Novel folding and stability defects cause a deficiency of human glutathione transferase omega 1

The polymorphic deletion of Glu-155 from human glutathione transferase omega1 (GSTO1-1) occurs in most populations. Although the recombinant ΔGlu-155 enzyme expressed in Escherichia coli is active, the deletion causes a deficiency of the active enzyme in vivo. The crystal structure and the folding/unfolding kinetics of the ΔGlu-155 variant were determined in order to investigate the cause of the rapid loss of the enzyme in human cells. The crystal structure revealed altered packing around the Glu-155 deletion, an increase in the predicted solvent-accessible area and a corresponding reduction in the buried surface area. This increase in solvent accessibility was consistent with an elevated Stern-Volmer constant. The unfolding of both the wild type and ΔGlu-155 enzyme in urea is best described by a three-state model, and there is evidence for the more pronounced population of an intermediate state by the ΔGlu-155 enzymes. Studies using intrinsic fluorescence revealed a free energy change around 14.4 kcal/mol for the wild type compared with around 8.6 kcal/mol for the ΔGlu-155 variant, which indicates a decrease in stability associated with the Glu-155 deletion. Urea induced unfolding of the wild type GSTO1-1 was reversible through an initial fast phase followed by a second slow phase. In contrast, the ΔGlu-155 variant lacks the slow phase, indicating a refolding defect. It is possible that in some conditions in vivo, the increased solvent-accessible area and the low stability of the ΔGlu-155 variant may promote its unfolding, whereas the refolding defect limits its refolding, resulting in GSTO1-1 deficiency.     

Honey bee colony collapse disorder is possibly caused by a dietary pyrethrum deficiency

Industrialized farming relies on bee keepers transporting hives to the vicinity of large areas of mono-crops for crop pollination. Hives are typically moved multiple times per growing season to satisfy the pollination need. A phenomenon wherein colonies of honey bees collapse in large numbers has been threatening crops in North America. Honey bees are hosts to at least two pathogenic mites; Varroa destructor and Acarapis woodi (a tracheal mite). Pyrethrums are a group of flowering plants which include Chrysanthemum coccineum, Chrysanthemum cinerariifolium, Chrysanthemum marschallii, and related species. These plants produce potent insecticides, also named pyrethrums, which are powerful mite toxins. We believe that a honey bee dietary deficiency of pyrethrums and other micro-nutrients from pyrethrum producing plants allows parasitic mites to either kill the honey bees directly or reduce honey bee resistance to other pathogens. Intermittent feeding of honey bees on pyrethrum producing plants might reverse or prevent colony collapse disorder.     

Heterodimerization,Altered Subcellular Localization,and Function of Multiple Zinc Transporters in Viable Cells Using Bimolecular Fluorescence Complementation

Zinc plays a crucial role in numerous key physiological functions. Zinc transporters (ZnTs) mediate zinc efflux and compartmentalization in intracellular organelles; thus, ZnTs play a central role in zinc homeostasis. We have recently shown the in situ dimerization and function of multiple normal and mutant ZnTs using bimolecular fluorescence complementation (BiFC). Prompted by these findings, we here uncovered the heterodimerization, altered subcellular localization, and function of multiple ZnTs in live cells using this sensitive BiFC technique. We show that ZnT1, -2, -3, and -4 form stable heterodimers at distinct intracellular compartments, some of which are completely different from their homodimer localization. Specifically, unlike the plasma membrane (PM) localization of ZnT1 homodimers, ZnT1-ZnT3 heterodimers localized at intracellular vesicles. Furthermore, upon heterodimerization with ZnT1, the zinc transporters ZnT2 and ZnT4 surprisingly localized at the PM, as opposed to their vesicular homodimer localization. We further demonstrate the deleterious effect that the G87R-ZnT2 mutation, associated with transient neonatal zinc deficiency, has on ZnT1, ZnT3, and ZnT4 upon heterodimerization. The functionality of the various ZnTs was assessed by the dual BiFC-Zinquin assay. We also undertook a novel transfection competition assay with ZnT cDNAs to confirm that the driving force for heterodimer formation is the core structure of ZnTs and not the BiFC tags. These findings uncover a novel network of homo- and heterodimers of ZnTs with distinct subcellular localizations and function, hence highlighting their possible role in zinc homeostasis under physiological and pathological conditions.     

