Biomedical subjects

H K Song

Publications and source records attributed to H K Song.

At least 55 records · Page 3Linked to original sources

PubMed1999

Crystal structure of a 16 kDa double-headed Bowman-Birk trypsin inhibitor from barley seeds at 1.9 A resolution.

The Bowman-Birk trypsin inhibitor from barley seeds (BBBI) consists of 125 amino acid residues with two inhibitory loops. Its crystal structure in the free state has been determined by the multiwavelength anomalous diffraction (MAD) method and has been refined to a crystallographic R-value of 19.1 % for 8.0-1.9 A data. This is the first report on the structure of a 16 kDa double-headed Bowman-Birk inhibitor (BBI) from monocotyledonous plants and provides the highest resolution picture of a BBI to date. The BBBI structure consists of 11 beta-strands and the loops connecting these beta-strands but it lacks alpha-helices. BBBI folds into two compact domains of similar tertiary structure. Each domain shares the same overall fold with 8 kDa dicotyledonous BBIs. The five disulfide bridges in each domain are a subset of the seven disulfide bridges in 8 kDa dicotyledonous BBIs. Two buried water molecules form hydrogen bonds to backbone atoms in the core of each domain. One interesting feature of this two-domain inhibitor structure is that the two P1 residues (Arg17 and Arg76) are approximately 40 A apart, allowing the two reactive-site loops to bind to and to inhibit two trypsin molecules simultaneously and independently. The conformations of the reactive-site loops of BBBI are highly similar to those of other substrate-like inhibitors. This structure provides the framework for modeling of the 1:2 complex between BBBI and trypsin.

Amino Acid Sequence↗

PubMed1999

Insights into eukaryotic multistep phosphorelay signal transduction revealed by the crystal structure of Ypd1p from Saccharomyces cerevisiae.

"Two-component" phosphorelay signal transduction systems constitute a potential target for antibacterial and antifungal agents, since they are found exclusively in prokaryotes and lower eukaryotes (yeast, fungi, slime mold, and plants) but not in mammalian organisms. Saccharomyces cerevisiae Ypd1p, a key intermediate in the osmosensing multistep phosphorelay signal transduction, catalyzes the phosphoryl group transfer between response regulators. Its 1.8 A structure, representing the first example of a eukaryotic phosphorelay protein, contains a four-helix bundle as in the HPt domain of Escherichia coli ArcB sensor kinase. However, Ypd1p has a 44-residue insertion between the last two helices of the helix bundle. The side-chain of His64, the site of phosphorylation, protrudes into the solvent. The structural resemblance between Ypd1p and ArcB HPt domain suggests that both prokaryotes and lower eukaryotes utilize the same basic protein fold for phosphorelay signal transduction. This study sheds light on the best characterized eukaryotic phosphorelay system.

Amino Acid Sequence↗

PubMed1999

Tracking alloreactive cell division in vivo.

BACKGROUND: The study of alloimmune responses has been limited by a lack of assays that can track the behavior of alloreactive lymphocytes in vivo. Here we utilize an experimental system that allows the identification and study of alloreactive CD4+ lymphocytes responding to major histocompatibility antigens in vivo. METHODS: Responder mouse lymphocytes were labeled with a fluorescein-based dye, adoptively transferred into irradiated allogeneic stimulator mice, and recovered at serial time points for analysis by flow cytometry. RESULTS: Discrete generations of CD4+ responder lymphocytes proliferating specifically in response to allogeneic MHC class II were distinguished by fluorescein intensity. Successive division of alloreactive CD4+ lymphocytes was traced up to six generations after 60 hr. CONCLUSIONS: This experimental system provides information on the division kinetics of alloreactive CD4+ cells. Other applications include immunophenotyping of alloreactive lymphocyte subsets. Further study of systems such as this will allow the detailed characterization of how alloimmune responses are initiated and proceed in vivo.

Animals↗

PubMed1999

I-Ag7-mediated antigen presentation by B lymphocytes is critical in overcoming a checkpoint in T cell tolerance to islet beta cells of nonobese diabetic mice.

