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Biomedical subjects

N P Wang

Publications and source records attributed to N P Wang.

At least 19 recordsLinked to original sources
PubMed1996

Uterine epithelioid leiomyosarcoma in a pig-tailed macaque.

During ultrasonographic examination following spontaneous abortion, an approximately 6-cm diameter mass of mixed echogenicity is detected in the uterine wall of a mature pig-tailed macaque (Macaca nemestrina). Grossly, the mass is associated with the uterine fundus, multilobulate and tan with red-black mottled foci on cut surface. Microscopically, the invasive mass is composed of poorly differentiated round cells arranged in sheets and bundles supported by a fine fibrous stroma. On immunohistochemical evaluation, neoplastic cells contain vimentin, desmin, muscle actin, and smooth muscle alpha-actin antigens and do not react with antibodies to low and high molecular-weight cytokeratins. Cell morphology and immunohistochemical results indicate a diagnosis of epithelioid leiomyosarcoma (clear cell variant).

Abortion, Veterinary↗
PubMed1996

Does alveolar soft-part sarcoma exhibit skeletal muscle differentiation? An immunocytochemical and biochemical study of myogenic regulatory protein expression.

There has been persistent controversy regarding the nature of cell differentiation in alveolar soft-part sarcoma (ASPS) since its first description in 1952. Some studies suggest that ASPS might represent an unusual variant of skeletal muscle tumor. Given the availability of new monoclonal antibodies to probe for skeletal muscle differentiation and the rapid advance in immunocytochemical techniques for deparaffinized, formalin-fixed tissue sections, we wished to test the proposed hypothesis that ASPS might represent a new type of rhabdomyosarcoma. Twelve archival samples of ASPS were retrieved, and we investigated the expression of two myogenic regulatory proteins, MyoD1 and myogenin, as well as other muscle-associated proteins, using sensitive immunocytochemical techniques. Despite the presence of desmin immunostaining in six ASPSs, no tumors were positive for either muscle actin or myoglobin. Most importantly, no specimen showed nuclear expression of MyoD1 or myogenin. In 11 tumors, however, there was considerable granular immunostaining in the tumor cell cytoplasm with the anti-MyoD1 monoclonal antibody 5.8A, a phenomenon observed in various nonmuscle normal and neoplastic tissues with this antibody. To analyze the exact nature of immunostaining of MyoD1 and desmin in ASPS, biochemical analyses using available fresh frozen tumor tissue were performed. Although a 53-kDa band was noted with antidesmin antibody on Western blot analysis, no specific protein band that corresponds to the 45-kDa MyoD1 was detected with antibody 5.8A. These results confirm the presence of desmin in ASPS but argue against authentic expression of MyoD1. They also suggest that the cytoplasmic immunostaining observed with anti-MyoD1 antibody 5.8A most likely represents a nonspecific cross-reaction with an unknown cytoplasmic antigen. Considering the master role that MyoD1 and myogenin play in skeletal muscle commitment and differentiation and the lack of expression of these two proteins in ASPS as determined immunocytochemically and biochemically, we think that the histogenesis of ASPS remains unknown.

Adolescent↗
PubMed1995

Expression of myogenic regulatory proteins (myogenin and MyoD1) in small blue round cell tumors of childhood.

The distinction of rhabdomyosarcoma (RMS) from other small blue round cell tumors of childhood, such as Ewing's sarcoma/peripheral primitive neuroectodermal tumor (pPNET) and neuroblastoma, continues to present a diagnostic challenge to pathologists. The recent recognition of the master role of myogenic regulatory proteins in skeletal muscle commitment and differentiation, and the availability of monoclonal antibodies to two of them (myogenin and MyoD1), has prompted us to test their diagnostic utility in routinely processed, formalin-fixed, and deparaffinized tissue. Preliminary studies had demonstrated that, with the use of heat-induced epitope retrieval techniques, expression of myogenin and MyoD1 could be documented specifically in nuclei of fetal skeletal muscle by the respective antibodies. We performed a retrospective immunohistochemical analysis on 72 cases of small blue round cell tumors, including 33 RMSs, 1 metastatic myogenous Wilms' tumor, 26 Ewing's sarcomas/pPNETs, and 12 neuroblastomas. Nuclear expression of myogenin and MyoD1 were both found in 30/33 non-overlapping cases of RMS, with no significant differences in the sensitivity with respect to histological subtypes, and in 1/1 case of myogenous Wilms' tumor. None of the neuroblastomas or Ewing's sarcomas/pPNETs demonstrated positive nuclear staining with either antibody. However, most of the neuroblastomas, and occasional Ewing's sarcomas/pPNETs, showed variable fibrillary, cytoplasmic immunoreactivity with antibody to MyoD1. We conclude that, with the use of microwave-based epitope retrieval, antibodies to myogenin and MyoD1 are both useful markers for the identification of RMS among other small blue round cell tumors of childhood, but antibodies to myogenin have technical advantages over those to MyoD1, as the latter may cross-react with an unknown cytoplasmic antigen in non-muscle cells and tumors.

