The raised midpoint potential of the [2Fe2S] cluster of cytochrome bc1 is mediated by both the Qo site occupants and the head domain position of the Fe-S protein subunit

We have previously reported that mutant strains of Rhodobacter capsulatus that have alanine insertions (+nAla mutants) in the hinge region of the iron sulfur (Fe-S) containing subunit of the bc(1) complex have increased redox midpoint potentials (E(m)) for their [2Fe2S] clusters. The alteration of the E(m) in these strains, which contain mutations far from the metal binding site, implied that the local environment of the metal center is indirectly altered by a change in the interaction of this subunit with the hydroquinone oxidizing (Q(o)) site [Darrouzet, E., Valkova-Valchanova, M., and Daldal, F. (2002) J. Biol. Chem. 277, 3464-3470]. Subsequently, the E(m) changes have been proposed to be predominantly due to a stronger or more stabilized hydrogen bonding between the reduced [2Fe2S] cluster and the Q(o) site inhabitant ubiquinone (Q) [Shinkarev, V. P., Kolling, D. R. J., Miller, T. J., and Crofts, A. R. (2002) Biochemistry 41, 14372-14382]. To further investigate this issue, Fe-S protein-Q interactions were monitored by electron paramagnetic resonance (EPR) spectroscopy and the findings indicated that the wild type and mutant proteins interactions with Q are similar. Moreover, when the Q(pool) was chemically depleted, the E(m) of the [2Fe2S] cluster in mutant bc(1) complexes remained more positive than a similarly treated native enzyme (e.g., the [2Fe2S] E(m) of the +2Ala mutant was 55 mV more positive than the wild type). These data suggest that the increased E(m) of the [2Fe2S] cluster in the +nAla mutants is in part due to the cluster's interaction with Q, and in part to additional factors that are independent of hydrogen bonding to Q. One such factor, the possibility of a different position of the Fe-S at the Q(o) site of the mutant proteins versus the native enzyme, was addressed by determining the orientation of the [2Fe2S] cluster in the membrane using EPR spectroscopy. In the case of the +2Ala mutant, the [2Fe2S] cluster orientation in the absence of inhibitor is different than that seen in the native enzyme. However, the +2Ala mutant cluster shared a similar orientation with the native enzyme when both samples were exposed to either stigmatellin or myxothiazol. In addition, Q(pool) extracted membranes of +2Ala mutant exhibited fewer overall orientations, with the predominant one being more similar to that observed in the non-Q-depleted membranes of the +2Ala mutant than the Q-depleted membranes of a wild-type strain. Therefore, additional component(s) that are independent of Q(o) site inhabitants and that originate from the newly observed orientations of the [2Fe2S] clusters in the +nAla mutants also contribute to the increased midpoint potentials of their [2Fe2S] clusters. While the molecular basis of these components remains to be determined, salient implications of these findings in terms of Q(o) site catalysis are discussed.     

Q fever and pneumonia in an area with a high livestock density: a large population-based study

Concerns about public health risks of intensive animal production in The Netherlands continue to rise, in particular related to outbreaks of infectious diseases. The aim was to investigate associations between the presence of farm animals around the home address and Q fever and pneumonia.Electronic medical record data for the year 2009 of all patients of 27 general practitioners (GPs) in a region with a high density of animal farms were used. Density of farm animals around the home address was calculated using a Geographic Information System. During the study period, a large Q fever outbreak occurred in this region. Associations between farm exposure variables and pneumonia or 'other infectious disease', the diagnosis code used by GPs for registration of Q fever, were analyzed in 22,406 children (0-17 y) and 70,142 adults (18-70 y), and adjusted for age and sex. In adults, clear exposure-response relationships between the number of goats within 5 km of the home address and pneumonia and 'other infectious disease' were observed. The association with 'other infectious disease' was particularly strong, with an OR [95%CI] of 12.03 [8.79-16.46] for the fourth quartile (>17,190 goats) compared with the first quartile (<2,251 goats). The presence of poultry within 1 km was associated with an increased incidence of pneumonia among adults (OR [95%CI] 1.25 [1.06-1.47]).A high density of goats in a densely populated region was associated with human Q fever. The use of GP records combined with individual exposure estimates using a Geographic Information System is a powerful approach to assess environmental health risks.     

