Biomedical subjects

Qi Cai

Publications and source records attributed to Qi Cai.

At least 19 recordsLinked to original sources

PubMed2006

Site-directed spin labeling measurements of nanometer distances in nucleic acids using a sequence-independent nitroxide probe.

In site-directed spin labeling (SDSL), local structural and dynamic information is obtained via electron paramagnetic resonance (EPR) spectroscopy of a stable nitroxide radical attached site-specifically to a macromolecule. Analysis of electron spin dipolar interactions between pairs of nitroxides yields the inter-nitroxide distance, which provides quantitative structural information. The development of pulse EPR methods has enabled such distance measurements up to 70 A in bio-molecules, thus opening up the possibility of SDSL global structural mapping. This study evaluates SDSL distance measurement using a nitroxide (designated as R5) that can be attached, in an efficient and cost-effective manner, to a phosphorothioate backbone position at arbitrary DNA or RNA sequences. R5 pairs were attached to selected positions of a dodecamer DNA duplex with a known NMR structure, and eight distances, ranging from 20 to 40 A, were measured using double electron-electron resonance (DEER). The measured distances correlated strongly (R2 = 0.98) with the predicted values calculated based on a search of sterically allowable R5 conformations in the NMR structure, thus demonstrating accurate distance measurements using R5. Furthermore, distance measurement in a 42 kD DNA was demonstrated. The results establish R5 as a sequence-independent probe for global structural mapping of DNA and DNA-protein complexes.

DNA↗

PubMed2006

Liver intestine-cadherin (CDH17) haplotype is associated with increased risk of hepatocellular carcinoma.

PURPOSE: Hepatocellular carcinoma (HCC), the most common form of liver cancer, is a leading cause of cancer death worldwide. We previously showed that aberrant mRNA splicing of the liver intestine-cadherin gene CDH17 in liver tissues was triggered by the specific constellation of two CDH17 single nucleotide polymorphisms (651T and IVS6+35G). CDH17 aberrant splicing was highly associated with tumor dissemination and shorter survival of HCC patients. Consequently, it is highly relevant to assess whether the presence of these single nucleotide polymorphisms in the general population represents a risk to the development of HCC. EXPERIMENTAL DESIGN: We conducted a case-control study including 164 HCC and 99 cirrhosis patients and 293 healthy controls. Genotyping was done by PCR and direct sequencing. Odds ratio (OR) and chi2 analysis were used to analyze genotypes and haplotypes. RESULTS: Genotypes 651TT [OR, 2.62; 95% confidence interval (95% CI), 1.34-5.03] and IVS6+35 GG (OR, 1.95; 95% CI, 1.04-3.62) were highly associated with HCC disease. The 651T (C>T) and IVS6+35G (A>G) alleles were also overrepresented in HCC patients and, in particular, the T-G haplotype was the most prevalent in HCC patients when compared with healthy controls (OR, 1.57; 95% CI, 1.167-2.109; P=0.004), which was in agreement with the aberrant splicing observed in tumor tissues. There was no significant difference in genotype and allele frequencies between cirrhosis patients and controls. CONCLUSION: The functional T-G haplotype of CDH17 (651 C>T and IVS6+35A>G) is a genetic susceptibility factor for the development of HCC in a Chinese population.

Alleles↗

PubMed2006

Axial heterogeneity of vasopressin-receptor subtypes along the human and mouse collecting duct.

Vasopressin and vasopressin antagonists are finding expanded use in mouse models of disease and in clinical medicine. To provide further insight into the physiological role of V1a and V2 vasopressin receptors in the human and mouse kidney, intrarenal localization of the receptors mRNA was determined by in situ hybridization. V2-receptor mRNA was predominantly expressed in the medulla, whereas mRNA for V1a receptors predominated in the cortex. The segmental localization of vasopressin-receptor mRNAs was determined using simultaneous in situ hybridization and immunohistochemistry for segment-specific markers, including aquaporin-2, Dolichos biflorus agglutinin, epithelial Na channels, Tamm Horsfall glycoprotein, and thiazide-sensitive Na(+)-Cl(-) cotransporter. Notably, V1a receptor expression was exclusively expressed in V-ATPase/anion exchanger-1-labeled alpha-intercalated cells of the medullary collecting duct in both mouse and human kidney. In cortical collecting ducts, V1a mRNA was more widespread and detected in both principal and intercalated cells. V2-receptor mRNA is diffusely expressed along the collecting ducts in both mouse and human kidney, with higher expression levels in the medulla. These results demonstrate heterogenous axial expression of both V1a and V2 vasopressin receptors along the human and mouse collecting duct. The restricted expression of V1a-receptor mRNA in intercalated cells suggests a role for this receptor in acid-base balance. These findings further suggest distinct regulation of renal transport function by AVP through V1a and V2 receptors in the cortex vs. the medulla.

