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Biomedical subjects

H Yu

Publications and source records attributed to H Yu.

At least 667 records · Page 37Linked to original sources
PubMed1993

Gene cloning and characterization of a GTP-binding Rab protein from mouse pituitary AtT-20 cells.

The Rab proteins constitute a family of Ras-like, low-molecular-weight GTP-binding proteins that are involved in vesicular transport in mammalian cells. Several members of this family have been localized to specific cellular compartments, indicating that they may control vesicular transfer between discrete portions of the endomembrane systems. To examine the complexity of the Rab proteins produced in cells with both regulated and constitutive secretory pathways, we used a PCR-based strategy to clone members of this gene family from rodent pituitary. Here, we report the identification and studies of one of the clones, RAB18. The full-length cDNA clone from a mouse pituitary AtT-20 cDNA library encodes a 23.5-kDa protein with four consensus GTP-binding domains. The deduced amino acid sequence is 42% and 40% identical to Rab2 and Sec4p, respectively, and contains sequence motifs characteristic of the Rab family. Northern blot analysis shows that the level of expression is high in the brain, moderate in the pituitary, and low in the liver. Rab18 may be involved in membrane traffic events during biogenesis and/or recycling steps of secretory vesicle components.

Amino Acid Sequence↗
PubMed1993

Osteoblast-like cells have a variable mixed population of purino/nucleotide receptors.

Osteoblast-like UMR 106.06 cells respond to extracellular application of nucleotides with a fast intracellular calcium pulse (latency of about 20 s, half-width of about 10 s), as measured with fluo-3 on a confocal laser scanning system. Cross-inhibition experiments at 50 microM show that, on a cell population basis, adenosine triphosphate (ATP) strongly inhibits the effect of uridine triphosphate (UTP) or 2-methylthio-ATP (2-MeSATP) applied within 2 min after the end of the ATP-induced pulse, while prior application of UTP or 2-MeSATP only weakly inhibits the ATP effect, and UTP and 2-MeSATP weakly inhibit each other. Furthermore, there are clear differences in cross-inhibition between individual cells. Our measurements provide strong evidence that these cells have at least two types of purino/nucleotide receptors, probably P2y and P2u, with a proportion that varies between individual cells.

Adenosine Triphosphate↗
PubMed1993

Ionization of bromouracil and fluorouracil stimulates base mispairing frequencies with guanine.

To test whether ionized base pairs influence polymerase-catalyzed misinsertion rates, we measured the efficiency of forming 5-bromouracil (B), 5-fluorouracil (F), and thymine base pairs with guanine and adenine as a function of pH using avian myeloblastosis reverse transcriptase. When B, F, and T were present as dNTP substrates, misincorporation efficiencies opposite G, normalized to incorporation of C opposite G, increased by about 20-, 13-, and 7-fold, respectively, as reaction pH increased from 7.0 to 9.5. Incorporation efficiencies to form the correct base pairs, B.A and F.A, normalized to T.A, decreased by 4- and 8-fold, respectively, with increasing pH. The effects of pH on misincorporation efficiencies were about 10-fold greater when B, F, and T were present as template bases. The relative misincorporation efficiencies of G opposite template B, F, and T, normalized to incorporation of A opposite B, F, and T, increased by about 430-, 370-, and 70-fold, respectively, as pH was increased from 6.5 to 9.5, while correct incorporation of A opposite template B and F decreased about 10-fold over the same pH range. Plots depicting incorrect and correct incorporation efficiencies versus pH were fit to a pH titration equation giving the fraction of ionized base as a function of pH. We conclude that avian myeloblastosis reverse transcriptase forms B.G and F.G mispairs in an ionized Watson-Crick conformation in preference to a neutral wobble structure containing favored keto tautomers of B or F. Although participation of disfavored enol tautomers in enzyme-catalyzed base mispair formation cannot be ruled out, the results are inconsistent with the "standard" disfavored tautomer model of mutagenesis. Instead, the data support a model in which ionization of halouracil bases is primarily responsible for B- and F-induced mutagenesis.

Base Sequence↗
PubMed1993

1H and 15N assignments and secondary structure of the Src SH3 domain.

The 1H and 15N sequential assignments of the Src SH3 domain have been determined through a combination of 2D and 3D Nuclear Magnetic Resonance (NMR) methods. The secondary structure of the protein has been identified based on long-range NOE patterns. The SH3 domain of Src consists largely of six beta-strands that form two anti-parallel beta-sheets.

