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Ten ingenane-type diterpenoids, including two undescribed compounds named as kansuininols A–B (1–2), were isolated from the roots of Euphorbia kansui. Their structures were elucidated by detailed interpretation of their spectroscopic data, especially 2D-NMR and HR-ESI-MS. Chemoreversal assays of these isolates were carried on adriamycin resistant human breast adenocarcinoma cell line MCF-7/ADR, and compounds 1–2 were identified as potent low-cytotoxic MDR modulators with greater chemoreversal ability than the positive drug verapamilti.

A new, efficient approach toward the preparation of 2-aryl-2,3-dihydrobenzofuran scaffolds through the Cu/SPDO-catalyzed [3 + 2] cycloaddition between quinone ester and styrene derivatives has been developed. The procedure features excellent enantioselectivities (up to 99% ee), high yields (up to 96%), and broad substrate tolerance. Additionally, asymmetric synthesis of natural corsifurans A and B from commercially available starting materials has also been achieved in two or three steps using this reaction as a key transformation.

We report herein a strategy to construct enantiopure inherently chiral macrocycles, ABCD-type heteracalix[4]-aromatics, through a catalytic enantioselective intramolecular C−N bond forming reaction. A chiral ligand-palladium complex was found to efficiently induce the inherent chirality of molecules during the macrocyclization process with ee values up to >99%. The resulting ABCD-type heteracalix[4]aromatics displayed excellent and pH triggered switchable electronic circular dichroism and circularly polarized luminescence properties.

Background Sepsis is a severe condition characterised by the body’s systemic inflammatory response to infection. The specific sepsis-related biomarkers should be used in clinical diagnosis, therapeutic response monitoring, rational use of antibiotics, and prognosis (risk stratification), etc. Results In this study, we investigated the expression level of Decoy Receptor 3 (DcR3) and the mechanism of high expression in sepsis patients. Septic cell model experiments were performed by treating human umbilical vein endothelial cells (HUVECs) and Jurkat cells with lipopolysaccharide (LPS), lipoteichoic acid (LTA) and zymosan, respectively. SP600125, SB203580 and ammonium pyrrolidinedithiocarbamate (PDTC) were used to inhibit JNK1/2, p38MAPK and NF-κB signalling pathways in septic cell model, respectively. These results showed that DcR3 levels were higher in sepsis group than control. DcR3 mRNA and protein levels in HUVECs were increased following treatment with LPS, LTA and zymosan, and also increased in Jurkat cells treated by LPS, but not by LTA or zymosan. When HUVECs were treated with the NF-κB inhibitor PDTC, DcR3 expression was decreased compared with controls. However, SP600125 and SB203580 had no effect on DcR3 mRNA or protein levels. Conclusions The results indicated that DcR3 secretion proceeded through the NF-κB signalling pathway in HUVECs.

Objectives This study aimed to investigate the factors affecting the quantity of DNA and RNA extractable from human formalin-fixed paraffin-embedded (FFPE) tissues stored for different lengths of time. Methods We randomly selected 20 FFPE specimens harvested from hysteromyoma patients with uterine fibroids during 2010, 2015, and 2017 at the Department of Pathology, Jiading District Central Hospital Affiliated Shanghai University of Medicine and Health Sciences. DNA and RNA extractions were performed using a DNA/RNA FFPE kit. DNA and RNA concentrations and their OD260/OD280 ratios were determined by a NanoDrop 2000 spectrophotometer. The human β-globin gene and aldehyde dehydrogenase-2 (ALDH2) gene were amplified from nucleic acids using a LightCycler 480 Real-Time PCR System, and PCR amplification products were electrophoresed on 1% agarose gels. Results Specimens that were stored for longer showed more degradation and a reduced concentration of DNA and RNA after nucleic acid extraction. However, there was no significant difference in DNA or RNA purity. β-globin and ALDH2 genes could be amplified from more than 99% of specimens. Conclusion We found that FFPE tissues stored for longer had a reduced quantity of extractable DNA and RNA. However, these tissues could be used for the analysis of some small target genes.

Objectives The purpose of this paper was to synthesize 4-amino-1,2,4-triazole Schiff base derivative and to evaluate its antitumor activity. Methods 2,4,6-Tris (4-formylphenoxy)-1,3,5-triazine was synthesized by nucleophilic substitution with reaction of p -hydroxybenzaldehyde with cyanuric chloride. Then, the intermediate was reacted with 1,2,4-triazole to form 4-amino-1,2,4-triazole Schiff base derivative by reduction and ammonification of aldehydes. The 4-amino-1,2,4-triazole Schiff base derivative was characterized by infrared spectroscopy, nuclear magnetic resonance, and elemental analyses. Finally, the anticancer effect of 4-amino-1,2,4-triazole Schiff base derivative was evaluated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay on a lung adenocarcinoma cell line (A549) and a human hepatoma cell line (Bel7402). Results The product synthesized in this study was confirmed to be 4-amino-1,2,4-triazole Schiff base derivative, and it significantly suppressed the proliferation of cancer cell lines in a dose-dependent manner. Conclusions We successfully prepared 4-amino-1,2,4-triazole Schiff base derivative and verified its antitumor activity.