Animals↗

PubMed2006

Effects of expression of p53 and Gadd45 on osmotic tolerance of renal inner medullary cells.

The response of renal inner medullary (IM) collecting duct cells (mIMCD3) to high NaCl involves increased expression of Gadd45 and p53, both of which have important effects on growth and survival of the cells. However, mIMCD3 cells, being immortalized by SV40, proliferate rapidly, which is known to sensitize cells to high NaCl, whereas IM cells in situ proliferate very slowly and survive much higher levels of NaCl. In the present studies, we have examined the importance of Gadd45 and p53 for survival of normal IM cells in their usual high-NaCl environment by using more slowly proliferating second-passage mouse inner medullary epithelial (p2mIME) cells and comparing cells from wild-type and gene knockout mice. Acutely elevating NaCl (and/or urea) reduces Gadd45a, but increases Gadd45b and Gadd45g mRNA, depending on the mix of NaCl and urea and the rate of increase of osmolality. Nevertheless, p2mIME cells from Gadd45b(-/-), Gadd45g(-/-), and Gadd45bg(-/-) mice survive elevation of NaCl (or urea) essentially the same as do wild-type cells. p53(-/-) Cells do not tolerate as high a concentration of NaCl (or urea) as p53(+/+) cells, but urinary concentrating ability of p53(-/-) mice is normal, as is the histology of inner medullas from p53(-/-) and Gadd45abg(-/-) mice. Thus although Gadd45 and p53 may play roles in osmotically stressed mIMCD3 cells, we do not find that their expression makes an important difference, either for Gadd45 in slower proliferating p2mIME cells or for Gadd45 or p53 in normal inner medullary epithelial cells in situ.

Animals↗

PubMed2006

Effects of water restriction on gene expression in mouse renal medulla: identification of 3betaHSD4 as a collecting duct protein.

To identify novel gene targets of vasopressin regulation in the renal medulla, we performed a cDNA microarray study on the inner medullary tissue of mice following a 48-h water restriction protocol. In this study, 4,625 genes of the possible approximately 12,000 genes on the array were included in the analysis, and of these 157 transcripts were increased and 63 transcripts were decreased by 1.5-fold or more. Quantitative, real-time PCR measurements confirmed the increases seen for 12 selected transcripts, and the decreases were confirmed for 7 transcripts. In addition, we measured transcript abundance for many renal collecting duct proteins that were not represented on the array; aquaporin-2 (AQP2), AQP3, Pax-8, and alpha- and beta-Na-K-ATPase subunits were all significantly increased in abundance; the beta- and gamma-subunits of ENaC and the vasopressin type 1A receptor were significantly decreased. To correlate changes in mRNA expression with changes in protein expression, we carried out quantitative immunoblotting. For most of the genes examined, changes in mRNA abundances were not associated with concomitant protein abundance changes; however, AQP2 transcript abundance and protein abundance did correlate. Surprisingly, aldolase B transcript abundance was increased but protein abundance was decreased following 48 h of water restriction. Several transcripts identified by microarray were novel with respect to their expression in mouse renal medullary tissues. The steroid hormone enzyme 3beta-hydroxysteroid dehydrogenase 4 (3betaHSD4) was identified as a novel target of vasopressin regulation, and via dual labeling immunofluorescence we colocalized the expression of this protein to AQP2-expressing collecting ducts of the kidney. These studies have identified several transcripts whose abundances are regulated in mouse inner medulla in response to an increase in endogenous vasopressin levels and could play roles in the regulation of salt and water excretion.

3-Hydroxysteroid Dehydrogenases↗

PubMed2006

[A modified cytogenetic study for multiple myeloma].

