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Biomedical subjects

Y Wu

Publications and source records attributed to Y Wu.

At least 991 records · Page 55Linked to original sources
PubMed1995

[Effect of antiserum against dynorphin A administered intrathecally on spinal cord injury (SCI) of rats and its significance].

The antagonistic effects of antisera against dynorphin A, beta-endorphin, and leu-enkephailin administered intrathecally on secondary SCI were observed and compared after moderate SCI using principle of antigen-antibody neutralization reaction. The protective effect of antiserum against dynorphin A was most prominent on secondary SCI and the effect was more prominent when administered at 24 hours following SCI than when administered at 0 hour four hours, one week or two weeks following SCI. That suggests increase of dynorphin A level in spinal cord tissue may play an active role in the early stage, but its harmful effect on secondary SCI will be more and more prominent after accumulation of excessive dynorphin A.

Animals↗
PubMed1995

[Papillary microcancer of the thyroid].

The aim of this study was to determine the outcome of patients operated for microscopic papillary cancer of the thyroid. Between 1962 to 1991, among 1103 patients operated for papillary thyroid carcinoma, 115 (11.5%) had a cancer with a diameter of 10 mm or less, no clinically detectable. 48 patients were diagnosed incidentally at the time of operation for other thyroid diseases, and 67 patients presented cervical lymph node metastases as the first sign. For patients diagnosed for other thyroid diseases, unilateral lobectomy with local neck dissection was performed in 35 cases, and in patients with cervical lymph node metastases unilateral lobectomy with locoregional neck dissection was performed in 56 cases. Mean overall follow-up was 12 years, and median follow-up time of 105 surviving patients was 10 years. 10-year survival rates were 100% in the patients diagnosed for other thyroid diseases, and 87% in patients with cervical lymph node metastases (P < 0.05). Out of 48 patients with no cervical lymph nodes, 44 were alive, and 4 died without recurrence or metastases. Out of the 67 patients with cervical lymph nodes, 5 died of the carcinoma. These results suggest that for patients diagnosed for other thyroid diseases, unilateral lobectomy with local neck dissection is sufficient, and for patients with cervical lymph node metastases unilateral lobectomy and locoregional neck dissection is necessary.

Adolescent↗
PubMed1994

The gene family of EF-hand calcium-binding proteins from the flagellum of Trypanosoma brucei.

The flagellum of Trypanosoma brucei contains calmodulin, and a separate family of antigenically related EF-hand calcium-binding proteins which we call calflagins. The following study evaluates the structure and genomic organization of the calflagin family. Genomic Southern blots indicated that multiple copies of calflagin genes occurred in T. brucei, and that all of these copies were contained in a single 23 kb XhoI-XhoI fragment on chromosomes 15 and 16 mRNAs of 1.2 and 1.6 kb were identified in bloodstream and procyclic life-cycle stages. Genomic fragments of 2.5 and 1.7 kb were cloned that encoded calflagin sequences. The calflagin genes were arranged tandemly along the genomic fragments. Three new members of the calflagin family were sequenced from a cDNA clone and the two genomic clones. Two unrelated families of 3' flanking sequences were downstream from the calflagin genes. An open reading frame that was unrelated to any calflagin sequence was at the 5' end of the 2.5 kb genomic fragment. The deduced amino acid sequences of the genomic clones (called Tb-24 and Tb-1.7g) were similar to the previously described Tb-17. Each encoded an approximately 24 kDa protein which contained three EF-hand calcium-binding motifs and one degenerate EF-hand motif. The cDNA encoded a protein (called Tb-44A) which was approximately twice as large as the other calflagins. The large size resulted from a nearly direct repeat of 186 amino acids. In general, variability among the T. brucei calflagins was greater than observed for related proteins from Trypanosoma cruzi. We demonstrate that this variability resulted from amino acid substitutions at the N-terminus, C-terminal extensions, and duplication of internal segments.

Amino Acid Sequence↗
PubMed1994

A unique protonated phosphate group in bone mineral not present in synthetic calcium phosphates. Identification by phosphorus-31 solid state NMR spectroscopy.