Membrane scaling is a critical challenge for membrane distillation (MD), especially when treating hypersaline brines. In this study, an anti-scaling membrane was developed with a liquid-like surface coating. This fabrication approach combined electrospun polyvinylidene fluoride (PVDF) membrane with surface fluorination without the addition of nanoparticles. Two fluoro silanes with the same alkyl-chain length but different hydrolysis reaction sites were investigated as the fluorination agents. Membrane characterizations indicated that surface modification with the single hydrolysis site fluoro silane (17-FAS-S) did not change the membrane morphology or structure. On the other hand, surface modification with the triple hydrolysis site fluoro silane (17-FAS-T) generated nanoparticles on the membrane surface, resulting in increased roughness. While both the 17-FAS-S and 17-FAS-T modified membranes exhibited excellent scaling resistance in the MD scaling tests, the 17-FAS-S membrane provided a much higher vapor flux than the 17-FAS-T one. Finally, the mechanism of scaling resistance and vapor permeability provided by the liquid-like modification was discussed.

A practical synthetic method has been developed for polyfunctionalized biaryls based on a facile one-pot nucleophilic aromatic substitution (SNAr) reaction and [5,5]- or [3,3]-sigmatropic rearrangement reaction cascade. Under mild basic conditions, N-arylhydroxylamines reacted with o-activated fluoro (het)arenes to form N,O-diarylhydroxylamine intermediates which underwent spontaneously selective [5,5]-sigmatropic rearrangement reaction to produce diverse functionalized 4-amino-4′-hydroxy-1,1′-biaryls. A sequential SNAr reaction and [3,3]-sigmatropic rearrangement took place between N-arylhydroxylamines and 2-fluoropyridine derivatives or 4-fluorobenzonitrile to afford functionalized 2-amino-2′-hydroxy-1,1′-biaryls. As invaluable and unique building blocks, the resulting biaryls were applied in the straightforward synthesis of N2,O2-coronarene, carbazole, aza- and diaza carbazole derivatives.

PepT1 can transport bacterial oligopeptide products and induce intestinal inflammation. Our aim was to investigate the mechanism of the small intestine injury induced by bacterial oligopeptide product muramyl dipeptide (MDP) which is transported by PepT1. We perfused the jejunum with a solution with or without MDP, or with a solution of MDP + Gly-Gly and explored the degree of inflammation to determine the role of PepT1–Nod2 signaling pathway in small intestine mucosa. MDP perfusion induced inflammatory cell accumulation and intestinal damage, accompanied by an increase in mucosal Nod2 and Rip2 transcript expression. NFκB activity and inflammatory cytokine expression, including serum levels of TNF-α, IL-1β, and IL-6, increased in the MDP group compared to the controls; these effects were reversed by perfusion of the nutritional dipeptide Gly-Gly. MDP can be transported through PepT1, causing inflammatory damage in the rat small intestine. Nod2–Rip2–NFκB signaling involved in the small intestinal inflammatory injury caused by MDP which is transported through PepT1.

Lanostane-type triterpenoids, inotolactones A and B, a drimane-type sesquiterpenoid, inotolactone C, and five known terpenoids 6β-hydroxy-trans-dihydroconfertifolin, inotodiol, 3β,22-dihydroxyanosta-7,9(11),24-triene, 3β-hydroxycinnamolide, and 17-hydroxy-ent-atisan-19-oic acid, were isolated from the submerged culture of chaga mushroom, Inonotus obliquus. Their structures were characterized by spectroscopic methods, including MS and NMR (1D and 2D) spectroscopic techniques. Inotolactones A and B, examples of lanostane-type triterpenoids bearing α,β-dimethyl, α,β-unsaturated δ-lactone side chains, exhibited more potent alpha-glucosidase inhibitory activities than the positive control acarbose. This finding might be related to the anti-hyperglycemic properties of the fungus and to its popular role as a diabetes treatment. In addition, a drimane-type sesquiterpenoid and an atisane-type diterpenoid were isolated from I. obliquus.

Two new polyketide derivatives, huaspenones C and D (1 and 2, resp.), were isolated from the cultures of an endophytic fungus Peyronellaea sp. HS-12, derived from the stems of Huperzia serrata. They share N-bearing furan-3(2H)-one backbone, and 2 has an unprecedented furo[3,2-c]pyridine skeleton. Their structures including the absolute configuration were elucidated by extensive spectroscopic analysis combined with quantum-chemical calculations. (2E,4E)-6-hydroxy-2-methylocta-2,4-dienoic acid (3), a key intermediate of the biosynthesis of 1 and 2, was also obtained from the endophyte.

