Chemical fingerprint analysis and quality assessment of Tibetan medicine Triphala from different origins by high-performance liquid chromatography.

INTRODUCTION: Although the Tibetan medicine Triphala (THL) is widely used in many countries, insufficient progress has been made in quality control. OBJECTIVES: The present study aimed to propose a methodology for quality control of THL based on HPLC fingerprinting combined with an orthogonal array design. METHODS: Seven identified peaks were used as indicators to examine the effects of temperature, extraction time, and solid-liquid ratio on the dissolution of active ingredients in THL. Fingerprint analysis was performed on 20 batches of THL from four geographical areas (China, Laos, Thailand, and Vietnam). For further chemometric assessment, analysis techniques including similarity analysis, hierarchical clustering analysis, principal component analysis, and orthogonal partial least squares discrimination analysis (OPLS-DA) were used to classify the 20 batches of samples. RESULTS: Fingerprints were established and 19 common peaks were identified. The similarity of 20 batches of THL was more than 0.9 and the batches were divided into two clusters. Four differential components of THL were identified based on OPLS-DA, including chebulinic acid, chebulagic acid, and corilagin. The optimal extraction conditions were an extraction time of 30 min, a temperature of 90°C, and a solid-liquid ratio of 30 mL/g. CONCLUSION: HPLC fingerprinting combined with an orthogonal array design could be used for comprehensive evaluation and quality assessment of THL, providing a theoretical basis for further development and utilization of THL.

GGC repeat expansion in NOTCH2NLC induces dysfunction in ribosome biogenesis and translation.

GGC repeat expansion in the 5' untranslated region (UTR) of NOTCH2NLC is associated with a broad spectrum of neurological disorders, especially neuronal intranuclear inclusion disease (NIID). Studies have found that GGC repeat expansion in NOTCH2NLC induces the formation of polyglycine (polyG)-containing protein, which is involved in the formation of neuronal intranuclear inclusions. However, the mechanism of neurotoxicity induced by NOTCH2NLC GGC repeats is unclear. Here, we used NIID patient-specific induced pluripotent stem cell (iPSC)-derived 3D cerebral organoids (3DCOs) and cellular models to investigate the pathophysiological mechanisms of NOTCH2NLC GGC repeat expansion. IPSC-derived 3DCOs and cellular models showed the deposition of polyG-containing intranuclear inclusions. The NOTCH2NLC GGC repeats could induce the upregulation of autophagic flux, enhance integrated stress response and activate EIF2α phosphorylation. Bulk RNA sequencing for iPSC-derived neurons and single-cell RNA sequencing (scRNA-seq) for iPSC-derived 3DCOs revealed that NOTCH2NLC GGC repeats may be associated with dysfunctions in ribosome biogenesis and translation. Moreover, NOTCH2NLC GGC repeats could induce the NPM1 nucleoplasm translocation, increase nucleolar stress, impair ribosome biogenesis and induce ribosomal RNA sequestration, suggesting dysfunction of membraneless organelles in the NIID cellular model. Dysfunctions in ribosome biogenesis and phosphorylated EIF2α and the resulting increase in the formation of G3BP1-positive stress granules may together lead to whole-cell translational inhibition, which may eventually cause cell death. Interestingly, scRNA-seq revealed that NOTCH2NLC GGC repeats may be associated with a significantly decreased proportion of immature neurons while 3DCOs were developing. Together, our results underscore the value of patient-specific iPSC-derived 3DCOs in investigating the mechanisms of polyG diseases, especially those caused by repeats in human-specific genes.

HSPA13 facilitates NF-κB-mediated transcription and attenuates cell death responses in TNFα signaling.

