Identification of the ataxin-1 interaction network and its impact on spinocerebellar ataxia type 1.

BACKGROUND: Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease caused by a polyglutamine expansion in the ataxin-1 protein. The pathogenic mechanism resulting in SCA1 is still unclear. Protein-protein interactions affect the function and stability of ataxin-1. METHODS: Wild-type and mutant ataxin-1 were expressed in HEK-293T cells. The levels of expression were assessed using real-time polymerase chain reaction (PCR) and Western blots. Co-immunoprecipitation was done in HEK-293T cells expressing exogenous wild-type and mutant ataxin-1 using anti-Flag antibody following by tandem affinity purification in order to study protein-protein interactions. The candidate interacting proteins were validated by immunoprecipitation. Chromatin immunoprecipitation and high-throughput sequencing and RNA immunoprecipitation and high-throughput sequencing were performed using HEK-293T cells expressing wild-type or mutant ataxin-1. RESULTS: In this study using HEK-293T cells, we found that wild-type ataxin-1 interacted with MCM2, GNAS, and TMEM206, while mutant ataxin-1 lost its interaction with MCM2, GNAS, and TMEM206. Two ataxin-1 binding targets containing the core GGAG or AAAT were identified in HEK-293T cells using ChIP-seq. Gene Ontology analysis of the top ataxin-1 binding genes identified SLC6A15, NTF3, KCNC3, and DNAJC6 as functional genes in neurons in vitro. Ataxin-1 also was identified as an RNA-binding protein in HEK-293T cells using RIP-seq, but the polyglutamine expansion in the ataxin-1 had no direct effects on the RNA-binding activity of ataxin-1. CONCLUSIONS: An expanded polyglutamine tract in ataxin-1 might interfere with protein-protein or protein-DNA interactions but had little effect on protein-RNA interactions. This study suggested that the dysfunction of protein-protein or protein-DNA interactions is involved in the pathogenesis of SCA1.

Persistency of Enlarged Autolysosomes Underscores Nanoparticle-Induced Autophagy in Hepatocytes.

The diverse biological effects of nanomaterials form the basis for their applications in biomedicine but also cause safety issues. Induction of autophagy is a cellular response after nanoparticles exposure. It may be beneficial in some circumstances, yet autophagy-mediated toxicity raises an alarming concern. Previously, it has been reported that upconversion nanoparticles (UCNs) elicit liver damage, with autophagy contributing most of this toxicity. However, the detailed mechanism is unclear. This study reveals persistent presence of enlarged autolysosomes in hepatocytes after exposure to UCNs and SiO2 nanoparticles both in vitro and in vivo. This phenomenon is due to anomaly in the autophagy termination process named autophagic lysosome reformation (ALR). Phosphatidylinositol 4-phosphate (PI(4)P) relocates onto autolysosome membrane, which is a key event of ALR. PI(4)P is then converted into phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 ) by phosphatidylinositol-4-phosphate 5-kinase. Clathrin is subsequently recruited by PI(4,5)P2 and leads to tubule budding of ALR. Yet it is observed that PI(4)P cannot be converted in nanoparticle-treated hepatocytes cells. Exogenous supplement of PI(4,5)P2 suppresses the enlarged autolysosomes in vitro. Abolishment of these enlarged autolysosomes by autophagy inhibitor relieves the hepatotoxicity of UCNs in vivo. The results provide evidence for disrupted ALR in nanoparticle-treated hepatocytes, suggesting that the termination of nanoparticle-induced autophagy is of equal importance as the initiation.

Giant Cellular Vacuoles Induced by Rare Earth Oxide Nanoparticles are Abnormally Enlarged Endo/Lysosomes and Promote mTOR-Dependent TFEB Nucleus Translocation.