Inflammatory and immune responses, as well as melatonin secretion, are affected by circadian regulation. Abnormal circadian rhythm of melatonin release has been reported to be associated with the later stages of sepsis; however, its role in the early stages of sepsis is unclear. We studied 11 septic and 11 non-septic patients in our intensive care unit (ICU). Peripheral blood was drawn at 4-h intervals on the first day, beginning at 2:00 p.m., over a total period of 24 h. Plasma levels of melatonin, tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) were measured by radioimmunoassay or enzyme-linked immunosorbent assay (ELISA). Messenger RNA levels of circadian genes Cry-1 and Per-2 were analyzed using quantitative real-time PCR. Results show the circadian rhythm of melatonin secretion was altered in the early stages of sepsis. The melatonin secretion acrophase occurred earlier in septic patients at 6:00 p.m., compared with at 2:00 a.m. in non-septic ICU patients. Compared with the non-septic group, both Cry-1 and Per-2 expression were significantly decreased while TNF-α and IL-6 expression were significantly increased in septic patients [TNF-α, 64.1 (43.6-89.1) vs. 11.4 (10.4-12.5) ng/ml; IL-6, 41.2 (35.7-50.8) vs. 19.1 (16-136.7) ng/ml; median (range), both P=0.04]. The peak concentrations of TNF-α and IL-6 were shown to be in concordance with the rhythm of melatonin secretion. The circadian rhythm of melatonin secretion and circadian gene expression were altered in the early stages of sepsis, which likely led to the changes in pro-inflammatory cytokine release. These findings shed light on the potential link between circadian rhythm and the progression of early-stage sepsis.

Evidence has shown that most hepatocellular carcinoma (HCC) cells are resistant to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. However, the molecular mechanisms underlying TRAIL-mediated apoptosis resistance are not well understood. In this study, we reported that downregulation of Decoy receptor 3 (DcR3) expression by lentiviral vectors carrying shRNA against DcR3 (LV-ShDcR3, shDcR3) in Huh7 both greatly enhanced TRAIL-mediated apoptosis and reduced cell proliferation capability. In addition, silencing DcR3 resulted in upregulation of the cell apoptotic regulators including Bid, caspase-3, and caspase-8. Caspase inhibitors inhibited shDcR3-mediated cell death, which indicated that downregulation of DcR3 expression in Huh7 cells increased TRAIL-induced caspase-dependent apoptotic cell death. Furthermore, although the knockdown of DcR3 altered the expression of some Bcl-2- and IAP-family proteins, this change was inhibited by pretreatment with a pancaspase inhibitor, which indicated the cytotoxic effect of shDcR3 was not due to the expression of these proteins. In contrast, shDcR3 significantly inhibited TRAIL-induced transcription factor nuclear κB (NF-κB) activation through the IκB kinase (IKK) pathway, as well as inhibited TRAIL-induced increases in FLICE-inhibitory protein long form (cFLIPL) expression at the transcriptional level. Silencing cFLIPL expression mimicked the cytotoxic effect of shDcR3 on TRAIL-mediated cell apoptosis. Moreover, overexpression of cFLIPL effectively prevented the increase in cell apoptosis in Huh7 cells co-treated with TRAIL and shDcR3. Taken together, our findings indicated that silencing DcR3 sensitizes TRAIL-mediated apoptosis in HCC cells by inhibiting NF-κB.

International audience; Roscovitine (CYC202) is often referred to as a "selective inhibitor of cyclin-dependent kinases." Besides its use as a biological tool in cell cycle, neuronal functions, and apoptosis studies, it is currently evaluated as a potential drug to treat cancers, neurodegenerative diseases, viral infections, and glomerulonephritis. We have investigated the selectivity of (R)-roscovitine using three different methods: 1) testing on a wide panel of purified kinases that, along with previously published data, now reaches 151 kinases; 2) identifying roscovitine-binding proteins from various tissue and cell types following their affinity chromatography purification on immobilized roscovitine; 3) investigating the effects of roscovitine on cells deprived of one of its targets, CDK2. Altogether, the results show that (R)-roscovitine is rather selective for CDKs, in fact most kinases are not affected. However, it binds an unexpected, non-protein kinase target, pyridoxal kinase, the enzyme responsible for phosphorylation and activation of vitamin B 6. These results could help in interpreting the cellular actions of (R)-roscovitine but also in guiding the synthesis of more selective roscovitine analogs.

Rubrerythrin (Rr) is a non-heme iron protein isolated from anaerobic sulfate-reducing bacteria. Rr is a dimeric molecule, each monomer contains a Fe(SCys)4 center in the C-terminal domain and a binuclear metal center in the N-terminal domain. Rr structures with different protein sources and/or preparation procedures have been studied. Two Rr crystal structures have been solved with significant differences in their binuclear metal centers. The first structure, which was obtained from expressed protein under aerobic conditions, has a diiron–oxo center. The second structure, which was obtained from native protein of Desulfovibrio vulgaris under aerobic conditions, has an Fe–Zn center with the zinc position differing from the corresponding iron position in the former structure by approximately 2 A. The crystal structures of Rr isolated from D. vulgaris (Hildenborough, NCIB 8303), the same as the second structured but prepared under anaerobic conditions, are reported in this paper. The binuclear metal center in these structures is an Fe–Zn center. When the crystal was exposed to air, the zinc atom moved gradually, approximately 2 A, accompanied by the entrance of a water molecule (or hydroxyl group) and changes in the binuclear metal center microenvironment. This finding can explain the differences between the two different structures. The results suggest that the zinc movement may be related to the enzymatic activity of Rr. Electronic supplementary material is available if you access this article at http://dx.doi.org/10.1007/s00775-002-0400-0. On that page (frame on the left side), a link takes you directly to the supplementary material.

