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Biomedical subjects

L Zhu

Publications and source records attributed to L Zhu.

At least 415 records · Page 23Linked to original sources
PubMed1994

Molecular cloning of a mammalian hyaluronidase reveals identity with hemopexin, a serum heme-binding protein.

Hyaluronan is the most abundant glycosaminoglycan of the extracellular matrix and is a critical substrate for cellular attachment and locomotion. Little is known about the class of enzymes, termed hyaluronidases, that are responsible for hyaluronan catabolism in mammals. We have determined a partial amino acid sequence from a purified preparation of porcine liver hyaluronidase and have used this information as the basis for cloning complementary DNA that encodes the corresponding protein. When expressed in a recombinant baculovirus system, the protein exhibited hyaluronidase activity in a substrate-gel assay. The deduced sequence of this mammalian hyaluronidase is that of a 459-amino-acid polypeptide bearing four potential N-glycosylation sites as well as a copy of a proposed hyaluronan binding motif. Remarkably, amino acid sequence comparisons and immunologic cross-reactivities strongly suggest that the cloned protein is identical to hemopexin, an abundant, heme-binding serum protein. Although hemopexin has not previously been reported to possess any enzymatic activity, it includes a conserved domain found in collagenases, stromelysins, and other enzymes that metabolize the extracellular matrix. We conclude that hemopexin is the predominant hyaluronidase expressed in mammalian liver.

Amino Acid Sequence↗
PubMed1994

The unusual structure of the human centromere (GGA)2 motif. Unpaired guanosine residues stacked between sheared G.A pairs.

The centromere of human chromosomes contains multiple repeats of the DNA sequence d(TGGAA)n. This sequence has the interesting property of pairing with itself to form stable duplexes. We have determined the solution structure of the unusual DNA duplex 5'-TGGAATGGAA:TGGAATGGAA-3' at atomic resolution. The duplex contains unpaired staggered guanosine residues, which co-stack by intercalation between sheared G.A and A.G base-pairs to form an interesting new structural motif, the GA-bracketed G-stack. The TGGAA repeat unit contains six "steps", four of which are not Watson-Crick base-pairs.

Adenosine↗
PubMed1994

E2F-4, a new member of the E2F gene family, has oncogenic activity and associates with p107 in vivo.

The E2F family of transcription factors controls the expression of genes that are involved in cell cycle regulation. E2F DNA-binding activity is found in complex with the retinoblastoma protein, pRb, and with the pRb-related p107 and p130. To date, cDNAs for three members of the E2F gene family have been isolated. However, all three E2Fs associate in vivo exclusively with pRb. We report here the cloning and functional analysis of a fourth E2F family member. E2F-4 encodes a 413-amino-acid protein with significant homology to E2F-1. E2F-4 antibodies recognize a 60-kD protein in anti-p107 immunoprecipitates, indicating that E2F-4 associates with p107 in vivo. Like the other E2Fs, E2F-4 requires DP-1 for efficient DNA binding and transcriptional activation of E2F site-containing promoters. Increased expression of E2F-4 and DP-1 in SaoS-2 osteosarcoma cells causes a shift from G1-phase cells to S and G2/M-phase cells, suggesting a role for E2F-4 in regulation of cell-cycle progression. We show that expression of E2F-4 and DP-1 together with an activated ras oncogene in rat embryo fibroblasts, causes transformation, indicating that E2F-4 has oncogenic activity.

Amino Acid Sequence↗
PubMed1994

Observation of coherent reaction dynamics in heme proteins.

Femtosecond laser pulses, resonant with Soret band of the nitric oxide complex of myoglobin (MbNO), were used to probe coherent, low-frequency nuclear motion of the heme group after photolysis. Distinct oscillations with periods of 430 and 150 femtoseconds were observed and are attributed to heme doming and iron-histidine motion, respectively. These results verify that the nuclear motion of the heme is strongly coupled to the ligand binding reaction and demonstrate that such motion is not determined by overdamped (diffusive) dynamics. The relative phases and frequencies of the nuclear motion of the photoproduct suggest that the coherence arises from impulsive electronic forces associated with the spin-state change of the heme iron atom and the depopulation of its dz2 orbital during the bond-breaking event.

