Biomedical subjects

Z Hu

Publications and source records attributed to Z Hu.

At least 19 recordsLinked to original sources

PubMed1999

A new antifungal peptide from the seeds of Phytolacca americana: characterization, amino acid sequence and cDNA cloning.

An antifungal peptide from seeds of Phytolacca americana, designated PAFP-s, has been isolated. The peptide is highly basic and consists of 38 residues with three disulfide bridges. Its molecular mass of 3929.0 was determined by mass spectrometry. The complete amino acid sequence was obtained from automated Edman degradation, and cDNA cloning was successfully performed by 3'-RACE. The deduced amino acid sequence of a partial cDNA corresponded to the amino acid sequence from chemical sequencing. PAFP-s exhibited a broad spectrum of antifungal activity, and its activities differed among various fungi. PAFP-s displayed no inhibitory activity towards Escherichia coli. PAFP-s shows significant sequence similarities and the same cysteine motif with Mj-AMPs, antimicrobial peptides from seeds of Mirabilis jalapa belonging to the knottin-type antimicrobial peptide.

Amino Acid Sequence↗

PubMed1999

The contribution of adjacent subunits to the active sites of D-3-phosphoglycerate dehydrogenase.

D-3-Phosphoglycerate dehydrogenase (PGDH) from Escherichia coli is allosterically inhibited by L-serine, the end product of its metabolic pathway. Previous results have shown that inhibition by serine has a large effect on Vmax and only a small or negligible effect on Km. PGDH is thus classified as a V-type allosteric enzyme. In this study, the active site of PGDH has been studied by site-directed mutagenesis to assess the role of certain residues in substrate binding and catalysis. These consist of a group of cationic residues (Arg-240, Arg-60, Arg-62, Lys-39, and Lys-141') that potentially form an electrostatic environment for the binding of the negatively charged substrate, as well as the only tryptophan residue found in PGDH and which fits into a hydrophobic pocket immediately adjacent to the active site histidine residue. Interestingly, Trp-139' and Lys-141' are part of the polypeptide chain of the subunit that is adjacent to the active site. The results of mutating these residues show that Arg-240, Arg-60, Arg-62, and Lys-141' play distinct roles in the binding of the substrate to the active site. Mutants of Trp-139' show that this residue may play a role in stabilizing the catalytic center of the enzyme. Furthermore, these mutants appear to have a significant effect on the cooperativity of serine inhibition and suggest a possible role for Trp-139' in the cooperative interactions between subunits.

Amino Acid Substitution↗

PubMed1999

Development of tissue-simulating optical phantoms: poly-N-isopropylacrylamide solution entrapped inside a hydrogel.

The average turbid optical properties of the N-isopropylacrylamide (NIPA) polymer solution entrapped inside a polyacrylamide hydrogel (called an NIPA/PAAM gel system) were studied using a multiwavelength oblique-incidence reflectometer. The turbidity of such a system can be drastically changed by simply switching the temperature from below the low critical solution temperature of the NIPA, around 33 degrees C, to above. The absorption coefficient and the reduced scattering coefficient were obtained as a function of wavelength for samples with selected NIPA and blue dextran concentrations. It is found that the scattering of the optical phantom comes from the NIPA polymer chains and the absorption from the blue dextran. The turbid optical properties of an NIPA/PAAM gel system can be tuned to simulate biological tissues at a specific wavelength by varying compositions of NIPA and blue dextran and further modified by controlling the temperature.

Acrylic Resins↗

PubMed1999

Efficiency of leukocyte removal by filters made of superfine glass fiber membranes.

