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Biomedical subjects

D Wang

Publications and source records attributed to D Wang.

At least 19 recordsLinked to original sources
PubMed1992

Structure of alpha 2-macroglobulin-protease complexes. Methylamine competition shows that proteases bridge two disulfide-bonded half-molecules.

alpha 2-Macroglobulin (alpha 2M) forms several different covalent complexes with proteases. These include unusual forms in which more than one of the four identical subunits of alpha 2M are cross-linked by amide bonds to more than one lysyl amino group of the bound protease. The structure of these complexes and the question of how the identical subunits are arranged to form two protease binding sites are matters of current controversy. The 185-kDa subunits are arranged into two disulfide-bonded half-molecules which are, in turn, noncovalently associated. We have provided evidence that, in the major multivalent cross-linked form, proteases can span the two half-molecules, forming a covalently bonded tetramer [Wang, D., Yuan, A. I., & Feinman, R. D. (1984) Biochemistry 23, 2807-2811]. An alternative theory has recently been proposed in which the major high molecular weight form has two bonds to protease that are within half-molecules--a multivalent cross-linked dimer [Sottrup-Jensen, L., Hansen, H. F., Pedersen, H. S., & Kristensen, L. (1990) J. Biol. Chem. 265, 17727-17737]. To resolve this conflict, experiments were carried out to determine the structure of one of the high molecular weight bands (band 3) seen on SDS-PAGE. Band 3 has anomalous migration, corresponding to markers of apparent molecular mass of 550 kDa (between the tetramer and dimer). In the experiments described here, reactions of thrombin with alpha 2M were run in the presence of methylamine, which competes for one of the two thrombin-alpha 2M covalent bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Disulfides↗
PubMed1992

Tissue-specific expression of a rat intestinal mucin-like peptide.

Expression of the gene for a rat intestinal mucin-like peptide (MLP) was studied by Northern-blot analyses of RNA prepared from a panel of rat tissues. Four probes (A-D) were constructed so as to span a 3.5 kb-long cDNA for rat MLP, and used for hybridization. Positive signals were obtained in intestine and colon, whereas lung, liver, stomach, submandibular gland and spleen were negative. The only transcript detected was approx. 9.5 kb in size. No mRNA splice variants were found. Hybridization in situ using probe B1, which corresponds to a cysteine-rich region near the C-terminus of MLP, confirmed that the gene for MLP is expressed by goblet cells of rat intestine and colon.

Animals↗
PubMed1992

Chemical evidence for the existence of activated G-actin.

Globular actin (G-actin) will polymerize to form filamentous actin (F-actin) under physiological ionic conditions, and is known to be regulated by univalent and bivalent cations, such as K+ and Mg2+. The current concept of this process involves four steps: activation, nucleation, elongation and annealing. Evidence for the existence of activated G-protein has been suggested by changes in the resistance to proteolysis [Rich & Estes (1976) J. Mol. Biol. 104, 777-792] and u.v.-light absorption [Rouayrenc & Travers (1981) Eur. J. Biochem. 116, 73-77]. More recently we [Liu et al. (1990) Biochem. J. 266, 453-459] have provided direct chemical evidence for extensive conformational changes during the transformation of G-actin into F-actin. In this study we now present direct chemical evidence for the existence of a short-lived species, an activated form of G-actin, which can be detected by changes in the accessibility of the free thiol groups on the G-actin molecule when modified by a specific thiol-group-targeted reagent, 7-dimethylamino-4-methyl-3-N-maleimidylcoumarin (DACM). The presence of K+ and/or Mg2+ ions caused a large increase in the accessibility of the thiol groups of Cys-217 and Cys-374, but not those of Cys-10 and Cys-257. Mg2+ effected relatively faster changes than did K+ ions. The results suggest that the function of these ions is to convert G-actin into an activated form, and further suggest that the change in conformation is mainly confined to the large domain. Such changes at least involve certain portions of the G-actin molecule that contain Cys-217 and Cys-374. On the other hand, little or no significant change could be observed in the small domain of G-actin as reflected by the accessibility of Cys-10. The bound nucleotide remained as ATP during the activation of G-actin and was hydrolysed to ADP on polymerization. The activated G-actin had a life-time of about 8 min or less depending on the concentration of G-actin. At higher protein concentration, its life-time was much shorter, probably owing to the earlier onset of polymerization, which apparently is governed by the concentration of the activated form. The life-time of this new species can be extended by lowering the temperature and is less affected by actin concentration. This new species is considered to be an activated form of G-actin, since polymerization renders all the thiol groups on actin inaccessible to the reagent DACM.