Structural analysis of new compound heterozygous variants in PEPD gene identified in a patient with Prolidase Deficiency diagnosed by exome sequencing

Prolidase Deficiency (PD) is an autosomal recessive rare disorder caused by loss or reduction of prolidase enzymatic activity due to variants in the PEPD gene. PD clinical features vary among affected individuals: skin ulcerations, recurrent infections, and developmental delay are common. In this study, we describe a 16-year-old boy with a mild PD phenotype comprising chronic eczema, recurrent infections and elevated IgE. Whole exome sequencing analysis revealed three PEPD variants: c.575T>C p.(Leu192Pro) inherited from the mother, and c.692_694del p.(Tyr231del) and c.1409G>A p.(Arg470His), both inherited from the father. The variant p.(Tyr231del) has been previously characterized by high-resolution X-ray structure analysis as altering protein dynamics/flexibility. In order to study the effects of the other two prolidase variants, we performed site directed mutagenesis purification and crystallization studies. A high-resolution X-ray structure could only be obtained for the p.(Arg470His) variant, which showed no significant structural differences in comparison to WT prolidase. On the other hand, the p.(Leu192Pro) variant led to significant protein destabilization. Hence, we conclude that the maternal p.(Leu192Pro) variant was likely causally associated with the proband´s disease, together with the known pathogenic paternal variant p.(Tyr231del). Our results demonstrated the utility of exome sequencing to perform diagnosis in PD cases with mild phenotype.     

CD36 Protein Influences Myocardial Ca2+ Homeostasis and Phospholipid Metabolism: CONDUCTION ANOMALIES IN CD36-DEFICIENT MICE DURING FASTING*

Sarcolemmal CD36 facilitates myocardial fatty acid (FA) uptake, which is markedly reduced in CD36-deficient rodents and humans. CD36 also mediates signal transduction events involving a number of cellular pathways. In taste cells and macrophages, CD36 signaling was recently shown to regulate store-responsive Ca²⁺ flux and activation of Ca²⁺-dependent phospholipases A₂ that cycle polyunsaturated FA into phospholipids. It is unknown whether CD36 deficiency influences myocardial Ca²⁺ handling and phospholipid metabolism, which could compromise the heart, typically during stresses. Myocardial function was examined in fed or fasted (18–22 h) CD36^−/− and WT mice. Echocardiography and telemetry identified conduction anomalies that were associated with the incidence of sudden death in fasted CD36^−/− mice. No anomalies or death occurred in WT mice during fasting. Optical imaging of perfused hearts from fasted CD36^−/− mice documented prolongation of Ca²⁺ transients. Consistent with this, knockdown of CD36 in cardiomyocytes delayed clearance of cytosolic Ca²⁺. Hearts of CD36^−/− mice (fed or fasted) had 3-fold higher SERCA2a and 40% lower phospholamban levels. Phospholamban phosphorylation by protein kinase A (PKA) was enhanced after fasting reflecting increased PKA activity and cAMP levels in CD36^−/− hearts. Abnormal Ca²⁺ homeostasis in the CD36^−/− myocardium associated with increased lysophospholipid content and a higher proportion of 22:6 FA in phospholipids suggests altered phospholipase A₂ activity and changes in membrane dynamics. The data support the role of CD36 in coordinating Ca²⁺ homeostasis and lipid metabolism and the importance of this role during myocardial adaptation to fasting. Potential relevance of the findings to CD36-deficient humans would need to be determined.     

