[Analysis of occupational health status of radiation workers in Guangdong Province].

Objective: To discuss the effect of low-dose ionizing radiation on the health of radiation workers, and provide a basis for occupational health risk assessment of radiation workers. Methods: In January 2020, 3165 radiation workers who performed radiation occupational health examinations in Guangzhou Prevention and Treatment Hospital for Occupational Disease from January 2017 to December 2019 were selected as the research objects, and compared and analyzed the health status of radiation workers with different examination types (pre-job, in-job and off-job) , types of work, gender, and length of service. Results: The off-job occupational radiological health examination was rare at 2.3% (74/3165) . The abnormal detection rate of chest radiographs, renal function, thyroid function, and blood routine of the radiation workers in-job group was higher than that of the pre-job group (P<0.05) . No statistical difference was found in the abnormal detection rate of the examination items during the in-job group and the off-job group (P>0.05) . The blood routine abnormality detection rate of medical application group and industrial application group were higher than those of nuclear fuel group (P<0.05) . The abnormal detection rate of blood pressure and renal function of male radiation workers was higher than that of females, while the abnormal detection rate of blood routine of females was higher than that of males (P<0.05) . The abnormal detection rate of electrocardiogram, chest radiograph, blood pressure, renal function, thyroid function, and blood routine of radiation workers increased with increasing working age (P<0.05) . Conclusion: Occupational health status of radiation workers is not optimistic. Radiation occupational health monitoring should be strengthened, special attention should be paid to off-job radiation occupational health examination, focusing on the sensitive indicators of sensitive personnel, improving radiation protection conditions, and effectively protecting the occupational health of radiation workers.

Prognostic significance of palliative gastrectomy in incurable advanced gastric cancer: a retrospective cohort study and meta-analysis.

OBJECTIVE: There is controversy regarding the role of palliative gastrectomy in patients with incurable advanced gastric cancer requiring surgical intervention. The present retrospective cohort study and meta-analysis aimed to determine whether palliative gastrectomy plus chemotherapy can prolong the survival of patients with incurable advanced gastric cancer requiring surgical intervention. PATIENTS AND METHODS: The data from 153 patients diagnosed with incurable advanced gastric cancer requiring surgical intervention at our institute between January 2000 and December 2012 were retrospectively reviewed. We analyzed the value of palliative gastrectomy and identified the potential prognostic factors. We also conducted a meta-analysis of 10 studies to validate our results. RESULTS: Multivariate analysis indicated that palliative gastrectomy was a favorable independent prognostic factor for prolonged overall survival in incurable advanced gastric cancer patients requiring surgical intervention (p=0.029). The median survival of patients who underwent palliative gastrectomy plus chemotherapy was significantly longer than that of those who underwent non-resection surgery plus chemotherapy (12 months vs. 9 months, p=0.020). The patients in the non-resection surgery plus chemotherapy group exhibited significantly shorter overall survival than those in the D1+ lymphadenectomy group, D2 lymphadenectomy group, or distal gastrectomy group (p=0.021, p=0.007, and p=0.006, respectively). Our meta-analysis revealed that gastrectomy plus chemotherapy improved long-term survival in incurable advanced gastric cancer patients (hazard ratio (HR): 0.48; 95% confidence interval (CI): 0.35-0.67; p<0.001). CONCLUSIONS: Palliative gastrectomy plus chemotherapy may improve overall patient survival compared with non-resection operations plus chemotherapy in incurable advanced gastric cancer patients requiring surgical intervention.

[Effect of low dose ionizing radiation on peripheral blood cells of radiation workers in nuclear power industry].

Objective: To investigate the effects of long-term ionizing radiation on peripheral blood cells of nuclear power workers. Methods: In March 2019, a total of 530 radiation exposed workers in the nuclear power industry who underwent in-service radiation occupational health examination in Guangzhou occupational disease prevention and control hospital in 2018 and with service age ≥1 year were selected as the radiation group. At the same time, 545 workers in nuclear power industry were selected as control group. According to the methods and requirements of GBZ 235-2011 ＂technical specification for occupational health monitoring of radiation workers＂ and GBZ 98-2017 ＂health requirements for radiation workers＂, the occupational health monitoring data were collected, and the change rules of peripheral blood cells in the two groups were analyzed. Results: Compared with the control group, the total number of WBC, NEUT, LYMP, Hb, MCV and MCHC in radiation group were lower than those in control group (P<0.05) , and the difference was statistically significant (P<0.05) . The MPV increased significantly (P<0.05) . Compared with the control group, the abnormal rate of WBC and Hb in the radiation group was higher than that in the control group (P<0.01) , but there was no significant difference in the abnormal rate of RBC and PLT (P>0.05) . Conclusion: Low dose ionizing radiation has a certain cumulative damage effect on peripheral blood cells of radiation workers in nuclear power industry. The change rules of different cell subtypes are different, and the changes of WBC and PLT appear earlier.