Actins↗
PubMed1992

A contiguous Not I restriction map of band q22.3 of human chromosome 21.

A contiguous high-resolution NotI restriction map of the distal region of the long arm of human chromosome 21 was constructed by three strategies: linking clones to identify adjacent pieces of DNA, partial digestion to identify neighboring fragments, and cell line polymorphisms to prove identity or adjacency of DNA fragments. Twenty-nine single-copy DNA probes and five linking clone probes were used to determine the order of 30 Not I fragments, covering 10 megabases of DNA in band q22.3. Smaller Not I fragments occur preferentially in this region, suggesting that band q22.3 is unusually rich in genes, since Not I sites occur almost exclusively in CpG islands. Comparison of the physical map and genetic maps in this region reveals a 10-fold higher than average recombination frequency.

Animals↗
PubMed1992

Mechanistic studies of transcription arrest at the adenovirus major late attenuation site. Comparison of purified RNA polymerase II and washed elongation complexes.

Transcription elongation in a nuclear extract in vitro is efficiently blocked by Sarkosyl at a specific site downstream of the adenovirus major late (ML) promoter at which regulated transcription arrest has also been observed in vivo. In the experiments reported here, we examined the response of the polymerase to the ML attenuation site in two assay systems: 1) purified RNA polymerase II (pol II) transcribing tailed templates and 2) elongation complexes formed on immobilized templates and then depleted of elongation factors by extensive washing. Efficient site-specific arrest occurred in both systems, demonstrating that recognition of the site is an intrinsic property of the polymerase. However, the elongation properties of washed elongation complexes and purified pol II were not equivalent. In particular, the efficiency of arrest of washed elongation complexes was influenced both by the promoter from which transcription was initiated and by DNA sequences upstream from the attenuation site that did not contribute to the arrest of purified pol II. The polymerase and washed elongation complexes both remained in stable ternary complexes at the ML site with a lifetime of hours; addition of the elongation factor SII to these complexes promoted resumption of elongation. The efficiency of arrest in both systems was dependent on the solution concentration of the nucleotide incorporated at +187 (just beyond the attenuation site), indicating that pausing is an important part of the arrest mechanism. Based on this and other findings, we argue that the polymerase assumes an altered, elongation-incompetent conformation when arrest occurs.

Adenoviridae↗
PubMed1992

Human intestinal mucin-like protein (MLP) is homologous with rat MLP in the C-terminal region, and is encoded by a gene on chromosome 11 p 15.5.

A cDNA specific for a human intestinal mucin (MLP) was amplified by PCR from cDNA of cultured human colonic adenocarcinoma cells, LS174T. The human cDNA shared high sequence homology with a corresponding rat intestinal mucin (MLP) cDNA in the 3' terminal region, and hybridized to the same mRNA (approximately 9.0 Kb) that was recognized by a probe for the MUC-2 human intestinal mucin gene. The gene encoding our human mucin peptide also mapped to chromosome 11 p 15.5, the known locus of MUC-2. Our findings suggest that human MLP and MUC-2 are encoded by the same gene and that rat and human intestinal mucin share a common C-terminal amino acid structure.

Adenocarcinoma↗
PubMed1992

cDNA for the carboxyl-terminal region of a rat intestinal mucin-like peptide.

When subjected to thiol reduction, purified intestinal mucins have been shown to undergo a decrease in molecular mass and to liberate a 118-kDa glycopeptide (Roberton, A. M., Mantle, M., Fahim, R. E. F., Specian, R., Bennick, A., Kawagishi, S., Sherman, P., and Forstner, J. F. (1989) Biochem. J. 261, 637-647). The latter has been called a putative "link" component because it is assumed to be important for disulfide bond-mediated mucin polymerization. Controversy exists as to whether the putative link is an integral mucin component or a separate mucin-associated glycopeptide. In the present study both NH2-terminal and internal amino acid sequences of the 118-kDa glycopeptide of rat intestinal mucin were used to generate opposing oligonucleotide primers for polymerase chain reaction. A specific 1.2-kilobase (kb) product was obtained, from which a 0.5-kb HindIII fragment was used as a probe to screen a lambda ZAP II cDNA library of rat intestine. A 2.6-kb cDNA (designated MLP 2677) was sequenced and revealed an open reading frame of 2.5 kb encoding 837 amino acids. The deduced amino acid sequence showed that the putative link peptide is equivalent to the carboxyl-terminal 689 amino acids of a larger peptide. Northern blots revealed a mRNA size of approximately 9 kb. Computer searches revealed no sequence homology with other proteins, but similarities were seen in the alignment of cysteine residues in the link and in several domains of human von Willebrand factor, as well as cysteine-rich areas of bovine and porcine submaxillary mucins and a frog skin mucin designated FIM-B.1. In keeping with earlier demonstrations of the presence of mannose in the 118-kDa glycopeptide, there were several (13) consensus sequences for attachment of N-linked oligosaccharides within the link domain. Further sequencing of MLP 2677 in a direction 5' to the codon specifying the NH2-terminal proline of the link has revealed a coding region for 148 amino acids, including a unique 75-amino acid domain rich in cysteine and proline, and a region containing 4.5-variable tandem repeats (each 11-12 amino acids) rich in serine, threonine, and proline. The presence of mucin-like tandem repeats suggests that the entire cysteine-rich link peptide represents the carboxyl-terminal region (75.5 kDa) of a mucin-like peptide (MLP). The latter is estimated to have a molecular mass of approximately 300 kDa.