The detailed chemical composition and microstructure of freshly deposited bone mineral, and how these properties change with maturation of the mineral, have been studied intensively and still remain controversial. For example, current analytical technology is inadequate for the unambiguous characterization of the monohydrogen phosphate ions in bone mineral. Using a differential cross polarization/magic angle spinning solid state nuclear magnetic resonance spectroscopy technique, we suppress the dominant orthophosphate (PO4-3) signal to reveal the spectra of the minor phosphate constituents. This method depends upon differences in the cross polarization time constants for phosphorus-31 nuclei in protonated and non-protonated phosphate ions. It is now possible for the first time to directly measure both the proportion of acid phosphate (HPO4-2) as well as the parameters which characterize its isotropic and anisotropic chemical shift. In bone from three species at several developmental stages, we have found a single type of acid phosphate species, identical in all of the specimens examined. The phosphorus-31 isotropic chemical shift of this acid phosphate group in bone mineral corresponds precisely with that of acid phosphate in octacalcium phosphate, and not with that of brushite. In contrast, the bone acid phosphate anisotropic chemical shift parameters are close to those of brushite, and differ significantly from those of octacalcium phosphate. The orthophosphate resonances of bone mineral, synthetic hydroxyapatite and synthetic octacalcium phosphate share identical chemical isotropic shifts, and similar chemical shift anisotropies. The implication of these results is that the intimate structure of the acid phosphate group in bone mineral is unique, and that none of the common synthetic calcium phosphates accounts well for all of the observed solid state phosphorus-31 NMR properties of bone mineral.

Animals↗
PubMed1994

Evolution of host cell RNA into efficient template RNA by Q beta replicase: the origin of RNA in untemplated reactions.

Q beta replicase can replicate a single molecule of certain species of RNA to 10(14) copies in minutes. This replication ability has been used for in vitro studies of molecular evolution and is currently being utilized as a method of amplifying RNAs that contain probe sequences. It has been observed that Q beta replicase can produce replicatable RNA even in the absence of exogenously added template RNA. The origin of this RNA has been ascribed either to contamination with replicatable RNA or to an ability of Q beta replicase to synthesize RNA de novo from the nucleotides present in the reaction. Technologies that employ Q beta replicase require a thorough understanding of the conditions that lead to this so-called spontaneous RNA production. We have created an expression system and purification method with which we produce gram quantities of highly purified Q beta replicase, and we have identified reaction conditions that prevent the amplification of RNA in assays that do not contain added RNA. However, when these reaction conditions are relaxed, spontaneous RNA replication is seen in up to 100% of the assays. To understand the origin of this RNA, we have cloned several spontaneously produced RNAs. Sequence analysis of one of these RNAs shows that it arose by the evolution of Escherichia coli tRNA into a replicatable template and not by de novo synthesis from nucleoside triphosphates in the reaction.

Allolevivirus↗
PubMed1994

Wild-type alternatively spliced p53: binding to DNA and interaction with the major p53 protein in vitro and in cells.

A p53 variant protein (p53as) generated from alternatively spliced p53 RNA is expressed in normal and malignant mouse cells and tissues, and p53as antigen activity is preferentially associated with the G2 phase of the cell cycle, suggesting that p53as and p53 protein may have distinct properties. Using p53as and p53 proteins translated in vitro, we now provide evidence that p53as protein has efficient sequence-specific DNA-binding ability. DNA binding by p53 protein is inefficient in comparison and requires activation. Furthermore, p53as and p53 proteins formed hetero-oligomers when co-translated in vitro, resulting in inactivation of p53as DNA-binding activity. Gel filtration indicated that p53as translated in vitro, like p53, formed tetramers. In support of a functional role of p53as in cells, p53as/p53 hetero-oligomers were coimmunoprecipitated from mouse cells, and both protein forms were detectable in nuclear extracts by electrophoretic mobility shift assays. These results suggest that the biochemical functions of p53 are mediated by interaction between two endogenous protein products of the wild-type p53 gene.

Alternative Splicing↗
PubMed1994

Retinoic acid is required for and potentiates differentiation of acute promyelocytic leukemia cells by nonretinoid agents.

