Biomedical subjects
Publications and source records attributed to Y Zhao.
Using the FTIR and 1H NMR methods, the structures, relative ratio and inventory ratio of segmented poly (urea) urethanes which are synthesized with different extenders (1,4-butandiol, ethylene diamine and their mixtures)have been studied successfully and the ratationship between the branch ratio and inventory ratio has been determined.
Usually, the emission spectra of terbium complexes exhibit 490 nm, 545 nm bands (green color) under the excitation of UV light, which is ascribed to the two 5D4 - 7F6, 5D4 - 7F5 transition. In this report, pink red fluorescence under the excitation of 300-370nm UV light is observed for a new 1, 10-phenanthroline-2-naphthoate complex of terbium. The fluorescence, FT-IR, ICP and X-ray fluorescence analysis were performed. The results showed that under the perturbation of the ligands field, the probability of 5D4 - 7F3 transition of terbium ion was greatly enhanced, while the probability of 5D4 - 7F5 transition was greatly decreased in the complex. Therefore, pink-red fluorescence was observed for the title complex.
The reagents 6-Cs-beta-CD with molecular recognition function were synthesized and confirmed. Both basic condition and enough illumination were necessary for obtaining stable and strong fluorescence. With 6-Cs-beta-CD as the host, cyclohexanol, cyclohexene, cyclohexane and bromocyclohexane as the guest, the recognition properties were studied. The affinity of 6-Cs-beta-CD and the guest is distinct with different hydrophobicity of the guest.
RHAMM is an oncogene that regulates signaling through ras and controls mitogen-activated protein kinase [extracellular signal-regulated protein kinase (ERK)] expression in embryonic murine fibroblasts. ERK is a dual-specificity kinase that controls expression of proteins relevant to tumorigenesis, proliferation, and motility. To assess whether RHAMM and ERK are involved in human breast tumor progression, we examined RHAMM, ras, and ERK expression in two cohorts of breast cancer patients using reverse transcription-PCR and immunocytochemistry. We show that overexpression of RHAMM in primary tumors of two patient cohorts was significantly prognostic of poor outcome in breast cancer progression. Furthermore, RHAMM overexpression occurred within subsets of tumor cells in the primary tumor, and this staining pattern was associated with lymph node metastases. The metastases exhibited a significantly higher level of staining for RHAMM than did the primary tumor. RHAMM expression strongly correlated with overexpression of both ras and ERK, although overexpression of either of these two signaling molecules was not by itself a prognostic indicator. These results identify a new parameter that is involved in lymph node metastasis of primary breast cancers and suggest that quantification of RHAMM overexpression may be a useful prognostic indicator for breast carcinoma progression.
We report a calculation of the electronic structure (density of states and molecular orbitals) of a new gene system-the plasmid shuttle vector pCRR1-performed at the ab initio Hartree-Fock level. To deal with the aperiodicity of the biopolymer, a vectorized version of a negative factor counting (NFC) program has been implemented. With this efficient tool, DNA molecules of up to 100 deoxynucleotides (approximately 10,000 basis functions at the ab initio level) can be calculated routinely. In our calculation the base sequence of the plasmid is explicitly taken into account and a standard helical structure is assumed. Minimum as well as 6-31G basis sets are used in the calculation, and solvation effects are explicitly included. The calculation shows that solvation does not have a significant effect on the electronic structure of the biopolymer. It is found that the frontier orbitals (HOMO and LUMO) are highly localized on the bases. The interpretation of this result in terms of the frontier molecular orbital theory is that the attack of radiation-induced radicals is mainly on the base moieties.