苓桂术甘汤对老年脾胃气虚型功能型消化不良患者胃蛋白酶原、胃泌素及血液流变学的影响

摘要 目的：研究苓桂术甘汤对老年脾胃气虚型功能型消化不良患者的临床治疗效果,并探讨治疗对患者胃蛋白酶原、胃泌素和血液流变学的影响。方法：选取2020年1月到2021年10月在我院接受治疗的老年脾胃气虚型功能型消化不良患者120例,按治疗方式分为对照组以及研究组,对照组患者给予常规药物治疗,研究组患者在对照组基础上加用苓桂术甘汤进行治疗,比较两组患者临床治疗疗效和治疗前后生活质量评分、中医症候积分、血清胃蛋白酶I和II、血清胃泌素-17、血液流变学。结果：（1）研究组患者临床治疗总有效率显著高于对照组（93.33 % vs 75.00 %,P<0.05）;（2）治疗后,两组患者的生活质量评分升高,而中医症候积分降低,且研究组优于对照组（P<0.05）;（3）两组患者治疗后血胃蛋白酶I和II、血清胃泌素-17均显著降低,并且治疗后研究组低于对照组（P<0.05）;（4）两组患者治疗后血液流变学指标（高切全血黏度、低切全血黏度和血浆黏度）均显著降低,并且研究组降低程度显著高于对照组（P<0.05）。结论：加用苓桂术甘汤治疗老年脾胃气虚型功能型消化不良可显著提高临床治疗疗效,改善患者生活质量,降低患者血清胃蛋白酶原、胃泌素以及血流变学指标表达。     

Pertilization and nutrition experiments with conifer seedlings in pots

Summary In conifer fertilization and nutrition experimentsPinus halepensis, P. radiata andP. maritima seedlings were grown in pots, filled with soil derived from mica schist and siliceous tertiary deposits and also in peat substrate in paperpots.Fertilization with P ofP. radiata andP. maritima seedlings growing in soil low in available P and N improved seedling height only in combination with N fertilization and fertilization with alone induced P deficiency symptoms. N fertilization with from 100 to 150 ppm (2.4 to 3.2 g N/kg, respectively) in the soil regardless of the form of N (NH₄ ⁺ or NO₃ ^–) applied in the summer or autumn together with application of 20 ppm P before sowing was the fertilization regime which produced the best seedlings.Fertilization of peat before sowingP. halepensis, P. radiata andP. maritima with omission of one of the nutrients N, P and K resulted in visible symptoms of N, P and K deficiency, respectively, in the seedlings. Comparative chemical analysis of needles from the three kinds of conifer seedlings with deficiency symptoms and healthy ones verified the visual symptoms of N and P deficiency but not so convincingly the K deficiency symptoms.     

Up-Regulation of Carbonic Anhydrase Isozyme IV in CNS Myelin of Mice Genetically Deficient in Carbonic Anhydrase II

Abstract: Carbonic anhydrase (CA) II is the major CA isozyme in the brain, where it participates in acid-base homeostasis, fluid transport, and myelin synthesis. The CA II deficiency [CA(II)D] mutation in the mouse results in structural changes in the glial cells in the CNS and in decreased susceptibility to seizures, but no detectable changes in myelin yield and ultrastructure. We compared the CA isozymes in brain and spinal cord fractions, as well as in purified myelin, between CA(II)D and control mice. CA(II)D resulted in a much lower total CA specific activity in all tissues examined but in higher CA IV specific activities in soluble and membrane-associated fractions and pure myelin. Western blots of purified myelin showed a band corresponding to CA IV in CA(II)D mice. This band was weak or undetectable in myelin samples from normal mice. Immunocytochemical staining demonstrated CA IV in oligodendrocytes and myelinated tracts in normal mouse brains and stronger staining of the same structures in brains of CA(II)D mutants. We conclude that CA(II)D mutation in the mouse up-regulates CNS CA IV. We speculate that this up-regulation could mitigate the effect of CA(II)D on myelin formation and maintenance.     

Mutations in the Hypoxanthine Guanine Phosphoribosyltransferase Gene (HPRT1) in Asian HPRT Deficient Families

Inherited mutation of hypoxanthine guanine phosphoribosyltransferase, (HPRT) gives rise to Lesch‐Nyhan syndrome or HPRT‐related gout. We have identified 34 mutations in 28 Japanese, 7 Korean, and 1 Indian families with the patients manifesting different clinical phenotypes, including two rare cases in female subjects, by the analysis of all nine exons of HPRT from the genomic DNA and reverse transcribed mRNA using PCR technique coupled with direct sequencing.