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Biomedical subjects

W K Dowjat

Publications and source records attributed to W K Dowjat.

At least 19 recordsLinked to original sources
PubMed2001

Alzheimer's disease presenilin-1 expression modulates the assembly of neurofilaments.

Mutations in presenilin-1 gene are responsible for the majority of early-onset familial Alzheimer's disease cases. The function of this protein and the mechanism underlying the pathogenicity of its mutations are still unclear. To elucidate the role of presenilin-1 in the Alzheimer's disease pathology, we tested two such mutations (P117L and M146L) for their effect in stably transfected mouse neuroblastoma cell lines. Over-expression of the wild-type presenilin-1 gene induced formation of a well-extended, orderly organized network consisting of neurofilaments assembled from the L and H subunits, while in cells with the mutant gene this network was markedly reduced to short filaments concentrated in structures resembling cups. Cells expressing the mutant gene displayed altered processing of the transgene protein and neurofilament-H, suggesting that presenilin-1 is the mediator of changes targeted at neurofilaments. The two different mutations produced similar alterations, implying that this is a common pathogenic mechanism. Presenilin-1, neurofilament-H and tau proteins showed co-localization as evidenced by confocal microscopy, suggesting a possible physiological connection between these three proteins. Presenilin-1 appears to influence assembly of the H subunit into neurofilaments and the subsequent formation of new neurites. Mutations impair this function of presenilin-1, resulting in inhibition of neurite outgrowth. That presenilin-1 influences the assembly of neurofilaments may represent a novel pathway through which presenilin-1 mutations are involved in Alzheimer's disease pathology. In this hypothesis, presenilin-1 mutations will be associated with aberrant sprouting leading to synaptic loss, a key neuropathological feature of Alzheimer's disease.

Alzheimer Disease↗
PubMed1999

Inhibition of neurite outgrowth by familial Alzheimer's disease-linked presenilin-1 mutations.

Two (P117L; M146L) familial Alzheimer's disease (FAD)-causing presenilin-1 (PS1) mutations have been tested fortheir effect in stably transfected mouse neuroblastoma (N2a) cell lines. The P117L mutation is associated with the earliest onset of AD reported so far (24 years), while the M146L is less pathogenic with the onset at about 43 years. Overexpression of wild-type (wt) PS1 gene was associated with the marked increase in the number and the length of neuritic outgrowths accompanied by accumulation of PS1 immunoreactivity in neurites. The highly pathogenic P117L mutation completely suppressed this effect and the pattern of PS1 immunolabeling resembled a cup structure with all immunoreactivity gathered at one pole of the cell. The effect of less pathogenic M146L mutation was similar, but not as pronounced. These findings suggest that one of the normal functions of PS1 may be the control of neurite outgrowth, and the inhibitory effect of two FAD-linked mutations stresses its importance in the cellular mechanism that leads to the development of Alzheimer's disease (AD).

Alzheimer Disease↗
PubMed1998

A novel Polish presenilin-1 mutation (P117L) is associated with familial Alzheimer's disease and leads to death as early as the age of 28 years.

The majority of early-onset familial Alzheimer's disease (FAD) is associated with mutations in the presenilin-1 (PS1) gene. We describe a novel Polish PS1 mutation of Pro117Leu, associated with the earliest average age of onset and death so far reported in a PS-linked, FAD kindred. Human kidney 293 and mouse neuroblastoma N2a cells were stably transfected with wild-type and PS1 P117L. There was a significant increase in the amyloid beta42/40 ratio in the N2a P117L PS1 transfected cells compared with N2a transfected with wild-type PS1. What role PS has in the pathogenesis of AD remains to be determined, however, the severity of the clinical picture associated with this PS1 mutation stresses the importance of presenilin.

Adult↗
PubMed1998

Cell-type-specific enhancement of amyloid-beta deposition in a novel presenilin-1 mutation (P117L).

The presenilin-1 (PS1) gene mutation (Pro117Leu), recently identified in a Polish family is characterized by the earliest reported onset (from 24-31 years) of Alzheimer disease (AD) and a very short duration of disease (4-6 years). The neuropathology of 2 subjects with this PS1 mutation (ages at death: 35 and 37 years) was compared to four Down syndrome (DS) patients (mean age at death: 62 years) and 4 sporadic AD patients (mean age at death: 79 years with a mean duration of disease of 18 years). The Polish familial AD (FAD) patients showed a marked increase in the amyloid burden of 2 6-fold in most areas of the brain. The entorhinal cortex was an exception where the amyloid burden was similar in each category of patient. Some brain regions of the Polish FAD patients showed a massive increase of amyloid, such as the molecular layer of the cerebellum where a 7- and 25-fold increase was noted, compared with DS and sporadic AD patients respectively. The cerebellar vessel amyloid burden was also greatly increased in the FAD patients, reflecting a vascular compartment specific increase of amyloid beta deposition. The presence of this PS1 mutation has an even greater effect on both vascular and parenchymal amyloid deposition, than the overexpression of the amyloid beta precursor protein present in DS patients, suggesting that PS mutations can be a critical factor determining amyloid deposition.

