DNA Synthesis Ability Research Articles

A Massively Parallel In Vivo Assay of TdT Mutants Yields Variants with Altered Nucleotide Insertion Biases.

Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase capable of template-independent extension of DNA. TdT's de novo DNA synthesis ability has found utility in DNA recording, DNA data storage, oligonucleotide synthesis, and nucleic acid labeling, but TdT's intrinsic nucleotide biases limit its versatility in such applications. Here, we describe a multiplexed assay for profiling and engineering the bias and overall activity of TdT variants with high throughput. In our assay, a library of TdTs is encoded next to a CRISPR-Cas9 target site in HEK293T cells. Upon transfection of Cas9 and sgRNA, the target site is cut, allowing TdT to intercept the double-strand break and add nucleotides. Each resulting insertion is sequenced alongside the identity of the TdT variant that generated it. Using this assay, 25,623 unique TdT variants, constructed by site-saturation mutagenesis at strategic positions, were profiled. This resulted in the isolation of several altered-bias TdTs that expanded the capabilities of our TdT-based DNA recording system, Cell HistorY Recording by Ordered InsertioN (CHYRON), by increasing the information density of recording through an unbiased TdT and achieving dual-channel recording of two distinct inducers (hypoxia and Wnt) through two differently biased TdTs. Select TdT variants were also tested in vitro, revealing concordance between each variant's in vitro bias and the in vivo bias determined from the multiplexed high throughput assay. Overall, our work and the multiplex assay it features should support the continued development of TdT-based DNA recorders, in vitro applications of TdT, and further study of the biology of TdT.

A massively parallel in vivo assay of TdT mutants yields variants with altered nucleotide insertion biases.

Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase capable of template-independent extension of DNA with random nucleotides. TdT's de novo DNA synthesis ability has found utility in DNA recording, DNA data storage, oligonucleotide synthesis, and nucleic acid labeling, but TdT's intrinsic nucleotide biases limit its versatility in such applications. Here, we describe a multiplexed assay for profiling and engineering the bias and overall activity of TdT variants in high throughput. In our assay, a library of TdTs is encoded next to a CRISPR-Cas9 target site in HEK293T cells. Upon transfection of Cas9 and sgRNA, the target site is cut, allowing TdT to intercept the double strand break and add nucleotides. Each resulting insertion is sequenced alongside the identity of the TdT variant that generated it. Using this assay, 25,623 unique TdT variants, constructed by site-saturation mutagenesis at strategic positions, were profiled. This resulted in the isolation of several altered-bias TdTs that expanded the capabilities of our TdT-based DNA recording system, Cell History Recording by Ordered Insertion (CHYRON), by increasing the information density of recording through an unbiased TdT and achieving dual-channel recording of two distinct inducers (hypoxia and Wnt) through two differently biased TdTs. Select TdT variants were also tested in vitro , revealing concordance between each variant's in vitro bias and the in vivo bias determined from the multiplexed high throughput assay. Overall, our work, and the multiplex assay it features, should support the continued development of TdT-based DNA recorders, in vitro applications of TdT, and further study of the biology of TdT.

Binding Modes of a Cytotoxic Dinuclear Copper(II) Complex with Phosphate Ligands Probed by Vibrational Photodissociation Ion Spectroscopy

The dinuclear copper complex bearing a 2,7-disubstituted-1,8-naphthalenediol ligand, [(HtomMe){Cu(OAc)}2](OAc), a potential anticancer drug able to bind to two neighboring phosphates in the DNA backbone, is endowed with stronger cytotoxic effects and inhibition ability of DNA synthesis in human cancer cells as compared to cisplatin. In this study, the intrinsic binding ability of the charged complex [(HtomMe){Cu(OAc)}2]+ is investigated with representative phosphate diester ligands with growing chemical complexity, ranging from simple inorganic phosphate up to mononucleotides. An integrated method based on high-resolution mass spectrometry (MS), tandem MS, and infrared multiple photon dissociation (IRMPD) spectroscopy in the 600-1800 cm-1 spectral range, backed by quantum chemical calculations, has been used to characterize complexes formed in solution and delivered as bare species by electrospray ionization. The structural features revealed by IRMPD spectroscopy have been interpreted by comparison with linear IR spectra of the lowest-energy structures, revealing diagnostic signatures of binding modes of the dinuclear copper(II) complex with phosphate groups, whereas the possible competitive interaction with the nucleobase is silenced in the gas phase. This result points to the prevailing interaction of [(HtomMe){Cu(OAc)}2]+ with phosphate diesters and mononucleotides as a conceivable contribution to the observed anticancer activity.

