枸橼酸莫沙必利对老年功能性消化不良患者胃功能的影响

目的:探讨枸橼酸莫沙必利分散片对老年功能性消化不良(FD)患者胃功能的影响。方法:将2012年3月~2014年3月我院79例老年FD患者(FD组)给予口服枸缘酸莫沙必利分散片,并随机抽取同一时期120例我院健康体检者为对照组,检测并比较两组的胃底气体的评分、延迟排空相、近端胃容积以及近端胃液体半排空时间。结果:FD组治疗前延迟排空相大于对照组,近端胃液体半排空时间小于对照组,差异有统计学意义(P0.05),两组的胃底气体的评分差异无统计学意义(P0.05);FD组治疗后延迟排空相小于治疗前,近端胃液体半排空时间大于治疗前,差异有统计学意义(P0.05);FD组治疗前0 min、5 min、15 min、30min、60 min的近端胃容积均小于对照组,差异均有统计学意义(P0.05);FD组治疗后各时刻近端胃容积大于治疗前,差异均有统计学意义(P0.05)。结论:老年FD患者采用枸缘酸莫沙必利分散片治疗可明显改善患者的胃功能,促进胃排空的速度,值得临床推广。     

A catalytic defect in mitochondrial respiratory chain complex I due to a mutation in NDUFS2 in a patient with Leigh syndrome

In this study, we investigated the pathogenicity of a homozygous Asp446Asn mutation in the NDUFS2 gene of a patient with a mitochondrial respiratory chain complex I deficiency. The clinical, biochemical, and genetic features of the NDUFS2 patient were compared with those of 4 patients with previously identified NDUFS2 mutations. All 5 patients presented with Leigh syndrome. In addition, 3 out of 5 showed hypertrophic cardiomyopathy. Complex I amounts in the patient carrying the Asp446Asn mutation were normal, while the complex I activity was strongly reduced, showing that the NDUFS2 mutation affects complex I enzymatic function. By contrast, the 4 other NDUFS2 patients showed both a reduced amount and activity of complex I. The enzymatic defect in fibroblasts of the patient carrying the Asp446Asn mutation was rescued by transduction of wild type NDUFS2. A 3-D model of the catalytic core of complex I showed that the mutated amino acid residue resides near the coenzyme Q binding pocket. However, the K_M of complex I for coenzyme Q analogs of the Asp446Asn mutated complex I was similar to the K_M observed in other complex I defects and in controls. We propose that the mutation interferes with the reduction of coenzyme Q or with the coupling of coenzyme Q reduction with the conformational changes involved in proton pumping of complex I.     

Crystal structure of the yeast His6 enzyme suggests a reaction mechanism

The Saccharomyces cerevisiae His6 gene codes for the enzyme phosphoribosyl-5-amino-1-phosphoribosyl-4-imidazolecarboxamide isomerase, catalyzing the fourth step in histidine biosynthesis. To get an insight into the structure and function of this enzyme, we determined its X-ray structure at a resolution of 1.30 A using the anomalous diffraction signal of the protein's sulphur atoms at 1.77 A wavelength. His6 folds in an (alpha/beta)8 barrel similar to HisA, which performs the same function in bacteria and archaea. We found a citrate molecule from the buffer bound in a pocket near the expected position of the active site and used it to model the open form of the substrate (phosphoribulosyl moiety), which is a reaction intermediate. This model enables us to identify catalytic residues and to propose a reaction mechanism where two aspartates act as acid/base catalysts: Asp134 as a proton donor for ring opening, and Asp9 as a proton acceptor and donor during enolization of the aminoaldose. Asp9 is conserved in yeast His6 and bacterial or archaeal HisA sequences, and Asp134 has equivalents in both HisA and TrpF, but they occur at a different position in the protein sequence.     

Characterization and localization of Rickettsia sp. in water beetles of genus Deronectes (Coleoptera: Dytiscidae)

In the present study, Rickettsia sp. was detected in four water beetles of the genus Deronectes ( Dytiscidae ) for the first time. Rickettsiae were found in 100% of examined specimens of Deronectes platynotus (45/45), 39.4% of Deronectes aubei (28/71), 40% of Deronectes delarouzei (2/5) and 33.3% of Deronectes semirufus (1/3). Analysis of 16S rRNA gene sequences revealed a phylogenetic relationship with rickettsial isolates of Limonia chorea ( Diptera ), tentatively classified as members of the basal ancestral group. Phylogenetic analysis of the gltA (citrate synthase) gene sequences showed that Deronectes symbionts were closest to bacterial symbionts from spiders. Ultrastructural examinations revealed typical morphological features and intracellular arrangements of rickettsiae. The distribution, transmission and localization of Rickettsia sp. in D. platynotus were studied using a diagnostic PCR assay and FISH. Eggs from infected females of D. platynotus were all Rickettsia -positive, indicative of a vertical transmission.     

Small molecule clearance in ultrafiltration/diafiltration in relation to protein interactions: Study of citrate binding to a Fab

Ultrafiltration/diafiltration (UFDF) is commonly utilized in the purification of recombinant proteins to concentrate and buffer exchange the product. It is often the final step in the purification process, placing the protein in its final formulation and clearing small molecules introduced in upstream purification steps. This article presents a case study of reduced small molecule clearance in ultrafiltration/diafiltration of an antigen‐binding fragment of a monoclonal antibody. Citrate, a commonly utilized small molecule in downstream processes, is shown to have reduced clearance due to specific interactions with the protein product. The study presents process solutions and utilizes a simple model to characterize clearance of small molecules which exhibit interactions with product protein. Biotechnol. Bioeng. 2009;102: 1718–1722. © 2008 Wiley Periodicals, Inc.     

Rapid down-regulation of mitochondrial fat metabolism in human muscle after training cessation is dissociated from changes in insulin sensitivity

The association between impairment in mitochondrial muscle fat oxidative capacity (OX_FA) and occurrence of insulin resistance was examined in 14 healthy trained men (age, 24 ± 4 yr) submitted to 4 weeks of training cessation. Training stop induced a significant decrease in mRNA levels of proteins involved in muscle fat metabolism, particularly PPARα (−58%, P < 0.01) and PGC-1α (−30%, P < 0.05), a 21% reduction in OX_FA (P < 0.01), and reduced fat oxidation during moderately intense exercise (P < 0.05). In contrast, there was no significant alteration in insulin sensitivity. In conclusion, decline in OX_FA is a rapid metabolic event following training cessation. It is involved in the regulation of whole body fat balance but not in the deterioration of insulin sensitivity.     

pH-Dependent Conformational Changes in Bacterial Hsp90 Reveal a Grp94-Like Conformation at pH 6 That Is Highly Active in Suppression of Citrate Synthase Aggregation

The molecular chaperone Hsp90 depends upon large conformational rearrangements for its function. One driving force for these rearrangements is the intrinsic ATPase activity of Hsp90, as seen with other chaperones. However, unlike other chaperones, structural and kinetic studies have shown that the ATPase cycle of Hsp90 is not conformationally deterministic. That is, rather than dictating the conformational state, ATP binding and hydrolysis shift the equilibrium between a preexisting set of conformational states in an organism-dependent manner. While many conformations of Hsp90 have been described, little is known about how they relate to chaperone function. In this study, we show that the conformational equilibrium of the bacterial Hsp90, HtpG, can be shifted with pH. Using small-angle X-ray scattering, we identify a two-state pH-dependent conformational equilibrium for apo HtpG. Our structural modeling reveals that this equilibrium is observed between the previously observed extended state and a second state that is strikingly similar to the recently solved Grp94 crystal structure. In the presence of nonhydrolyzable 5′-adenylyl-β,γ-imidodiphosphate, a third state, which is identical with the solved AMPPNP-bound structure from yeast Hsp90, is populated. Electron microscopy confirmed the observed conformational equilibria. We also identify key histidine residues that control this pH-dependent equilibrium; using mutagenesis, we successfully modulate the conformational equilibrium at neutral pH. Using these mutations, we show that the Grp94-like state provides stronger aggregation protection compared to the extended apo conformation in the context of a citrate synthase aggregation assay. These studies provide a more detailed view of HtpG's conformational dynamics and provide the first linkage between a specific conformation and chaperone function.     

Inhibition of heat- and chemical-induced aggregation of various proteins reveals chaperone-like activity of the acute-phase component and serine protease inhibitor human α1-antitrypsin

In vitro chaperone-like activity of the serpin family member and plasma acute-phase component human α₁-antitrypsin (AAT) has been shown for the first time. Results of light-scattering experiments demonstrated that AAT efficiently inhibits both heat- and chemical-induced aggregation of various test proteins including alcohol dehydrogenase, aldolase, carbonic anhydrase, catalase, citrate synthase, enolase, glutathione S-transferase, l-lactate dehydrogenase, and β_L-crystallin. The results suggest that the unique metastable serpin architecture enables dual function, protease inhibiton as well as chaperone activity and highlight the serpin superfamily as a possible source of additional intra- and extracellular chaperones (e.g. α₁-antichymotrypsin). The present finding is surprising in the light of the well-known role of mutated forms of AAT and other serpins in the pathogenesis of diseases called serpinopathies that featured with aberrant conformational transitions and consequent self-aggregation of serpin proteins.     

Charge-Rich Regions Modulate the Anti-Aggregation Activity of Hsp90

Protein aggregation can have dramatic effects on cellular function and plays a causative role in many human diseases. In all cells, molecular chaperones bind to aggregation-prone proteins and hinder aggregation. The ability of a protein to resist aggregation and remain soluble in aqueous solution is linked to the physical properties of the protein. Numerous physical studies demonstrate that charged atoms favor solubility. We note that many molecular chaperones possess a substantial negative charge that may allow them to impart solubility on aggregation-prone proteins. Hsp90 is one such negatively charged molecular chaperone. The charge on Hsp90 is largely concentrated in two highly acidic regions. To investigate the relationship between chaperone charge and protein solubility, we deleted these charge-rich regions and analyzed the resulting Hsp90 constructs for anti-aggregation activity. We found that deletion of both charge-rich regions dramatically impaired Hsp90 anti-aggregation activity. The anti-aggregation role of the deleted charge-rich regions could be due to net charge or sequence-specific features. To distinguish these possibilities, we attached an acid-rich region with a distinct amino acid sequence to our double-deleted Hsp90 construct. This charge rescue construct displayed effective anti-aggregation activity indicating that the net charge of Hsp90 contributes to its anti-aggregation activity.