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Biomedical subjects

M Luo

Publications and source records attributed to M Luo.

At least 127 records · Page 7Linked to original sources
PubMed1996

Injury induces rapid changes in hepatocyte nuclear factor-1: DNA binding.

BACKGROUND: Transcriptional regulation in the liver plays a critical role in mediating the acute phase response to injury. The molecular mechanisms driving these transcriptional events, however, are poorly defined in vivo. The liver-specific transcription factor hepatocyte nuclear factor (HNF)-1 binds to the 5' upstream region of many acute phase genes. To explore the connection between injury and transcriptional regulatory mechanisms, we investigated the effect of injury on HNF-1 binding activity. METHODS: Liver nuclear extracts were prepared from animals after burn or anesthetized sham burn injury. HNF-1 binding activity, affinity, and off rate were assessed by electrophoretic mobility shift analysis. RESULTS: HNF-1 binding activity decreased by 28% 1 1/2 hours after injury. The dissociation constant for HNF-1 increased from 0.6 nm to 11.8 nm at 1 1/2 hours after burn injury partly because of an increase in off rate for the HNF-1: DNA complex. CONCLUSIONS: Burn injury leads to a significant decrease in HNF-1 binding activity as a result of decreased affinity of HNF-1 for DNA. These injury-induced alterations in binding of a liver-specific transcription factor for its DNA binding site represent a mechanism for rapidly modulating acute phase gene transcription in vivo.

Animals↗
PubMed1996

Carcinoma of the minor salivary glands: results of surgery and combined therapy.

PURPOSE: This article reports the results of treatment of 7 histologic types of minor salivary gland carcinoma. PATIENTS AND METHODS: Of 256 cases, local excision was used in 194, extended excision in 62, excision accompanied by neck dissection in 98, and surgery with adjunctive radiation therapy or chemotherapy in 101. Forty-five of the 98 neck dissection cases had cervical node metastases. RESULTS: Survival rates were analyzed for 233 patients. The 3-year survival rate was (167 of 215) (77.67%), the 5-year survival rate was 134 of 187 (71.76%), and the 10-year survival rate was 79 of 113 (69.91%). CONCLUSIONS: These data confirm that surgery is still the mode of choice in the treatment of minor salivary gland carcinoma, but a combination with radiation therapy or chemotherapy is advisable in some patients.

Adenocarcinoma↗
PubMed1995

4-(Acetylamino)-3-hydroxy-5-nitrobenzoic acid.

The 4-(acetylamino)-3-hydroxy-5-nitrobenzoic acid molecule, C9H8N2O6, a designed inhibitor for the influenza virus neuraminidase protein, crystallizes as hydrogen-bonded dimers. The dihedral angles of the substituent groups with respect to the planar phenyl moiety are 5.0 (3) degrees for the carboxyl group, 45.0 (2) degrees for the nitro group and 37.3 (1) degrees for the acetylamino substituent. The crystal structure is stabilized by intermolecular hydrogen bonding.

Antiviral Agents↗
PubMed1995

Crystallization and preliminary X-ray analysis of two density populations of feline calicivirus.

Two density populations of full-size feline calicivirus (FCV), the intact infectious particles (P(H)) and the empty capsids (P(L)), have been crystallized using the hanging-drop method. Exposed to high-intensity synchrotron radiation, P(H) and P(L) crystals were shown to diffract X-rays to about 3.0 and 5.5 A resolution, respectively. The P(H) crystal belongs to an orthorhombic crystal system with unit-cell dimensions a = 889.0, b = 995.0, c = 436.6 A. Based on the V(M) value (3.4 A(3) Da(-l)), it was estimated that one crystallographic asymmetric unit of P(H) crystals contains the unique content of an entire virus particle, not necessarily from the same particle. This implies the presence of 60-fold non-crystallographic redundancy. The particle orientation was obtained from a locked rotation function.

Journal Article↗
PubMed1995

Structure-based inhibitors of influenza virus sialidase. A benzoic acid lead with novel interaction.

Influenza virus sialidase is a surface enzyme that is essential for infection of the virus. The catalytic site is highly conserved among all known influenza variants, suggesting that this protein is a suitable target for drug intervention. The most potent known inhibitors are analogs of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en), particularly the 4-guanidino derivative (4-guanidino-Neu5Ac2en). We utilized the benzene ring of 4-(N-acetylamino)benzoic acids as a cyclic template to substitute for the dihydropyran ring of Neu5Ac2en. In this study several 3-(N-acylamino) derivatives were prepared as potential replacements for the glycerol side chain of Neu5Ac2en, and some were found to interact with the same binding subsite of sialidase. Of greater significance was the observation that the 3-guanidinobenzoic acid derivative (equivalent to the 4-guanidino grouping of 4-guanidino-Neu5Ac2en), the most potent benzoic acid inhibitor of influenza sialidase thus far identified (IC50 = 10 microM), occupied the glycerol-binding subsite on sialidase as opposed to the guanidino-binding subsite. This benzoic acid derivative thus provides a new compound that interacts in a novel manner with the catalytic site of influenza sialidase.