Histidine↗
PubMed1994

Interaction of c-Myc with the pRb-related protein p107 results in inhibition of c-Myc-mediated transactivation.

The product of the c-myc proto-oncogene, c-Myc, is a sequence-specific DNA binding protein with an N-terminal transactivation domain and a C-terminal DNA binding domain. Several lines of evidence indicate that c-Myc activity is essential for normal cell cycle progression. Since the abundance of c-Myc during the cell cycle is constant, c-Myc's activity may be regulated at a post-translational level. We have shown previously that the N-terminus of c-Myc can form a specific complex with the product of the retinoblastoma gene, pRb, in vitro. These data suggested a model in which pRb, or pRb-related proteins, regulate c-Myc activity through direct binding. We show here that the pRb-related protein p107, but not pRb itself, forms a specific complex with the N-terminal transactivation domain of c-Myc in vivo. Binding of p107 to c-Myc causes a significant inhibition of c-Myc transactivation. Expression of c-Myc releases cells from a p107-induced growth arrest, but not from pRb-induced growth arrest. Our data suggest that p107 can control c-Myc activity through direct binding to the transactivation domain and that c-Myc is a target for p107-mediated growth suppression.

Binding Sites↗
PubMed1994

The solution structure of the r(gcg)d(TATACCC):d(GGGTATACGC) Okazaki fragment contains two distinct duplex morphologies connected by a junction.

Okazaki fragments are important intermediates in DNA replication. Chimeric duplexes that are structurally equivalent to Okazaki fragments also occur during reverse transcription of RNA retroviruses. Such duplexes consist of an RNA-DNA chimeric strand base-paired to a pure DNA strand; hence they have a hybrid duplex "left half" covalently linked to a "right half" that is pure DNA. We have determined the solution structure of the synthetic Okazaki fragment r(gcg)d(TATACCC):d(GGGTATACGC) by means of two-dimensional NMR, restrained molecular dynamics and full relaxation matrix simulation of the two-dimensional nuclear Overhauser effect spectra at various mixing times. The large negative x-displacement and large positive inclination in the hybrid section of the duplex are structural characteristics similar to those found in pure hybrid duplexes. However, the DNA sugar puckers and the width and depth of the minor groove in the pure DNA section are more like B-form DNA, especially beyond the junction. Thus, this Okazaki fragment duplex assumes a conformation in solution that is a chimeric mixture of hybrid-form (H-form) and B-form structures and the overall molecule cannot be classified as either an A-form or a B-form duplex. The co-existence of these two different conformations in a single duplex gives rise to a structural discontinuity with a bend of approximately 18.1 (+/- 0.4) degrees at the junction between the hybrid and DNA segments that may be important for reverse transcriptase binding and RNase H cleavage of such molecules. Despite the fact that the solution structure is quite different from the all A-form structure reported recently for the exact same molecule in the crystalline state, a surprising number of local helical parameters were found to be quite similar to those reported for the crystal structure.

Base Sequence↗
PubMed1994

Reversal of terminal differentiation mediated by p107 in Rb-/- muscle cells.

The terminal differentiation of mammalian muscle cells requires the tumor suppressor retinoblastoma protein (Rb). Unlike their wild-type counterparts, multinucleated myotubes from mouse cells deficient in Rb (Rb-/-) were induced by serum to re-enter the cell cycle. Development of the myogenic phenotype in Rb-/- cells correlated with increased expression of p107, which interacted with myogenic transcription factors. Serum-induced cell cycle reentry, on the other hand, correlated with decreased p107 expression. Thus, although p107 could substitute for Rb as a cofactor for differentiation, it could not maintain the terminally differentiated state in Rb-/- myotubes.