The C-terminal segment of copper-containing nitrite reductase from Achromobacter cycloclastes (AcNiR) has been found essential for maintaining both the quaternary structure and the enzyme activity of AcNiR. C-terminal despentapeptide AcNiR (NiRc-5) and desundecapeptide AcNiR (NiRc-11) are two important truncated mutants whose activities and stability have been affected by residue deletion. In this study, the two mutants were crystallized using the hanging drop vapor diffusion method. Crystals of NiRc-5 obtained at pH 5.0 and 6.2 both belonged to the P2(1)2(1)2(1) space group with unit cell parameters a=99.0 A, b=117.4 A, c=122.8 A (pH 5.0) and a=98.9A, b=117.7A, c=123.0A (pH 6.2). NiRc-11 was crystallized in two crystal forms: the tetragonal form belonged to the space group P4(1) with a=b=96.0A and c=146.6A; the monoclinic form belonged to the space group P2(1) with a=86.0A, b=110.1A, c=122.7A, and beta=101.9 degrees. The crystallizing behaviors of the two mutants differed from that of the native enzyme. Such change in combination with residue deletion is also discussed here.

The three-dimensional structures of brain pyridoxal kinase and its complex with the nucleotide ATP have been elucidated in the dimeric form at 2.1 and 2.6 A, respectively. Results have shown that pyridoxal kinase, as an enzyme obeying random sequential kinetics in catalysis, does not possess a lid shape structure common to all kinases in the ribokinase superfamily. This finding has been shown to be in line with the condition that pyridoxal kinase binds substrates with variable sizes of chemical groups at position 4 of vitamin B(6) and its derivatives. In addition, the enzyme contains a 12-residue peptide loop in the active site for the prevention of premature hydrolysis of ATP. Conserved amino acid residues Asp(118) and Tyr(127) in the peptide loop could be moved to a position covering the nucleotide after its binding so that its chance to hydrolyze in the aqueous environment of the active site was reduced. With respect to the evolutionary trend of kinase enzymes, the existence of this loop in pyridoxal kinase could be classified as an independent category in the ribokinase superfamily according to the structural feature found and mechanism followed in catalysis.

A so-called "green protein" has been purified from a moderate halophilic eubacterium, Bacillus halodenitrificans (ATCC 49067), under anaerobic conditions. The protein, which might play an important role in denitrification, dissociates mainly into two components after exposure to air: a manganese superoxide dismutase (GP-MnSOD) and a nucleoside diphosphate kinase. As a first step in elucidating the overall structure of the green protein and the role of each component, the 2.8-A resolution crystal structure of GP-MnSOD was determined. Compared with other manganese dismutases, GP-MnSOD shows two significant characteristics. The first is that the entrance to its substrate channel has an additional basic residue-Lys38. The second is that its surface is decorated with an excess of acidic over basic residues. All these structural features may be related to GP-MnSOD's high catalytic activity and its endurance against the special cytoplasm of B. halodenitrificans. The structure of GP-MnSOD provides the basis for recognizing its possible role and assembly state in the green protein.

Zinc finger motif widely exists in protein structure, which can play different roles in different proteins. RecR is an important recombination mediator protein (RMP) in the RecFOR pathway and zinc finger motif is the most conserved domain in RecR protein. However, the function of this zinc finger motif in RecR is unclear. Here, we have studied the structures of the single cysteine and double cysteines mutation within the zinc finger motif in Thermoanaerobacter tengcongensis RecR (TTERecR). We have also studied the DNA binding ability as well as TTERecO protein binding ability of single, double and even triple cysteines mutation of the zinc finger motif, and the mutants do not alter DNA binding by RecR nor the interaction between RecR and RecO. The function of TTERecR zinc finger motif is to maintain the stability of the three-dimensional structure.

The crystal structure of R-Phycoerythrin (R-PE) from Polysiphonia urceolata has been refined to a resolution of 1.9 A, based on the atomic coordinates of R-PE determined at 2.8 A resolution, through the use of difference Fourier method and steorochemistry parameters restrained refinement with model adjustment according to the electron density map. Crystallographic R-factor of the refined model is 0.195 (Rfree = 0.282) from 8-1.9 A. High resolution structure of R-PE showed precise interactions between the chromophores and protein residues, which explained the spectrum characteristic and function of chromophores. Four chiral atoms of phycourobilin (PUB) were identified as C(4)-S, C(16)-S, C(21)-S, and C(20)-R. In addition to the coupling distances of 19 A to 45 A between the chromophores which were observed and involved in the energy transfer pathway, high resolution structure of R-PE suggested other pathways of energy transfer, such as the ultrashort distance between alpha140a and beta155. It has been proposed that aromatic residues in linker proteins not only influence the conformation of chromophore, but may also bridge chromophores to improve the energy transfer efficiency.