Patients with Hermansky-Pudlak syndrome (HPS) exhibit moderate to mild hypopigmentation of the skin, hair, and eyes. To understand the inherent basis for this reduced pigmentation, pure cultures of melanocytes were derived using skin biopsies obtained from four patients with HPS. A nucleotide lesion in the HPS gene was identified in these individuals. Expression of HPS mRNA, parameters of melanin synthesis, characteristics in ultrastructural morphology, and expression of melanocyte-specific proteins were assessed in HPS melanocytes. The patients' cells appeared microscopically hypopigmented, and melanin content ranged from 0% to 50% of that for normal melanocytes. In cell lysates of HPS melanocytes, tyrosine hydroxylase activity was within the normal range, but in intact HPS melanocytes, it was almost half that of normal melanocytes. HPS melanocytes also appeared refractory to stimulators of melanization, eg, a combination of isobutyl methylxanthine and cholera toxin (IBMX/CT). HPS melanocytes contained many morphologically normal melanosomes, mostly Stage II with a few Stage I or III. After dihydroxyphenylalanine (DOPA) incubation, there appeared to be an equal number of Stage II and III melanosomes with the addition of a moderate number of Stage IV melanosomes. A characteristic ultrastructural feature of most HPS melanocytes was a variety of unusual cellular structures. These aberrancies include the following: (a) large membrane-bound complexes containing membranous chambers, unpigmented, and pigmented melanosomes, irregular deposits of DOPA reaction products, and granular/amorphous material sometimes resembling the cytoplasm; and (b) DOPA-positive rings delineated on either side by limiting membranes. The expression of tyrosinase-related protein-1 and granulophysin, a 40-kd membrane protein originally identified as a component of platelet-dense bodies that are undetectable in HPS, was assessed by light microscopy immunofluorescence. For both proteins, HPS melanocytes exhibited a large granular pattern of expression throughout the cell, which seems to correlate with the large membrane complexes observed ultrastructurally. These observations support the hypothesis that the HPS gene product is involved in organellogenesis. We propose that in the melanocyte, the HPS gene product regulates in part the trafficking of melanocyte-specific proteins from the trans-Golgi network to preformed premelanosomes.
Stored sera from asymptomatic hepatitis B virus (HBV) carriers and hepatitis B virus surface antigen-positive hepatocellular carcinoma (HCC) patients were tested for HBV subtypes, such as subtype determinants d, y, w, r and also antigenic determinants isoleucine (i) and threonine (t) by direct S gene nucleotide sequencing. Significant changes in minor i and t determinants in hepatocellular carcinoma patients with adr hepatitis B carriers were seen. The adr subtype with t determinant was present in 14/25 (56%) of HCC patients compared with only two of 28 (7%) in asymptomatic hepatitis B carriers (P<0.001). However, the adr subtype with i determinant was present in nine of 25 (36%) of the HCC patients and also present in 24/28 (86%) of asymptomatic carriers (P<0.001). No significant changes were seen with the adw subtypes. These results show that i and t minor determinant changes are more common with adr subtypes associated with HCC than with the adw subtype. Whether these subtle changes are pathologically relevant or only a polymorphism of hepatitis B genotypes will depend on subsequent follow-up studies.
In order to search for tumor cells apoptosis inducer, the apoptosis effects and mechanism of tea polyphenols were studied. Tea polyphenols are active compounds purified from tea. The apoptosis effects of tea polyphenols were observed on the human promyelocytic leukemic cells (HL-60) by an MTT reduction test, DNA agarose gel electrophoresis and a transmission electronic microscopy technique. After HL-60 cells were treated by tea polyphenols in 250 microg/ml for 5 h, DNA extracted from HL-60 cells showed a typical internucleosomal DNA degradation, i.e. DNA ladder and apoptotic vehicles were observed. The effects of tea polyphenols were shown to be similar to their cytotoxic activity in HL-60 cells. These results suggest that the mechanism of antitumor action of tea polyphenols is related to its apoptosis-inducing activity.
Soluble guanylate cyclase (sGC) is a heterodimeric hemoprotein composed of alpha1 and beta1 subunits. sGC is activated by nitric oxide (NO) and therefore plays a central role in NO signal transduction. Activation of sGC by NO is believed to be mediated by the interaction between NO and the heme of sGC. Spectroscopic and kinetic studies have shown that the heme of sGC is in a unique environment. Characterization of the heme environment is critical to the understanding of the mechanism of NO activation. To approach this goal, the beta1 N-terminal fragment consisting of residues 1-385 [beta1(1-385)] of sGC was expressed in E. coli. beta1(1-385) was then purified to homogeneity in two steps by DEAE ion exchange and gel filtration chromatography. Purified beta1(1-385) was found to contain a stoichiometric amount of heme. The UV-visible spectrum of beta1(1-385) is almost identical to that of the native heterodimeric sGC purified from bovine lung. beta1(1-385) binds both NO and CO, leading to a shift in the Soret maximum from 431 nm to 398 and 423 nm, respectively. These spectral shifts are identical to those observed with heterodimeric sGC purified from bovine lung. These results suggest that the heme in the beta1(1-385) is similar to that in the heterodimeric sGC. Therefore, for the first time, the heme binding region of sGC has been unambiguously localized to the N-terminal region of the beta1 subunit. Our data also suggest that the N-terminal region of the beta1 subunit of sGC is itself sufficient for heme binding.