Amino Acid Sequence↗
PubMed1993

1H and 15N assignments and secondary structure of the PI3K SH3 domain.

The sequential 1H and 15N assignments of the SH3 domain of human phosphatidyl inositol 3'-kinase (PI3K) were determined by a combination of homonuclear and heteronuclear NMR experiments. With the exception of several protons belonging to lysine and proline residues, all proton and proton-bearing amide nitrogen resonances were assigned. Based on the sequential nuclear Overhauser effects (NOEs), 3JNH-C alpha H coupling constants and locations of slowly exchanging amide protons, we determined that the secondary structures of the protein consists of six beta-strands, two beta-turns and four short helices. Additional long range NOEs indicate that these beta-strands form two antiparallel beta-sheets. The topology of secondary structural elements of the PI3K SH3 domain is similar to those of the SH3 domains from c-Src and alpha-spectrin, suggesting that the SH3 family has a common tertiary structural motif.

Amino Acid Sequence↗
PubMed1993

Separation of sugars by ion-exclusion chromatography on a cation-exchange resin.

A method for the separation of N-acetylmannosamine and N-acetylglucosamine is described, which consists of chromatography of the two sugars on a column (30 x 1 cm) of the cation-exchange resin, Dowex 50W-X2, in borate buffer at pH 7.8. N-Acetylmannosamine is eluted near the void volume, while N-acetylglucosamine emerges in a more retarded position. It is postulated that the separation occurs as a result of the combined effects of ion exclusion and gel permeation. Thus, in borate solution, N-acetylmannosamine presumably exists largely as a negatively charged complex and is therefore excluded from the sulfonated polystyrene matrix, while N-acetylglucosamine occurs mainly as the free sugar in the equilibrium mixture and, being a neutral compound, has free access to the porous resin. The proposed mechanism for the separation was supported by the finding that glucose and glucose 6-phosphate could also be separated on a column of the same resin, with water as the eluent.

Acetylglucosamine↗
PubMed1993

Structure of the PI3K SH3 domain and analysis of the SH3 family.

Src homology 3 (SH3) domains, which are found in many proteins involved in intracellular signal transduction, mediate specific protein-protein interactions. The three-dimensional structure of the SH3 domain in the p85 subunit of the phosphatidylinositol 3-kinase (PI3K) has been determined by multidimensional NMR methods. The molecule consists of four short helices, two beta turns, and two antiparallel beta sheets. The beta sheets are highly similar to corresponding regions in the SH3 domain of the tyrosine kinase Src, even though the sequence identity of the two domains is low. There is a unique 15 amino acid insert in PI3K that contains three short helices. There are substantial differences in the identity of the amino acids that make up the receptor site of SH3 domains. The results suggest that while the overall structures of the binding sites in the PI3K and Src SH3 domains are similar, their ligand binding properties may differ.

Amino Acid Sequence↗
PubMed1993

Amino-aromatic interaction between histidine 197 of the neurokinin-1 receptor and CP 96345.

Substance P is a peptide neurotransmitter that binds to the neurokinin-1 receptor and is involved in pain transmission and neurogenic inflammation. Recently, a non-peptide substance P antagonist (CP 96345) has been shown to be effective in animal models of pain and inflammation. An understanding of the molecular interactions responsible for ligand binding will be critical for the development of more specific and selective antagonists for the neurokinin-1 receptor and for the discovery of new antagonists for related G-protein-coupled receptors. Here we report that histidine at position 197 in the fifth transmembrane helix of the human neurokinin-1 receptor binds specifically to CP 96345 but not to peptide agonists. Substitution of His 197 by different amino acids and analysis of structural analogues of antagonists suggest that His 197 is involved in an amino-aromatic interaction with the benzhydryl moiety of CP 96345.

Amino Acid Sequence↗
PubMed1993

Interleukin-1 alpha induces a sustained increase in cytosolic free calcium in cultured rabbit osteoclasts.

Using laser confocal microscopy and a Ca2+ indicator, fluo-3, we find that osteoclasts respond to IL-1 alpha with a sustained increase (4 to 40 min) in cytosolic free calcium ([Ca2+]i). The delay time (T1/2) varies from 2 to 8 minutes. These effects cannot be mimicked by application of cAMP and can be blocked by removal of Ca2+ from the bathing medium. Pre-incubation with indomethacin (30 microM) does not block the IL-1 alpha effect on [Ca2+]i. We employ a 'micropipette' method and a 'pieces of cover slip' method to isolate single osteoclasts. Our results provide evidence that IL-1 alpha directly stimulates osteoclasts via a [Ca2+]i signalling pathway. We postulate that this slowly developing, long-lasting increase in [Ca2+]i in osteoclasts may account for the stimulatory effect of IL-1 alpha on osteoclastic bone resorption in conjunction with signalling molecules from osteoblasts.

