Biomedical subjects
Publications and source records attributed to M Zhang.
Ecliptasaponin C (1), a new triterpenoid glucoside, was isolated together with daucosterol (2) and stigmasterol-3-O-glucoside (3) from Eclipta alba. Based on spectral analysis, chemical evidence and hydrolytic results, it's structure was deduced as 3-beta-O-beta-D-glucopyranosyl-19beta-hydroxy olean-12-ene-28-oic acid 28-O-beta-D-glucopyranoside, Compounds 2 and 3 were obtained from Eclipta for the fist time.
Explore the source record for details and available documents.
Virtually all cells respond to heat stress by increased expression or induction of one or more of the highly conserved cellular stress response proteins, heat shock proteins (HSPs). Here, we report the unusual property of rat Nb2-11 cells, a prolactin-dependent pre-T-cell line, to display reduced HSP expression following exposure to elevated temperature. After heat stress (41 degrees C, 1 h), there was no evidence of inducible members of the 70 kDa HSP family, a response common to other cell culture and tissue systems. Moreover, expression of constitutive members of the HSP70 and HSP90 families decreased during the heat stress, apparently reflecting a decrease in mRNA stability. Gel shift assays revealed that heat shock factor (HSF) was activated in spite of the lack of expression of inducible HSP70 transcripts, although its DNA binding rapidly deteriorated. Immunoblotting, using an antibody specific to HSF1, indicated that proteolysis of HSF1 may be responsible for this rapid termination of heat shock element binding. CCAAT binding, a component of constitutive HSP70 expression, was also reduced by heat stress in Nb2-11 cells and may account for the decline in constitutive HSP70 expression. Prolactin pretreatment prevented the fragmentation of HSF1, protected heat shock element and CCAAT binding, prevented the decline in constitutive HSP70 and HSP90 expression, and restored a modest expression of inducible HSP70 following heat treatment. Results of this study describe a unique regulatory defect in HSP expression in Nb2-11 cells, possibly a common characteristic of other hormone-dependent tumors.
Explore the source record for details and available documents.
The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, small platelets, eczema, recurrent infections, and immunodeficiency. Besides the classic WAS phenotype, there is a group of patients with congenital X-linked thrombocytopenia (XLT) who have small platelets but only transient eczema, if any, and minimal immune deficiency. Because the gene responsible for WAS has been sequenced, it was possible to correlate the WAS phenotypes with WAS gene mutations. Using a fingerprinting screening technique, we determined the approximate location of the mutation in 13 unrelated WAS patients with mild to severe clinical symptoms. Direct sequence analysis of cDNA and genomic DNA obtained from patient-derived cell lines showed 12 unique mutations distributed throughout the WAS gene, including insertions, deletions, and point mutations resulting in amino acid substitutions, termination, exon skipping, or splicing defects. Of 4 unrelated patients with the XLT phenotype, 3 had missense mutations affecting exon 2 and 1 had a splice-site mutation affecting exon 9. Patients with classic WAS had more complex mutations, resulting in termination codons, frameshift, and early termination. These findings provide direct evidence that XLT and WAS are caused by mutations of the same gene and suggest that severe clinical phenotypes are associated with complex mutations.
An enzyme which converts D-dopachrome into 5,6-dihydroxyindole has recently been isolated from rat liver. Enzymatic D-dopachrome conversion has been observed in extracts from all tissues examined of several species, including man. We have now cloned and sequenced a 628 bp long cDNA encoding the enzyme provisionally called D-dopachrome tautomerase. The cDNA was isolated by 3' and 5' rapid amplification and cloning of cDNA ends (RACE) from rat liver cells using degenerate oligonucleotide primers, deduced from the N-terminal peptide sequence of D-dopachrome tautomerase. The cDNA contains an open reading frame encoding 118 amino acids. Edman degradation of intact and of trypsin degraded D-dopachrome tautomerase fragments gave information on and corroborated 67% of the deduced protein sequence. A homology search in the EST database found a human cDNA encoding a peptide sharing 66% homology with the rat enzyme. The rat D-dopachrome tautomerase shares 27% homology with the rat macrophage migration inhibitory factor (MIF).
The intercellular messenger nitric oxide is produced through the action of nitric oxide synthases, a class of enzymes that is regulated by calcium-calmodulin (CaM). In this work, the interaction of CaM with a 23-amino-acid residue synthetic peptide, encompassing the CaM-binding domain of constitutive rat cerebellar nitric oxide synthase (cNOS), was investigated by various NMR methods. Cadmium-113 NMR studies showed that binding of the cNOS peptide increased the affinity of CaM for metal ions and induced interdomain cooperativity in metal ion binding as earlier observed for complexes of CaM with myosin light chain kinase (MLCK) peptides. By using specific isotopically labeled [13C]methyl-Met and selenomethionine-substituted CaM in two-dimensional proton-detected 13C and 77Se NMR studies, we obtained evidence for the involvement of the Met residues of CaM in the binding of the cNOS peptide. These residues form two hydrophobic surface areas on CaM, and they are also involved in the binding of other target proteins. A nitroxide spin-labeled version of the cNOS peptide caused broadening only for NMR resonances in the N-terminal half of CaM, showing that the peptide binds with a C to N orientation to the N- and C-terminal domains of CaM. pH titration experiments of CaM dimethylated with [13C]formaldehyde show that Lys-75 (and Lys-148) experience a large increase in pKa upon peptide binding; this indicates an unraveling of part of the helical linker region of CaM upon cNOS peptide binding. Taken together, our data show that the cNOS and MLCK peptides bind in a closely analogous fashion to CaM.