Child↗
PubMed1995

Purification and characterization of [3H]mepyramine (histamine H1 antagonist)-binding protein from rat liver: a highly homologous protein with cytochrome P450 2D.

A protein having a high-affinity binding site for [3H]mepyramine (MBP) was purified to homogeneity from rat liver membranes. The purified MBP has a single type of binding site for [3H]mepyramine with Kd value of 18.5 nM, and its molecular weight was determined to be 56,000 by SDS polyacrylamide gel electrophoresis. Amino acid sequences of twelve tryptic peptides derived from MBP are highly homologous with those of rat debrisoquine 4-hydroxylase (cytochrome P450 2D1) and other rat P450 2D subfamily members. In immunoblotting analysis, an antibody against rat P450 2D1 stained a band corresponding to MBP with Mr of 56,000; its migration position was clearly different from that of rat P450 2D1. Substrates and inhibitors of debrisoquine 4-hydroxylase potently displace [3H]-mepyramine binding to MBP. Quinine and quinidine showed 400 and 80 times, respectively, higher affinity for MBP than for debrisoquine 4-hydroxylase. These results suggest that MBP is a novel P450 2D family member.

Amino Acid Sequence↗
PubMed1995

Determination of age-related changes of cytochrome P-450 sensitive to mepyramine in rat hepatic microsomes.

The cytochrome P-450 (Cyt P-450) sensitive to mepyramine (HP-450) in liver microsome is a protein that possesses properties of both H1 receptor and cytochrome P-450. We used [3H]mepyramine radioligand binding assay and enzymological technique to study the kinetic properties of HP-450 and Cyt P-450. Age-related changes in the Bmax, Kd of HP-450 and Cyt P-450 in rat hepatic microsome were demonstrated. The levels of Bmax for both HP-450 and the Cyt P-450 dramatically increased in the postnatal period from the second to the eighth week, and reached maximum steady status during the eighth to tenth week. The values of Bmax of HP-450 correlated well with the content of Cyt P-450 in rat liver microsome (r = 0.625). The affinity of HP-450 (Kd) decreased with the growth of rats during postnatal. No sexual-related difference was observed in this experiment.

Aging↗
PubMed1993

Tumor suppressor activity of RB and p53 genes in human breast carcinoma cells.

Breast cancer is the most common cancer of women in Western countries. Various genetic alterations have been implicated in its development. Two tumor suppressor genes, the retinoblastoma susceptibility gene (RB) and the gene encoding the p53 protein, are frequently found to be deleted or mutated in breast cancer cell lines and primary tumor samples. Breast carcinoma cell lines MDA-MB468 and BT549 both harbor partial RB gene deletions as well as point mutations of their p53 genes, thus providing an excellent model system for testing the roles played by these two genes in the oncogenesis of breast cancer. Single copies of wild-type RB or p53 were delivered to these cells by retrovirus-mediated gene transfer. Restoration of RB expression in cells reduced their ability to grow in soft agar and their tumorigenicity in nude mice, although no significant alteration of growth rate in culture could be detected. Introduction of wild-type p53 into these cells exerted a similar effect on the neoplastic phenotypes. This effect occurred even in the presence of their endogenous mutated p53 alleles, which argues for the phenotypical dominance of the wild-type p53 over mutated p53 during human oncogenesis. The ability of RB and p53 genes to suppress the tumorigenicity of breast carcinoma cells provides functional evidence that deletion or mutational inactivation of tumor suppressor genes represents an important step in the genesis of breast cancer.