Epidemiological,clinical and laboratory features of acute Q fever in a cohort of hospitalized patients in a regional hospital,Israel, 2012-2018

IntroductionAcute Q fever is endemic in Israel, yet the clinical and laboratory picture is poorly defined.MethodsA retrospective study reviewing the medical records of acute Q fever patients, conducted in a single hospital in the Sharon district, Israel. Serum samples from suspected cases were preliminary tested by a qualitative enzyme immunoassay (EIA). Confirmatory testing at the reference laboratory used an indirect immunofluorescence assay (IFA). Positive cases were defined as fever with at least one other symptom and accepted laboratory criteria such as a single-phase II immunoglobulin G (IgG) antibody titer ≥1:200. Cases not fulfilling these criteria and in which acute Q fever was excluded, served as a control group.ResultsBetween January 2012 and May 2018, 484 patients tested positive. After confirmatory testing, 65 (13.4%) were positive for acute Q fever (with requisite clinical picture), 171 (35.3%) were definitely not infected, the remaining 248 were excluded because of past/chronic/undetermined infection. The average age was 58 years and 66% were males. Most resided in urban areas with rare animal exposure. Pneumonia was seen in 57% of cases and a combination with headache/hepatitis was highly suggestive of acute Q fever diagnosis. Syncope/presyncope, fall and arthritis were more common in acute Q fever cases. Laboratory indexes were similar to the control group, except for erythrocyte sedimentation rate (ESR) which was more common and higher in the study group.ConclusionAcute Q fever in the Sharon district could be better diagnosed by using a syndromic approach in combination with improved rapid diagnostic testing.     

High endemicity of Q fever in French Guiana: A cross sectional study (2007–2017)

Q fever (QF) is a zoonosis caused by Coxiella burnetii (Cb). French Guiana (FG) had a high incidence but no data have been published since 2006. The objective of this study was to update the incidence and epidemiological data on QF in FG. A retrospective study of all FG Q fever serodiagnosis between 2007 and 2017 was carried out. Among the 695 patients included, the M/F sex-ratio was 2.0 and the median age of 45.3 years (IQR 33.7–56.3). The annual QF incidence rate was 27.4 cases (95%CI: 7.1–47.7) per 100,000 inhabitants ranging from 5.2 in 2007 to 40.4 in 2010. Risk factors associated with Q fever compared to general population were male gender, being born in mainland France, an age between 30 to 59 years-old and a residence in Cayenne and surroundings. The incidence of QF in FG remains high and stable and the highest in the world.     

Thermostability of manganese- and iron-superoxide dismutases from Escherichia coli is determined by the characteristic position of a glutamine residue.

The structurally homologous mononuclear iron and manganese superoxide dismutases (FeSOD and MnSOD, respectively) contain a highly conserved glutamine residue in the active site which projects toward the active-site metal centre and participates in an extensive hydrogen bonding network. The position of this residue is different for each SOD isoenzyme (Q69 in FeSOD and Q146 in MnSOD of Escherichia coli). Although site-directed mutant enzymes lacking this glutamine residue (FeSOD[Q69G] and MnSOD[Q146A]) demonstrated a higher degree of selectivity for their respective metal, they showed little or no activity compared with wild types. FeSOD double mutants (FeSOD[Q69G/A141Q]), which mimic the glutamine position in MnSOD, elicited 25% the activity of wild-type FeSOD while the activity of the corresponding MnSOD double mutant (MnSOD[G77Q/Q146A]) increased to 150% (relative to wild-type MnSOD). Both double mutants showed reduced selectivity toward their metal. Differences exhibited in the thermostability of SOD activity was most obvious in the mutants that contained two glutamine residues (FeSOD[A141Q] and MnSOD[G77Q]), where the MnSOD mutant was thermostable and the FeSOD mutant was thermolabile. Significantly, the MnSOD double mutant exhibited a thermal-inactivation profile similar to that of wild-type FeSOD while that of the FeSOD double mutant was similar to wild-type MnSOD. We conclude therefore that the position of this glutamine residue contributes to metal selectivity and is responsible for some of the different physicochemical properties of these SODs, and in particular their characteristic thermostability.     

