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Biomedical subjects

E Wang

Publications and source records attributed to E Wang.

At least 415 records · Page 23Linked to original sources
PubMed1985

Monoclonal antibodies to epitopes on different regions of the 200 000 dalton neurofilament protein. Probes for the geometry of the filament.

Neurofilaments in mammalian nervous tissues have three subunit proteins. These subunit proteins have apparent molecular masses of 200 (NF200), 150 (NF150) and 68 (NF68) kD. Biochemical assembly studies have indicated that the NF68 protein forms the core of the filament and that the other two proteins are associated proteins. Electron microscopy immunolocalization studies have been performed previously on isolated filaments and on filaments from neurons in culture, and have confirmed the localization of NF68 as a core filament protein and NF200 as a peripheral protein. We have raised two monoclonal antibodies to the NF200 components. Using immunogold labelled protein A, we have been able to localize these antibodies to tissue sections of adult cerebellum at the EM level. With this method, we have found that one of the monoclonal antibodies (NF2) shows a linear arrangement of gold particles directly on the filament, whereas the second monoclonal antibody (NF111) reacts with the filaments to give a periodic arrangement of gold particles. By immunoblotting against chymotryptic fragments of the NF200 protein, we have found that the mAB-NF111 reacts solely with a 160 kD piece, whereas the other monoclonal antibody reacts with both the 160 kD piece and the 40 kD piece. The latter piece was shown to be associated to the filament by binding studies with iodinated NF68. Thus the EM localization studies and the biochemical studies indicate that the two monoclonal antibodies react with different parts of the NF200 molecule, one binding to a part of the molecule which is located closer to the filament, and one to a more peripheral part of the molecule.

Animals↗
PubMed1985

Overflow urinary incontinence due to carbamazepine.

We describe the first well documented case of overflow urinary incontinence owing to prolonged carbamazepine treatment for temporal lobe seizures. Carbamazepine increased the bladder capacity to 1,700 ml. and was accompanied by symptoms of urgency and frequency. After carbamazepine was discontinued and the patient was given primidone the voiding symptoms disappeared. Post-voiding catheterization of the bladder showed minimal residual urine volume. In a review of urological adverse reactions of anticonvulsant drugs that are effective in the management of temporal lobe seizures we found that only primidone and phenobarbital have not been convincingly associated with such side effects. However, phenytoin and clonazepam have been linked with urinary incontinence, and valproic acid with enuresis.

Adolescent↗
PubMed1985

A 57,000-mol-wt protein uniquely present in nonproliferating cells and senescent human fibroblasts.

Mouse monoclonal antibody, S-30, was produced from hybridoma preparation from mice injected with the cytoskeleton extract of an in vitro aged culture of human fibroblasts derived from a 66-yr-old donor. The antibody stains positively the nuclei of the nonproliferating cells present predominantly in the senescent cultures of five selected fibroblast strains derived from donors of different age groups, whereas a negative reaction is observed in the cultures of their young counterparts. In the intermediate stage of the in vitro life span of these cell strains, a heterogeneous positive reaction for staining with S-30 antibody is observed in different subfractions of cell cultures. However, the expression of S-30 can be induced in the young fibroblasts at the early stage of their life by prolonged culturing to confluence. This induced expression of S-30 nuclear staining can be depleted upon subculturing at low cell density. Immunoelectron microscopy with colloidal gold-protein A complex demonstrates that the S-30 proteins are present in the nuclear plasma and at the region of nuclear envelope in a clustered arrangement. Immunoprecipitation of [3H]leucine labeled cell specimens shows that the antibody S-30 reacts with a protein with a molecular weight of approximately 57,000.

Animals↗
PubMed1985

Are cross-bridging structures involved in the bundle formation of intermediate filaments and the decrease in locomotion that accompany cell aging?