RIP1 has emerged as a master regulator in TNFα signaling that controls two distinct cellular fates: cell survival versus programmed cell death. Because the default response of most cells to TNFα is NF-κB­mediated inflammation and survival, a specific mechanism must exist to control the divergence of signaling outcome. Here, we identify HSPA13 as a transcription-independent checkpoint to modulate the role of RIP1 in TNFα signaling. Through specific binding to TNFR1 and RIP1, HSPA13 enhances TNFα-induced recruitment of RIP1 to TNFR1, and consequently promotes downstream NF-κB transcriptional responses. Meanwhile, HSPA13 attenuates the participation of RIP1 in cytosolic complex II and prevents cells from programmed death. Loss of HSPA13 shifts the transition of RIP1 from complex I to complex II and promotes both apoptosis and necroptosis. Thus, our study provides compelling evidence for the cellular protective function of HSPA13 in fine-tuning TNFα responses.

Hepatic connective tissue growth factor expression and regulation differ between non-steatotic and non-alcoholic steatotic livers from brain-dead donor.

Accurate evaluation of liver steatosis is required from brain-dead donors (BDDs) with nonalcoholic fatty liver disease (NAFLD). Our purposes were to investigate expression and regulation of connective tissue growth factor (CTGF) expression in livers from human and rat after brain death, and further evaluate its potential application. NAFLD and brain death models were established in rats. LX2 cells were cultured under hypoxia/reoxygenation. CTGF protein and mRNA levels were measured in liver samples from BDDs of human and rat by immunohistochemistry and reverse transcription-quantitative polymerase chain reaction. YAP-regulated CTGF expression was investigated in LX2 cells via YAP small interfering RNA and Verteporfin treatment. Blood CTGF level from BDDs was measured by enzyme-linked immunosorbent assay. After brain death, CTGF, transforming growth factor-ß and YAP were overexpressed in non-alcoholic steatotic liver, whereas CTGF was downregulated in non-steatotic liver. Time-series analysis revealed that CTGF and YAP expression was comparable, as confirmed by inhibited YAP expression in LX2 cells. CTGF level and NAFLD activity were linearly correlated. CTGF expression and regulation differ between non-steatosis and nonalcoholic steatosis livers from BDDs. CTGF may be an important factor to evaluate graft quality from BDDs with NAFLD.

SAF-189s, a potent new-generation ROS1 inhibitor, is active against crizotinib-resistant ROS1 mutant-driven tumors.

The ROS1 fusion kinase is an attractive antitumor target. Though with significant clinical efficacy, the well-known first-generation ROS1 inhibitor (ROS1i) crizotinib inevitably developed acquired resistance due to secondary point mutations in the ROS1 kinase. Novel ROS1is effective against mutations conferring secondary crizotinib resistance, especially G2032R, are urgently needed. In the present study, we evaluated the antitumor efficacy of SAF-189s, the new-generation ROS1/ALK inhibitor, against ROS1 fusion wild-type and crizotinib-resistant mutants. We showed that SAF-189s potently inhibited ROS1 kinase and its known acquired clinically resistant mutants, including the highly resistant G2032R mutant. SAF-189s displayed subnanomolar to nanomolar IC50 values against ROS1 wild-type and mutant kinase activity and a selectivity vs. other 288 protein kinases tested. SAF-189s blocked cellular ROS1 signaling, and in turn potently inhibited the cell proliferation in HCC78 cells and BaF3 cells expressing ROS1 fusion wild-type and resistance mutants. In nude mice bearing BaF3/CD74-ROS1 or BaF3/CD74-ROS1G2032R xenografts, oral administration of SAF-189s dose dependently suppressed the growth of both ROS1 wild-type- and G2032R mutant-driven tumors. In a patient-derived xenograft model of SDC4-ROS1 fusion NSCLC, oral administration of SAF-189s (20 mg/kg every day) induced tumor regression and exhibited notable prolonged and durable efficacy. In addition, SAF-189s was more potent than crizotinib and comparable to lorlatinib, the most advanced ROS1i known against the ROS1G2032R. Collectively, these results suggest the promising potential of SAF-189s for the treatment of patients with the ROS1 fusion G2032R mutation who relapse on crizotinib. It is now recruiting both crizotinib-relapsed and naive ROS1-positive NSCLC patients in a multicenter phase II trial (ClinicalTrials.gov Identifier: NCT04237805).

miR-425-5p suppresses tumorigenesis and DDP resistance in human-prostate cancer by targeting GSK3ß and inactivating the Wnt/ß-catenin signaling pathway.