Elafin is an elastase inhibitor with a unique structure, not related to the serpin family, which includes the neutrophil elastase inhibitor. The gene was identified in this laboratory by subtractive hybridization between RNAs from human mammary tumor-derived cells and cDNAs from normal human mammary epithelial cells. Elafin is consistently expressed in normal mammary epithelial cells, but is down-regulated in most breast tumor cell lines. Restriction fragment analysis detected no gross deletions or rearrangement of the gene in any of the tumor cell lines examined. The elafin gene was cloned, and both the cDNA and the promoter region were sequenced. A major positive upstream promoter element was identified by chloramphenicol acetyltransferase assay and deletion analysis, active in normal cell extracts but not in extracts of tumor cells. These results demonstrate that differential expression of elafin in normal mammary epithelial cells and breast tumor cells is regulated at the transcriptional level. Cell synchronization experiments demonstrated that elafin mRNA is down-regulated in S phase in normal cells. These results suggest that elafin may act as an inhibitor of cell cycle progression.
Calmodulin (CaM) is a ubiquitous Ca(2+)-binding protein that can regulate a wide variety of cellular events. The protein contains 9 Met out of a total of 148 amino acid residues. The binding of Ca2+ to CaM induces conformational changes and exposes two Met-rich hydrophobic surfaces which provide the main protein-protein contact areas when CaM interacts with its target enzymes. Two-dimensional (1H,13C)-heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy was used to study selectively 13C-isotope labelled Met methyl groups in apo-CaM, Ca(2+)-CaM and a complex of CaM with the CaM-binding domain of skeletal muscle Myosin Light Chain Kinase (MLCK). The resonance assignment of the Met methyl groups in these three functionally different states were obtained by site-directed mutagenesis (Met-->Leu). Chemical shift changes indicate that the methyl groups of the Met residues are in different environments in apo-, calcium-, and MLCK-bound-CaM. The T1 relaxation rates of the individual Met methyl carbons in the three forms of CaM indicate that those in Ca(2+)-CaM have the highest mobility. Our results also suggest that the methyl groups of the unbranched Met sidechains in general are more flexible than those of aliphatic amino acid residues such as Leu and Ile.
The synthetic dodecameric RNA fragment rUAAGGAGGUGAU resembles a region upstream of the initiation site in prokaryotic mRNAs whereas the pyrimidine-rich complementary strand is identical to the last 12 nucleotides of Escherichia coli 16 S rRNA. The complex thus serves as a model for the Shine-Dalgarno interaction which is required for proper initiation of translation. The crystal structure of rUAAGGAGGUGUA.rAUCACCUCCUUA has been determined at 2.6 A resolution and refined against 2957 1 sigma(F) structure amplitudes to an R-value of 0.195. The unit cell of the triclinic crystals contains two double-stranded RNA molecules. The conformation of the two duplexes is similar, with a root-mean-square deviation of 0.683 A between equivalent atoms, and resembles calf thymus A-DNA as determined by X-ray fiber diffraction methods. Both molecules from continuous helices that penetrate the entire crystal, but the dinucleotide step in between dodecameric duplexes has an unusual geometry with a negative twist angle. The long helices cross over each other in a characteristic manner by inserting the backbone of one molecule into the minor groove of another. These contacts are stabilized by several direct intermolecular hydrogen bonds most of which are mediated by 2'-hydroxyl groups of the ribose sugars suggesting a general mode for the interaction between RNA molecules which is different from DNA-DNA interactions.
Domain 5 (D5) is a small hairpin structure within group II introns. A bimolecular assay system depends on binding by D5 to an intron substrate for self-splicing activity. In this study, mutations in D5 identify two among six nearly invariant nucleotides as being critical for 5' splice junction hydrolysis but unimportant for binding. A mutation at another site in D5 blocks binding. Thus, mutations can distinguish two D5 functions: substrate binding and catalysis. The secondary structure of D5 may resemble helix I formed by the U2 and U6 small nuclear RNAs in the eukaryotic spliceosome. Our results support a revision of the previously proposed correspondence between D5 and helix I on the basis of the critical trinucleotide 5'-AGC-3' present in both. We suggest that this trinucleotide plays a similar role in promoting the chemical reactions for both splicing systems.
Explore the source record for details and available documents.
