Efficacy and safety of iodine-125 particle implantation for treatment of bone metastatic tumor pain: a retrospective analysis.

OBJECTIVE: Patients with advanced tumors often suffer from spinal metastatic tumor pain. The current drugs are less effective and have side effects. The objective was to explore the efficacy of iodine-125 particle implantation in the treatment of bone metastatic tumor pain. PATIENTS AND METHODS: In a retrospective study, a total of 27 patients with bone metastatic tumors who could not receive surgery or radiotherapy and chemotherapy were analyzed. All patients received conventional treatment, with the visual analog scale (VAS) of >3 points, and the daily onset pain of >3 times. All patients received CT-guided iodine-125 particle implantation to treat local painful lesions. VAS scores were recorded before treatment (T0) and 1 day (T1), 7 days (T2), 30 days (T3), 90 days (T4), and 180 days (T5) after treatment. Kaplan-Meier analytical method was used to calculate the local control rate (LCR) and survival rate (SR). RESULTS: All patients successfully completed the CT-guided iodine-125 particle implantation. There was no significant difference in VAS scores before and 1 day after surgery. However, compared with pre-operation, the VAS scores decreased at 7, 30, 90, and 180 days after surgery. The postoperative follow-up was 6-38 months, with a median of 16 months; the LCR at 1, 2, and 3 years after the follow-up were 87%, 51%, and 21%, respectively, and the SR was 84%, 43%, and 16%, respectively. Moreover, no serious adverse reactions were observed. CONCLUSIONS: Iodine-125 particle implantation was effective in the treatment of bone metastatic tumor pain without serious complications, and hence, can be used clinically.

[T cell immune profiling of systemic light chain amyloidosis patients].

Objective: To investigate the characteristics of T cell immunophenotype and its relationship with clinical manifestation in patients with systemic light chain amyloidosis (AL) . Methods: The peripheral blood mononuclear cells from 36 patients with AL were collected and analyzed by multicolor flow cytometry, and the expression of surface antigen CD3, CD56, CD4, CD8, CD25, CD45RA, CD28, CD57 and nuclear antigen FOXP3 were examined. Samples from 28 age-matched healthy donors (HD) were also examined. Patients were divided by Mayo 2012 staging system and the difference between immunophenotype of Ⅰ-Ⅱ and Ⅲ-Ⅳ stage patients were analyzed. The correlations between the proportion of T-cell subpopulation and clinical manifestations in λ light chain type AL patients were analyzed. Results: The differences in the peripheral total T cells and T cell subsets, including CD4(+), CD8(+), regulatory T cells, and natural killer T cells were not significantly between AL and HD. The ratio of CD57(+) cells in CD8(+) T cells was lower in AL than in HD, and there was no significantly difference in the rate of CD45RA(+) and CD28(+)cells between these two groups. No differences were found in the ratio of total T cells or T cell subsets between stages Ⅰ-Ⅱ and Ⅲ-Ⅳaccording to the standard of Mayo 2012. Within λ light chain type AL patients, peripheral CD8(+) T cell ratio was positively correlated with 24-hour urine protein and creatinine level and negatively correlated with estimated glomerular filtration rate (eGFR) . Conclusion: The overall T cell distribution in the periphery is not significantly different between AL patients and age-matched healthy donors. However, the percentages of CD8(+) T cells are positively correlated with renal injury, indicating the importance of CD8(+) T cell subset in the prognostic evaluation of renal involvement.

[Correlation between chest CT features and clinical characteristics of patients with bronchiectasis].

