Copper efflux transporters ATP7A and ATP7B: Novel biomarkers for platinum drug resistance and targets for therapy.

Platinum-based chemotherapy agents are widely used in the treatment of various solid malignancies. However, their efficacy is limited by drug resistance. Recent studies suggest that copper efflux transporters, which are encoded by ATP7A and ATP7B, play an important role in platinum drug resistance. Over-expressions of ATP7A and ATP7B are observed in multiple cancers. Moreover, their expressions are associated with cancer prognosis and treatment outcomes of platinum-based chemotherapy. In our review, we highlight the roles of ATP7A/7B in platinum drug resistance and cancer progression. We also discuss the possible mechanisms of platinum drug resistance mediated by ATP7A/7B and provide novel strategies for overcoming resistance. This review may be helpful for understanding the roles of ATP7A and ATP7B in platinum drug resistance. © 2018 IUBMB Life, 70(3):183-191, 2018.

Human cytomegalovirus UL49 encodes an early, virion-associated protein essential for virus growth in human foreskin fibroblasts.

Despite recent results of deletion experiments showing that open reading frame (ORF) UL49 of human cytomegalovirus (HCMV) is essential, the expression, function and functional location of its encoded protein remain unknown. We generated an antibody specific for pUL49 to investigate the protein product encoded by the UL49 ORF and identified its function in HCMV-infected host foreskin fibroblasts. A bacterial artificial chromosome (BAC) of HCMV strain Towne (pRV-Towne) and the UL49-deleted mutant pRV-delUL49Towne were used to observe virus growth by plaque assay. Using a UL49-protein-binding antibody, we located pUL49 in the fibroblast cytoplasm. pUL49 exhibited expression kinetics resembling those of the class ß-2 proteins and was detected in the virion tegument. Following deletion of UL49 ORF, the virus failed to replicate, but it could be recovered by addition of pUL49 from pCDNA3.1 (+)-UL49. Our findings indicate that UL49 ORF is essential for HCMV replication in host foreskin fibroblasts.

MicroRNA 375 mediates the signaling pathway of corticotropin-releasing factor (CRF) regulating pro-opiomelanocortin (POMC) expression by targeting mitogen-activated protein kinase 8.

Pro-opiomelanocortin (POMC) is a common precursor of melanocortin-related peptides in the pituitary and primarily regulated by corticotropin- releasing factor (CRF). Our results show that miR-375 is highly expressed in the mouse pituitary gland and located specifically in the intermediate lobe of pituitary. The functional studies show that the forced inhibition of endogenous miR-375 in AtT-20 mouse pituitary tumor cells and in the intermediate lobe of the pituitary gland significantly increases POMC expression, whereas miR-375 overexpression down-regulates POMC expression and ACTH secretion stimulated by CRF. This function of miR-375 is accomplished by its binding to the 3'-UTR of mitogen-activated protein kinase kinase kinase-8. Our results here have demonstrated that miR-375 acts as a negative regulating molecule mediating the signaling pathway of CRF and affecting POMC expression by targeting mitogen-activated protein kinase kinase kinase-8, which subsequently down-regulates ERK1/2 phosphorylation and nerve growth factor-induced clone B (NGFI-B) transcription activity. Taken together, our results show that miR-375 is a novel negative regulator of POMC expression and related hormone secretion.

[The expression of peripheral Th1 and Th17 cells in inflammatory bowel disease and its potential clinical value].

OBJECTIVE: To explore the probable role of Th1 and Th17 cells in the pathogenesis of inflammatory bowel disease (IBD). METHODS: The peripheral blood mononuclear cells (PBMCs) from peripheral blood specimens were collected in the study, including 40 healthy controls, 42 ulcerative colitis (UC) and 39 Crohn's disease (CD). The proportion of Th1 and Th17 cells in the PBMCs was detected with flow cytometry after stimulated by PMA and ionomycin. The result and the clinical data were analyzed. RESULT: The Th1 cell expression was increased in CD (38.32 ± 16.18)% and UC group (34.23 ± 11.60)%, compared with the controls (24.58 ± 10.02)% (P < 0.01). During the convalescence, the Th1 expression in the CD and UC groups in vivo was significantly reduced without difference between the two groups (P > 0.05) . In the IBD group , significant difference in the frequency of Th17 cells could be found between the CD group (2.51 ± 1.59)% and the UC group (4.15 ± 2.75)%, while the Th17 cells were increased in both groups, compared with the controls (1.44 ± 0.73)% (P < 0.05) . Obvious difference in the frequency of Th17 cells could be found between patients at different activity stages and remission stages. The proportion of Th17 cells were higher in the UC patients than that in the CD patients (P < 0.01) . The Th17/Th1 ratio of CD patients, UC patients were 0.08 ± 0.06, 0.14 ± 0.11, which were both higher that in the controls (0.07 ± 0.06). Significant difference could be found between the UC group and the CD group (P < 0.01). CONCLUSIONS: The higher proportion of Th1 and Th17 cells are detected in the peripheral blood of IBD patients, which is correlated closely to the activity of the disease. Th1 and Th17 cells may play an important role in the pathogenesis of IBD.