let-7i inhibits proliferation and migration of bladder cancer cells by targeting HMGA1.

BACKGROUND: Let-7 is one of the earliest discovered microRNAs(miRNAs) and has been reported to be down-regulated in multiple malignant tumors. The effects and molecular mechanisms of let-7i in bladder cancer are still unclear. This study was to investigate the effects and potential mechanisms of let-7i on bladder cancer cells. METHODS: Total RNA was extracted from bladder cancer cell lines. The expression levels of let-7i and HMGA1 were examined by quantitative real-time PCR. Cell viability was detected using the CCK-8 and colony formation assays, while transwell and wound healing assays were used to evaluate migration ability. Luciferase reporter assay and western blot were used to confirm the target gene of let-7i. RESULTS: Compared with the SV-40 immortalized human uroepithelial cell line (SV-HUC-1), bladder cancer cell lines T24 and 5637 had low levels of let-7i expression, but high levels of high mobility group protein A1 (HMGA1) expression. Transfection of cell lines T24 and 5637 with let-7i mimic suppressed cell proliferation and migration. Luciferase reporter assay confirmed HMGA1 may be one of the target genes of let-7i-5p. Protein and mRNA expression of HMGA1 was significantly downregulated in let-7i mimic transfected cell lines T24 and 5637. CONCLUSIONS: Up-regulation of let-7i suppressed proliferation and migration of the human bladder cancer cell lines T24 and 5637 by targeting HMGA1. These findings suggest that let-7i might be considered as a novel therapeutic target for bladder cancer.

[Risk factors associated with systemic inflammatory response syndrome after flexible ueteroscopic lithotripsy based on enhanced recovery after surgery].

Objective: To investigate the risk factors of systemic inflammatory response syndrome (SIRS) in patients undergoing flexible ureteroscopic lithotripsy based on enhanced recovery after surgery (ERAS). Methods: The clinical data of 243 kidney stone cases who underwent flexible ureteroscopic lithotripsy based on ERAS in the First Affiliated Hospital of Wannan Medical College from January 2016 to December 2017 were analyzed retrospectively. The cases were divided into two groups according to whether they had SIRS after surgery: SIRS group (26 cases) and non-SIRS group (217 cases). The age, gender, laterality of kidney stone, history of previous kidney stone surgery, degree of hydronephrosis, multiple kidney stones, length of operation time, white blood cell count of preoperative urine routine, result of preoperative urine culture, use of preoperative antibiotics, diabetes and other chronic diseases in the groups were collected and analyzed. Results: SIRS occurred in 26 cases in this study, which accounted for 10.7% (26/243). Multivariate analysis found that, moderate and severe hydronephrosis (OR=6.711, P=0.008), stone burden ≥2 cm (OR=10.353, P<0.001), length of operation time ≥ 60 min (OR=5.583, P=0.011), white blood cell count of preoperative urine routine ≥25×10(6)/L (OR=6.195, P=0.005), positive preoperative urine culture (OR=4.216, P=0.011), diabetes and other chronic diseases (OR=4.532, P=0.006) were the independent risk factors for postoperative SIRS (P<0.05). Conclusions: The occurrence of SIRS after flexible ureteroscopic lithotripsy based on ERAS is closely correlated with hydronephrosis, stone burden, length of operation time, white blood cell count of preoperative urine routine, positive preoperative urine culture, diabetes and other chronic diseases.

[Treatment strategies of complex lesions in patients with acute Stanford type A dissection of important branches involvement].

Acute Stanford type A aortic dissection with important branches involved is more complex, could lead to organ malperfusion syndrome even organ failure. The understanding of pathological anatomy, classification, staging, and the pathophysiological change has increasingly mature, but not complete. In addition, the treatment strategy for complex lesions is diversified, some questions may not reach consensus. Fully understanding of the anatomical and pathophysiology is very important for surgeons to choose reasonable treatment strategy. As the rapid development of the basic research, imaging techniques and the concept of surgery procedures, the manage technique of Stanfrod type A dissection and branch vessels at the same time is getting seriously, the related issues also need further discussions.