Adult↗
PubMed1997

Presenilin-1 is associated with Alzheimer's disease amyloid.

Mutations in presenilin (PS)-1 and -2, located on chromosome 14 and 1 respectively, are the major association with early-onset familial Alzheimer's disease (FAD). FAD has also been linked to mutations in the amyloid beta precursor protein (beta PP), and the presence of the apolipoprotein E4 allele is a risk factor for late-onset AD. The role of PS in FAD and in sporadic AD is unclear. We previously reported the presence of a PS-1 carboxyl-terminal epitope in neuritic plaques (Wisniewski T, Palha JA, Ghiso J, Frangione B: S182 protein in Alzheimer's disease neuritic plaques. Lancet 1995, 346:1366). In the present study, we examined a number of biochemically different cerebral and systemic amyloidoses, finding the PS-1 carboxy epitope only in association with amyloid beta (A beta) lesions. We confirm the presence of this epitope ultrastructurally in neuritic plaques. In addition, biochemical and amino acid sequence data are presented for an association of the 18-kd carboxy fragment of PS-1 with neuritic plaques with a start at residue 300. Three of the proteins with linkage to AD have now been found as components of neuritic plaques. It remains to be determined whether all of these proteins are involved in the same or different pathological pathway(s) and which of these proteins is the most important for the common, late-onset form of AD.

Adult↗
PubMed1996

DNA and RNA strand scission by copper, zinc and manganese superoxide dismutases.

Copper/zinc (Cu/ZnSOD) and manganese (MnSOD) superoxide dismutases which catalyze the dismutation of toxic superoxide anion, O(2-)-, to O2 and H2O2, play a major role in protecting cells from toxicity of oxidative stress. However, cells overexpressing either form of the enzyme show signs of toxicity, suggesting that too much SOD may be injurious to the cell. To elucidate the possible mechanism of this cytotoxicity, the effect of SOD on DNA and RNA strand scission was studied. High purity preparations of Cu/ZnSOD and MnSOD were tested in an in vitro assay in which DNA cleavage was measured by conversion of phage phi X174 supercoiled double-stranded DNA to open circular and linear forms. Both types of SOD were able to induce DNA strand scission generating single- and double-strand breaks in a process that required oxygen and the presence of fully active enzyme. The DNA strand scission could be prevented by specific anti-SOD antibodies added directly or used for immunodepletion of SOD. Requirement for oxygen and the effect of Fe(II) and Fe(III) ions suggest that cleavage of DNA may be in part mediated by hydroxyl radicals formed in Fenton-type reactions where enzyme-bound transition metals serve as a catalyst by first being reduced by superoxide and then oxidized by H2O2. Another mechanism was probably operative in this system, since in the presence of magnesium DNA cleavage by SOD was oxygen independent and not affected by sodium cyanide. It is postulated that SOD, by having a similar structure to the active center of zinc-containing nucleases, is capable of exhibiting non-specific nuclease activity causing hydrolysis of the phosphodiester bonds of DNA and RNA. Both types of SOD were shown to effectively cleave RNA. These findings may help explain the origin of pathology of certain hereditary diseases genetically linked to Cu/ZnSOD gene.

Animals↗
PubMed1996

Peroxidase deficiency of nickel-transformed hamster cells correlates with their increased resistance to cytotoxicity of peroxides.

Using a procedure aimed at isolation of genes that are inactivated during nickel-induced carcinogenesis in Chinese hamster cells, a homolog of genes encoding human and mouse heme containing peroxidases has been cloned. Northern blot analysis of normal cultured fibroblasts and two nickel-transformed cell lines confirmed that this gene was expressed in normal but not in transformed cells. Nickel-transformed cells also tested negative for peroxidase activity using a sensitive fluorescence assay. Cultured embryo cells or fibroblasts that express peroxidase activity and their nickel-transformed peroxidase-deficient counterparts were employed to investigate the role of peroxidase-catalyzed processes in cytotoxicity induced by tert-butyl hydroperoxide or cumene hydroperoxide. It has been found that peroxidase-deficient cells were significantly more resistant to cytotoxic effect of these compounds suggesting that cytotoxic effect of hydroperoxides may be mediated in part by free radicals generated in the course of peroxidase-catalyzed reactions.

