Immunospecific protein of 34.5 kDa from DNA-protein cross-links induced by cis- and trans-diamminedichloroplatinum

Cis-diamminedichloroplatinum(II) (cis-DDP) is one of the most often used anticancer drugs. It is generally accepted that the antitumor activity of the drug results from its interactions with DNA. Trans-diamminedichloroplatinum(II) (trans-DDP) also binds to DNA effectively, but is clinically ineffective. In the present work the lymphocyte nuclear proteins that participate in DNA-protein cross-links induced by cis- and trans-DDP are investigated. In lymphocytes which are incubated without platinum compounds there are DNA-binding proteins in the range of 45-71 kDa. It is shown that additional proteins of 28, 30, 34.5, 45 and 120 kDa are cross-linked with DNA in lymphocytes after 2-h incubation with cis-DDP at concentrations of 0.1 and 0.5 mM. Trans-DDP does not bind additional proteins to DNA after the same incubation time. Electrophoretic analysis shows that trans-DDP binds much more of the same nuclear proteins to DNA than cis-DDP after 12-h incubation. In this study a test for the identification of 34.5 kDa protein is also undertaken. This protein appears in the samples obtained after 12-h incubation of lymphocytes with cis- and trans-DDP at 0.5 and 1 mM, especially. The protein of 34.5 kDa from cross-links induced by 1 mM trans-DDP is recognized by antibodies against the protein of the same molecular weight from the nuclear matrix of the lymphocytes. The results obtained here are discussed in relation to the biological activity of diamminedichloroplatinum isomers.     

Evaluation of the human melanoma targeting properties of radiolabeled alpha-melanocyte stimulating hormone peptide analogues

The purpose of this study was to evaluate the human MC1 receptor-mediated melanoma targeting properties of two metal cyclized alpha-MSH peptide analogues, (188)Re-(Arg(11))CCMSH and (188)Re-CCMSH. Initially, the presence and density of the MC1 receptor were determined on a bank of human melanoma cell lines. All eight human melanoma cell lines tested in this study displayed the MC1 receptor at a density of 900 to 5700 receptors per cell. Receptor affinity and biodistribution properties of (188)Re-(Arg(11))CCMSH and (188)Re-CCMSH were evaluated in a cultured TXM13 human melanoma-xenografted Scid mouse model. Biodistribution results demonstrated that 3.06 +/- 0.68 %ID/g of (188)Re-(Arg(11))CCMSH accumulated in the tumors 1 h postinjection and greater than 65% of the activity at 1 h postinjection remained in the tumors at 4 h after dose administration. Whole body clearance of (188)Re-(Arg(11))CCMSH was very rapid, with approximately 82% of injected dose cleared through urinary system at 4 h postinjection. There was very little activity in blood and major organs such as liver, lung, and muscle except for the kidney. (188)Re-CCMSH exhibited similar tumor uptake and retention in TXM13 human melanoma-xenografted Scid mice as (188)Re-(Arg(11))CCMSH. However, the kidney uptake value of (188)Re-CCMSH was two times higher than that of (188)Re-(Arg(11))CCMSH. The results of this study indicate that the MC1 receptor is present on the surface of a large number of human melanoma cells, which makes the MC1 receptor a good imaging or therapeutic target. Moreover, the biodistribution properties of (188)Re-(Arg(11))CCMSH and (188)Re-CCMSH highlight their potential as therapeutic agents for human melanoma.     

Effects of electron transport inhibitors on iron reduction in Aeromonas hydrophila strain KB1

The aim of this study was to determine the influence of respiratory chain inhibitors upon iron (III) reduction in Aeromonas hydrophila strain KB1. Optimal conditions of the reduction process were established by determining the amount of biomass, optimal pH, temperature and substrate concentration. The obtained results allowed us to determine Hill equation coefficients (K(m)=1.45+/-0.18 mM; V(max)=83.40+/-2.70 microM/min, and h=0.7+/-0.03). The value of h points to Michaelis-like kinetics of the process. The substrate concentration used in our study was such as to allow the maximum iron reduction rate. The reaction was mesophilic. The participation of electron carriers in the iron reduction process was investigated using respiratory chain inhibitors. Rotenone and capsaicin were used to study Q sites of the respiratory chain complex I. Dicumarol was used as an inhibitor of the quinone loop, while quinacrine was used to inhibit alloxazine centers. Additionally, complex III inhibitors, such as antimycin A, myxothiazole and 2-heptyl-4-hydroxy-quinoline N-oxide (HQNO) were used. Azide was used to inhibit complex IV. The observed inhibition of iron reduction by rotenone and capsaicin may suggest the existence of Q sites in formate reductase, analogous to those in complex I. Inhibition of quinones, isoalloxazine centers and complex III suggests participation of these carriers in the electron transport during iron reduction. Lack of inhibition of iron reduction by azide suggests that complex IV does not participate in this process.     

