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H K Song

Publications and source records attributed to H K Song.

At least 19 recordsLinked to original sources
PubMed1997

Crystal structure of carboxylesterase from Pseudomonas fluorescens, an alpha/beta hydrolase with broad substrate specificity.

BACKGROUND: A group of esterases, classified as carboxylesterases, hydrolyze carboxylic ester bonds with relatively broad substrate specificity and are useful for stereospecific synthesis and hydrolysis of esters. One such carboxylesterase from Pseudomonas fluorescens is a homodimeric enzyme, consisting of 218-residue subunits. It shows a limited sequence similarity to some members of the alpha/beta hydrolase superfamily. Although crystal structures of a number of serine esterases and lipases have been reported, structural information on carboxylesterases is very limited. This study was undertaken in order to provide such information and to understand a structural basis for the substrate specificity of this carboxylesterase. RESULTS: In this study, the crystal structure of carboxylesterase from P. fluorescens has been determined by the isomorphous replacement method and refined to 1.8 A resolution. Each subunit consists of a central seven-stranded beta sheet flanked by six alpha helices. The structure reveals the catalytic triad as Ser 114-His 199-Asp 168. The structure of the enzyme in complex with the inhibitor phenylmethylsulfonyl fluoride has also been determined and refined to 2.5 . The inhibitor is covalently attached to Ser 114 of both subunits, with the aromatic ring occupying a hydrophobic site defined by the aliphatic sidechains of Leu23, Ile58, Ile70, Met73 and Val170. No large structural changes are observed between the free and inhibitor-bound structures. CONCLUSIONS: Carboxylesterase from P. fluorescens has the alpha/beta hydrolase fold and the Ser-His-Asp catalytic triad. The active-site cleft in each subunit is formed by the six loops covering the catalytic serine residue. Three of the active-site loops in each subunit are involved in a head-to-head subunit interaction to form a dimer; it may be these extra structural elements, not seen in other esterases, that account for the inability of carboxylesterase to hydrolyze long chain fatty acids. As a result of dimerization, the active-site clefts from the two subunits merge to form holes in the dimer. The active-site clefts are relatively open and thus the catalytic residues are exposed to the solvent. An oxyanion hole, formed by nitrogen atoms of Leu23 and Gln115, is present in both the free and inhibitor-bound structures. An open active site, as well as a large binding pocket for the acid part of substrates, in P. fluorescens carboxylesterase may contribute to its relatively broad substrate specificity.

Anions↗
PubMed1997

Crystal structure of thermostable alpha-amylase from Bacillus licheniformis refined at 1.7 A resolution.

alpha-Amylases (alpha-1,4-glucan-4-glucanohydrolase, E.C.3.2.1.1) catalyze the cleavage of alpha-1, 4-glucosidic linkages of starch components, glycogen, and various oligosaccharides. Thermostable alpha-amylases from Bacillus species are of great industrial importance in the production of corn syrup or dextrose. Thermostable alpha-amylase from Bacillus licheniformis, a monomeric enzyme with molecular mass of 55,200 Da (483 amino acid residues), shows a remarkable heat stability. This enzyme provides an attractive model for investigating the structural basis for thermostability of proteins. The three-dimensional structure of thermostable alpha-amylase from Bacillus licheniformis has been determined by the multiple isomorphous replacement method of X-ray crystallography. The structure has been refined to a crystallographic R-factor of 19.9% for 58,601 independent reflections with F0 > 2 sigma F0 between 8.0 and 1.7 A resolution, with root mean square deviations of 0.013 A from ideal bond lengths and 1.72 degrees from ideal bond angles. The final model consists of 469 amino acid residues and 294 water molecules. Missing from the model are the N- and C-termini and the segment between Trp182 and Asn192. Like other alpha-amylases, the polypeptide chain folds into three distinct domains. The first domain (domain A), consisting of 291 residues (from residue 3 to 103 and 207 to 396), forms a (beta/alpha)8-barrel structure. The second domain (domain B), consisting of residues 104 to 206, is inserted between the third beta-strand and the third alpha-helix of domain A. The third C-terminal domain (domain C), consisting of residues 397 to 482, folds into an eight-stranded antiparallel beta-barrel. Neither calcium ion nor chloride ion is located near the active site. This study reveals the architecture of the thermostable alpha-amylase from Bacillus licheniformis. By homology with other alpha-amylases, important active site residues can be identified as Asp231, Glu261, and Asp328, which are all located at the C-terminal end of the central (beta/alpha)8-barrel. Since many of the stabilizing and destabilizing mutations obtained so far fall in domain B or at its border, this region of the enzyme appears to be important for thermostability. The factors responsible for the remarkable thermostability of this enzyme may be increased ionic interactions, reduced surface area, and increased packing interactions in the interior.

Amino Acid Sequence↗
PubMed1997

The crystal structure of a triacylglycerol lipase from Pseudomonas cepacia reveals a highly open conformation in the absence of a bound inhibitor.

BACKGROUND: . Lipases, a family of enzymes which catalyze the hydrolysis of triglycerides, are widely distributed in many organisms. True lipases are distinguished from esterases by the characteristic interfacial activation they exhibit at an oil-water interface. Lipases are one of the most frequently used biocatalysts for organic reactions performed under mild conditions. Their biotechnological applications include food and oil processing and the preparation of chiral intermediates for the synthesis of enantiomerically pure pharmaceuticals. Recent structural studies on several lipases have provided some clues towards understanding the mechanisms of hydrolytic activity, interfacial activation, and stereoselectivity. This study was undertaken in order to provide structural information on bacterial lipases, which is relatively limited in comparison to that on the enzymes from other sources. RESULTS: . We have determined the crystal structure of a triacylglycerol lipase from Pseudomonas cepacia (PcL) in the absence of a bound inhibitor using X-ray crystallography. The structure shows the lipase to contain an alpha/beta-hydrolase fold and a catalytic triad comprising of residues Ser87, His286 and Asp264. The enzyme shares several structural features with homologous lipases from Pseudomonas glumae (PgL) and Chromobacterium viscosum (CvL), including a calcium-binding site. The present structure of PcL reveals a highly open conformation with a solvent-accessible active site. This is in contrast to the structures of PgL and PcL in which the active site is buried under a closed or partially opened 'lid', respectively. CONCLUSIONS: . PcL exhibits some structural features found in other lipases. The presence of the Ser-His-Asp catalytic triad, an oxyanion hole, and the opening of a helical lid suggest that this enzyme shares the same mechanisms of catalysis and interfacial activation as other lipases. The highly open conformation observed in this study is likely to reflect the activated form of the lipase at an oil-water interface. The structure suggests that the interfacial activation of bacterial lipases involves the reorganization of secondary structures and a large movement of the lid to expose the active site. This is similar to the mechanism described for other well characterized fungal and mammalian lipases.

Amino Acid Sequence↗
PubMed1997

In vivo MR microscopy of the human skin.

The requirements for imaging the skin are dictated by the organ's layered structure, which extends only a few millimeters from the surface and thus demands extremely high resolution in this direction. While less critical, resolution in the remaining two dimensions determines whether the skin's accessory structures can be resolved. The problem is compounded by short transverse relaxation times, in particular of the dermis, the structure of most clinical interest. In this work images of the normal human skin were obtained in vivo at voxel sizes as small as 19 x 78 x 800 microm3, by means of customized 3D gradient and partial flip-angle spin-echo pulse sequences and very small transmit/receive coils on a 1.5T clinical imager equipped with high-power whole-body gradients. Structures resolved include hair follicles and the sublayers of the dermis. The very short time constant for the major component (91%) for transverse relaxation in the dermis (T2* approximately 10 ms) suggests the potential of substantial gains in achievable signal-to-noise ratio by shortening the echo time.

Adipose Tissue↗
PubMed1997

A single-scan imaging technique for measurement of the relative concentrations of fat and water protons and their transverse relaxation times.

A two-component chemical-shift-imaging technique is described from which fat and water images can be obtained in a single scan and in the presence of an inhomogeneous field. In addition, the method provides transverse relaxation rates R2 and R2' separately for each of the spectral components. The method is a combination and extension of the GESFIDE [gradient echo sampling of FID and echo, J. Ma and F. W. Wehrli, J. Magn. Reson. B 111, 61 (1996)] and the multipoint Dixon techniques. It is based on sampling the descending and ascending portions of a Hahn spin echo with a train of gradient echoes which are spaced at one-half of the chemical-shift modulation period. Processing of the complex echo data, involving an automated phase unwrapping algorithm, affords relative amplitudes and transverse relaxation rates of the two spectral components. An additional benefit of the method is its superior signal-to-noise ratio resulting from echo summation. Applications targeted and illustrated involve MRI osteodensitometry of trabecular bone in the presence of varying fractions of hematopoietic and fatty bone marrow.

Adult↗
PubMed1997

Effects of single and repeated administration of sulthiame on amygdaloid kindled seizures in rats.

In this study, we assessed the anti-convulsive effects of sulthiame (SUL) in amygdaloid (AM) kindled rats. Electrodes were implanted into the left AM of adult male Wistar rats. The animals were kindled at the after-discharge (AD) threshold. Upon completion of kindling, a generalized seizure triggering threshold was determined. The drugs were administered intraperitoneally in rats which reproducibly exhibited generalized convulsions at the near-threshold stimulation. Single administration of SUL (25-200 mg; n = 7-9) reduced the forelimb clonus (FCL) duration, but only the highest dose significantly regressed the secondarily generalized convulsion. During repeated administration of SUL, 50 mg/kg for 8 days, FCL duration was significantly alleviated until the fifth treatment day. With the dose of 200 mg/kg, significant suppression of secondary generalization was noted only until the second test day. On the other hand, significant reductions of FCL and AD duration were preserved afterwards. The anti-convulsive effects of SUL indicated in this study were not comparable to those of other standard anti-epileptic drugs reported from our laboratory.

Amygdala↗
PubMed1997

Acute Myelitis after hepatitis B vaccination.

We report a case of myelitis after plasma-derived hepatitis B vaccination. The patient was a 31-year-old man who presented with progressive sensory symptoms in extremities that developed 2 weeks after a third vaccination. MRI of the cervicothoracic region revealed swelling and T2 high signal at the level of C4 to C5 cord, and isolated enhancement in the posterior columns between C4 and C5 cord. The significance of MRI findings and HLA haplotype of the patient will be briefly discussed.

Acute Disease↗
PubMed1996

Dorsal dermis of the scaleless (sc/sc) embryo directs normal feather pattern formation until day 8 of development.

We have examined the ability of the scaleless (sc/sc) backskin dermis (6 to 16 days of incubation) to regulate pattern formation using the presumptive scutate scale epidermis from 11-day normal embryos as the responding tissue. Prior to 8 days of incubation the sc/sc backskin dermis is able to induce hexagonally patterned and uniformly oriented feather germs in normal epidermis. This ability is lost during day 8 and follows a central to lateral gradient. Such gradients are characteristic of normal feather development in the spinal tract. We discuss the change in the inductive ability of the sc/sc dermis in relation to the stabilization of the feather pattern, which occurs all at once throughout the dorsal dermis at 7.5-8 days of development. After day 8 until day 10, the sc/sc backskin dermis only supports the formation of sporadic, unpatterned feather germs; thereafter it will not support feather formation.

Animals↗
PubMed1996

Fast 3D large-angle spin-echo imaging (3D FLASE).

A rapid steady-state 3D spin-echo imaging pulse sequence, based on the principle of nutating the spins by an angle greater than 90 degrees, has been designed and implemented on a clinical 1.5-T whole-body MR scanner. The pulse sequence, denoted fast large-angle spin-echo (FLASE), has been optimized for high-resolution imaging of tissues with short T2 and T2*. Features of FLASE include a minimum-phase Shinnar-Le Roux excitation pulse and distribution of phase- and slice-encoding gradients before and after the 180 degrees refocusing pulse to minimize the critical time delay between inversion and restoration of the residual longitudinal magnetization and for minimizing echo time. A Bloch equation analysis, corroborated by experimental data, shows FLASE signal-to-noise to be superior to its closest analog, 3D rapid spin-echo excitation (RASEE) (Jara et al., Magn Reson Medicine 29, 528 (1993)), and 3D gradient-recalled acquisition in steady state (GRASS). It is demonstrated that with judicious RF phase-cycling and steady state operation, FLASE can produce high-quality microimages free of intravoxel phase dispersion from susceptibility-induced background gradients. The performance of the method is exemplified with ultra high-resolution images of trabecular bone in vitro and in vivo in the human calcaneus and wrist at voxel sizes as low as 98 x 98 x 200 microns3. Finally, the contrast behavior of refocused FLASE can be altered by disrupting the steady state analogous to gradient echo imaging.

Bone and Bones↗
PubMed1996

Crystal structure analyses of uncomplexed ecotin in two crystal forms: implications for its function and stability.

Ecotin, a homodimeric protein composed of 142 residue subunits, is a novel serine protease inhibitor present in Escherichia coli. Its thermostability and acid stability, as well as broad specificity toward proteases, make it an interesting protein for structural characterization. Its structure in the uncomplexed state, determined for two different crystalline environments, allows a structural comparison of the free inhibitor with that in complex with trypsin. Although there is no gross structural rearrangement of ecotin when binding trypsin, the loops involved in binding trypsin show relatively large shifts in atomic positions. The inherent flexibility of the loops and the highly nonglobular shape are the two features essential for its inhibitory function. An insight into the understanding of the structural basis of thermostability and acid stability of ecotin is also provided by the present structure.

Amino Acid Sequence↗
PubMed1996

Reverse transcriptase-polymerase chain reaction for prostate-specific antigen may be a prognostic indicator in breast cancer.

Among women with node-negative breast cancer and small tumours, it is important to identify those with tumours that will recur, so that they may receive adjuvant therapy, while sparing those with tumours that will not recur the hazards of adjuvant treatment. A reverse transcriptase-polymerase chain reaction (RT-PCR) for prostate-specific antigen (PSA) may be used to identify circulating metastatic cells in patients with prostate cancer. Approximately 30% of breast cancer cells also produce PSA. Therefore, we tested the PSA RT-PCR assay on blood specimens from women with breast cancer. We evaluated 78 women at Mount Sinai Medical Center with histologically confirmed breast cancer. Venous blood (5 cm3) from the women was collected in ethylene diaminetetraacetic acid (EDTA)-treated collection tubes and approximately 400 ng of RNA from each sample was subjected to an RT-PCR. We were able to detect the amplified PSA fragment in 18 of 78 women with breast cancer; 7 of the 18 women with the PSA fragment had localised, small, node-negative tumours, both oestrogen receptor (ER) positive and ER negative. We could not detect the amplified PSA fragment in 20 normal women and 22 normal men. We conclude that PSA RT-PCR may be a useful method for determining the presence of circulating metastatic cells in some women with node-negative breast cancer, and therefore the potential for these women to develop recurrent disease and thus benefit from adjuvant therapy.

Adult↗
PubMed1996

In-vitro and in-vivo antibacterial activity of LB10517, a novel catechol-substituted cephalosporin with a broad antibacterial spectrum.

LB10517 is a new injectable cephalosporin with a broad spectrum of antibacterial activity against Gram-positive and Gram-negative bacteria. LB10517 inhibited 90% of methicillin-susceptible Staphylococcus aureus (MSSA) at 0.25 mg/L (MIC90), and was 8-fold more active than cefpirome. LB10517 was two- or four-fold more active than cefpirome against methicillin-susceptible Staphylococcus epidermidis (MSSE), Streptococcus pyogenes and Enterococcus faecalis. Both methicillin-resistant S. aureus (MRSA) and methicillin-resistant S. epidermidis (MRSE) were highly resistant to all compounds. LB10517 activity against most Enterobacteriaceae was comparable to or greater than that of cefpirome, and it also showed high activity against Pseudomonas aeruginosa. In a mouse septicaemia model, LB10517 exhibited excellent protective effects. In a respiratory tract infection model, the protective effect of LB10517 was comparable to that of cefpirome and ceftazidime. It was highly stable to hydrolysis by beta-lactamases, showing better physiological efficiency for TEM-9 than the other test compounds. LB10517 had the most potent antibacterial activity against beta-lactamase producing resistant strains. LB10517 did not induce beta-lactamase production in Enterobacter cloacae 1194E.

Animals↗
PubMed1995

Crystal structure of an uncleaved alpha 1-antitrypsin reveals the conformation of its inhibitory reactive loop.

The crystal structure of a recombinant human alpha 1-antitrypsin, in the uncleaved and uncomplexed state, has been determined by X-ray crystallographic methods and refined to an R-factor of 18.4% for 8.0-3.46 A data with good stereochemistry. This structure provides the first view at the inhibitory loop and the central beta-sheet A of the uncleaved alpha 1-antitrypsin. The reactive loop takes a distorted helical conformation and no pre-insertion of two residues in the reactive loop into the beta-sheet A is observed. The present structure is largely in agreement with the model predicted by Engh, Wright, and Huber [Prot. Eng. 3 (1990) 469-477].

Binding Sites↗
PubMed1995

Crystallization, molecular replacement solution, and refinement of tetrameric beta-amylase from sweet potato.

Sweet potato beta-amylase is a tetramer of identical subunits, which are arranged to exhibit 222 molecular symmetry. Its subunit consists of 498 amino acid residues (Mr 55,880). It has been crystallized at room temperature using polyethylene glycol 1500 as precipitant. The crystals, growing to dimensions of 0.4 mm x 0.4 mm x 1.0 mm within 2 weeks, belong to the tetragonal space group P4(2)2(1)2 with unit cell dimensions of a = b = 129.63 A and c = 68.42 A. The asymmetric unit contains 1 subunit of beta-amylase, with a crystal volume per protein mass (VM) of 2.57 A3/Da and a solvent content of 52% by volume. The three-dimensional structure of the tetrameric beta-amylase from sweet potato has been determined by molecular replacement methods using the monomeric structure of soybean enzyme as the starting model. The refined subunit model contains 3,863 nonhydrogen protein atoms (488 amino acid residues) and 319 water oxygen atoms. The current R-value is 20.3% for data in the resolution range of 8-2.3 A (with 2 sigma cut-off) with good stereochemistry. The subunit structure of sweet potato beta-amylase (crystallized in the absence of alpha-cyclodextrin) is very similar to that of soybean beta-amylase (complexed with alpha-cyclodextrin). The root-mean-square (RMS) difference for 487 equivalent C alpha atoms of the two beta-amylases is 0.96 A. Each subunit of sweet potato beta-amylase is composed of a large (alpha/beta)8 core domain, a small one made up of three long loops [L3 (residues 91-150), L4 (residues 183-258), and L5 (residues 300-327)], and a long C-terminal loop formed by residues 445-493. Conserved Glu 187, believed to play an important role in catalysis, is located at the cleft between the (alpha/beta)8 barrel core and a small domain made up of three long loops (L3, L4, and L5). Conserved Cys 96, important in the inactivation of enzyme activity by sulfhydryl reagents, is located at the entrance of the (alpha/beta)8 barrel.

Amino Acid Sequence↗
PubMed1994

Pattern formation in chick feather development: distribution of beta 1-integrin in normal and scaleless embryos.

We have examined the immunolocalization of beta 1-integrin during feather development in the spino-lumbar tract of the backskin from normal and scaleless chick embryos. beta 1-integrin appears during early feather development in three distinct phases which correspond to important developmental events. The first phase (5-5 1/2 days of incubation; Hamburger and Hamilton [H.H.] stage 27) represents the period prior to the formation of dermis. During this phase, beta 1-integrin antiserum labels mesenchymal cells located in the central region of the spino-lumbar tract where the initiation site for feather development is located. The second phase (5 1/2-7 1/2 days of incubation; H.H. stages 28-32) corresponds to the period during which dermis is formed. The cells that make up the dermis are readily distinguished by their lack of beta 1-integrin immunostaining. The third phase (7 1/2-10 days of incubation; H.H. stages 33-36) begins with the sudden appearance of beta 1-integrin in the central and lateral regions of the dermis. The pattern of beta 1-integrin immunostaining in scaleless backskin becomes different from that of normal backskin during this phase. In normal backskin the dermal condensations of feather germs are not labeled with the beta 1-integrin antiserum. This produces a heterogeneous immunostaining pattern very similar to the pattern seen for Type I collagen (Mauger et al. [1982] Dev. Biol. 94:93-105). In contrast, homogeneous immunostaining is observed in the dermis of scaleless backskin. The initial time of appearance, manner of appearance, and pattern of integrin expression in the third phase suggest that beta 1-integrin may be involved in the stabilization of the feather pattern. We also observed the appearance of beta 1-integrin on the epidermal basal cells during the time of feather follicle formation. The beta 1-integrin antiserum reacts strongly with the baso-lateral surfaces of normal basal cells, yet the basal surfaces of the scaleless basal cells are unstained. This lack of immunostaining along the basal surfaces of the scaleless basal cells may relate to the abnormal adhesion between the epidermis and dermis in scaleless backskin.

Animals↗
PubMed1994

Histopathologic changes in the olfactory epithelium in mice after exposure to sulfur dioxide.

To investigate the effects of sulfur dioxide (SO2) on olfactory epithelium, an experiment was performed with 56 mice from the same colony. Experimental animals were divided into three groups consisting of a 30-min exposure group (group 1), a 60-min exposure group (group 2), and a 120-min exposure group (group 3). The olfactory mucosa in these mice were studied by light microscopy immediately, and after 24 h, 48 h, or 72 h exposure to 20 ppm of SO2. Edema, loss of cilia, epithelial thinning, and epithelial desquamation in the olfactory epithelium were observed in groups 2 and 3. The basal lamina and the connective tissue were well preserved throughout the entire mucosa. Injuries to olfactory epithelium became severer with exposure time. These changes were further pronounced 24 h after exposure. Regenerated epithelia were not observed in any group. Scanning electron microscopic findings were consistent with light microscopic findings. Olfactory epithelial surface were consistent with light microscopic findings. Olfactory epithelial surface was sloughed off and revealed, underlining intact basal lamina. The results of this study suggest that early lesions of olfactory epithelium after exposure to SO2 may be primarily degenerative.

Administration, Inhalation↗
PubMed1993

Crystallization and preliminary X-ray crystallographic analysis of chitinase from barley seeds.

Chitinase from barley seeds has been crystallized at room temperature using polyethylene glycol as precipitant. The crystal is monoclinic, belonging to the space group P2(1), with unit cell parameters of a = 69.43 A, b = 44.55 A, c = 81.41 A, and beta = 111.95 degrees. The asymmetric unit seems to contain two molecules of chitinase with a corresponding crystal volume per protein mass (VM) of 2.25 A3/Da and a solvent content of 45% by volume. The crystal diffracts to at least 2.0 A with X-rays from a rotating anode source and is very stable in the X-ray beam. X-ray data have been collected to better than 2.2 A Bragg spacing from a native crystal.

Chitinases↗