Animals↗
PubMed1994

Nickel-induced transformation of human cells causes loss of the phosphorylation of the retinoblastoma protein.

The retinoblastoma (Rb) protein (pRb) has been studied in various crystalline NiS-transformed cell clones derived from the human osteoblast cell line, HOS TE-85. The parental HOS cells were not able to proliferate in soft agar medium, but they acquired this property following treatment with crystalline NiS. The pRb was found only in the hypophosphorylated form in 8 of 9 nickel-transformed clones examined, whereas in the parental cells the pRb appeared in both phosphorylated and unphosphorylated forms. Neither Rb gene expression nor its phosphorylation was affected by acute nickel treatments of HOS cells. The nickel-transformed HOS clones expressed the major regulators of Rb phosphorylation, cyclin E and cdk-2, at levels similar to those of the parental cells. In coimmunoprecipitation assays with cell lysates from the transformed clones that exhibited the hypophosphorylated form of pRb, the Rb protein failed to form a complex with simian virus 40 large T-antigen, indicating a lack of functional activity. When a plasmid containing the normal Rb gene was transfected into these nickel-transformed cells, it restored the Rb phosphorylation pattern observed in parental cells and the cells acquired a normal phenotype (i.e., they were no longer able to grow in soft agar). This suggested that a mutation was induced in nickel-transformed cells that affected the ability of the Rb protein to be phosphorylated and function normally, and this mutation allowed the human nickel-transformed cells to acquire anchorage-independent growth.

Antigen-Antibody Reactions↗
PubMed1989

In vitro proliferation of murine spleen cells: inhibition by monoclonal antibodies to L3T4 and Lyt-2 T cell markers or intracellular cyclic adenosine monophosphate.

Proliferation of normal (not immunized intentionally) murine spleen cells was elicited with concanavalin A, supernatant fluid from cultures of EL-4 cells, human recombinant interleukin 2 (IL-2), or a mixture of phorbol ester and calcium ionophore A23187. IL-2-induced proliferation was inhibited by membrane-permeable dibutyryl cyclic adenosine monophosphate (cAMP) or by the adenylate cyclase activator forskolin. Consistent with these observations was the finding that stimulation with IL-2 decreased and forskolin increased the intracellular content of cAMP. IL-2-induced proliferation, as well as that induced by concanavalin A or phorbol-ionophore mixture, was inhibited by monoclonal antibodies specific for L3T4 or Lyt-2 cell surface markers. This inhibition was observed even when antibodies were added several hours after exposure of cells to IL-2. Notably, antibodies did not alter the intracellular content of cAMP. Thus, the experimental data failed to establish a functional linkage between the inhibitory effect of antibodies and the regulatory effect of the adenylate cyclase system. However, our results provide a rational basis for the postulation that antibodies, upon binding to their corresponding ligands, generate a negative signal that interferes with IL-2-induced proliferation. Therefore, L3T4 and Lyt-2 molecules appear to play an important role in the regulation of lymphocyte proliferation.

Animals↗
PubMed1989

In vitro proliferation of murine spleen cells: I. Strain variation of response to medium from cultures of EL-4 cells.

Proliferative responses of murine lymphoid cells were elicited in vitro with supernatant fluid from cultures of EL-4 thymoma cells stimulated with phorbol ester. It was demonstrated that such responses depend on IL-2 contained in the supernatant fluid, but reflect co-stimulation with IL-2 and phorbol ester. Striking differences in the magnitude of proliferative responses of spleen, splenic T lymphocytes, thymus and bone marrow cells from various strains were observed. Three classes of responders could be identified. The differences in responsiveness, at least in part, reflected differences in the frequency of responsive cells and were genetically controlled by codominant alleles of two independent somatic genes.

Animals↗
PubMed1988

Transfer by pro gene transfection of tumor promoter-sensitive phenotype to promotion-insensitive JB6 cells.

