Biomedical subjects

M Y Liu

Publications and source records attributed to M Y Liu.

At least 127 records · Page 7Linked to original sources

PubMed1991

Direct evidence of the metal-free nature of sirohydrochlorin in desulfoviridin.

We have obtained direct evidence that the majority of the sirohydrochlorin chromophore in the dissimilatory sulfite reductase desulfoviridin from Desulfovibrio gigas, is not associated with any metal. The evidence comes from resonance Raman measurements of native and deuterated samples of desulfoviridin. The breathing mode v4 (or v4*) at 1336 cm-1 in the native enzyme is downshifted to 1326 cm-1 upon deuteration. This mode is not sensitive to deuteration if a metal is present at the center of the chromophore inside protein or in solution. The results also establish the existence of exchangeable core hydrogen(s) at the pyrrolic nitrogen(s).

Desulfovibrio↗

PubMed1991

The 2.3 angstrom X-ray structure of nitrite reductase from Achromobacter cycloclastes.

The three-dimensional crystal structure of the copper-containing nitrite reductase (NIR) from Achromobacter cycloclastes has been determined to 2.3 angstrom (A) resolution by isomorphous replacement. The monomer has two Greek key beta-barrel domains similar to that of plastocyanin and contains two copper sites. The enzyme is a trimer both in the crystal and in solution. The two copper atoms in the monomer comprise one type I copper site (Cu-I; two His, one Cys, and one Met ligands) and one putative type II copper site (Cu-II; three His and one solvent ligands). Although ligated by adjacent amino acids Cu-I and Cu-II are approximately 12.5 A apart. Cu-II is bound with nearly perfect tetrahedral geometry by residues not within a single monomer, but from each of two monomers of the trimer. The Cu-II site is at the bottom of a 12 A deep solvent channel and is the site to which the substrate (NO2-) binds, as evidenced by difference density maps of substrate-soaked and native crystals.

Alcaligenes↗

PubMed1991

Amino acid sequence of nitrite reductase: a copper protein from Achromobacter cycloclastes.

The amino acid sequence of the copper-containing nitrite reductase (EC 1.7.99.3) from Achromobacter cycloclastes strain IAM 1013 has been determined by using peptides derived from digestion with Achromobacter protease I (Lys), Staphylococcus aureus V8 protease (Glu), cyanogen bromide, and BNPS-skatole in acetic acid. The subunit contains 340 amino acids. The identity of the first seven amino acids is tentative. The sequence has been instrumental in the X-ray structure determination of this molecule; in conjunction with the X-ray structure, ligands to a type I copper atom and a type II copper atom (one of each per subunit) have been identified. Comparison of the sequence to those of multi-copper oxidases such as ascorbate oxidase, laccase, and ceruloplasmin [Messerschmidt, A., & Huber, R. (1990) Eur. J. Biochem. 187, 341-352] reveals that each of two domains seen in the X-ray structure is similar to the oxidases and also to the small blue copper-containing proteins such as plastocyanin. The combination of sequence and structural similarity to ascorbate oxidase and sequence similarity to ceruloplasmin leads to a plausible model for the domain structure of ceruloplasmin.

Alcaligenes↗

PubMed1991

Structural and functional approach toward a classification of the complex cytochrome c system found in sulfate-reducing bacteria.

Following the discovery of the tetraheme cytochrome c3 in the strict anaerobic sulfate-reducing bacteria (Postgate, J.R. (1954) Biochem. J. 59, xi; Ishimoto et al. (1954) Bull. Chem. Soc. Japan 27, 564-565), a variety of c-type cytochromes (and others) have been reported, indicating that the array of heme proteins in these bacteria is complex. We are proposing here a tentative classification of sulfate- (and sulfur-) reducing bacteria cytochromes c based on: number of hemes per monomer, heme axial ligation, heme spin state and primary structures (whole or fragmentary). Different and complementary spectroscopic tools have been used to reveal the structural features of the heme sites.

Amino Acid Sequence↗

PubMed1991

Inhibition of pulmonary surfactant function by meconium.

