OBJECTIVETo determine the frequency paired-box domain 5 (PAX5) gene alterations in B-lineage acute lymphoblastic leukemia (B-ALL) harboring 9p abnormalities and its implication for clinical prognosis.

METHODSBacterial artificial chromosomes RP11-344B23 and RP11-652D9 encompassing the PAX5 gene were selected. DNA was extracted with conventional method and labeled with fluorescein by nicking transition. Fluorescence in situ hybridization (FISH) was used to determine the rearrangement or deletion of the PAX5 gene in B-ALL harboring chromosome 9p abnormalities. Clinical and laboratory features of patients were analyzed.

RESULTSFifty cases were analyzed with FISH. Complete deletion was observed in 23 patients (46%), partial deletion was observed in 2 patients (4%), and rearrangement was detected only in 1 case. The total frequency of abnormalities was 52% (26/50). No significant difference was found in clinical features of patients with or without PAX5 rearrangement or deletion.

CONCLUSIONThe frequency of PAX5 gene alterations in B-ALL harboring 9p abnormalities was 52%. However, no significant difference was found between patients with and without PAX5 alterations.

Acute Disease ; Adolescent ; Adult ; Child ; Chromosomes, Human, Pair 9 ; genetics ; Female ; Gene Rearrangement ; Humans ; In Situ Hybridization, Fluorescence ; Leukemia, B-Cell ; genetics ; Male ; Middle Aged ; PAX5 Transcription Factor ; genetics ; Sequence Deletion ; Young Adult

OBJECTIVETo investigate leptin mRNA expressions in subcutaneous (SC) and omental (OM) adipose tissues of patients with nonalcoholic fatty liver disease (NAFLD), and their relationships with insulin resistance (IR), blood leptin, blood triglyceride, total blood cholesterol, blood glucose, body weight index and waist-hip ratio.

METHODSSC and OM adipose tissues were obtained from 10 obese and 11 nonobese NAFLD patients and from 11 obese and 13 nonobese patients without NAFLD, who served as controls. Leptin mRNA expression levels in the subcutaneous and omental adipose tissues were measured using SYBR Green I quantitative real-time PCR. IR was estimated using homeostasis assessment (HOMA). The levels of plasma leptin and insulin were measured using ELISA.

RESULTSThe relative mRNA expression of leptin, HOMA-IR and blood leptin levels in NAFLD differed significantly from those of the controls (P < 0.05). The leptin/GAPDH ratio of the obese and nonobese NAFLD and control cases were 1.32 +/- 0.12, 0.99 +/- 0.05, 1.10 +/- 0.09, 0.87 +/- 0.13 respectively. The expression levels of SC and OM adipose leptin mRNA in NAFLD patients were positively correlated with HOMA-IR (r=0.72, P < 0.05), blood leptin (r=0.69, P < 0.05), blood triglyceride (r=0.32, P < 0.05), body weight index (r=0.57, P < 0.05) and waist-hip ratio (r=0.50, P <0.05).

CONCLUSIONThe primary reason for high levels of blood leptin is high leptin mRNA expression in adipose tissues; in both obese and nonobese patients with NAFLD; high levels of blood leptin and the leptin mRNA expression in adipose tissues and IR exist. These findings suggest that leptin resistance exists in patients with NAFLD and leptin resistance is positively correlated with NAFLD, the same as in insulin resistance.

Adipose Tissue ; metabolism ; Adult ; Aged ; DNA, Complementary ; Fatty Liver ; genetics ; metabolism ; Female ; Gene Expression ; Humans ; Leptin ; genetics ; metabolism ; Male ; Middle Aged ; RNA

BACKGROUNDAlthough flunarizine has been widely used for migraine prophylaxis with clear success, the mechanisms of its actions in migraine prophylaxis are not completely understood. The aim of this study was to investigate the effects of flunarizine on tetrodotoxin-resistant Na(+) channels and high-voltage activated Ca(2+) channels of acutely isolated mouse trigeminal ganglion neurons.

METHODSSodium currents and calcium currents in trigeminal ganglion neurons were monitored using whole-cell patch-clamp recordings. Paired Student's t test was used as appropriate to evaluate the statistical significance of differences between two group means.

RESULTSBoth tetrodotoxin-resistant sodium currents and high-voltage activated calcium currents were blocked by flunarizine in a concentration-dependent manner with the concentration producing half-maximal current block values of 2.89 µmol/L and 2.73 µmol/L, respectively. The steady-state inactivation curves of tetrodotoxin-resistant sodium currents and high-voltage activated calcium currents were shifted towards more hyperpolarizing potentials after exposure to flunarizine. Furthermore, the actions of flunarizine in blocking tetrodotoxin-resistant sodium currents and high-voltage activated calcium currents were use-dependent, with effects enhanced at higher rates of channel activation.