Prostate cancer (PCa) represents the most frequently diagnosed cancer in men. Cisplatin, also known as cis-diamminedichloroplatinum (DDP), is a standard chemotherapeutic agent used to treat PCa, and DDP resistance remains one important obstacle in DDP-based chemotherapy. In our research, we found miR-425-5p was down-regulated in PCa and even lower in DDP-resistant PCa determined by quantitative polymerase chain reaction; in contrast, GSK3ß mRNA expression was upregulated in PCa and even higher in DDP-resistant PCa. Moreover, there was a modest but significant inverse correlation between the expression of GSK3ß mRNA and miR-425-5p. Functional experiments showed that miR-425-5p mimic inhibited DDP resistance as evidenced by a promoted apoptosis rate (flow cytometry) and suppressed cell viability (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay) and expressions of MDR1 andMRP1 (western blotting) in DU145/DDP and PC3/DDP cells. Luciferase reporter assay and RNA immunoprecipitation identifiedGSK3ß was a potential target of miR-425-5p. The effect ofmiR-425-5pmimic on DDP resistance was partially reversed by pcDNA-GSK3ß. Mechanically, miR-425-5p mimic reduced expression of ß-catenin, cyclin D1 and C-myc, which was further blocked when GSK3ß overexpressed. In vivo experiments, recovery of GSK3ß prevented xenograft tumor growth and DDP resistance in the presence of miR-425-5p mimic. To sum up, miR-425-5p upregulation might sensitize human PCa to DDP by targeting GSK3ß and inactivating the Wnt/ß-catenin signaling pathway.

A genetic link between CXCR5 and IL2RA gene polymorphisms and susceptibility to multiple sclerosis.

OBJECTIVES: Multiple sclerosis (MS) is a T-cell-mediated disease of the central nervous system that develops in individuals possessing a complex susceptibility trait. We explored relationship between gene polymorphisms in MS. METHODS: To identify the associations of CXCR5 and IL2RA gene polymorphisms with susceptibility to MS, we recruited 263 MS patients from the Han nationality and 138 from the Hui nationality as MS group and 284 healthy volunteers from the Han nationality and 156 from the Hui nationality as controls. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was employed to test gene polymorphisms of IL2RA (rs2104286 and rs12722489). Sequenom MassARRAY system was applied to analyze genotyping of CXCR5 (rs3922). RESULTS: The genotypes and allele frequency distributions at the loci of IL2RA rs2104286 and rs12722489 showed significant differences between the MS and control groups. The gene polymorphisms at the loci of IL2RA rs2104286 and rs12722489 may increase the onset risk of MS. IL2RA-rs2104286 showed a positive relationship with CXCR5-rs3922. The same relationship was also observed between IL2RA-rs12722489 and CXCR5-rs3922. The genotypes and allele frequencies of loci of rs2104286 and rs12722489 were significantly different in MS clinical subtypes and severity (EDSS score). Additionally, CAC and TGC haplotype at rs3922-rs12722489-rs2104286 may reduce the risk of MS, while CGT and TGT haplotypes increase the risk. CONCLUSION: The gene polymorphisms at the loci of IL2RA rs2104286 and rs12722489 are closely associated with susceptibility to MS in the Han and Hui nationalities.

Non-viral liposome-mediated transfer of brain-derived neurotrophic factor across the blood-brain barrier.

