Silencing the PIK3CA Gene Enhances the Sensitivity of Childhood Leukemia Cells to Chemotherapy Drugs by Suppressing the Phosphorylation of Akt

PURPOSE: This study aimed to investigate the effects of PIK3CA on the sensitivity of acute B lymphocytic leukemia cells (Nalm-6 cells) to chemotherapy drugs. MATERIALS AND METHODS: Children's normal B lymphocytes and Nalm-6 cells were cultured. Nalm-6 cells were transfected with PIK3CA siRNA (siPIK3CA group) or its negative control (PIK3CA-Control group). Normal Nalm-6 cells were named Mock group. Nalm-6 cells transfected by PIK3CA siRNA were treated with Akt inhibitor (siPIK3CA+Akti-1/2 group). mRNA and protein expression was detected by qRT-PCR and Western blot. Proliferation and sensitivity to chemotherapeutic drugs was detected by MTT assay. Cell cycle and apoptosis was explored by low cytometry. Transwell assay was performed to test invasion. RESULTS: PIK3CA mRNA (p=0.008) and protein (p=0.006) expression was higher in Nalm-6 cells than that in normal B lymphocytes. Compared with the Mock group and PIK3CA-Control group, Nalm-6 cells of the siPIK3CA group had lower OD495 values (all p < 0.05) and invasion cell numbers (p=0.03 and p=0.025), as well as a higher proportion of G0/G1 phase cells (p=0.020 and p=0.022), percentage of apoptosis (p=0.016 and p=0.022), and inhibition rate (all p < 0.05). pAkt expression in the siPIK3CA group (p=0.026 and p=0.031) and siPIK3CA+Akti-1/2 group (p=0.019 and p=0.023) was lower than that in the Mock group. CONCLUSION: PIK3CA silencing inhibited Nalm-6 cell proliferation and invasion, and promoted their apoptosis and sensitivity to chemotherapeutic drugs, potentially through regulation of the PI3K/AKT signaling pathway.

Clinical characteristics of patients with delayed hepatolenticular degeneration / 中华内科杂志

Objective To evaluate the clinical manifestations, metal metabolism, imaging characteristics and treatment response in patients with delayed Wilson disease (WD). Methods Patients with untreated WD (40 with delayed onset and 40 with non?delayed onset) were enrolled. Twenty healthy people were included as normal controls. All patients were evaluated with modified Young scale neural symptom scores, grade of Child liver function and mental symptoms rating scale, magnetic resonance imaging (MRI) scan, magnetic sensitive imaging (susceptibility weighted imaging, SWI), metal metabolism. Corrected phase (CP) was measured at SWI. After 2 week treatment, neurologic symptoms, liver function, and metal metabolism were reviewed. Results The total score of neurological symptoms in WD patients with delayed onset was lower than that of non?delayed onset (13.00 ± 6.87 vs. 21.13 ± 5.53, P=0.033). The scores of SCL?90 and HAMA depression scales in patients with delayed onset were lower than those of non?delayed onset. On T2 weighted imaging, areas including substantia nigra and thalamus, the caudate nucleus, globus pallidus, putamen presented high signal rate in patients with delated onset than those with non?delayed (P=0.022, 0.037, 0.022, 0.037, 0.029 respectively). The SWI CP values of cangbai sphere and shell nucleus in patients with delayed onset were lower than those with non?delayed onset. Patients with delayed onset had higher urinary copper than those with non?delayed onset before and after treatment (P=0.040, 0.036). After treatment, the score of abnormal tremor and gait in patients with delayed onset was decreased (P=0.037, 0.044), while as the occurrence of neurological symptoms was increased by 10%, and the liver function level in patients with delayed WD was decreased in 3 cases. Conclusions The brain of WD patients with delayed onset is mainly composed of metal deposits, however the cell damage is not apparent. Clinical symptoms are characterized by significant liver injury, but relatively mild neurological and psychiatric symptoms. Patients with delayed WD have higher urinary copper excretion than those with non?delayed WD. Chelating agents improves the neurological symptoms in patients with delayed onset.