Animals↗
PubMed1994

Structure and expression of the human slow twitch skeletal muscle troponin I gene.

The contractile protein troponin I is encoded by a multigene family whose members are expressed differentially in various classes of muscle fibers. In vertebrates, the "slow" isoform of troponin I is expressed during early heart and skeletal muscle development but is restricted to slow twitch skeletal muscle in the adult. This diverse expression pattern offers an opportunity to study the regulation of a single gene within different developmental contexts. To initiate such studies, we have cloned the gene encoding the human slow twitch skeletal muscle isoform of troponin I and have identified 5'-flanking sequences required for its expression in skeletal muscle cells. The slow troponin I gene spans 12.5 kilobases and is divided into nine exons. In contrast to many muscle-specific genes, the troponin I promoter does not contain consensus CCAAT or TATA elements. Moreover, the sequence from -9 to +11 resembles an "initiator element" previously shown to direct transcription of some tissue-specific genes lacking TATA boxes (Smale, S. T., and Baltimore, D. (1989) Cell 57, 103-113; Brand, N. J., Petkovich, M., and Chambon, P. (1990) Nucleic Acids Res. 18, 6799-6806; Weis, L., and Reinberg, D. (1992) FASEB J. 6, 3300-3309). A transcriptional fusion construct, comprising 4.2 kilobases of troponin I 5'-flanking DNA linked to the bacterial chloramphenicol acetyl-transferase gene, exhibited cell type-specific and developmentally regulated expression. A muscle-specific enhancer regulated slow troponin I promoter activity.

Adult↗
PubMed1994

Sequencing of a cDNA encoding the human fast-twitch skeletal muscle isoform of troponin I.

A cDNA encoding the human fast-twitch skeletal muscle isoform of troponin I (TnIfast) has been sequenced. This cDNA is 701 base pairs in length, and encodes a protein of 182 amino acids. TnIfast is the last of the three known human TnI isoforms to be sequenced. Comparison of the deduced human TnIfast protein sequence with a variety of troponin I isoforms from other species has revealed a high degree of cross-species sequence conservation between TnIfast proteins.

Amino Acid Sequence↗
PubMed1994

Serum-induced vascular smooth muscle cell elastolytic activity through tyrosine kinase intracellular signalling.

In previous studies, we related increased elastolytic activity in pulmonary arteries (PA) with endothelial injury to the later development of PA hypertension in rats. As the mechanism causing the increased PA elastase was unknown, we hypothesized that serum factors which are accessible to vascular smooth muscle cells (SMC) following endothelial injury stimulate their elastolytic activity. To test this, we developed an in vitro assay in which we added [3H]-elastin to cultured vascular SMC after 24 h serum starvation and monitored elastolysis following a further 24 h incubation with fetal bovine serum (FBS). We observed that serum induced increased elastolytic activity in both PA and aorta-derived SMC but not in endothelial cells or SMC with low basal levels of elastolytic activity. Maximum stimulation of SMC elastolytic activity occurred with a concentration as low as 1% FBS and despite elastase inhibitors in serum, suggesting that the activity is confined to the immediate pericellular region where enzyme concentration is high. Serum-stimulated elastolytic activity was not reproduced by growth factors or cytokines known to be associated with vascular disease or to induce release of elastases in other cells. The serum inducing elastolytic activity was heat and acid labile. It was associated with increased elastin adhesion to the 67 kD elastin binding protein on SMC surfaces and was prevented by tyrosine kinase inhibitors but not protein kinase C or A inhibitors. Our studies therefore suggest a mechanism whereby serum induction of SMC elastase requires signalling through the elastin binding protein and activation of tyrosine kinase.

Animals↗
PubMed1994

Membrane association of phospholipase C encoded by the norpA gene of Drosophila melanogaster.