[BRPF2, a novel transcript of BRPF1 and their expression in mice tissues].

In order to identify a novel transcript of BRPF1 (BRPF2), a clone separated from mouse cDNA library was sequenced and submitted to GenBank. The expressions of BRPF1 and BRPF2 in different mice tissues were detected using RT-PCR and Northern blotting assays. The preliminary protein functions and conservative domains were analyzed by bioinformatic methods. The results indicated that BRPF2 was a novel transcript of BRPF1. Both BRPF1 and BRPF2 transcripts could be detected in most mice tissues, including liver, embryo, epididymis, testis, ovary and muscle. However, only BRPF2 transcript could be detected in the spleen. BRPF1 mRNA encoded 1 246 aa and the predicted molecular mass was 140 kDa, while BRPF2 encoded 442 aa, partly owing to the absence of a new stop codon. The results of CDD analysis suggested that BRPF2 lost the bromodomain and the PWWP domain compared to BRPF1. Because the bromodomain and the PWWP domain are the critical structures of BRPF1 to interact with histones and recruit the other transcriptional factors, BRPF2 (without these two critical domains) may serve as a negative regulatory factor of BRPF1 and be involved in the chromosome remodeling and transcriptional regulating.

Bis[µ-N,N'-bis(quinolin-8-yl)pyridine-2,6-dicarboxamido]dizinc(II) dichloromethane disolvate.

The title compound, [Zn(2)(C(25)H(15)N(5)O(2))(2)]·2CH(2)Cl(2), is a dinuclear double-helical complex which lies on a crystallographic twofold axis. In the complex, both ligands are partitioned into two tridentate domains which allow each ligand to bridge both metal centres. Each Zn(II) atom is six-coordinated in a distorted octahedral environment formed by two amide N atoms, two quinoline N atoms and two pyridine N atoms from two different ligand molecules, with the central pyridine ring, unusually, bridging two Zn(II) atoms. The deprotonated ligand is not planar, the amide side chains being considerably twisted out from the plane of the central pyridine ring.

[Mutation analysis of the MMACHC gene in a pedigree with methylmalonic aciduria].

OBJECTIVE: To identify the mutation of the methylmalonic aciduria (cobalamin deficiency) CblC type, with homocystinuria (MMACHC) gene in a pedigree with methylmalonic aciduria. METHODS: The MMACHC gene mutation was detected using polymerase chain reaction (PCR) and DNA sequencing. The MMACHC gene of 50 healthy people was also sequenced as control. RESULTS: A new mutation of 146_154 del CCTTCCTGG was found in the patient and his father, and was absent in the controls. CONCLUSION: A new mutation (146_154 del CCTTCCTGG) in the MMACHC gene was detected in a Chinese family with methylmalonic aciduria.

[Studies of effect of prosaposin on cell proliferation, cell apoptosis and its possible molecular mechanism].

pcDNA3.1 in NIH3T3 and Psap-Myc in NIH3T3 cell strains were used as cell models in order to study the effect of prosaposin on cell proliferation, cell apoptosis and its possible molecular mechanism. MTT assay and Annexin V/PI apoptosis kit were used to detect the effect of prosaposin on cell proliferation and cell apoptosis induced by se-rum-starvation stress, respectively. Western blotting was conducted to detect the phosphorylative level of PI3K/Akt pathway, and real-time PCR was carried out to explore the expression of the genes regulated by PI3K/Akt pathway. Prosaposin pro-tein was proved to activate the PI3K/Akt signal pathway, upregulate the phosphorylative activity of Akt at Serine 473, downregulate the expression of P27(Kip1) gene, upregulate the expression of Cyclin D1 gene and then promote the G1/S tran-sition, and upregulate the expression of survival genes cIAP1 and cIAP2 and then prevent cell apoptosis. These findings suggest that the growth promotion and anti-apoptotic activity of prosaposin may be partly through the PI3K/Akt signal pathway and its downstream targeted genes.

Poly[[tetra-aqua-(µ(6)-2,2'-diiodo-biphenyl-4,4',5,5'-tetra-carboxyl-ato)dizinc(II)] dihydrate].

