Angle-resolved photoemission extended fine-structure study of the structure of p(2 x 2)K/Ni(111).
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Journal Article↗
Biomedical subjects
Z Huang
Publications and source records attributed to Z Huang.
A fluorometric microplate-based assay of submicrogram monomeric actin by inhibition of deoxyribonuclease I.
Inhibition of bovine pancreatic deoxyribonuclease I (DNase I) has been a standard method for specific quantitation of monomeric (G-) actin. The aim of this work is to substantially enhance the sensitivity of this type of G-actin assay by using a fluorescent dye, thiazole orange (TO), which has a high optical absorption, moderate DNA affinity, and large fluorescence enhancement upon binding to DNA. The high fluorescence output and moderate affinity of TO's DNA complex produce a large fluorescence decrease when the complex is disrupted by DNase I, thus permitting a highly sensitive detection of G-actin by its inhibition on the fluorescence decrease. The results show that the dynamic responsiveness of a fluorometric G-actin assay can be improved by lowering the concentration of DNA and affinity of the labeling dye, as long as the fluorescence signal of the dye-DNA complex is sufficient for instrumental detection. With a DNA concentration of 6.0 micrograms/ml and TO dye labeling, as little as 20 ng of G-actin can be reliably detected in a fluorescence microplate scanner. This sensitivity also appears to be the utmost detection limit of any G-actin assay that is based on DNase I inhibition.
Actins↗
One-step affinity isolation of recombinant protein using the baculovirus/insect cell expression system.
We have developed two baculovirus transfer vectors which allow single-step affinity isolation of recombinant proteins after expression in insect cells. Using these vectors, recombinant proteins are synthesized as fusions with glutathione-S-transferase and are amenable to enrichment from a crude insect cell lysate using glutathione affinity agarose. After affinity isolation, glutathione-S-transferase can be cleaved from the recombinant polypeptides of interest at an engineered thrombin cleavage site. We used this approach to successfully isolate glutathione-S-transferase, the human low density lipoprotein receptor, two large polypeptides containing cytoplasmic domains of the cystic fibrosis transmembrane conductance regulator (CFTR), and the full-length CFTR. The approach has potential advantages over prokaryotic overexpression of foreign polypeptides, including: (i) eukaryotic post-translational modification of expressed protein, (ii) increased solubility of recombinant fusion proteins synthesized in insect cells leading to increased affinity yield under mild conditions, and (iii) production of large and/or complex polypeptides which might be difficult to purify from prokaryotic cells. The method also allows enrichment of recombinant protein representing a small fraction (less than 5%) of total insect cell protein produced and provides a general method for eukaryotic protein synthesis and isolation which is independent of the particular protein being expressed.
Animals↗
Spotted fever group rickettsial infection in south-eastern Australia: isolation of rickettsiae.
Flinders Island spotted fever (FISF), a spotted fever group (SFG) rickettsial disease first described in 1991, occurs in south-eastern Australia. The isolation of the aetiological agent is described for the first time having been obtained from the blood of two patients. An additional 22 cases are also reported. Of these patients four had positive initial serology, and 20 showed seroconversion (using Rickettsia australis as antigen). Acute phase blood specimens taken from seven patients caused neonatal mice to seroconvert to R. australis and a blood specimen from one of these patients (and one other) yielded rickettsiae. A field survey for possible reservoir and vector animals on Flinders Island, Tasmania and in Gippsland, Victoria (both in south-eastern Australia) yielded 217 vertebrates and 1445 invertebrate ectoparasites, mostly ticks. Ixodes cornuatus from humans and dogs in Gippsland produced seroconversion to SFG rickettsia when inoculated into mice but no invertebrate pools from Flinders Island produced seroconversion in mice. Haemolymph from an individual I. cornuatus removed from a human in Gippsland, yielded a SFG rickettsia on tissue culture. Sera from several species of native vertebrates, especially the bush rat, Rattus fuscipes, were positive for antibodies to SFG rickettsia.
Adolescent↗
Theory for two-photon excitation in pattern photobleaching with evanescent illumination.
