Biological Functions and Mechanism of Casein KinaseHrr25 / 中国生物化学与分子生物学报

Hrr25 is a member of the casein kinase 1 family in Saccharomyces cerevisiae and has serine / threonine protein kinase activity. It can function by phosphorylating a variety of proteins. The substrate proteins of Hrr25 include autophagy related proteins, COPII (coat protein complexes II) vesicle coat proteins Sec24 and Sec23, eukaryotic translation initiation factor 6, γ- Tubulin tub4, and extender complex protein 1, etc. In addition, Hrr25 can also interact with meiotic recombinant protein Rec8, Nucleoporin Nup53, transcription regulator Crz1, and transcription activator Haa1, etc. A variety of interacting proteins of Hrr25 enable it to play a role in autophagy, vesicular transport, microtubule assembly, meiosis, mitosis, DNA repair, ribosomal biogenesis, weak organic acid stress and other biological processes. In order to better understand the action mechanism of Hrr25 in various biological processes and the relationship between various biological processes, this paper summarizes the biological functions and action mechanism of Hrr25, and the potential significance of its research, so as to provide a theoretical basis for the further research of Hrr25.

Influences of vesicular transport inhibition on proliferation and store-operated calcium entry in rat endothelial progenitor cells / 中国病理生理杂志

AIM:To investigate the effects of vesicular transport inhibition on the proliferation and regulation of store-operated calcium entry ( SOCE) in rat endothelial progenitor cells ( EPCs) .METHODS:EPCs were isolated from the rats with density-gradient centrifugation and confirmed via double fluorescence staining with acLDL-DiI and FITC-UEA-I.After inhibition of vesicular transport with brefeldin A ( BFA) , the proliferation of EPCs was measured by CCK-8 assay and real-time cell analyzer instrument, apoptosis was analyzed by flow cytometry, and the expression of ADP-ribosylation factor GTPase-activating protein 1 (ARFGAP1), a key protein to vesicular transport, was also detected.SOCE was ob-served under laser scanning confocal microscope after the vesicular transport was inhibited, and the protein expression of SOCC complex was determined by Western blot.Furthermore, the influences of vesicular transport inhibition on the expres-sion of transient receptor potential channel 1 ( TRPC1 ) and SOCE were examined with a RNA interference method.RE-SULTS:The acLDL-DiI and FITC-UEA-I double positive rate of the cells was 82.53%±6.12%.BFA insult significantly inhibited the proliferation of EPCs and down-regulated the expression of ARFGAP1, and no influence on the apoptosis of the EPCs was observed, suggesting that vesicular transport of EPCs was inhibited.Vesicular transport inhibition remarkably down-regulated the expression of TRPC1 and decreased SOCE level.No evident difference in the level of SOCE between siTRPC1 group and siTRPC1+BFA group, in which the cells were pretreated with siTRPC1 before BFA addition, was ob-served.CONCLUSION:Vesicular transport inhibition in EPCs reduces the proliferation of EPCs and decreases SOCE lev-el through down-regulation of TRPC1.

Regulation of retinoic acid on permeability of retinal pigment epithelial barrier in lens-induced myopia / 中华实验眼科杂志

Background Retinal retinoic acid (RA) plays an important role in the formation of the lensinduced myopia.However,it is not clear how RA transfer the myopic signal to choroid throughout the retinal pigment epithelium (RPE) barrier.Objective The aim of this study was to investigate the effect of all-trans retinoic acid (atRA) on the barrier of RPE in lens-induced myopic eye of guinea pig.Methods Thirty left eyes of 30 21-dayold clean guinea pigs were randomized into normal control group and the model group.The models of out of focus were induced by covering of-6.00 D concave lens on the left eyes for 15 days.Radius of corneal curvature was measured using corneal curvimeter,and diopeter of the guinea pig was examined by mydriatic optometry.The length of ocular axis was detected by A-sonography.The animals were sacrificed and the retinas of the left eyes were isolated for the culture and passage of RPE cells.The third generation of cells were incubated Millcell-PET microporous film,and atRA at the concentration of 1 × 10-6,1 × 10-7,1 × 10-8 and 1 × 10-9 mol/L was added to the micropore respectively for 12 hours,and the micropores with equal-solvent served as negative control group.Methyl thiazolyl tetrazolium (MTT)colorimetric method was used to detect the survival rate of the cells.Subsequently,the transepithelial electrical resistance (TER) of the monolayer cells was determined with CN10-EVOM2 resistance measuring meter.The vesicular transport change of RPE membrane in different groups was evaluated by FM1-43 fluorescence staining.Results The mean diopter was (-2.20±0.95) D in the models,and that in the controls was (+ 1.15 ±0.30) D,with a significant difference between them (t =14.57,P＜ 0.01).The axial length was (8.24 ± 0.09) mm in the models and it was significantly longer than (7.81±0.05) mm in the controls (t=17.20,P＜0.01).RPE cells grew well to form a monolayer in Millcell culture pool after one week.After 24 hours of the atRA treatment,the survival rate of RPE cells reduced gradually with the increase of atRA concentration with the highest rate in the 1 × 10-9 mol/L atRA group (93.3 ％) and followed by the 1 × 10-8 mol/L atRA group (88.2％).More than half of the cells dead in the 1 × 10-6mol/L and 1 × 10-7mol/L atRA groups (53.8％ and 47.1％).Significant differences in the TER value and fluorescence staining intensity of the cells were seen among the various groups (F =43.89,P =0.00 ; F =26.13,P =0.00),with the maximal values in the 1 × 10-8mol/L atRA group.The FM1-43 fluorescence located on the cellular membrane and cytoplasm.Conclusions AtRA can increase the functional state of tight junction and vesicular transport,which regulated the RPE cell barrier in the guinea pig.