A multi-functional role for the MCM8/9 helicase complex in maintaining fork integrity during replication stress

The minichromosome maintenance (MCM) 8/9 helicase is a AAA+ complex involved in DNA replication-associated repair. Despite high sequence homology to the MCM2-7 helicase, a precise cellular role for MCM8/9 has remained elusive. We have interrogated the DNA synthesis ability and replication fork stability in cells lacking MCM8 or 9 and find that there is a functional partitioning of MCM8/9 activity between promoting replication fork progression and protecting persistently stalled forks. The helicase function of MCM8/9 aids in normal replication fork progression, but upon persistent stalling, MCM8/9 directs additional downstream stabilizers, including BRCA1 and Rad51, to protect forks from excessive degradation. Loss of MCM8 or 9 slows the overall replication rate and allows for excessive nascent strand degradation, detectable by increased markers of genomic damage. This evidence defines multifunctional roles for MCM8/9 in promoting normal replication fork progression and genome integrity following stress.

Estradiol exerts a neuroprotective effect on SH-SY5Y cells through the miR-106b-5p/TXNIP axis.

Alzheimer's disease (AD) is a neurodegenerative disease. Thioredoxin and thioredoxin-interacting protein (TXNIP) complexes help sustain cell oxidation/reduction balance. In the present study, we verified the neuroprotective role of estradiol against amyloid-beta 42 in SH-SY5Y cells through inhibiting TXNIP expression, promoting cell viability and DNA synthesis ability, inhibiting cell apoptosis, and affecting caspase and Bax/Bcl-2 apoptotic signaling. miR-106b-5p could bind to TXNIP 3'-untranslated region to inhibit the expression level of TXNIP. Within SH-SY5Y cells, miR-106b-5p inhibition repressed cell viability and DNA synthesis ability and promoted cell apoptosis through caspase and Bax/Bcl-2 apoptotic signaling, while miR-106b-5p overexpression or TXNIP knockdown exerted the opposite effects on SH-SY5Y cells; TXNIP knockdown remarkably attenuated the roles of miR-106b-5p inhibition. In conclusion, estradiol treatment on SH-SY5Y cells downregulates TXNIP expression and upregulates miR-106b-5p expression. miR-106b-5p exerts a neuroprotective effect on SH-SY5Y cells by promoting cell proliferation and inhibiting cell apoptosis through targeting TXNIP.

Study of the mechanism by which dinaciclib induces apoptosis and cell cycle arrest of lymphoma Raji cells through a CDK1-involved pathway.

ObjectiveThis study aimed to identify and evaluate the mechanism by which apoptosis and cell cycle arrest were induced by dinaciclib in lymphoma Raji cells.MethodsThe colony formation assay was used to detect cell proliferation of Raji cells. Cell cycle arrest and cell apoptosis were determined by flow cytometry and TUNEL assays, respectively. Protein expression related to the Raji cell state was evaluated by Western blot. The Raji/Dinaciclib drug‐resistant cell line was established, where the regulating functions of CDK1‐involved pathway were verified. In addition, the effect of dinaciclib in vivo was examined in orthotopically implanted tumors in nude mice.ResultsCell apoptosis was induced, and DNA synthesis ability was decreased in a time‐dependent manner in dinaciclib‐treated lymphoma Raji cells. Furthermore, the cell cycle was found to be blocked in the G2/M Phase. Further study indicated that CDK1‐involved pathway played a key regulatory role in this process. It was revealed by cell transfection that the expression of cell cycle proteins was downregulated after treatment with dinaciclib through a CDK1‐involved pathway, which eventually led to apoptosis. Knockdown of CDK1 restored the sensitivity of the Raji/Dinaciclib cells to dinaciclib. Xenograft model of nude mice showed that dinaciclib treatment in vivo could effectively inhibit tumor growth, consistent with the experiment results mentioned before.ConclusionIn this study, we clarified the mechanisms through which dinaciclib induces Raji cell apoptosis and blocks the cell cycle through a CDK1‐involved pathway, which supported that dinaciclib had potential values in the treatment of lymphoma.