Although the translational diffusion coefficients of proteins embedded in, or tightly bound to, natural and artificial cell membranes have been extensively studied, very little is known about the diffusion of proteins weakly bound to membranes. One method for measuring the diffusion coefficients of weakly bound species is to combine evanescent interference patterns with fluorescence pattern photobleaching recovery (TIR-FPPR). However, this technique is intrinsically limited in that the maximum post-bleach fluorescence change, normalized to the pre-bleach fluorescence, is 0.22. This limitation severely restricts the precision of the data and the applicability of the method. In the theoretical work described herein, it is shown that the maximum fractional fluorescence change in TIR-FPPR with two-photon bleaching and observation would be 0.36. Thus, the use of two-photon excitation in TIR-FPPR would significantly improve the signal-to-noise ratio and would provide more precise measurements of the translational diffusion of proteins weakly bound to surfaces.
Diffusion↗
Cyclic hexapeptide analogs of somatostatin containing bridge modifications. Syntheses and conformational analyses.
The cyclic hexapeptide c[Pro6-Phe7-D-Trp8-Lys9-Thr10-Phe11] displays higher bioactivity than native somatostatin in inhibiting the release of growth hormone. The superscript numbers refer to the location of the residues in native somatostatin. To investigate the structural role played by the Phe11-Pro6 bridging region, we have synthesized a series of cyclic hexapeptide analogs of somatostatin incorporating peptidomimetics and retro-inverso modifications at the bridging region. Among them, two analogs contain the retro-inverso modification mAla6-gPhe11 at the bridging region, and five analogs contain 2-aminocyclopentane carboxylic acid (2-Ac5c) and 1-aminocyclopentane carboxylic acid (1-Ac5c) as proline mimetics. The conformational preferences of these analogs have been studied using 1H-NMR and computer simulations. All of these analogs maintain conformations similar to those of the parent cyclic hexapeptide around the Phe7-D-Trp8-Lys9-Thr10 tetrapeptide region consisting of a beta II' turn. However, they display different conformational features around the bridging region. The R-mAla analog and the five Ac5c analogs show only a trans amide bond for Phe11-Pro6 in the bridging region, while the S-mAla analog displays a cis/trans isomerization for the same amide linkage in the bridging region. The R-mAla and the five Ac5c analogs do not bind to the somatostatin receptor, while the S-mAla analog displays a high binding activity. Applying our recently proposed model for bioactivity of somatostatin analogs, we examined the structure-bioactivity relationships for these somatostatin analogs. This investigation provides valuable insight into the structural role played by the bridging region.
Amino Acid Sequence↗
Time-dependent blockade of neurogenic plasma extravasation in dura mater by 5-HT1B/D agonists and endopeptidase 24.11.
1. The delayed effects of stimulating the trigeminal ganglion unilaterally in rats and guinea-pigs were assessed by measuring the leakage of radiolabelled albumin from blood into the dura mater at intervals for up to 120 min after a 5 min stimulation period (5 Hz, 0.6 mA, 5 ms). 2. [125I]-albumin (50 microCi kg-1) was injected i.v. as a tracer 0, 20, 50, 80 or 110 min after stimulation and the animals were killed 10 min later. Extravasation of plasma protein developed for up to 90 min poststimulation. 3. To examine the mechanism underlying delayed plasma protein extravasation, CP-93,129 (5-HT1B receptor agonist, 460 nmol.kg-1), sumatriptan (5-HT1B/D receptor agonist, 24 nmol kg-1), or neutral endopeptidase 24.11 (1 nmol kg-1) were administered 45 or 75 min after trigeminal stimulation and 5 min before radiolabelled albumin. The extravasation response was reduced at 45 min. CP-23,129 also blocked extravasation when injected 25 min after capsaicin administration (1 mumol kg-1). 4. If the tracer was injected 5 min prior to electrical trigeminal stimulation, endopeptidase 24.11 (1 nmol kg-1) given 10 min before stimulation blocked the leakage (as reported previously for CP-93,129 or sumatriptan). All three compounds blocked the leakage when administered 30 min (but not 60 min) poststimulation in this paradigm. 5. The data support the previously made contention that neuropeptide release from sensory fibres mediates the plasma extravasation response following trigeminal ganglion stimulation, and that release and plasma leakage continue many minutes beyond the stimulation period. Hence, drugs that inhibit neuropeptide release (CP-93,129, sumatriptan), or enhance breakdown of neuropeptide mediators(endopeptidase 24.11) block the delayed extravasation response. Extravasation developing later than 45 min poststimulation was not neurogenically mediated.