Genetic diagnosis of four combined deficiency of factor Ⅴ and Ⅷ patients / 中华检验医学杂志

Objective To investigate the genetic diagnosis and molecular pathogenesis of four patients with combined deficiency of coagulation factor Ⅴ and Ⅷ and their family members. Methods The APPT, FT, FⅤ: C, FⅧ: C were detected for phenotypic diagnosis. Thrombin generation assay was applied to determine the generation condition of thrombin in patients and healthy controls. Cenomic DNA was extracted from peripheral blood using the TianGen RelaxCene Blood DNA System;amniotic fluid DNA was extracted with phenol-ethyl ether method. The LMAN1 and MCFD2 genes were analyzed by PCR. Gene mutations were detected with nucleotid sequences by using end-labeling dideoxy method. Results The APTT of Proband 1 was significantly prolonged to 88. 2s and her PT was prolonged to 19. 6 s. The combined deficiency was identified with FⅧ (FⅧ: C 24. 2% ) and FV(FⅤ: C 9. 1% ). Proband 2 and 3 were sisters. The coagulation studies revealed that both of them had prolonged APTT (71.6 s and 74.6 s respectively) and PT (22. 1 s and 18. 3 s respectively). The combined deficiency of FⅤ (FⅤ: C 7. 6% and 14. 5% respectively) and FⅧ( FⅧ: C 25% and 19.6% respectively) were identified. Proband 4 was detected to have the prolonged APTT (70.3 s),PT (18.2 s) and the deficiency of FⅤ(FⅤ: C 9. 4% ) and FⅧ (15. 7% ). The remaining phenotype indicators test of the 4 probands were normal. The diagnosis for the 4 probands was combined deficiency of factor Ⅴ and Ⅷ. The proband 1 was detected to have compound heterozygous mutations in LMAN1 gene while having the LMAN1 and MCFD2 direct gene sequencing. One mutation was a small insertion located on exon 8 [ nt912insA (X71661. 1)] that resulted in p. 305frameshiftX20 and her mother was detected to have the same heterozygous mutation on the the locus. The other mutation was located on exon 11: nt1366C > CT ( X71661. 1 ) , p. 456Arg > Stop which was inherited from her father. Amniocyte DNA was detected to have only one heterozygous mutaion [nt1366C > CT (X71661. 1) , 456Arg > Stop] inherited from the father. No mutation in MCFD2 gene was found in proband 1 and her parents. The analysis of the MCFD2 gene in proband 2 and 3 revealed a novel homozygous single base substitution (nt411T>C) in exon 4, which results in the exchange of the amino acid isoleucine by the amino acid threonine at amino acid position 136 (p. Ile136Thr). Sequencing of the whole LMAN1 gene showed that the proband 4 had one homozygous nonsence mutation in the exon 5 of the LMAN1 ( nt615C >T,p. 202 Arg> Stop). All of the 4 probands with combined deficiency of FⅤ and FⅧ showed declined endogenous thrombin potential in the thrombin generation tests. Conclusion The combined deficiency of FⅤ and FⅧ in the proband 1 results from the compound heterozygous mutations ( nt1366C > CT and nt912insA) in LMAN1 gene, which are inherited from her parents respectively. The prenatal genetic investigation for the patient mother with preganency indicates that the fetus is a female carrier with one mutation (nt1366C > CT) inherited from the father. The homozygous missence mutation ( nt411T > C, p. Ile136Thr) in the MCFD2 gene accounts for the proband 2 and 3. The daughter of the proband 2 is a carrier with a heterozygous mutation inherited from her mother. The homozygous nonsence mutation in the LMAN1 gene of the proband 4 results in the deficency of F Ⅴ and FⅧ.

Expression and significance of Livin, Survivln in CIN and cervical carcinoma / 中国医师杂志

suitable tumor markers and new targets for gene therapy of cervical carcinoma.

Vesículas de transporte de proteínas e lipídeos em histoplasma capsulatum e outros ascomicetos / Transport vesicle proteins and lipids in histoplasma capsulatum and other ascomycetes

Secreção vesicular de macromoléculas foi demonstrada recentemente em Cryptococcus neoformans levantando a hipótese da possibilidade desse mecanismo de transporte vesicular ocorrer também em ascomicetos. Neste trabalho, analisamos se o fungo Histoplasma capsulatum e outros ascomicetos com importância clínica produzem vesículas e utilizam essas estruturas na secreção de macromoléculas. Microscopia eletrônica de transmissão (MET) evidenciou secreção trans-celular de vesículas na fase leveduriforme. Análises proteômicas e de lipídeos revelaram uma gama de macromoléculas envolvidas em diversos processos celulares, como por exemplo: metabolismo, sinalização e virulência. Os resultados demonstraram que H. capsulatum utiliza-se de um transporte secretório celular vesicular trans-membrana para promover virulência...

MET de sobrenadantes de cultura de Candida albicans, Candida parapsilosis, Sporothrix schenckii, e Saccharomyces cerevisiae evidenciaram a presença de vesículas, confirmando a hipótese de que os ascomicetos similarmente produzem vesículas. Anticorpos presentes em pool de soros de pacientes com histoplasmose, reagiram com moléculas presentes nas vesículas isoladas, ressaltando a possível associação dos produtos vesiculares no processo de patogênese. Nossos resultados corroboram a proposta de que a secreção vesicular é um mecanismo comum em fungos para o transporte de macromoléculas relacionadas à virulência, sendo este um promissor alvo em novas linhas terapêuticas...