Expression of miR-31 in rectal cancer patients and its effect on proliferation ability of rectal cancer cells SW837.

OBJECTIVE: This study aimed to investigate the expression of miR-31 in rectal cancer patients and its effect on the proliferation and invasion ability of human rectal cancer cells SW837. PATIENTS AND METHODS: 55 rectal cancer cancerous tissue specimens and 55 corresponding adjacent tissue (tissue adjacent to carcinoma) specimens were collected from rectal cancer patients treated in The First Hospital of Jilin University from March 2014 to March 2015. Real Time-quantitative Polymerase Chain Reaction was used for detecting the expression level of miR-31 in cancerous tissue and corresponding adjacent tissues. Differences in the expression of miR-31 were compared between the two groups. Different miR-31 expression vectors were established and rectal cancer cells SW837 were transfected. MTT was used for detecting the proliferation ability of the cells in the miR-31-mimics group, miR-31-inhibitor group and miR-control group. RESULTS: The expression level of miR-31 was significantly higher in rectal cancer tissues than that in the adjacent tissues (p<0.05). The expression of miR-31 was higher in the miR-31-mimics group (23.6±4.6) than that in the miR-control group (1.63±0.65), while the expression of miR-31 was lower in the miR-31-inhibitor group (0.65±0.23) than that in the miR-control group. The proliferation ability of cells at the 6th, 12th, 24th, 48th, and 72nd hours was higher in the miR-31-mimics group than in the miR-31-inhibitor group, while that of cells was significantly lower in the miR-31-inhibitor group than in the miR-control group, with statistically significant differences (p<0.05). The number of invasive membrane cells (cell membrane number) counted under a microscope was (84.2±10.6) cells in the miR-31-mimics group, (12.3±4.1) cells in the miR-31-inhibitor group, and (45.2±10.6) cells in the miR-control group. The invasion ability in vitro of SW837 cells significantly increased after the overexpression of miR-31 (p<0.05). CONCLUSIONS: miR-31 is increasingly expressed in rectal cancer. Low expression of miR31 can inhibit the proliferation and invasion ability of the cells. MiR-31 is expected to become a current biotherapeutic target.

A combined partially threaded cancellous lag screw for achieving maximum compressive force without compromising pullout strength.

OBJECTIVE: The partially threaded cancellous lag screw (PTLS) could not provide maximum compressive force (C(MAX)) for compression due to compromised pullout strength (POS). The combined partially threaded cancellous lag screw (CPTLS) could provide higher C(MAX) than PTLS. However, the change of POS at the point of C(MAX) when using CPTLS for compression has never been explored. The aim of this study was to determine whether POS decreased at the point of C(MAX) during CPTLS compression for different bone mineral densities (BMDs). MATERIALS AND METHODS: Three synthetic cancellous bone blocks were used for this study, and the BMDs were 0.12 g/cm³, 0.16 g/cm³, and 0.20 g/cm³, respectively. 20 pilot holes with 3.2 mm diameters were prepared equably in each block. A CPTLS was inserted through the custom-designed measuring device into a pilot hole manually until failure for measuring C(MAX), and the pullout test was done with the identical CPTLS for measuring POS. RESULTS: The C(MAX) and POS of the CPTLS were not significantly different in the three specimens, and the ratios of the mean C(MAX) to the mean POS were very similar in the three specimens (0.98 in the 0.12 g/cc specimen, 1.01 in the 0.16 g/cc specimen and 0.98 in the 0.20 g/cc specimen). CONCLUSIONS: C(MAX) is achieved without a decrease in POS during CPTLS compression independent of the BMD.

The new shank construct of lag screw improves the maximum compression force for internal fixations: preliminary results.