Primaquine, an 8-aminoquinoline, is the only drug which cures the dormant hypnozoites of persistent liver stages from P. vivax. Increasing resistance needs the discovery of alternative pathways as drug targets to develop novel drug entities. Deoxyhypusine hydroxylase (DOHH) completes hypusine biosynthesis in eukaryotic initiation factor (eIF-5A) which is the only cellular protein known to contain the unusual amino acid hypusine. Modified EIF-5A is important for proliferation of the malaria parasite. Here, we present the first successful cloning and expression of DOHH from P. vivax causing tertiary malaria. The nucleic acid sequence of 1041 bp encodes an open reading frame of 346 amino acids. Histidine tagged expression of P. vivax DOHH detected a protein of 39.01 kDa in E. coli. The DOHH protein from P. vivax shares significant amino acid identity to the simian orthologues from P. knowlesi and P. yoelii strain H. In contrast to P. falciparum only four E-Z-type HEAT-like repeats are present in P. vivax DOHH with different homology to phycocyanin lyase subunits from cyanobacteria and in proteins participating in energy metabolism of Archaea and Halobacteria. However, phycocyanin lyase activity is absent in P. vivax DOHH. The dohh gene is present as a single copy gene and transcribed throughout the whole erythrocytic cycle. Specific inhibition of recombinant P. vivax DOHH is possible by complexing the ferrous iron with zileuton, an inhibitor of mammalian 5-lipoxygenase (5-LOX). Ferrous iron in the active site of 5-LOX is coordinated by three conserved histidines and the carboxylate of isoleucine673. Zileuton inhibited the P. vivax DOHH protein with an IC50 of 12,5 nmol determined by a relative quantification by GC/MS. By contrast, the human orthologue is only less affected with an IC50 of 90 nmol suggesting a selective iron-complexing strategy for the parasitic enzyme. OA Förderung 2013

RecR is an important recombination mediator protein in the RecFOR pathway. RecR together with RecO and RecF facilitates RecA nucleoprotein filament formation and homologous pairing. Structural and biochemical studies of Thermoanaerobacter tengcongensis RecR (TTERecR) and its series mutants revealed that TTERecR uses the N-N dimer as a basic functional unit to interact with TTERecO monomer. Two TTERecR N-N dimers form a ring-shaped tetramer via an interaction between their C-terminal regions. The tetramer is a result of crystallization only. Hydrophobic interactions between the entire helix-hairpin-helix domains within the N-terminal regions of two TTERecR monomers are necessary for formation of a RecR functional N-N dimer. The TTERecR N-N dimer conformation also affects formation of a hydrophobic patch, which creates a binding site for TTERecO in the TTERecR Toprim domain. In addition, we demonstrate that TTERecR does not bind single-stranded DNA (ssDNA) and binds double-stranded DNA very weakly, whereas TTERecOR complex can stably bind DNA, with a higher affinity for ssDNA than double-stranded DNA. Based on these results, we propose an interaction model for the RecOR:ssDNA complex.

Crystal structures of the AHL-lactonase AidH in complex with substrate and product are reported at high resolution and a catalytic mechanism is proposed for the metal-independent AHL-lactonase., Many pathogenic bacteria that infect humans, animals and plants rely on a quorum-sensing (QS) system to produce virulence factors. N-Acyl homoserine lactones (AHLs) are the best-characterized cell–cell communication signals in QS. The concentration of AHL plays a key role in regulating the virulence-gene expression and essential biological functions of pathogenic bacteria. N-Acyl homoserine lactonases (AHL-lactonases) have important functions in decreasing pathogenicity by degrading AHLs. Here, structures of the AHL-lactonase from Ochrobactrum sp. (AidH) in complex with N-­hexanoyl homoserine lactone, N-hexanoyl homoserine and N-­butanoyl homoserine are reported. The high-resolution structures together with biochemical analyses reveal convincing details of AHL degradation. No metal ion is bound in the active site, which is different from other AHL-lactonases, which have a dual Lewis acid catalysis mechanism. AidH contains a substrate-binding tunnel between the core domain and the cap domain. The conformation of the tunnel entrance varies with the AHL acyl-chain length, which contributes to the binding promiscuity of AHL molecules in the active site. It also supports the biochemical result that AidH is a broad catalytic spectrum AHL-lactonase. Taken together, the present results reveal the catalytic mechanism of the metal-independent AHL-lactonase, which is a typical acid–base covalent catalysis.

Pyridoxal kinase (ATP:pyridoxal 5'-phosphotransferase; EC 2.7.1.35) is a key enzyme in the transformation of vitamin B(6) to pyridoxal-5'-phosphate. Pyridoxal-5'-phosphate is the crucial cofactor required by numerous enzymes involved in the metabolism of amino acids and the synthesis of many neurotransmitters. Pyridoxal kinase from sheep brain was crystallized in an orthorhombic form using the hanging-drop vapour-diffusion method with sodium citrate as the precipitant. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 59.8, b = 94.4, c = 128.2 A, and diffract to a resolution of 2.1 A. Crystals were transferred into a soaking liquid without citrate and two heavy-atom derivatives were prepared.