Patients with acute promyelocytic leukemia (APL) associated with the t(15;17) translocation and fusion of the promyelocytic leukemia (PML) and retinoic acid receptor-alpha (RAR-alpha) genes achieve complete remission but not cure with all-trans retinoic acid (RA), NB4, a cell line derived from a patient with t(15;17) APL that undergoes granulocytic differentiation when treated with pharmacologic doses of RA, was used as a model for differentiation therapy of APL. We found that NB4 cells are resistant to differentiation by nonretinoid inducers such as hexamethylene bisacetamide (HMBA), butyrates, vitamin D3, or hypoxanthine, all of which can induce differentiation in the commonly used HL60 leukemia cell line. Preexposure of NB4 cells to low concentrations of RA for a period as short as 30 minutes abolished resistance to nonretinoids and potentiated differentiation. Sequential RA and HMBA treatment yielded maximal differentiation by 3 days of drug exposure, whereas the effect of RA alone peaked after 6 days and yielded a smaller percentage of differentiated cells. RA also reversed NB4 cell resistance to butyrates and allowed for synergistic differentiation by these agents. Pretreatment with HMBA before exposure to RA failed to stimulate differentiation. Sequential RA/HMBA treatment also markedly increased the extent of differentiation of primary cultures of bone marrow and peripheral blood mononuclear cells from three APL patients. In one case RA/HMBA treatment overcame resistance to RA in vitro. Together, these results suggest that intermittent low doses of RA followed by either HMBA or butyrates may be a useful combination in the treatment of APL. This clinical strategy may help prevent or overcome RA resistance in APL.

Acetamides↗
PubMed1994

A lag in intracellular degradation of mutant alpha 1-antitrypsin correlates with the liver disease phenotype in homozygous PiZZ alpha 1-antitrypsin deficiency.

Liver injury in PiZZ alpha 1-antitrypsin (alpha 1-AT) deficiency probably results from toxic effects of the abnormal alpha 1-AT molecule accumulating within the ER of liver cells. However, only 12-15% of individuals with this same genotype develops liver disease. Therefore, we predicted that other genetic traits that determine the net intracellular accumulation of the mutant alpha 1-AT molecule would also determine susceptibility to liver disease. To address this prediction, we transduced skin fibroblasts from PiZZ individuals with liver disease or without liver disease with amphotropic recombinant retroviral particles designed for constitutive expression of the mutant alpha 1-AT Z gene. Human skin fibroblasts do not express the endogenous alpha 1-AT gene but presumably express other genes involved in postsynthetic processing of secretory proteins. The results show that expression of human alpha 1-AT gene was conferred on each fibroblast cell line. Compared to the same cell line transduced with the wild-type alpha 1-AT M gene, there was selective intracellular accumulation of the mutant alpha 1-AT Z protein in each case. However, there was a marked delay in degradation of the mutant alpha 1-AT Z protein after it accumulated in the fibroblasts from ZZ individuals with liver disease ("susceptible hosts") as compared to those without liver disease ("protected hosts"). Appropriate disease controls showed that the lag in degradation in susceptible hosts is specific for the combination of PiZZ phenotype and liver disease. Biochemical characteristics of alpha 1-AT Z degradation in the protected hosts were found to be similar to those of a common ER degradation pathway previously described in model experimental cell systems for T-cell receptor alpha subunits and asialoglycoprotein receptor subunits, therefore, raising the possibility that the lag in degradation in the susceptible host is a defect in this common ER degradation pathway. Thus, these data provide evidence that other genetic traits that affect the fate of the abnormal alpha 1-AT Z molecule, at least in part, determine susceptibility to liver disease. These data also validate a system for elucidating the biochemical/genetic characteristics of these traits and for examining the relevance to human disease of pathways for protein degradation in the ER.

Amino Acid Sequence↗
PubMed1994

Two-dimensional DNA electrophoresis in mutation detection.

Accurate detection of gene mutations is important in many areas of biology and medicine. In fundamental studies of mutagenesis it is often necessary to assess all possible mutations, either spontaneous or induced by genotoxic agents, in a particular gene or gene sequence to explain a given cellular or physiological endpoint. In molecular medicine comprehensive detection of all possible mutations in a disease gene is required before clinical genetic testing becomes feasible. Of the many mutation detection methods currently available none is capable of scanning for all possible mutations in a cost-effective manner. Here we show that by two-dimensional DNA electrophoretic separation, on the basis of both size and base pair sequence, in principle all mutations in a given gene can be detected. This is illustrated by some data on 2-D electrophoresis of 10 exons of the cystic fibrosis gene.

Cystic Fibrosis↗
PubMed1994

Localization of the high and low affinity [3H]ryanodine binding sites on the skeletal muscle Ca2+ release channel.