In the title compound, {[Zn(2)(C(16)H(4)I(2)O(8))(H(2)O)(4)]·2H(2)O}(n), two crystallographically independent Zn(II) atoms are each located on a twofold rotation axis. Both Zn(II) atoms are in distorted octa-hedral coordination geometries: one is coordinated by six O atoms from four carboxyl-ate groups, while the other is coordinated by two carboxyl-ate groups and four water mol-ecules. The tetra-carboxyl-ate ligand mol-ecules connect the Zn(II) atoms, completing a three-dimensional metal-organic framework. O-H⋯O hydrogen bonds link the metal-organic framework with the uncoord-inated water mol-ecules.

[Identification and expression of human cytomegalovirus UL148 mRNA of low-passage clinical isolates in Guangzhou].

OBJECTIVE: To research the structure of the unique gene UL148 of the human cytomegalovirus (HCMV) clinical strain in Guangzhou, analyze the relation between its structure polymorphism and HCMV congenital infection, investigate the function of its expression products, and try to reveal the pathogenic mechanism of the gene in HCMV infection in newborns. METHODS: Urine samples were collected from 10 newborns with HCMV infection and inoculated on human fetal lung cells. The viral DNA was isolated, and UL148 gene fragment was amplified and identified. After TA clone and gene sequencing, total RNA of virus was extracted and the mRNA expression of UL148 gene was identified by using RT-PCR. RESULTS: The UL148 gene complete sequence was determined. A specific 582 bp length DNA fragment was amplified by RT-PCR, which confirmed the expression of UL148 gene. CONCLUSIONS: UL148 gene exists in the low-passage clinical HCMV isolate from Guangzhou. It is confirmed that open reading frame of UL148 has genetic structure with mRNA expression.

ATP7B rs9535826 is associated with gastrointestinal toxicity of platinum-based chemotherapy in nonsmall cell lung cancer patients.

AIMS: Platinum-based chemotherapy is considered as the first-line treatment for nonsmall cell lung cancer (NSCLC) patients. However, platinum resistance and toxicity are major obstacles to its clinical applications. The two P-type ATPases ATP7A and ATP7B have been identified to play an essential role in the transport of platinum. Their genetic polymorphisms may affect the treatment outcome and toxicity of platinum. In this study, we aimed to investigate the association of ATP7A and ATP7B genetic polymorphisms with clinical outcome and toxicity of platinum-based chemotherapy in NSCLC patients. SUBJECTS AND METHODS: Four hundred and twenty-seven NSCLC patients were enrolled. All patients have accepted platinum-based chemotherapy for at least two cycles. ATP7A (rs2227291 and rs6622665) and ATP7B (rs1061472 and rs9535826) polymorphisms were genotyped by allele-specific matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Chemotherapeutic response, overall survival time, and hematological and gastrointestinal toxicity were recorded and their associations with genetic factors were evaluated. RESULTS: ATP7A rs2227291 and rs6622665 deviated from Hardy-Weinberg equilibrium. Therefore, the two single-nucleotide polymorphisms were not taken into consideration. For ATP7B polymorphism, ATP7B rs9535826 was associated with gastrointestinal toxicity, and the GG genotype showed lower gastrointestinal toxicity (odds ratio = 0.30; 95% confidence interval = 0.10-0.90; P = 0.031). CONCLUSION: The genotypes of ATP7B gene may be novel and significant biomarkers for predicting the gastrointestinal toxicity of platinum-based chemotherapy in NSCLC patients.

Gene expression and single nucleotide polymorphism of ATP7B are associated with platinum-based chemotherapy response in non-small cell lung cancer patients.