Evaluating the functional activity of the subcortical nuclei in Wilson's disease using resting state functional magnetic resonance imaging / 中华神经科杂志

Objective To evaluate functional activity of the subcortical nuclei in Wilson's disease (WD) using resting state functional MRI (rs-fMRI),and to evaluate damage to the functional conjunction in the extracorticospinal tract in WD patients.Methods Twenty-two patients with WD (between January 2015 and January 2016),including 18 with cerebral type and 4 with hepatic type,and 20 age-matched healthy controls were enrolled.Neurological symptoms were scored using the modified Young Scale.Patients with cerebral type WD were divided into 4 subgroups.All study subjects underwent rs-fMRI of the brain.The values of amplitude of low-frequency fluctuation (ALFF) and regional homogeneity (REHO) in the thalamus,caudate nucleus,putamen and globus pallidus were determined.The relationships between rsfMRI metrics and clinical status were evaluated.Results ALFF values were lower in the caudate nucleus,putamen and right thalamus of WD patients than in controls (t =-3.07,-3.00,-3.12,-2.46,-2.20;P =0.005,0.006,0.004,0.020,0.036),while REHO values were lower in the left caudate nucleus and left thalamus of WD patients (t =-2.38,-2.16;P =0.025,0.040).In the caudate nucleus (P =0.032,0.029,0.023),thalamus (P =0.022,0.041,0.035) ALFF values were lower in group 4 than in other groups.REHO values of the putamen (P =0.040,0.017,0.040) and thalamus (P =0.024,0.029 7,0.041) were higher in group 4 than in other groups.ALFF values in the caudate nucleus (t =-0.29,P=0.037),and thalamus (t =-1.77,P =0.042) were lower,and REHO values in the caudate nncleus (t =-1.46,P =0.040) were lower,in patients of cerebral type than in hepatic type patients.Conclusions The damage to the functional activity of the subcortical nuclei may occur in the WD patients.The functional activity of nuclei may be different between hepatic and cerebral type patients.Damage to the activity of neurons in the putamen and thalamus may correlate with psychiatric symptoms in WD patients.

The clinical analysis of Huntington disease：a case study from 12 genetic diagnosis families / 中国神经精神疾病杂志

Objective To investigate the clinical manifestation, inherited pattern and the related factor of Hunting?ton disease families. Method The clinical data from 12 HD families was collected from 2013-2014. Patients received the genetic test and neurological evaluation including motor, cognitive and problem of behavior. Results There were 12 patients having the IT15 gene dynamic mutations, including 1 Juvenile Huntington disease patient and 3 pre-symptomat?ic mutant gene carriers. The average CAG repeats of these patients was between the range of 40 to 60, and the average on?set age ranged from 13 to 54 year-old. Positive family history and genetic anticipation could be observed. Patients pre?sented with different clinical manifestations at the early stage while had typical chorea movements, declined cognitive and psychiatric symptoms at the late stage of the illness. Conclusions There are typical triad symptoms in the late stage but not in the early stage nor pre-symptom stage illness. Clinical manifestation and the neuroimaging are both of great ref?erence value, and the genetic test is essential for final diagnosis.

The mechanism for neurological symptom deterioration in Wilson' s disease during the treatment of D-penicillamine / 中华神经科杂志

Objective To explore the mechanism of the secondary deterioration of neurological symptoms in Wilson' s disease (WD) at early stage of treatment using D-penicillamine. Methods Forty non-treated WD patients, 32 of encephalic and 8 hepatic type respectively, were enrolled in the study. Their neural symptoms were scored using modified Young grade. Cerebrospinal fluid (CSF) copper, serum copper, urinary copper, neuron specific enolase (NSE) in CSF and the albumin ratio CSF/serum (AR) were measured at the same time. After 3 months of treatment with D-penicillamine, neural symptoms of patients were scored again. All dates were analyzed. Results After 3 months of treatment with D-penicillamine, 15 patients (46. 9%) developed a secondary deterioration in neurological symptoms. The concentration of copper and the NSE in CSF of patients whose neural symptom was increasingly deteriorated. The serum copper declined after treatment((0. 37± 0. 09) vs (0. 25 ± 0. 08) mg/L, t = 3. 17, P < 0. 05). The 24 hours urinary copper of patients whose symptoms had deteriorated was much lower than that of patients who had not. No significant change was found in AR ratio before and after the treatment (9. 53 ± 3.18vs12.24±3.17) in the worsened group (t=1.45, P>0. 05). Conclusions The degree of the injury in the neural system and the dose of penicillamine may affect the deterioration of the neural symptom.