The activity of selected glycosidases in salivary gland tumors

The monitoring of the patients after salivary gland tumors surgery is an important clinical issue. Still imperfect diagnostic procedures also remain a challenge for searching new sensitive and specific biomarkers of neoplastic processes in salivary glands. The aim of the presented study was an the assessment of the activity of HEX, with its isoforms HEX-A and HEX-B, GLU, GAL, MAN and FUC in salivary gland tumor tissues in comparison to a healthy salivary gland tissues taken during autopsy. A group of 42 patients with benign and malignant salivary gland tumors, aged 25-65 were examined. Fragments of salivary gland tumor tissue, fragments of healthy tissue removed during autopsy, blood serum and saliva were collected from patients with salivary gland tumors and healthy volunteers. In salivary gland tumor tissue the activity of HEX, HEX-A, HEX-B, GAL, FUC was considerably higher than in comparison to healthy salivary gland tissue and ascending trend of activity of GLU, MAN was also noticed. The activity of all lysosomal exoglycosidases in blood serum in patients with salivary gland tumors was considerably higher in comparison to healthy volunteers blood serum. The considerably higher activity of HEX, HEX-A, GLU, GAL, MAN, FUC and descending trend of activity of HEX-B were noticed in saliva of patients with salivary gland tumors in comparison to healthy volunteers. The assessment of HEX in blood serum and saliva of patients with salivary gland tumor can be possibly used in diagnostics and monitoring of salivary glands tumors.     

KaKs_Calculator 2.0： A Toolkit Incorporating Gamma-Series Methods and Sliding Window Strategies

We present an integrated stand-alone software package named KaKs_Calculator 2.0 as an updated version.It incorporates 17 methods for the calculation of nonsynonymous and synonymous substitution rates;among them,we added our modified versions of several widely used methods as the gamma series including γ-NG,γ-LWL,γ-MLWL,γ-LPB,γ-MLPB,γ-YN and γ-MYN,which have been demonstrated to perform better under certain conditions than their original forms and are not implemented in the previous version.The package is readily used for the identification of positively selected sites based on a sliding window across the sequences of interests in 5' to 3' direction of protein-coding sequences,and have improved the overall performance on sequence analysis for evolution studies.A toolbox,including C++ and Java source code and executable files on both Windows and Linux platforms together with a user instruction,is downloadable from the website for academic purpose at https://sourceforge.net/projects/kakscalculator2/.     

MKR转基因小鼠糖尿病发病特点的初步探讨

目的观察骨骼肌胰岛素样生长因子-1受体及胰岛素受体的功能缺失(Mouse overexpressing adominant-negative IGF-I receptor specifically in muscle,MKR mouse)转基因2型糖尿病小鼠的血糖及其生长情况。方法由美国国立卫生研究院(NIH)授权使用引进的MKR鼠,经自然交配后繁殖的子代作为研究对象,制备尾组织基因组DNA,经聚合酶链反应得到扩增产物,1%琼脂糖凝胶电泳观察鉴定。同时对MKR鼠子代出生后的血糖、体重、胰岛素定时检测。同期采用昆明种小鼠或C57野生型小鼠进行对照。结果MKR鼠所有子代鼠均会出现长度为330 bp的DNA片段,表明该MKR鼠后代能稳定遗传。新生MKR鼠自出生3周开始,即出现显著的血糖增高,5周以后血糖则较稳定地维持在高水平。与对照组小鼠同期的血糖比较,差异具有非常显著性意义(P<0.01)。其体重增长随年龄的增长而减慢,至4月龄时体重基本稳定,但无下降趋势。MKR鼠在2月龄时即表现出显著的高胰岛素血症及糖耐量低下,与对照组小鼠同期比较,亦具有非常显著性意义(P<0.01)。MKR鼠在生长过程中无自然死亡发生。结论MKR鼠是至今为止一种非常良好的2型糖尿病的动物模型。     

O2-binding albumin thin films: solid membranes of poly(ethylene glycol)-conjugated human serum albumin incorporating iron porphyrin