Brain-derived neurotrophic factor (BDNF) plays an important role in the repair of central nervous system injury, but cannot directly traverse the blood-brain barrier. Liposomes are a new type of non-viral vector, able to carry macromolecules across the blood-brain barrier and into the brain. Here, we investigate whether BDNF could be transported across the blood-brain barrier by tail-vein injection of liposomes conjugated to transferrin (Tf) and polyethylene glycol (PEG), and carrying BDNF modified with cytomegalovirus promoter (pCMV) or glial fibrillary acidic protein promoter (pGFAP) (Tf-pCMV-BDNF-PEG and Tf-pGFAP-BDNF-PEG, respectively). Both liposomes were able to traverse the blood-brain barrier, and BDNF was mainly expressed in the cerebral cortex. BDNF expression in the cerebral cortex was higher in the Tf-pGFAP-BDNF-PEG group than in the Tf-pCMV-BDNF-PEG group. This study demonstrates the successful construction of a non-virus targeted liposome, Tf-pGFAP-BDNF-PEG, which crosses the blood-brain barrier and is distributed in the cerebral cortex. Our work provides an experimental basis for BDNF-related targeted drug delivery in the brain.

Influence of artificial luminous environment and TCM intervention on development of myopia rabbits

OBJECTIVE@#To explore the influence of artificial luminous environment and preventive function of traditional Chinese medicine (TCM) intervention based on "Theory of yin-yang clock" on myopia.@*METHODS@#A total of 45 New Zealand young rabbits were randomly divided into 5 groups, 9 for each group. Control group was exposed in natural light. Fluorescent group and full spectrum group were exposed in fluorescent light and full spectrum light, on which basis fluorescent TCM group and full spectrum TCM group were added with "Rizhong Yinyang Formulas", respectively. Optical parameters were measured and the influence of different lights on the serum and retinal dopamine (DA) levels as well as the retinal histopathological tissues was observed.@*RESULTS@#The spectrum of fluorescent light mainly focused at 420-490 nm with the peak value of wavelength near 450 nm, whereas that of full spectrum was wider (400-800 nm) with the peak value near 600 nm. After 4 and 12 weeks, fluorescent group was evidently lower in serum and retinal DA levels (P0.05). Histopathological observation showed that there was significant difference in pigment epithelium layer, photoreceptor and nerve fiber layer between fluorescent group and control group, but the difference among the test groups was not significant.@*CONCLUSIONS@#Fluorescent light has certain influence on retinal histological construction and visual performance. However, TCM intervention may have some degree of protective function on retina.

Influence of artificial luminous environment and TCM intervention on development of myopia rabbits

Objective:To explore the influence of artificial luminous environment and preventive function of traditional Chinese medicine (TCM) intervention based on “Theory of yin-yang clock” on myopia.Methods:A total of 45 New Zealand young rabbits were randomly divided into 5 groups, 9 for each group. Control group was exposed in natural light. Fluorescent group and full spectrum group were exposed in fluorescent light and full spectrum light, on which basis fluorescent TCM group and full spectrum TCM group were added with “Rizhong Yinyang Formulas”, respectively. Optical parameters were measured and the influence of different lights on the serum and retinal dopamine (DA) levels as well as the retinal histopathological tissues was observed.Results: The spectrum of fluorescent light mainly focused at 420-490 nm with the peak value of wavelength near 450 nm, whereas that of full spectrum was wider (400-800 nm) with the peak value near 600 nm. After 4 and 12 weeks, fluorescent group was evidently lower in serum and retinal DA levels (P0.05). Histopathological observation showed that there was significant difference in pigment epithelium layer, photoreceptor and nerve fiber layer between fluorescent group and control group, but the difference among the test groups was not significant.Conclusions:Fluorescent light has certain influence on retinal histological construction and visual performance. However, TCM intervention may have some degree of protective function on retina.

Influence of artificial luminous environment and TCM intervention on development of myopia rabbits