Study on the Effect of Immunosuppressive Agent FK506 on Growth and Migration of Lung Cancer Cell

背景与目的FK506（他克莫司，tacrolimus）是一种大环内酯类的新型免疫抑制剂。研究报道FK506对多种肿瘤细胞具有增殖抑制作用。本研究旨在观察FK506对肺癌细胞增殖和迁移的作用，并探讨其可能的机制。方法体外培养A549和H1299细胞，采用CCK-8法测定FK506对A549和H1299细胞增殖的作用；EDU标记法检测DNA合成；流式细胞术检测细胞的周期分布情况；Transwell和细胞划痕实验检测细胞的体外迁移能力；Western blot技术检测P27、RB1、CDK4、CDK6和MMP9蛋白的表达。结果FK506可抑制A549和H1299细胞的增殖、诱导细胞周期G0期/G1期阻滞；与对照组比较，FK506处理后A549和H1299细胞迁移能力明显降低，且呈剂量依赖性。此外，与对照组相比，FK506处理组中P27和RB1表达上调，而CDK4、CDK6和MMP9表达显著下降。结论FK506对人肺癌A549和H1299细胞的增殖和迁移能力有明显的抑制作用，其机制可能与上调p27表达、抑制CDK4、CDK6和MMP-9表达有关。

The effect of eleven-nineteen lysine-rich leukemia gene 2 on proliferation of prostate cancer cell C4-2

Objective To observe the level of eleven-nineteen lysine-rich leukemia gene 2 (ELL2) in human prostate cancer tissues and its effect on proliferation of prostate cancer cell C4-2. Methods The expression of ELL2 in human prostate cancer tissues was analyzed by immunohistochemical method, and the relationship between ELL2 and malignancy degree of prostate cancer was evaluated. The human prostate cancer cell line C4-2 was stimulated with synthetic androgen R1881 to detect its response to androgen at different time points. ELL2-small interfering RNA (siRNA) and overexpression of ELL2-bearing plasmid were transfected into C4-2 cells respectively, then cell proliferation was evaluated by methyl thiazol tetrazolium (MTT) assay. The DNA synthesis ability of cells was assessed by 5-Bromo-2’-deoxy-uridine (BrdU) incorporation assay. Results The expression of ELL2 was significantly lower in human prostate cancer than that of benign specimen, and decreased with the increase of malignancy of prostate cancer. The correlation analysis between ELL2 and prostate cancer Gleason score reveal a negative correlation result (r=0.836, P=0.000). The protein expression of ELL2 was significantly increased in 48 h and increased by (38.27±10.11)% (t=7.573, P=0.005) and (58.34±13.70)% (t=8.519, P=0.003) at 24 h and 48 h, respectively. The expression of ELL2 protein in the two RNA interference groups were significantly decreased, and the decreased rate were (56.38±11.26)% (t=10.010, P=0.002) and (65.27±12.10)% (t=10.790, P=0.002), respectively. While the ELL2 protein expression in the overexpression group was significantly increased at (80.23±20.94)% (t=7.662, P=0.005). The proliferation ability of C4-2 cells was significantly increased (39.28±8.52)% (t=11.300, P=0.000) and (48.2±7.87)% (t=15.000, P=0.000) in the two RNA interference groups, respectively, compared with the control group. While its proliferation ability decreased (42.74±10.38)% (t=10.090, P=0.000) in ELL2-bearing plasmid. Transfection of ELL2-specific interfering RNA increased the ability of DNA synthesis, while the transfection of ELL2-bearing plasmid cells had lower DNA synthesis ability. Conclusion ELL2 is lowly expressed in human prostate cancer tissues and negatively correlated with the malignant degree. ELL2 can inhibit the proliferation of prostate cancer cells to a certain extent. Key words: Prostate cancer; Eleven-nineteen lysine-rich leukemia gene 2; Proliferation

Resveratrol inhibits adventitial fibroblast proliferation and induces cell apoptosis through the SIRT1 pathway.