Animals↗
PubMed1991

The retinoblastoma gene product regulates progression through the G1 phase of the cell cycle.

The RB gene product is a nuclear phosphoprotein that undergoes cell cycle-dependent changes in its phosphorylation status. To test whether RB regulates cell cycle progression, purified RB proteins, either full-length or a truncated form containing the T antigen-binding region, were injected into cells. Injection of either protein early in G1 inhibits progression into S phase. Co-injection of anti-RB antibodies antagonizes this effect. Injection of RB into cells arrested at G1/S or late in G1 has no effect on BrdU incorporation, suggesting that RB does not inhibit DNA synthesis in S phase. These results indicate that RB regulates cell proliferation by restricting cell cycle progression at a specific point in G1 and establish a biological assay for RB activity. Neither co-injection of RB with a T antigen peptide nor injection into cells expressing T antigen prevents cells from progressing into S phase, which supports the hypothesis that T antigen binding has functional consequences for RB.

Animals↗
PubMed1991

[3H]mepyramine binding sites, histamine H1-receptors, in bovine retinal blood vessels.

The presence of histamine H1-receptors in the bovine retinal blood vessels was studied with a [3H]mepyramine binding assay. The membranes of purified vessels obtained from bovine retinas showed specific [3H]mepyramine binding sites with a dissociation constant (KD) of 2.78 +/- 0.32 nM. This was similar to values obtained from the retinal neuronal fractions. The binding capacity (Bmax) was 53.8 +/- 1.7 fmol/mg protein, which was about a half that of the retinal neuronal fractions (108.9 +/- 3.1 fmol/mg protein). Some H1-antagonists proved to be potent competitors for [3H]mepyramine binding sites in bovine retinal blood vessels. These results indicate that histamine H1-receptors exist in the retinal blood vessels which may be involved in the physiological and the pathological responses of blood circulation in retinas.

Animals↗
PubMed1991

Histamine and its synthesis in mammalian retinas.

Histamine (HA) content and histidine decarboxylase (HDC) activity were studied in the sensory retinas of the rat, guinea-pig, rabbit, dog, monkey, bovine and pig. Using a highly sensitive and specific method developed for the determination of histamine using high-performance liquid chromatography, HA was detected in the retinas of all species examined. HA content ranged from 11 (bovine) to 540 pmole/g wet tissue (dog), and HDC activity ranged from 2 (rabbit) to 150 (dog) fmol HA synthesized/min/mg protein. With the exception of the dogs, the retinal values were one order lower than those in the brain in all species examined. The canine retinas had the high HA and HDC values among the species examined, and were approximately equal to those in the brain. However, the ratios of HDC activity to HA content ranged from 0.04/min to 0.51/min. These values resembled those in the brain and were much higher than those in mast cells. This suggests that histamine in the retinas exists mainly in the neurons even though the actual amounts are small and may be involved in the physiological and the pathological responses in mammalian retinas.

Animals↗
PubMed1991

[Preliminary evaluation of inoculation of hepatitis B vaccine in normal adult subjects].

A total of 107 subjects in population without HBsAg, anti-HBs and anti-HBc were inoculated with hepatitis B vaccine in month 0, 1, 6, respectively and followed up for three years. Seven months after the administration of first dose of vaccine response rate of anti-HBs was 94.4%, of which 68.2% had a S/N ratio greater than 10. Three years later the response rate of anti-HBs dropped to 75.7% and the rate of S/N ratio 10 fell to 46.7%, 20 subjects turned negative for anti-HBs and one turned positive for HBsAg. During the same period, of 124 adults who were hepatitis B virus markers negative and were not inoculated, 14 became HBsAg-positive (ALT increased in 3 of them) and 3 turned anti-HBs positive.

Adolescent↗
PubMed1990

Two distinct and frequently mutated regions of retinoblastoma protein are required for binding to SV40 T antigen.