OBJECTIVE: To evaluate the effect of modified culture method used to cytogenetic analysis and the clinically significance of chromosomal abnormalities to multiple myeloma (MM). METHODS: Mononuclear cells were isolated from bone marrow aspirate of 20 MM patients; and then cultured for 3 days without any cytokines, and 6 days in the presence of IL-6 (10 ng/mL) and GM-CSF (30 ng/mL) before RHG banding analysis; the remained part of aspirates were treated directly. Eight cases of iron deficiency anemia were taken as control. RESULTS: The experiment was failure in 2 cases because of blood clot, and another 2 cases could be analyzed only by direct method due to inadequate cells. The karyotype abnormalities were found from 4 cases of 16 available patients. Of them, three cases had complex karyotypes. The abnormalities were detected after 6 days culture with addition of cytokines. No abnormalities were detected from those groups of directly analysis and 3 day culture. Meantime, the clinical data showed that the patients with cytogenetic abnormalities were in stage III, and had a high percentage of MM cells (25%-56%) in their bone marrow, and also poor responses to prior chemotherapy. No cytogenetic abnormalities were found from control individuals in all groups. CONCLUSION: Extended culture in the presence of cytokines could improve the efficiency of cytogenetic analysis to MM. Complex karyotype was common cytogenetic abnormalities in MM patients with poor response to chemotherapy.

Aged↗

PubMed2006

[Partial deletion of chromosome 13 long arm in multiple myeloma and its clinical significance].

OBJECTIVE: To explore the specific locus deletion of the long arm of chromosome 13 and its relationship with the clinical behavior and prognosis of multiple myeloma (MM). METHODS: FISH analysis was performed on bone marrow smears from 68 patients with MM to study the deletion of Rb-1 gene and locus 13q14.3 on chromosome 13. The statistic value of its effect on clinical features were determined. RESULTS: 35 out of the 68 (51%) cases were found with deletion of chromosome 13; deletion of Rb-1 gene were found in 29 (43%) cases; deletion of locus 13q14.3 were found in 23 out of 44 (52%) cases; the analysis results were same in 66% of the cases (29/44) with the above two probes. Chi-square test showed that partial deletion of chromosome 13 was associated with clinical behavior, early chemotherapy response and 1 year survival. CONCLUSION: Deletion of Rb-1 gene and locus 13q14.3 were both common cytogenetic changes in MM patients with effect on the biological behavior of the disease.

Adult↗

PubMed2006

[Two novel germline mutations of MLH1 in hereditary nonpolyposis colorectal cancer family].

OBJECTIVE: To explore germline mutations of MLH1 in hereditary nonpolyposis colorectal cancer (HNPCC), and to investigate the pathobiology of novel detectable mutations of MLH1. METHOD: RNA was extracted from the peripheral blood of 12 patients from 12 different families fulfilling the Amsterdam II Criteria of HNPCC. Germline mutations of MLH1 were determined by RT-PCR with gene specific primers, heat-resistance reverse transcriptase and long-template PCR polymerase, followed by cDNA sequencing analysis. PCR-Genescan analysis was used to further investigate microsatellite instability with a panel of 5 microsatellite markers (BAT26, BAT25, D5S346, D2S123 and Mfd15), along with immunohistochemistry staining to detect the expression of MLH1 protein in the tumor tissues. RESULTS: Four germline mutations were found in 4 patients, 2 of which were previously reported GTT-->GAT mutation at codon 384 of exon 12, and the other two were novel mutations: CGC-->TGC at codon 217 of exon 8 and CCG-->CTG at codon 581 of exon 16. Two tumors with the novel mutations had high frequency microsatellite instability showing more than 2 instable loci (RER + phenotype), and both tumors lost their MLH1 protein expression. CONCLUSION: The two novel germline mutations of MLH1 identified in this study, i.e. CGC-->TGC at codon 217 of exon 8 and CCG-->CTG at codon 581 of exon 16, are very likely to have pathological significance.

Adaptor Proteins, Signal Transducing↗

PubMed2006

[Evaluation of the subsets of lymphocytes and their activated status in patients with myelodysplastic syndrome].