Objectives: Platinum-based chemotherapy is first-line treatment for non-small cell lung cancer (NSCLC) patients. The efficacy is limited by drug resistance. Recent studies suggest that ATP7B, a copper efflux transporter, may be involved in platinum resistance. However, the clinical significance of ATP7B expression in NSCLC is controversial. Moreover, the effects of single nucleotide polymorphisms (SNPs) in ATP7B gene on the response to platinum-based chemotherapy are scarcely understood. The aim of our study is to evaluate the clinical value of ATP7B in NSCLC patients and explore the interrelationships between ATP7B SNPs and protein expression, and their association with chemotherapy response. Materials and Methods: A total of 247 NSCLC patients were recruited in this study. Among them, 158 patients who received platinum-based chemotherapy were used to explore the interrelationships between ATP7B SNPs, protein expression and chemotherapy response, while 89 patients who underwent surgical resection were used to further investigate the association between ATP7B SNPs and expression level. We genotyped 15 SNPs of ATP7B by Sequenom MassARRAY and determined ATP7B protein levels by immunohistochemistry. Results: Patients with ATP7B-negative tumors had improved chemotherapeutic response (p=0.025) and better overall survival (p=0.044) compared with the patients with ATP7B-positive tumors. The multivariate Cox regression analysis revealed that ATP7B expression was an independent prognostic factor (HR=0.639, 95%CI=0.424-0.962, p=0.032). Moreover, we found that the rs9526814 GG genotype was significantly associated with favorable response to platinum-based chemotherapy when compared with TT+TG genotypes (OR=0.362, 95CI%=0.140-0.935, p=0.036). Mechanistically, rs9526814 GG genotype showed a strong trend towards reduced expression level of ATP7B compared with the TT+TG genotypes (p= 0.048). Conclusion: Our findings indicate that ATP7B rs9526814 may contribute to platinum resistance by influencing ATP7B gene expression and can be used as a potential biomarker to predict the sensitivity of platinum-based chemotherapy in NSCLC patients.

[A study on the association between polymorphism of the interleukin-4 receptor alpha gene and bronchial asthma in a population of Han nationality].

OBJECTIVE: To detect the polymorphism of Ile50Val in the interleukin-4 receptor (IL-4R) alpha gene in a population of Han nationality, and to investigate the association between the polymorphism of Ile50Val and bronchial asthma. METHODS: The Ile50Val polymorphism of the IL-4R alpha gene was genotyped by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) in 103 asthmatic subjects and 62 controls of Han nationality who were from the First Affiliated Hospital of Jinan University in the year 2002 - 2004. RESULTS: The frequencies of the genotypes (Ile50/Ile50, Ile50/Val50, Val50/Val50) were respectively 32.0% (n = 33), 35.0% (n = 36) and 33.0% (n = 34) in the 103 asthmatic subjects and respectively 29.0% (n = 18), 37.1% (n = 23) and 33.9% (n = 21) in the 62 controls. No significant difference was found in the distributions of the genotypes (Ile50/Ile50, Ile50/Val50, Val50/Val50) between the asthmatic subjects and controls (chi(2) = 0.172, P > 0.05). The frequencies of alleles (Ile50, Val50) were respectively 49.5% and 50.5% in the asthmatic subjects and respectively 47.6% and 52.4% in the controls. No significant difference was observed in the allele frequency between the two groups (chi(2) = 0.116, P > 0.05). CONCLUSION: It is suggested that the Ile50Val polymorphism of IL-4R alpha gene exists in this population of Han nationality, but is not associated with bronchial asthma.

Construction of an effective m1 GS ribozyme targeting HCMV UL97 mRNA segment in vitro.

A sequence-specific M1GS ribozyme (M1-T3) was constructed by covalently linking an oligonucleotide (guide sequence,GS) to the 3' terminus of M1 RNA ,the catalytic subunit of RNase P from Escherichia coli. The engineered ribozyme is targeted to the mRNA sequence encoding a protein kinase (UL97) of HCMV and could effectively cleave the mRNA segment in vitro. Further studies about the significance of some structural elements in the M1 GS (e.g. the 3' CCA tail sequence and a bridge sequence between the 3' terminus of M1 RNA and the 5' terminus of the GS) were carried out. The results showed that the bridge sequence of 88 nucleotides in a mutated M1 GS (i.e. M1-T3*) dramatically increased the cleavage activity to the substrate in vitro. Moreover, the 3'CCA tail sequence was confirmed to be a necessary element for the cleavage activity of M1 GS ribozyme. These data we got in the study will help in understanding the interaction between the M1 GS RNA and its substrate,and will markedly facilitate the research of a general gene targeting agent for anti-HCMV applications.

Prognosis of psychomotor and mental development in premature infants by early cranial ultrasound.

BACKGROUND: It is of high incidence of brain injuries in premature infants, so it is necessary to diagnose and treat the brain injury early for neonatal clinical practice. We are aimed to investigate the relationship between early postnatal cranial ultrasonography and psychomotor and mental development in prematrue infants at the age of 12 months. METHODS: Two-hundred and eight premature infants were selected and underwent follow-up from January, 2007 to November, 2012. Cranial ultrasonography was performed on them. The developmental outcomes of these premature infants at the age of 12 months were assessed by the psychomotor developmental index (PDI) scale and mental development index (MDI). The relationship between ultrasonic gray-scale value and PDI and MDI was analyzed. RESULTS: The worse prognosis for psychomotor and mental development was associated with the gestational age, Apgar score(1 min), gender, chorioamnionitis, duration of mechanical ventilation and duration of mechanic ventilation. The differences between the prognosis of psychomotor and mental development, and peri-intraventricular hemorrhage (PIVH) and periventricular white matter damage (PWMD), were statistically significant (P<0.05). There were also significant differences between the early postnatal ultrasonic gray-scale value and prognoses of both psychomotor development and mental development (P<0.05). There were negative correlations between ultrasonic gray-scale and both PDI and MDI (r=-0.753, P<0.05; r=-0.764, P<0.05). CONCLUSIONS: The early postnatal cranial ultrasonography can assist to predict the prognosis of psychomotor and mental development for premature infants. The higher grade of PIVH and PWMD was associated with the worse prognosis of psychomotor and mental development.