BACKGROUND AND OBJECTIVES: To demonstrate the application of leukocyte removal filters made of a new type of filter material - superfine glass fiber - for depleting leukocytes in SAGM red cell suspensions and preventing nonhemolytic transfusion reactions. MATERIALS AND METHODS: The extent of leukocyte depletion and red cell recovery was based on cell counts. Trace leukocytes were counted in a 50-microl Nageotte counting chamber or by using a flow cytometer. The chemical stability of the glass fiber membranes was studied by plasma emission spectrometer and by measuring the ion content and weighing nonvolatile matter in water extract. The structural stability of the glass fiber membranes was studied by a micropore-filter membrane method. RESULTS: Leukocyte removal filters made of superfine glass fiber membranes removed more than 99.0% of leukocytes in SAGM red cell suspensions prepared from 400 ml whole blood. Red cell recovery exceeded 90%, and the total number of residual leukocytes was less than 5x10(6). A water extract of the glass fiber membranes contained only traces of Si4+ and Ca2+ and less than 2 mg/100 ml of nonvolatile matter. No broken or loose fibers were found in the filters. Scanning electron microscopy showed that the web structure of the glass fiber membranes was instrumental in trapping and holding leukocytes. CONCLUSION: A filter made of glass fiber membranes is effective in leukocyte depletion.

Adenine↗

PubMed1998

New preparation method of intestinal pressure-controlled colon delivery capsules by coating machine and evaluation in beagle dogs.

A new method for preparing large amounts of pressure-controlled colon delivery capsules (PCDCs) which employs a pharmaceutical coating machine, Hicoater-mini, has been developed. In contrast to our original method for preparing PCDCs where the inner surfaces of gelatin capsule were coated with the water-insoluble polymer ethylcellulose (EC), PCDC were directly prepared by coating the capsular shaped suppositories with EC. As a model drug, fluorescein (FL) was used in this study. FL powder was suspended with the suppository base, polyethylene glycol (PEG) 1000, at 50 degreesC, and was hardened in the capsular shape the sizes of which were #0 and #2. The capsular shaped suppositories were coated with 5% w/v ethanolic EC (7G grade) solution by a coating machine. By increasing the coating time from 55 to 75 min, the mean coating thickness of #0 PCDCs increased from 141+/-7 to 211+/-4 micrometer. In the case of #2 PDDCs, the mean coating thickness increased from 102+/-3 to 110+/-5 micrometer by increasing the coating time from 35 min to 40 min. Several kinds of #0 PCDCs having the mean EC coating membrane thickness of 141+/-7 micrometer (type 1), 166+/-4 micrometer (type 2), 188+/-4 micrometer (type 3), 211+/-4 micrometer (type 4) as well as #2 PCDCs having thickness of 102+/-3 micrometer (type 5) and 110+/-5 micrometer (type 6) were used for in vivo evaluation using beagle dogs. After oral administration of the test preparations containing 30 mg of FL, blood samples were obtained from the jugular vein and plasma FL levels were measured. The first appearance time, Ti, of FL in the plasma was used as a parameter for the estimation of the release time of FL from PCDCs in the gastrointestinal tract. The mean Ti of #0 PCDCs were 2.3+/-0.5 for type 1, 3.3+/-0.5 for type 2, 4.8+/-1.0 for type 3 and 7.8+/-1.7 h for type 4 preparations while the mean Ti of #2 PCDCs were 3.2+/-0.4 for type 5 and 3.8+/-0.4 h for type 6, respectively. There were good correlations between EC coatings.

Animals↗

PubMed1998

Protein trans-splicing by a split intein encoded in a split DnaE gene of Synechocystis sp. PCC6803.

A split intein capable of protein trans-splicing is identified in a DnaE protein of the cyanobacterium Synechocystis sp. strain PCC6803. The N- and C-terminal halves of DnaE (catalytic subunit alpha of DNA polymerase III) are encoded by two separate genes, dnaE-n and dnaE-c, respectively. These two genes are located 745,226 bp apart in the genome and on opposite DNA strands. The dnaE-n product consists of a N-extein sequence followed by a 123-aa intein sequence, whereas the dnaE-c product consists of a 36-aa intein sequence followed by a C-extein sequence. The N- and C-extein sequences together reconstitute a complete DnaE sequence that is interrupted by the intein sequences inside the beta- and tau-binding domains. The two intein sequences together reconstitute a split mini-intein that not only has intein-like sequence features but also exhibited protein trans-splicing activity when tested in Escherichia coli cells.

Amino Acid Sequence↗

PubMed1998

Direct Observation of Polymer Network Structure in Macroporous N-Isopropylacrylamide Gel by Raman Microscopy.

The Raman microscopy technique is used to characterize the temperature-induced evolution of the pore structures of a macroporous N-isopropylacrylamide (NIPA). The gel is synthesized using a suspension of 45 wt % toluene and 55 wt % pregel NIPA solution. The intensity of the band due to the CH2 bending vibration, centered at 1445 cm-1, is used to monitor the distribution of the polymer chain density in the lateral plane. It is found that the macroporous gel consists of water-rich areas, which can be interpreted as the pores, and polymer-rich areas. At room temperature, the average sizes of the pores and the width of polymer-rich areas are 75 µm and 20 µm, respectively. Both the pores and their surrounding polymer-rich areas have random geometry, as demonstrated by the Raman microimaging. With increased temperature the size of the pores decreases. This process is accompanied by a narrowing of the polymer-rich areas. At higher temperatures polymer chains bunch together and this process accelerates rapidly near the volume phase transition temperature (34 degreesC). Above 36 degreesC, the pore sizes become too small to be resolved using Raman microscopy.

Journal Article↗

PubMed1998

Cloning and expression of a group IV cytosolic Ca2+-dependent phospholipase A2 from rat pancreatic islets. Comparison of the expressed activity with that of an islet group VI cytosolic Ca2+-independent phospholipase A2.

Stimulation of pancreatic islets with glucose induces phospholipid hydrolysis and accumulation of nonesterified arachidonic acid, which may play signaling or effector roles in insulin secretion. Of enzymes that catalyze phospholipid hydrolysis, islet beta-cells express low molecular weight secretory phospholipases A2 (PLA2) and a Group VI, Ca2+-independent PLA2 (iPLA2). Previous studies indicate that islets also express a protein recognized by antibodies against a Group IV, cytosolic, Ca2+-dependent PLA2 (cPLA2). To further examine the possible expression of cPLA2 by islets, we screened a rat islet cDNA library with a probe that recognizes cPLA2 sequence, and isolated a full-length cPLA2 cDNA. The rat islet cPLA2-deduced amino acid sequence is 96% identical to those of human and mouse cPLA2. Transfection of COS-7 cells with cPLA2 cDNA in an expression vector induced expression of Ca2+-dependent PLA2 activity and of a protein recognized by anti-cPLA2 antibody. Comparison of recombinant islet cPLA2 and iPLA2 activities expressed in transfected COS-7 cells indicated that iPLA2 but not cPLA2 is stimulated by ATP. Both activities are similarly sensitive to inhibition by arachidonyltrifluoromethyl ketone, but iPLA2 is more effectively inhibited by a haloenol lactone suicide substrate than cPLA2. RT-PCR experiments with RNA from purified islet beta-cells and from an alpha-cell-enriched population prepared by fluorescence-activated cell-sorting indicated that cPLA2 mRNA is more abundant in the beta-cell population. Immunoblotting analyses indicate that islets express cPLA2-immunoreactive protein, and that interleukin-1 does not affect its expression. The cPLA2 is thus one of at least three classes of PLA2 enzymes with distinct properties expressed in beta-cells.

Amino Acid Sequence↗

PubMed1998

A topA intein in Pyrococcus furiosus and its relatedness to the r-gyr intein of Methanococcus jannaschii.

A new intein coding sequence was found in a topA (DNA topoisomerase I) gene by cloning and sequencing this gene from the hyperthermophilic Archaeon Pyrococcus furiosus. The predicted Pfu topA intein sequence is 373 amino acids long and located two residues away from the catalytic tyrosine of the topoisomerase. It contains putative intein sequence blocks (C, E, and H) associated with intein endonuclease activity, in addition to intein sequence blocks (A, B, F, and G) that are necessary for protein splicing. This DNA topoisomerase I intein is most related to a reverse gyrase intein from the methanogenic Archaeon Methanococcus jannaschii. These two inteins share 31% amino acid sequence identity and, more importantly, have the same insertion sites in their respective host proteins. It is suggested that these two inteins are homologous inteins present in structurally related, but functionally distinct, proteins, with implications on intein evolution and intein homing.

Amino Acid Sequence↗

PubMed1998

Beta3 integrins are upregulated after vascular injury and modulate thrombospondin- and thrombin-induced proliferation of cultured smooth muscle cells.

BACKGROUND: Treatment with an antibody that binds beta3 integrins (abciximab; c7E3 Fab) at the time of coronary angioplasty decreases the need for repeat revascularization. Two potential mechanisms have been proposed to explain this effect: (1) inhibition of platelet aggregation or (2) interruption of ligand binding to beta3 integrins on the smooth muscle cell (SMC) surface. We examined the latter hypothesis by determining (1) if beta3 integrin expression is upregulated after vascular injury in the baboon, (2) if 7E3 binds beta3 integrins on cultured SMC, and (3) if beta3 integrin activation plays a role in proliferation of cultured SMC. METHODS AND RESULTS: Results demonstrated that immunostaining for beta3 integrins was present in the neointima 1 week after balloon withdrawal injury of baboon brachial arteries and that beta3 integrin expression colocalized with alpha-actin-positive cells. In contrast, staining for beta3 integrins was undetectable in contralateral uninjured brachial arteries. 7E3 bound to cultured human aortic SMC with an affinity (KD=3.3 nmol/L) similar to 7E3 binding to endothelial cells or platelets. Cotreatment with 7E3 partially inhibited thrombospondin-induced or alpha-thrombin-induced proliferation but not PDGF-induced or serum-induced proliferation. CONCLUSIONS: In summary, these studies demonstrate that vascular cell beta3 integrin expression is increased after injury, that 7E3 binds to cultured SMC with high affinity, and that beta3 activation is important for thrombospondin-induced or alpha-thrombin-induced proliferation. These results support the hypothesis that beta3 integrins play a role in SMC growth responses after balloon injury.

Abciximab↗

PubMed1998

Evaluation of intestinal pressure-controlled colon delivery capsule containing caffeine as a model drug in human volunteers.

The delivery ability of a pressure-controlled colon delivery capsule (PCDC) containing caffeine as a test drug was evaluated after oral administration to healthy male human volunteers. The driving force causing PCDC disintegration in the intestinal tract is the physiological luminal pressure which results from peristalsis. Three kinds of PCDCs having different thickness of a water-insoluble polymer membrane was prepared by coating the inner surface of the gelatin capsules with ethylcellulose (EC). The mean thickness were 40 +/- 1 (SE) for type 1, 44 +/- 1 for type 2 and 50 +/- 1 micron for type 3 PCDC, respectively. Caffeine was dissolved with a suppository base (PEGs 400 and 1000) and the capsules were filled. Doses were 15, 45 or 75 mg. After blank saliva samples were obtained, test preparations were orally administered to the volunteers and saliva samples were collected for 1 min intervals hourly from 1 to 10 h in the fasted state study, and from 1 to 20 h and at 25 h in the fed state study. Caffeine concentrations in the saliva samples were analyzed by HPLC. The maximum salivary caffeine excretion rate increased as the oral caffeine dose increased. The maximum salivary caffeine excretion rate increased predominantly compared to the pre-dose level in 75 mg dose study. Therefore, all following studies were performed with this dose. The first appearance time of caffeine into the saliva, TI, was used as a parameter to estimate the disintegration time of test preparations in the gastrointestinal tract. The mean TI of types 1, 2, and 3 PCDCs were 3.0 +/- 0.4, 4.0 +/- 0.4 and 4.5 +/- 0.3 h, respectively. After oral administration of 75 mg caffeine in pain gelatin capsule as a reference preparation, caffeine appeared in the saliva within 0.5 h. The mean hardness of the PCDCs were 1.05 +/- 0.10 (type 1), 1.55 +/- 0.06 (type 2) and 2.08 +/- 0.15 newton (type 3), respectively. There were good correlations between three parameters: EC coating membrane thickness, hardness and TI (determination coefficient r2 = 0.935 between TI and thickness, r2 = 0.998 between thickness and hardness, r2 = 0.958 between hardness and TI). The effect of food intake on the delivery ability was examined with type 3 PCDCs. Food intake prolonged the mean TI, from 4.5 +/- 0.3 to 7.8 +/- 1.3 h. This increase is thought to be ascribed to prolonged gastric emptying time. Comparison with reported colon arrival times indicates that the type 3 PCDC functions in colon delivery of caffeine and is thought to be applicable to other drugs.

Adult↗

PubMed1998

Design and synthesis of monocyclic beta-lactams as mechanism-based inhibitors of human cytomegalovirus protease.

Mechanism based inhibitors of HCMV protease have been designed based on the monocyclic beta-lactam nucleus, which have been shown to acylate the viral enzyme in a time dependent manner. SAR in a series of monocyclic beta-lactam N-ureas, has defined the size and relative stereochemistry of the C-3 substituent producing a low micromolar inhibitor 17b with good aqueous stability and selectivity over the mammalian serine proteases.

Drug Stability↗

PubMed1998

Prenatal expression of inwardly rectifying potassium channel mRNA (Kir4.1) in rat brain.

The levels and cellular localization of the mRNA encoding the inwardly rectifying potassium ion channel Kir4.1 were investigated in the embryonic rat brain by Northern blots and in situ hybridization. This transcript was absent at embryonic day 13 (E13), whereas it was clearly present in E14-15 preparations, principally in the neuroepithelium of the cerebral cortex, thalamus, and hypothalamus. At later embryonic stages (E17-20), Kir4.1 mRNA levels increased and expanded to the mantle zone, such as the cortical plate, hippocampus, thalamus, and hypothalamus. The early appearance of Kir4.1 mRNA in various brain regions suggests an involvement of the channel in cell proliferation, migration and differentiation in the rat CNS.

Animals↗

PubMed1998

Crystallization and preliminary crystallographic analyses of pokeweed antiviral protein from seeds.

Pokeweed antiviral protein from seeds (PAP-S) is a ribosome inactivating protein which has lowest toxicity and highest inhibition activity as opposed to other pokeweed antiviral proteins and its three potential glycosylation sites (10, 44, 255) were shown to bind to N-acetylglucosamine. Good quality crystals of PAP-S were grown at high protein concentration (100 mg ml-1) and high temperature (306 K). The crystals have space group I222 and cell parameters a = 78.7, b = 85.2 and c = 93.0 A. An X-ray diffraction data set with resolution up to 1.8 A was collected. This high-resolution data will help to locate the sugars bound to the protein and provide accurate structural data for understanding structure-function relationships of PAP-S.

Antiviral Agents↗

PubMed1998

In vivo time course of morphological changes and DNA degradation during the degeneration of castration-induced apoptotic prostate cells.

The in vivo time course of the morphological changes and DNA degradation in castration-induced apoptotic prostate cells was studied from the earliest to the latest stage of the degeneration process. To study this problem, we first induced apoptotic prostate cells in rats by castration for 3 days and then promptly and continuously blocked the death of healthy prostatic cells in the castrated rats by in vivo testosterone replacement. Because testosterone replacement could not stop the irreversible lysis of already damaged prostate cells, apoptotic cells at different stages of the degeneration process were eliminated sequentially from the prostate after the healthy prostate cells had been protected. Prostate cells at the earliest stage of apoptosis at the time when the castrated rats received testosterone replacement disappeared last. By tracing the morphological and DNA degradation of apoptotic cells after hormone treatment, we estimated the time course of prostate cell death from the early to the final stage. In the morphological evolution of apoptotic prostate cells, the clumping of nuclear chromatin, the degeneration of cytoplasm and the involution of the cell surface occurred and progressed simultaneously, resulting in the rapid formation of apoptotic bodies that were gradually digested by other cells. The DNA ladders of apoptotic cells were progressively cleaved into a mononucleosomal subunit that was further degraded at an additional site, generating a heterogeneous population of small nucleotides. The final digestion of DNA fragments occurred within the apoptotic bodies. The whole course of prostate cell death after castration took about 44 h.

Animals↗

PubMed1998

Steroid hormones and their receptors in the brain.

Steroid hormones regulate several important functions of the brain by altering the expression of particular genes through their receptors. First in this paper the localization of glucocorticoid receptor immunoreactivity and mRNA in the brain was examined. Second biphasic effects of glucocorticoid on the hippocampus was described and particular emphasis was given on the apoptosis. Third the significance of estrogen receptor in the sexually dimorphic areas was discussed. These results suggest that steroids modulate the gene expression along with the alteration of cell structures in a different manner in a tissue-specific pattern.

Animals↗

PubMed1998

The single-nucleocapsid nucleopolyhedrovirus of Buzura suppressaria encodes a P10 protein.

The p10 gene of Buzura suppressaria single-nucleocapsid nucleopolyhedrovirus (BusuNPV) was identified by virtue of its localization downstream from the Autographa californica (Ac) MNPV p26 homologue. The BusuNPV p10 gene encodes a protein of 94 amino acids. The amino acid sequence contains domains characteristic of baculovirus P10 proteins, e.g. a coiled-coil domain, a proline-rich motif and a positively charged C terminus. The highest amino acid homologies were found with the Spodoptera littoralis (Spli) NPV and Spodoptera exigua (Se) MNPV P10 proteins. An AcMNPV recombinant expressing the BusuNPV P10 formed fibrillar structures in the cytoplasm of Spodoptera frugiperda cells. BusuNPV P10 could not fully replace AcMNPV P10 in its nuclear disintegration function, since polyhedra were not efficiently liberated from infected cells late in infection. The BusuNPV p26 gene encodes a protein of 263 amino acid residues with 70% amino acid similarity with SeMNPV P26. Downstream of the BusuNPV p10 gene, the gene for the occlusion-derived virus protein ODVP-6e is located. This is unlike the situation in many other NPVs, including SeMNPV, where the p10 gene neighbours the p74 gene. The data presented here suggest that although the p10 gene is not conserved in sequence, evolutionary pressure preserves the structure of P10 and hence its function. These data also indicate that all NPVs, MNPVs as well as SNPVs, contain this gene.

Amino Acid Sequence↗

PubMed1998

Altered growth of transgenic tobacco lacking leaf cytosolic pyruvate kinase.

Previously, we reported that transformation of tobacco (Nicotiana tabacum L.) with a vector containing a potato cytosolic pyruvate kinase (PKc) cDNA generated two plant lines specifically lacking leaf PKc (PKc-) as a result of co-suppression. PKc deficiency in these primary transformants did not appear to alter plant development, although root growth was not examined. Here we report a striking reduction in root growth of homozygous progeny of both PKc- lines throughout development under moderate (600 microE m-2 s-1) or low (100 microE m-2 s-1) light intensities. When both PKc- lines were cultivated under low light, shoot and flower development were also delayed and leaf indentations were apparent. Leaf PK activity in the transformants was significantly decreased at all time points examined, whereas root activities were unaffected. Polypeptides corresponding to PKc were undetectable on immunoblots of PKc- leaf extracts, except in 6-week-old low-light-grown PKc- plants, in which leaf PKc expression appeared to be greatly reduced. The metabolic implications of the kinetic characteristics of partially purified PKc from wild-type tobacco leaves are discussed. Overall, the results suggest that leaf PKc deficiency leads to a perturbation in source-sink relationships.

Cytosol↗