This study was purposed to investigate the clinical significance of the amount and activated status of T cell subsets, B cells, NK cells in peripheral blood from patients with myelodysplastic syndrome (MDS). The proportion of T cells, B cells, NK cells in peripheral blood from 30 patients with MDS and their surface activation markers of CD28, CD45RA, CD45RO, CD69, HLA-DR were analyzed by flow cytometry. Twenty-two patients were in the low risk group (RA + RAS) while eight patients were in the high risk group (RAEB + RAEBT). The result showed that the amounts of T cells (CD3+ cells) in peripheral blood from patients with MDS were lower than those in control group. The amounts of naive CD4+ cells (CD4+ CD45RA+ cells) in MDS patients were lower than those in control. The expression rates of early activation marker (CD69) and late activation marker (HLA-DR) on CD3+ cells in MDS patients were significantly higher than those in control. The abnormalities of the immunologically competent cells were mainly observed in the low risk group (RA + RAS), and were characterized by the high expression rates of CD69+ and HLA-DR+ on CD3+ cells, the decrease of B cell amounts. The amount abnormalities of T cell subsets were mainly observed in high risk group (RAEB + RAEBT), and were characterized by the decrease of CD3+ cells and CD3+ CD4+ CD8- cells (Th cells) amounts without high expression of the CD69 and HLA-DR, the decrease of NK cells amounts. It is concluded that there are the abnormalities of T cell subsets and function in the patients with MDS and may change with disease progression, so the measurement of amount and activated status of T cell subsets in peripheral blood from MDS patients can have predictive role for diagnosis of disease progression and guide of therapy.

Adolescent↗

PubMed2006

[Prognostic factor analysis of 77 old patients with acute myelogenous leukemia].

BACKGROUND & OBJECTIVE: The manifestations of old acute myelogenous (AML) patients have their special biological and clinical characteristics, with lower response rate to therapy and shorter survival time. This study was to investigate the prognostic factors of elderly patients with AML retrospectively. METHODS: 77 patients aged> or =60 years with AML from 1994 to 2005 were admitted to our study and all the possible prognostic factors were analyzed with Kaplan-Meier survival analysis. The significant factors were further analyzed by Cox proportional hazard model analysis. RESULTS: Seventy-two patients were evaluated. The patients aged 60-70 (median survival time was 350 days) had significantly longer survival time than those aged more than 70 (median survival time is 60 days)(P<0.001), which their CR ratios were 71.4% and 29.4% (P=0.001). The patients with performance status 0 or 1 (median survival time was 402 days) had significantly longer survival time than those with performance status 2, 3 or 4 (median survival time was 31 days)(P<0.001), which their CR ratios were 75% and 15% (P<0.001). The patients with primary AML (median survival time was 98 days) had significantly longer survival time than those with secondary AML (median survival time was 32 days)(P=0.007), which their CR ratios were 50% and 0% (P=0.023). The patients treated with sub-standard dosage of anthracycline (median survival time was 293 days) had significantly longer survival time than those treated with reduced dosage of anthracycline (median survival time was 35 days)(P=0.006), which their CR ratios were 63.6% and 33.3% (P=0.02). The patients with bone marrow blast cell ratio< or =50% (median survival time was 98 days ) had significantly longer survival time than those with bone marrow blast cell ratio >50% (median survival time was 55 days)(P=0.006). The patients with favorable karyotype (median survival time was 293 days) had significantly longer survival time than those with unfavorable or normal karyotype (median survival time was 31 days)(P=0.005). The patients without CD34 expression (median survival time was 201 days) had significantly longer survival time than those with CD34 expression(median survival time was 36 days)(P<0.001). The patients with the peripheral blood white blood cell count (PBWBC)>10x10(9)/L (50%) had significantly higher CR ratio than those with PBWBC< or =10x10(9)/L (25%)(P=0.043). The patients received chemotherapy (50%) had significantly higher CR ratio than those received supportive therapy (0%)(P=0.001). In the stepwise COX proportional hazard regression model, all the seven factors related to OS remained independent and significant. CONCLUSIONS: Factors, including age >70, PS 2 to 4, percentage of blasts in bone marrow >50%, secondary AML, unfavorable karyotype, expression of CD34, lower dosage.

Age Factors↗

PubMed2005

The procapsid binding domain of phi29 packaging RNA has a modular architecture and requires 2'-hydroxyl groups in packaging RNA interaction.