Manganese superoxide dismutase (GP-MnSOD), a component of the so-called `green protein' (green protein complex) from the facultative anaerobic halodenitrifier Bacillus halodenitrificans , has been crystallized using the hanging-drop vapor diffusion method. Crystals have unit-cell parameters a = b =93.4 A, c =65.0 A, and belong to the space group P4 3 2 1 2. Preliminary analysis indicates there is one monomer in each asymmetric unit. The structural information from this enzyme will enrich our knowledge on its high catalytic activity and its possible role in green protein complex.

Sepsis, a common deadly systemic infection caused by a variety of pathogens, has some clinical symptoms similar to the systemic inflammatory response syndrome (SIRS), a whole-body non-infectious inflammatory reaction to severe insults, such as burn, trauma, hypotensive shock and so on. Treatment of sepsis depends mainly on anti-microbial, while remedy for SIRS might require steroids that could possibly enhance the spread of microbes. Unfortunately, it is very difficult to distinguish these two completely different serious conditions without blood culture, which takes days to grow and identify causative pathogens. We examined a biomarker, serum decoy receptor 3 (DcR3), was evaluated for its utility in the differential diagnosis between sepsis and SIRS.Serum DcR3 level in 118 healthy controls, 24 sepsis patients and 43 SIRS patients, was quantitatively measured by enzyme-linked immunosorbent assay (ELISA).The serum DcR3 was significantly increased in sepsis patients compared with SIRS patients and healthy controls (6.11±2.58 ng/ml vs 2.62±1.46 ng/ml, and 0.91±0.56 ng/ml, respectively, p0.001). The areas under the receiver operating characteristic curve of DcR3 for the normal vs. SIRS, normal vs. sepsis and SIRS vs. sepsis were 0.910 (0.870-0.950), 0.992 (0.984-1.000) and 0.896 (0.820-0.973), respectively. In addition, the DcR3 exhibited a positive correlation coefficient with APACHE II score, a most commonly used index for the severity of sepsis (r=0.556, p=0.005).The serum DcR3 has a potential to serve as a new biomarker for sepsis with its high specificity and sensitivity.

Type I DNA methyltransferases contain one specificity subunit (HsdS) and two modification subunits (HsdM). The electron microscopy model of M.EcoKI-M2S1 methyltransferase shows a reasonable closed state of this clamp-like enzyme, but the structure of the open state is still unclear. The 1.95 A crystal structure of the specificity subunit from Thermoanaerobacter tengcongensis (TTE-HsdS) shows an unreported open form inter-domain orientation of this subunit. Based on the crystal structure of TTE-HsdS and the closed state model of M.EcoKI-M2S1, we constructed a potential open state model of type I methyltransferase. Mutational studies indicated that two α-helices (aa30-59 and aa466-495) of the TTE-HsdM subunit are important inter-subunit interaction sites in the TTE-M2S1 complex. DNA binding assays also highlighted the importance of the C-terminal region of TTE-HsdM for DNA binding by the TTE-M2S1 complex. On the basis of structural analysis, biochemical experiments and previous studies, we propose a dynamic opening and closing mechanism for type I methyltransferase.

Methylmalonyl-CoA is an important metabolic intermediate that exists in several key degradative and biosynthetic pathways.1 The pathway responsible for the degradation of branched amino acids and odd chain fatty acids involves three steps. In the first step, a biotindependent carboxylation produces the S-epimer of methylmalonyl-CoA. The third step, the B12-dependent methylmalonyl-CoA mutase converts the R-epimer of methylmalonyl-CoA to succinyl-CoA.1 This key epimerization between the two steps is carried out by the essential enzyme methylmalonyl-CoA epimerase (MMCE: EC number 5.1.99.1), which catalyzes the conversion of (2S)methylmalonyl-CoA to (2R)-methylmalonyl-CoA. In prokaryotes, MMCE participated in autotrophic CO2 fixation via the 3-hydroxypropionate pathway, in propionate fermentation, the regeneration of glyoxylate and in the biosynthesis of polyketide antibiotics.2–6 In animals, defects of this pathway can result in a buildup of methylmalonic acid, which causes severe acidosis and also damages the central nervous system.7,8 In this study, we determined the crystal structure of TTE0360 from Thermoanaerobacter tengcongensis to 2.0 A resolution using single-wavelength anomalous dispersion (SAD) by short cryo-soaking with a high concentration of potassium iodide.9,10 In GenBank database, the gene tte0360 is predicted to be glyoxalase I, a member of glyoxalase superfamily. However, the TTE0360 structure is very different than the released structures of glyoxalase I. A search for the 3D homologs using DALI11 shows that TTE0360 has significant structural similarity with the MMCE from Propionibacterium shermanii,12 which is also a member of glyoxalase superfamily. The fold type of the TTE0360 molecule belongs to the vicinal-oxygen-chelate superfamily (VOC).13 The similarities of structure folds, dimer formation, and active sites between TTE0360 and MMCE, suggest that the protein TTE0360 may be the MMCE from T. tengcongensis.