The pathophysiology of meconium aspiration is marked by lung hyperinflation because of airway obstruction, which is often followed by an acute pneumonitis with classic lung injury characteristics. Surfactant dysfunction may contribute to this latter pulmonary pathophysiology. We sought to determine to what extent meconium itself might contribute to a functional surfactant deficiency. Specimens of newborn infants' first meconium were collected and pooled. Serial dilutions of the meconium were then added to various concentrations of calf lung surfactant extract, a mixture with the surface properties of natural surfactant that is used clinically to treat neonatal respiratory distress syndrome, and the dynamic surface activity of these mixtures was studied with a pulsating bubble surfactometer. At surfactant concentrations of less than or equal to 1.5 mg/ml, even 6500-fold dilutions of meconium-inhibited surface tension lowering ability (10 +/- 2 mN/m vs 1 +/- 0.1 mN/m for controls, p less than 0.05). Moreover, this inhibitory activity resided in both the chloroform-soluble and the aqueous phases of meconium and appeared to be additive in nature. However, at sufficiently high concentrations of surfactant, even large amounts of meconium were unable to affect surface tension lowering properties. Thus meconium inhibits surfactant function in a manner that is dependent on the surfactant concentration, suggesting the possible utility of exogenous surfactant therapy in some cases of meconium aspiration.

Animals↗

PubMed1991

The genome of human herpesvirus 6: maps of unit-length and concatemeric genomes for nine restriction endonucleases.

More than 50 fragments resulting from complete digestion of the DNA of human herpesvirus 6 (HHV-6, strain U1102) with BamHI, EcoRI, HindIII, KpnI, NruI, SalI or SmaI have been isolated as clones in M13, plasmid, cosmid and lambda vectors. Using these clones, maps have been constructed for the fragments produced by nine restriction enzymes from unit-length virus genomes and from their concatemeric precursors. The unit-length genome is a linear, double-stranded molecule of 161.5 kbp composed of a central segment of a largely unique sequence of 141 kbp (U) with a sequence of 10 kbp duplicated in the same orientation at both 'left' and 'right' genomic termini (i.e. 'left' and 'right' copies of the direct repeat; DRL and DRR). Adopting as standard an orientation in which the major capsid protein gene is 'left' of the gene for alkaline exonuclease, then the 'right' genome termini and DRL. U junctions occur close to or within repetitive (GGGTTA)n sequences. Repetitions of short sequence motifs are present in at least two other regions of the genome. One of these regions consists of a simple repeat (TC/G) of approximately 1.5 kbp in length and is unstable as clones in bacterial vectors. The second region is stably maintained in such vectors and consists of a tandem array of at least 25 copies of a 110 bp sequence containing a single KpnI site. Comparisons of fragments arising from unit-length DNA with those from virus DNA from the nuclei of infected cells have shown that the concatemeric junctions in intracellular DNA contain head-to-tail dimers of the terminal duplications (i.e. ...U1.DRR1.DRL2.U2...). The gross structure established here for the genome from the U1102 isolate of HHV-6 resembles closely that suggested by Pellett and his colleagues for the Z29 isolate and differs from that of the five previously characterized human herpesviruses. This structure of HHV-6 DNA bears a superficial resemblance to that proposed for DNA from channel catfish virus and equine cytomegalovirus.

Base Sequence↗

PubMed1991

Voltage gating of the mitochondrial outer membrane channel VDAC is regulated by a very conserved protein.

Soluble protein preparations obtained from the mitochondrial fractions of three very different organisms, Neurospora crassa, rat, and potato, were discovered to greatly enhance the voltage sensitivity of the mitochondrial outer membrane channel, VDAC. The active ingredient, referred to as the VDAC modulator, increased the rate of voltage-dependent channel closure by approximately 10-fold. The modulator from one species increased the closing rate of VDAC channels from all three species. The activity is pronase sensitive and not mimicked by another negatively charged protein, BSA. The highly conserved property of this modulator suggests an important physiological role in regulating mitochondrial function.

Animals↗

PubMed1991

Inhibition of pulmonary surfactant function by phospholipases.

