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Biomedical subjects

M Y Liu

Publications and source records attributed to M Y Liu.

At least 91 records · Page 5Linked to original sources
PubMed1995

Characterization of Epstein-Barr virus DNase and its interaction with the major DNA binding protein.

Bacterially expressed Epstein-Barr virus (EBV) DNase was purified to 98% purity and used as the source for characterization of the enzyme activities. Complete digestion of DNA by EBV DNase yielded 5'-monophosphate nucleosides as the final products. During the logarithmic phase of the reaction, EBV DNase acted processively on dsDNA but distributively on ssDNA. Both 5' to 3' and 3' to 5' exonuclease activities were present, although the former was shown to be 10-fold stronger. No significant discrepancy was seen in the liberation of end-labeled nucleotides by DNase when substrates with 5'-protruding, blunt, or 3'-protruding ends were used. EBV DNase was demonstrated also to have an endonuclease activity using supercoiled plasmid DNA as substrate. Two preferential dsDNA cleavage sites were mapped on pBS-TR, a pBlueScript vector containing one copy of the EBV terminal repeat; both are in vector sequences. Finally, an N-terminally truncated EBV major DNA binding protein, but not EA-D, was shown to inhibit EBV DNase activity. This inhibitory effect may due to direct protein-protein interactions between EBV DNase and the major DNA binding protein. The biological significance of these characteristics is discussed.

Antigens, Viral↗
PubMed1995

Siroamide: a prosthetic group isolated from sulfite reductases in the genus Desulfovibrio.

While isolating siroheme from enzymes or whole cells of Desulfovibrio species, it was discovered that the main product after metal removal and esterification was not the octamethyl ester derivative of sirohydrochlorin, but a monoamide, heptamethyl ester derivative. The structure of this derivative was established by mass spectrometry and NMR. Nuclear Overhauser enhancement measurements in combination with chemical shift analogy arguments indicate that the 2(1)-acetate has been stereospecifically amidated. Other cellular sources of siroheme were investigated, but only the octamethyl ester derivative was found, with no traces of the amide derivative. The results suggest that, in Desulfovibrio, the physiologically active prosthetic group may be an amidated form of siroheme.

Desulfovibrio↗
PubMed1995

Surgical management of epilepsy associated with developmental anomalies of the brain.

BACKGROUND: Disorders as the central nervous system forms during the pre-, peri-, or postnatal periods may cause developmental anomalies of the brain. Such maldevelopment, ranging from slight to severe disturbances, appears to be the mechanism for causing childhood seizure disorders. However, the surgical treatment of these lesions responsible for intractable partial seizures remains undefined. METHOD: Twenty-two patients (8 males, 14 females) with chronic seizure disorders associated with structural lesions of the brain related to developmental anomalies were surgically treated (1983 to 1990). Imaging studies, intraoperative findings, and histology showed 12 lesions in the temporal lobe, 1 in the frontal lobe, and 9 of multilobular involvement. All patients had preoperative prolonged electroencephalography with sphenoidal electrodes. Sixteen patients had intraoperative electrocorticography. All underwent craniotomy and total excision of the epileptogenic zone unless the speech or motor cortex was involved. RESULTS: Results of postoperative seizure control during the follow-up period (mean 5.5 years) were excellent (seizure-free) in 12 patients (including four with complete and eight with incomplete excision of the structural lesion), good in seven, fair in two, and poor in one. CONCLUSION: Seizure control is possible for patients with total excision of the epileptogenic zone, even including those with incomplete excision of a structural lesion caused by developmental brain anomalies.

Adolescent↗
PubMed1995

Isolation and preliminary characterization of a soluble nitrate reductase from the sulfate reducing organism Desulfovibrio desulfuricans ATCC 27774.

Desulfovibrio desulfuricans ATCC 27774 is a sulfate reducer that can adapt to nitrate respiration, inducing the enzymes required to utilize this alternative metabolic pathway. Nitrite reductase from this organism has been previously isolated and characterized, but no information was available on the enzyme involved in the reduction of nitrate. This is the first report of purification to homogeneity of a nitrate reductase from a sulfate reducing organism, thus completing the enzymatic system required to convert nitrate (through nitrite) to ammonia. D. desulfuricans nitrate reductase is a monomeric (circa 70 kDa) periplasmic enzyme with a specific activity of 5.4 K(m) for nitrate was estimated to be 20 microM. EPR signals due to one [4Fe-4S] cluster and Mo(V) were identified in dithionite reduced samples and in the presence of nitrate.