The phi29 packaging RNA (pRNA) is an essential component in the phi29 bacteriophage DNA packaging motor, the strongest biomolecular motor known today. Utilizing Mg2+-dependent intermolecular base pairing interactions between two 4-nucleotide loops within the pRNA procapsid binding domain, multiple copies of pRNA form a ring-shaped complex that is indispensable for packaging motor function. To understand pRNA structural organization and pRNA/pRNA interaction, studies were carried out on pRNA closed dimers, the simplest functional pRNA complex believed to be the building blocks for assembling the oligomeric ring. Tertiary folding and interactions in various pRNA mutants were evaluated based on measured closed dimer affinity that is directly linked to the proper positioning of the interacting loops. The data revealed that the procapsid binding domain contains two autonomous modules that are capable of interacting noncovalently to form a fully active species in pRNA/pRNA interaction. Deleting the 2'-hydroxyl groups in one of the interacting loops weakens the dimer affinity by 125-fold, suggesting potential tertiary interactions involving these 2'-hydroxyl groups. The results provide evidence that nonbase functional groups are involved in pRNA folding and interaction and lead to a simple model that describes the pRNA monomer configuration in terms of three arms spanning a hinge. The functional constructs developed here will aid biophysical and biochemical investigations of pRNA structure and function, as well as developments of pRNA-based technology for nanoscience and gene therapy.

Bacillus Phages↗

PubMed2005

High NaCl increases TonEBP/OREBP mRNA and protein by stabilizing its mRNA.

Hypertonicity increases mRNA and protein abundance of the transcription factor tonicity-responsive enhancer/osmotic response element binding protein (TonEBP/OREBP), contributing to increased transcription of downstream osmoprotective genes. Previously, this was attributed to increased transcription of TonEBP/OREBP because no change was found in its mRNA stability. However, there is no direct evidence for increased transcription, and the 3'-untranslated region (UTR) of TonEBP/OREBP contains numerous adenylate/uridylate-rich elements, which can modulate RNA stability. Therefore, we have reinvestigated the effect of hypertonicity on TonEBP/OREBP mRNA stability. We find that, in mouse inner medullary collecting duct cells, raising osmolality from 300 to 500 mosmol/kgH(2)O by adding NaCl increases TonEBP/OREBP mRNA to a peak of 2.3-fold after 4 h, followed by a decline. TonEBP/OREBP protein increases to a sustained peak of 3.0-fold at 8 h. To determine the stability of TonEBP/OREBP mRNA, we measured the rate of its decrease after inhibiting transcription with actinomycin D, finding that it is stabilized for 6 h after addition of NaCl. This stabilization is sufficient to explain the increase in mRNA without any change in transcription. To investigate how hypertonicity stabilizes TonEBP/OREBP mRNA, we tested luciferase reporters containing parts of the TonEBP/OREBP mRNA UTR. Inclusion of both the 5'- and 3'-UTR increases reporter activity, consistent with mRNA stabilization. Surprisingly, however, it is the 5'-UTR that stabilizes; the 3'-UTR, by itself, decreases reporter activity. We concluded that 1) hypertonicity stabilizes TonEBP/OREBP mRNA, contributing to its increase, and 2) stabilization depends on the presence of the 5'-UTR.

3' Untranslated Regions↗

PubMed2005

Clinicopathological and molecular genetic analysis of HNPCC in China.

AIM: To explore the clinicopathological and molecular genetic features of hereditary nonpolyposis colorectal cancer (HNPCC) in Chinese population. METHODS: We collected 16 Chinese HNPCC families from Wenzhou, Zhejiang Province, China. Tumor tissues and peripheral white blood cells were studied using microdissection, microsatellite analysis, immunostaining of hMSH2 and hMLH1 proteins and direct DNA sequencing of hMSH2 and hMLH1 genes. RESULTS: (1) A total of 50 patients had CRC. Average age at diagnosis of the first CRC was 45.7 years; 40.9% and 28.7% of the CRCs were located proximal to the splenic flexure and in the rectum, respectively. Thirty-eight percent of the colorectal cancer patients had synchronous and metachronous CRC. 34.4% and 25% of the CRCs were poor differentiation cancer and mucinous adenocarcinoma, respectively. Fourteen extracoloni tumors were found, and the hepatic cancer was the most common tumor type. Twenty-one patients whose median survival time was 5.7 years died during 1-23 years. Twenty-nine patients have survived for 1-28 years, 58.6%, 41.4% and 24.1% patients have survived for more than 5, 10 and 15 years, respectively; (2) All nine tumor-tissues showed microsatellite instability (MSI) at more than two loci. Four tumor-tissues lost hMSH2 protein expression and one lost hMLH1 protein expression. Three pathological germline mutations were identified from five genetically analyzed families; two of three mutations had not been reported previously as they were a transition from C to A in exon 14 (codon 743) of hMSH2 and a TTC deletion in exon 14 (codon 530) of hMLH1. CONCLUSION: Chinese HNPCC have specific clinicopathological features, such as early onset, propensity to involve the proximal colon, and high frequency of multiple CRCs, liver cancer more frequent than endometrial cancer. Chinese HNPCC showed relatively frequent germline mutation of mismatch repair (MMR) genes that correlated closely with high-level MSI and loss of expression of MMR genes protein.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Increased reactive oxygen species contribute to high NaCl-induced activation of the osmoregulatory transcription factor TonEBP/OREBP.