Many nanomaterials are reported to disrupt lysosomal function and homeostasis, but how cells sense and then respond to nanomaterial-elicited lysosome stress is poorly understood. Nucleus translocation of transcription factor EB (TFEB) plays critical roles in lysosome biogenesis following lysosome stress induced by starvation. The authors previously reported massive cellular vacuolization, along with autophagy induction, in cells treated with rare earth oxide (REO) nanoparticles. Here, the authors identify these giant cellular vacuoles as abnormally enlarged and alkalinized endo/lysosomes whose formation is dependent on macropinocytosis. This vacuolization causes deactivation of mammalian target of rapamycin (mTOR), a TFEB-interacting kinase that resides on the lysosome membrane. Subsequently, TFEB is dephosphorylated at serine 142 and translocated into cell nucleus. This nucleus translocation of TFEB is observed only in vacuolated cells and it is critical for maintaining lysosome homeostasis after REO nanoparticle treatment, as knock-down of TFEB gene significantly compromises lysosome function and enhances cell death in nanoparticle-treated cells. Our results reveal that cellular vacuolization, which is commonly observed in cells treated with REOs and other nanomaterials, represents a condition of profound lysosome stress, and cells sense and respond to this stress by facilitating mTOR-dependent TFEB nucleus translocation in an effort to restore lysosome homeostasis.

Establishment of evanescent wave fiber-optic immunosensor method for detection bluetongue virus.

The evanescent wave fiber immunosensors (EWFI) technique was developed for the real-time rapidly sensitive and specific detection of the monoclonal antibody 3E2 of BTV. The outer-core protein VP7 of BTV was labled on the surface of the exposed fiber-optic core. The monoclonal antibody 3E2 of BTV VP7 were added and then the goat ant-rat IgG conjugated with Cy3 was captured. After the 532nm pulse (excitation source) reached the fiber probe, evanescent wave was generated, which excited the Cy3 bound to the immuno-complex and produced the fluorescent signal, which was changed into electrical signals read through computer. The preliminary results suggested that a detection limit of 10ng/ml was measured for the monoclonal antibody 3E2, which is equal to the sensitivity of ELISA. The 3E2 sample was specifically detected through the EWFI assay in 15min, and the fiber can be recycled at least ten times through TEA solution condition. This developed EWFI was a real-time rapidly sensitive and specific way for the detection of BTV antibodies.

Modified Zhenwu Tang delays chronic renal failure progression by modulating oxidative stress and hypoxic responses in renal proximal tubular epithelial cells.

Background: Tubulointerstitial fibrosis (TIF) is a critical pathological feature of chronic renal failure (CRF), with oxidative stress (OS) and hypoxic responses in renal proximal tubular epithelial cells playing pivotal roles in disease progression. This study explores the effects of Modified Zhenwu Tang (MZWT) on these processes, aiming to uncover its potential mechanisms in slowing CRF progression. Methods: We used adenine (Ade) to induce CRF in rats, which were then treated with benazepril hydrochloride (Lotensin) and MZWT for 8 weeks. Assessments included liver and renal function, electrolytes, blood lipids, renal tissue pathology, OS levels, the hypoxia-inducible factor (HIF) pathway, inflammatory markers, and other relevant indicators. In vitro, human renal cortical proximal tubular epithelial cells were subjected to hypoxia and lipopolysaccharide for 72 h, with concurrent treatment using MZWT, FM19G11, and N-acetyl-l-cysteine. Measurements taken included reactive oxygen species (ROS), HIF pathway activity, inflammatory markers, and other relevant indicators. Results: Ade treatment induced significant disruptions in renal function, blood lipids, electrolytes, and tubulointerstitial architecture, alongside heightened OS, HIF pathway activation, and inflammatory responses in rats. In vivo, MZWT effectively ameliorated proteinuria, renal dysfunction, lipid and electrolyte imbalances, and renal tissue damage; it also suppressed OS, HIF pathway activation, epithelial-mesenchymal transition (EMT) in proximal tubular epithelial cells, and reduced the production of inflammatory cytokines and collagen fibers. In vitro findings demonstrated that MZWT decreased apoptosis, reduced ROS production, curbed OS, HIF pathway activation, and EMT in proximal tubular epithelial cells, and diminished the output of inflammatory cytokines and collagen. Conclusion: OS and hypoxic responses significantly contribute to TIF development. MZWT mitigates these responses in renal proximal tubular epithelial cells, thereby delaying the progression of CRF.

Effect of Reduced INR in Early Pregnancy on the Occurrence of Preeclampsia: A Retrospective Cohort Study.