Objective: To explore the influence of artificial luminous environment and preventive function of traditional Chinese medicine (TCM) intervention based on "Theory of yin-yang clock" on myopia. Methods: A total of 45 New Zealand young rabbits were randomly divided into 5 groups, 9 for each group. Control group was exposed in natural light. Fluorescent group and full spectrum group were exposed in fluorescent light and full spectrum light, on which basis fluorescent TCM group and full spectrum TCM group were added with "Rizhong Yinyang Formulas", respectively. Optical parameters were measured and the influence of different lights on the serum and retinal dopamine (DA) levels as well as the retinal histopathological tissues was observed. Results: The spectrum of fluorescent light mainly focused at 420-490 nm with the peak value of wavelength near 450 nm, whereas that of full spectrum was wider (400-800 nm) with the peak value near 600 nm. After 4 and 12 weeks, fluorescent group was evidently lower in serum and retinal DA levels (. P0.05). Histopathological observation showed that there was significant difference in pigment epithelium layer, photoreceptor and nerve fiber layer between fluorescent group and control group, but the difference among the test groups was not significant. Conclusions: Fluorescent light has certain influence on retinal histological construction and visual performance. However, TCM intervention may have some degree of protective function on retina.

Expression heterogeneity research of ITGB3 and BCL-2 in lung adenocarcinoma tissue and adenocarcinoma cell line.

OBJECTIVE: To analyze expression heterogeneity of Integrin beta 3 (ITGB3) and B-cell lymphoma 2 (BCL-2) in lung adenocarcinoma tissue and adenocarcinoma cell line and further provide theoretical direction for molecular biological research of lung adenocarcinoma. METHODS: Tissue microarray was used to observe relation among expression, heterogeneitpy and clinical characteristics of ITGB3 and BCL-2 in lung cancer. RESULTS: ITGB3 and BCL-2 increased significantly in A549 cells in CAFs group withß-actin as control; the expression level of BCL-2 also increased in ITGB3 transfected cells with GFP plasmid transfected A549 cells as control; immunohistochemistry staining showed that positive rates of ITGB3, ITGB1 and BCL-2 in normal lung tissues were 0, the positive rates in lung adenocarcinoma were 7.04%, 84.51% and 4.23%, respectively; in the results of immunohistochemistry staining, the expression of Girdin protein in lung adenocarcinoma was homogeneous, however protein expression of ITGB3, ITGB1 and BCL-2 showed different patterns in the same location with significant heterogeneity; majority of ITGB3, ITGB1 or BCL-2 positive tissue showed heterogeneity that expression in trailing edge was higher than that of trailing edge in lung adenocarcinoma tissue, the patients with BCL-2 heterogeneity showed higher lymph node metastasis ratio and lower clinical stage (P<0.05); and the expression of ITGB3 and the clinical characteristics of patients were not significant related (P>0.05). CONCLUSIONS: Expression of ITGB3 and BCL-2 in lung adenocarcinoma and adenocarcinoma cell line showed heterogeneity that expression in trailing edge was higher than that of trailing edge, which may play an important role in promoting tumor lymph node metastasis and vascular invasion, and provides a new research direction for exploration of lung adenocarcinoma metastasis mechanism.

Population pharmacokinetics of digoxin in elderly patients.

This study was aimed at determining the population pharmacokinetics of digoxin and identifying factors that explain pharmacokinetic variability in elderly patients. The data of 142 elderly patients and 448 samples were collected after repetitive oral digoxin. Blood samples were drawn at various times after administration. Population pharmacokinetic analysis was performed using nonlinear mixed effects modelling program (NONMEM). A one-compartment model with first-order absorption and elimination was selected as the base model. The influence of demographic characteristics, biochemical and haematological indices as well as other commonly used co-medications were explored. The typical values with interindividual variability for apparent clearance (CL/F) and apparent volume of distribution (V/F) were 8.9 L h(-1) (43.2 %) and 420 L (65.8 %), respectively. The residual variability was 31.6 %. CL/F decreased significantly with renal function, total body weight, calcium channel blockers or spironolactone co-therapy and symptom with congestive heart failure. The median parameter estimates from a nonparametric bootstrap procedure were comparable and within 5 % of the estimates from NONMEM. These results provide important information for clinicians to optimize digoxin regimens in elderly patients.

[Effects of nitrification inhibitors DCD and DMPP on cinnamon soil' s gross nitrogen mineralization and nitrification rates].