Atherosclerosis is one of the most important causes of cardiovascular disease and studies have showed that adventitial fibroblasts, which are considered to be the most common cell type of the vascular adventitia, are involved in the development of early atherosclerotic plaques. Resveratrol is a plant polyphenolic compound confirmed to have anti-atherosclerotic and cardioprotective effects. The aim of the present study was to investigate the effects of resveratrol on adventitial fibroblasts in vitro and to clarify the underlying mechanism. Adventitial fibroblasts were isolated from the thoracic aorta of 8-week-old SPF Sprague-Dawley rats. Following pre-treatment with different concentrations of resveratrol, cell viability, DNA synthesis ability, cell apoptosis and cell migration ability were assessed in vitro. Through transfection with small interfering (si)RNA targeting sirtuin 1 (SIRT1), the role of the SIRT1 pathway in these processes was evaluated. Western blot analysis was used to assess the protein expression of SIRT1. It was demonstrated that resveratrol inhibited the cell viability, DNA synthesis and migratory ability of the adventitial fibroblasts, and induced cell apoptosis in a concentration-dependent manner in vitro. These effects were partly through the SIRT1 pathways. siRNA targeting SIRT1 successfully reversed the antiproliferative, antimigratory and pro-apoptotic effects of resveratrol on adventitial fibroblasts. In conclusion, the data showed that resveratrol inhibited cell viability, DNA synthesis and cell migration, and induced cell apoptosis in the rat adventitial fibroblasts in vitro through the SIRT1 signaling pathway. As the activation and migration of adventitial fibroblasts contributes to the early development of atherosclerosis, this may be a mechanism underlying the anti-atherosclerotic effect of resveratrol.

Autophagy stimulates apoptosis in HER2‐overexpressing breast cancers treated by lapatinib

HER2-overexpressing breast cancers often show hyperactivation of the HER2/AKT/mTOR signaling pathway. Lapatinib is an oral dual tyrosine kinase inhibitor (TKI) that targets both EGFR and HER2 to inhibit the proliferation of breast cancer cells. However, it is obscure whether and how lapatinib could induce autophagy in breast cancer cells, an important cell response with drug treatment. In this study, we investigated the apoptosis and the autophagy in the HER2-overexpressing breast cancer cells BT474 and AU565 treated with lapatinib, and further examined their relationship. Lapatinib inhibited the proliferation and the rate of DNA synthesis in HER2-positive cells, as observed by MTT, colony formation and EDU assays. Lapatinib not only induced apoptosis accompanied by an increased expression of cleaved Caspase-3 and cleaved PARP, but it also induced autophagy in vitro, as confirmed by electron microscopy (EM), acridine orange (AO) staining and LC3-II expression. Meanwhile, lapatinib inhibited the phosphorylation of HER2, AKT, mTOR, and p70S6K, whereas that of AMPK was activated. When the cells were pre-incubated with 3-Methyladenine (3-MA), the specific autophagy inhibitor, the growth inhibitory ratio and apoptosis rate were frustrated, whereas colony formation and DNA synthesis ability were encouraged. In addition, 3-MA application could up-regulate Caspase-3 and PARP expression, compared with the treatment with lapatinib alone. The addition of 3-MA could attenuate the inhibitory role on HER2/AKT/mTOR pathway and the active role on AMPK that was raised by lapatinib. Therefore, lapatinib simultaneously induced both apoptosis and autophagy in the BT474 and AU565 cells, and in these settings, autophagy facilitates apoptosis.