A non-aqueous capillary electrophoretic method was established for the determination of berberine in Rhizoma coptidis and its preparations. The effects of organic solvent and the concentrations of sodium acetate were studied, which showed that berberine in extracts of traditional Chinese medicine can be separated successfully in a buffer solution of 75 mmol/L of sodium acetate in methanol containing 1 mol/L of acetic acid. The simple and rapid method was linear in the range 25-200 microgram/mL of berberine and had a good reproducibility, with the relative standard deviation below 2%. Non-aqueous capillary electrophoresis is a satisfactory system for the analysis of alkaloids in traditional Chinese medicine.

Tetrahydropalmatine in Rhizoma corydalis and its preparations were separated and determined with no pretreatment in the buffer solution of 50 mmol/L of sodium acetate in methanol containing 2 mol/L acetic acid.

The human light chain of the motor protein dynein, Dnlc2A, is also a novel TGF-beta-signaling component, which is altered with high frequency in epithelial ovarian cancer. It is an important mediator of dynein and the development of cancer, owing to its ability to bind to the dynein intermediate light chain (DIC) IC74 and to regulate TGF-beta-dependent transcriptional events. Here we report the 2.1-A crystal structure of Dnlc2A using single anomalous diffraction. The proteins form a homodimer in solution and interact mainly through the helix alpha(2), strand beta(3), and the loop following this strand in each protein to generate a 10-stranded beta-sheet core. The surface of the beta-sheet core is mainly positively charged and predicted (by software PPI-Pred) to be the site that interacts with other partners. At the same time, the residues 79-82, 88, and 90 of each molecule formed two holes in the core. Residue 89 of each molecule, which is crucial for the DIC binding function of Dnlc2A, is within the holes. On the basis of these observations, we propose that the homodimer is the structural and functional unit maintained by hydrogen bonding interactions and hydrophobic packing, and that the patch of the surface of the beta-sheet core is the main area of interaction with other partners. Furthermore, the two holes would be the key sites to interact with IC74.

Pyridoxal kinase (PDXK) catalyzes the phosphorylation of pyridoxal, pyridoxamine, and pyridoxine in the presence of ATP and Zn2+. This constitutes an essential step in the synthesis of pyridoxal 5′-phosphate (PLP), the active form of vitamin B6, a cofactor for over 140 enzymes. (R)-Roscovitine (CYC202, Seliciclib) is a relatively selective inhibitor of cyclin-dependent kinases (CDKs), currently evaluated for the treatment of cancers, neurodegenerative disorders, renal diseases, and several viral infections. Affinity chromatography investigations have shown that (R)-roscovitine also interacts with PDXK. To understand this interaction, we determined the crystal structure of PDXK in complex with (R)-roscovitine, N6-methyl-(R)-roscovitine, and O6-(R)-roscovitine, the two latter derivatives being designed to bind to PDXK but not to CDKs. Structural analysis revealed that these three roscovitines bind similarly in the pyridoxal-binding site of PDXK rather than in the anticipated ATP-binding site. The pyridoxal pocket has thus an unexpected ability to accommodate molecules different from and larger than pyridoxal. This work provides detailed structural information on the interactions between PDXK and roscovitine and analogs. It could also aid in the design of roscovitine derivatives displaying strict selectivity for either PDXK or CDKs.

Cyclophilins (CyPs) are a widespreading protein family in living organisms and possess the activity of peptidyl-prolyl cis–trans isomerase (PPIase), which is inhibited by cyclosporin A (CsA). The human nuclear cyclophilin (hCyP33) is the first protein which was found to contain two RNA binding domains at the amino-terminus and a PPIase domain at the carboxyl-terminus. We isolated the hCyP33 gene from the human hematopoietic stem/progenitor cells and expressed it in Escherichia coli, and determined the crystal structure of the C domain of hCyP33 at 1.88 A resolution. The core structure is a β-barrel covered by two α-helices. Superposition of the structure of the C domain of hCyP33 with the structure of CypA suggests that the C domain contains PPIase active site which binds to CsA. Furthermore, C domain seems to be able to bind with the Gag-encoded capsid (CA) of HIV-1 and may affect the viral replication of HIV-1. A key residue of the active site is changed from Ala-103-CypA to Ser-239-hCyP33, which may affect the PPIase domain/substrates interactions.

To understand the processes involved in the catalytic mechanism of pyridoxal kinase (PLK),1 we determined the crystal structures of PLK.AMP-PCP-pyridoxamine, PLK.ADP.PLP, and PLK.ADP complexes. Comparisons of these structures have revealed that PLK exhibits different conformations during its catalytic process. After the binding of AMP-PCP (an analogue that replaced ATP) and pyridoxamine to PLK, this enzyme retains a conformation similar to that of the PLK.ATP complex. The distance between the reacting groups of the two substrates is 5.8 A apart, indicating that the position of ATP is not favorable to spontaneous transfer of its phosphate group. However, the structure of PLK.ADP.PLP complex exhibited significant changes in both the conformation of the enzyme and the location of the ligands at the active site. Therefore, it appears that after binding of both substrates, the enzyme-substrate complex requires changes in the protein structure to enable the transfer of the phosphate group from ATP to vitamin B(6). Furthermore, a conformation of the enzyme-substrate complex before the transition state of the enzymatic reaction was also hypothesized.