CONCLUSIONBlockades of these currents might help explain the peripheral mechanism underlying the preventive effect of flunarizine on migraine attacks.

Animals ; Calcium ; metabolism ; Cells, Cultured ; Female ; Flunarizine ; pharmacology ; Male ; Mice ; Patch-Clamp Techniques ; Sensory Receptor Cells ; drug effects ; metabolism ; Sodium ; metabolism ; Trigeminal Ganglion ; cytology ; drug effects ; metabolism

OBJECTIVETo detect the expression of Toll-like receptor 9 (TLR9) in ovarian cancer, and to explore their clinical significance.

METHODSWestern blot method and immunohistochemical staining were used to examine the expression of TLR9 in the ovarian cancer, paracancerous tissues and normal ovarian tissues, obtained during operation from 30 ovarian carcinoma patients and 30 normal non-tumor patients. The relationships of TLR9 with pathological grade, clinical stage, and metastasis of ovarian cancer were statistically analyzed.

RESULTSThe percentage of positive cells expressing TLR9 protein in human ovarian cancer tissues, paracancerous tissues and normal ovarian tissues were 80.0%, 36.7% and 20.0%, respectively. The protein expression level of TLR9 was gradually descending (P < 0.01). The highly expressed TLR9 significantly correlated with the degree of tumor differentiation, an advanced FIGO stage and lymph node metastasis. The Western blot results showed that TLR9 protein expression in ovarian cancer, paracancerous tissues and normal ovarian tissues were 0.803 ± 0.072, 0.411 ± 0.087 and 0.113 ± 0.065, respectively. The expression of TLR9 in ovarian cancer was significantly higher than that in normal tissue and paracancerous tissues (P < 0.01).

CONCLUSIONSTLR9 has a higher expression in ovarian cancer tissues. TLR9 expression has a close relationship with pathological grades of ovarian cancer, suggesting that TLR9 plays an important role in the development and progression of ovarian cancer through immunologic mechanisms.

Adult ; Blotting, Western ; Carcinoma, Endometrioid ; metabolism ; pathology ; Case-Control Studies ; Cystadenocarcinoma, Mucinous ; metabolism ; pathology ; Cystadenocarcinoma, Serous ; metabolism ; pathology ; Female ; Humans ; Immunohistochemistry ; Lymphatic Metastasis ; Neoplasm Staging ; Ovarian Neoplasms ; metabolism ; pathology ; Ovary ; metabolism ; Toll-Like Receptor 9 ; metabolism

AIMTo identify genes related to the human testis development by substrate hybridization technique.

METHODSA human testis cDNA microarray was constructed and hybridized with probes prepared from human adult and fetal testes and spermatozoa mRNAs by reverse transcription reactions. The differentially expressed genes were sequenced. And a newly identified cullin-3 (CUL-3) transcript variant (designated cul-3b) was bio-informatically analyzed with an online GenBank database. Multi-tissue reverse transcription polymerase chain reaction (RT-PCR) was used to determine the tissue expression profile of cul-3b.

RESULTSCul-3b, a novel CUL-3 transcript variant, was identified. The expression level of cul-3b in adult testes was 3.79-fold higher than that in fetal ones. Cul-3b differed from cul-3 (including NM_003590 and AY337761) in the opening reading frame and had three internal ribosomal entry sites IRESes in the 5'-UTR. These led to a 24 amino acid (aa) truncation at N-terminus of CUL-3b as compared with CUL-3 and a more motivated expression pattern of cul-3b under some strict circumstances. Additionally, cul-3b expressed ubiquitously in human tissues according to multi-tissue RT-PCR.

CONCLUSIONCul-3b is a novel transcript variant of CUL-3, which may be important not only for the development of human testis but also for that of other organs.

Base Sequence ; Cell Cycle Proteins ; genetics ; Cullin Proteins ; genetics ; Humans ; Molecular Sequence Data ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

AIMTo identify the genes specifically expressed in human adult and fetal testes and spermatozoa.

METHODSA human testis cDNA microarray was established. Then mRNAs of human adult and fetal testis and spermatozoa were purified and probes were prepared by a reverse transcription reaction with mRNA as the template. The microarray was hybridized with probes of adult and fetal testes and spermatozoa. The nucleic acid sequences of differentially expressed genes were determined and homologies were searched in the databases of GenBank.