Animals↗
Microbial transformations of ferulic acid by Saccharomyces cerevisiae and Pseudomonas fluorescens.
Saccharomyces cerevisiae (dry baker's yeast) and Pseudomonas fluorescens were used to convert trans-ferulic acid into 4-hydroxy-3-methoxystyrene in 96 and 89% yields, respectively. The metabolites were isolated by solid-phase extraction and analyzed by thin-layer chromatography and high-performance liquid chromatography. The identities of the metabolites were determined by 1H- and 13C-nuclear magnetic resonance spectroscopy and by mass spectrometry. The mechanism of the decarboxylation of ferulic acid was investigated by measuring the degree and position of deuterium incorporated into the styrene derivative from D2O by mass spectrometry and by both proton and deuterium nuclear magnetic resonance spectroscopies. Resting cells of baker's yeast reduced ferulic acid to 4-hydroxy-3-methoxyphenylpropionic acid in 54% yield when incubations were under an argon atmosphere.
Biotransformation↗
Glucose-dependent insulinotropic polypeptide stimulated insulin release from a tumor-derived beta-cell line (beta TC3).
The beta TC3 tumor cell line was examined for the presence of functional glucose-dependent insulinotropic polypeptide (GIP) receptors. Increasing amounts of natural porcine GIP decreased the binding of HPLC-purified [125I]GIP to beta TC3 cells in a concentration-dependent manner. Displacement of GIP was significant at concentrations as low as 500 pM, and the radioligand was fully displaced at 100 nM. GIP(1-30) produced a displacement of [125I]GIP comparable with that produced by GIP(1-42), and glucagon yielded 20% displacement at a concentration of 1 microM but was without effect at 100 mM. Incubation of beta TC3 cells in the presence of glucose concentrations of 2-20 mM yielded a concentration-dependent stimulation of immunoreactive insulin (IRI) release. GIP and glucagon-like peptide-I(7-36) amide (tGLP-I) at concentrations of 1 nM or greater significantly stimulated IRI release in the presence of 2 mM glucose. The threshold glucose concentration for GIP-stimulated IRI release from beta TC3 cells was 0.5 mM, and maximal potentiation of IRI release by GIP occurred at 5 mM glucose. Somatostatin significantly inhibited GIP-stimulated IRI release in the presence of 5 mM glucose. It is concluded that beta TC3 cells have functional GIP receptors and may provide a useful model for the study of IRI secretion at the cellular level.
Animals↗
A novel fluorogenic substrate for detecting alkaline phosphatase activity in situ.
We describe here the in situ detection of alkaline phosphatase (APase) activity with a new fluorogenic substrate, 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (CPPCQ). CPPCQ is very soluble and colorless. APase converts it into a rapidly precipitating product, whose strong fluorescence marks the sites of APase activity. The detected APase was either a probing enzyme anchored to epidermal growth factor (EGF) receptors of fixed human epidermoid carcinoma cell line (A431) by biotinylated EGF and streptavidin-APase conjugates or an endogenous marker existing in a fixed canine kidney cell line (MDCK). With CPPCQ staining, the EGF receptors and the endogenous APase were both visualized by fluorescence microscopy as contrasting, photostable, and well-resolved fluorescent stains. The EGF receptor staining was specific since it could be blocked by excessive unlabeled EGF. In contrast, fluorescein-labeled EGF failed to specifically stain the EGF receptors under the same fluorescent microscope. The endogenous APase staining with CPPCQ was sensitive to heating, levamisole and L-homoarginine, showing an APase tissue specificity of the liver/bone/kidney type. Therefore, CPPCQ appears to be a novel substrate dye for sensitive fluorescence APase histochemistry.
Alkaline Phosphatase↗
Oligonucleotide analogs with dimethylenesulfide, -sulfoxide, and -sulfone groups replacing phosphodiester linkages.