Objective: To analyze the features of chest CT imaging in adult patients with bronchiectasis and explore its correlation with clinical characteristics. Methods: From January 2010 to December 2017, patients with bronchiectasis diagnosed by chest high-resolution CT (HRCT) and aged at or above 18 years old in 5 general hospitals of Shandong province were included in the study. The correlations between the HRCT imaging features and etiology, clinical manifestations, lung function, sputum culture, prognosis and other characteristics were analyzed. Results: There were 410 bronchiectasis patients included in the study. The chest HRCT imaging of bronchiectasis were divided into three types, including columnar 46.8%, cystic 45.9% and varicose 7.3%, respectively. The HRCT imaging score was [6.0 (4.0, 7.0)]. In addition, the most common etiology of bronchiectasis was idiopathic (262, 69.3%). The proportion of idiopathic bronchiectasis in cystic bronchiectasis patients was significantly higher than that in columnar and varicose bronchiectasis (71.8% vs 58.3%, 50.0%; both P<0.017). Compared with columnar bronchiectasis, patients with cystic bronchiectasis were more likely to suffer from clinical manifestations such as cough, dyspnea, fever and wet rales (P<0.017). Compared with patients with HRCT scores of 1 to 4, patients with scores ≥8 were more likely to suffer from cough, dyspnea, fever, wet rales and clubbing (P<0.017). The proportions of pulmonary ventilatory dysfunction were significantly greater in patients with cystic bronchiectasis and varicose bronchiectasis than columnar bronchiectasis (86.7%, 86.7% vs 51.0%; both P<0.017). The HRCT scores were significantly negatively correlated with pulmonary function (P<0.001). The number of acute exacerbations, hospitalizations, and bronchiectasis severe index scores in patients with cystic bronchiectasis were significantly higher than those with columnar bronchiectasis (P<0.017). There was a significantly positive correlation between HRCT scores and the number of acute exacerbations, hospitalizations and the bronchiectasis severity index scores (P<0.001). The mortality of patients with cystic and varicose bronchiectasis was significantly higher than that of patients with columnar bronchiectasis (9.0%, 10.0% vs 2.1%; both P<0.017). Compared with patients with HRCT scores of 1 to 4, patients with scores ≥8 had a higher mortality rate (15.9% vs 0.9%; P<0.017). Conclusions: There is a correlation between HRCT findings and clinical manifestations in patients with bronchiectasis. The clinical manifestations, lung function and prognosis of patients with cystic bronchiectasis are worse than those of the columnar bronchiectasis; the higher the HRCT scores are, the worse the clinical manifestations, lung function and prognosis of the patients are.

[Effect of skin soft tissue expansion on repair of large area of scars on extremities].

Objective: To investigate the effect of skin soft tissue expansion on repair of large area of scars on extremities. Methods: Twenty-five patients with large area of scars on extremities were admitted to our department from June 2007 to October 2014. There were 14 males and 11 females, aged 4 to 36 years. Operations were performed under local infiltration anesthesia or general anesthesia. In the first stage, 1 to 5 cylindrical expanders with capacities of 250 to 600 mL were placed at left or right sides or at upper or lower parts of the scars. In the second stage, scars of 21 patients were repaired with expanded transverse propulsive and lateral flaps, and scars of 4 patients were repaired with expanded perforator flaps whose pedicles were perforators of brachial artery, superior ulnar collateral artery, or posterior interosseous artery according to areas and shapes of the scars. The secondary wound areas ranged from 13 cm×7 cm to 34 cm×18 cm after dissolution or excision of scars. The areas of flaps ranged from 13 cm×7 cm to 20 cm×12 cm. The donor sites were sutured directly. The flaps after operation and follow-up of patients were observed and recorded. Results: All expanded flaps survived after operation. And the superficial distal part of flap whose pedicle was perforator of posterior interosseous artery in one patient was with necrosis, and other flaps survived well. During follow-up of 3 to 15 months after operation of the second stage, color and texture of flaps were similar to surrounding skin, while extremities of donor sites were thinner and auxiliary incisional scars formed after expansion. Conclusions: Expanded flap is a good way to repair large area of scar on extremities. Bilateral skin of scar is the first choice of donor site of expanded flap. If there isn't enough skin for expanding on bilateral sides, expanded perforator flap designed at upper or lower part of the scar is another choice to repair the scar.

[Study on analgesic effect and mechanism of cinobufagin on rats with bone cancer pain].