OBJECTIVE: The treatment of osteoporotic intra-articular fractures with AO lag screws remained challenging due to insufficient compression. Several strategies to improve the compressive ability of lag screws have been evaluated. However, the effect of the shank construct on the compressive ability of lag screw has never been explored. The aim of this study was to determine the effect of a shank construction the compressive ability of lag screw for different bone mineral densities (BMDs). MATERIALS AND METHODS: Three synthetic cancellous bone blocks were used for this study, including 0.12 g/cc, 0.16 g/cc, and 0.20 g/cc. 24 pilot holes with 3.2 mm diameters were drilled equably in each block. An AO lag screw and a combined lag screw with the newly designed compound shank construct were inserted through the custom-designed measuring device into a pilot hole by hand until failure, and the maximum compressive force (CMAX) was determined. RESULTS: Among three densities specimens CMAX of the combined lag screw was significantly higher than that of the AO lag screw (p < 0.001), and the mean CMAX difference value of the two screws in a specimen increased as the BMD increased. The CMAX of two screws increased as the BMD increased (p < 0.001), and the amplification of the CMAX generated by the combined lag screw was higher than that generated by the AO lag screw when the BMD increased. CONCLUSIONS: The newly designed compound shank construct improves the compressive ability of lag screws independent of the BMD.

Structure of killer cell immunoglobulin-like receptors and their recognition of the class I MHC molecules.

The recognition of class I MHC molecules by killer cell immunoglobulin-like receptors (KIR) constitutes an integral part of immune surveillance by the innate immune system. To understand the molecular basis of this recognition, the structures of several members of this superfamily have been determined. Despite their functional diversity, members of this superfamily share many conserved structural features. A central question is how these receptors recognize their ligands. The recent determination of the crystal structure of KIR2DL2 in complex with HLA-Cw3 has revealed the molecular mechanisms underpinning this interaction, which ultimately modulates the cytolytic activity of natural killer cells. While the recognition of MHC molecules by KIR is characterized by a number of unique features, some unexpected similarities with T-cell receptor recognition of MHC molecules are also observed. The detailed interactions between KIR2DL2 and HLA-Cw3 and their functional implications will be reviewed here.

The structure of a human type III Fcgamma receptor in complex with Fc.

Fcgamma receptors mediate antibody-dependent inflammatory responses and cytotoxicity as well as certain autoimmune dysfunctions. Here we report the crystal structure of a human Fc receptor (FcgammaRIIIB) in complex with an Fc fragment of human IgG1 determined from orthorhombic and hexagonal crystal forms at 3.0- and 3.5-A resolution, respectively. The refined structures from the two crystal forms are nearly identical with no significant discrepancies between the coordinates. Regions of the C-terminal domain of FcgammaRIII, including the BC, C'E, FG loops, and the C' beta-strand, bind asymmetrically to the lower hinge region, residues Leu(234)-Pro(238), of both Fc chains creating a 1:1 receptor-ligand stoichiometry. Minor conformational changes are observed in both the receptor and Fc upon complex formation. Hydrophobic residues, hydrogen bonds, and salt bridges are distributed throughout the receptor.Fc interface. Sequence comparisons of the receptor-ligand interface residues suggest a conserved binding mode common to all members of immunoglobulin-like Fc receptors. Structural comparison between FcgammaRIII.Fc and FcepsilonRI.Fc complexes highlights the differences in ligand recognition between the high and low affinity receptors. Although not in direct contact with the receptor, the carbohydrate attached to the conserved glycosylation residue Asn(297) on Fc may stabilize the conformation of the receptor-binding epitope on Fc. An antibody-FcgammaRIII model suggests two possible ligand-induced receptor aggregations.

Recognition of IgG by Fcgamma receptor. The role of Fc glycosylation and the binding of peptide inhibitors.