Journal Article↗
PubMed1995

The product of the pleiotropic Escherichia coli gene csrA modulates glycogen biosynthesis via effects on mRNA stability.

The carbon storage regulator gene, csrA, modulates the expression of genes in the glycogen biosynthesis and gluconeogenesis pathways in Escherichia coli and has been cloned, mapped and sequenced (T. Romeo, M. Gong, M.Y. Liu, and A.M. Brun-Zinkernagel, J. Bacteriol. 175:4744-4755, 1993; T. Romeo and M. Gong, J. Bacteriol. 175:5740-5741, 1993). We have now conducted experiments that begin to elucidate a unique mechanism for csrA-mediated regulation. Steady-state levels of glgC transcripts, encoding ADP-glucose pyrophosphorylase, were elevated by up to sixfold in a csrA::kanR mutant and were less than 6.5% of wild-type levels in a strain containing pCSR10 (csrA+), as shown by S1 nuclease protection analysis. The rate of chemical decay of these transcripts after adding rifampin to cultures was dramatically reduced by the csrA::kanR mutation. Deletion studies of a glgC'-'lacZ translational fusion demonstrated that the region surrounding the initiation codon was important for csrA-mediated regulation and indicated that neither csrA-mediated regulation nor stationary phase induction of glgC expression originates at the level of transcript initiation. Cell-free (S-200) extracts containing the CsrA gene product potently and specifically inhibited the in vitro transcription-translation of glg genes. The deduced amino acid sequence of CsrA was found to contain the KH motif, which characterizes a subset of diverse RNA-binding proteins. The results indicate that CsrA accelerates net 5'-to-3' degradation of glg transcripts, potentially through selective RNA binding.

Amino Acid Sequence↗
PubMed1995

Protective effects of propranolol on experimentally head-injured mouse brains.

Catecholamines have a deleterious effect on the traumatized brain. Propranolol, a beta-adrenergic blocker, can act on the vasomotor centers within the hypothalamus and brain stem to reduce the activity of the sympathetic nerves and the cerebral metabolic need for oxygen and glucose. This study investigated the effects of racemic (d,l) propranolol on the neurologic recovery of mice with head-injuries. Male BALB-C mice (n = 110) received a 1,100 g-cm head injury from a 50 g weight dropped from a height of 22 cm resulting in immediate loss of consciousness in all animals and some deaths. The survivors were divided into treatment (n = 46) and placebo (n = 45) groups. The treatment group received an intraperitoneal injection of propranolol 2.5mg/kg immediately after the head injury. The placebo group received the same amount of 0.9% normal saline by injection in the same time interval. Neurologic status was evaluated 1 hour after injury. A group of 50 mice without head injury was also tested to evaluate the performance of normal mice. All three groups of mice were then decapitated. Mice with depressed fractures of the skull (n = 1) or intracranial hematomas (n = 1) after head injury were excluded from the study. Small pieces of brain parenchyma were used to check the water content by gravimetric method. The treatment group showed better neurologic recovery than the placebo group by both the string test scoring system and grip test time. The brain edema was significantly reduced at the temporal lobes in the treatment group as compared to the placebo group.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Antagonists↗
PubMed1994

Replacement of methionine as the axial ligand of Achromobacter cycloclastes cytochrome c554 at high pH values revealed by absorption, EPR and MCD spectroscopy.

Cytochrome c554 from the denitrifying bacterium Achromobacter cycloclastes is a monoheme class II c-type cytochrome with a His-Met axial coordination at neutral pH. The amino acid composition and the N-terminal sequence of the cytochrome have been determined. Subsequent determination of the pH-dependence of the redox potential and examination of the EPR and MCD spectra of ferricytochrome c554 revealed a new form at high pH values made apparent with both spectroscopies. These observations are consistent with the presence of lysine as the axial ligand for which methionine substitutes at high pH values.

Alcaligenes↗
PubMed1994

Characterization and partial purification of the VDAC-channel-modulating protein from calf liver mitochondria.