Five different fibroblast strains derived from donors of a wide range of ages were used for investigation of senescence-associated changes in the organization of intermediate filaments (IFs) and the activity of cell locomotion. Results of immunofluorescence microscopy demonstrate that, in large and flat in vitro aged fibroblasts, vimentin-containing IFs are distributed as unusually organized large bundles. Electron microscopic examination shows that these large bundles are indeed composed of filaments of 8-10 nm. Such a profile of large bundles is rarely seen in young fibroblasts whose IFs are usually interdispersed among microtubules. Within the large filament bundles of senescent fibroblasts, cross-bridge-like extensions are frequently observed along the individual IFs. Immunogold labeling with antibody to one of the cross-bridging proteins, p50, further illustrates the abundance of interfilament links within the IF bundles. The senescence-related increase in interfilament association was also supported by the results of co-precipitation between vimentin and an associated protein of 50,000 D. Time-lapse cinematographic studies of cell locomotion reveal that accompanying aging, fibroblasts have a significantly reduced ability to translocate across a solid substratum. These results led me to suggest that the increased interfilament links via cross-bridges may in part contribute to the mechanism that orchestrates the formation of large filament bundles. The presence of enormous bundles in the cytoplasm may physically impede the efficiency of locomotion for these nondividing cells.

Aging↗
PubMed1985

Rapid disappearance of statin, a nonproliferating and senescent cell-specific protein, upon reentering the process of cell cycling.

Statin, a 57,000-D protein characteristically found in nonreplicating cells, was identified by a monoclonal antibody produced by hybridomas established from mice injected with extracts of in vitro aged human fibroblasts (Wang, E., 1985, J. Cell Biol., 100:545-551). Fluorescence staining with the antibody shows that the expression of statin disappears upon reinitiation of the process for cell replication. The rapid de-expression is observed in fibroblasts involved in the in vitro wound-healing process, as well as in cells that have been subcultured after trypsinization and replated from a confluent culture. Kinetic analysis shows that 50% of the cell population lose their statin expression at 12 h after replating, before the actual events of mitosis. Immunogold labeling with highly purified antibodies localizes the protein at the nuclear envelope in nonreplicating cells, but not in their replicating counterparts. Immunoblotting analysis confirms the disappearance of statin in cells that have reentered the cycling process. Using the technique of flow cytometry to examine the large number of nonreplicating fibroblasts in confluent cultures, we have found that statin is mostly expressed in those cells showing the least amount of DNA content, whose growth is blocked at the G0/G1 stage of the cell cycle. This close correlation is rapidly altered once the cells are released from the confluent state. These results suggest that the expression of statin may be regulated by a fine mechanism controlling the transition from the nonreplicating to the replicating state, and that the protein is structurally associated with the nuclear envelope.

Aged↗
PubMed1985

Electron microprobe analysis of human labial gland secretory granules in cystic fibrosis.

X-ray microanalysis of freeze-dried labial gland cryosections revealed that Na concentration was doubled and the Ca/S concentration ratio was decreased in secretory granules of labial glands from patients with cystic fibrosis (CF) when compared with glands from normal subjects. Other results suggested that the decrease in the Ca/S concentration ratio resulted from an increase in S concentration. These findings imply that mucous granules in labial saliva showed a CF-related increase in Na and S content, and such changes would be expected to affect the rheology of the mucus after exocytosis. In contrast with a previous study in human parotid glands, no evidence was found for CF-related changes in cytoplasmic or nuclear Na, K, and Ca concentrations. Significant elemental differences were found between secretory granules and nuclei and cytoplasm of control cells.

Adolescent↗
PubMed1985

Application of a unique monoclonal antibody as a marker for nonproliferating subpopulations of cells of some tissue.

A monoclonal antibody (clone S-30), directed to a protein of 57,000 daltons, was developed from the fusion of mouse myeloma cells and the spleen cells of mice injected with cytoskeletal extracts of fibroblasts that have been aged in in vitro culturing conditions according to a schedule of serial passaging (Cristofalo VJ, Charpentier R: J Tissue Culture Meth 6:117, 1981; Wang E: J Cell Biol, submitted). The staining activity of S-30 antibody was observed exclusively in the nuclei of nonproliferating senescent fibroblasts, but not in their young counterparts. Immunolocalization of S-30 antibody in frozen tissues from various sites reveals the positive staining reaction in the nuclear envelope region in those cells that are at the final stage of differentiation and are no longer replicating. These tissue sites include epithelial cells of the suprabasal layer of epidermis, hair sheath, and tongue, a subpopulation of fibroblasts in the dermis, chondrocytes, hepatocytes, and cells of cardiac muscle. The absence of S-30 staining activity was noted in tissues such as simple epithelium located in the gastrointestinal tract and kidney, and keratinocytes in the basal layer. These results suggest that the S-30 antibody can be used as a marker for nonproliferating cells both in cultured fibroblasts and in some tissues. It seems that the mechanism that controls the cessation of cell proliferation is related, in part, to the postmitotic expression of the 57,000 dalton protein.

