzed the quantitative relationships between the expression of selected phenotypes and tumorigenicity, suggest that the elevated expression of myc and TGF-alpha mRNAs interact in their associations with tumor yield. These results suggest that elevated myc expression sensitizes hepatic epithelial cells to the possible tumorigenic action of TGF-alpha. This observation may explain why the correlation between the qualitative expression of TGF-alpha and tumorigenicity, described here, is not perfect. Conventionally applied markers of transformation in hepatocytes in vivo and in cultured liver epithelial cells in vitro that we studied -histochemical expression of GGT, ability to grow in medium containing low levels of calcium, and ability to grow in soft agar- clonally segregated with tumorigenicity poorly in liver epithelial cells transformed in vitro. We conclude that these phenotypes are not adequate markers for determining the lineage of hepatic epithelial neoplasms (including, probably hepatocellular cancers arising in vivo). This study appears to be the first to attempt to analyze clonally the association of these markers with tumorigenicity, and to quantify the sensitivity, specificity, and predictive value of the associations. Our study suggests that the relatively weak associations of these phenotypes with tumorigenicity may be related only to their stronger associations with expression of TGF-alpha, or to some other property that is strongly associated with tumorigenicity. Expression of TGF-alpha is more strongly associated with expression of GLC, for example, than is the GLC phenotype with tumorigenicity. At least for GLC, autocrine stimulation by TGF-alpha is likely, since EGF increases growth of WB cells in low calcium medium. This observation may explain the perfect correlation between expression of TGF-alpha and GLC. EGF also stimulates lactate dehydrogenase, pyruvate kinase, and glucose 6-phosphate dehydrogenase in WB cells. However, quantitative correlation between GGT activity and TGF-alpha is less strong. Thus, our data from these studies suggest that the tumorigenic phenotype of cultured hepatic epithelial cells is intimately dependent on the expression of the TGF-alpha gene, possibly producing autocrine stimulation of growth via the cells' EGF receptors. This is the most simple view of the potential relationship between TGF-alpha expression and tumorigenicity in liver epithelial cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

The role of inflammation in nonspecific abdominal aortic aneurysm disease.

The predominant pathologic feature of abdominal aortic aneurysm is elastin destruction, and elastin destruction may be mediated by inflammation. In this investigation serial sections of abdominal aortic aneurysm specimens were selectively stained to study the relationship between inflammation and elastin degradation. In addition, soluble aortic extracts were examined for the presence of immunoglobulins. An inflammatory infiltrate was present in 8 of 10 of the abdominal aortic aneurysm specimens examined. The infiltrate was mononuclear, commonly located at the junction of the media and adventitia; it did not codistribute with loss of elastin. The presence of an inflammatory component in abdominal aortic aneurysm was associated with a large amount of immunoglobulin in soluble extracts from aneurysmal tissue compared to atherosclerotic and normal control extracts. This study further characterizes the microscopic pathology of abdominal aortic aneurysm and describes the presence of immunoglobulin in soluble tissue extracts. In addition, the possible role of inflammation in abdominal aortic aneurysm as it relates to protease expression is detailed.

Aorta, Abdominal↗