The mitochondrial channel, VDAC, mediates metabolic flux across the mitochondrial outer membrane. When reconstituted into planar phospholipid membranes, VDAC is voltage-dependent, existing in multiple conformational states with different selectivities and permeabilities. At low membrane potentials, these channels are in the open state and are anion-selective. VDAC channels switch to lower-conductive closed states at high membrane potentials. The VDAC modulator, a soluble mitochondrial protein, has been demonstrated to dramatically increase the voltage dependence of VDAC channels and induce the channels to enter closed states even at low membrane potentials. We have isolated and partially purified this modulating protein and the activity is associated with a 54 kDa protein on SDS-PAGE. Under native reduced conditions the activity eluted around 100 kDa from a gel filtration column. As little as 200 ng/ml of the partially purified protein was sufficient to modulate reconstituted VDAC channels. This protein had a pI of 5.1. A second activity with a pI of 4.8 was far more potent, making VDAC-channel-containing membranes virtually non-conductive in some experiments. The effects of both modulator activities could be completely reversed by the addition of pronase. Simple perfusion of the chamber did not reverse the effect of the modulator on VDAC. By controlling the gating of VDAC channels, the VDAC modulator could play an important role in regulating cellular metabolism.

Animals↗
PubMed1994

A simple method for diagnosing traumatic occlusion of the vertebral artery at the craniovertebral junction.

STUDY DESIGN: A simple method, thin-slice contrast-enhanced computed tomographic (CT) scan, was evaluated as a first-line rapid screening procedure for diagnosing traumatic occlusion of the vertebral artery at the craniovertebral junction. OBJECTIVES: This procedure was performed in patients suffering from injuries of the high cervical spine or the craniovertebral junction. Vertebral angiography was undertaken after this procedure to confirm the diagnosis. SUMMARY OF BACKGROUND DATA: Well-enhanced vertebral arteries can be demonstrated clearly in normal subjects by using this method. Occlusion of the vertebral artery produces delayed contrast enhancement of the proximal artery and abrupt interruption of its course. METHODS: Thin-slice CT scan was performed from the atlas to the occipital condyles after an intravenous injection of angiograffin. The plane of the CT scan was aligned in a parallel manner to the upper margin of the atlas. The CT scanning was repeated until the contrast enhancement of the vertebral arteries became faint. RESULTS: Occlusion of the unilateral vertebral artery was successfully diagnosed in a patient with Jefferson's fracture by using the present technique. A subsequent vertebral angiography confirmed the diagnosis. CONCLUSIONS: The present technique should be performed as a routine screening procedure in patients suffering from injuries to the craniovertebral junction.

Atlanto-Axial Joint↗
PubMed1994

Thiol/disulfide formation associated with the redox activity of the [Fe3S4] cluster of Desulfovibrio gigas ferredoxin II. 1H NMR and Mössbauer spectroscopic study.

Desulfovibrio gigas ferredoxin II (FdII) is a small protein (alpha 4 subunit structure as isolated; M(r) approximately 6400 per subunit; 6 cysteine residues) containing one Fe3S4 cluster per alpha-subunit. The x-ray structure of FdII has revealed a disulfide bridge formed by Cys-18 and Cys-42 approximately 13 A away from the center of the cluster; moreover, the x-ray structure indicates that Cys-11 forms a disulfide bridge with a methanethiol. In the oxidized state, FdIIoxm the 1H NMR spectra, exhibit four low-field contact-shifted resonances at 29, 24, 18, and 15.5 ppm whereas the reduced state, FdIIR (S = 2), yields two features at +18.5 and -11 ppm. In the course of studying the redox behavior of FdII, we have discovered a stable intermediate, FdIIint, that yields 1H resonances at 24, 21.5, 21, and 14 ppm. This intermediate appears in the potential range where the cluster (E'0 approximately -130 mV) is reduced from the [Fe3S4]1+ to the [Fe3S4]0 state. FdIIint is observed during reductive titrations with dithionite or hydrogen/hydrogenase or after partial oxidation of FdIIR by 2,6-dichlorophenolindophenol or air. Our studies show that a total of three electrons per alpha-subunit are transferred to FdII. Our experiments demonstrate the absence of a methanethiol-Cys-11 linkage in our preparations, and we propose that two of the three electrons are used for the reduction of the disulfide bridge. Mössbauer (and EPR) studies show that the Fe3S4 cluster of FdIIint is at the same oxidation level as FdIIox, but indicate some changes in the exchange couplings among the three ferric sites. Our data suggest that the differences in the NMR and Mössbauer spectra of FdIIox and FdIIint result from conformational changes attending the breaking or formation of the disulfide bridge. The present study suggests that experiments be undertaken to explore an in vivo redox function for the disulfide bridge.