Aged↗
PubMed1984

Activities of the initial enzymes of glycerolipid and sphingolipid synthesis in lung microsomes from rats exposed to air or 85% oxygen.

Lungs of adult rats exposed to 85% oxygen undergo extensive cellular reorganization; therefore, to investigate changes in lipid metabolism the initial enzymes of glycerolipid and sphingolipid synthesis were measured in lung microsomes. After 1 week of O2 treatment, the specific activity of the glycerol 3-phosphate acyltransferase increased to nearly twice that of the controls and remained elevated for the 3 weeks of study. Serine palmitoyl-transferase activities were approximately the same for both groups. These results suggest that in addition to cellular proliferation caused by hyperoxia there are also selective changes in glycerolipid synthesis, which may explain the decreased sphingomyelin content of lung and lamellar bodies.

Acyltransferases↗
PubMed1984

Enzymology of long-chain base synthesis by liver: characterization of serine palmitoyltransferase in rat liver microsomes.

Serine palmitoyltransferase [palmitoyl-CoA:L-serine C-palmitoyltransferase (decarboxylating) EC 2.3.1.50] catalyzes the initial and committed step in the biosynthesis of the long-chain bases of sphingolipids. A simple assay, based upon the incorporation of [3H]serine into the chloroform-soluble product 3-ketosphinganine, has been developed and demonstrated to be valid for analyzing this enzyme in rat liver microsomes. More than 75% of the serine palmitoyltransferase of rat liver was associated with the microsomal subfraction. The dependencies of activity on the incubation time, pH, temperature, other assay components (e.g., dithiothreitol, EDTA, and pyridoxal 5'-phosphate), and the concentrations of microsomal protein, L-serine, and palmitoyl-CoA were investigated. The requirement of pyridoxal 5'-phosphate for activity was established by formation of the apoenzyme by dialysis against cysteine, and recovery of full activity upon reconstitution with the coenzyme. Activities with fatty acyl-CoA's of varying alkyl chain length were distributed nearly symmetrically around a maximum at 16 carbons (palmitoyl-CoA) for the fully saturated substrates. Less activity was obtained with the CoA thioesters of cis-unsaturated fatty acids, but trans-9-hexadecenoyl-CoA yielded essentially the same activity as palmitoyl-CoA. Hence, this enzyme is capable of initiating the synthesis of the major long-chain bases, as well as compounds that may constitute the unidentified bases reported in analyses of mammalian sphingolipids.

Acyl Coenzyme A↗
PubMed1984

Increased organization of cytoskeleton accompanying the aging of human fibroblasts in vitro.

Fibroblastic cells of human origin have a limited lifespan in culture. One of the senescence-associated phenotypic changes is an increase in the abundance of cytoplasmic filaments. Human skin fibroblasts (strain 0011), derived from an 8-week-old male fetus, were passaged according to a predetermined schedule and examined at successive population doubling levels. In young rapidly growing cultures, fluorescence microscopy with NBD-Phallacidin shows a normal organization of the actin-containing fibers, microtubules and intermediate filaments, as has been described previously. At stages close to the end of the in vitro lifespan of the cell strain, large flat fibroblasts are the predominant cell type in culture. These large senescent fibroblasts contain numerous prominent actin fibers traversing the entire long axis of the cytoplasm. The fibers are often located adjacent to each other and appear to form a sheet on the ventral side of the cytoplasm. Staining of senescent cells with anti-tubulin antibody reveals an increase in the abundance of microtubules per cell and the distribution pattern is altered through the increase in the number of organization centers. Intermediate filaments are also more abundant and display tightly packed fibrillar sheets or bundles. Electron microscopic studies have confirmed the increased organization of microfilaments into bundles in senescent cells. These results suggest that during in vitro senescence, the increase in cell size is correlated with increased organization of the cytoskeleton. The presence of a rigid cytoskeletal structure may contribute in part to the inability of the senescent cell to replicate.

Cell Survival↗
PubMed1984

Identification of glial filament protein and vimentin in the same intermediate filament system in human glioma cells.