RESULTSA novel human testis-specific gene, PKH-T, was identified by hybridizing adult and fetal testis and spermatozoa probes with a human testis cDNA microarray. The cDNA of PKH-T was 1 069 bp in length. The cDNA sequence of this clone was deposited in the Genbank (AY303972) and PKH-T was also determined as Interim GenSymbol (Unigene, HS.38041). PKH-T contained most PKH conserved motif. The 239 amino acid sequences deduced from the 719 bp open reading frame (ORF) had a homology with the gene PKH (U89606). PKH-T was specifically and strongly expressed in the testis. Comparison of the differential expressions of PKH and PKH-T in testes of different developmental stages indicated that PKH-T was expressed in the adult testis and spermatozoa, while PKH, in the adult, fetal and aged testes. PKH-T had no expression in the testis of Sertoli cell only and partially spermatogenic arrest patients.

CONCLUSIONPKH-T is a gene highly expressed in adult human testis and spermatozoa. It may play an important role in spermatogenesis and could be related to male infertility.

Adult ; Amino Acid Sequence ; DNA, Complementary ; biosynthesis ; genetics ; Female ; Gene Expression Regulation, Enzymologic ; genetics ; Humans ; Infertility, Male ; genetics ; Isoenzymes ; biosynthesis ; genetics ; Male ; Molecular Sequence Data ; Oligonucleotide Array Sequence Analysis ; Pregnancy ; Pyridoxal Kinase ; biosynthesis ; genetics ; RNA Splicing ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sertoli Cells ; metabolism ; Spermatogenesis ; genetics ; Spermatozoa ; metabolism ; Testis ; embryology ; enzymology ; growth & development ; Tissue Distribution

OBJECTIVETo identification and analysis Aichi virus from diarrhea and normal children in Lanzhou, and discuss the relationship between Aichi virus and Infant Diarrhea.

METHODSAccording to the literature published data, Using RT-PCR method to amplified Aichi virus 3CD fragment and the positive products were sequenced and determined, and made the alignment analysis between the nucleotide sequences of the amplified fragment with the known sequence of this virus.

RESULTSThere was 1 case detection of Aichi virus in the 46 hospitalized children with diarrhea and 299 children with diarrhea out-patients specifically, Overall detection rate was 0.06%, and there was no Aichi virus was detected in normal control children. 2 viral 3CD gene and the known reference strains of nucleotide sequences were 97%, while phylogenetic analysis showed that genotype of 2 viral belongs to the B.

CONCLUSIONSThere existed B Genotype of Aichi virus in China, and more research is needed to clarified the etiology and epidemiology of Aichi virus characteristics.

Child ; China ; Diarrhea ; virology ; Feces ; virology ; Humans ; Infant ; Kobuvirus ; classification ; genetics ; isolation & purification ; Molecular Sequence Data ; Phylogeny ; Picornaviridae Infections ; virology

Objective To investigate the modulation of sensitization of dural afferent nerve endings on high voltage active calcium currents (HVA-ICa) of primary sensory neurons from rat trigeminal ganglion. Methods Male adult SD rats were randomly divided into normal saline treatment group (NS,n=8) and proinflammatory agent plus calcitontin gene-related peptide infusion group (IS+CGRP,n=8).One h after the infusion,the effect of CGRP on currents of voltage-gated calcium channel (VGCC) in neurons acutely isolated from trigeminal ganglion was recorded using a conventional whole-cell recording patch clamp technique. Results The peak level of calcium current in the IS+CGRP group ( [-80.48±4.43] pA/pF) was significantly increased as compared with that in the NS group ([-49.5±5.18] pA/pF); the half-activation voltage (Va1/2) in the activation curve of calcium current in the IS+CGRP group ([-20.9±0.4] mV) moved 4.7 mV to the hyperpolarization direction as compared with that in the NS group ([-16.2±0.5] mV) with significant difference (P＜0.05); the half-inactivation voltage (Vi1/2) in the activation curve of calcium current in the IS+CGRP group ([-12.4±0.2] mV) moved 10.1 mV to the depolarization direction as compared with that in the NS group ([-22.5 ±0.3] mV) with significant difference (P＜0.05). Conclusion Sensitization of dural afferent nerve endings facilitates peripheral sensitization of primary sensory neurons, with an outstanding performance as increment of calcium current.