Objective: To evaluate the analgesic effects of cinobufagin (CBG) on cancer-induced bone pain in rat and study the role of the muscarinic receptor M4 subtype (M4 mAChR) in its involvement. Methods: A total of 100 Female Sprague-Dawley rats were randomly divided into 5 groups (n=20): Sham group (group S), Cancer group (group A), Normal saline + CBG vehicle solution group (group ANS), Normal saline + 1 mg/kg CBG group (group ANC) and Tropicamide + 1 mg/kg CBG group (group ATC). Rats in group S were injected 10 µl Hank's solution into the left tibia medullar cavity, while rats in group A, ANS, ANC, and ATC were injected Walker 256 mammary cancer cells (10 µl, 2×10(7) cells/ml) into the same place. On day 9 post-inoculation rats in group ANS, ANC, and ATC were respectively received Saline (0.9%, 15 µl, i.t.), Saline (0.9%, 15 µl, i.t.)and 10 nmol of M4 mAChR blocker Tropicamide. After 10 min, ANS group, ANC group and ATC group were intraperitoneally injected with CBG vehicle solution, 1 mg/kg CBG and 1 mg/kg CBG. Model rats in each group were tested three times average as its basis pain threshold before injection cancer cells (T(0)). Mechanical withdrawal thresholds were measured on left hind paws, before 20 min (T(1)) and after 10 min (T(2)), 30 min (T(3)), 60 min (T(4)), 90 min (T(5)) and 120 min (T(6)) intrathecal injection. Left L4-L6 spinal dorsal horn and DRG were removed for determination of the expression of CaM-dependent kinaseⅡa (CaMKⅡa) and pCaMKⅡa by Western Blot after 60 min drug delivery. Results: At each time point from T(1) to T(6), the mechanical pain thresholds of group S were (8.69±0.45), (8.63±0.44), (8.65±0.39), (8.84±0.23), (8.80±0.14), (8.75±0.14) g, respectively, and the mechanical pain thresholds of group A were (6.37±0.30), (6.42±0.13), (6.29±0.17), (6.25±0.22), (6.34±0.33), (6.36±0.34) g, the difference was statistically significant (t=-16.41, -23.47, -30.25, -17.35, -19.52, -22.56, all P<0.01). At each time point from T(3) to T(5), the mechanical pain thresholds of the ANS group were (6.42±0.32), (6.39±0.34), (6.26±0.32) g, respectively, and the mechanical pain thresholds of the ANC group were (7.29±0.34), (7.81±0.15), (7.54±0.19) g, the difference was statistically significant (t=13.52, 14.22, 17.33, all P<0.01). At each time point from T(3) to T(5), compared with the ANC group, the mechanical pain threshold of the ATC group decreased (6.55±0.23), (6.84±0.46), (6.80±0.43) g, and the difference was statistically significant (t=-12.69, -11.26, -10.33, all P<0.01). At the time of T(4), the expressions of pCaMKⅡa in the spinal dorsal horn of each group were (0.67±0.05), (1.64±0.12), (1.57±0.14), (0.78±0.09), (1.39±0.11), respectively, and the expressions of pCaMKⅡa in DRG of each group were (1.65±0.39), (3.59±0.17), (3.43±0.32), (2.17±0.34), (2.95±0.23). The differences were statistically significant (F=179.89, 198.76, both P<0.01). Compared with the S group, the expression of pCaMⅡa was up-regulated in group A. Compared with ANS group, the expression of pCaMKⅡ a was down-regulated in ANC group. Compared with ANC group, the expression of pCaMK Ⅱ a was up-regulated in ATC group. The expression of CaMKⅡa in spinal dorsal horn and DRG was not statistically significant (F=1.25, 2.79, both P>0.05). Conclusions: These results demonstrated that M4mAChR participated in mediating the alleviation of hyperalgesia by cinobufagin in rats with bone cancer pain, and its mechanism may be related to pCaMKⅡa/CaMKⅡa signaling pathway.

[Expression and distribution of programmed death receptor 1 and T cell immunoglobulin mucin 3 in breast cancer microenvironment and its relationship with clinicopathological features].