We have used a human glioma cell line (U-251MG) to study the expression and cytoplasmic organization of vimentin (decamin) and the glial filament protein (GFP). Four clones of the parental U-251 cultures were isolated and found to express GFP from 1-2% to 99% of the cells in the population. Double immunofluorescence microscopy with antibodies to vimentin and GFP has shown that, in all four clonal cell lines, vimentin-containing filaments are expressed in most cells as an organized network and, in GFP-positive cells, GFP and vimentin are associated with the same filament network. Immunoelectron microscopy with specific antibodies labeled with colloidal gold particles of various sizes shows that GFP and vimentin are localized in the same filaments. These findings confirm in vitro studies of the copolymerization of subunits of different biochemical nature into the same intermediate filament and suggest the in vivo probability of the coassembly of GFP and vimentin from a possible soluble pool of monomers.

Astrocytoma↗
PubMed1984

Unphosphorylated gelsolin is localized in regions of cell-substratum contact or attachment in Rous sarcoma virus-transformed rat cells.

Regions associated with cell-substratum contact or attachment in Rous sarcoma virus (RSV)-transformed rat fibroblasts (RR1022 cells) were identified by reflection-interference microscopy. Electron microscopy of such regions revealed the presence of discrete membrane-associated structures composed of a paracrystalline lattice of hexagons and pentagons to which actin filaments appear to be attached. Staining of actin by biotin-labeled heavy meromyosin showed that transformed cells, unlike normal fibroblasts, lack prominent actin fibers, and that, instead, much of the fluorescence is concentrated in loci corresponding to locations of transient association between the cell and the substratum. In stationary cells, such loci were found in rosette formation, predominantly in the region beneath the nucleus. In cells engaged in active movement, such as during migration into a wound, the actin-containing spots were concentrated in the region of the leading edge. A similar pattern of staining was observed with antibody to gelsolin, a 91,000-dalton Ca2+-dependent actin filament-shortening protein. Since the action of gelsolin on actin is reversible and dependent on physiologically relevant changes in calcium concentration, the localization of gelsolin, together with actin-bundling proteins such as alpha-actinin, in the regions containing many small microfilament bundles on the ventral side of cytoplasm suggests that gelsolin may be a component of the mechanism for the disassembly and assembly of actin during the dissolution and reformation of structures for cell-substratum contact during cell locomotion. Regulation of gelsolin activity was not dependent on protein phosphorylation, as shown by lack of 32P-incorporation into gelsolin in either transformed or normal fibroblasts.

Actins↗
PubMed1984

Interferon suppresses pinocytosis but stimulates phagocytosis in mouse peritoneal macrophages: related changes in cytoskeletal organization.

Treatment of thioglycolate-elicited macrophages with mouse beta-interferon markedly reduces pinocytosis of horseradish peroxidase and fluorescein isothiocyanate (FITC)-dextran but stimulates phagocytosis of IgG-coated sheep erythrocytes. Experiments with FITC-dextran have revealed that the overall decrease in pinocytosis is due to a nearly complete inhibition of pinocytosis in a large fraction of interferon-treated macrophages. In the remaining cells pinocytosis continues at a rate similar to that in untreated control cells. A considerable reduction in the number of cells pinocytosing FITC-dextran was observed within 12 h from the beginning of interferon treatment. Measurement of the overall level of pinocytic activity with horseradish peroxidase showed a progressive decline through 72 h of treatment. In the interferon-sensitive subpopulation, there were marked changes in cytoskeletal organization. Microtubules and 10-nm filaments were aggregated in the perinuclear region while most of the peripheral cytoplasm became devoid of these cytoskeletal structures as observed by fluorescence and electron microscopy. In addition, interferon treatment of macrophages appeared to disrupt the close topological association between bundles of 10-nm filaments and organelles such as mitochondria, lysosomes, and elements of the Golgi apparatus and endoplasmic reticulum. Such alterations in the distribution of microtubules and 10-nm filaments were not seen in the interferon-insensitive subpopulation. We have investigated the mechanism of the interferon-induced enhancement of phagocytic activity by binding IgG-coated sheep erythrocytes to mouse peritoneal macrophages at 4 degrees C and then initiating a synchronous round of ingestion by warming the cells to 37 degrees C. Thioglycolate-elicited macrophages that had been treated with mouse beta-interferon ingested IgG-coated erythrocytes faster and to a higher level than control cells in a single round of phagocytosis. In interferon-treated cultures, phagocytic cups became evident within 30 s of the shift of cultures from 4 degrees to 37 degrees C, whereas in control cultures, they appeared in 2 min. Cytochalasin D, an inhibitor of actin assembly and polymerization, abolished phagocytic activity in both control and beta-interferon-treated macrophages. However, to inhibit phagocytosis completely in thioglycolate-elicited interferon-treated macrophages, twice as much cytochalasin D was required in the treated as in control cultures.(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗
PubMed1984

Metabolism of vitamin B-6 by human liver.