The structure of the catalytically inactive mutant (C215S) of the human protein-tyrosine phosphatase 1B (PTP1B) has been solved to high resolution in two complexes. In the first, crystals were grown in the presence of bis-(para-phosphophenyl) methane (BPPM), a synthetic high-affinity low-molecular weight nonpeptidic substrate (Km = 16 microM), and the structure was refined to an R-factor of 18. 2% at 1.9 A resolution. In the second, crystals were grown in a saturating concentration of phosphotyrosine (pTyr), and the structure was refined to an R-factor of 18.1% at 1.85 A. Difference Fourier maps showed that BPPM binds PTP1B in two mutually exclusive modes, one in which it occupies the canonical pTyr-binding site (the active site), and another in which a phosphophenyl moiety interacts with a set of residues not previously observed to bind aryl phosphates. The identification of a second pTyr molecule at the same site in the PTP1B/C215S-pTyr complex confirms that these residues constitute a low-affinity noncatalytic aryl phosphate-binding site. Identification of a second aryl phosphate binding site adjacent to the active site provides a paradigm for the design of tight-binding, highly specific PTP1B inhibitors that can span both the active site and the adjacent noncatalytic site. This design can be achieved by tethering together two small ligands that are individually targeted to the active site and the proximal noncatalytic site.
PfPK4, a protein kinase gene from the human malarial parasite Plasmodium falciparum, has been cloned utilizing oligonucleotide probing. The gene encodes a protein of a predicted length of 1123 amino acids, and within this amino acid sequence all the conserved regions characteristic of protein kinases can be identified. The catalytic kinase domain possesses highest identities (34-37%) with eukaryotic initiation factor-2alpha (eIF-2alpha) kinases, especially haem-regulated inhibitory (HRI) protein kinases. There are two kinase inserts in PfPK4, located at positions common to eIF-2alpha kinases. The first insert separates kinase subdomains IV and VI by 559 amino acids, and the second subdomains VII and VIII by 41 amino acids. Both inserts are larger than their homologues in eIF-2alpha kinases. The sequence of PfPK4 has one putative haemin-binding site. The recombinant protein, expressed in Escherichia coli, phosphorylates a synthetic peptide representing a substrate of eIF-2alpha kinases. Autophosphorylation and substrate phosphorylation are inhibited by haemin. Thus PfPK4 appears to be the first protozoan protein kinase related to eIF-2alpha kinases and might be the first non-mammalian HRI kinase. Western blots indicated that the protein is expressed as major forms of 80 and 90 kDa. Whereas the 80 kDa form is present throughout the intraerythrocytic development and in merozoites, the two 90 kDa forms are only found in mature parasites. One of the latter is also present in the membrane fraction of erythrocytes harbouring segmenters. Confocal microscopy detected the protein distributed throughout the trophozoite, whereas it was found in discrete foci (punctate distribution) in segmenters. PfPK4 co-localizes with P. falciparum 83 kDa antigen/apical membrane antigen-1 at the apical complex in segmenters and merozoites, but does not co-localize with rhoptry-associated protein-1.