Recently determined crystal structures of the complex between immunoglobulin constant regions (Fc) and their Fc-respective receptors (FcR) have revealed the detailed molecular interactions of this receptor-ligand pair. Of particular interest is the contribution of a glycosylation at Asn(297) of the C(H)2 domain of IgG to receptor recognition. The carbohydrate moieties are found outside the receptor.Fc interface in all receptor.Fc complex structures. To understand the role of glycosylation in FcR recognition, the receptor affinities of a deglycosylated IgG1 and its Fc fragment were determined by solution binding studies using surface plasmon resonance. The removal of carbohydrates resulted in a non-detectable receptor binding to the Fc alone and a 15- to 20-fold reduction of the receptor binding to IgG1, suggesting that the carbohydrates are important in the function of the FcgammaRIII. Structurally, the carbohydrates attached to Asn(297) fill the cavity between the C(H)2 domains of Fc functioning equivalently as a hydrophobic core. This may stabilize a favorable lower hinge conformation for the receptor binding. The structure of the complex also revealed the dominance of the lower hinge region in receptor.Fc recognition. To evaluate the potential of designing small molecular ligands to inhibit the receptor function, four lower hinge peptides were investigated for their ability to bind to the receptor FcgammaRIII. These peptides bind specifically to FcgammaRIII with affinities 20- to 100-fold lower than IgG1 and are able to compete with Fc in receptor binding. The results of peptide binding illustrate new ways of designing therapeutic compounds to block Fc receptor activation.

[Study of the adsorption properties in chromatographic separation of the citric acid].

A new method studying the change of thermodynamic properties at different temperatures to screen suitable stationary phases for chromatographic separation of the citric acid (CA) on an analytical column has been developed. The applied method is mainly based on using CA with a series of solute concentrations (0 g/L-250 g/L) as mobile phase to seriously overpass the unloaded stationary phase to find the breakthrough curves of CA. The adsorption isotherms were calculated from the retention times of breakthrough curve. The bed porosity of column was calculated from the elution time of a peak of the blue dextran as non-adsorbed tracer. The stationary phase, which has been selected through this method, for the separation of CA from fermentation broth, showed a very high adsorption capacity for CA, even under very low concentrations, so it can even be used in the separation and purification process of minute amounts of CA. The separation effect of this stationary phase for citric acid can be greatly enhanced through the technique of change of temperature. This new separation process can shorten the old process path, improve the yield of finished products more than 10%, and reduce the cost more than 10%. At the same time, the pollution of the production of citric acid can be reduced. It turned out that this method will play an important role in the synthesis of stationary phase for the separation of citric acid and the study of its structure.

Overview of protein folds in the immune system.

The rapid advancement of X-ray crystallography and nuclear magnetic resonance techniques in recent years has resulted in the solution of macromolecular structures at an unprecedented rate. This review aims at providing a comprehensive description of structures and folds related to the function of the immune system. Focus is placed on immunologically relevant proteins such as immunoreceptors and major histocompatibility complexes. Information is also provided regarding protein structure data banks.

Conformational plasticity revealed by the cocrystal structure of NKG2D and its class I MHC-like ligand ULBP3.

NKG2D is known to trigger the natural killer (NK) cell lysis of various tumor and virally infected cells. In the NKG2D/ULBP3 complex, the structure of ULBP3 resembles the alpha1 and alpha2 domains of classical MHC molecules without a bound peptide. The lack of alpha3 and beta2m domains is compensated by replacing two hydrophobic patches at the underside of the class I MHC-like beta sheet floor with a group of hydrophilic and charged residues in ULBP3. NKG2D binds diagonally across the ULBP3 alpha helices, creating a complementary interface, an asymmetrical subunit orientation, and local conformational adjustments in the receptor. The interface is stabilized primarily by hydrogen bonds and hydrophobic interactions. Unlike the KIR receptors that recognize a conserved HLA region by a lock-and-key mechanism, NKG2D recognizes diverse ligands by an induced-fit mechanism.

Crystal structure of the extracellular domain of a human Fc gamma RIII.

Fc receptors play a major role in immune defenses against pathogens and in inflammatory processes. The crystal structure of a human immunoglobulin receptor, FcgammaRIIIb, has been determined to 1.8 A resolution. The overall fold consists of two immunoglobulin-like domains with an acute interdomain hinge angle of approximately 50 degrees. Trp-113, wedged between the N-terminal D1 and the C-terminal D2 domains, appears to further restrict the hinge angle. The putative Fc binding region of the receptor carries a net positive charge complementary to the negative-charged receptor binding regions on Fc. A 1:1 binding stoichiometry between the receptor and Fc was measured by both the equilibrium and nonequilibrium size-exclusion chromatography. Two separate parallel dimers are observed in the crystal lattice, offering intriguing models for receptor aggregation.