I257E was obtained by site directed mutagenesis of nitrite reductase from Achromobacter cycloclastes . The mutant has no enzyme activity. Its crystal structure determined at 1.65 A resolution shows that the side-chain carboxyl group of the mutated residue, Glu257, coordinates with the type 2 copper in the mutant and blocks the contact between the type 2 copper and its solvent channel, indicating that the accessibility of the type 2 copper is essential for maintaining the activity of nitrite reductase. The carboxylate is an analog of the substrate, nitrite, but the distances between the type 2 copper and the two oxygen atoms of the side-chain carboxyl group are reversed in comparison to the binding of nitrite to the native enzyme. In the mutant, both the type 2 copper and the Ne atom on the imidazole ring of its coordinated residue His135 move in the substrate binding direction relative to the native enzyme. In addition, an EPR study showed that the type 2 copper in the mutant is in a reduced state. We propose that mutant I257E is in a state corresponding to a transition state in the enzymatic reaction.

Earthworm fibrinolytic enzyme component A (EFEa) from Eisenia fetida is a strong fibrinolytic enzyme that not only directly degrades fibrin, but also activates plasminogen. Proteolytic assays further revealed that it cleaved behind various P1 residue types. The crystal structure of EFEa was determined using the MIR method and refined to 2.3A resolution. The enzyme, showing the overall polypeptide fold of chymotrypsin-like serine proteases, possesses essential S1 specificity determinants characteristic of elastase. However, the beta strand at the west rim of the S1 specificity pocket is significantly elongated by a unique four-residue insertion (Ser-Ser-Gly-Leu) after Val217, which not only provides additional substrate hydrogen binding sites for distal P residues, but also causes extension of the S1 pocket at the south rim. The S2 subsite of the enzyme was partially occluded by the bulky side-chain of residue Tyr99. Structure-based inhibitor modeling demonstrated that EFEa's S1 specificity pocket was preferable for elastase-specific small hydrophobic P1 residues, while its accommodation of long and/or bulky P1 residues was also feasible if enhanced binding of the substrate and induced fit of the S1 pocket were achieved. EFEa is thereby endowed with relatively broad substrate specificity, including the dual fibrinolysis. The presence of Tyr99 at the S2 subsite indicates a preference for P2-Gly, while an induced fit of Tyr99 was also suggested for accommodation of bigger P2 residues. This structure is the first reported for an earthworm fibrinolytic enzyme component and serine protease originating from annelid worms.

Icariin in Chinese traditional medicine was determined by an accurate and simple capillary zone electrophoresis method which used 30 mmol/L of borate solution containing 10% of acetonitrile as running buffer and needed no pretreatment. Copyright © 1999 John Wiley & Sons, Ltd.

The crystal structure of allophycocyanin from red algae Porphyra yezoensis (APC-PY) at 2.2-A resolution has been determined by the molecular replacement method. The crystal belongs to space group R32 with cell parameters a = b = 105.3 A, c = 189.4 A, alpha = beta = 90 degrees, gamma = 120 degrees. After several cycles of refinement using program X-PLOR and model building based on the electron density map, the crystallographic R-factor converged to 19.3% (R-free factor is 26.9%) in the range of 10.0 to 2.2 A. The r.m.s. deviations of bond length and angles are 0.015 A and 2.9 degrees, respectively. In the crystal, two APC-PY trimers associate face to face into a hexamer. The assembly of two trimers within the hexamer is similar to that of C-phycocyanin (C-PC) and R-phycoerythrin (R-PE) hexamers, but the assembly tightness of the two trimers to the hexamer is not so high as that in C-PC and R-PE hexamers. The chromophore-protein interactions and possible pathway of energy transfer were discussed. Phycocyanobilin 1alpha84 of APC-PY forms 5 hydrogen bonds with 3 residues in subunit 2beta of another monomer. In R-PE and C-PC, chromophore 1alpha84 only forms 1 hydrogen bond with 2beta77 residue in subunit 2beta. This result may support and explain great spectrum difference exists between APC trimer and monomer.

Based on the extensive structural comparisons among the determined structures of the different species and crystal forms of insulin and its derivatives in our laboratory, it was suggested that the binding interaction with the receptor molecule should take place mainly on an amphipathic surface of the insulin molecule. In the middle of this amphipathis surface, there was a hydrophobic surface with an area of about 150 A 2 , while the polar and charged groups distributing around the hydrophobic surface constructed a hydrophilic zone. The hydrophobic surface was usually covered by the extended B-chain C-terminal peptides with great mobility. The angle between the proposed binding interaction surface and the surface of dimerization was about 20°. The results from studies on structures of A1-( l -Trp) insulin and A1-( d -Trp) insulin confirmed the interaction mechanism model we proposed.