TGF-ß upregulates the translation of USP15 via the PI3K/AKT pathway to promote p53 stability.

Crosstalk between transforming growth factor beta (TGF-ß) signaling and p53 has a critical role in cancer progression. TGF-ß signals via Smad and non-Smad pathways. Under normal conditions, wild-type p53 forms a complex with Smad2/3 and co-activates transcription of a variety of tumor suppressor genes, resulting in tumor suppressive effects. Thus, p53 stability is essential in progression of tumor suppressive responses mediated by TGF-ß signaling. However, it remains unknown whether p53 stability is regulated by TGF-ß. In the current study, we identify that USP15 binds to and stabilizes p53 through deubiquitination in U2OS and HEK293 cells. TGF-ß promotes the translation of USP15 through activation of mammalian target of rapamycin by the phosphoinositide 3-kinase/AKT pathway. Upregulation of USP15 translation links the crosstalk between TGF-ß signaling and p53 stability, allowing this cytokine to have a critical role in cancer progression.

EB-virus latent membrane protein 1 potentiates the stemness of nasopharyngeal carcinoma via preferential activation of PI3K/AKT pathway by a positive feedback loop.

Our previous study reported that Epstein-Barr virus(EBV)-encoded latent membrane protein 1 (LMP1) could induce development of CD44(+/High) stem-like cells in nasopharyngeal carcinoma (NPC). However, the molecular mechanisms that underlie modulation of cancer stem cells (CSCs) in NPC remain unclear. Here, we show that LMP1 induced CSC-like properties through promotion of the expression of epithelial-mesenchymal transition-like cellular markers and through alterations in differentiation markers. Furthermore, LMP1 activated and triggered phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway, which subsequently stimulated expression of CSC markers, development of side population and tumor sphere formation. This suggests that PI3K/AKT pathway has an important role in the induction and maintenance of CSC properties in NPC. Similarly, PI3K/AKT pathway was also activated by phosphorylase in LMP1-induced CD44(+/High) cells. In addition, LMP1 greatly increased expression of miR-21 and downregulated expression of the miR-21 target, PTEN. Overexpression of miR-21 by transfection of miR-21 mimics into LMP1-transformed cells led to phosphorylase-mediated activation of the PI3K/AKT pathway and induction of CSCs. On the contrary, phosphorylation of the PI3K/AKT pathway and the expression of CSC were reversed by an miR-21 inhibitor. The specific inhibitor (Ly294002) of PI3K/AKT pathway significantly decreased expression of miR-21 and CSC markers and upregulated the expression of PTEN, which indicates that miR-21 and PTEN are the downstream effectors of PI3K/AKT and that expression of these two effectors are related to the development of NPC CSCs. Taken together, our novel findings indicate that LMP1, PI3K/AKT, miR-21 and PTEN constitute a positive feedback loop and have a key role in LMP1-induced CSCs in NPC.

The activation of G protein-coupled receptor 30 (GPR30) inhibits proliferation of estrogen receptor-negative breast cancer cells in vitro and in vivo.

There is an urgent clinical need for safe and effective treatment agents and therapy targets for estrogen receptor negative (ER-) breast cancer. G protein-coupled receptor 30 (GPR30), which mediates non-genomic signaling of estrogen to regulate cell growth, is highly expressed in ER--breast cancer cells. We here showed that activation of GPR30 by the receptor-specific agonist G-1 inhibited the growth of ER--breast cancer cells in vitro. Treatment of ER--breast cancer cells with G-1 resulted in G2/M-phase arrest, downregulation of G2-checkpoint regulator cyclin B, and induction of mitochondrial-related apoptosis. The G-1 treatment increased expression of p53 and its phosphorylation levels at Serine 15, promoted its nuclear translocation, and inhibited its ubiquitylation, which mediated the growth arrest effects on cell proliferation. Further, the G-1 induced sustained activation and nuclear translocation of ERK1/2, which was mediated by GPR30/epidermal growth factor receptor (EGFR) signals, also mediated its inhibition effects of G-1. With extensive use of siRNA-knockdown experiments and inhibitors, we found that upregulation of p21 by the cross-talk of GPR30/EGFR and p53 was also involved in G-1-induced cell growth arrest. In vivo experiments showed that G-1 treatment significantly suppressed the growth of SkBr3 xenograft tumors and increased the survival rate, associated with proliferation suppression and upregulation of p53, p21 while downregulation of cyclin B. The discovery of multiple signal pathways mediated the suppression effects of G-1 makes it a promising candidate drug and lays the foundation for future development of GPR30-based therapies for ER- breast cancer treatment.