Development of a recombinant bacterial expression system for the active form of a human transforming growth factor beta type II receptor ligand binding domain.

Expression systems have been designed to test the suitability of expressing the high cysteine containing extracellular domain (residues 1-136) of human transforming growth factor beta type II receptor (TbetaRII). Receptor expressed using a baculovirus system was functional following both enzymatic deglycosylation and elimination of the N-terminal 22 amino acids by protease degradation. Bacterial expression of a TbetaRII lacking the 26 N-terminal amino acids retained the ability to bind its ligand, TGF-beta1. Receptor expressed in bacteria was sensitive to proteolytic degradation at residue Lys98 but a K98T mutation eliminated degradation and did not disrupt binding. Although several different forms of TbetaRII were expressed, only a fusion with glutathione S-transferase gave soluble TbetaRII, which was purified at a yield of 0.1 mg/10 L of bacterial growth. N-Terminal truncations of TbetaRII (residues 22-136 or 27-136) could be refolded from inclusion bodies and purified to an active form with an efficiency of 10%.

Crystal structure of an NK cell immunoglobulin-like receptor in complex with its class I MHC ligand.

Target cell lysis is regulated by natural killer (NK) cell receptors that recognize class I MHC molecules. Here we report the crystal structure of the human immunoglobulin-like NK cell receptor KIR2DL2 in complex with its class I ligand HLA-Cw3 and peptide. KIR binds in a nearly orthogonal orientation across the alpha1 and alpha2 helices of Cw3 and directly contacts positions 7 and 8 of the peptide. No significant conformational changes in KIR occur on complex formation. The receptor footprint on HLA overlaps with but is distinct from that of the T-cell receptor. Charge complementarity dominates the KIR/HLA interface and mutations that disrupt interface salt bridges substantially diminish binding. Most contacts in the complex are between KIR and conserved HLA-C residues, but a hydrogen bond between Lys 44 of KIR2DL2 and Asn 80 of Cw3 confers the allotype specificity. KIR contact requires position 8 of the peptide to be a residue smaller than valine. A second KIR/HLA interface produced an ordered receptor-ligand aggregation in the crystal which may resemble receptor clustering during immune synapse formation.

Mass-spectrometry assisted heavy-atom derivative screening of human Fc gamma RIII crystals.

A heavy-atom screening method aided by mass spectrometry is described here. Using mass spectrometry, several heavy-atom compounds have been screened in order to obtain potential phasing derivatives for the crystals of a human immunoglobulin Fc receptor, Fc gamma RIII. Of these, HgCl(2), trimethyllead acetate (TMLA), KAu(CN)(2), K(2)PtCl(4) and PbAc(2) reacted with Fc gamma RIII in solution, whereas KAuCl(4), ethylmercuric thiosalicylate (EMTS) and Na(2)WO(4) did not. To validate the mass-spectrometry results, these heavy-atom compounds were also used to soak crystals of Fc gamma RIII and crystallographic data were collected after soaking. The calculated R(iso) indicated that HgCl(2), TMLA, K(2)PtCl(4) and PbAc(2) were likely to form derivatives, whereas KAu(CN)(2) and Na(2)WO(4) were not. The anomalous difference Patterson maps calculated for the HgCl(2) and TMLA derivative data sets were of good quality and can readily be interpreted by hand. In general, the number of binding sites obtained from the crystallographic phase refinement of the derivatives agrees with those obtained from the mass spectrometry, suggesting that mass spectrometry can be applied for rapid searching of suitable heavy-atom derivatives for X-ray crystallography.

Reconstitution of bacterial expressed human CD94: the importance of the stem region for dimer formation.