Previous studies have shown that respiratory failure associated with disorders such as acute pancreatitis correlates well with increased levels of phospholipase A2 (PLA2) in lung lavages and that intratracheal administration of PLA2 generates an acute lung injury. In addition, bacteria such as Pseudomonas have been shown to secrete phospholipase C (PLC). We studied the effects of these phospholipases on pulmonary surfactant activity using a pulsating bubble surfactometer. Concentrations greater than or equal to 0.1 unit/ml PLA2 destroyed surfactant biophysical activity, increasing surface tension at minimum bubble size from less than 1 to 15 mN/m. This surfactant inactivation was predominantly related to the effect of lysophosphatidylcholine on the surface film, although the fatty acids released with higher PLA2 concentrations also had a detrimental effect on surfactant function. Similarly, as little as 0.1 unit PLC increased the surface tension at minimal size of an oscillating bubble from less than 1 to 15 mN/m, an effect that could be mimicked by the addition of dipalmitin to surfactant in the absence of PLC. Moreover, lower, noninhibitory concentrations (0.01 unit/ml) of PLA2 and PLC increased the sensitivity of surfactant to other inhibitory agents, such as albumin. Thus, relatively low concentrations of PLC and PLA2 can cause severe breakdown of surfactant function and may contribute significantly to some forms of lung injury.

Animals↗

PubMed1991

Pulmonary surfactant will secure free airflow through a narrow tube.

Well functioning pulmonary surfactant is necessary to ensure alveolar stability. It is proposed that surfactant is also required to keep the finest cylindrical airways open, thereby securing an unrestricted flow of air to and from the alveoli. If the surfactant is inadequate in quality or quality there is a risk that liquid will accumulate in the most marrow section of the airway and form a blocking column. To study that possibility special glass capillaries were used. The glass capillaries were heated and extended to make a short section very narrow. In the lumen of that section a minute volume (1 microliter) of liquid was deposited, which formed a blocking column. When pressure was raised on one side of the column, it forced the liquid to move away from the narrow section. Pressure dropped to zero as air could pass, and if the liquid column consisted of calf lung surfactant extract (CLSE), pressure remained at zero because a new liquid column did not form. If, on the other hand, the liquid column consisted of saline solution it would repeatedly reform as soon as it had been pressed out of the capillary's narrow section. The same occurred if the CLSE suspension forming the liquid column was very dilute or contained inhibiting proteins. These observations did not require that the capillary consisted of the material glass; they were also noted when the narrow tube was outlined by epithelium.

Air Pressure↗

PubMed1991

Detection of human herpesvirus 6 DNA in T and B cell lymphomas.

DNA extracted from tissues of 109 patients with lymphoproliferative disorders, including 40 T-cell lymphomas, 40 B-cell lymphomas, 8 Hodgkin's disease, 13 hyperplasia, 5 infectious mononucleosis, 2 histiocytic medullary reticulosis and 1 Kikuchi's disease, and peripheral blood lymphocytes (PBL) from 17 healthy donors were investigated for the presence of human herpesvirus 6 (HHV-6) DNA sequence by polymerase chain reaction (PCR) and Southern hybridization. The procedures for DNA amplification were composed of the first round of PCR using a pair of primers LL and LR, which were contained in the HHV-6 major capsid gene, and the second round of nested PCR using an internal pair of primers LLN and LRN. Results showed that the prevalence of HHV-6 DNA sequence in human tissues was similar in both B and T cell lymphoma patients and normal adults, and viral heterogeneity was detected in the amplified DNA sequence from local strain of Taiwan as compared to that of U1102 strain from Uganda.

DNA, Viral↗

PubMed1991

Analysis of Epstein-Barr virus DNA in nude mouse-passaged nasopharyngeal carcinoma tissues.

DNAs extracted from two series of nude mouse-passaged nasopharyngeal carcinoma (NPC) tissues, NPC-295 and -306, and P3HR1 cells were digested with restriction enzyme Bam HI, then electrophoresed on agarose gel and analysed for the Epstein-Barr virus (EBV) DNA fragments by Southern hybridization with 27 radioactive Bam HI DNA fragments and the Eco RI Dhet DNA fragment from cloned EBV DNA of B95-8 cell line. Constant hybridization patterns were revealed in these experiments and the results suggest a stable existence of EBV DNA including replicative form and episomal form in these tissues even after a long time of passage. Neither deletion in YH region nor in I region could be detected when using Bam HI Y, H and I as probes, suggesting the EBV in these tissues are "more complete" viruses. When Bam HI F was used as a probe, an extra Bam HI site was revealed in the analysis of the DNA from NPC-295 suggesting the polymorphism of DNA present in this region. Restriction maps of EBV DNAs in both nude mouse-passaged NPC tissues are proposed. The results suggest that nude mouse-passaged NPC tissue is a good model to study the structure of EBV DNA in tumor tissue.