Objective: To explore the expression and distribution of programmed death receptor 1 (PD-1) and T-cell immunoglobulin mucin 3 (TIM-3) in breast cancer microenvironment and analyze the their correlation with the clinicopathological features. Methods: The specimens of tumor tissue and adjacent tissues from 30 patients with infiltrative breast cancer who were diagnosed as breast cancer from June 2016 to May 2017 in The First Hospital of Jiaxing were collected, and the specimen were divided into two parts along the center. After embedding and cryosectioning, the expression and distribution of PD-1 and TIM-3 protein in tumor tissues were observed by immunofluorescence staining. Another part of the specimen was cut and digested, and non-continuous density gradient centrifugation was used to extract tumor-infiltrating lymphocytes (TILs), real-time quantitative PCR (qRT-PCR) was used to detect the mRNA expression of PD-1 and TIM-3 in TILs. Meanwhile, the protein expression was determined by Western blotting. The relationship between the expression of PD-1 and TIM-3 and pathological parameters of breast cancer was analyzed with correlation analysis. Results: Immunofluorescence results showed that more PD-1 and TIM-3 positive cells were observed in the tumor tissues compared with the tumor-adjacent tissues. The qRT-PCR showed that the expression of PD-1 and TIM-3 mRNA in TILs were both significantly higher than those in paracancerous tissues (3.09±0.38 vs 1.26±0.23, 3.42±0.31 vs 1.57±0.29, t=4.16, 4.37, both P<0.05). At the protein level, the expression of PD-1 and TIM-3 in tumor tissue lymphocytes(0.66±0.08, 0.80±0.11) was significantly higher than those in cancerous tissues(0.10±0.01, 0.26±0.02) (t=6.79, 4.57, both P<0.05). There were significant differences in the expression of PD-1, TIM-3 mRNA in the TILs between the different tumor histological grades, tumor sizes, lymph node metastasis (t=2.22-2.99, all P<0.05). Correlation analysis showed that there was a significant positive correlation between the expression of PD-1 and TIM-3 in tumor tissues (r=0.616, P<0.01). Conclusions: In the breast cancer microenvironment, PD-1, TIM-3-mediated signaling pathway plays an important role in the occurrence and development of breast cancer, it provides a new basis for the combination therapy of breast cancer.

[Relationship between C-C chemokine receptor type 2 and P38 mitogen-activated protein kinase signaling pathway in the spinal cord of rats with bone cancer pain].

Objective: To investigate the relationship between C-C chemokine receptor type 2(CCR2) and P38 mitogen-activated protein kinase (P38MAPK) signaling pathway in the spinal cord of rats and further clarify the mechanism of bone cancer pain (BCP). Methods: A total of 92 healthy female SD rats, of which 60 were subjected to behavioral tests using a ciliary mechanical stimulation needle. SD rats were randomly divided into six groups: sham operation group (group S), bone cancer pain group (group B), sham operation + DMSO solvent group (group SD), bone cancer pain + DMSO solvent group (group BD), sham operation + RS102895 CCR2 inhibitor group (group SR), bone cancer pain + RS102895 CCR2 inhibitor group (group BR), and Von Frey was used in the behavioral test. Another 32 SD rats were randomly divided into the following 8 groups (n=4): sham operation group (group S), bone cancer pain 5 d group (group B5), bone cancer pain 9 d group (group B9), bone cancer pain 14 d group (group B14), bone cancer pain + DMSO solvent group (group BD), bone cancer pain + RS102895 CCR2 inhibitor 0.5 h group (group BR0.5 h), bone cancer pain + RS102895 CCR2 inhibitor 4 h group (group BR4 h), bone cancer pain + RS102895 CCR2 inhibitor 12 h group (group BR12 h). Western blot was used to detect the expression of P38, p-P38 and CCR2 in spinal cord of rats. Results: At day 5, 7, 9, 14, 21 post-injection, mechanical withdrawal thresholds of group S were(30.9±1.5), (31.9±1.2), (32.0±1.1), (31.6±1.5), (32.2±1.4)g respectively, the mechanical withdrawal thresholds of group B were( 26.4±0.7), (24.4±0.8), (21.4±0.8), (13.5±0.4), (9.9±0.2)g respectively, the mechanical withdrawal thresholds in group B decreased obviously versus group S, and the differences were statistically significant(t=-13.177, -16.660, -23.778, -35.574, -48.401, all P<0.01). At day 9 post-injection, the mechanical withdrawal thresholds in SD, BD, SR and BR groups were (32.4±1.7), (19.4±1.1), (32.1±1.3), (26.3±1.0) g respectively, the difference was statistically significant (F=224.681, P<0.01), and the mechanical withdrawal thresholds in group BD decreased obviously versus group SD, while the mechanical withdrawal thresholds in group BR increased obviously versus group BD. The expression levels of p-P38 in spinal cord of group S, group B5, group B9 and group B14 were(0.08±0.03), (0.20±0.05), (0.40±0.17), (0.65±0.14)respectively, the expression levels of CCR2 were(0.08±0.04), (0.18±0.05), (0.30±0.09), (0.58±0.07)respectively, the difference was statistically significant(F=19.123, 40.746, all P<0.01), and the expression of p-P38 and CCR2 in group B9 were showed a significant up-regulation versus group S. The expression levels of p-P38 in spinal cord of group BD, group BR0.5 h, group BR4 h and group BR12 h were (0.57±0.06), (0.17±0.11), (0.03±0.01), (0.25±0.11)respectively, and the difference was statistically significant(F=29.582, P<0.01). The expression of p-P38 in group BR0.5 h, BR4 h, BR12 h showed a significant down-regulation versus group BD. Conclusion: CCR2 in the spinal cord may be involved in the development of bone cancer pain by activating P38MAPK signaling pathway in rats.