By using 15N pool dilution technique in combining with in situ soil cultivation, this paper studied the effects of nitrification inhibitors dicyandiamide (DCD) and 3,4-dimethylpyrazole phosphate (DMPP) on the gross nitrogen (N) mineralization and nitrification rates in a saline-alkali cinnamon soil in North China. The experiment was carried out in a maize-wheat rotation field in Yuncheng City of Shanxi Province, and three treatments were installed, i.e., urea, urea + DCD, and urea + DMPP. In the first two weeks after fertilization, DCD and DMPP made the gross N mineralization rate and gross N nitrification rate decreased by 25.5% and 7.3%, and by 60.3% and 59.1%, respectively, with a significant difference in the gross N mineralization rate but less difference in the gross N nitrification rate between the effects of DCD and DMPP. However, significant difference was observed in the gross N nitrification rate between the effects of DCD and DMPP after seven weeks of fertilization. The gross N mineralization and nitrification rates and the NH4+ and NO3-consumption rates after two weeks of fertilization were 7.2-10.0, 5.5-21.5, 9.1-12.2, and 5.1-8.4 times of those before fertilization, respectively, possibly due to the stimulating effect of N fertilization. DCD and DMPP made the fertilizer urea N more maintained in NH(4+)-N form and less accumulated in NO(3-)-N form in soil. The decreases of the gross N mineralization and nitrifications rate in the test soil due to the effects of the inhibitors would benefit the reduction of N2O emission from the soil.

[KRAS and epidermal growth factor receptor gene mutation in non-small cell lung cancer].

OBJECTIVE: To study KRAS and epidermal growth factor receptor (EGFR) mutations in primary non-small cell lung cancer (NSCLC) and their association with the effects of targeted therapy. METHODS: Gene mutations of KRAS and EGFR in both primary tumors and local lymph node metastases from 150 patients with NSCLC were analyzed by direct sequencing. Twelve of the patients were given gefitinib as neoadjuvant therapy after EGFR-TKI sensitive mutations had been detected in biopsies of mediastinal lymph node metastases. RESULTS: Two primary tumors and 10 metastases were identified to have KRAS mutations, while 35 primary tumors and 44 metastases were found to have EGFR mutations. KRAS and EGFR mutation status was different between primary tumors and corresponding metastases in 6.7% (10/150) and 8.67% (13/150) patients, respectively. One patient with no TKI sensitive mutations in the primary tumor showed disease progression with gefitinib therapy. CONCLUSIONS: Our results suggest that a considerable proportion of NSCLC in Chinese patients showed discrepancy in KRAS and EGFR mutation status between primary tumors and corresponding metastases. This observation may have important implications for the use of targeted TKI therapy in the treatment of NSCLC patients.

QTL mapping for test weight by using F(2:3) population in maize.

Test weight is an important trait in maize breeding. Understanding the genetic mechanism of test weight is important for effective selection of maize test weight improvement. In this study, quantitative trait loci (QTL) for maize test weight were identified. In the years 2007 and 2008, a F(2:3) population along with the parents Chang7-2 and Zheng58 were planted in Zhengzhou, People's Republic of China. Significant genotypic variation for maize test weight was observed in both years. Based on the genetic map containing 180 polymorphic SSR markers with an average linkage distance of 11.0 cM, QTL for maize test weight were analysed by mixed-model composite interval mapping. Five QTL, including four QTL with only additive effects, were identified on chromosomes 1, 2, 3, 4 and 5, and together explained 25.2% of the phenotypic variation. Seven pairs of epistatic interactions were also detected, involving 11 loci distributed on chromosomes 1, 2, 3, 4, 5 and 7, respectively, which totally contributed 18.2% of the phenotypic variation. However, no significant QTL x environment (QxE) interaction and epistasis x environment interaction effects were detected. The results showed that besides the additive QTL, epistatic interactions also formed an important genetic basis for test weight in maize.

A new QTL for resistance to Fusarium ear rot in maize.