To investigate the effect of reduced early-pregnancy activated partial thrombin time (APTT), prothrombin time (PT), and international standardized ratio (INR) on the risk of preeclampsia. A total of 8549 pregnant women with singleton births were included. Early pregnancy APTT, PT, and INR levels, with age, birth, prepregnancy body mass index, fibrinogen (FBG), thrombin time (TT), D-dimer (DD2), antithrombin III (ATIII), fibrin degradation products (FDP) as confounders, generalized linear model of APTT, the relative risk of PT and INR when INR reduction. After adequate adjustment for confounders, the relative risk of preeclampsia was 0.703 for every 1 s increase in plasma PT results in early pregnancy, and for every 0.1 increase in plasma INR results, the relative risk of preeclampsia was 0.767. With a PT less than the P25 quantile (<11 s), the relative risk of preeclampsia was 1.328. The relative risk of preeclampsia at an INR less than the P25 quantile (<0.92) was 1.24. There was no statistical association between APTT on the risk of preeclampsia. The relative risk of preeclampsia is strongly associated with a decrease in PT and INR in early pregnancy. PT and INR in early pregnancy were a potential marker in the risk stratification of preeclampsia. Focusing on reduced PT and INR levels in early pregnancy can help to identify early pregnancies at risk for preeclampsia.

Reticulocyte hemoglobin content associated with the risk of iron deficiency anemia.

Background/Objective: Reticulocyte hemoglobin content (MCHr) was recognized as a rapid and reliable marker for investigating iron deficiency (ID). We hypothesized that MCHr was associated with the risk of iron deficiency anemia in adults. Methods: This is a dual-center case-control study. A total of 806 patients and healthy individuals were recruited from Ruijin Hospital and Xinhua Hospital affiliated to Shanghai Jiaotong University School of Medicine between January 2021 and December 2021. The participants were categorized into iron deficiency anemia (IDA) group (n = 302), non-IDA group (n = 366), and healthy control group (n = 138). According to the MCHr level, the participants were divided into two groups, i.e. normal MCHr (≥25 pg) and decreased MCHr (<25 pg) group. Multivariate logistic regression analysis and adjusted subgroup analysis were conducted to estimate the relative risk between MCHr and IDA, with confounding factors including age, sex, hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), Hematocrit (HCT), serum iron (Fe), ferritin (Ferrit), and total iron binding capacity (TIBC). Results: Compared with the non-IDA, the MCHr level with IDA decreased significantly. ROC curve analysis showed that MCHr had the largest area under the AUC curve. After comprehensive adjustment for confounding factors, individuals with normal level of MCHr exhibited a decreased risk of IDA (OR = 0.68 [0.60, 0.77], P < 0.01), while the risk of IDA was up to 5 times higher for those with decreased MCHr. Conclusion: Our findings supported the hypothesis that MCHr was associated with the risk of IDA in adults and could serve as an indicator of IDA severity. MCHr holds clinical value as an auxiliary diagnostic indicator, providing valuable insights into whether invasive examinations are warranted in the assessment of IDA.

Investigation of the relationship between changes in maternal coagulation profile in the first trimester and the risk of developing preeclampsia.

Normal pregnancy is a hypercoagulable state with an increase in coagulation factor levels and a decrease in natural anticoagulation. However, a higher hypercoagulable state with prolonged activated partial thromboplastin time (APTT), prothrombin time (PT), increased D-dimer, and mean platelet volume is seen in women with preeclampsia at the time of onset. In addition, endothelial dysfunction occurs before the clinical symptoms of preeclampsia. Therefore, we undertook this study to investigate the coagulation profile in the first trimester in women who developed preeclampsia later. A total of 853 pregnant women with singleton births at the Obstetrics and Gynecology Hospital of Fudan University between January 2021 and December 2021 were included in this case-control study. In the comparison with the controls (n = 531), the mean value of D-dimer, APTT, thrombin time (TT), antithrombin (AT)), and fibrin degradation products (FDP) was significantly lower in preeclamptic women at the time of diagnosis (n = 322). The changes in the coagulation profile were not associated with the severity or the time of onset. The reduced values of D-dimer, AT, and FDP, and increased values of TT were also observed in the first trimester in women who developed preeclampsia later and were not associated with the severity, or the time of onset of preeclampsia. After adjusting for maternal age and BMI, the values of D-dimer and AT in the first trimester were correlated to the risk of developing preeclampsia. Our findings suggest that there is an abnormal maternal response to the hemostatic system in early gestational age in women who developed preeclampsia later and measuring the coagulation profile could be an additional predictive marker of preeclampsia.

A novel Pro126His beta propeller mutation in integrin alphaIIb causes Glanzmann thrombasthenia by impairing progression of pro-alphaIIbbeta3 from endoplasmic reticulum to Golgi.

BACKGROUND: Glanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by lack of platelet aggregation in response to most physiological agonists and caused by either a lack or dysfunction of the platelet integrin alphaIIbbeta3 (glycoprotein IIb/IIIa). PATIENTS: Mucocutaneous bleeding manifestations and platelet dysfunction consistent with GT were observed in a 20-year-old proband of a Chinese family. OBJECTIVES: To determine the molecular basis of GT and characterize the mutation by in vitro expression studies. RESULTS: Analysis of the patient's platelets by fluorescence-activated cell sorting demonstrated the presence of trace amounts of beta3, exposed on her platelet surface, but a complete absence of alphaIIbbeta3. Sequence analysis revealed a novel C470A transversion in exon 4 of the alphaIIb gene predicting a Pro126His alteration in the blade 2 of the alphaIIb beta propeller domain. The proband was homozygous for the mutation, the mother and the father were heterozygous, whereas 100 healthy subjects lacked this transversion. Chinese hamster ovary cells cotransfected with cDNAs of mutated alphaIIb and wild-type beta3 failed to express alphaIIbbeta3 on the cell surface as shown by FACS. Western blot analysis of the cell lysates showed no detectable mature alphaIIb. Immunoprecipitation with antibody against beta3 demonstrated pro-alphaIIb in the cells expressing the mutant alphaIIbbeta3, indicating pro-alphaIIbbeta3 complex formation. Intracellular immunofluorescence studies demonstrated the pro-alphaIIbbeta3 complex that co-localized with an ER marker, but showed minimal co-localization with a Golgi marker. CONCLUSIONS: A novel Pro126His mutation in alphaIIb compromised transport of the pro-alphaIIbbeta3 complex from the endoplasmic reticulum to the Golgi, leading to intracellular retention. The impaired alphaIIbbeta3 transport is responsible for the thrombasthenia in this patient.

[Molecular mechanisms of Glanzmann thrombasthenia caused by alphaII b P126H mutation: an in vitro experiment].

OBJECTIVE: To explore the molecular mechanisms of Glanzmann thrombasthenia caused by alpha IIb P126H mutation. METHODS: Eukaryotic vector of alpha IIb P126H was constructed by PCR site-directed mutagenesis and then co-transfected with eukaryotic vector PCDM8 II a expressing the subunit beta3 into human renal epithelial cells of the line 293& and Chinese hamster ovarian cancer cells of the line CHO after sequencing. The membrane expression of alpha IIb P126H mutant was analyzed by flow cytometry and the whole expression was confirmed by Western blotting. The alpha IIb P126H mutant subcellular localization was determined by laser confocal scanning microscopy. RESULTS: The 293T cells cotransfected with cDNAs of mutated alpha IIb and wild-type beta3 failed to express alpha II bbeta3 on the cell surface as shown by FACS. Western blotting of the cell lysate showed no detectable mature alpha IIb. Immunofluorescence studies demonstrated proa II bbeta3 complex colocalized with an endoplasmic reticulum (ER) marker, but showed minimal colocalization with an Golgi marker. CONCLUSION: The P126H mutation in alpha IIb prevents transport of the pro-alpha II bbeta3 complex from ER to the Golgi body, thus hindering its maturation and surface expression. The impaired alpha II bp33 transport is responsible for the thrombasthenia.