Combination of nifedipine and subtherapeutic dose of cyclosporin additively suppresses mononuclear cells activation of patients with rheumatoid arthritis and normal individuals via Ca(2+) -calcineurin-nuclear factor of activated T cells pathway.

Abnormal Ca(2+) -mediated signalling contributes to the pathogenesis of rheumatoid arthritis (RA). However, the potential implication of calcium channel blocker in RA remained unknown. We hypothesized that nifedipine, an L-type calcium channel blocker, combined with a calcineurin inhibitor, could suppress T cell activation via targeting different level of the Ca(2+) signalling pathway. The percentage of activated T cells and the apoptotic rate of mononuclear cells (MNCs) was measured by flow cytometry. The MNC viability, cytokine production, cytosolic Ca(2+) level and activity of the nuclear factor of activated T cells (NFAT) were measured by enzyme-linked immunosorbent assay (ELISA). The NFAT-regulated gene expression, including interleukin (IL)-2, interferon (IFN)-γ and granulocyte-macrophage colony-stimulating factor (GM-CSF), was measured by real-time polymerase chain reaction (PCR). We found that the percentage of activated T cells in anti-CD3 + anti-CD28-activated MNC was higher in RA patients. High doses of nifedipine (50 µM) increased MNCs apoptosis, inhibited T cell activation and decreased T helper type 2 (Th1) (IFN-γ)/Th2 (IL-10) cytokine production in both groups. The Ca(2+) influx was lower in anti-CD3 + anti-CD28-activated MNC from RA patients than healthy volunteers and suppressed by nifedipine. When combined with a subtherapeutic dose (50 ng/ml) of cyclosporin, 1 µM nifedipine suppressed the percentage of activated T cells in both groups. Moreover, this combination suppressed more IFN-γ secretion and NFAT-regulated gene (GM-CSF and IFN-γ) expression in RA-MNCs than normal MNCs via decreasing the activity of NFATc1. In conclusion, we found that L-type Ca(2+) channel blockers and subtherapeutic doses of cyclosporin act additively to suppress the Ca(2+) -calcineurin-NFAT signalling pathway, leading to inhibition of T cell activity. We propose that this combination may become a potential treatment of RA.

Piezoelectric driven non-toxic injector for automated cell manipulation.

Stimulated by state-of-the-art robotic and computer technology, Intra Cytoplasmic Sperm Injection (ICSI) automation aims to scale and seamlessly transfer the human hand movements into more precise and fast movements of the micro manipulator. Piezo-drill cell injection, a novel technique using piezo-driven pipettes with a very small mercury column, has significantly improves the survival rates of ICSI process. It is found that complications are due, in large part, to toxicity of mercury and the damage to the cell membrane because of the lateral tip oscillations of injector pipette. In this paper, a new design of piezo-driven cell injector is proposed for automated suspended cell injection. This new piezo-driven cell injector design centralizes the piezo oscillation power on the injector pipette which eliminates the vibration effect on other parts of the micromanipulator. Detrimental lateral tip oscillations of the injector pipette are attenuated to a desirable level even without the help of mercury column. This mercury-free injector can sublime the piezoelectric driven injection technique to completely non-toxic level with great research and commercial application in gene injection, in-vitro fertilization, ICSI and drug development.

Retrospective analysis of thyroid nodules by clinical and pathological characteristics, and ultrasonographically detected calcification correlated to thyroid carcinoma in South China.