Distribution of different genotypes of hereditary ataxia in south China / 中国组织工程研究

BACKGROUND: It is of great importance to study the genotype distribution of hereditary ataxia in understanding its epidemiologic rule and pathogenetic pathway.OBJECTIVE: To analyze the distribution of different genotype of hereditary ataxia in south China.DESIGN: A case-control observation.SETTING: Department of Neurology, the First Affiliated Hospital of Sun Yat-sen University.PARTICIPANTS: Forty-three patients (26 males and 17 females) with hereditary ataxia from 36 families and 38 patients with sporadic hereditary ataxia (24 males and 14 females) were selected from the Outpatient Clinic of Neurogenetics, Department of Neurology, the First Affiliated Hospital of Sun Yat-sen University between September 1998 and September 2002. At the same time, 60 healthy individuals from the patients' families and 44randomly-selected healthy physical examinees were taken as controls. All the participants were enrolled voluntarily.METHODS: The fragments of trinucleotide repeats at different sites of mutant genes were amplified with polymerase chain reaction (PCR), and then the lengths were calculated with polyacrylamide gel electrophoresis and imaging analytical software. The repeated numbers of trinucleotide repeats in all the normal and abnormal amplified alleles were calculated respectively.MAIN OUTCOME MEASURES: Different genotype distribution in patients with hereditary ataxia.RESULTS: All the subjects were involved in the analysis of results. Of the detected patients with hereditary ataxia, the Machado-Joseph disease/spinocerebellar ataxia (SCA) 3 was the most common type of autosomal dominant SCA in South China, which was 42.0%, and was followed by SCA2 (7.4%), SCA1 (4.9%), SCA7 (3.7%), SCA6 (2.5%), SCA12 (1.2%).No patient was detected to have SCA8 SCA 10, SCA 17 dentatorubropallidoluysian atrophy (DRPLA) and Friedreich ataxia (FRDA).CONCLUSION: Autosomal dominant SCA3 is the most familiar genotype in South China. Clinical detection of hereditary ataxia should be done firstly aiming at the SCA3 genotype.

Effects of the combined use of benazepril and valsartan on apoptosis in the kidney of rats with adriamycin-induced nephritic glomerulosclerosis / 华中科技大学学报（医学）（英德文版）

The effects of the combined use of angiotensin converting enzyme inhibitor (ACEI) benazepril and angiotensin II type 1 receptor antagonist (AT1RA) valsartan on apoptosis and the expression of apoptosis-related proteins Fas and FasL in the kidney of rats with adriamycin-induced nephritic glomerulosclerosis was investigated. Uninephrectomy and the injection of adriamycin induced the rat model of glomerulosclerosis. Benazepril (6 mg/kg), valsantan (20 mg/kg), or benazepril (3 mg/kg) plus valsantan (20 mg/kg) was respectively delivered daily by gavage to the rats in three treatment groups for 12 weeks. Apoptosis was examined by means of terminal-deoxynucleotidyl transferase mediated d-UTP nick end labeling (TUNEL). Immunohistochemistry was adopted to detect the expression of Fas and FasL. Software of pathological analysis quantitated the levels of Fas and FasL. The results showed that as compared with those in the control group, the kidneys in the model group had more severe glomerulosclerosis, much more apoptotic cells and higher levels of expression of Fas and FasL. The degree of glomerulosclerosis, the number of apoptotic cells and the levels of expression of Fas and FasL were reduced by benazepril and valsartan. The combined use of benazepril and valsartan had the best therapeutic effect. It was concluded that benazepril and valsartan could suppress the excessive apoptosis of kidney cells by lowering the expression of the apoptosis-related proteins Fas and FasL, so as to postpone the process of glomerulosclerosis. The combined use of benazepril and valsartan has better therapeutic effect.

Molecular genetic diagnosis and clinical characteristics of spinocerebellar ataxia type 6 / 临床神经病学杂志

Objective To study the molecular genetic diagnosis and clinical characteristics of spinocerebellar ataxia type 6 (SCA6).Methods 43 patients with autosomal dominant SCA from 36 families and 38 sporadic SCA patients were enrolled in the study. SCA6 (CAG)n dynamic mutations were detected by polymerase chain reaction (PCR). Abnormal allele fragments were sequenced and repeated numbers were calculated. The clinical data of two cases with SCA6 were analyzed.Results CAG repeat of normal SCA6 allele ranged from 10 to 13. CAG repeat of abnormal SCA6 allele expanded to 25 in one familial patient and 24 in one sporadic patient in our study. The basic characteristics of these SCA6 patients were slowly progressive cerebellar ataxia, nystagmus and dysarthria.Conclusion Diagnosis of SCA6 can be confirmed by detection of abnormal CAG repeat expansion. There is no obvious difference of clinical features between SCA6 and other SCA subtypes.