Human CD94 is a subunit of the disulfide-linked, heterodimeric natural killer (NK) cell surface receptor CD94/NKG2. This receptor, a member of the C-type lectin superfamily, participates in regulating NK cell directed lysis through interaction with the major histocompatibility antigen HLA-E. Two forms of CD94 were expressed using a bacterial expression system and refolded in vitro. One form, residues 34-179, designated S34, corresponds to the entire extracellular region of the receptor, including a 23-residue stem region, and the other, residues 51-179, designated E51, corresponds only to the putative carbohydrate recognition domain of the receptor. The refolded full-length S34 protein existed as a noncovalent dimer initially but formed an interchain disulfide bond upon storage for several months. In contrast, the stemless construct, E51, existed largely as a monomeric form. The stem region of S34, residues 34-56, is sensitive to proteolysis and its absence results in dissociation of the dimer. This suggests that the residues in the stem region of CD94 help to stabilize the dimeric conformation.

Natural killer cell recognition of HLA class I molecules.

Human NK cells express multiple receptors that interact with HLA class I molecules. These receptors belong to one of two major protein superfamilies, the immunoglobulin superfamily or the C type lectin superfamily. The killer cell immunoglobulin-like receptor (KIR) family predominantly recognise classical HLA class I molecules and different family members interact with discrete HLA class I allotypes. The solution of the crystal structure of KIR2DL2 in complex with its ligand, HLA-Cw3 has provided the molecular details of a KIR/class I interaction. The interaction site spans both the alpha1 and alpha2 helices of class I and the KIR makes direct contact with peptide residues 7 and 8. The allotype specificity of KIR2DL2 for HLA-Cw3 is the result of a single hydrogen bond from Lys44 of the KIR to Asn80 of HLA-C as all other HLA-C residues that contact KIR are conserved. The lectin-like CD94/NKG2 receptors specifically interact with the non-classical class I molecule, HLA-E. Cell surface expression of HLA-E is dependent on the expression of other class I molecules as they are the major source of HLA-E binding peptides in normal cells. Consequently recognition of HLA-E by the CD94/NKG2 receptors allows NK cells to indirectly monitor the expression of a broad array of class I molecules. While the molecular interactions underlying ligand recognition by both KIR and CD94/NKG2 receptors are likely to be distinct, recognition of class I by both families of receptors appears peptide dependent. This suggest that cells that lack class I and also those that are impaired in their ability to load class I molecules with peptide will become targets for NK-mediated destruction.

Stoichiometric arginine binding in the oxygenase domain of inducible nitric oxide synthase requires a single molecule of tetrahydrobiopterin per dimer.

In addition to its catalytic roles, the nitric oxide synthase (NOS) cofactor tetrahydrobiopterin (H4B) is required for substrate binding and for stabilization of the dimeric structure. We expressed and purified the core of the iNOS oxygenase domain consisting of residues 75-500 (CODiNOS) in the presence (H4B+) and absence (H4B-) of this cofactor. Both forms bound stoichiometric amounts of heme (>0.9 heme per protein subunit). H4B- CODiNOS was unable to bind arginine, gave an unstable ferrous carbonyl adduct, and was a mixture of monomer and dimer. H4B+ CODiNOS bound arginine, gave a stable ferrous carbonyl adduct, and was exclusively dimeric. The H4B cofactor content of this species was only one per dimer yet this was sufficient to form two competent arginine binding sites as determined by optical stoichiometric titrations.

Crystal structure of the HLA-Cw3 allotype-specific killer cell inhibitory receptor KIR2DL2.

Killer cell inhibitory receptors (KIR) protect class I HLAs expressing target cells from natural killer (NK) cell-mediated lysis. To understand the molecular basis of this receptor-ligand recognition, we have crystallized the extracellular ligand-binding domains of KIR2DL2, a member of the Ig superfamily receptors that recognize HLA-Cw1, 3, 7, and 8 allotypes. The structure was determined in two different crystal forms, an orthorhombic P212121 and a trigonal P3221 space group, to resolutions of 3.0 and 2.9 A, respectively. The overall fold of this structure, like KIR2DL1, exhibits K-type Ig topology with cis-proline residues in both domains that define beta-strand switching, which sets KIR apart from the C2-type hematopoietic growth hormone receptor fold. The hinge angle of KIR2DL2 is approximately 80 degrees, 14 degrees larger than that observed in KIR2DL1 despite the existence of conserved hydrophobic residues near the hinge region. There is also a 5 degrees difference in the observed hinge angles in two crystal forms of 2DL2, suggesting that the interdomain hinge angle is not fixed. The putative ligand-binding site is formed by residues from several variable loops with charge distribution apparently complementary to that of HLA-C. The packing of the receptors in the orthorhombic crystal form offers an intriguing model for receptor aggregation on the cell surface.