Neurofilaments are an important structural component of the axonal cytoskeleton and are made of neuronal intermediate filament (nIF) proteins. During axonal development, neurofilaments undergo progressive changes in molecular composition. In mammals, for example, highly phosphorylated forms of the middle- and high-molecular-weight neurofilament proteins (NF-M and NF-H, respectively) are characteristic of mature axons, whereas nIF proteins such as alpha-internexin are typical of young axons. Such changes have been proposed to help growing axons accommodate varying demands for plasticity and stability by modulating the structure of the axonal cytoskeleton. Xefiltin is a recently discovered nIF protein of the frog Xenopus laevis, whose nervous system has a large capacity for regeneration and plasticity. By amino acid identity, xefiltin is closely related to two other nIF proteins, alpha-internexin and gefiltin. alpha-Internexin is found principally in embryonic axons of the mammalian brain, and gefiltin is expressed primarily in goldfish retinal ganglion cells and has been associated with the ability of the goldfish optic nerve to regenerate. Like gefiltin in goldfish, xefiltin in Xenopus is the most abundantly expressed nIF protein of mature retinal ganglion cells. In the present study, we used immunocytochemistry to study the distribution of xefiltin during optic nerve development and regeneration. During development, xefiltin was found in optic axons at stage 35/36, before they reach the tectum at stage 37/38. Similarly, after an orbital crush injury, xefiltin first reemerged in optic axons after the front of regeneration reached the optic chiasm, but before it reached the tectum. Thus, during both development and regeneration, xefiltin was present within actively growing optic axons. In addition, aberrantly projecting retinoretinal axons expressed less xefiltin than those entering the optic tract, suggesting that xefiltin expression is influenced by interactions between regenerating axons and cells encountered along the visual pathway. These results support the idea that changes in xefiltin expression, along with those of other nIF proteins, modulate the structure and stability of actively growing optic axons and that this stability is under the control of the pathway which growing axons follow.
A new procedure for combining direct methods with the solvent-flattening technique is proposed for phasing single isomorphous replacement (SIR) or one-wavelength anomalous scattering (OAS) data of proteins. The new procedure differs from the previous one [Zheng, Zheng, Gu, Mo, Fan & Hao (1997). Acta Cryst. D53, 49-55] in that the direct method not only provides input phases to but also accepts feedback phases from solvent flattening, thus forming an iterative process for breaking the ambiguities and refining the values of phases. The new procedure was tested with the experimental SIR data of the known structure ribonuclease Sa. For the strongest 1000 of the total 7264 reflections, the mean F(obs)-weighted phase error is 7.5 and 9.4 degrees lower than that of the previous procedure and that of solvent flattening alone, respectively.
A procedure is described for rapid, high-confidence identification of proteins using matrix-assisted laser desorption/ionization tandem ion trap mass spectrometry in conjunction with a genome database searching strategy. The procedure involves excision of copper-stained bands or spots from electrophoretic gels, in-gel trypsin digestion of the proteins, single-stage mass spectrometric analysis of the resultant mixture of tryptic peptides, followed by tandem ion trap mass spectrometric analysis of selected individual peptides, and database searching of the relevant genomic database using the program PepFrag. The scheme provides sensitive, real-time protein identification as well as facile identification of modifications. A single operator can unambiguously identify 5-10 proteins/day from an organism whose genome is known at a level of > 0.5 pmol of protein loaded on a gel. The utility of the technique was demonstrated by the identification and characterization of a band from a human HTLV-I preparation and 11 different proteins from a yeast RNA polymerase II C-terminal repeat domain-affinity preparation. The technology has great potential for postgenome biological science, where it promises to facilitate the dissection and anatomy of macromolecular assemblages, the definition of disease state markers, and the investigation of protein targets in biological processes such as the cell cycle and signal transduction.
The title compounds, (Xylp beta (1-->4))nXylp beta-3,4-DNP (n = 0-4) have been made by selective anomeric deprotection of peracetylated xylose oligosaccharides with hydrazine, followed by formation of the trichloroacetimidate, uncatalysed reaction with 3,4-dinitrophenol, and Zemplén deacetylation. The values of k(cat)/K(m) for 3,4-dinitrophenol release from these substrates by xylanase III of Streptomyces cyaneus, expressed in Escherichia coli, increase with increasing n up to n = 2 and then slightly decrease. Since it is known from previous work that in its normal host, the enzyme is produced constitutively at low levels and excreted, these results suggest that the biological function of the enzyme may be to produce small molecule inducers, predominantly xylotriose, from the non-reducing end of the xylan. Activity on cellooligosaccharide glycosides (Glcp beta (1-->4))nGlcp beta-3,4-DNP (n = 0-3) was detected, at a rate about two-and-a-half orders of magnitude less than that observed on the corresponding xylooligosaccharides, indicating that the enzyme is a true xylanase.