The retinoblastoma susceptibility gene (RB) encodes a phosphoprotein of 110 kd (pp110RB) that forms specific complexes with SV40 T antigen and the transforming proteins of several other DNA tumor viruses. Interaction with RB is thought to contribute to transformation by these viruses as demonstrated by genetic analyses. To help understand the function of these interactions, the regions of RB that are involved in binding to T have been mapped. An in vitro protein synthesis system capable of producing full-length RB protein has been developed to facilitate the mapping study. A 5- to 10-fold increase in translational efficiency in the reticulocyte lysate was obtained when the 5' non-coding region of RB mRNA was replaced with that of beta-globin mRNA or a plant viral RNA, alfalfa mosaic virus (AMV) RNA4. A series of mutated RB polypeptides produced from this system were assayed for T binding. Two non-contiguous regions of the RB protein, amino acid residues 394-571 and 649-773, were found to be necessary for binding to T: mutations in either region abolished T-RB complex formation. These results are consistent with the finding that, in all the cases analyzed so far, mutated RB proteins in human tumor cells also failed to bind to T antigen due to deletions including at least one of the two required regions. Thus the regions of RB defined in vitro as necessary for interaction with T might be physiologically relevant as well, and might play a fundamental role in normal RB protein function.

Antigens, Polyomavirus Transforming↗
PubMed1990

DNA-binding activity of retinoblastoma protein is intrinsic to its carboxyl-terminal region.

The retinoblastoma (RB) gene encodes a nuclear phosphoprotein with a molecular weight of 110,000 (pp110RB) associated with DNA-binding activity. This sequence-nonspecific DNA binding activity was further studied by Southwestern and DNA-cellulose chromatography using purified fusion proteins expressed in Escherichia coli. Three fusion proteins, containing amino acids 612-775, 776-928, and 612-928 of pp110RB, bound to DNA; the binding affinity of the latter was approximately 20-fold higher than those of either smaller region. Other regions of pp110RB had no detectable binding activity, indicating that the carboxyl-terminal region of the RB protein is the major domain responsible for interacting with DNA. Since several potential phosphorylation sites reside within this region, isoforms of RB protein from cellular lysates with various degrees of phosphorylation were compared with respect to their DNA-binding affinity. The hyperphosphorylated form was eluted from DNA-cellulose columns at 0.1-0.3 M NaCl, whereas the hypophosphorylated form appeared in the eluates only at salt concentrations of 0.4-0.7 M, implying that phosphorylation of RB protein may affect its DNA-binding activity. That pp110RB can bind DNA intrinsically, and that this activity can be modulated by phosphorylation, is consistent with the proposed regulatory role of the RB protein in cell growth and differentiation.

Blotting, Southern↗
PubMed1990

Expression of the human retinoblastoma gene product pp110RB in insect cells using the baculovirus system.

The product of the retinoblastoma susceptibility gene (RB) was overproduced in cultured insect cells using the baculovirus expression system. Upon insertion of the cloned human RB complementary DNA sequence into the viral genome downstream of the promoter of the polyhedrin gene, full-length RB protein with an apparent molecular weight of 110,000 was expressed in the insect cells. This protein was found to be phosphorylated, located in the nuclei of the infected cells, and immunologically indistinguishable from pp110RB of human cells as assayed by several anti-RB antibodies. Following cell disruption and a one-step immunoaffinity chromatographic purification, 6-12 mg of soluble pp110RB with approximately 95% purity were obtained per liter of infected suspension culture. Characterization of the two known biochemical properties of RB protein showed that this purified protein from insect cells behaved similarly to the authentic human pp110RB. First, it bound to DNA, and second, it could form a specific complex with SV40 T antigen in vitro. Prompt translocation of the protein from cytoplasm to nucleus after microinjection further indicated that the purified RB protein may be active. The availability of soluble, intact, and presumably active pp110RB in large quantity represents a significant advance for studying the biochemical and biophysical properties of the RB gene product as well as its potential biological function in cancer suppression.

Animals↗
PubMed1989

Characterization of histamine H1-receptors on astrocytes in primary culture: [3H]mepyramine binding studies.

The characteristics of histamine H1-receptors on astrocytes from the cerebral cortex of newborn rats in primary culture were analyzed with a [3H]mepyramine binding assay, and compared with those in the cerebral cortex. The apparent dissociation constant (KD) of [3H]mepyramine binding, the apparent inhibition constants (Ki) of various H1-ligands for [3H]mepyramine binding and the stereoselectivity of d- and l-chlorpheniramine for the inhibition of [3H]mepyramine binding to receptors on cultured astrocytes and to receptors in the brain tissue were very similar, indicating that these receptors are identical. The apparent density of H1-receptors (Bmax) on astrocytes was 262 +/- 60 fmol/mg protein, which was comparable to that in the brain tissue (194 +/- 24 fmol/mg protein). The development of H1-receptors on cultured astrocytes resembled the postnatal development of the receptors in the rat brain. These results suggest that astrocytes could be one of the main targets of the central histaminergic system.