Adult↗

PubMed1990

Purification and characterization of desulfoferrodoxin. A novel protein from Desulfovibrio desulfuricans (ATCC 27774) and from Desulfovibrio vulgaris (strain Hildenborough) that contains a distorted rubredoxin center and a mononuclear ferrous center.

A new type of non-heme iron protein was purified to homogeneity from extracts of Desulfovibrio desulfuricans (ATCC 27774) and Desulfovibrio vulgaris (strain Hildenborough). This protein is a monomer of 16-kDa containing two iron atoms per molecule. The visible spectrum has maxima at 495, 368, and 279 nm and the EPR spectrum of the native form shows resonances at g = 7.7, 5.7, 4.1 and 1.8 characteristic of a high-spin ferric ion (S = 5/2) with E/D = 0.08. Mössbauer data indicates the presence of two types of iron: an FeS4 site very similar to that found in desulforedoxin from Desulfovibrio gigas and an octahedral coordinated high-spin ferrous site most probably with nitrogen/oxygen-containing ligands. Due to this rather unusual combination of active centers, this novel protein is named desulfoferrodoxin. Based on NH2-terminal amino acid sequence determined so far, the desulfoferrodoxin isolated from D. desulfuricans (ATCC 27774) appears to be a close analogue to a recently discovered gene product from D. vulgaris (Brumlik, M.J., and Voordouw, G. (1989) J. Bacteriol. 171, 49996-50004), which was suggested to be a rubredoxin oxidoreductase. However, reduced pyridine nucleotides failed to reduce the desulforedoxin-like center of this new protein.

Amino Acid Sequence↗

PubMed1990

Regulation of the hexaheme nitrite/nitric oxide reductase of Desulfovibrio desulfuricans, Wolinella succinogenes and Escherichia coli. A mass spectrometric study.

Dissimilatory nitrite reduction, carried out by hexaheme proteins, gives ammonia as the final product. Representatives of this enzyme group from 3 bacterial species can also reduce NO to either ammonia or N2O. The redox regulation of the nitrite/nitric oxide activities is discussed in the context of the denitrifying pathway.

Ascorbic Acid↗

PubMed1990

Purification and characterization of two proteins with inorganic pyrophosphatase activity from Desulfovibrio vulgaris: rubrerythrin and a new, highly active, enzyme.

The inorganic pyrophosphatase activity of a soluble extract from the strict anaerobe, sulfate-reducing, Desulfovibrio vulgaris, is readily resolved into two peaks. After purification, two active proteins with very dissimilar properties are obtained. One is the non-heme iron-containing rubrerythrin, with a specific activity of 350 pyrophosphate hydrolyzed, min-1, mg protein-1. The other, a protein of Mr = 39,000, with a specific activity of 12,000.

Amino Acid Sequence↗

PubMed1990

Hexaheme nitrite reductase from Desulfovibrio desulfuricans. Mössbauer and EPR characterization of the heme groups.

Mössbauer and EPR spectroscopy were used to characterize the heme prosthetic groups of the nitrite reductase isolated from Desulfovibrio desulfuricans (ATCC 27774), which is a membrane-bound multiheme cytochrome capable of catalyzing the 6-electron reduction of nitrite to ammonia. At pH 7.6, the as-isolated enzyme exhibited a complex EPR spectrum consisting of a low-spin ferric heme signal at g = 2.96, 2.28, and 1.50 plus several broad resonances indicative of spin-spin interactions among the heme groups. EPR redox titration studies revealed yet another low-spin ferric heme signal at g = 3.2 and 2.14 (the third g value was undetected) and the presence of a high-spin ferric heme. Mössbauer measurements demonstrated further that this enzyme contained six distinct heme groups: one high-spin (S = 5/2) and five low-spin (S = 1/2) ferric hemes. Characteristic hyperfine parameters for all six hemes were obtained through a detailed analysis of the Mössbauer spectra. D. desulfuricans nitrite reductase can be reduced by chemical reductants, such as dithionite or reduced methyl viologen, or by hydrogenase under hydrogen atmosphere. Addition of nitrite to the fully reduced enzyme reoxidized all five low-spin hemes to their ferric states. The high-spin heme, however, was found to complex NO, suggesting that the high-spin heme could be the substrate binding site and that NO could be an intermediate present in an enzyme-bound form.

Desulfovibrio↗

PubMed1990

Spin-equilibrium and heme-ligand alteration in a high-potential monoheme cytochrome (cytochrome c554) from Achromobacter cycloclastes, a denitrifying organism.