湖北省神农架林区野猪分布与传播非洲猪瘟风险分析

野猪是非洲猪瘟(ASF)传播的重要媒介,神农架林区野猪种群数量高、分布广,调查野猪分布对ASF防控有着重要意义.本研究首先通过布设红外相机117台、样线287条调查神农架林区野猪实体和痕迹位点后,应用最大熵模型预测林区野猪分布,再根据野猪、居民区、交通要道的空间分布数据,应用核密度估计法分析神农架林区各地野猪传播ASF...     

鱼腥藻苯丙氨酸脱氨酶的基因克隆、表达及最适反应pH改造

【目的】表达鱼腥藻苯丙氨酸脱氨酶(AvPAL),并经分子改造降低其最适反应pH。【方法】PCR克隆AvPAL编码基因,并在大肠杆菌中表达,用Ni2+亲和层析柱和凝胶柱纯化重组蛋白。利用GETAREA软件筛选与催化残基距离较近的暴露于酶分子表面的氨基酸位点,将其突变为带电性质不同的氨基酸,并对突变体进行酶学性质研究。【结果】在大肠杆菌中成功表达了AvPAL,纯化后得到电泳纯的重组酶。突变体E75Q和E75R的最适反应pH从8.5分别偏移到7.5和7.0。E75Q在pH 7.5时的比酶活较原酶提高了25%,在pH 6.5–9.5之间酶的稳定性良好,其最适反应温度为50 °C,在此温度下保温1 h酶活无显著变化。在最适反应条件下,E75Q的kcat/Km值较原酶提高了26.6%。【结论】改变AvPAL酶分子中起路易斯碱作用的关键氨基酸残基(质子受体)附近与之有相互作用的氨基酸的带电性质,降低了AvPAL的最适反应pH,提升了其在医疗领域的应用前景。     

Protein candidates for Q fever serodiagnosis

The discriminatory diagnosis of Q fever remains difficult because of the unspecific clinical presentations of the disease. Additionally, the diagnosis is often delayed because serodiagnosis is not sensitive enough in the early stages of the disease when the immune response is not yet efficient. Similarly, the diagnosis of Q fever endocarditis can only be performed in approximately 35%, mainly via serology, which was a criterion postulated by Duke. Owing to the discriminatory diagnosis of Q fever and the high number of tests requested, we focused on expressing several proteins for ELISA studies with Coxiella burnetii-infected sera. Previously, we selected a list of 31 candidates [Sekeyova et?al. (2009) Eur J Clin Microbiol Infect Dis 28: 287-295], of which we have successfully cloned and expressed 21. Finally, 15 recombinant proteins were prescreened with the sera of patients with acute Q fever and Q fever endocarditis, respectively. Sera from a control group were also screened. The nine most immunoreactive proteins from the first assay were tested with the sera from a larger group of patients. Our study identified CBU_0092 as the best marker of acute Q fever but failed to isolate a highly specific and sensitive marker of Q fever endocarditis.     

基于结构B因子分析指导的头孢菌素C酰化酶动力学稳定性改造

【目的】筛选Pseudomonas sp.SE83 acy Ⅱ定点饱和突变库,获得动力学稳定性提高的头孢菌素C(CPC)酰化酶突变体,并对突变酶进行初步的结构-功能关系分析。【方法】靶标酶Pseudomonas sp.SE83 acy Ⅱ与Pseudomonas diminuta N176具有较高的同源性,通过分析N176的结构B因子,构建CPC酰化酶SE83定点饱和突变库;基于pH指示剂显色法,采用Biomek FX~P自动工作站建立CPC酰化酶高通量筛选方法,获得优良突变酶,对其活性、稳定性等酶学性质进行表征;利用SWISS-MODEL对突变体进行同源建模,探讨突变体结构与功能的关系。【结果】通过B因子分析和同源结构比对,共找出9个靶标位点;经过3轮筛选,发现R218及K226位点突变显著提高酶的热稳定性,其中最显著的R218Q和K226V在40°C的半衰期分别为野生型的3.77和2.77倍,催化效率k_(cat)/K_m分别为野生型的1.8和3.1倍。同源建模分析表明氢键作用和疏水相互作用的增加可能是突变体稳定性提高的原因。【结论】B因子指导的酶分子改造是一种高效可靠的动力学稳定性改造策略,突变体R218Q和K226V均可提高CPC酰化酶的稳定性和催化效率,对进一步的CPC酰化酶分子改造具有一定的参考价值和指导意义。     