The signaling pathways leading to high NaCl-induced activation of the transcription factor tonicity-responsive enhancer binding protein/osmotic response element binding protein (TonEBP/OREBP) remain incompletely understood. High NaCl has been reported to produce oxidative stress. Reactive oxygen species (ROS), which are a component of oxidative stress, contribute to regulation of transcription factors. The present study was undertaken to test whether the high NaCl-induced increase in ROS contributes to tonicity-dependent activation of TonEBP/OREBP. Human embryonic kidney 293 cells were used as a model. We find that raising NaCl increases ROS, including superoxide. N-acetylcysteine (NAC), an antioxidant, and MnTBAP, an inhibitor of superoxide, reduce high NaCl-induced superoxide activity and suppress both high NaCl-induced increase in TonEBP/OREBP transcriptional activity and high NaCl-induced increase in expression of BGT1mRNA, a transcriptional target of TonEBP/OREBP. Catalase, which decomposes hydrogen peroxide, does not have these effects, whether applied exogenously or overexpressed within the cells. Furthermore, NAC and MnTBAP, but not catalase, blunt high NaCl-induced increase in TonEBP/OREBP transactivation. N(G)-monomethyl-l-arginine, a general inhibitor of nitric oxide synthase, has no significant effect on either high NaCl-induced increase in superoxide or TonEBP/OREBP transcriptional activity, suggesting that the effects of ROS do not involve nitric oxide. Ouabain, an inhibitor of Na-K-ATPase, attenuates high NaCl-induced superoxide activity and inhibits TonEBP/OREBP transcriptional activity. We conclude that the high NaCl-induced increase in ROS, including superoxide, contributes to activation of TonEBP/OREBP by increasing its transactivation.

Carrier Proteins↗

PubMed2005

[Inhibitory effect of RNA interference on chronic myeloid leukemia bcr/abl oncogene expression].

OBJECTIVE: To investigate the inhibitory effect of RNA interference on chronic myeloid leukemia (CML) bcr/abl oncogene expression. METHODS: The small interference RNAs (siRNAs) were synthesized in vitro. K562 cells stably expressing bcr/abl gene were transfected with the siRNA by electroporation, both the non-transfected cells and non-specific siRNAs transfected cells were taken as controls. The enhanced green fluorescent protein (EGFP) plasmid was used as positive control and the transfection efficiency was detected by flow cytometry. Inhibitory effect of siRNAs was demonstrated by real-time quantitative RT-PCR and Western blots. Cell proliferation was measured by MTT assay and apoptosis by Annexin V-FITC assay. RESULTS: The transfection efficiency was about 70%. The synthesized siRNAs inhibited CML bcr/abl oncogene expression at both mRNA and protein levels. siRNAs could inhibit K562 cell proliferation to 47% and 56% at 24 h and 48 h after transfection, respectively, and induce cell apoptosis from 1.00% in control group to 15.05% and 19.4% at 24 h and 48 h respectively. CONCLUSION: At the cell level, inhibition of CML bcr/abl oncogene expression by chemically synthesized siRNAs provides the new method for anti-leukemia study.

Apoptosis↗

PubMed2004

Pax2 expression occurs in renal medullary epithelial cells in vivo and in cell culture, is osmoregulated, and promotes osmotic tolerance.