Structure of CD94 reveals a novel C-type lectin fold: implications for the NK cell-associated CD94/NKG2 receptors.

The crystal structure of the extracellular domain of CD94, a component of the CD94/NKG2 NK cell receptor, has been determined to 2.6 A resolution, revealing a unique variation of the C-type lectin fold. In this variation, the second alpha helix, corresponding to residues 102-112, is replaced by a loop, the putative carbohydrate-binding site is significantly altered, and the Ca2+-binding site appears nonfunctional. This structure may serve as a prototype for other NK cell receptors such as Ly-49, NKR-P1, and CD69. The CD94 dimer observed in the crystal has an extensive hydrophobic interface that stabilizes the loop conformation of residues 102-112. The formation of this dimer reveals a putative ligand-binding region for HLA-E and suggests how NKG2 interacts with CD94.

Crystal structure of formate dehydrogenase H: catalysis involving Mo, molybdopterin, selenocysteine, and an Fe4S4 cluster.

Formate dehydrogenase H from Escherichia coli contains selenocysteine (SeCys), molybdenum, two molybdopterin guanine dinucleotide (MGD) cofactors, and an Fe4S4 cluster at the active site and catalyzes the two-electron oxidation of formate to carbon dioxide. The crystal structures of the oxidized [Mo(VI), Fe4S4(ox)] form of formate dehydrogenase H (with and without bound inhibitor) and the reduced [Mo(IV), Fe4S4(red)] form have been determined, revealing a four-domain alphabeta structure with the molybdenum directly coordinated to selenium and both MGD cofactors. These structures suggest a reaction mechanism that directly involves SeCys140 and His141 in proton abstraction and the molybdenum, molybdopterin, Lys44, and the Fe4S4 cluster in electron transfer.

Characterization of crystalline formate dehydrogenase H from Escherichia coli. Stabilization, EPR spectroscopy, and preliminary crystallographic analysis.

The selenocysteine-containing formate dehydrogenase H (FDH) is an 80-kDa component of the Escherichia coli formate-hydrogen lyase complex. The molybdenum-coordinated selenocysteine is essential for catalytic activity of the native enzyme. FDH in dilute solutions (30 microg/ml) was rapidly inactivated at basic pH or in the presence of formate under anaerobic conditions, but at higher enzyme concentrations (>/=3 mg/ml) the enzyme was relatively stable. The formate-reduced enzyme was extremely sensitive to air inactivation under all conditions examined. Active formate-reduced FDH was crystallized under anaerobic conditions in the presence of ammonium sulfate and PEG 400. The crystals diffract to 2.6 A resolution and belong to a space group of P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions a = b = 146.1 A and c = 82.7 A. There is one monomer of FDH per crystallographic asymmetric unit. Similar diffraction quality crystals of oxidized FDH could be obtained by oxidation of crystals of formate-reduced enzyme with benzyl viologen. By EPR spectroscopy, a signal of a single reduced FeS cluster was found in a crystal of reduced FDH, but not in a crystal of oxidized enzyme, whereas Mo(V) signal was not detected in either form of crystalline FDH. This suggests that Mo(IV)- and the reduced FeS cluster-containing form of the enzyme was crystallized and this could be converted into Mo(VI)- and oxidized FeS cluster form upon oxidation. A procedure that combines anaerobic and cryocrystallography has been developed that is generally applicable to crystallographic studies of oxygen-sensitive enzymes. These data provide the first example of crystallization of a substrate-reduced form of a Se- and Mo-containing enzyme.