Molecular mapping of genes Yr64 and Yr65 for stripe rust resistance in hexaploid derivatives of durum wheat accessions PI 331260 and PI 480016.

KEY MESSAGE: This manuscript reports two new genes ( Yr64 and Yr65 ) for effective resistance to stripe rust and usefulness of their flanking SSR markers for marker-assisted selection. Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most important diseases of wheat worldwide and resistance is the best control strategy. Durum wheat accessions PI 331260 and PI 480016 were resistant to all tested Pst races. To transfer the resistance genes to common wheat and map them to wheat chromosomes, both accessions were crossed with the stripe rust-susceptible spring wheat 'Avocet S'. Resistant F3 plants with 42 chromosomes were selected cytologically and by rust phenotype. A single dominant gene for resistance was identified in segregating F4 lines from each cross. F6 populations for each cross were developed from single F5 plants and used for genetic mapping. Different genes from PI 331260 and PI 480016 were mapped to different loci in chromosome 1BS using simple sequence repeat markers. The gene from PI 331260 was flanked by Xgwm413 and Xgdm33 in bin 1BS9-0.84-1.06 at genetic distances of 3.5 and 2.0 cM; and the gene from PI 480016 was flanked by Xgwm18 and Xgwm11 in chromosome bin C-1BS10-0.50 at 1.2 and 2.1 cM, respectively. Chromosomal locations and race and allelism tests indicated that the two genes are different from previously reported stripe rust resistance genes, and therefore are named as Yr64 from PI 331260 and Yr65 from PI 480016. These genes and their flanking markers, and selected common wheat lines with the genes should be valuable for diversifying resistance genes used in breeding wheat cultivars with stripe rust resistance.

Effects of dielectric values of human body on Specific Absorption Rate (SAR) following 800 MHz radio frequency exposure to ingestible wireless device.

In order to assess the compliance of Ingested Wireless Device (IWD) within safety guidelines, the Specific Absorption Rate (SAR) and near fields of IWD in two realistic human body models whose dielectric values are increased from the original by +/-10 and +/-20% are studied using the Finite-Difference Time-Domain (FDTD) method. The radiation characteristics of the IWD in the human body models with original and changed dielectric values are compared. Simulations are carried out at 13 scenarios where the IWD is placed at center positions of abdomens in the two models at the operation frequency of 800 MHz. Results show that variation of radiation intensity near the surface of abdomen is around 1.6 dB within 20% variation of dielectric values at the frequency of 800 MHz. Electric fields in the anterior of the human body models are higher than those in the posterior for all scenarios. SAR values increase as the conductivities of human body tissues increase and usually decrease as the increase of relative permittivities of human body tissues increase. The effect of the dielectric values of human body on SAR is orientation, human body and frequency dependent. An increment up to 20% in conductivities and relative permittivities alone or simultaneously always causes a SAR variation less than 20%. As far as the compliance of safety was concerned, the IWD was safe to be used at the input power less than 9.3 mW according to IEEE safety standards.

Polyphyllin D exerts potent anti-tumour effects on Lewis cancer cells under hypoxic conditions.

Paris polyphylla has been used to treat cancer in China for many years and components of the plant, such as polyphyllin D, may have potent antiproliferative effects in vitro. To investigate the potential antitumour effects of polyphyllin D on cancer cells under hypoxia, Lewis lung cancer cells and mouse tracheal epithelial cells were cultured with or without polyphyllin D under normoxic and hypoxic conditions. Proliferation and apoptosis of cells were assayed. Real-time reverse transcription-polymerase chain reaction was used to quantify the expression of hypoxia-inducible factor 1 alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) mRNA. Polyphyllin D decreased cell proliferation, increased apoptosis and inhibited expression of HIF-1alpha and VEGF mRNAs in Lewis cells. These effects were greater under hypoxic than normoxic conditions. Polyphyllin D did not show a cytotoxic effect in non-tumour cells (mouse skin fibroblasts and tracheal epithelial cells). These results suggest that polyphyllin D potentially has anticancer effects in vitro under hypoxia.