Epidemiological investigation and physician awareness regarding the diagnosis and management of Q fever in South Korea, 2011 to 2017

BackgroundIn South Korea, the number of Q fever cases has rapidly increased since 2015. Therefore, this study aimed to characterize the epidemiological and clinical features of Q fever in South Korea between 2011 and 2017.Methods/Principal findingsWe analyzed the epidemiological investigations and reviewed the medical records from all hospitals that had reported at least one case of Q fever from 2011 to 2017. We also conducted an online survey to investigate physicians’ awareness regarding how to appropriately diagnose and manage Q fever. The nationwide incidence rate of Q fever was annually 0.07 cases per 100,000 persons. However, there has been a sharp increase in its incidence, reaching up to 0.19 cases per 100,000 persons in 2017. Q fever sporadically occurred across the country, with the highest incidences in Chungbuk (0.53 cases per 100,000 persons per year) and Chungnam (0.27 cases per 100,000 persons per year) areas. Patients with acute Q fever primarily presented with mild illnesses such as hepatitis (64.5%) and isolated febrile illness (24.0%), whereas those with chronic Q fever were likely to undergo surgery (41.2%) and had a high mortality rate (23.5%). Follow-up for 6 months after acute Q fever was performed by 24.0% of the physician respondents, and only 22.3% of them reported that clinical and serological evaluations were required after acute Q fever diagnosis.ConclusionsQ fever is becoming an endemic disease in the midwestern area of South Korea. Given the clinical severity and mortality of chronic Q fever, physicians should be made aware of appropriate diagnosis and management strategies for Q fever.     

Primary quinone (QA) binding site of bacterial photosynthetic reaction centers: mutations at residue M265 probed by FTIR spectroscopy

In the primary quinone (Q(A)) binding site of Rb. sphaeroides reaction centers (RCs), isoleucine M265 is in extensive van der Waals contact with the ubiquinone headgroup. Substitution of threonine or serine for this residue (mutants M265IT and M265IS), but not valine (mutant M265IV), lowers the redox midpoint potential of Q(A) by about 100 mV (Takahashi et al. (2001) Biochemistry 40, 1020-1028). The unexpectedly large effect of the polar substitutions is not due to reorientation of the methoxy groups as similar redox potential changes are seen for these mutants with either ubiquinone or anthraquinone as Q(A). Using FTIR spectroscopy to compare Q(A)(-)/Q(A) IR difference spectra for wild type and the M265 mutant RCs, we found changes in the polar mutants (M265IT and M265IS) in the quinone C[double bond]O and C[double bond]C stretching region (1600-1660 cm(-1)) and in the semiquinone anion band (1440-1490 cm(-1)), as well as in protein modes. Modeling the mutations into the X-ray structure of the wild-type RC indicates that the hydroxyl group of the mutant polar residues, Thr and Ser, is hydrogen bonded to the peptide C[double bond]O of Thr(M261). It is suggested that the mutational effect is exerted through the extended backbone region that includes Ala(M260), the hydrogen bonding partner to the C1 carbonyl of the quinone headgroup. The resulting structural perturbations are likely to include lengthening of the hydrogen bond between the quinone C1[double bond]O and the peptide NH of Ala(M260). Possible origins of the IR spectroscopic and redox potential effects are discussed.     

反应器进水管生物膜中喹啉降解菌的分离鉴定及降解特性

【背景】喹啉是一类高毒、致癌且难降解的含氮杂环化合物,本实验室建立了一个长期高效运行的反硝化喹啉降解生物反应器。【目的】从反应器进水管富集的生物膜中筛选有氧条件下降解喹啉的菌株。【方法】通过以喹啉为唯一碳源的培养基来富集、分离、纯化菌株;利用16S rRNA基因的序列分析鉴定分离株的系统发育地位;比较不同pH及温度条件下菌株的喹啉降解特性。【结果】经鉴定,4株分离物Q1、Q3、Q7和Q8分别属于Sphingobium、Massilia、Rhodococcus和Dyadobacter属。降解实验表明,以上菌株均能在48 h内实现50 mg/L喹啉的完全去除,但各自表现出不同的降解特性,其中Q1、Q3和Q8在降解过程中都检测到了喹啉降解产物2-羟基喹啉的积累。降解喹啉的Sphingobium、Massilia和Dyadobacter属菌株尚未见报道。【结论】从喹啉降解生物反应器的进水管内分离的4株喹啉降解菌可为设计处理含喹啉工业废水的反应器提供新菌种资源,对于完善喹啉生物降解机理研究具有实际意义。     

Detection of nucleoside Q precursor in methyl-deficient E.coli tRNA.