OBJECTIVE: To investigate the clinical and pathological characteristics of thyroid nodules, as well as to evaluate the significance of ultrasonographically detected thyroid calcification in the diagnosis of thyroid carcinomas. METHODS: Retrospective data were studied from 1,051 consecutive patients who underwent a thyroidectomy in the Provincial Hospital of Fujian Medical University in South China between January 2003 and July 2006 for nodular thyroid disease. Complete sonographical information before surgery was only collected from 758 of the 1,051 patients. RESULTS: Among the 1,051 patients, benign lesions were found in 857 (81.54%) patients, of whom 612 (71.41%) were nodular goiter; malignant lesions were found in 194 (18.46%) patients, in whom benign thyroid lesions were also found in 85 (43.81%) patients. A total of 48 patients suffered from microcarcinomas, of whom 37 patients had benign lesions; these 37 accounted for 43.53 and 77.08%, respectively, of the 85 malignant cases with benign lesions and the 48 cases with microcarcinomas. In the 758 patients who underwent thyroid ultrasonography before surgery, intrathyroidal calcifications were apparent in 243 patients (32.06%). The incidence of calcification was significantly higher in patients with thyroid carcinoma (54.17%) than in those with benign lesions (26.87%; p < 0.005). Detection of calcification in thyroid lesions by ultrasound had a sensitivity of 32.38% and a specificity of 87.35%, with an OR of 3.31 (95% CI, 2.24-4.63), positive likelihood ratio of 2.56, negative likelihood ratio of 0.77 and a kappa value of 0.23. CONCLUSION: Thyroid carcinoma, especially microcarcinoma, often coexists with benign thyroid disease. Calcification detected by thyroid ultrasound represents a risk factor for malignancy, but is of limited use as a sole marker of malignancy.

The correlation between neurotoxicity, aggregative ability and secondary structure studied by sequence truncated Abeta peptides.

Aggregated beta-amyloid (Abeta) peptides are neurotoxic and cause neuronal death both in vitro and in vivo. Although the formation of a beta-sheet structure is usual required to form aggregates, the relationship between neurotoxicity and the Abeta sequence remains unclear. To explore the correlation between Abeta sequence, secondary structure, aggregative ability, and neurotoxicity, we utilized both full-length and fragment-truncated Abeta peptides. Using a combination of spectroscopic and cellular techniques, we demonstrated that neurotoxicity and aggregative ability are correlated while the relationship between these characteristics and secondary structure is not significant. The hydrophobic C-terminus, particularly the amino acids of 17-21, 25-35, and 41-42, is the main region responsible for neurotoxicity and aggregation. Deleting residues 17-21, 25-35 or 41-42 significantly reduced the toxicity. On the other hand, truncation of the peptides at either residues 22-24 or residues 36-40 had little effect on toxicity and aggregative ability. While the N-terminal residues 1-16 may not play a major role in neurotoxicity and aggregation, a lack of N-terminal fragment Abeta peptide, (e.g. Abeta17-35), does not display the neurotoxicity of either full-length or 17-21, 25-35 truncated Abeta peptides.

Purification and characterization of a chymotrypsin inhibitor from the venom of Ophiophagus hannah (King Cobra).

A chymotrypsin inhibitor from the venom of Ophiophagus hannah was isolated by a combination of ion-exchange chromatography and reverse phase HPLC. Amino acid sequence analysis revealed that this protein consists of 58 amino acids, six of these being cysteine residues and is highly homologous to Kunitz-type protease inhibitors. ESI-mass spectrum showed that the protein had a mass of 6493, which is in agreement with that predicted from its primary structure. In contrast to P1 Leu, Met, Phe, Trp, and Tyr appearing in other chymotrypsin inhibitors, a P1 Asn in the novel inhibitor may cause a weak binding (Ki = 3.52 microM) with chymotrypsin. Phylogenetic analysis suggests that the functional variations of the chymotrypsin inhibitor and other Kunitz-type inhibitors probably distinguish from dendrotoxins by accelerated evolution.

Protein phosphatase 1 regulation by inhibitors and targeting subunits.