Role of phosphate and kinetic characteristics of complete iron release from native pig spleen ferritin-Fe.

The kinetics for complete iron release showing biphasic behavior from pig spleen ferritin-Fe (PSFF) was measured by spectrophotometry. The native core within the PSFF shell consisted of 1682 hydroxide Fe3+ and 13 phosphate molecules. Inhibition kinetics for complete iron release was measure by differential spectrophotometry in the presence of phosphate; the process was clearly divided into two phases involving a first-order reaction at an increasing rate of 46.5 Fe3+/PSFF/min on the surface of the iron core and a zero-order reaction at a decreasing rate of 6.67 Fe3+/PSFF/min inside the core. The kinetic equation [C(PSFF-Fe3+)max - C(PSFF-Fe3+)t](1/2) = Tmax - Tt gives the transition time between the two rates and represents the complex kinetic characteristics. The rate was directly accelerated twofold by a mixed reducer of dithionite and ascorbic acid. These results suggest that the channel of the PSFF shell may carry out multiple functions for iron metabolism and storage and that the phosphate strongly affects the rate of iron release.

A sesquiterpene lactone glucoside from Ixeris denticulata f. pinnatipartita.

An extract from the entire Ixeris denticulata f. pinnatipartita plant afforded the guaianolide sesquiterpene lactone glucoside, 8 beta,15-dihydroxy-1(10),3,11(13)-guaiatrien-12,6-olide-15-O- glucopyranoside, along with the known flavonoids luteolin-7-O-glucoside and luteolin-7-O-glucuronide-6'-methyl ester; their structures were determined by spectroscopic methods. Ixerin Y inhibited the growth of human breast cancer MCF7 and MDA468 cell lines.

H2-uptake activity, spectra, reduction potentials, and kinetics of iron release on the surface of iron core from Azotobacter vinelandii bacterial ferritin.

Bacterial ferritin from Azotobacter vinelandii (AvBFo) has a function in H2 uptake. The Fe3+ reduction on the surface of the iron core from AvBFo is accompanied simultaneously by H2 uptake, with a maximum activity of H2 uptake of 450 H2/AvBFo. A reduction potential of -402 mV for iron reduction on the surface of the core is found. A shift to the red the protein absorbance peaks ranging from 280 to 290 nm is observed between pH 5 and 9 under 100% H2 reduction. The reduction potential for iron release becomes negative at a rate of 0.025 mV/Fe2+ released. The kinetics of iron release on the surface of the core is a first-order reaction.

[A survey of commercial Chinese crude drug shasheng (radix Adenophorae and Glehniae)].

The commercial crude drug of Radix Adenophorae bought from 15 provinces and autonomous regions, and Radix Glehniae bought from 17 provinces and autonomous regions were surveyed. The original plants of 60 samples of Radix Adenophorae were identified as 9 species and 4 subspecies of genus Adenophora. The main species are Adenophora stricta, A. stricta subsp. sessilifolia, A. potaninii and A. hunanensis. Sixty two samples of Radix Glehniae were all identified as the roots of Glehnia littoralis.

Effects of nickel ions on polymerase activity and fidelity during DNA replication in vitro.

Nickel is a genotoxic carcinogen. However, the mechanisms of nickel-induced genotoxicity are not well understood. We have investigated the effects of Ni2+ ions on DNA polymerase activity and the fidelity of DNA replication in vitro. The effect of Ni2+ on different DNA polymerases is quite variable. The amount of enzyme inhibition and degree of alteration in replication fidelity induced by Ni2+ are dependent both on the polymerase and its associated 3'-5' exonuclease activity. Some polymerases, such as E. coli DNA polymerase I, AMV reverse transcriptase and human DNA polymerase alpha, can utilize Ni2+ as a weak substitute for Mg2+ during DNA replication. Other polymerases are very sensitive to inhibition by Ni2+ and the IC50 can vary by an order of magnitude. T4 polymerase is relatively insensitive to inhibition by Ni2+, although the sensitivity is enhanced in the absence of added Mg2+, and Ni preferentially inhibits the 3'-5' exonuclease function of T7 DNA polymerase. The fidelity and processivity of DNA polymerases may be either increased or decreased by Ni ions in a polymerase dependent manner. The inhibition DNA polymerase activity and altered replication fidelity may contribute significantly to Ni-induced mutagenesis and genotoxicity in vivo.