B cell-deficient nonobese diabetic (NOD) mice are protected from the development of spontaneous autoimmune diabetes, suggesting a requisite role for Ag presentation by B lymphocytes for the activation of a diabetogenic T cell repertoire. This study specifically examines the importance of B cell-mediated MHC class II Ag presentation as a regulator of peripheral T cell tolerance to islet beta cells. We describe the construction of NOD mice with an I-Ag7 deficiency confined to the B cell compartment. Analysis of these mice, termed NOD BCIID, revealed the presence of functionally competent non-B cell APCs (macrophages/dendritic cells) with normal I-Ag7 expression and capable of activating Ag-reactive T cells. In addition, the secondary lymphoid organs of these mice harbored phenotypically normal CD4+ and CD8+ T cell compartments. Interestingly, whereas control NOD mice harboring I-Ag7-sufficient B cells developed diabetes spontaneously, NOD BCIID mice were resistant to the development of autoimmune diabetes. Despite their diabetes resistance, histologic examination of pancreata from NOD BCIID mice revealed foci of noninvasive peri-insulitis that could be intentionally converted into a destructive process upon treatment with cyclophosphamide. We conclude that I-Ag7-mediated Ag presentation by B cells serves to overcome a checkpoint in T cell tolerance to islet beta cells after their initial targeting has occurred. Overall, this work indicates that the full expression of the autoimmune potential of anti-islet T cells in NOD mice is intimately regulated by B cell-mediated MHC class II Ag presentation.

Animals↗

PubMed1999

Contribution of the innate immune system to autoimmune diabetes: a role for the CR1/CR2 complement receptors.

B lymphocytes are required for diabetogenesis in nonobese diabetic (NOD) mice. The complement component of the innate immune system regulates B cell activation and tolerance through complement receptors CR1/CR2. Thus, it is important to assess the contribution of complement receptors to autoimmune diabetes in NOD mice. Examination of the lymphoid compartments of NOD mice revealed striking expansion of a splenic B cell subset with high cell surface expression of CR1/CR2. This subset of B cells exhibited an enhanced C3 binding ability. Importantly, long-term in vivo blockade of C3 binding to CR1/CR2 prevented the emergence of the CR1/CR2(hi) B cells and afforded resistance to autoimmune diabetes in NOD mice. These findings implicate complement as an important regulatory element in controlling the T cell-mediated attack on islet beta cells of NOD mice.

Animals↗

PubMed1999

A direct method for the calculation of alloreactive CD4+ T cell precursor frequency.

BACKGROUND: Direct measurement of the precursor frequency of alloreactive CD4+ T cells has been impossible due to the lack of a specific means of determining the absolute number of daughter cells generated with each division in a repertoire of stimulated T cells. METHODS: Responder lymphocytes were fluorescently labeled and adoptively transferred into irradiated allogeneic stimulator mice or incubated in vitro with irradiated stimulator splenocytes. After a 65- to 70-hr stimulation period, responder cells were analyzed by flow cytometry. RESULTS: The precursor frequency of dividing CD4+ T cells was determined both in vivo and in vitro. The observed number of alloreactive daughter cells generated with each round of division was used to calculate the frequency of alloantigen-specific CD4+ T cells. CONCLUSIONS: A novel method for the direct calculation of the frequency of alloreactive CD4+ T cells is described. This technique allows the determination of changes in the frequency of alloreactive T cells that might underlie tolerance to alloantigens.

Animals↗

PubMed1999

Crystal structure of deoxycytidylate hydroxymethylase from bacteriophage T4, a component of the deoxyribonucleoside triphosphate-synthesizing complex.

Bacteriophage T4 deoxycytidylate hydroxymethylase (EC 2.1.2.8), a homodimer of 246-residue subunits, catalyzes hydroxymethylation of the cytosine base in deoxycytidylate (dCMP) to produce 5-hydroxymethyl-dCMP. It forms part of a phage DNA protection system and appears to function in vivo as a component of a multienzyme complex called deoxyribonucleoside triphosphate (dNTP) synthetase. We have determined its crystal structure in the presence of the substrate dCMP at 1.6 A resolution. The structure reveals a subunit fold and a dimerization pattern in common with thymidylate synthases, despite low (approximately 20%) sequence identity. Among the residues that form the dCMP binding site, those interacting with the sugar and phosphate are arranged in a configuration similar to the deoxyuridylate binding site of thymidylate synthases. However, the residues interacting directly or indirectly with the cytosine base show a more divergent structure and the presumed folate cofactor binding site is more open. Our structure reveals a water molecule properly positioned near C-6 of cytosine to add to the C-7 methylene intermediate during the last step of hydroxymethylation. On the basis of sequence comparison and crystal packing analysis, a hypothetical model for the interaction between T4 deoxycytidylate hydroxymethylase and T4 thymidylate synthase in the dNTP-synthesizing complex has been built.