Regulation of protein phosphatase 1 (PP1) by protein inhibitors and targeting subunits has been previously studied through the use of recombinant protein expressed in Escherichia coli. This preparation is limited by several key differences in its properties compared with native PP1. In the present study, we have analyzed recombinant PP1 expressed in Sf9 insect cells using baculovirus. Sf9 PP1 exhibited properties identical to those of native PP1, with respect to regulation by metals, inhibitor proteins, and targeting subunits, and failure to dephosphorylate a phosphotyrosine-containing substrate or phospho-DARPP-32 (Dopamine and cAMP-regulated phosphoprotein, M(r) 32,000). Mutations at Y272 in the beta12/beta13 loop resulted in a loss of activity and reduced the sensitivity to thiophospho-DARPP-32 and inhibitor-2. Mutations of Y272 also increased the relative activity toward a phosphotyrosine-containing substrate or phospho-DARPP-32. Mutation of acidic groove residues caused no change in sensitivity to thiophospho-DARPP-32 or inhibitor-2, but one mutant (E252A:D253A:E256R) exhibited an increased K(m) for phosphorylase a. Several PP1/PP2A chimeras were prepared in which C-terminal sequences of PP2A were substituted into PP1. Replacement of residues 274-330 of PP1 with the corresponding region of PP2A resulted in a large loss of sensitivity to thiophospho-DARPP-32 and inhibitor-2, and also resulted in a loss of interaction with the targeting subunits, spinophilin and PP1 nuclear targeting subunit (PNUTS). More limited alterations in residues in beta12, beta13, and beta14 strands highlighted a key role for M290 and C291 in the interaction of PP1 with thiophospho-DARPP-32, but not inhibitor-2.

Characterization of the inhibition of protein phosphatase-1 by DARPP-32 and inhibitor-2.

Phospho-DARPP-32 (where DARPP-32 is dopamine- and cAMP-regulated phosphoprotein, Mr 32,000), its homolog, phospho-inhibitor-1, and inhibitor-2 are potent inhibitors (IC50 approximately 1 nM) of the catalytic subunit of protein phosphatase-1 (PP1). Our previous studies have indicated that a region encompassing residues 6-11 (RKKIQF) and phospho-Thr-34, of phospho-DARPP-32, interacts with PP1. However, little is known about specific regions of inhibitor-2 that interact with PP1. We have now characterized in detail the interaction of phospho-DARPP-32 and inhibitor-2 with PP1. Mutagenesis studies indicate that within DARPP-32 Phe-11 and Ile-9 play critical roles, with Lys-7 playing a lesser role in inhibition of PP1. Pro-33 and Pro-35 are also important, as is the number of amino acids between residues 7 and 11 and phospho-Thr-34. For inhibitor-2, deletion of amino acids 1-8 (I2-(9-204)) or 100-204 (I2-(1-99)) had little effect on the ability of the mutant proteins to inhibit PP1. Further deletion of residues 9-13 (I2-(14-204)) resulted in a large decrease in inhibitory potency (IC50 approximately 800 nM), whereas further COOH-terminal deletion (I2-(1-84)) caused a moderate decrease in inhibitory potency (IC50 approximately 10 nM). Within residues 9-13 (PIKGI), mutagenesis indicated that Ile-10, Lys-11, and Ile-13 play critical roles. The peptide I2-(6-20) antagonized the inhibition of PP-1 by inhibitor-2 but had no effect on inhibition by phospho-DARPP-32. In contrast, the peptide D32-(6-38) antagonized the inhibition of PP1 by phospho-DARPP-32, inhibitor-2, and I2-(1-120) but not I2-(85-204). These results indicate that distinct amino acid motifs contained within the NH2 termini of phospho-DARPP-32 (KKIQF, where italics indicate important residues) and inhibitor-2 (IKGI) are critical for inhibition of PP1. Moreover, residues 14-84 of inhibitor-2 and residues 6-38 of phospho-DARPP-32 share elements that are important for interaction with PP1.

Circumvention of tumor multidrug resistance by a new annonaceous acetogenin: atemoyacin-B.

AIM: To explore the effect of atemoyacin-B (Ate) on overcoming multidrug resistance (MDR). METHODS: Bullatacin (Bul) was used as a positive control. Cytotoxic effects of Bul and Ate were studied with cell culture of human MDR breast adenocarcinoma cells, MCF-7/Dox and human KBv200 cells, and their parental sensitive cell lines MCF-7 and KB. Cytotoxicity was determined by tetrazolium (MTT) assay. The function of P-glycoprotein (P-gp) was examined by Fura 2-AM assay. Cellular accumulation of doxorubicin (Dox) was determined by fluorescence spectrophotometry. Apoptosis was measured by flow cytometry. RESULTS: IC50 of Ate for MCF-7/Dox, MCF-7, KBv200, and KB cells were 122, 120, 1.34, and 1.27 nmol.L-1, respectively. IC50 of Bul for MCF-7/Dox, MCF-7, KBv200, and KB cells were 0.60, 0.59, 0.04, and 0.04 nmol.L-1, respectively. The cytotoxicities of Bul and Ate to MDR cells were similar to those to parental sensitive cells. Bul and Ate markedly increased cellular Fura-2 and Dox accumulation in MCF-7/Dox cells, but not in MCF-7 cells. The rates of apoptosis in MDR cells were similar to those in sensitive cells induced by Ate. CONCLUSION: There was no cross-resistance of P-gp positive MCF-7/Dox and KBv200 cell lines to Bul and Ate as compared with their sensitive P-gp negative MCF-7 and KB cell lines. The mechanism of the circumvention of MDR was associated with the decrease of P-gp function and the increase of cellular drug accumulation in MDR cells.