Anti-Infective Agents↗
PubMed1995

Differentiation between homoeologous chromosomes 1A of wheat and 1Am of Triticum monococcum and its recognition by the wheat Ph1 locus.

In most allopolyploid plants, only homogenetic chromosome pairing occurs in meiosis, as a result of the recognition of genome differentiation by the genetic system regulating meiotic chromosome pairing. The nature of differentiation between chromosomes of closely related genomes is examined here by investigating recombination between wheat chromosome 1A and the closely related homoeologous chromosome 1Am of Triticum monococcum. The recognition of the differentiation between these chromosomes by the Ph1 locus, which prevents heterogenetic chromosome pairing in wheat, is also investigated. Chromosomes 1A and 1Am are shown to be colinear, and it is concluded that they are differentiated "substructurally." This substructural differentiation is argued to be recognized by the Ph1 locus. In the absence of Ph1, the distribution and frequencies of crossing over between the 1A and 1Am homoeologues were similar to the distribution and frequencies of crossing over between 1A homologues. The cytogenetic and evolutionary significance of these findings is discussed.

Journal Article↗
PubMed1995

Benzoic acid inhibitors of influenza virus neuraminidase.

A strategy was developed to design non-carbohydrate inhibitors of influenza virus neuraminidase. Using an iterative cycle of modeling, synthesis, biological testing and X-ray crystallography structure determination, a series of inhibitors based on benzoic acid were produced. The refined structures of three compounds complexed with neuraminidase are reported. The results demonstrate the success of this structure-based drug-design strategy.

Journal Article↗
PubMed1995

Structure of a monoclonal anti-ICAM-1 antibody R6.5 Fab fragment at 2.8 A resolution.

The specific binding of the monoclonal murine anti-intercellular adhesion molecule-1 (anti-ICAM-1) antibody, R6.5, inhibits the attachment of neutrophils to endothelium and prevents the attachment of major group human rhinovirus (HRV) to ICAM-1. This binding interferes with the host immune system and, as a result, the R6.5 antibody has been developed as a therapeutic anti-inflammatory and perhaps anti-HRV agent. The variable-region amino-acid sequence of R6.5 was determined from the anti-ICAM-1 cDNA. The crystallization conditions of the Fab fragment of R6.5 were established and the three-dimensional structure was determined by X-ray crystallography. The crystal space group is orthorhombic P2(1)2(1)2(1), a = 40.36, b = 137.76, c = 91.32 A, and the highest resolution of recorded reflections is 2.7 A. The molecular-replacement method using known Fab structures was employed to solve the R6.5 Fab structure. The final R-factor is 18.8% for a total of 3320 non-H protein atoms, 39 water molecules and 10 606 unique reflections. The protein exhibits the typical immunoglobulin fold. The surface contour of the antigen-combining site of the R6.5 antibody has a wide groove which resembles more the structure of an anti-polypeptide antibody than the structure of an anti-protein antibody.

Journal Article↗
PubMed1995

Structures of aromatic inhibitors of influenza virus neuraminidase.

Neuraminidase (NA), a surface glycoprotein of influenza virus, is a potential target for design of antiinfluenza agents. The crystal structure of influenza virus neuraminidase showed that in the active site 11 residues are universally conserved among all strains known so far. Several potent inhibitors based on the carbohydrate compound 2-deoxy-2,3-didehydro-D-N-acetylneuraminic acid (DANA) have been shown to bind to the conserved active site and to reduce virus infection in animals when administered by nasal spray. Inhibitors of this type are, however, rapidly excreted from physiological systems and may not be effective in order to provide long-time protection. A new class of specific NA inhibitors, which are benzoic acid derivatives, has been designed on the basis of the three-dimensional structure of the NA-DANA complex and modeling of derivatives of 4-(acetylamino)benzoic acid in the NA active site. Intermediates were synthesized and were shown to moderately inhibit the NA activity and to bind to the NA active site as predicted. These rudimentary inhibitors, 4-(acetylamino)-3-hydroxy-5-nitrobenzoic acid, 4-(acetylamino)-3-hydroxy-5-aminobenzoic acid, and 4-(acetylamino)-3-aminobenzoic acid, and their X-ray structures in complexes with N2 (A/Tokyo/3/67) and B/Lee/40 neuraminidases have been analyzed. The coordinates of such inhibitors complexed with NA were used as the starting model for further design of more potent benzoic acid inhibitors. Because the active site residues of NA are invariant, the designed aromatic inhibitors have the potential to become an antiviral drug against all strains of influenza virus.

Aminobenzoates↗
PubMed1995

A sialic acid-derived phosphonate analog inhibits different strains of influenza virus neuraminidase with different efficiencies.