Amino Acid Sequence↗
PubMed1992

Configurational pattern discrimination responsible for dishabituation in common toads Bufo bufo (L.): behavioral tests of the predictions of a neural model.

Recently, a neural model of visual pattern discrimination for stimulus-specific habituation was developed, based on previous behavioral studies which demonstrated that toads exhibit a dishabituation hierarchy for different worm-like stimuli. The model suggests that visual objects are represented by temporal coding and predicts that the dishabituation hierarchy changes when the stimulus/background contrast direction is reversed or the stimulus size is varied. The behavioral experiments reported in this paper were designed to test these predictions. (1) For a pair of stimuli from the contrast reversal prediction, the experimental results validated the theory. (2) For a pair of stimuli from the size reduction prediction, the experimental results failed to validate the theory. Further experiments concerning size effects suggest that configural visual pattern discrimination in toads exhibits size invariance. (3) Inspired by the Groves-Thompson account of habituation, we found that dishabituation by a second stimulus has a separate process from habituation to a first stimulus. This paper serves as an example of a fruitful dialogue between experimentation and modeling, crucial for understanding brain functions.

Animals↗
PubMed1992

Modeling the dishabituation hierarchy: the role of the primordial hippocampus.

We present a neural model for the organization and neural dynamics of the medial pallium, the toad's homolog of mammalian hippocampus. A neural mechanism, called cumulative shrinking, is proposed for mapping temporal responses from the anterior thalamus into a form of population coding referenced by spatial positions. Synaptic plasticity is modeled as an interaction of two dynamic processes which simulates acquisition and both short-term and long-term forgetting. The structure of the medial pallium model plus the plasticity model allows us to provide an account of the neural mechanisms of habituation and dishabituation. Computer simulations demonstrate a remarkable match between the model performance and the original experimental data on which the dishabituation hierarchy was based. A set of model predictions is presented, concerning mechanisms of habituation and cellular organization of the medial pallium.

Animals↗
PubMed1992

Fiberoptic examination of the nasal cavity and nasopharynx in children.

In this prospective study, a flexible fiberoptic nasolaryngoscope with color video camera was used to examine the nasal cavity and nasopharynx in 180 pediatric patients. The relative size of the adenoid tissue was judged by endoscopy, which lead to a classification into 3 types according to the distance from the vomer to the adenoid tissue. The condition of the nasopharyngeal orifice of the Eustachian tube was also described and differentiated into 3 types relating to the condition of adenoid tissue. Assessment was performed by correlating these measurements with the tympanogram, lateral X-ray and clinical complaints. The authors conclude that: (1) fiberoptic examination allows direct visualization of the size and condition of the adenoid tissue, as well as of the condition of the nasopharyngeal orifice of the Eustachian tube. (2) The size of the adenoid tissue correlates very well with the nasal obstruction complaints as well as with the type of tympanogram. (3) The condition of the nasopharyngeal orifice of the Eustachian tube significantly corresponds with the type of tympanogram. (4) For the indication of adenoidectomy, fiberscopy gives more accurate information than standard X-ray. (5) With a correct choice of premedication and local anesthesia, it is a minor invasive technique which is very well tolerated by children. It is possible in all cases, provided it is performed by a skilled endoscopist and preceded by careful explanation to the child. (6) Finally, thanks to the possibility of direct visualization of the fiberscopic image via a monitor, it allows a better explanation of the indication for adenoidectomy to the child's parents.