A c-type monoheme cytochrome c554 (13 kDa) was isolated from cells of Achromobacter cycloclastes IAM 1013 grown anaerobically as a denitrifier. The visible absorption spectrum indicates the presence of a band at 695 nm characteristic of heme-methionine coordination (low-spin form) coexisting with a minor high-spin form as revealed by the contribution at 630 nm. Magnetic susceptibility measurements support the existence of a small contribution of a high-spin form at all pH values, attaining a minimum at intermediate pH values. The mid-point redox potential determined by visible spectroscopy at pH 7.2 is +150 mV. The pH-dependent spin equilibrum and other relevant structural features were studied by 300-MHz 1H-NMR spectroscopy. In the oxidized form, the 1H-NMR spectrum shows pH dependence with pKa values at 5.0 and 8.9. According to these pKa values, three forms designated as I, II and III can be attributed to cytochrome c554. Forms I and II predominate at low pH values, and the 1H-NMR spectra reveal heme methyl proton resonances between 40 ppm and 22 ppm. These forms have a methionyl residue as a sixth ligand, and C6 methyl group of the bound methionine was identified in the low-field region of the NMR spectra. Above pH 9.6, form III predominates and the 1H-NMR spectrum is characterized by down-field hyperfine-shifted heme methyl proton resonances between 29 ppm and 22 ppm. Two new resonances are observed at congruent to 66 ppm and 54 ppm, and are taken as indicative of a new type of heme coordination (probably a lysine residue). These pH-dependent features of the 1H-NMR spectra are discussed in terms of the heme environment structure. The chemical shifts of the methyl resonances at different pH values exhibit anti-Curie temperature dependence. In the ferrous state, the 1H-NMR spectrum shows a methyl proton resonance at -3.9 ppm characteristic of methionine axial ligation. The electron-transfer rate between ferric and ferrous forms has been estimated to be smaller than 2 x 10(4) M-1 s-1 at pH 5. EPR spectroscopy was also used to probe the ferric heme environment. A prominent signal at gmax congruent to 3.58 and the overall lineshape of the spectrum indicate an almost axial heme environment.

Alcaligenes↗

PubMed1990

Formamidines as synergists of cypermethrin in susceptible and pyrethroid resistant house flies (Diptera: Muscidae).

Mechanisms of formamidine synergism of pyrethroid insecticides were investigated in the house fly, Musca domestica L. A bioassay method was developed to show formamidine synergism of cypermethrin in adult house flies. Flies were exposed to a residue of cypermethrin with and without formamidine for 30 min and then transferred to a clean container. Mortality was recorded 24 h later. Synergism of cypermethrin occurred in flies exposed simultaneously to cypermethrin plus formamidines and in flies exposed to cypermethrin either before or after exposure to a formamidine. Synergism ranged up to 11.8-fold and was greater in susceptible than in resistant house flies. A monomeric derivative of amitraz (BTS 27271) was the most active synergist, chlordimeform was intermediate, and amitraz was least active. Synergism of cypermethrin also occurred in flies injected with octopamine and then exposed to cypermethrin, suggesting that formamidines may be acting as octopamine agonists. Measurements of the effects of formamidines on uptake of cypermethrin showed that BTS 27271 increased uptake less than chlordimeform and that amitraz had almost no effect. Both target site and behavioral effects are discussed as possible mechanisms of formamidine synergism of cypermethrin and other pyrethroids.

Amidines↗

PubMed1990

Identification, cloning, and expression of the major capsid protein gene of human herpesvirus 6.

DNA sequence analysis of part of the human herpesvirus 6 (HHV-6) genome led to the identification of an open reading frame with amino acid sequence homology to the major capsid proteins (MCP) of other HHVs. DIAGON analysis showed that the closest homology was with human cytomegalovirus. Plasmids were constructed which were shown to express the HHV-6 MCP as either the entire open reading frame or as portions of it, and the recombinant-produced proteins were used to raise antisera. The antisera were shown by immunofluorescence to react with HHV-6-infected lymphoblastoid cells and in Western blots with a 135-kilodalton protein specific to HHV-6-infected cells. The recombinant protein expressed from the entire HHV-6 MCP gene was detected only weakly in Western blot assays with normal HHV-6-positive human sera as a probe.

Amino Acid Sequence↗