[Cloning and expression of a gene encoding shortened cecropin A-melittin hybrid in E.coli].

According to the studies of David Andreu and other scientists, we designed and synthesized a gene encoding shortened cecropin A-melittin hybrid. This hybrid consists of CA(1-7) and M(5-12). The gene was inserted into pGEMEX-1 in the site of EcoRI and BamHI. A positive clone was obtained by hybridization and DNA sequencing and was expressed in JM109(DE3).

[Genetic mutations of homocysteine metabolism related enzymes in patients with ischemic stroke].

To study genetic mutations of methylenetetrahydrofolate reductase (MTHFR) C677T and cystathionine-beta-synthase (CBS) T833C related to homocysteine metabolism in patients with ischemic stroke, the MTHFR gene C677T gene mutation and the CBS T833C gene mutation were detected by PCR-RFLP or ARMS method in 74 patients with ischemic stroke and 83 normal people for control. Results showed that the frequencies of MTHFR T homogenetic type (2.7%) , heterogenetic type (51.4%) and T allele (28.4%) in ischemic group were higher than those in control group (1.2%, 39.8% and 21.1%, respectively). The frequencies of CBS C homogenetic type (13.5%) and C allele (43.9%) in ischemic group were higher than those in control group (6.0% and 38.0%, respectively). Multiple Logistic Regression analysis showed that together with the T allele in MTHFR, the C allele in CBS and age were related to ischemic stroke (P<0.05). The odds ratios (OR) of the T allele in MTHFR C677T and the C allele in CBS T833C were 1.74 (95%CI 1.06-2.86) and 1.73 (95%CI 1.07-2.81) respectively. The study revealed that the genetic mutations of MTHFR C677T, CBS T833C,were related with the ischemic stroke. The genetic mutations of MTHFR C677T and CBS T833C may be genetic factors for ischemic stroke.

[The structure and function analysis of duplicate genes in Merlin strains of human cytomegalovirus].

OBJECTIVE: To determine the genes in which exist overlapping ORF in Merlin strains of human cytomegalovirus, and to reveal their structure and functional characteristics. METHODS: We search for overlapping genes of ORF in HCMV Merlin strains' whole genome by Bioinformatics methods, analyzing coding sequence CDS and starting and ending sites of ORF, calculating the length of CDS and ORF, analyzing the molecular weight of encoding protein, overlapping length and coding direction of protein, identifying overlapping sequences and overlapping types, analyzing the expression phase of overlapping genes and the function of proteins. RESULTS: There were 39 overlapping ORF genes in HCMV Merlin strains, accounting for 23% of total genes. Among these 39 genes, there are 13 IE genes, 9 E genes and 17 L genes, which can be divided into 16 contigs. There are 11 contigs when two genes overlap, with 3 contigs in three genes overlapping, and 2 contigs in four genes overlapping. The functions of overlapping genes are widely. CONCLUSION: We found that there are a lot of complex overlapping genes in HCMV Merlin strains, which are basis for further study of the transcription and translation mechanism of overlapping genes.

[Identification of ubiquitously expressed transcript as the potential interactor of hepatitis B virus polymerase].

OBJECTIVE: To investigate the function of hepatitis B virus polymerase (HBV Pol) in the viral life cycle by screening the proteins interacting with HBV polymerase. METHODS: The HBV Pol gene was constructed into the pGBKT7 vector. GAL4 yeast two-hybrid system was used to screen the human liver cDNA library to obtain proteins which interacted with HBV Pol. GST-pull down assay was applied to confirm the protein interactions. RESULTS: Ubiquitously expressed transcript (UXT) was selected by the yeast two-hybrid system. GST-pull down assay confirmed the in vitro interaction between HBV Pol and UXT. CONCLUSIONS: UXT is a potential interactor of HBV Pol, and this protein interaction may provide clues of the function of HBV Pol in HBV life cycle.