Aminopyridines↗
PubMed1989

Divalent cations increase the binding capacity of the [3H]mepyramine binding site, a possible histamine H1 receptor, in rat liver membranes.

The [3H]mepyramine binding to rat liver membranes was about 2-fold higher in the presence of 2 mM CaCl2 than in the absence. However, the [3H]mepyramine binding to rat brain membranes was not affected by the presence of 2 mM CaCl2. Scatchard analysis showed that 2 mM CaCl2 did not change the affinity (KD) of liver membranes to [3H]mepyramine, but increased the binding capacity (Bmax). CaCl2 had a half maximal effect (ED50) at 3 microM and a maximal effect at concentrations of more than 10 microM. Other divalent cations, Mg++, Mn++, Sr++ and Ba++, also increased the [3H]mepyramine binding, whereas monovalent cations, Na+, K+, Li+ and Rb+, had no effect.

Aminopyridines↗
PubMed1988

Histamine H1-receptor in the retina: species differences.

Histamine H1-receptors in membranes of the various mammalian retinas were studied by [3H]mepyramine binding assay. Specific [3H]mepyramine bindings to bovine, pig, dog and human retinas were observed with the dissociation constants (KD), 3.8 +/- 1.2 nM, 1.8 +/- 0.6 nM, 2.6 +/- 0.6 nM and 3.0 +/- 0.9 nM, respectively, which were similar to those found in brains. But there was no detectable specific binding in the guinea-pig and rabbit retinas. The number of binding sites (Bmax) ranged from negligible value to 290.7 +/- 51.7 fmole/mg protein(human retina). Some H1-antagonists acted as potent agents in competing with [3H]mepyramine binding to bovine and pig retinas. These results indicated that histamine H1-receptors exist in some mammalian retina and have similar characteristics to those in brain membranes, but they distributes in the wide difference of the binding capacities among the species, while in brain variations were smaller.

Animals↗
PubMed1988

Solubilization, characterization and partial purification of [3H]mepyramine-binding protein, a possible histamine H1 receptor, from rat liver membrane.

[3H]Mepyramine binding protein, a possible subtype of histamine H1 receptors, was solubilized from rat liver membrane with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and Tween 60 as detergents and glycerol as an enhancer of solubilization. The optimal concentration of CHAPS was 10 mM and that of glycerol was 20% or more (v/v). The molecular weight of the [3H]mepyramine binding protein-detergent complex was determined to be 670K by Sepharose CL-4B gel filtration and 800K by sucrose density gradient sedimentation. By target size analysis, the molecular weights of both the membrane-bound and solubilized [3H]mepyramine binding protein were determined to be 162K. These values are similar to those of other well-characterized H1-receptor proteins, though slightly different. Simultaneous computerized analysis of the data obtained by [3H]mepyramine binding to the solubilized [3H]mepyramine binding protein indicated the presence of a single binding site with a KD value of 19.0 +/- 5.6 nM and a binding capacity (Bmax) of 6.6 +/- 2.1 pmole/mg protein. The Ki value of cold mepyramine for [3H]mepyramine binding to the solubilized receptor was 20 +/- 4 nM, whereas those of diphenhydramine, d-chlorpheniramine and triprolidine were all 2.9 +/- 0.8 microM, or about 150 times that of mepyramine. These data on the molecular and binding characteristics of the solubilized protein reported here suggest that there is a subtype of histamine H1 receptor in rat liver membrane. The solubilized preparation retained 90% and 75% of its [3H]mepyramine binding activity after storage at -80 degrees C and 4 degrees C, respectively, for 20 days. The solubilized [3H]mepyramine binding protein was purified 30-fold by Sepharose CL-4B gel filtration, Bio Gel HTP hydroxylapatite, Octyl Sepharose 4B and hydroxylapatite HPLC column chromatographies.

Aminopyridines↗