32P-Labeled tRNAAsn was isolated from methyl-deficient E. coli tRNA. Nucleotide sequence analysis showed that tRNAAsn contains three derivatives of the Q nucleoside, possibly Q precursors, in addition to guanosine in the first position of the anticodon. One of the Q precursors was isolated on a large scale. Its UV spectra were identical with those of normal Q, indicating that 7-deazaguanosine structure having a side chain at position C-7 is complete in the Q precursor. No radioactivity was incorporated into Q or Q precursors from either [methyl-14C]methionine, [1-14C]methionine or [U-14C]methionine, showing that methionine was not directly involved in the formation of Q.     

Effect of activating and inactivating mutations of GS-and Gi2-alpha protein subunits on growth and differentiation of 3T3-L1 preadipocytes

Previous investigations have demonstrated that both G_s- and the G_i-family of GTP-binding proteins are implicated in differentiation of the 3T3-L1 preadipocyte. In order to further analyze the role of G_sα vs. G_i2α, which are both involved in adenylate cyclase modulation, we transfected undifferentiated 3T3-L1 cells with two sets of G-protein cDNA: the pZEM vector with either wild type, the activating (i.e., GTP-ase inhibiting) R201C-G_sα or the inactivating G226A(H21a)-G_sα point mutations, or the pZIPNeoSV(X) retroviral vector constructs containing the G_i2α wild type or the missense mutations R179E-G_i2α, Q205L-G_i2α, and G204A(H21a)-G_i2α. The activating [R201C]G_sα-mutant did not significantly affect the differentiation process, i.e., increase in the steady-state levels of G-protein subunits, gross appearance, or insulin-elicited deoxy-glucose uptake into 3T3-Ll adipocytes, despite a marked initial increase in hormone-elicited adenylate cyclase activity. The [H21a]G_sα-mutant, on the other hand, enhanced the degree of differentiation slightly, as evidenced by an augmented production of lipid vesicles and insulin-stimulated deoxy-glucose uptake. However, an expected increase in mRNA for hormone-sensitive lipase was not seen. Secondly, it appeared that both activating [R179E]G_i2α or [Q205L]G_i2α mutants reduced cell doubling time in non-confluent 3T3-L1 cell cultures, while [H21a]G_i2α slowed proliferation rate. Furthermore, it seemed that cell proliferation, as evidenced by thymidine incorporation, ceased at a much earlier stage prior to cell confluency when cultures were transfected with the [R179E]G_i2α or [Q205L]G_i2α mutants. Upon differentiation with insulin, dexamethasone, and iBuMeXan, the following cell characteristics emerged: the [R179E]G_i2α and [Q205L]G_i2α mutants consistently enhanced adenylate cyclase activation and cAMP accumulation stimulated by isoproterenol and corticotropin over controls. Deoxy-glucose uptake was also super-activated by the [R179E]G_i2α and [Q205L]G_i2α mutants. Finally, steady-state levels of hormone sensitive lipase mRNA were dramatically increased by [R179E]G_i2α and [Q205L]G_i2α over differentiated controls. The inactivating [H21a]G_i2α-mutant obliterated all signs of preadipocyte differentiation. It is concluded that G_i2 plays a positive and much more important role than G_s in 3T3-L1 preadipocyte differentiation. Cyclic AMP appears to play no role in this process. J. Cell. Biochem. 64:242–257. © 1997 Wiley-Liss, Inc.     