The blastocyst stage of embryo development is characterized by a fluid-filled cavity called the blastocoel. Blastocoel formation requires vectorial Na+ and Cl- transport and the accompanying osmotic accumulation of fluid. We found under conditions of low external Cl- that inhibitors of Cl- transport mechanisms inhibited blastocoel expansion, indicating a possible transcellular route for Cl- uptake across the outer epithelial layer (the trophectoderm). Using the Cl--sensitive fluorophore, N-(6-methoxyquinolyl)acetoethyl ester, we found that Cl- efflux from the blastocoel can occur via pathways with properties that resemble both HCO-3/Cl- exchange and Cl- channels, as well as by another yet uncharacterized pathway. In contrast, Cl- re-uptake into Cl--depleted blastocoels (the physiologically relevant direction for Cl- transport during blastocoel expansion) occurred only via the channel-like mechanism. Patch-clamp recordings detected a component of current carried by apical Cl- channels. Intracellular pH measurements during external Cl- removal detected HCO-3/Cl- exchange activity in collapsed blastocysts but little in intact blastocysts, suggesting predominantly basolateral HCO-3/Cl- exchange activity. This was corroborated by the immunolocalization of the AE2 isoform of HCO-3/Cl- exchanger to the basolateral surface of the trophectoderm. Thus, it appears that Cl- transport into the blastocoel may occur via apical Cl- channels, while efflux also involves a basolateral HCO-3/Cl- exchanger.
Specific tolerance to discordant xenogeneic donors can be achieved by grafting of fetal pig thymic and liver tissue (FP THY/LIV) to T cell and NK cell-depleted, thymectomized (ATX) mice. Mouse CD4+ T cells develop in FP THY/LIV grafts, and demonstrate remarkably normal immune function, including host-restricted responses to keyhole limpet hemocyanin. We have therefore studied the role of host MHC class II in the development of mouse T cells in FP THY/LIV grafts by comparing their development in ATX MHC class II-deficient (IIKO) and wild-type (H-2b) mice. Mouse CD4+ T cells repopulated T/NK cell-depleted, ATX IIKO mice after grafting with FP THY/LIV, indicating that pig MHC can positively select mouse CD4 cells. Expression of TCR, MHC class I, Qa-2, heat-stable Ag, and CD45RB among double-positive and CD4 single-positive (SP) graft thymocytes in wild-type recipients was similar to that in normal mouse thymi, whereas CD4 SP thymocytes in grafts of IIKO mice showed increased Qa-2 and decreased heat-stable Ag expression, suggesting an increased level of maturity. Double-positive cells in grafts of IIKO mice also expressed higher than normal levels of Qa-2. Deletion within the grafts of Vbeta3+, Vbeta5.1/5.2+, and Vbeta11+ but not Vbeta6+, Vbeta7+, or Vbeta8.1/8.2+ mouse CD4 SP thymocytes in ATX IIKO mice demonstrated that swine leukocyte Ag participates in negative selection of the T cell repertoire. Therefore, porcine MHC mediates positive and negative selection of mouse thymocytes, but host class II MHC molecules also regulate thymocyte maturation in xenogeneic thymic grafts.
Transcription promoter of the aldolase B gene was previously shown to be centered on an initiation region of DNA replication in rat hepatoma cells in vivo. Here, we defined an essential region required for replication in a plasmid form upon transfection. Deletion analyses around the origin region revealed that the proximal 200 bp promoter was necessary, but not sufficient for replication as flanking sequence restored replication activity. Therefore, the 200 bp region seemed to cooperate with the flanking sequence to play an important role in replication. Electrophoretic mobility shift assays using nuclear extracts from synchronously growing hepatoma cells showed that some protein factors bound to this region in a cell cycle-regulated manner. Since transcription of the aldolase B gene is repressed in the hepatoma cells, the cell cycle-regulated protein-binding is considered to be involved in regulation of replication initiation.