Chromium(III) bound to DNA templates promotes increased polymerase processivity and decreased fidelity during replication in vitro.

Carcinogenic chromium [Cr(VI)] compounds are reduced intracellularly to DNA- and protein-reactive chromium(III) species. However, the role of Cr(III) ions in chromium-induced genotoxicity remains unclear. We have investigated the effects of chromium(III) binding on DNA replication and polymerase processivity in vitro. Chromium ions bind slowly and in a dose-dependent manner to DNA. Micromolar concentrations of free chromium inhibit DNA replication, but if the unbound chromium is removed by gel filtration, the rate of DNA replication by polymerase I (Klenow fragment) on the chromium-bound template is increased greater than 6-fold relative to the control. This increase is paralleled by as much as a 4-fold increase in processivity and a 2-fold decrease in replication fidelity. These effects are optimum when very low concentrations of chromium ions are bound to the DNA [3-4 Cr(III) ions per 1000 nucleotide phosphates]. Increased concentrations of chromium lead to the production of DNA-DNA cross-links and inhibition of polymerase activity. These results suggest that low levels of DNA-bound chromium(III) ions may contribute to chromium mutagenesis and carcinogenesis by altering the kinetics and fidelity of DNA replication.

Effects of chromium(III) on DNA replication in vitro.

A number of metal compounds are important environmental carcinogens; however, the molecular mechanisms of metal-induced genotoxicity are not yet understood. Chromium, for example, is substantially mutagenic in vivo and has been shown to decrease the DNA replication fidelity in vitro. But the mechanism of chromium-induced mutagenesis is unknown and the role of replication fidelity in chromium-induced carcinogenesis is unclear. We have used in vitro DNA replication assays to investigate the effects of chromium ions on DNA polymerase activity preliminary to studying their role in chromium-induced mutagenesis. Biologically active M13mp2 DNA was replicated with purified DNA polymerases in the presence of micromolar amounts of chromium with or without the normal divalent cation, magnesium. Nucleotide incorporation kinetics were determined and sequence specific pausing was analyzed by primer-extension. Our results have demonstrated an unexpected polymerase activation by low (0.5-5.0 microns) concentrations of chromium (III), although higher concentrations of chromium are increasingly inhibitory. The increased incorporation seem at low chromium(III) concentrations is the result of increased enzyme processivity and is not polymerase specific. The possible relationship between processivity and metal-ion mutagenesis is discussed.

Effect of magnesium on nickel-induced genotoxicity and cell transformation.

Raising the extracellular level of magnesium ions inhibited nickel-induced DNA strand breaks, DNA-protein crosslinks, sister chromatid exchanges, chromosomal aberrations and cell transformation. Carcinogenic nickel ions preferentially damaged centromeres and other heterochromatic regions of Chinese hamster ovary cell chromosomes. Elevation of extracellular magnesium levels prevented the effects of nickel on heterochromatin and inhibited cell transformation, but did not substantially reduce the DNA damage induced by nickel in euchromatic regions. This study suggests that heterochromatic DNA damage may be important to the nickel-induced neoplastic transformation process.

Some properties of the nickel-containing hydrogenase of chemolithotrophically grown Rhizobium japonicum.

The uptake hydrogenase of chemolithotrophically grown Rhizobium japonicum was purified to apparent homogeneity with a final specific activity of 69 mumol of H2 oxidized per min per mg of protein. The procedure included Triton extraction of broken membranes and DEAE-cellulose and Sephacryl S-200 chromatographies. The purified protein contained two polypeptides separable only by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They comigrated on native polyacrylamide gels and sucrose density gradients. The molecular weights were ca. 60,000 and 30,000. Densitometric scans of the sodium dodecyl sulfate gels indicated a molar ratio of 1.03 +/- 0.03. Antiserum was developed against the 60-kilodalton polypeptide for use in hydrogenase detection by an enzyme-linked immunosorbent assay. The antiserum did not cross-react with the 30-kilodalton polypeptide. Native gel electrophoresis of Triton-extracted cells grown in the presence of 63Ni showed comigration of the hydrogenase and radioactive Ni.