Reversal of the Apoptotic Resistance of Non-Small-Cell Lung Carcinoma towards TRAIL by Natural Product Toosendanin.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively triggers cancer cell death via its association with death receptors on the cell membrane, but exerts negligible side effects on normal cells. However, some non-small-cell lung carcinoma (NSCLC) patients exhibited resistance to TRAIL treatment in clinical trials, and the mechanism varies. In this study, we described for the first time that toosendanin (TSN), a triterpenoid derivative used in Chinese medicine for pain management, could significantly sensitize human primary NSCLC cells or NSCLC cell lines to TRAIL-mediated apoptosis both in vitro and in vivo, while showing low toxicity against human primary cells or tissues. The underlying apoptotic mechanisms involved upregulation of death receptor 5 (DR5) and CCAAT/enhancer binding protein homologous protein, which is related to the endoplasmic reticulum stress response, and is further associated with reactive oxygen species generation and Ca2+ accumulation. Surprisingly, TSN also induced autophagy in NSCLC cells, which recruited membrane DR5, and subsequently antagonized the apoptosis-sensitizing effect of TSN. Taken together, TSN can be used to sensitize tumors and the combination of TRAIL and TSN may represent a useful strategy for NSCLC therapy; moreover, autophagy serves as an important drug resistance mechanism for TSN.

Differential ERK activation during autophagy induced by europium hydroxide nanorods and trehalose: Maximum clearance of huntingtin aggregates through combined treatment.

Accelerating the clearance of intracellular protein aggregates through elevation of autophagy represents a viable approach for the treatment of neurodegenerative diseases. In our earlier report, we have demonstrated the enhanced degradation of mutant huntingtin protein aggregates through autophagy process induced by europium hydroxide nanorods [EHNs: Eu(III)(OH)3], but the underlying molecular mechanism of EHNs mediated autophagy was unclear. The present report reveals that EHNs induced autophagy does not follow the classical AKT-mTOR and AMPK signaling pathways. The inhibition of ERK1/2 phosphorylation using the specific MEK inhibitor U0126 partially abrogates the autophagy as well as the clearance of mutant huntingtin protein aggregates mediated by EHNs suggesting that nanorods stimulate the activation of MEK/ERK1/2 signaling pathway during autophagy process. In contrast, another mTOR-independent autophagy inducer trehalose has been found to induce autophagy without activating ERK1/2 signaling pathway. Interestingly, the combined treatment of EHNs and trehalose leads to more degradation of mutant huntingtin protein aggregates than that obtained with single treatment of either nanorods or trehalose. Our results demonstrate the rational that further enhanced clearance of intracellular protein aggregates, needed for diverse neurodegenerative diseases, may be achieved through the combined treatment of two or more autophagy inducers, which stimulate autophagy through different signaling pathways.

Enhanced transdermal delivery of epidermal growth factor facilitated by dual peptide chaperone motifs.

TD1, a peptide chaperone consisting of the sequence ACSSSPHKHCG, has been shown to facilitate transdermal delivery for protein molecules via either co-administration or the fusion approach. We previously reported that a single TD1 motif, fused to the N-terminus of human epidermal growth factor (hEGF) can significantly enhance the transdermal efficiency of the recombinant EGF protein. In an effort to further increase the transdermal efficiency, we have created EGF fusion proteins harboring dual TD1 motifs: TD1-hEGF-TD1, containing one TD1 motif at both the N- and the Cterminus, and TD1-TD1-hEGF, containing two tandem TD1 motifs at the N-terminus. Both TD1-hEGF-TD1 and TD1- TD1-hEGF proteins, expressed in Escherichia coli and purified to apparent homogeneity, exhibited biological activity similar to unmodified hEGF, as revealed by their relative abilities to stimulate fibroblast growth, promote fibroblast migration, and activate the MAP kinase signaling cascade. On the other hand, both TD1-hEGF-TD1 and TD1-TD1-hEGF proteins exhibited a transdermal efficiency enhancement. The improvement was >5-fold compared to unmodified hEGF and 3-fold over the hEGF fusion protein with only one TD1 motif attached. These findings provided proof-of-concept for improving transdermal delivery of protein actives through rational protein design.

Transdermal delivery of human epidermal growth factor facilitated by a peptide chaperon.

Peptide chaperon TD1 was discovered to facilitate several proteins' transdermal delivery via topical co-administration. To design a practical, safe system for advanced transdermal peptide, a novel method was carried out. Human epidermal growth factor (hEGF) was selected as the model biological agent and a fusion protein: TD1-hEGF was designed. Study showed that TD1-hEGF not only had the similar bioactivity with native hEGF, but also possessed considerable higher transdermal ability than hEGF and a co-administration of TD1 and hEGF. These results provided convincing evidence for the advantages of TD1-hEGF in cosmetic and medical applications. Moreover, the fusion pattern between the cargoes and TD1 offered a new approach to facilitate other hydrophilic drugs' transdermal delivery for therapeutic application.

Identification of compound heterozygous mutations in the ITGA2B gene in a Chinese patient with Glanzmann thrombasthenia.

BACKGROUND: Glanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by the tendency to hemorrhage and the inability of platelets to aggregate in response to agonists. GT is caused by a defect of the platelet glycoprotein IIb/IIIa complex. The objective of this study was to describe the clinical features and the genetic cause of GT in a 6-year-old girl from south China. METHODS: A three-generation family was studied. The proband patient aged 6 years and her parents undertook examinations of platelet counts, blood film, bleeding time, platelet aggregation, and flow cytometry. All coding exons of the ITGA2B and ITGB3 genes were amplified by polymerase chain reaction (PCR), and direct sequencing was performed for mutational screening on the patient and normal controls consisted of 52 healthy blood donors. Reverse transcription PCR was conducted to test for exon skipping. RESULTS: The proposita patient showed dispersing platelets, prolonged bleeding time, and severely reduced platelet aggregation in response to the physiological agonists adenosine diphosphate (ADP), epinephrine, collagen, and ristocetin. Flow cytometric measurements showed that the contents of alphaIIb and beta3 were significantly decreased. Sequencing results demonstrated two different types of heterozygous mutations existed in the alphaIIb gene (c.2930delG and IVS15-1delG). The compound mutations were also confirmed in the patient's mother and father separately. CONCLUSIONS: The alphaIIbbeta3 deficiency of the proband was caused by two compound ITGA2B mutations, which were first reported in Chinese GT patients. The IVS15-1delG was first confirmed to cause an exon skipping.

MR molecular imaging of thrombus: development and application of a Gd-based novel contrast agent targeting to P-selectin.

Molecular imaging of thrombus formation at initial stage requires a robust thrombus-specific contrast agent with high sensitivity. In this study, we report a novel P-selectin-targeted paramagnetic molecular imaging agent and the agent's potential to sensitively detect occult microthrombi on the intimal surface of endothelium. Platelet clots and blood clots targeted in vitro with paramagnetic nanoparticles presented a highly detectable, homogeneous T1-weighted contrast enhancement that was improved with increasing gadolinium level. In vivo contrast enhancement under part of circulation conditions was assessed in dogs. The micro-thrombi around the femoral vein of dog demonstrated higher signal intensities than the control clots and the adjacent muscle. Histology was performed on regions likely to contain thrombus as indicated by MRI. These results suggest that molecular imaging of P-selectin-targeted paramagnetic nanoparticles can provide sensitive detection and localization of P-selectin and may allow for early, direct identification of microthrombi, leading to early diagnosis.

Ser234Leu missense mutation in the A1 domain of factor V causing moderate factor V deficiency in a Chinese family.

AIMS: To investigate the molecular defects in a Chinese pedigree with inherited factor V (FV) deficiency. METHODS: Laboratory studies including activated partial thromboplastin time (APTT), prothrombin (PT), and thrombin time (TT) were tested in a patient and his family members. FV antigen (FV:Ag) and FV activity (FV:C) were measured by both ELISA and one-stage clotting assays. All the exons, exon-intron boundaries and promoter regions of FV gene were analysed by direct sequencing. The detected mutations were introduced independently by site-directed mutagenesis into a pMT2/FV mammalian expression plasmid containing the full-length FV cDNA and the wild-type and mutant FV proteins were expressed in COS-7 and CHO cells. RESULTS: The proposita, a 52-year-old Chinese man, had no spontaneous bleeding syndrome. It was found that he had prolonged APTT and PT, 52 s and 22.8 s, respectively, a FV:C of 5.5% and a FV:Ag of 33.1%. Gene analysis showed the proposita was a compound heterozygote of FV mutations, carrying Ser234Leu and Arg413Cys. The FV antigen and activity levels of the Ser234Leu and Arg413Cys mutants are lower than wild type both in cell lysates and in culture media. Protein degradation inhibitor experiment in transfected COS-7 cells showed that Ser234Leu and Arg413Cys degraded intracellularly through the lysosomal pathway. CHO cells expressing either the wild-type or the mutant FV were subjected to immunofluorescence staining with the indicated antibodies and organelle markers, indicating that Ser234Leu and Arg413Cys can be transported to Golgi partially. CONCLUSIONS: We identified the molecular pathological mechanism of the novel C785T mutation causing type I inherited FV deficiency for the first time.