The Ca2+ release channel of skeletal muscle sarcoplasmic reticulum is modulated in a biphasic manner by the plant alkaloid ryanodine and there are two distinct binding sites on this channel for ryanodine. The Ca2+ release channel is a homotetramer with a subunit of 5037 amino acids. The ability of sarcoplasmic reticulum membranes to bind [3H]ryanodine to the high affinity site is lost upon proteolysis with trypsin. [3H]Ryanodine, however, bound before proteolysis remains bound after trypsin digestion. If the high affinity site is first occupied with [3H]ryanodine and then 100 microM ryanodine is added to occupy the low affinity sites, almost all of [3H]ryanodine bound to the high affinity site remains bound after proteolysis. Proteolysis causes the solubilized Ca2+ release channel containing bound [3H]ryanodine to undergo four discrete shifts in sedimentation (30 S-->28 S-->26 S-->19 S-->14 S). Polypeptides having apparent molecular masses of 76, 66, 56, 45, 37, and 27 kDa can be identified in the 14 S complex. The 76-, 56-, 45-, and 27-kDa polypeptides have been partially sequenced from the NH2 terminus. In addition, the 76-, 66-, and 27-kDa fragments are recognized by an antibody to the last 9 amino acids at the carboxyl terminus of the skeletal muscle ryanodine receptor and the 76-, 66-, and 37-kDa fragments are recognized by an antibody to a peptide matching the sequence 4670-4685. The 56-kDa and the 45-kDa fragments are not Ca2+ release channel fragments. Both high and low affinity ryanodine binding sites are found in the 14 S complex and are, therefore, most likely located between Arg-4475 and the carboxyl terminus.

Amino Acid Sequence↗
PubMed1994

Viral induction of co-stimulatory activity on antigen-presenting cells bypasses the need for CD4+ T-cell help in CD8+ T-cell responses.

BACKGROUND: CD4+ T-cell help is critical for cytotoxic (CD8+) T-lymphocyte responses to many antigens, such as viruses, minor histocompatibility antigens and allogeneic major histocompatibility antigens. However, the nature of such help is still a mystery: cytokines such as interleukin-2 may be involved but cell-cell contact may also be necessary. As some viruses can induce CD8+ T-cell responses in the absence of CD4+ T cells, we asked whether these viruses and CD4+ cells share a pathway for helping the CD8+ T-cell response. RESULTS: We show here that the H2N2 subtype of influenza virus, which elicits a CD4+ T-cell-independent anti-viral CD8+ T-cell response in vitro, induces expression of the co-stimulatory molecule B7-2, but not of B7, on the cell surface of antigen-presenting cells. In contrast, the H1N1 subtype of influenza virus, which requires CD4+ T-cell help to elicit CD8+ T-cell responses under the same conditions, does not induce B7-2 expression. We also find that CD4+ T cells can induce expression of B7-2 on antigen-presenting cells. In both cases, the induced B7-2 is necessary for the clonal expansion and functional maturation of CD8+ T cells. CONCLUSION: Our results support the view that the induction of co-stimulatory activity on antigen-presenting cells by CD4+ T cells can substitute for the requirement for exogenous interleukin-2 in CD8+ T-cell help. Viruses that can induce co-stimulatory activity on antigen-presenting cells thus induce a CD4+ T-cell-independent CD8+ T-cell response. These findings could explain the reported differences in the requirements for CD4+ T cells in CD8+ T-cell responses.

Animals↗
PubMed1994

Heme protein dynamics studied by phosphorescence of an external phosphorescent probe molecule.

The rate constant for quenching, kq, of the phosphorescence of 6-bromo-2-naphthyl sulfate (BNS) by cytochromes c, cytochrome c peroxidase, catalase, and myoglobin has been measured as a function of temperature and solvent viscosity. In aqueous solution at pH 7.0 for cytochromes c and myoglobin the value of kq is nearly equal to the rate constant for diffusional intermolecular contact, which is estimated from the value of kq for microperoxidase-11. For cytochrome c peroxidase and catalase kq is at least 350 times smaller than the rate of diffusional quenching, which shows that quenching of BNS phosphorescence occurs predominantly over the short distance between donor and acceptor. The mechanism for cytochrome c and myoglobin is found not to involve static quenching, deep penetration of BNS into the globin, or unfolding of the protein to allow contact between heme and BNS. It is concluded that quenching occurs by interaction of BNS with the exposed heme edge and by surface insertion of BNS into the protein to a depth sufficient for quenching by the unexposed heme. The effect of rapid-diffusional enhancement on kq is small. From a comparison of the results for the heme proteins, a model emerges that describes cytochrome c and myoglobin as having dynamic surfaces. Sufficient fluctuations persist to allow penetration of polyatomic probe molecules into the protein matrix, but the dynamics and/or interior microenvironment acts to increase resistance with increasing depth of penetration.