Two sets of experimental protocols are given for the synthesis of 3',5'-bis-homodeoxyribonucleosides, building blocks for the synthesis of oligodeoxynucleotide analogs where the -O-PO2-O- groups are replaced by -CH2-S-CH2-, -CH2-SO-CH2-, and -CH2-SO2-CH2- units. Conditions are presented for joining these building blocks to create short nucleic acid analogs. Since isosteric, achiral, and non-ionic analogs of natural oligonucleotides stable to both enzymatic and chemical hydrolysis, such molecules have potential application as probes in the laboratory, in studies of the biological function of individual genes, and as "antisense" oligonucleotide analogs.
Acrylates↗
Structure and function of the receptor for parathyroid hormone and parathyroid hormone-related protein.
The receptor for parathyroid hormone (PTH) and PTH-related protein (PTHrP) is a member of a subfamily of G-protein-coupled receptors. This subfamily includes receptors for calcitonin, secretin, vasoactive intestinal polypeptide, glucagon, and related peptides, growth hormone-releasing hormone, and pituitary adenylate cyclase activating peptide. These receptors couple agonist occupancy to activation of adenylate cyclase and, in some cases, to increases in Cai2+, but the molecular basis of signalling is unclear Mutagenesis studies of recombinant PTH/PTHrP receptors indicates that large portions of the third intracellular loop and C-terminal tail can be deleted and/or mutated without major loss of receptor-G-protein interaction, as evidenced by high affinity ligand binding and signal transduction. However, specific determinants in these domains appear to modulate the efficiency of effector activation. Further studies are needed to define the contact sites for PTH/PTHrP receptor-G-protein interaction.
Amino Acid Sequence↗
Structural requirements for tissue factor pathway inhibitor interactions with factor Xa and heparin.
Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type protease inhibitor, which inhibits factor Xa directly and in a factor Xa dependent manner inhibits the factor VIIa/tissue factor catalytic complex. Altered forms of recombinant TFPI (rTFPI) were tested for their ability to inhibit human factor Xa and bovine gamma-carboxyglutamate (Gla)-domainless factor Xa in the presence and absence of calcium ions, heparin, phospholipids, and factor Va. Sequential deletions of the positively charged C-terminus of TFPI produces proteins that have decreasing inhibitory activity against factor Xa as well as decreasing affinity for heparin-agarose. Deletion of the C-terminus distal to Leu181, which eliminates the third Kunitz-type domain, results in the loss of heparin-agarose binding at physiological ionic strength. Furthermore, the entire C-terminal polypeptide, including at least a portion of the third Kunitz-type domain, appears to be involved in heparin binding. Residues 230-241 probably form an alpha helix in which Lys231 and Arg237 within the Kunitz domain and Lys240 and Lys241 could provide a positively charged surface epitope capable of binding heparin. Heparin and Ca2+ together, but not individually, enhance the rate of factor Xa inhibition by full-length TFPI. The effect of heparin is concentration dependent and biphasic (maximal between 0.1 and 1.0 unit/ml) suggesting that the acceleration of factor Xa inhibition occurs at least in part through a 'template' mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Malignant fibrous histiocytoma of the ascending colon in a child.
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Child↗
Pharmacological profile of NPC 17742 [2R,4R,5S-(2-amino-4,5-(1, 2-cyclohexyl)-7-phosphonoheptanoic acid)], a potent, selective and competitive N-methyl-D-aspartate receptor antagonist.