Animals↗
PubMed1993

ATP induces an intracellular calcium pulse in osteoclasts.

Adenosine triphosphate (ATP; 50 microM) induces a large, fast, transient increase in intracellular calcium ([Ca2+]i) in rabbit osteoclasts, as measured with fluo-3 on a confocal laser scanning system. The [Ca2+]i increase is most intense in the nuclei of these multinuclear cells, indicating that Ca2+ release is occurring just outside the nuclei. ATP produces a much larger effect than adenosine, indicating that these cells have P2 purinergic receptors. Ca(2+)-free bathing medium and the Ca2+ channel blocker Cd2+ both inhibit but do not block the effect, showing that internal Ca2+ release is involved, but that either the steady state Ca2+ influx or a change in influx is important in modulating the effect. Thapsigargin also inhibits the effect. The ATP effect is repeatable with no change in bathing medium, implying that this signalling pathway has a mechanism for adaptation to progressively higher levels of ATP.

Adenosine Triphosphate↗
PubMed1993

Separation of N-acetylglucosamine and N-acetylmannosamine by chromatography on Sephadex in borate buffer.

Several procedures have been used previously for the separation of N-acetylglucosamine and N-acetylmannosamine, which are based on the difference in strength of the borate complexes of the two N-acetylhexosamines and include paper chromatography on borate-treated paper, paper electrophoresis in borate buffer, and anion-exchange chromatography of the borate complexes. In the present study, we have observed that the two sugars, despite their identical size in noncomplexed form, may also be separated by gel chromatography in borate buffer. Nearly complete resolution was obtained by chromatography on a column (1.5 x 117 cm) of Sephadex G-15, which was eluted at room temperature with 0.27 M sodium borate, pH 7.8 (prepared from H3BO3 by addition of NaOH), at a flow rate of 10 ml/h. This procedure complements existing methods for the separation of N-acetylmannosamine and N-acetylglucosamine and has the advantage that it can be carried out on a relatively large preparative scale.

Acetylglucosamine↗
PubMed1993

The surface properties of lung 36 kDa Ca(2+)-dependent phospholipid-binding protein.

The intrinsic surface activity of a 36 kDa rabbit lung calcium-dependent phospholipid-binding protein (PLBP), a member of the annexin family of such proteins, at the air/water interface has been determined from measurements of surface tension of aqueous solutions, and surface concentration of 14C-labeled PLBP adsorbed from aqueous solution in the absence and presence of Ca2+. It was also possible to spread insoluble monolayers of PLBP to determine surface pressure vs. surface concentration isotherms, as well as surface elasticity and surface viscosity as a function of frequency from electrocapillary wave diffraction measurements. PLBP has been shown to exhibit significant intrinsic surface activity at the air/water interface, comparable to a variety of other hydrophobic proteins known to be quite surface active. In all cases, surface properties were enhanced by the presence of Ca2+, particularly the degree of surface viscoelasticity at close-packing in the monolayer. This is believed to reflect changes in protein conformation at the surface.

Animals↗
PubMed1993

(R)- and (S)-8-acetyl-2-(dipropylamino)tetralin (LY-41): two novel 5-HT1A receptor agonists.

Interactions between central 5-HT1A receptors and the enantiomers of LY-41, a 2-aminotetralin derivative related to 8-OH-DPAT (8-hydroxy-2-(dipropylamino)tetralin), were studied. Both enantiomers of LY-41 behaved as potent 5-HT1A receptor agonists in rats, inducing the 5-HT behavioural syndrome, decreasing body temperature and inhibiting the cage-leaving response. The behavioural syndrome and the hypothermia were antagonized by the 5-HT1A receptor antagonist, (S)-UH-301. The LY-41 enantiomers also reduced brain 5-HTP accumulation in rats treated with a decarboxylase inhibitor. The pharmacology of the enantiomers of LY-41 appeared similar to that of 8-OH-DPAT. However, it is noteworthy that the stereoselective interaction of 5-HT1A receptors with LY-41 was opposite to that of 8-OH-DPAT. Thus, (R)-8-OH-DPAT was more potent than (S)-8-OH-DPAT, whereas (S)-LY-41 appeared to be more potent than (R)-LY-41.