Animals↗
PubMed1994

Avian erythroblastosis virus E26: only one (myb) of two cell-derived coding regions is necessary for oncogenicity.

The oncogene hypothesis postulates that mutated cellular genes, termed proto-onc genes, function as cancer genes because they are related to retroviral onc genes. However, in contrast to retroviral onc genes, mutated proto-onc genes from cancers are not sufficient for carcinogenesis. Therefore, it has been proposed that mutated proto-onc genes depend on other proto-onc genes for carcinogenesis. Since the oncogene of the avian leukemia virus E26 includes coding regions derived from two cellular proto-onc genes, proto-myb and proto-ets, this hybrid gene has been proposed to be a model for two-gene-carcinogenesis. Here we set out to test this proposal. For this purpose myb and ets deletion mutants of cloned E26 provirus were prepared, and the corresponding viruses, produced by transfected primary chicken embryo cells, were tested for leukemogenicity in newborn chickens. It was found that an ets deletion mutant was just as leukemogenic as the wild-type virus and that a myb deletion mutant lacked leukemogenicity completely. To eliminate the possibility that our E26 myb deletion mutant failed to be leukemogenic because it failed to replicate, the virus was titered by a quantitative polymerase chain reaction (PCR) method. By this method, E26 from the plasma of infected chickens was first allowed to reverse-transcribe viral RNA to cDNA in vitro, and then the cDNA concentration was determined from the lowest dilution that gave a positive signal after amplification of E26 cDNA by the PCR method. Virus titers of about 10(5) per ml were found for wild type and for myb and ets deletion mutants of E26. It is concluded that the ets region is not essential for carcinogenesis, and E26 derives transforming function from overexpression of its proto-myb coding region via the retroviral promoter. Thus, E26 is a single-hit carcinogen and, like all other oncogenic retroviruses, is not a model for two-gene-carcinogenesis. Viral ets probably reflects a genetic accident that transduced sequences of proto-ets together with proto-myb in generating E26.

Alpharetrovirus↗
PubMed1994

T cell costimulation by B7/BB1 induces CD8 T cell-dependent tumor rejection: an important role of B7/BB1 in the induction, recruitment, and effector function of antitumor T cells.

A successful antitumor T cell immune response involves induction, recruitment, and effector function of T cells. While B7/BB1 is known as a major costimulatory molecule in the induction of T cell responses, its role in T cell recruitment and effector function is still unclear. In this study, we show that introducing a major costimulatory molecule B7/BB1 into a major histocompatibility complex class II-negative tumor cell line, J558, results in a drastic reduction of its tumorigenicity. The tumor rejection depends on CD8 T cells but not CD4 T cells. However, unlike the previous reports on melanoma cell lines, B7/BB1-transfected J558 cells fail to induce cross-protection against parental J558 cells. The B7/BB1-transfected (J558-B7), but not untransfected J558 cells (J558-Neo) induce a CD8 T cell-dominant inflammatory response, and the T cells isolated from the tumor infiltrating lymphocytes (TIL) are polyclonal in terms of their T cell receptor V beta usage. Most surprisingly, the freshly prepared TIL have a potent, CD8 T cell-mediated cytotoxicity on tumor cells without any in vitro stimulation. The cytotoxic T lymphocyte (CTL) activity can be blocked by anti-CD8 monoclonal antibody (mAb). Interestingly, the CTL lyse J558-B7 about 10- to 80-fold more efficiently than untransfected J558-Neo cells. This preferential lysis cannot be attributed to recognition of B7/BB1-derived antigen by the T cells. This finding, together with the lack of the cross-protection between the J558-B7 and J558-Neo, suggests that B7/BB1 can also function at the effector phase of CTL responses. This notion is confirmed by our findings that the lysis of J558-B7 can be blocked by anti-B7 mAbs. Taken together, our results indicate that not only can the B7/BB1 molecule function as a costimulatory molecule at the initiation of immune response, it can also play a major role in T cell recruitment and effector function. This conclusion has significant implications for immunotherapy of tumors.

Animals↗