[Molecular mechanisms of Glanzmann thrombasthenia caused by alpha II b L721R and Q860X compound heterozygous mutation].

OBJECTIVE: To explore the molecular mechanisms of Glanzmann thrombasthenia caused by alpha II b L721R and Q860X compound heterozygous mutation. METHODS: All exons and exon-intron boundaries of alpha II b and beta3 gene were amplified by PCR and analyzed by direct DNA sequencing. Gene polymorphisms were excluded by direct DNA sequencing. Alpha II b L721R and Q860X mutants expressing vectors were constructed by in vitro site-directed mutagenesis. The expression of alpha II b L721R and Q860X mutants on transfected cell membrane were analyzed by flow cytometry and the whole expression level was confirmed by Western blot. The subcellular localizations of alpha II b L721R and Q860X mutants were determined by immunofluorescent confocal scanning microscopy. RESULTS: The alpha II b compound heterozygous mutations, T2255G (L721R) and C2671T (Q860X), were identified in the proband, the former being inherited from the maternal side and the latter the paternal side. The 293T cells cotransfected with mutated alpha II b L721R and wild-type beta3 expression plasmids expressed 2.1% of normal amount of alpha II b on the cell surface as shown by FACS, in contrast to 31.9% of normal amount of alpha II b on the cells cotransfected with cDNAs of mutated alpha II b Q860X and wildtype beta3 expression plasmids. Western blot of the cell lysates showed no detectable mature alpha II b in cells lysates with L721R mutant. While, truncated alpha II b protein was detected in cell lystes with Q860X mutant. Immunofluorescence studies demonstrated that both L721R and Q860X mutant pro-alpha II bbeta33 complex colocalized in endoplasmic reticulum, but a little in Golgi. CONCLUSIONS: The L721R and Q860X mutations of alpha II b prevent transport of the pro-alpha II bbeta3 complex from the endoplasmic reticulum to the Golgi, hindering its maturation and surface expression. The impaired alpha II bbeta3 transport is responsible for the thrombasthenia.

[Analysis of clinical features and genotype in three Chinese pedigrees with Glanzmann thrombasthenia].

OBJECTIVE: To study the clinical feature and alpha II b beta 3 gene mutations of three Glanzmann thrombasthenia (GT) pedigrees. METHODS: Platelet counts (BPC), blood film, bleeding time, platelet aggregation and flow cytometry were used for phenotype diagnosis of all the patients. All the exons of alpha II b and beta 3 genes were amplified by polymerase chain reaction (PCR) and direct sequencing was performed for mutational screening. One hundred and three healthy blood donors were as normal controls. RESULTS: Three probands showed normal BPC, defective platelets aggregation, prolonged bleeding time and significantly reduced platelet aggregation to ADP, epinephrine, and collagen, while relatively normal aggregation to ristocetin. Flow cytometry showed platelet surface expressed alpha II b beta 3 was strongly reduced in proband 1 and proband 3 and mildly reduced in the amount of surface expressed alpha II b beta 3 (63%) in proband 2. Sequencing results showed that proband 1 had a G10A homozygous mutation in alpha II b, and a G1412T homozygous mutation in beta3. Compound heterozygous mutations in beta3, G1199A and 1525delC were identified in proband 2. No mutations in alpha II b beta 3 gene were identified in proband 3. CONCLUSIONS: Compound homozygous mutations, GI0A in alpha II b and G1412T in beta3, lead to GT in proband 1. Compound heterozygous mutations in beta3, G1199A and 1525delC, lead to GT in proband 2. The mutations of G10A, G1412T and 1525delC were reported for the first time in GT patients.