Analysis of the gene mutation and clinical characteristic of hereditary spinocerebellar ataxia type 7 / 临床神经病学杂志

Objective To study the gene mutation and clinical characteristic of hereditary spinocerebellar ataxia type 7 (SCA7).Methods The SCA7 (CAG) trinucleotide repeat mutations were detected by polymerase chain reaction(PCR) and polyacrylamide gel electrophoresis technique in 24 patients with autosomal dominant SCA from 15 families, 20 sporadic SCA patients and 41 normal persons from the same family and 30 healthy persons from different family,the abnormal allele fragments were sequenced by ABI 373 DNA sequencing machine.Results 24 patients with SCA had CAG repeat numbers of SCA 7 allele from 9~18.Normal alleles of SCA 7 had CAG repeat number from 9 to 19. One sporadic SCA patient had one abnormal SCA 7 allele with the CAG repeat expanded to 63 repeats, being confirmed by DNA sequencing.Conclusion CAG expansions were pathogenic cause of SCA 7. The technique of gene mutation detection could provide an effective way for the prediction of asymptomatic and genetic counseling,which was a basis for gene typing.

Study on polymorphisms and mutations in the promoter region of WD gene / 中国实用内科杂志

Objective To screen polymorphisms and mutations in the promoter region of WD gene.Methods DNA from peripheral blood was obtained from 71 subjects of 36 family (48 WD patients,23 patients first-degree relatives) and 20 healthy people from Feb.2001 to Feb.2004.DNA sequence of the genes was analyzed by PCR amplification and direct genomic sequencing.Results There were three polymorphisms at positions-190,-78,+260(transcription start site as +1) of the promoter region of WD gene.Normal controls,WD patients and patients’ first-degree relatives all showed the polymorphisms;three of 48 WD patients presented C→T base substitution mutations at the same position -183:two were homozygous mutation,while the other was heterozygous.Normal control subjects and patients' relatives didn’t show this kind of mutation.Conclusion It suggests that the mutation of the promoter region is one of WD pathogenesis.

The Molecular Biological Study of Hepatolenticular Degeneration / 中山大学学报(医学科学版)

【Objective】 To investigate the molecular basis of hepatolenticular degeneration (Wilson disease, WD) and to attempt to construct the feasibility of gene diagnosis in the disease. 【Methods】 We have performed the molecular biological study on this disease for 10 years by molecular geneti c techniques. 【Results】 ①Location of WD gene in Ch inese: Using pairwise linkage analysis and multipoint linkage analysis method, w e constructed a genetic map of DNA markers within D13q14.2-3 which refined the location of WD gene by restriction fragment length polymorphism(RFL P) and microsatellite polymorphism analysis; ②Screen for mutations of WD gene in Chinese people: we detected the structure of 21 exons of WD ge ne in 45 patients from 39 pedigrees by PCR-SSCP(Single strand conformation poly morphism) and PCR-DNA sequencing technology, found a new mutation in exon 5 and nuclcotide sequence analysis showed it is a T insertion. We also conformed the Arg778Leu in exon 8, the highest frequence mutation point in Chinese people, wit h mutation rate 22.8% in total;③Carrier detection and presymptomatic diagnosi s of WD: Based on DNA recombination technology, we peformed successfully the gen e diagnosis in all individuals of 79 families with WD and built up a helpful spe cific enzyme cut method (PCR-Msp1) to detect the carrier and presympomatic patients in Chinese pe ople with WD. 【Conclusion】 These results showed that the location of WD gene within D13q14.2-3 is the same in Chinese as in Caucasians, but the g ene high m utation point,the gene diagnosis method and its pathogenesis are markly different.

Detection of Wilson disease gene expression in transfected Menkes disease patients fibroblast / 中国神经精神疾病杂志

Objective 　Determination of Wilson disease gene mRNA expression in human fibroblast cell strain (Me32aT22/2L) by reverse transcription-polymerase chain reaction (RT-PCR). Methods　Using lipofection reagent, the plasmid vector carrying the Wilson disease gene (pRc/CMV-WD) was transferred into Me32aT22/2L cultured in serum free complement medium. RT-PCR was used to determine WD mRNA expression in Me32aT22/2L. Results　 Wilson disease gene expression was detected in Me32aT22/2L, while no specific signals were detected in untransfected fibroblast. Conclusions　It demonstrated that Me32aT22/2L strain could express the Wilson disease gene, suggesting that Wilson disease gene transfer might develop a new approach to study Wilson disease.