Acoustic Impedance Tests↗
PubMed1992

Procedures for the efficient purification of pea seed-borne mosaic virus and its genomic RNA.

An efficient protocol for the purification of pea seed-borne mosaic potyvirus (PSbMV) particles was developed. This led to the purification of 10 PSbMV isolates by a single procedure. Virus aggregation during purification did not occur and consequently, high virus yield was consistently obtained. The virus thus purified was suitable for preparing viral genomic RNA, although conventional methods for RNA extraction resulted in RNA degradation. An alternative method was adopted which yielded reproducibly full length and infectious RNA. This was applied to three isolates of PSbMV and the RNA used to direct complementary DNA synthesis which in turn yielded nearly full length cDNA products.

Fabaceae↗
PubMed1992

Reaction rate enhancement by surface diffusion of adsorbates.

Ligands can be captured by a surface target through either direct bulk diffusion or surface diffusion following reversible adsorption to the surface. We have solved a steady state boundary value problem for a perfect sink disk target in the surface, taking into account bulk and surface diffusion coefficients D and Ds and adsorption/desorption kinetic rate constants ka and kd at non-target regions. Solutions have been successfully found by numerical computation. The results show that the rate of capture from the surface depends non-linearly on Ds, D, ka, kd and geometrical dimensions. In particular, we demonstrate that not only is the non-target region equilibrium constant Keq (= ka/kd) important in determining the rate of capture from the surface, but so are the kinetic rate constants ka and kd separately. In all cases, the surface adsorption/diffusion combination enhances the total rate of capture. The results should be useful for predicting reaction rates of biological membrane bound receptor clusters and substrate-immobilized enzymes.

Adsorption↗
PubMed1992

Immunological analysis of chemoattractive proteins from earthworm to garter snakes.

1. Two sets of polyclonal antibodies to two highly purified prey-derived snake-attractive proteins, a low molecular weight (3000) protein and a 20,000 mol. wt protein, were generated in rabbits. 2. They are immunospecific for their respective purified immunogens and do not cross-react with each other. 3. Eight prey-derived proteins that elicit attack by garter snakes (Thamnophis sirtalis) from earthworms (Lumbricus terrestris) were analyzed with these antibodies, and can be assigned to three distinct groups on the basis of their antigenic properties. 4. Unfolding or denaturation of the low molecular weight protein did not alter its antigenic activity to its polyclonal antibodies, suggesting the antigenic epitopes contain contiguous amino acid sequences. 5. In contrast, unfolding of the 20,000 mol. wt protein resulted in a loss of its binding with antibodies, suggesting that the epitope of this protein contains noncontiguous amino acid sequences. 6. The snake-attractivity of the 20,000 mol. wt protein could not be neutralized by reacting it with its antiserum, suggesting that the antigenic determinant (the epitope) of the antigen is not an integral part of the attractive domain of the ES20 protein. 7. In contrast, the attractivity of the purified low molecular weight protein could be neutralized by the polyclonal antibodies.

Animals↗
PubMed1992

Dimethylthiourea ameliorates acute lung injury induced by phorbol myristate acetate in dogs.

BACKGROUND AND METHODS: The protective effects of dimethylthiourea, a potent scavenger of hydroxy radical (.OH) and hydrogen peroxide, in experimental lung injury in large animals remain controversial. The present study was designed to determine whether dimethylthiourea can ameliorate the acute lung injury produced in dogs by phorbol myristate acetate. Six dogs were infused with dimethylthiourea (0.75 g/kg in saline) for 1.5 hrs, beginning 1 hr before an i.v. bolus injection of phorbol myristate acetate (17 micrograms/kg); six dogs received phorbol myristate acetate (17 micrograms/kg) alone; and six dogs were infused with saline alone. Hemodynamic changes, arterial oxygenation, and the development of lung edema were monitored for 4 hrs after phorbol myristate acetate injection to assess the extent of lung damage. RESULTS: As compared with the dogs that received phorbol myristate acetate alone, the edematous lung damage was significantly reduced in those dogs that received dimethylthiourea as well as phorbol myristate acetate. In the dimethylthiourea-treated dogs, the lung wet/dry weight ratios were smaller (p less than .01); protein concentrations in lung lavage fluid were lower (p less than .01); the decrease in PaO2 was significantly reduced (p less than .01); and there were significant reductions in the alveolar-arterial oxygen tension difference (P[A-a]O2) (p less than .01) and shunt (Qsp/Qt) (p less than .05). Also, dimethylthiourea significantly lowered the increased mean pulmonary arterial pressure levels during the second half of the experiment. CONCLUSIONS: These experimental data suggest that dimethylthiourea is capable of reducing the neutrophil-mediated lung injury produced by the release of hydroxy radical and/or hydrogen peroxide in dogs exposed to phorbol myristate acetate.