2R,4R,5S-(2-amino-4,5-(1,2-cyclohexyl)-7-phosphonoheptanoic acid) (NPC 17742), the most potent isomer of the mixture 2-amino-4,5-(1,2-cyclohexyl)-7-phosphonoheptanoic acid (NPC 12626), was evaluated for activity in tests associated with receptors for excitatory amino acids. In receptor binding assays, NPC 17742 was selective for the N-methyl-D-aspartate (NMDA) receptor with a potency comparable to that of D(-, -3-(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid. Like (+/-)cis-4-phosphono-methyl-2-piperidine carboxylic acid (CGS 19755) and (+/-)(E)-2-amino-4-methyl-5-phosphono-3-penteneoic acid (CGP 37849), NPC 17742 competitively inhibited NMDA-induced enhancement of 1-[(2-thienyl)cyclohexyl]piperidine binding to the NMDA receptor ionophore and partially inhibited [3H]glycine binding to strychnine-insensitive sites. In contrast, NPC 17742 and CGP 37849 inhibited Mg(++)-stimulated 1-[(2-thienyl)cyclohexyl]piperidine binding in a noncompetitive fashion. In voltage-clamped Xenopus oocytes expressing excitatory amino acid receptors, NPC 17742 (pKB = 6.91) was equipotent with CGP 37849 (pKB = 7.17) in inhibiting NMDA-induced inward currents. Likewise, NPC 17742 (ED50 = 2.68 mg/kg) was equipotent with CGP 37849 and CGS 19755 in blocking NMDA-induced convulsions, but was less potent than these two compounds in the maximal electroshock test. Unlike CGP 37849 or CGS 19755, NPC 17742 potently antagonized seizures induced by pentylenetetrazol. In a model of global ischemia, low doses of NPC 17742 given either before or after ischemic result were effective in blocking damage to hippocampal CA1 neurons. The pharmacologic responses to NPC 17742 occurred at doses 30- to 300-fold lower than the acute lethal dose.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acids↗
Effect of bovine prothrombin fragment 1 on the translational diffusion of phospholipids in Langmuir-Blodgett monolayers.
Previous work has shown that bovine prothrombin fragment 1 binds to supported planar membranes composed of phosphatidylcholine and phosphatidylserine in a Ca(2+)-specific manner (Tendian et al. (1991) Biochemistry 30, 10991; Pearce et al. (1992) Biochemistry 31, 5983-5995). In the present work, fluorescence pattern photobleaching recovery has been used to examine the effect of membrane-bound fragment 1 on the translational diffusion coefficients of two fluorescent phospholipids in fluid-like phosphatidylserine/phosphatidylcholine Langmuir-Blodgett monolayers. The results show that saturating concentrations of fragment 1, in the presence of Ca2+, reduce the diffusion coefficient of nitrobenzoxadiazolyl-conjugated phosphatidylserine (NBD-PS) and nitrobenzoxadiazolyl-conjugated phosphatidylcholine (NBD-PC) by factors of approximately four and two, respectively. Ca2+ or fragment 1 alone do not have a statistically significant effect on NBD-PS or NBD-PC diffusion. In addition, a nonspecific protein (ovalbumin) does not change the diffusion coefficients of the fluorescent phospholipids either in the absence or presence of Ca2+. The fractions of the fluorescent phospholipids that are laterally mobile are approximately 0.9 for all samples. These results are interpreted with several models for possible mechanisms by which extrinsically bound proteins might retard phospholipid diffusion in membranes.
4-Chloro-7-nitrobenzofurazan↗
UK-14,304, R(-)-alpha-methyl-histamine and SMS 201-995 block plasma protein leakage within dura mater by prejunctional mechanisms.
Intravenous administration of an alpha-adrenoceptor agonist, UK-14,304, a histamine H3 receptor agonist, R(-)-alpha-methyl-histamine (alpha-MeHA) or SMS 201-995 (a synthetic octapeptide analogue of somatostatin), blocked plasma protein (125I-albumin) extravasation within rat and/or guinea pig dura mater following unilateral electrical trigeminal ganglion stimulation or capsaicin administration. The extravasation caused by the administration of the neuropeptide mediator, substance P, was not inhibited by any of the three compounds. Blockade by UK-14,304 was completely antagonized by pretreatment with the highly selective alpha 2-antagonist, idazoxan, as was alpha-MeHA by pretreatment with the highly selective histamine H3 antagonist, thioperamide. Taken together, the results are consistent with blockade by prejunctional alpha 2, histamine H3 and probably somatostatin receptors which may be coupled to inhibition of neuropeptide release. Because 5-HT1-like agonists, which are useful for treating migraine and related headaches, share similar inhibitory properties in this in vivo model, the significance of prejunctional alpha 2, histamine H3 and somatostatin receptors to treatment of vascular headaches is suggested.
Adrenergic alpha-Agonists↗
Anomalous discrete symmetry.
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Journal Article↗