A phosphonate analog of N-acetyl neuraminic acid (PANA) has been designed as a potential neuraminidase (NA) inhibitor and synthesized as both the alpha (ePANA) and beta (aPANA) anomers. Inhibition of type A (N2) and type B NA activity by ePANA was approximately a 100-fold better than by sialic acid, but inhibition of type A (N9) NA was only ten-fold better than by sialic acid. The aPANA compound was not a strong inhibitor for any of the NA strains tested. The crystal structures at 2.4 A resolution of ePANA complexed to type A (N2) NA, type A (N9) NA and type B NA and aPANA complexed to type A (N2) NA showed that neither of the PANA compounds distorted the NA active site upon binding. No significant differences in the NA-ePANA complex structures were found to explain the anomalous inhibition of N9 neuraminidase by ePANA. We put forward the hypothesis that an increase in the ePANA inhibition compared to that caused by sialic acid is due to (1) a stronger electrostatic interaction between the inhibitor phosphonyl group and the active site arginine pocket and (2) a lower distortion energy requirement for binding of ePANA.

Binding Sites↗
PubMed1995

Crystallization and preliminary X-ray diffraction studies of the cartilage link protein from bovine trachea.

Cartilage extracellular matrix link protein, having molecular mass of approximately 40 kDa, is a metalloprotein that binds divalent cations and is only soluble in low ionic strength solutions. The link protein was purified from bovine trachea and has been crystallized by a vapor diffusion method using PEG 3350 as precipitant. The crystal symmetry is P1, and the unit cell dimensions are a = 43.55, b = 53.11, c = 60.10 A, alpha = 90.44, beta = 106.21, gamma = 101.51 degrees. The VM of 1.8 A3/Da is consistent with the presence of two molecules of the link protein in the asymmetric unit. The crystals diffract X-rays from a synchrotron source to 1.7 A resolution.

Animals↗
PubMed1995

A strategy for theoretical binding constant, Ki, calculations for neuraminidase aromatic inhibitors designed on the basis of the active site structure of influenza virus neuraminidase.

Neuraminidase (NA) is one of the two major surface antigens of influenza virus. It plays an indispensable role in the release and spread of progeny virus particles during infection. NA inhibitors reduce virus infection in animals. To improve the clinical efficacy of NA inhibitors, we have begun the design of non-carbohydrate inhibitors based on the active site structure of NA. The approach is an iterative process of ligand modeling and electrostatic calculations followed by chemical synthesis of compounds, biological testing, and NA-inhibitor complex structure determination by X-ray crystallography. A strategy has been developed to calculate Ki for newly designed inhibitors. The calculations using the DelPhi program were performed for carbohydrate inhibitors and three preliminary benzoic acid inhibitors of neuraminidase (BANA) that have been synthesized and shown to bind to the active site of NA in the crystal structure. The calculated Kis of these inhibitors have an enlightening agreement with their in vitro biological activities. This demonstrates that the calculations produce informative results on the affinity of modeled inhibitors. GRID maps were also calculated and several pockets were identified for accepting possible new ligands. The calculated Kis for newly designed ligands suggest that these potential compounds will have high inhibitory activities.

Aminobenzoates↗
PubMed1994

Structure of influenza virus neuraminidase B/Lee/40 complexed with sialic acid and a dehydro analog at 1.8-A resolution: implications for the catalytic mechanism.

Neuraminidase is one of the two glycoprotein spikes protruding from the influenza virus membrane. We have determined by X-ray crystallography the native structure of B/Lee/40 neuraminidase (NA) and the structures of its crystals soaked with a substrate, N-acetylneuraminyllactose (NANL), and an inhibitor, 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) at 1.8-A resolution. NANL was hydrolyzed by the crystalline NA to generate the product N-acetylneuraminic acid (NANA, also known as sialic acid), which is still able to bind to NA. In the difference Fourier map of the presumed NA-NANA complex, the moiety bound in the active site had a distorted boat conformation of NANA, but there is no significant electron density for O2. The structure of the bound moiety is not identical to that of chemically synthesized DANA soaked into NA crystals. Prolonged incubation of NANA with NA in solution at room temperature produced only a trace amount of DANA as detected by NMR. On the basis of our studies, a mechanism is proposed for the enzymatic hydrolysis by influenza virus neuraminidase.

Binding Sites↗
PubMed1994

A reliable amplification technique with single-sided specificity for the isolation of 5' gene-regulating regions.

A simple and efficient method is described for the isolation of extension fragments of known DNA sequences by polymerase chain reaction (PCR) using a single specific primer. With this method, size-selected genomic DNA fragments are ligated to a plasmid vector (pGEM-4Z) which contains sequencing primers and the population of chimeric plasmids is used for transforming Escherichia coli. DNA is extracted from an aliquot of the resulting mini-library and PCR performed using a sequence-specific primer and either of the standard sequencing primers of the plasmid vector. This method appears to be more versatile than inverse PCR (IPCR), since: (i) the DNA sequence needed as the specific primer can be as short as about 20 nucleotides (nt); (ii) the DNA templates to be used in PCR are available in high amount, thus facilitating all manipulations; and (iii) if relinearization of the DNA by restriction enzyme digestion is desired before the PCR reaction, many restriction sites can be chosen from the vector polylinker. Using this method, we have isolated the genomic 5' region of the carrot bifunctional dihydrofolate reductase-thymidylate synthase-encoding gene dhfr-ts using a 21-nt sequence of the 5' region of the dhfr-ts cDNA clone as the specific primer.

Base Sequence↗