5-Hydroxytryptophan↗
PubMed1993

Apoptosis and hematopoiesis in murine fetal liver.

The fetal mouse liver (FL) is an organ of intense, but transient, hematopoietic activity during mid-gestation, with erythropoiesis being predominant during days 11 through 16. It therefore seemed reasonable to expect that hematopoietic cytokines, such as erythropoietin (epo), interleukin-3 (IL-3), and stem cell factor (SCF), may play important roles in maintaining a homeostatic balance of erythropoiesis and apoptosis in liver during ontogeny. First, we determined the effects of these growth factors on hematopoiesis by measuring colony formation and hemoglobin synthesis of cultured FLs. Secondly, we determined the protection from apoptosis afforded by these cytokines, using electrophoretic analysis of DNA and by flow cytometry of FL cells deprived in culture of epo, IL-3, and SCF. Erythropoietin was necessary and alone sufficient for hemoglobin synthesis in colony-forming units-erythroid colonies, but IL-3 was a required cofactor to obtain maximal development of burst-forming units-erythroid colonies. SCF alone caused little colony formation in methylcellulose cultures of FLs, but when combined with epo and IL-3, it had dramatic effects both on the number of colonies and their size. Secondly, indices of apoptosis were determined by measuring DNA fragmentation caused by endogenous nuclease activity in apoptotic cells. Liver cells from cultures without cytokines showed the extensive degradation of DNA to low molecular weight nucleosomal oligomers, which is characteristic of apoptosis. Protection from apoptosis afforded by epo directly corresponded to the level of erythropoiesis in FLs of different gestational age. Erythropoietin was by far the most critical cytokine in sparing FL cells from apoptosis. Analyses of agarose gels showed that SCF and IL-3 alone had no apparent effect in reducing the amount of DNA in fragments, and when combined with epo they had no more protective effect than that provided by epo alone. However, using the more sensitive flow cytometric determination of cells with subdiploid amounts of DNA, SCF, and IL-3 alone had measurable protective effects that were less than those caused by epo. Thus, we show that normal, untransformed cells of the developing hematopoietic system not only require cytokines for proliferation and differentiation, but they have an initial and absolute requirement of them for protection from apoptosis.

Animals↗
PubMed1993

Human PCK1 encoding phosphoenolpyruvate carboxykinase is located on chromosome 20q13.2.

Cytoplasmic liver phosphoenolpyruvate carboxykinase (GTP) (PEPCK) catalyzes a rate-limiting step in gluconeogenesis. Primers derived from the rat liver PEPCK sequence were used to amplify a portion of the human liver cDNA and to screen a YAC library of human genomic DNA. The sequences of human and rat PEPCK cDNA differed at 16% of the nucleotides compared (45/291). Analysis of a human/rodent hybrid mapping panel demonstrated concordant segregation of PCK1 with chromosome 20. Fluorescence in situ hybridization with YAC DNA further localized PCK1 to subband 20q13.2.

Animals↗
PubMed1993

Organisation of the tomato polyphenol oxidase gene family.

We report the isolation and characterization of seven nuclear genes encoding polyphenol oxidase (PPO) in tomato (Lycopersicon esculentum cv. VFNT Cherry). The seven genes (PPOs A, A', B, C, D, E and F) fall into three structural classes (I, II, and III) based on Eco RI and Hind III restriction fragment length polymorphisms (RFLP). RFLP mapping and PFGE analysis demonstrated that the genes reside on chromosome 8, and may be clustered within a 165 kb region. Phage insert mapping demonstrated PPO E and PPO F (both class III), and PPOs B, D and A (classes I, II and I respectively) are grouped within separate 12.4 kb clusters. The complete nucleotide sequence was determined for each gene. Comparison to cDNAs revealed that the PPOs lack introns. A transcript of about 2 kb is expected for each PPO. Each PPO possesses a region encoding a transit peptide characteristic of polypeptides targeted to the thylakoid lumen. Predicted precursor polypeptides range in mass from 66 to 71 kDa and predicted mature polypeptides range from 57 to 62 kDa. All the PPOs encode two putative copper-binding sites characteristic of bacterial, fungal and mammalian tyrosinases. Five of the seven PPOs possess divergent DNA sequences in their 5' promoter regions. These flanking sequence differences may regulate the differential expression of PPO genes.

Amino Acid Sequence↗