Confirm polymorphisms in exon 18 of Wilson disease gene in Chinese / 中国神经精神疾病杂志

Objective 　To screen for gene mutation of exon 18 in Chinese patients with Wilson disease. Methods　PCR-SSCP was used to screen exon 18 in 45 Wilson disease patients among 39 Chinese families and 10 normal controls. Those with abnormality were further analyzed by necleotide sequence analysis. Results　There were 16 mobility shift with two different styles in exon 18. All abnormal mobility shifts were sequence analysed. No gene mutation was found. Conclusions　Our result suggest that, contrary to findings in Caucasians, exon 18 is not a frequent mutation point in Chinese patients with Wilson disease.

The study of sequence and structure of intron 8 in Wilson disease gene / 中国神经精神疾病杂志

Objective 　To study the sequence and structure of intron 8 in WD gene in order to further understand the relationship between intron 8 and WD. Methods　We utilized polymerase chain reaction (PCR) to the amplification of exon 8-intron 8-exon 9 which were then sequenced by a dideoxy chain termination methon in 10 normal controls and 32 members of 11 families(20 WD patients and 10 of their relative). The results were analyzed by the computer. Results　The sequence of intron 8 was 703 bp with the G　+　C content of 42.7%. There were one short tandom repeats, 7 direct and inverted repeats in it. An open reading frame coded with 82aa was found at 323 base pairs of downstream of a TATAbox. There were two DNA polymorphisms at 408 and 487 nucleotides. The sequence analysis showed that the 5end has the sequence of 5-GTAAC, 3end has the sequence of CCTAG-3, and branchpoint of 5-TTTCGA-3.Conclusions　The sequences and structures of intron 8 in WD familiess members are not different from normal controls. Our data suggest that the WD gene intron 8 might not play an important role in the pathogenesis of WD.

Study on correlation between gene Arg778Leu mutation and clinical manifestation in Wilson’s disease / 中华神经科杂志

0 05) Conclusions Arg778Leu mutation was related to the onset age in patients with WD, but not to patients′ sex, first symptoms and copper metabolic disturbance Arg778Leu mutation is suggested to postpone the onset age of WD patients

Values of accessory examination in the diagnosis of hepatolenticular degeneration / 临床神经病学杂志

0.9 mg/L in 6 patients.Kayser-Fleischer ring were found in 85.5 % of all the patients. The abnormal hepatic function in the liver type HLD was more common than that of in the brain type. The liver injury was detectable by B mote ultrasonic wave in different type HLD.MRI examination was taken in 79 patients, 65 of them had showed the symmetry abnormal signs in basal ganglia. Conclusions CP has independent diagnostic values when its content ≤0.08 g/L. Kayser-Fleischer ring is an excellent discriminatory test for the diagnosis of HLD patients with neurological or psychiatric symptom. 24 h urine copper is the best single screening test because it increases over 100 ?g/24 h in all patients who were taken the test. The B mote ultrasonic wave test for the liver and MRI for brain were helpful in detecting the damage in the liver and brain.

Construction and expression of mouse liver-specific expression vector Kbpala/Alb-STBXATP7B / 中国病理生理杂志

AIM: To construct the liver-specific transgene vectors encoding wild-type as well as most common disease mutant STBXATP7B cDNA under the control of mouse albumin promoter and to explore their expression. METHODS: Two Kbpala/Alb-STBXATP7B mutants containing the Arg778Leu and His1069Gln mutations were constructed using site-directed mutagenesis system plus site-subcloning technique. The vectors expressed wild-type and mutants of human STBXATP7B in mouse liver cells were obtained and transiently transfected into BRL and BHK cell lines. Western blotting analysis was utilized to detect the expression of human STBXATP7B. RESULTS: Enzyme analysis and sequencing analysis confirmed that the target genes were STBXATP7B and in right position. The results of Western blotting showed that the gene products were expressed specifically in liver cells. CONCLUSION: The Kbpala/Alb-STBXATP7B vectors were constructed successfully and the liver-specific expression of human STBXATP7B proteins were conformed.