Understanding the inheritance of resistance to Fusarium ear rot is a basic prerequisite for an efficient resistance breeding in maize. In this study, 250 recombinant inbred lines (RILs) along with their resistant (BT-1) and susceptible (N6) parents were planted in Zhengzhou with three replications in 2007 and 2008. Each line was artificially inoculated using the nail-punch method. Significant genotypic variation in response to Fusarium ear rot was detected in both years. Based on a genetic map containing 207 polymorphic simple sequence repeat (SSR) markers with average genetic distances of 8.83 cM, the ear rot resistance quantitative trait loci (QTL) were analyzed by composite interval mapping with a mixed model (MCIM) across the environments. In total, four QTL were detected on chromosomes 3, 4, 5, and 6. The resistance allele at each of these four QTL was contributed by resistant parent BT-1, and accounted for 2.5-10.2% of the phenotypic variation. However, no significant epistasis interaction effect was detected after a two-dimensional genome scan. Among the four QTL, one QTL with the largest effect on chromosome 4 (bin 4.06) can be suggested to be a new locus for resistance to Fusarium ear rot, which broadens the genetic base for resistance to the disease and can be used for further genetic improvement in maize-breeding programs.

The enzymatic reaction-induced configuration change of the prosthetic group PQQ of methanol dehydrogenase.

Methanol dehydrogenase is a heterotetrameric enzyme containing the prosthetic group pyrroloquinoline quinone (PQQ), which catalyzes the oxidation of methanol to formaldehyde. The crystal structure of methanol dehydrogenase from Methylophilus W3A1, previously determined at high resolution, exhibits a non-planar configuration of the PQQ ring system and lends support for a hydride transfer mechanism of the enzymatic reaction catalyzed by the enzyme. To investigate why PQQ is in the C5-reduced form and to better understand the catalytic mechanism of the enzyme, three structures of this enzyme in a new crystal form have been determined at higher resolution. Two of the three crystals were grown in the presence of 1 and 50 mM methanol, respectively, both structures of which show non-planar configurations of the PQQ ring system, confirming the previous conclusion; the other was crystallized in the presence of 50 mM ethanol, the structure of which displays a planar ring system for PQQ. Comparison of these structures reveals that the configuration change of PQQ is induced by the enzymatic reaction. The reaction takes place and the C5-reduced PQQ intermediate is produced when the enzyme co-crystallizes with methanol, but the enzymatic reaction does not take place and the PQQ ring retains a planar configuration of the oxidized orthoquinone form when ethanol instead of methanol is present in the crystallization solution.

Expression, crystallization and preliminary X-ray analysis of an anomeric inverting agarase from Pseudoalteromonas sp. CY24.

AgaB from Pseudoalteromonas sp. CY24 is a novel agarase that hydrolyzes agarose to generate products with inverted anomeric configuration and that has been proposed to have a larger catalytic cleft than other ß-agarases. Here, the expression, purification, crystallization and data collection of AgaB in both wild-type and selenomethionine-substituted forms is described. The crystals of wild-type AgaB diffracted to 1.97 Šresolution and belonged to space group C222(1). The selenomethionine derivative crystallized in space group I222. The phasing problem was solved by the multiwavelength anomalous dispersion (MAD) method. These results will facilitate detailed structural and enzymatic analysis of AgaB.

4-[6,8-Dibromo-2-(2-chloro-5-nitro-phen-yl)-1,2,3,4-tetra-hydro-quinazolin-3-yl]cyclo-hexa-nol.

The title compound, C(20)H(20)Br(2)ClN(3)O(3), was synthesized by the condensation reaction of 2-chloro-5-nitro-benzaldehyde with 4-(2-amino-3,5-dibromo-benzyl-amino)cyclo-hexa-nol in a methanol solution. There are two independent mol-ecules in the asymmetric unit and in one mol-ecule the atoms of the cyclo-hexane ring are disordered over two sets of sites with refined occupancies of 0.657 (12) and 0.343 (12). The dihedral angle between the two benzene rings is 89.5 (2)° in one mol-ecule and 82.9 (2)° in the other. In the crystal structure, inter-molecular N-H⋯O and O-H⋯O hydrogen bonds link the mol-ecules into chains propagating along [01].