Desulfovibrio↗
PubMed1994

Functional analysis of the amino terminus of Epstein-Barr virus deoxyribonuclease.

A cDNA coding for the Epstein-Barr virus (EBV) alkaline deoxyribonuclease (DNase) was expressed in Escherichia coli using the T7 phage system designed to allow expression of potentially lethal proteins. Induction of protein synthesis from the gene yielded a peptide with a molecular weight of approximately 52 kDa, consistent with the predicted open reading frame of EBV BGLF5. A high level of nuclease activity was detected in crude cell extracts, and the activity was neutralized by sera from nasopharyngeal carcinoma patients with high titers of anti-DNase antibodies. A series of deletion clones truncated at the amino or carboxyl terminus was constructed and expressed to define the regions responsible for the nuclease activity of EBV DNase. All the mutated molecules lost their activities even though their expression levels were comparable to that of the full-length DNase. To determine the exact role of the amino terminus of EBV DNase, mutants with small deletions were expressed. While three mutants with deletions of amino acid residues 11-30, 16-28, or 23-28 failed to show any detectable nuclease activities, one mutant in which the first 8 amino acids were replaced by the first 12 amino acid residues of the T7 major capsid protein contributed by the vector was enzymatically active. To further define the importance of the amino-terminal region, full-length DNase with point mutations was generated among residues 23-29 by site-directed mutagenesis and expressed in the same system. Assays of the DNase activity of these mutants revealed that the mutation of residue 29 was fully active, and mutations in 24-27 retained 50% activity. Nevertheless, the mutation at residue 23 resulted in a complete loss of activity and the mutation at residue 28 resulted in loss of 70% activity. These results suggest the biological importance of the amino terminus of the EBV DNase, especially residues 23-28.

Amino Acid Sequence↗
PubMed1994

Localization and specificity of cytochromes and other electron transfer proteins from sulfate-reducing bacteria.

Recently data have accumulated concerning the electron transfer chains of sulfate-reducing bacteria in general and of the genus Desulfovibrio in particular. Because of the ever growing number of newly discovered individual redox proteins, it has become essential to try to assign them to physiologically relevant chains. This work presents some new data concerning the localization of these proteins within the bacterial cell and the specificity of electron transfer between the three types of hydrogenases which have been found so far in Desulfovibrio, namely the iron-only, the iron-nickel and the iron-nickel-selenium enzymes. The iron-only hydrogenase reduces cytochromes which have bis-histidinyl heme ligation or histidinyl-methionyl heme ligation. In contrast, the iron-nickel and iron-nickel-selenium hydrogenases cannot reduce cytochromes having a His-Met heme ligation, but are very active toward the cytochromes having a bis-histidinyl ligand. This observation has been used to demonstrate that the tetraheme cytochrome c3 can exchange electrons with the monoheme cytochrome c553. No clear specificity has been established for the reaction of hydrogenases toward the hexadecaheme cytochromes from either D vulgaris or D gigas.

Amino Acid Sequence↗
PubMed1993

Aerobic metabolism of carbon reserves by the "obligate anaerobe" Desulfovibrio gigas.

The sulfate-reducing bacterium, Desulfovibrio gigas, is shown by in vivo 31P-NMR to be capable of generating NTP from the utilization of internal carbon reserves both in anaerobic and in aerobic conditions. Acetate, glycerol and ethanol are the major end-products, but the production of alcohols decreases strongly when oxygen is present. When the glycolytic pathway is inhibited with fluoride, NTP levels decrease drastically but can be remarkably restored when an electron acceptor, such as oxygen, is provided. Our data are in favour of a NADH-linked electron transfer chain enabling transfer of reducing power derived from polyglucose to oxygen which provides this so-called "strict anaerobe" with the capability of surviving to oxic environments.