The enzymes that metabolize vitamin B-6 were analyzed in liver biopsy samples from five patients without hepatic disease by using methods optimized for small samples. Pyridoxal kinase (EC 2.7.1.35) activities were 11.2 +/- 3.6 nmol/minute per gram of tissue and 0.16 +/- 0.05 nmol/minute per milligram of soluble protein (mean +/- SD); a clear dependence of the activity on zinc as the divalent cation was observed. Pyridoxine (pyridoxamine) 5'-phosphate oxidase (EC 1.4.3.5) activities, when using N-(5'-phosphopyridoxyl)-[3H]tryptamine as the substrate, were 0.64 +/- 0.22 pmol/minute per milligram of protein and 47 +/- 19 pmol/minute per gram of tissue. The activities were 63 +/- 18% lower when riboflavin 5'-phosphate was omitted from the assay; hence, it appears the oxidase is only partially saturated with its cofactor. The pyridoxal 5'-phosphate hydrolase(s) activities at alkaline pH were 282 +/- 183 nmol/minute per gram of tissue and 4.0 +/- 3.2 nmol/minute per milligram of particulate protein. Pyridoxal was rapidly oxidized to pyridoxic acid (28.1 +/- 19.8 nmol/minute per gram of tissue and 0.37 +/- 0.24 nmol/minute per milligram of soluble protein) by soluble enzyme(s), and the rate was unaffected by pyridine nucleotides. These experiments constitute the first quantitative analyses of the enzymes responsible for metabolizing vitamin B-6 in human liver, and provide data for interpreting the pharmacokinetics of B-6 utilization by humans, as well as methods for investigating diseases with aberrant metabolism of this nutrient.

Alcohol Oxidoreductases↗
PubMed1984

In situ hybridization and translocation breakpoint mapping. I. Nonidentical 22q11 breakpoints for the t(9;22) of CML and the t(8;22) of Burkitt lymphoma.

In situ chromosomal hybridization of a probe for part of the lambda light chain constant region (C lambda) has demonstrated that the 22q11 breakpoints of chronic myelogenous leukemia (CML) t(9;22) and Burkitt lymphoma t(8;22) are not identical. For CML, the breakpoint is distal to the IGLC genes, whereas for Burkitt lymphoma it is proximal. The study provides direct evidence for regional assignment of the IGLC gene cluster to 22q11.

Burkitt Lymphoma↗
PubMed1984

Growth rate of control and beta-interferon-treated human fibroblast populations over the course of their in vitro life span.

The estimated growth rates for five lines of human fibroblasts (Human Genetic Mutant Cell Repository designations 0011, 2936B , 0038A , 2912A , and 3529), during the first few population doublings after establishment of the lines, were within the expected range, i.e., equivalent to doubling times from 16 to 28 hr, with a mean of 20 hr. The lines were derived from donors aged 8 fetal weeks, 20 days, 9 years, 26 years, and 66 years, respectively. The growth rates of the five lines declined as an exponential function of the population doubling level in all cases. The rate of decline of the growth rate varied for different lines and appeared to be related to the life span of the lines, which in turn was related to donor age. After 30 population doublings, the population doubling times had increased 1.3, 1.3, 1.7, 3.4, and 4.7 times for the five cell lines of the corresponding replicative life spans of 65, 57, 56, 31, and 28 population doublings. Sensitivity of the fibroblast lines to the cell growth-inhibitory effect of beta-interferon was independent of the population doubling level of the lines, i.e., interferon depressed the population growth rate of low population doubling level cells as much as that of middle or late population doubling level cells for any given line. However, the fibroblast lines showed differences in their sensitivity to the cell growth-inhibitory effect of interferon, probably as an expression of genotypic differences among the lines.

Adult↗