Characterization of the interaction between DARPP-32 and protein phosphatase 1 (PP-1): DARPP-32 peptides antagonize the interaction of PP-1 with binding proteins.

The catalytic subunit of PP-1 (PP-1C) is potently inhibited (IC50, approximately 1 nM) by DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, M(r) 32,000), inhibitor-1, and inhibitor-2. The NH2-terminal 50 amino acid residues of DARPP-32 and inhibitor-1 are similar, and phosphorylation of a common threonine residue (Thr-34/Thr-35) is necessary for inhibition of PP-1C. We have characterized further the interaction between DARPP-32 and PP-1C. Using synthetic peptides derived from the NH2-terminal region of DARPP-32, residues 6-11, RKKIQF, have been shown to be required for inhibition of PP-1C. Peptides containing this motif were able to antagonize the inhibition of PP-1C by phospho-DARPP-32 and phosphoinhibitor-1. The inhibition of PP-1C by inhibitor-2, but not by okadaic acid, microcystin, or calyculin A, was also attentuated by these antagonist peptides. These results together with results from other studies support a model in which two subdomains of phospho-DARPP-32 interact with PP-1C. The region encompassing phospho-Thr-34 appears to interact with the active site of the enzyme blocking enzyme activity. The region encompassing the RKKIQF motif binds to a domain of PP-1C removed from the active site. Amino acid sequence analysis indicates that basic and hydrophobic features of the RKKIQF motif are conserved in the binding domains of certain PP-1C targeting proteins, suggesting that interaction of inhibitor proteins and targeting proteins may be mutually exclusive.

Three-dimensional structure of the catalytic subunit of protein serine/threonine phosphatase-1.

The crystal structure of mammalian protein phosphatase-1, complexed with the toxin microcystin and determined at 2.1 A resolution, reveals that it is a metalloenzyme unrelated in architecture to the tyrosine phosphatases. Two metal ions are positioned by a central beta-alpha-beta-alpha-beta scaffold at the active site, from which emanate three surface grooves that are potential binding sites for substrates and inhibitors. The carboxy terminus is positioned at the end of one of the grooves such that regulatory sequences following the domain might modulate function. The fold of the catalytic domain is expected to be closely preserved in protein phosphatases 2A and 2B (calcineurin).

Mechanism of inhibition of protein phosphatase 1 by DARPP-32: studies with recombinant DARPP-32 and synthetic peptides.

The mechanism of inhibition of protein phosphatase-1 catalytic subunit (PP-1c) by recombinant DARPP-32 and synthetic peptides was studied. DARPP-32 was expressed in Escherichia coli as a non-fusion protein using a pEt-3a plasmid, purified to homogeneity and shown to have physicochemical properties similar to those of the protein purified from bovine brain. Recombinant DARPP-32 phosphorylated on threonine-34 by cAMP-dependent protein kinase inhibited PP-1c with an IC50 approximately 0.5 nM, comparable to that obtained with bovine DARPP-32. Non-phosphorylated DARPP-32, and mutated forms in which threonine-34 was replaced by an alanine or a glutamic acid, inhibited PP-1c with an IC50 approximately 1 microM. Surface plasmon resonance analysis showed binding of PP-1c to nonphospho- and phospho-DARPP-32-(8-38) synthetic peptides with apparent Kd values of 1.2 and 0.3 microM, respectively, supporting the existence of an interaction between non-phosphorylated DARPP-32 and PP-1c that is increased by phosphorylation of DARPP-32 at threonine-34. These results suggest a model in which DARPP-32 interacts with PP-1c by at least two low affinity sites, the combination of which is responsible for the high affinity (nM) inhibition.