Amino Acid Sequence↗

PubMed1999

Cutting edge: alloimmune responses against major and minor histocompatibility antigens: distinct division kinetics and requirement for CD28 costimulation.

Comparative study of alloimmune responses against major and minor histocompatibility Ags has been limited by the lack of suitable assays. Here, we use a bioassay that permits tracking of alloreactive CD4+ T cell populations as they proliferate in response to major or minor histocompatibility Ags in vivo. Division of alloreactive CD4+ T cells proceeded more rapidly in response to major histocompatibility Ags than minor Ags, although CD4+ T cells alloreactive to minor Ags had a similar capacity to divide successively up to eight times after stimulation. Allorecognition of minor histocompatibility Ags was highly dependent on CD28 costimulation, with the frequency of CD4+ T cells proliferating in response to minor Ags in the absence of CD28 costimulation reduced up to 20-fold. These findings highlight differences in signaling processes that lead to allorecognition of major and minor histocompatibility Ags and have implications on the design of interventions aimed at abrogating these responses.

Animals↗

PubMed1999

A thermostable xylose isomerase from Thermus caldophilus: biochemical characterization, crystallization and preliminary X-ray analysis.

A highly thermostable xylose isomerase from Thermus caldophilus has been expressed in Escherichia coli. The purified enzyme has an optimum temperature of 363 K. It has been crystallized at room temperature using ammonium sulfate as a precipitant. The crystal belongs to the orthorhombic space group P212121, with unit-cell parameters a = 84.35, b = 123.60, c = 140.24 A. The presence of one molecule of tetrameric xylose isomerase in the asymmetric unit gives a crystal volume per protein mass (Vm) of 2.1 A3 Da-1 and a solvent content of 41% by volume. The crystals initially showed diffraction to 1.7 A Bragg spacing with synchrotron X-rays, and a set of native data extending to 2.3 A resolution has been collected.

Aldose-Ketose Isomerases↗

PubMed1999

In vivo micro-imaging using alternating navigator echoes with applications to cancellous bone structural analysis.

In micro-magnetic resonance imaging of cancellous bone architecture, involuntary subject motion even on a sub-millimeter scale is detrimental and generally precludes accurate quantification of architectural parameters. In this work a navigator-assisted three-dimensional spin-echo technique is described and evaluated for imaging at 137 microm resolution in humans. The method is based on gradient navigator echoes following the spin-echo readout for sensing translational displacements alternately in x- and y-directions with a spatial resolution of 273 microm and a temporal resolution of 0.2 sec. The technique is shown to improve micro-images of the distal forearm significantly and to enhance accuracy and reproducibility of bone volume fraction, transverse contiguity, and tubularity, parameters introduced in prior work to characterize the trabecular network. It is further shown that a fourfold reduction in navigator sampling time, along with zero-filling, improves the accuracy of the navigator correction while reducing the minimum pulse repetition time or gradient heating. The data indicate that navigator-assisted micro-imaging is capable of effectively correcting sub-millimeter displacements in micro-imaging.

Adult↗

PubMed1999

Crystallization and preliminary X-ray crystallographic analysis of deoxycytidylate hydroxymethylase from bacteriophage T4.

Deoxycytidylate hydroxymethylase from bacteriophage T4 is a homodimeric enzyme in which each polypeptide chain consists of 246 amino-acid residues. It has been crystallized in the presence of its substrate, deoxycytidine monophosphate, at room temperature using sodium citrate as precipitant. The crystals are monoclinic, belonging to space group C2, with unit-cell parameters a = 174.22, b = 53.12, c = 75.17 A, beta = 115.29 degrees. The asymmetric unit contains one homodimer, with a corresponding Vm of 2.65 A3 Da-1 and solvent content of 54%. Native diffraction data to 1.6 A resolution have been collected from two crystals using synchrotron radiation.

Bacteriophage T4↗

PubMed1999

Crystallization and preliminary X-ray crystallographic analysis of the protease inhibitor ecotin in complex with chymotrypsin.