Severe mutations within the norpA gene of Drosophila abolish the photoreceptor potential and render the fly blind by deleting phospholipase C, an essential component of the phototransduction pathway. To study the membrane association of phospholipase C, we have utilized biochemical assays of phospholipase C activity, which predominant measurable phospholipase C activity in head homogenates has been shown to be encoded by norpA, as well as antisera generated against the major gene product of norpA to examine its subcellular distribution before and during phototransduction. We find that both phospholipase C activity and the norpA protein are predominantly associated with membrane fractions in heads of both light- and dark-adapted flies. Moreover, phospholipase C activity as well as norpA protein can be easily extracted from membrane preparations of light- or dark-adapted flies using high salt, indicating that the norpA protein is peripherally localized on the membrane. These data suggest that the norpA encoded phospholipase C of Drosophila is a permanent peripheral membrane protein. If this is indeed the case, then it would mean that the reversible redistribution of phospholipase C from the cytosol to the membrane, as observed in epidermal growth factor receptor stimulation of mammalian phospholipase C gamma, is not a universal mechanism utilized by all types of phosphatidylinositol-specific phospholipase C.

Animals↗
PubMed1994

The relationship of dietary animal protein and electrolytes to blood pressure: a study on three Chinese populations.

The relationship of diet to blood pressure was studied in a total of 705 men and women aged 40-59 from three Chinese population samples having different mean blood pressure and dietary sodium and animal protein intake. Two groups were farmers from Shanxi in northern China, and Guangxi in southern China, and the third were fishermen from Zhejiang, eastern China. Three 24-hour dietary recalls were done for each participant. Serum and overnight urine amino acids were measured in random subsamples. Determination of electrolytes in three 24-hour urine specimens was done in an additional sample of 59 men in each population. Results of multiple or stepwise regression showed: 1) in the pooled group, individual intake of sodium was positively associated with systolic blood pressure; 2) when stratifying by median calcium intake, a positive association of dietary sodium or sodium/potassium was found only in the group with calcium intake lower than the median; 3) daily intake of animal protein, urinary sulphate and certain serum and urine amino acids formed from protein metabolism, were inversely associated with blood pressure.

Adult↗
PubMed1994

Posttranscriptional effector domains in the Rev proteins of feline immunodeficiency virus and equine infectious anemia virus.

By systematically dissecting the Rev proteins of feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV), we have identified within each a short peptide that is functionally interchangeable with the effector domains found in Rev-like proteins from other retroviruses. The active sequences from FIV and EIAV differ in several respects from other known effectors and may represent a distinct class of effector domain.

Amino Acid Sequence↗
PubMed1994

The endogenous vascular elastase that governs development and progression of monocrotaline-induced pulmonary hypertension in rats is a novel enzyme related to the serine proteinase adipsin.

We showed previously a cause and effect relationship between increased activity of an endogenous vascular elastase (EVE) and experimentally induced pulmonary hypertension in rats. We now report the isolation and characterization of EVE. Degenerate oligonucleotides synthesized to homologous sequences in serine elastases were used in a PCR with rat pulmonary artery (PA) cDNA. The PCR product hybridized to a 1.2-kb mRNA and the intensity of hybridization was threefold increased in RNA from rat hypertensive PA at a timepoint when EVE activity was increased. The PCR product was used to screen a cDNA library and sequences obtained encoded rat adipsin. We then used immunoaffinity to purify EVE. An antibody to the elastin-binding protein was used to remove this competitor of elastase from the PA extract and the elastolytic activity increased 100-fold. The enzyme was purified using an antibody that recognizes NH2-terminal sequences of serine proteinases and the eluate was further purified using an antibody raised against recombinant adipsin. A single band at 20 kD immunoreactive with the adipsin antibody was resolved as an active enzyme on an elastin substrate gel. Immunogold labeling with an antibody to an adipsin peptide sequence localized EVE to PA smooth muscle cells. This is the first isolation of EVE; it appears to be a novel enzyme related to the serine proteinase adipsin originally found in adipose tissue.

Amino Acid Sequence↗