Transfection of activated promotion sensitivity genes (pro genes) confers on insensitive (P-) cells susceptibility to induction of anchorage-independent growth by tumor-promoting phorbol esters. Promotion-sensitive (P+) JB6 cell variants, from which activated pro-1 and pro-2 were cloned, respond to 12-O-tetradecanoylphorbol-13-acetate (TPA) and various nonphorbol tumor promoters with anchorage-independent transformation that is irreversible 60-80% of the time. Anchorage-independent (Tx) clonal lines derived from these TPA-induced agar colonies were also tumorigenic in nude mice. This report has addressed the question of whether the phenotypes associated with parental P+ cells are transferred by transfection of activated pro-1 and pro-2. Clonal lines were established after transfection of JB6 P- cells with activated pro-1 or pro-2, induction of anchorage-independent colony formation by TPA, and growth of individual agar colonies to yield clonal transfectant lines. The lines so derived from transfected populations included Tx, P+, and P- lines, reflecting irreversible neoplastic transformation and greater and lesser degrees of preneoplastic progression, respectively. The anchorage-independent transfectants were found to be tumorigenic. Since untransfected P- cells subjected to the same single-cycle TPA treatment and cloning in agar yielded no anchorage-independent and few P+ transfectants, the appearance of P+ and Tx transfectants after pro-1 and pro-2 transfection is therefore likely to be due to the transfected pro genes. Indirect assay of pro gene uptake by quick-blot hybridization of transfectant cell DNA with the vectors into which pro genes had been cloned confirmed the association of transferred P+ and Tx phenotypes with the presence of the transfected DNA. Finally, assay of the sensitivity of P+ pro-1 and pro-2 transfectants to transformation by TPA at various concentrations showed that transfection with pro-1 or pro-2 conferred about equal responses that were somewhat lower than those observed with parental P+ controls. Taken together these data indicate that promotion-insensitive JB6 cells need only an activated pro gene and TPA exposure to become neoplastically transformed.

Animals↗
PubMed1988

Comparison of P+-active and -inactive pro-1 homologues from human nasopharyngeal carcinoma cells.

Eleven pro-1 homologous clones from human nasopharyngeal carcinoma cell line CNE2 were studied with respect to their ability to transfer sensitivity to tumor promoter-induced neoplastic transformation (P+ activity), their molecular structure, and their homology to both mouse pro-1 and each other. Restriction mapping and Southern analysis of CNE2 pro-1 homologous clones revealed three structural classes, all of which showed P+ activity. The approximate limits of mouse pro-1 hybridizing sequences within CNE2 clones from each structural class were identified, and some of these minimum homologous sequences were tested for P+ activity by transfection into P- cells. For all classes, a strong association between P+ activity and pro-1 homology was observed. This finding implies that, for CNE2 clones to be P+-active, structural homology to mouse pro-1 is required. The minimum P+-active sequence thus far identified was a 2.6-kbp EcoRI-SstI fragment. One clone was inactive, even though it was indistinguishable from active clones of the same class by restriction mapping, Southern analysis, and electron microscope examination of heteroduplexes formed by an active and an inactive clone. This raises the possibility that discrete changes, involving a few nucleotides rather than gross rearrangement, may determine the P+ activation of these pro-1-homologous sequences.

Blotting, Southern↗
PubMed1987

Lifespan extension of basal cell nevus syndrome fibroblasts by transfection with mouse pro or v-myc genes.

Dermal fibroblasts from patients with the autosomal dominant cancer-prone disease Basal Cell Nevus Syndrome (BCNS) exhibit a serum dependence, anchorage dependence and in vitro lifespan (about 20 population doublings or less) similar to those of fibroblasts from normal age-, race- and sex-matched controls. Transfection with v-myc or with an activated mouse pro-I gene (which specifies sensitivity to promotion of neoplastic transformation in JB6 mouse epidermal cells) specifically conferred partial immortality on the BCNS fibroblasts by substantially extending their population doubling levels by more than 19 population doublings. This suggests that either v-myc or pro-I gene can cooperate with BCNS gene(s) to produce an extension of lifespan or partial immortality. However, the transfected BCNS fibroblasts that escaped senescence were anchorage-dependent even after exposure to the tumor promoters 12-O-tetradecanoyl-phorbol-13-acetate (TPA), epidermal growth factor (EGF) or platelet-derived growth factor (PDGF). These observations indicate that BCNS fibroblasts differ from their normal counterparts in susceptibility to extended growth and may therefore be pre-neoplastic. It is clear that they require more than an activated pro or myc gene for progression to the tumor cell phenotype.

Adult↗
PubMed1986

Anti-Thy-1 response of H-2f/H-2r heterozygotes: an unusual case of genetic control.

Anti-Thy-1 responsiveness of H-2r homozygous and H-2f/H-2r heterozygous mice was studied. Good responsiveness appeared to be independent of H-2 phenotype of responder but was influenced by the phenotype of the donor. These results were incompatible with the concept of Ir-Thy-1 genes controlling the response to cell-free Thy-1 in these mice. In contrast the results were indicative of the response to the cell-bound form of the Thy-1 antigen. It is proposed that good anti-Thy-1 response may reflect the presence of clones capable of recognizing the Thy-1 antigen in the context of or in association with incompatible class I H-2 molecules.

Animals↗