Comparative analyses of both glycosylated and nonglycosylated neu differentiation factor (NDF) isoforms revealed significant similarities and differences of their overall structures and functions. Biophysical analyses confirmed that all NDF isoforms are monomeric, but have an extended ellipsoidal shape in solution. All full-length NDFs are similar in secondary and tertiary structures and they contain no alpha-helix but are abundant in beta-strand structures. A small NDF fragment containing only the epidermal growth factor domain is also rich in beta-strand structures, but exhibits tertiary structure different from the long NDF forms. Monoclonal antibodies that selectively recognize epidermal growth factor domains of human NDF-alpha and -beta can specifically bind the respective NDF-alpha and -beta isoforms independent of NDF origins. Western blot analysis and quantitative binding assays further identify that an NDF preparation produced naturally from Rat1-EJ cells contains both alpha and beta isoforms in a 3 to 2 ratio. In receptor-binding competition experiments, human and rat NDF-beta isoforms have higher affinity than NDF-alpha isoforms. NDF-beta isoforms can dramatically enhance the stimulation of DNA synthesis for transfected NIH3T3 cells that overexpress HER-3 and HER-4 receptors, while NDF-alpha isoforms can only stimulate proliferation of HER-4-transfected cells with lower activity. Taken together, NDF-alpha and -beta isoforms share similar gross protein conformations but are biologically distinct.
The arginase (ARG)-encoding gene (arg) of Coccidioides immitis, a human fungal pathogen, was cloned and sequenced. Both the genomic and cDNA sequences are provided. The transcription start point and poly(A) sites were confirmed. The arg gene, which was located on chromosome II of C. immitis by Southern hybridization, is a single-copy gene with two introns and a 966-bp ORF which translates a 322-aa protein of 35.1 kDa. The deduced ARG protein showed 44% identity and 68% similarity to the Saccharomyces cerevisiae ARG.
MyoD belongs to a family of helix-loop-helix proteins that control myogenic differentiation. Transfection of various non-myogenic cell lines with MyoD transforms them into myogenic cells. In normal embryonic development MyoD is upregulated at the time when the hypaxial musculature begins to form, but its role in the function of adult muscle remains to be elucidated. In this study we examined the cellular locations of MyoD protein in normal and abnormal muscles to see whether the presence of MyoD protein is correlated with a particular cellular behaviour and to assess the usefulness of MyoD as a marker for satellite cells. Adult rats were anaesthetised and their tibialis anterior or soleus muscles either denervated, tenotomised, freeze lesioned, lesioned and denervated, or lesioned and tenotomised. At various intervals after the operations the rats were killed and their muscles removed, snap frozen, and sectioned with a cryostat along with muscles from unoperated neonatal and adult rats. The sections were processed for immunohistochemistry using a rabbit affinity-purified antibody to recombinant MyoD. MyoD proved to be an excellent marker for active satellite cells; satellite cells in neonatal and regenerating muscles contained high levels of MyoD protein. MyoD positive cells were not observed in the muscles of old adults, in which the satellite cells are fully quiescent. MyoD immunoreactivity was rapidly lost from satellite cell nuclei after they fused into myotubes and was not detected in either sub-synaptic or non-synaptic nuclei of mature fibers. Denervation, and to a lesser extent tenotomy, of lesioned muscles induced expression of MyoD in myotubal nuclei. Denervation of normal muscles also upregulated MyoD in muscle fiber nuclei, an effect which was maximal after 3 days. We conclude that MyoD protein is neurally regulated in both myotubes and muscle fibers.
Developing muscles contain at least two types of myoblasts. Early myoblasts are the first myoblast to form and are the only myoblasts present during primary myotube formation. By the time secondary myotube formation begins, early myoblasts are rare and late myoblasts are common. The late myoblasts have been postulated to give rise to secondary myotubes. While this is generally accepted, it is unclear whether late myoblasts also contribute to the growth of primary myotubes. One study has produced evidence that myoblasts present during secondary myogenesis selectively fuse with each other or with secondary myotubes, but not with primary myotubes (Harris et al. [1989a] Development 107:771-784). However, the sizes of primary myotubes increase during secondary myotube formation. We have therefore re-examined the question of whether primary myotubes absorb new nuclei during secondary myotube formation. Pregnant rats were given a single intraperitoneal injection of 5 mg of 5-bromodeoxyuridine (BrdU) on one embryonic day (from E13 to E19) and their embryos removed on E20. The brominated-nuclei were labelled with an antibody to BrdU and the myotubes were marked with anti-myosin antibodies. Double labelled sections from the soleus, tibialis anterior, and extensor digitorum longus muscles were examined with a confocal microscope. The numbers and locations of labelled nuclear profiles in primary and secondary myotubes were counted and recorded. The results show: (1) that primary myotubes absorb nuclei at all stages of development, including the period of secondary myotube formation; (2) that in the early stages of secondary myotube formation, more myoblasts fuse with primary than secondary myotubes whereas this situation is reversed by the end of secondary myotube formation; and (3) that the nuclei added to primary and secondary myotubes during the early stages of their formation are located within the middle of E20 muscles. The nuclei added to growing myotubes are preferentially located at the ends of the muscles.
CM101 (previously called GBS toxin), a new anticancer polysaccharide that induces inflammatory reactions in neovasculature of tumors, does not cause similar reactions in neovasculature of healing wounds. It appears that treatment with CM101 will not interfere with normal wound healing in cancer patients.
Explore the source record for details and available documents.