OBJECTIVETo investigate the function of central auditory, Speech-perception-in-noise test (SPIN test) was used to assess that whether temporal lobe epilepsy patients have central auditory processing disorders.

METHODSFour audiological test were performed in 9 patients with temporal lobe epilepsy and 19 age-matched normal hearing persons as controls, which include pure tone audiometry, acoustic immittance measurement, ABR (auditory brainstem response) and SPIN test. All the temporal lobe epilepsy patients were performed by CT or MRI and diagnosed by the neurologists prior to the test. The testing materials of speech and noise in SPIN test was recorded in independent tracks and the S/N was identified by -25 dB by pre-experiment. In the test, every words were read twice and both the results were recorded.

RESULTSIn the ABR test, only wave V latency showed longer values in the left ear, other waves like I, III and inter-peak intervals had'nt shown significant statistical differences in both ears of temporal lobe epilepsy patients. However, in the SPIN test, there's no significant statistical differences in both left and right ears of the controls by the first-time hearing (P = 0.107), and the differences showed by the second-time hearing (P = 0.048); but in the comparison of both ears in temporal lobe epilepsy patients, both time of hearing showed no significant statistical differences with P = 0.174 and P = 0.163. In additional, the SPIN recognition score of the temporal lobe epilepsy patients, whether in the fist or second time hearing, whether in the monaural or binaural models, presented significant statistical differences compared to the controls (P = 0.000).

CONCLUSIONSThere was no statistically significant difference in pure tone audiometry and ABR test for all the groups. But the speech recognition score obtained from SPIN test of tempol lobe epilepsy patients is lower than the normal persons, which has statistically significant difference. (P < 0.001) Then we can come to the conclusion that Temporal lobe epilepsy patients had central auditory dysfunctions and SPIN test is a sensitive way to assess this abnormal.

Adult ; Audiometry, Pure-Tone ; Auditory Perception ; Case-Control Studies ; Epilepsy, Temporal Lobe ; diagnosis ; physiopathology ; Evoked Potentials, Auditory, Brain Stem ; Female ; Hearing Tests ; Humans ; Male ; Middle Aged ; Noise ; Speech Perception ; Young Adult

BACKGROUNDRNA interference using short hairpin RNA (shRNA) can mediate sequence-specific inhibition of gene expression in mammalian cells. A vector-based approach for synthesizing shRNA has been developed recently. Overexpression of P-glycoprotein (P-gp), the MDR1 gene product, confers multidrug resistance (MDR) to cancer cells. In this study, we reversed MDR using shRNA expression vectors in a multidrug-resistant human breast cancer cell line (MCF-7/AdrR).

METHODSThe two shRNA expression vectors were constructed and introduced into MCF-7/AdrR cells. Expression of MDR1 mRNA was assessed by RT-PCR, and P-gp expression was determined by Western Blot and immunocytochemistry. Apoptosis and sensitization of the breast cancer cells to doxorubicin were quantified by flow cytometry and methyl thiazolyl tetrazolium (MTT) assays, respectively. Cellular daunorubicin accumulation was assayed by laser confocal scanning microscopy (LCSM). Statistical significance of differences in mean values was evaluated by Student's t tests. P < 0.05 was considered statistically significant.

RESULTSIn MCF-7/AdrA cells transfected with MDR1-A and MDR1-B shRNA expression vectors, RT-PCR showed that MDR1 mRNA expression was reduced by 40.9% (P < 0.05), 30.1% (P < 0.01) (transient transfection) and 37.6% (P < 0.05), 28.0% (P < 0.01) (stable transfection), respectively. Western Blot and immunocytochemistry showed that P-gp expression was significantly and specifically inhibited. Resistance against doxorubicin was decreased from 162-fold to 109-fold (P < 0.05), 54-fold (P < 0.01) (transient transfection) and to 108-fold (P < 0.05), 50-fold (P < 0.01) (stable transfection). Furthermore, shRNA vectors significantly enhanced the cellular daunorubicin accumulation. The combination of shRNA vectors and doxorubicin significantly induced apoptosis in MCF-7/AdrR cells.

CONCLUSIONSshRNA expression vectors effectively reduce MDR expression in a sustained fashion and can restore the sensitivity of drug-resistant cancer cells to conventional chemotherapeutic agents.

ATP-Binding Cassette, Sub-Family B, Member 1 ; analysis ; antagonists & inhibitors ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Survival ; drug effects ; Daunorubicin ; pharmacokinetics ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Flow Cytometry ; Genes, MDR ; Genetic Vectors ; Humans ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection