Studies on adrenomedullin: a novel hypotensive peptide.
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Adrenomedullin↗
Biomedical subjects
J Tang
Publications and source records attributed to J Tang.
[Observational and experimental studies on relationship between domestic pigs and epidemic hemorrhagic fever investigation].
Observational and experimental studies on the relationship between domestic pigs and epidemic hemorrhagic fever (EHF) were carried out during 1986 to 1994 with reverse passive hemagglutination (RPHA), reverse passive hemagglutination inhibition (RPHI), horse-radish peroxidase-staphylococcal protein A (HRP-SPA), indirect immunofluorescence assay (IFA) and reverse transcript polymerase chain reaction (RT-PCR) to understand the roles of domestic pigs played in the epidemic areas. Results showed domestic pigs could infect epidemic hemorrhagic fever virus (EHFV) naturally or experimentally, and EHFV could disseminate to many organs of the animals and caused transient pathologic changes in them. EHFV could duplicate within the animal bodies and be excreted out of the bodies with their excreta. Proportion of inapparent infection of EHF in people closely exposed to domestic pigs was significantly higher than in those without it. It indicated domestic pigs maybe played an important role in EHF as a reservoir host.
Animals↗
[Angiotensin II increasing the inhibitory effects of electroacupuncture on the nociceptive discharge of neurons in the spinal dorsal horn].
Experiments were performed on 53 rats. The nociceptive discharges of spinal dorsal horn neurons were extracellularly recorded by glass microelectrodes and tested for the effects of angiotensin II (A II) on the nociceptive discharges of spinal dorsal horn neurons and AII on the inhibitory effects of electroacupuncture (EA). The results showed as follows: (a) The nociceptive discharges of spinal dorsal horn neurons might be inhibited (25/35) or facilitated (10) with A II (50-500 ng/20 ul) applied to the surface of dorsal spinal cord (L2-3); (b) The facilitory effects (5/5) were observed with A II (2 ug/20 ul) applied to the surface of dorsal spinal cord; (c) The inhibitory effects of EA (20 Hz, 2-4 V, 5 min) could be increased with A II (250 mg/20 ul, 8/9). Therefore, A II may modulate the nociceptive response of spinal dorsal horn neurons and influence the effects of EA, and different effects can be produced when the different dosage of A II is applied to the surface of dorsal horn.
Angiotensin II↗
[Flexor tendon repair: timing of surgery and sheath management].
Effect of timing of repair and management of sheath on functions of injuried tendons was evaluated in a clinical relevant model using 60 white leghorn chickens. The flexor digitorum profundus (FDP) tendons of the long toes were cut and were repaired at 1, 4, 8, 14 and 20 days after injury. The sheath on the left foot was closed and that on the right excised. The tendon excursion, toe motion, morphology of the sheath and tendon healing were studied 6 weeks after tendon repair. Tendon functions were the best in primary repair, and they got worse as the delay became longer. Repair in the late stage produced significantly worse tendon functions. Sheath closure with remarkable tension worsened tendon healing. We conclude that the optimal timing for tendon repair is at the primary stage. Delayed tendon repair can be performed within 3 weeks to one month after injury, but the repair is preferably done in initial several days of delay. Direct sheath closure is not advocated in tendon repair in the delayed period.
Animals↗
Interference of electromagnetic waves in dynamic metabolism.
Life is a continuous process of the dynamic metabolism. The influence of electromagnetic waves on the process of metabolism cannot be neglected. Here a new theory of electromagnetic interference in the dynamic metabolism of life is proposed. The statistical dynamic equations of ion and free radical in the biochemical reaction radiated by electromagnetic waves are given. The intensity of electromagnetic interference could be described with an interference factor. Good agreement can be seen between the calculated and measured results for a famous experiment of radio-frequency radiation-induced calcium ion efflux enhancement.
Animals↗
Discriminating PCR artifacts using directed heteroduplex analysis (DHDA).
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Animals↗
Technical report. Optimizing probe selection in directed heteroduplex analysis using HDprobe 1.1.
Directed heteroduplex analysis (DHDA) has proven to be a powerful technique for rapid geotyping in human populations. This strategy should also have widespread utility in differentiating closely related organisms of medical and public health importance through identification of DNA sequence polymorphisms. Identifying an optimal probe sequence for use in DHDA has required empirical testing of both the positive and negative strands of a number of potential probes. To identify optimal probes more efficiently, a computer program has been developed that predicts the number of potential stable and unstable mismatches between a probe and its target sequences in DHDA. This information can then be used to predict--from among a group of potential probes--which one will be the most successful in differentiating closely related homologues of a targeted gene sequence. This approach was tested on a number of probe and target sequences derived from the mitochondrial NADH dehydrogenase subunit 4 gene of the West African black fly, Simulium damnosum sensu lato. The number of unstable mismatches predicted to occur in a given heteroduplex by the computer program was found to be important in differentiating closely related species. Therefore, this strategy is useful in identifying informative probes in the development of new DHDA-based assays.
Animals↗
Antisense oligodeoxynucleotide phosphorothioate complementary to Gag mRNA blocks replication of human immunodeficiency virus type 1 in human peripheral blood cells.
Gene-expression modulator 91 (GEM91) is a 25-nt antisense oligodeoxynucleotide phosphorothioate complementary to the Gag mRNA of human immunodeficiency virus type 1 (HIV-1). Cellular uptake and intracellular distribution of GEM91 within cells suggest that this oligomer is readily available for antisense activity. GEM91 inhibited HIV-1 replication in a dose-dependent and sequence-specific manner. In a comparative study, 2 microM GEM91 was as effective as 5 microM 3'-azido-3'-deoxythymidine in blocking virus replication during the 28-day treatment of an HIV-1-infected T-cell line. GEM91 also completely inhibited (> 99%) the growth of three different HIV-1 isolates in primary lymphocytes and prevented the cytopathic effect of the virus in primary CD4+ T cells. Similarly, treatment with GEM91 for 3 weeks of HIV-1/BaL-infected primary macrophages blocked virus replication. Based on GEM91 anti-HIV-activity, safety, and pharmacokinetic profile in animals, a clinical trial was started using this compound as an antisense oligonucleotide drug for the treatment of the acquired immunodeficiency syndrome.
Antigens, CD↗
Proline-rich domain and glycosylation are not essential for the enzymic activity of bile salt-activated lipase. Kinetic studies of T-BAL, a truncated form of the enzyme, expressed in Escherichia coli.
We have expressed and purified a truncated recombinant human milk bile salt-activated lipase (T-BAL) from the T7 expression system in Escherichia coli. This T-BAL contains the N-terminal 538 residues of the 722-residue native enzyme. The purified T-BAL, when assayed with PANA (p-nitrophenyl acetate), had a specific activity of 64 +/- 2 units/mg (n = 4), as compared to 52 units/mg for the native enzyme. Because the recombinant T-BAL expressed in E. coli is not glycosylated, these results indicated that the highly glycosylated C-terminal region of BAL is not essential for catalytic function. Heat inactivation patterns of native BAL and T-BAL were found to be similar, further suggesting that the folding of T-BAL is similar to that of the catalytic domain of the native enzyme. With the availability of a sufficient amount of recombinant T-BAL, the specificity and kinetics of T-BAL and native BAL were compared. Fluorescence studies of T-BAL indicated that it has a slightly higher affinity for the monomeric form of taurocholate with a dissociation constant (KA) of 0.32 mM, compared with the reported 0.37 mM for the native enzyme. Further kinetic analysis indicated that there are enzyme specificity changes revealed with the use of PANA and PANB (p-nitrophenyl butyrate) as substrates. When assayed in the presence of taurocholate, T-BAL has a higher turnover rate constant with p-nitrophenyl acetate than with p-nitrophenyl butyrate, which was found to be in contrast to native BAL.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Membrane association and oligomeric organization of the alpha and beta subunits of mouse meprin A.
Meprins are oligomeric cell surface metalloproteinases of the "astacin family." They consist of two types of subunits (alpha and beta), which are evolutionarily related and whose cDNA sequences predict a similar domain structure. The present work shows that reducing agents solubilized meprin alpha subunits (approximately 90%), but not beta subunits, from mouse kidney brush border membranes. In addition, immunoblotting of membranes or purified meprins with an antibody raised to the alpha subunit epidermal growth factor-like domain, predicted to be near the COOH terminus from the cDNA-deduced amino acid sequence, indicated that this domain is not present in the mature alpha subunit. By contrast, an epitope predicted to be near the COOH terminus of the beta subunit was present in the mature form of beta. When meprins were solubilized from brush border membranes by papain, the size of the alpha subunit (approximately 90 kDa) did not change, while the beta subunit decreased from 110 to 90 kDa with concomitant loss of the COOH-terminal epitope. These data indicate that beta is a type I transmembrane protein, while alpha does not transverse the membrane and its association is dependent on disulfide bonds. The oligomeric organization of purified meprin A (EC 3.4.24.18), examined by sedimentation equilibrium analysis and native gradient gel electrophoresis, is that of disulfide-bridged dimers which aggregate noncovalently to form higher molecular weight complexes, predominantly tetramers. Western blotting of ICR kidney brush border membrane proteins identified alpha 2 homodimers and alpha beta heterodimers. Treatment of mouse or rat kidney brush border membranes with 7 M urea solubilized alpha 2, but not alpha beta dimers. Thus, the mature alpha subunit exists in alpha 2 and alpha beta disulfide-linked dimers which form tetramers, and the alpha 2 homodimers associate with the membrane through noncovalent interactions with alpha beta.
Amino Acid Sequence↗
Proteolytic processing mechanisms of a miniprecursor of the aspartic protease of human immunodeficiency virus type 1.
The infectivity of the human immunodeficiency virus (HIV) depends upon correct proteolytic processing of viral polyprotein precursors, the Pr55gag and Pr160gag-pol polyproteins. The processing is mediated spontaneously by the viral protease unit (PR) contained within the Pr160gag-pol precursor. However, little is known about the mechanism of this process. The expression in Escherichia coli and the isolation of a 14-kDa HIV-1 PR "miniprecursor" with Ala28 mutated to serine has permitted study of the mechanism for cleavage at the N-terminus of the protease. The miniprecursor is active against a synthetic peptide substrate, and its specific activity is near that of the mutant mature protease. The rate of conversion of radiolabeled precursor to mature protease is quantitated by measuring the amounts of the two radiolabeled proteins separated by SDS-PAGE. The apparent first-order conversion rate constant, kapp, is dependent on miniprecursor concentration indicating a second-order reaction and suggesting an interdimeric processing mechanism. A significant first-order rate constant is observed when the plot of kapp versus initial precursor concentration is extrapolated to zero. This observation suggests the presence of an alternative processing mechanism involving a single active precursor dimer. The presence of both mechanisms is an advantage for the virus to ensure processing under various conditions.
Amino Acid Sequence↗
Microarterial anastomosis using a noncontact diode laser versus a control study.
A series of direct carotid end-to-end laser anastomosis vs. direct manual suture was carried out on a series of 70 Wistar rats (mean weight 260 g). Both common carotids (0.8-1.2 mm) were sectioned and repaired. The left side (n = 70) was submitted to laser-assisted microvascular anastomosis (LAMA) performed by means of a diode laser device (wavelength 830 nm and power output 3 W in continuous wave) without chromophore. The right side (n = 70) underwent a control manual suture (CMA). The diode laser energy was delivered into a micromanipulator coupled to a Zeiss operating microscope with a focused spot of 300 microns in diameter. After placement of three 10.0 stitches for edge coaptation, the LAMA was achieved using laser shots (average 3) of 500 mW power, 4.5 s duration, and 700 W/cm2 irradiance each. The CMA was performed by means of six 10.0 stitches. The good vascular flow was confirmed by Doppler spectral analysis (n = 466) carried out from day 0 to day 90. Light and scanning electron microscopy (n = 82) showed that re-endothelialization after LAMA was gaining ground on day 3, whereas collagenous network developed in the media scar by day 10. In contrast, after CMA the arterial repair was delayed on day 20, inducing a media fibrotic scar. The patency rate was 93% in both anastomoses. The shorter operating time (13 min for LAMA vs. 22 min for CMA) and the noncontact laser technique are the main intraoperative advantages. The technical benefits of the diode laser are pointed out.
Anastomosis, Surgical↗
Relationships of human immunodeficiency virus protease with eukaryotic aspartic proteases.
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Amino Acid Sequence↗
Polysialic acid regulates growth cone behavior during sorting of motor axons in the plexus region.
Removal of polysialic acid (PSA) from N-CAM during the time when chick motoneuron axons are segregating into target-specific fascicles at the base of the limb was previously shown to result in motoneuron projection errors. Here, it is established that these errors are associated with altered growth cone behavior in the plexus. In contrast to control embryos, in which individual axons were observed to exhibit dramatic changes in direction and extensive divergence, axonal trajectories following the removal of PSA were relatively straight. To determine whether enhanced axon-axon fasciculation following PSA removal had prevented growth cones from responding appropriately to guidance cues at the base of the limb, we also examined the role of L1, a major mediator of axon-axon fasciculation in this system. Anti-L1 reversed the effects of PSA removal on both growth cone trajectories and projection errors. These results indicate that PSA plays a permissive role, attenuating axon-axon interactions in the plexus and thereby allowing the axonal reorganization that is essential for the formation of specific motoneuron projections.
Animals↗
Engineering aspartic proteases to probe structure and function relationships.
Recently, protein engineering has been used to interconvert homodimeric and homologous single-chain aspartic proteases, with some success. The independent folding of the domains of these proteases has also permitted the engineering of domain-rearranged protease zymogens and the use of individual domains as probes for structural denaturation. In addition, site-directed mutagenesis has provided insights into the catalytic mechanism and specificity of this family of proteases.
Aspartic Acid Endopeptidases↗
Large-scale synthesis, purification, and analysis of oligodeoxynucleotide phosphorothioates.
Synthesis of oligonucleotides has been carried out on 1-, 2-, and 5-mmol scales using an appropriately modified automated, commercially available DNA synthesizer. The reaction cycles were optimized to obtain efficient coupling (> or = 97%). The synthesized oligonucleotide was purified by preparative reversed-phase liquid chromatography, followed by detritylation and desalting to obtain the oligonucleotides in the Na+ form. The purified oligonucleotides were characterized by 31P NMR, mass spectrometry, capillary gel electrophoresis, and ion-exchange high-performance liquid chromatography. By using these protocols, a 25-mer oligodeoxyribonucleotide can be synthesized on a 1-, 2-, or 5-mmole scale to obtain approximately 2.4, 4.8, or 12 g of purified product.
Base Sequence↗
Cellular uptake of oligodeoxynucleotide phosphorothioates and their analogs.
The uptake and intracellular distribution of oligonucleotide phosphorothioates (S-oligonucleotides) and their analogs by various cells lines were studied using internally 35S-labeled oligonucleotides. Intracellular accumulation starts in the first 2 hr and reaches a maximum at about 16 hr. A decrease of intracellular concentration of the S-oligonucleotide was observed after 16 hr, probably due to a significant efflux transport. Cellular uptake was dependent on the extracellular concentration. The intracellular concentration was significantly higher than that in the medium. The uptake and the intracellular distribution were different in the various cell lines studied. Comparative studies of the uptake of the S-oligonucleotide and various 3' end-modified S-oligonucleotides show that end modified S-oligonucleotides with a hydrophobic group significantly increases the uptake. The introduction of methylphosphonothioate linkages at the 3' end of the S-oligonucleotide also resulted in an increase in uptake.
3T3 Cells↗
Characterization and cilofungin inhibition of solubilized Aspergillus fumigatus (1,3)-beta-D-glucan synthase.
(1,3)-beta-D-Glucan synthase, a major cell wall synthesis enzyme, is the target of antifungal drugs of the lipopeptide class. Aspergillus fumigatus (1,3)-beta-D-glucan synthase was prepared and its activity was measured by incorporation of [14C]glucose from UDP-[U-14C]glucose into an insoluble polymer in the presence of alpha-amylase. Solubilization of the (1,3)-beta-D-glucan synthase was attempted with several detergents, and the maximum percent solubilization was obtained with a polyoxyethylene ether detergent, W-1. Up to 70% of enzyme activity and 50% of total protein were recovered when 1-mg/ml membrane preparations were extracted with 0.045% W-1 at 4 degrees C overnight. Confirmation of the presence of a (1,3)-beta-D-glucose polymer synthesized by this glucan synthase was done by three methods. The first was enzymatic end product degradation by alpha-amylase (no degradation) and beta-glucanase (85 to 95% degradation). The second was gas chromatography-mass spectroscopy analysis of the partially methylated alditol acetate derivatives prepared from total carbohydrate polymers present in the sample. This method identified the presence of (1,3)- and (1,2)-glucosidic linkages. The third was high-performance anion exchange chromatography of radioactive oligosaccharides. This method allowed differentiation of the newly synthesized, radioactive polymers from the contaminating carbohydrates already present in the preparation. The results showed that the polymer synthesized comprised oligosaccharides consistent with beta-(1,3)-linked sugars. Maximal inhibition of the (1,3)-beta-D-glucan synthase by the lipopeptide antifungal agent cilofungin was 80%. Dose-response experiments with this inhibitor showed that the solubilized enzyme was maximally inhibited at a cilofungin concentration of 1.25 microgram/ml and showed <5% inhibition at 0.02 microgram/ml. The apparent K(m) (K(m app)) for the solubilized glucan synthase was 400 +/- 80 microM, and the apparent K(i) (K(i app)) for cilofungin was 0.19 +/- 0.03 microM. Inhibition of A.fumigatus (1,3)-beta-D-glucan synthase with cilofungin was noncompetitive, as it was for the Candida albicans (1,3)-beta-D-glucan synthase.
Antifungal Agents↗