Ecotin, a homodimeric protein composed of 142-residue subunits, is a novel protease inhibitor present in the periplasm of Escherichia coli. It shows a broad inhibitory specificity towards a group of serine proteases and binds two molecules of protease to form a tetrameric complex in a distinct chelation mechanism. The ecotin-chymotrypsin complex has been crystallized in the triclinic space group P1 with unit-cell parameters a = 57.29, b = 57.39, c = 79.75 A, alpha = 91.49, beta = 88.63 and gamma = 112.45 degrees. The asymmetric unit contains the whole tetrameric complex, consisting of two molecules of chymotrypsin bound to the ecotin dimer, with a corresponding crystal volume per protein mass (VM) of 2.58 A3 Da-1 and a solvent fraction of 48.9%. The crystals diffract beyond 2.0 A with Cu Kalpha X-rays and are very stable in the X-ray beam. Native X-ray data have been collected from a crystal to approximately 2.0 A Bragg spacing.

Bacterial Proteins↗

PubMed1999

Crystallization and preliminary X-ray analysis of Saccharomyces cerevisiae Ypd1p, a key intermediate in phosphorelay signal transduction.

Ypd1p, a 167-residue protein from Saccharomyces cerevisiae, plays a key role in osmosensing phosphorelay signal transduction. It forms part of a multistep phosphorelay system which also includes Sln1p hybrid histidine kinase and two response regulators, Ssk1p and Skn7p. It has been overexpressed in soluble form in Escherichia coli with a His6-tag at its C-terminus. The recombinant protein has been crystallized at room temperature using ammonium sulfate and lithium sulfate as precipitants. Native diffraction data have been collected to 2.3 A using synchrotron radiation. The crystals are triclinic, belonging to the space group P1, with unit-cell parameters a = 65.78, b = 66.74, c = 65.75 A, alpha = 106.60, beta = 106.48, gamma = 115. 53 degrees. The asymmetric unit contains four molecules of the monomeric recombinant Ypd1p, with a corresponding Vm of 2.75 A3 Da-1 and a solvent content of 55.3%.

Crystallization↗

PubMed1999

Crystallization and preliminary X-ray analysis of a complex between the Bowman-Birk trypsin inhibitor from barley and porcine pancreatic trypsin.

A 1:2 complex between the Bowman-Birk trypsin inhibitor from barley seeds and porcine pancreatic trypsin has been crystallized at 291 K using polyethylene glycol as precipitant. The crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 67. 10, b = 88.38 and c = 203.65 A. The asymmetric unit contains two monomers of the complex, with a corresponding Vm of 2.41 A3 Da-1 and a solvent content of 49%. Native data to 2.2 A resolution have been collected at 100 K using synchrotron X-rays.

Crystallization↗

PubMed1999

Testosterone 5 alpha-reductase inhibitors, menaquinone 7 produced by a Bacillus and phenazine methosulfate.

Menaquinone 7 (MW: 649, C46H64O2), a natural electron acceptor for steroid ring A dehydrogenations, produced by Bacillus sp. SNU-299, was isolated as a rat prostate testosterone 5 alpha-reductase inhibitor with an IC50 value of 4.0 x 10(-5) M from the cultured broth. Phylloquinone was as active as the purified microbial metabolite with an IC50 value of 6.6 x 10(-4) M. On the basis of this evidence, the inhibitory activities of electron carriers, menadione, phenazine methosulfate, and 2,6-dichlorophenolindophenol, for rat prostate testosterone 5 alpha-reductase were tested, and the IC50 values were 3.1 x 10(-6) M, 4.9 x 10(-8) M, 8.9 x 10(-5) M, respectively. A product of the 5 alpha-reductase enzyme reaction and an electron and proton carrier, NADP+, inhibited the 5 alpha-reduction by rat prostate testosterone 5 alpha-reductase with an IC50 value of 9.2 x 10(-5) M. However, the inhibition effect of a proton carrier, carbonylcyanide-m-chlorophenylhydrazone, for rat prostate testosterone 5 alpha-reductase was substantially inactive.

5-alpha Reductase Inhibitors↗

PubMed1999

Choledochal cyst associated the with anomalous union of pancreaticobiliary duct (AUPBD) has a more grave clinical course than choledochal cyst alone.

OBJECTIVE: Since choledochal cyst is frequently associated with the anomalous union of pancreaticobiliary duct (AUPBD), AUPBD has been regarded to be the etiologic factor of choledochal cyst. However, the clinical significance of AUPBD an patients with choledochal cyst has not been clearly defined. Therefore, to clarify the significance of AUPBD in choledochal cyst patients, we compared the clinical features of patients with choledochal cyst according to the presence or absence of AUPBD. METHODS: Among 52 cases which were diagnosed as choledochal cyst out of 5,037 ERCP referrals between August 1990 and December 1996, we selected 44 cases, in which the pancreaticobiliary junction was clearly visualized on cholangio-pancreaticography. These cases were divided into AUPBD-present group (n = 28) and AUPBD-absent group (n = 16). Clinical features were compared between the two groups. Furthermore, in AUPBD-present group, clinical data were also analyzed according to Kimura's classification of AUPBD. RESULTS: In our study, AUPBD was associated with choledochal cyst in 28 (64%) cases. AUPBD was found only in type I and IV according to Todani's classification of choledochal cyst. There were no significant differences between the AUPBD-present group and the AUPBD-absent group in the incidence of gallstone disease, while the incidence of acute inflammation was 93% (26/28) in the AUPBD-absent group (p < 0.01). Carcinoma developed only in the AUOBD-present group (9/28, 32%) (p < 0.05). Pancreatic disorders (i.e. pancreatic stone, pancreatitis or pancreatic cancer) occurred in 12 of 28 cases in the AUPBD-present group (43%), while only in 1 of 16 cases in the AUPBD-absent group (6%) (p < 0.05). CONCLUSION: AUPBD associated with choledochal cyst may have implications not only as a possible etiologic factor but also as an important factor that may affect the clinical course, surgical planning and prognosis. In cases with choledochal cyst, we should make an effort to evaluate the presence of AUPBD.

Adolescent↗

PubMed1998

Preliminary X-ray crystallographic analysis of Bowman-Birk trypsin inhibitor from barley seeds.

Bowman-Birk trypsin inhibitor from barley seeds has been crystallized at room temperature using polyethylene glycol as precipitant. The crystal is tetragonal, belonging to the space group P41212 (or P43212), with unit cell parameters of a = b = 62.48 and c = 94.63 A. The asymmetric unit contains one molecule of Bowman-Birk trypsin inhibitor with corresponding crystal volume per protein mass (Vm) of 2.89 A3 Da-1 and the solvent content of 57% by volume. The crystals diffract to at least 1.9 A Bragg spacing upon exposure to synchrotron X-rays. X-ray data to 1.9 A have been collected from a native crystal.

Crystallography, X-Ray↗

PubMed1998

Kunitz-type soybean trypsin inhibitor revisited: refined structure of its complex with porcine trypsin reveals an insight into the interaction between a homologous inhibitor from Erythrina caffra and tissue-type plasminogen activator.

The Kunitz-type trypsin inhibitor from soybean (STI) consists of 181 amino acid residues with two disulfide bridges. Its crystal structures have been determined in complex with porcine pancreatic trypsin in two crystal forms (an orthorhombic form at 1.75 A resolution and a tetragonal form at 1.9 A) and in the free state at 2.3 A resolution. They have been refined to crystallographic R-values of 18.9%, 21.6% and 19.8%, respectively. The three models of STI reported here represent a significant improvement over the partial inhibitor structure in the complex, which was previously determined at a nominal resolution of 2.6 A by the multiple isomorphous replacement method. This study provides the first high-resolution picture of the complex between a Kunitz-type proteinase inhibitor with its cognate proteinase. Many of the external loops of STI show high B-factors, both in the free and the complexed states, except the reactive site loop whose B-factors are dramatically reduced upon complexation. The reactive site loop of STI adopts a canonical conformation similar to those in other substrate-like inhibitors. The P1 carbonyl group displays no out-of-plane displacement and thus retains a nominal trigonal planar geometry. Modeling studies on the complex between a homologous Kunitz-type trypsin inhibitor DE-3 from Erythrina caffra and the human tissue-type plasminogen activator reveal a new insight into the specific interactions which could play a crucial role in their binding.

Amino Acid Sequence↗