Poly(ethylene glycol) (PEG)-conjugated human serum albumin (HSA) incorporating the tetrakis(alpha,alpha,alpha,alpha-o-amidophenyl)porphinatoiron(II) derivative (FeP) [PEG(HSA-FeP)] is a unique plasma protein-based O2 carrier as a red blood cell substitute. The aqueous solution of PEG(HSA-FeP) [mw of PEG: 2-kDa (PEG2) or 5-kDa (PEG5)] was evaporated on a glass surface to produce a red-colored solid membrane. Scanning electron microscopy observations revealed that the PEG2(HSA-FeP) membrane consisted of two parts: (i) a surface layer made of a fibrous component (10 microm thickness), and (ii) a bottom layer of an amorphous phase (5 microm thickness). The condensed solution provided a thick membrane (70 microm), which also has the amorphous bottom layer. On the other hand, the PEG5(HSA-FeP) produced homogeneous membrane made of the fibrous component. The FeP active sites in the solid membrane formed very stable O2-adduct complexes at 37 degrees C with a half-lifetime of 40 h. The O2-binding affinity of the PEG2(HSA-FeP) membrane (P1/2 = 40 Torr, 25 degrees C) was 4-fold lower than that in aqueous solution, which is kinetically due to the low association rate constant. The membrane was soluble again in water and organic solvents (ethanol and chloroform) without deformation of the secondary structure of the protein. The addition of hyaluronic acid gave a free-standing flexible thin film, and it can also bind and release O2 as well. These O2-carrying albumin membranes with a micrometer-thickness would be of significant medical importance for a variety of clinical treatments.     

Synthesis and evaluation of novel gonadotropin-releasing hormone receptor-targeting peptides

The purpose of this study was to develop novel radiolabeled gonadotropin-releasing hormone (GnRH) receptor-targeting peptides for breast cancer imaging. Three novel 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated GnRH peptides were designed and synthesized. The radiometal chelator DOTA was conjugated to the epsilon or alpha amino group of D-lysine, or the epsilon amino group of L-lysine via an Ahx {aminohexanoic acid} linker to generate DOTA-Ahx-(D-Lys(6)-GnRH1), DOTA-Ahx-(D-Lys(6)-GnRH2) and DOTA-Ahx-(L-Lys(6)-GnRH3), respectively. The conjugation of the DOTA to the epsilon amino group of D-lysine (rather than alpha amino group of D-lysine nor epsilon amino group of L-lysine) maintained the nanomolar GnRH receptor binding affinity. The IC(50) values of DOTA-Ahx-(D-Lys(6)-GnRH1), DOTA-Ahx-(D-Lys(6)-GnRH2) and DOTA-Ahx-(L-Lys(6)-GnRH3) were 36.1 nM, 10.6 mM and 4.3 mM, respectively. Since only DOTA-Ahx-(D-Lys(6)-GnRH1) displayed nanomolar receptor binding affinity, the specific GnRH receptor binding of (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1) was determined in human GnRH receptor membrane preparations. Furthermore, the biodistribution and tumor imaging properties of (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1) were examined in MDA-MB-231 human breast cancer-xenografted nude mice. (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1) exhibited specific GnRH receptor binding and rapid tumor uptake (1.76 ± 0.58% ID/g at 0.5 h postinjection) coupled with fast whole-body clearance through the urinary system. The MDA-MB-231 human breast cancer-xenografted tumor lesions were clearly visualized by single photon emission computed tomography (SPECT)/CT at 1 h postinjection of (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1). The profound impact of DOTA position on the binding affinity of the GnRH peptide provided a new insight into the design of novel radiolabeled GnRH peptides. The successful imaging of MDA-MB-231 human breast cancer-xenografted tumor lesions using (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1) suggested its potential as a novel imaging probe for human breast cancer imaging.     

Inhibition of histone deacetylases 1 and 6 enhances cytarabine-induced apoptosis in pediatric acute myeloid leukemia cells

Background

Pediatric acute myeloid leukemia (AML) remains a challenging disease to treat even with intensified cytarabine-based chemotherapy. Histone deacetylases (HDACs) have been reported to be promising therapeutic targets for treating AML. However, HDAC family members that are involved in chemotherapy sensitivities remain unknown. In this study, we sought to identify members of the HDAC family that are involved in cytarabine sensitivities, and to select the optimal HDACI that is most efficacious when combined with cytarabine for treating children with AML.

Methodology

Expression profiles of classes I, II, and IV HDACs in 4 pediatric AML cell lines were determined by Western blotting. Inhibition of class I HDACs by different HDACIs was measured post immnunoprecipitation. Individual down-regulation of HDACs in pediatric AML cells was performed with lentiviral shRNA. The effects of cytarabine and HDACIs on apoptosis were determined by flow cytometry analysis.

Results

Treatments with structurally diverse HDACIs and HDAC shRNA knockdown experiments revealed that down-regulation of both HDACs 1 and 6 is critical in enhancing cytarabine-induced apoptosis in pediatric AML, at least partly mediated by Bim. However, down-regulation of HDAC2 may negatively impact cytarabine sensitivities in the disease. At clinically achievable concentrations, HDACIs that simultaneously inhibited both HDACs 1 and 6 showed the best anti-leukemic activities and significantly enhanced cytarabine-induced apoptosis.

Conclusion

Our results further confirm that HDACs are bona fide therapeutic targets for treating pediatric AML and suggest that pan-HDACIs may be more beneficial than isoform-specific drugs.