Acetates↗
PubMed1993

Purification and characterization of an NADH-rubredoxin oxidoreductase involved in the utilization of oxygen by Desulfovibrio gigas.

An NADH--rubredoxin oxidoreductase previously isolated from Desulfovibrio gigas [LeGall, J. (1968) Ann. Inst. Pasteur 114, 109-115] has now been fully purified and further characterized. It contains two subunits of 27 kDa and 32 kDa. With two mid-point redox potentials of -295 mV and -325 mV, this FMN- and FAD-containing protein can induce the specific reduction of D. gigas rubredoxin. In contrast, rubredoxins from the other Desulfovibrio species or desulforedoxin from D. gigas show very low reaction rates with the same enzyme. The phylogenetic significance of the narrow specificity of the enzyme toward the rubredoxin from the same organism is discussed. The purified enzyme has NADH oxidase activity with H2O2 as a final product of O2 reduction. The reaction is half-inhibited by 4.2 microM p-chloromercuribenzoate, whereas cyanide and azide are not significant inhibitors in this reaction. The role of this protein as a part of the enzymic equipment that allows the formation of ATP in the presence of oxygen from the degradation of carbon reserves is discussed.

Chloromercuribenzoates↗
PubMed1993

Rubredoxin oxidase, a new flavo-hemo-protein, is the site of oxygen reduction to water by the "strict anaerobe" Desulfovibrio gigas.

A rubredoxin-oxygen oxidoreductase, a homodimer with a molecular weight of 43 kDa per monomer, was found to be a component of an electron transfer chain that couples the reduction of oxygen to water with NADH oxidation. This FAD-containing protein appears to contain a new type of heme group. The electron transfer chain is not inhibited by cyanide and azide. In contrast, CO decreases NADH oxidation rate and also induces release of the prosthetic groups from the native terminal reductase.

Chromatography, Gel↗
PubMed1993

Isolation and characterization of flavoredoxin, a new flavoprotein that permits in vitro reconstitution of an electron transfer chain from molecular hydrogen to sulfite reduction in the bacterium Desulfovibrio gigas.

A new FMN-containing flavoprotein isolated from Desulfovibrio gigas provided maximum coupling efficiency for the reduction of bisulfite from molecular H2. This protein, which is distinct from flavodoxin and for which the name flavoredoxin is proposed, is required for reconstitution of an electron transfer chain between hydrogenase and bisulfite reductase. A Ca(2+)-binding protein functions as a modulator in the presence of Ca2+ in the process. The finding of a membrane-bound cytochrome c with a molecular weight of 104,000 Da that is also active in this electron transfer chain provides an explanation for the energetic linkage between periplasmic and cytoplasmic proteins in this sulfate-reducing bacterium.

Amino Acid Sequence↗
PubMed1993

Electrochemical studies of the hexaheme nitrite reductase from Desulfovibrio desulfuricans ATCC 27774.

The electron-transfer kinetics between three different mediators and the hexahemic enzyme nitrite reductase isolated from Desulfovibrio desulfuricans (ATCC 27774) were investigated by cyclic voltammetry and by chronoamperometry. The mediators, methyl viologen, Desulfovibrio vulgaris (Hildenborough) cytochrome c3 and D. desulfuricans (ATCC 27774) cytochrome c3 differ in structure, redox potential and charge. The reduced form of each mediator exchanged electrons with nitrite reductase. Second-order rate constants, k, were calculated on the basis of the theory for a simple catalytic mechanism and the results, obtained by cyclic voltammetry, were compared with those obtained by chronoamperometry. Values for k are in the range 10(6)-10(8) M-1 s-1 and increase in the direction D. desulfuricans cytochrome c3-->D. vulgaris cytochrome c3-->methyl viologen. An explanation is advanced on the basis of electrostatic interactions and relative orientation between the partners involved. Chronoamperometry (computer controlled) offers advantages over cyclic voltammetry in the determination of homogeneous rate constants (faster, more accurate and better reproducibility). Direct, unmediated electrochemical responses of the hexaheme nitrite reductase were also reported.

Cytochrome c Group↗