Pax2 is a transcription factor that is crucial for kidney development, and it is also expressed in the normal adult kidney, where its physiological function is unknown. In the present study, we find by cDNA microarray analysis that Pax2 expression in second-passage mouse inner-medullary epithelial cells is increased by a high NaCl concentration, which is significant because NaCl levels are normally high in the inner medulla in vivo, and varies with urinary concentration. Furthermore, a high NaCl concentration increases Pax2 mRNA and protein expression in mouse inner medullary collecting duct (mIMCD3) cells, and its transcriptional activity. Pax2 mRNA and protein expression is high in normal adult mouse renal inner medulla but much lower in renal cortex. Pax2 protein is present in collecting duct cells in both renal medulla and cortex and in thin descending limbs of Henle's loop in inner medulla. Treating Brattleboro rats with desamino-Cys-1,d-Arg-8 vasopressin, which increases inner-medullary NaCl concentration, causes a 4-fold increase in inner-medullary Pax2 protein. Treatment with furosemide, which decreases inner-medullary NaCl, reduces inner-medullary Pax2 mRNA and protein. Pax2-specific short interfering RNA increases high NaCl concentration-induced activation of caspase-3 and apoptotic bodies in mIMCD3 cells. We thus conclude that (i) Pax2 is expressed in normal renal medulla, (ii) its expression is regulated there by the normally high and variable NaCl concentration, and (iii) it protects renal medullary cells from high NaCl concentration-induced apoptosis.

Animals↗

PubMed2004

Cells adapted to high NaCl have many DNA breaks and impaired DNA repair both in cell culture and in vivo.

Acute exposure of cells in culture to high NaCl damages DNA and impairs its repair. However, after several hours of cell cycle arrest, cells multiply in the hypertonic medium. Here, we show that, although adapted cells proliferate rapidly and do not become apoptotic, they nevertheless contain numerous DNA breaks, which do not elicit a DNA damage response. Thus, in adapted cells, Mre11 exonuclease is mainly present in the cytoplasm, rather than nucleus, and histone H2AX and chk1 are not phosphorylated, as they normally would be in response to DNA damage. Also, the adapted cells are deficient in repair of luciferase reporter plasmids damaged by UV irradiation. On the other hand, the DNA damage response activates rapidly when the level of NaCl is reduced. Then, Mre11 moves into the nucleus, and H2AX and chk1 become phosphorylated. Renal inner medullary cells in vivo are normally exposed to a variable, but always high, level of NaCl. As with adapted cells in culture, inner medullary cells in normal mice exhibit numerous DNA breaks. These DNA breaks are rapidly repaired when the NaCl level is decreased by injection of the diuretic furosemide. Moreover, repair of DNA breaks induced by ionizing radiation is inhibited in the inner medulla. Histone H2AX does not become phosphorylated, and repair synthesis is not detectable in response to total body irradiation unless NaCl is lowered by furosemide. Thus, both in cell culture and in vivo, although cells adapt to high NaCl, their DNA is damaged and its repair is inhibited.

Animals↗

PubMed2004

[Clinical analysis and molecular genetic study of hereditary nonpolyposis colorectal cancer kindreds].

OBJECTIVE: To study the clinicopathological and molecular genetic characteristics of hereditary nonpolyposis colorectal cancer (HNPCC), to enable the early diagnosis and to evaluate the treatment. METHODS: We analyzed 12 families of HNPCC from Wenzhou, Zhejiang province, China. Mismatch repair genes hMSH2 and hMLH1 expression and microsatellite instability of tumor tissue were studied using microdissection, microsatellite analysis, immunohistochemical staining and Gene Scan analysis. Direct DNA sequencing of hMSH2 and hMLH1 were performed subsequently. RESULTS: Altogether 32 patients with colorectal cancer were recognized in 12 HNPCC families, with the median age of 45.2 years (75.0% before the age of 50 years). The proximal tumors accounted for 51.1%, while multiple colorectal cancers accounted for 34.4%. Poor differentiation cancers occupied half of the patients (53.1%). And 68.8% of the patients had the tumor of Dukes A and B. Among 12 HNPCC families, 7 cases in 6 HNPCC families developed extracolonic cancer. 13 cases died during follow up of 1 - 23 years. The median survival time was 6.4 years. 19 alive cases followed up from 1 to 28 years. All tumors (9/9) displayed microsatellite instability, with the half losing hMSH2 or hMLH1 expression. In the 5 genetic analyzed kindreds 3 possessed germline mutation. Two of three mutations have not been reported in the worldwide database previously. CONCLUSION: HNPCC showed distinct clinicopathological characteristics. Microsatellite instability analysis and immunohistochemical staining might be the effective screening methods before direct DNA sequencing for the detection of mutation in mismatch repair genes. It is important to analyze the members of affected families.

Adaptor Proteins, Signal Transducing↗