Acute Disease↗
PubMed1992

Early embryo invasion as a determinant in pea of the seed transmission of pea seed-borne mosaic virus.

Seed transmission of an isolate of pea seed-borne mosaic virus (PSbMV) in several pea genotypes has been studied. Cross-pollination experiments showed that pollen transmission of PSbMV did not occur and accordingly, virus was not detected in pollen grains by ELISA or electron microscopy. Comparative studies between two pea cultivars, one with a high incidence of seed transmission and one with none, showed that PSbMV infected the floral tissues (sepals, petals, anther and carpel) of both cultivars, but was not detected in ovules prior to fertilization. Virus was detected equally well in seed coats of the progeny in both cultivars. Analysis of virus incidence and concentration in pea seeds of different developmental stages demonstrated that in the cultivar with a high incidence of seed transmission, PSbMV directly invaded immature embryos, multiplied in the embryonic tissues and persisted during seed maturation. In contrast, the cultivar without seed transmission did not show invasion of immature embryos by the virus; there was no evidence for virus multiplication or persistence during embryo development and seed maturation. Hence seed transmission of PSbMV resulted from direct invasion of immature pea embryos by the virus and the block to seed transmission in the non-permissive cultivar probably occurred at this step.

Blotting, Northern↗
PubMed1992

Cloning, sequencing, and expression of Na(+)-H+ antiporter cDNAs from human tissues.

Two types of Na(+)-H+ antiporter with different sensitivities to amiloride analogues have been identified in mammalian plasma membranes. A human Na(+)-H+ antiporter cDNA was obtained by Sardet and co-workers (C. Sardet, L. Counillon, A. Franchi, and J. Pouysségur. Cell 56: 271-280, 1989) using mutant mouse fibroblasts lacking Na(+)-H+ antiporter transformed with human genomic DNA. However, the amiloride sensitivity of this cloned Na(+)-H+ antiporter was not precisely determined. Furthermore, the reported cDNA sequence may be a chimera of human and mouse genes. Hence we isolated a Na(+)-H+ antiporter cDNA actually expressed in human tissues and characterized its amiloride sensitivity. Our 4 kb cDNA obtained from human kidney cortex contained the identical open reading frame to that previously reported and the entire 3' terminus, which was quite different from that reported. This discrepancy was not due to differences in tissue-specific expression because cDNAs from different human tissues were identical, and single bands were observed under high stringency on Northern blots of various human tissues. Na(+)-H+ antiporter activity of mutant mouse fibroblasts deficient in Na(+)-H+ antiporter activity transfected with the cloned cDNA was very sensitive to amiloride and 5-N substituted analogues of amiloride. Thus the cloned cDNA represents the NHE-1 isoform of the Na(+)-H+ antiporter.

Amiloride↗
PubMed1992

Air embolism-induced lung injury in isolated rat lungs.

Pulmonary air embolism causes physical obstruction of microvasculature and leads to permeability changes, release of mediators, and injury to lung tissue. In this study we employed an isolated perfused rat lung model to investigate the primary and secondary effects produced by infusion of air into the pulmonary artery. Infusion of various doses of air (0.10-0.25 ml) over a 1-min period produced a dose-dependent increase in pulmonary arterial pressure and lung weight gain. In contrast, when a constant air dose was administered over various periods of time (0.25 ml over 0.5-8.0 min), the pulmonary arterial pressure rose to the same extent regardless of the infusion rate, whereas the lung weight gain increased proportionately with the rate of infusion. Total vascular resistance rose from 1.41 +/- 0.04 to 5.04 +/- 0.09 mmHg.ml-1.min in rats given 0.25 ml air over 1 min (n = 14, P less than 0.001), with greater than or equal to 90% of this increase occurring in the arterial segments. Both thromboxane B2 and endothelin concentrations also increased in the perfusate, suggesting their involvement in this increased resistance. Furthermore the pulmonary filtration coefficient increased from 0.21 +/- 0.05 to 1.28 +/- 0.26 g.min-1.cmH2O-1.100 g (n = 8, P less than 0.001), and the protein concentration in lung lavage fluid also rose, indicating lung injury. Leukocyte counts in the perfusate were unaffected by embolization, but chemiluminescent activity was increased, indicating a possible role for activated leukocytes in lung injury induced by air emboli.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