Serine 776 of ataxin-1 is critical for polyglutamine-induced disease in SCA1 transgenic mice

Polyglutamine-induced neurodegeneration in transgenic mice carrying the spinocerebellar ataxia type 1 (SCA1) gene is modulated by subcellular distribution of ataxin-1 and by components of the protein folding/degradation machinery. Since phosphorylation is a prominent mechanism by which these processes are regulated, we examined phosphorylation of ataxin-1 and found that serine 776 (S776) was phosphorylated. Residue 776 appeared to affect cellular deposition of ataxin-1[82Q] in that ataxin-1[82Q]-A776 failed to form nuclear inclusions in tissue culture cells. The importance of S776 for polyglutamine-induced pathogenesis was examined by generating ataxin-1[82Q]-A776 transgenic mice. These mice expressed ataxin-1[82Q]-A776 within Purkinje cell nuclei, yet the ability of ataxin-1[82Q]-A776 to induce disease was substantially reduced. These studies demonstrate that polyglutamine tract expansion and localization of ataxin-1 to the nucleus of Purkinje cells are not sufficient to induce disease. We suggest that S776 of ataxin-1 also has a critical role in SCA1 pathogenesis.     

Erinacine Q,a new erinacine from Hericium erinaceum,and its biosynthetic route to erinacine C in the basidiomycete

Erinacines as cyathane-xylosides are known to have potent stimulating activity for nerve-growth-factor synthesis. Our search for new cyathane metabolites from a liquid culture of Hericium erinaceum YB4-6237 resulted in the isolation of a new erinacine named erinacine Q (1). NMR spectrometry and a chemical derivation from erinacine P (2) determined the compound to be a derivative in which the formyl group of erinacine P had been reduced to the hydroxymethyl group. To clarify the biosynthetic relationship between erinacine Q and the others, [1'-13C]erinacine Q ([1'-13C]-1) was chemically derived from [1'-13C]erinacine P ([1'-13C]-2) which had been prepared by feeding [1-13C]-D-glucose to the basidiomycete. The biotransformation of labeled erinacine Q into [1'-13C]erinacine C ([1'-13C]-5) via [1'-13C]erinacine P in this basidiomycete was demonstrated by NMR spectrometry.     

基因局部改组技术在ubiA基因突变中的应用

辅酶Q(coenzyme Q,CoQ)作为线粒体呼吸链中的递氢体具有较高的学术及应用价值.由ubiA基因编码的4-羟苯甲酸聚异戊二烯转移酶(UbiA)是大肠杆菌CoQ生物合成途径的限速步骤,但通过系统突变对其结构进行研究鲜有报道.[目的]应用化学合成的随机序列寡核苷酸,对ubiA基因编码活性区域的DNA序列进行随机突变...     

Influence of Environmental Factors and Air Composition on the Emission of [alpha]-Pinene from Quercus ilex Leaves

We studied the emission of [alpha]-pinene from Quercus ilex leaves. Only the abaxial side of the hypostomatous Q. ilex leaf emits [alpha]-pinene. Light induced photosynthesis and [alpha]-pinene emission. However, the response of photosynthesis to dark-to-light transitions was faster than that of [alpha]-pinene, suggesting that ATP controls the emission. The emission was higher at 30 than at 20[deg]C, whereas photosynthesis did not change. Therefore, the relationship between photosynthesis and [alpha]-pinene emission does not always hold. When CO2 was removed from the air, transpiration was stimulated but photosynthesis and [alpha]-pinene emission were inhibited. [alpha]-Pinene inhibition was more rapid under low O2. When CO2 in the air was increased, photosynthesis was stimulated and transpiration was reduced, but [alpha]-pinene emission was unaffected. Therefore, the emission depends on the availability of photosynthetic carbon, is not saturated at ambient CO2, and is not dependent on stomatal opening. The pattern of [alpha]-pinene emission from Q. ilex is different from that of plants having specialized structures for storage and emission of terpenes. We suggest that [alpha]-pinene emitted by Q. ilex leaves is synthesized in the chloroplasts and shares the same biochemical pathway with isoprene emitted by isoprene-emitting oak species.     

A natural focus of sandfly fever in the Republic of Afghanistan]

The serological study of persons contacting dengue-like fever in 1987 in Afghanistan (in Rukha, Parwān Province) revealed that in 74% of cases an increase in the titers of antibodies to Sicilian and Neapolitan sandfly [correction of mosquito-borne] fever viruses was registered. Considering that such diseases appeared here for a number of years and were linked in time with the activity of sandflies [correction of mosquitoes] of the species Phlebotomus papatasii, the suggestion was made on the existence of a stable natural focus of sandfly [correction of mosquito-borne] fevers in the region of Rukha.