Biochemical analysis of active site mutations of human polymerase η

DNA polymerase η (pol η) plays a critical role in suppressing mutations caused by the bypass of cis–syn cyclobutane pyrimidine dimers (CPD) that escape repair. There is evidence this is also the case for the oxidative lesion 7,8-dihydro-8-oxo-guanine (8-oxoG). Both of these lesions cause moderate to severe blockage of synthesis when encountered by replicative polymerases, while pol η displays little no to pausing during translesion synthesis. However, since lesion bypass does not remove damaged DNA from the genome and can possibly be accompanied by errors in synthesis during bypass, the process is often called ‘damage tolerance’ to delineate it from classical DNA repair pathways. The fidelity of lesion bypass is therefore of importance when determining how pol η suppresses mutations after DNA damage. As pol η has been implicated in numerous in vivo pathways other than lesion bypass, we wanted to better understand the molecular mechanisms involved in the relatively low-fidelity synthesis displayed by pol η. To that end, we have created a set of mutant pol η proteins each containing a single amino acid substitution in the active site and closely surrounding regions. We determined overall DNA synthesis ability as well as the efficiency and fidelity of bypass of thymine–thymine CPD (T–T CPD) and 8-oxoG containing DNA templates. Our results show that several amino acids are critical for normal polymerase function, with changes in overall activity and fidelity being observed. Of the mutants that retain polymerase activity, we demonstrate that amino acids Q38, Y52, and R61 play key roles in determining polymerase fidelity, with substation of alanine causing both increases and decreases in fidelity. Remarkably, the Q38A mutant displays increased fidelity during synthesis opposite 8-oxoG but decreased fidelity during synthesis opposite a T–T CPD.

Calcitonin gene-related peptide released from endothelial progenitor cells inhibits the proliferation of rat vascular smooth muscle cells induced by angiotensin II

We have recently demonstrated that endothelial progenitor cells (EPCs) inhibit AngII-induced proliferation of vascular smooth muscle cells (VSMCs) by inactivating MAPKs and NF-κB signaling pathway and reducing expression of oncogene c-myc and c-fos. The inhibitory effect of EPCs on VSMCs is associated with paracrine mechanism. However, the potential mechanism of EPCs on the regulation of AngII-induced proliferation of VSMCs was unknown. Calcitonin gene-related peptide (CGRP) could inhibit AngII-induced proliferation and transformation of VSMCs. However, it has not been known whether CGRP released from EPCs is a potential regulator in regulation of AngII-induced proliferation of VSMCs. Early endothelial progenitor cell-conditioned medium(E-EPC-CM) was pre-incubated with functional blocking antibodies against CGRP for 1 h or VSMCs was preteated with CGRP(837)(CGRP receptor antagonist) for 1 h before VSMCs were pretreated with CM for 30 min. DNA synthesis ability, total protein levels, cell survival, signal transduction, and expressions of c-myc and c-fos of VSMCs induced by AngII (10(-6)mol/l) were detected to assess the role of CGRP in AngII-induced proliferation of VSMCs. E-EPC-CM could significantly inhibit AngII-induced DNA synthesis ability, total protein levels, cell survival, phosphorylation of ERK, JNK, p38, p65, and expressions of c-myc and c-fos compared with the control group(P < 0.05). However, Pretreatment with anti-CGRP antibody and CGRP(837) could significantly weaken the inhibitory effect of E-EPC-CM on proliferation of VSMCs induced by AngII (P < 0.05). EPCs exert anti-proliferative effects on VSMCs mediated by the release of CGRP.

Knockdown of MED19 by Lentivirus-Mediated shRNA in Human Osteosarcoma Cells Inhibits Cell Proliferation by Inducing Cell Cycle Arrest in the G0/G1 Phase

MED 19 is a subunit of the mediator complex, which is a coactivator of RNA polymerase II and also interacts with the downstream coding region of many genes. However, the role of MED19 in osteosarcoma is unknown. In the present study, we applied lentivirus-mediated short hairpin RNA (shRNA)-triggered RNA interference to downregulate MED19 expression in human osteosarcoma SaOS-2 and U2OS cells. Knockdown of MED19 expression was confirmed by real-time PCR and Western blot. It was found that silencing of MED19 resulted in decreased cell viability, colony formation capacity, and DNA synthesis ability in both cells, as well as the G0/G1 phase cell cycle arrest. These results implied that MED19 played an important role in cell growth and cell cycle progression of human osteosarcoma cells. MED19 may be an attractive candidate for the therapeutic target in osteosarcoma.

Effect of endothelial progenitor cells on the proliferation of co-cultured vascular smooth muscle cells

To explore the effect of endothelial progenitor cells (EPCs) on the proliferation of co-cultured rat vascular smooth muscle cells (VSMCs). Mononuclear cells were isolated from fresh cord blood by 6% hydroxyethyl starch (HES) and density gradient centrifugation. Isolated mononuclear cells were cultured in EGM-2 medium supplemented with 20% fetal bovine serum (FBS), VEGF,bFGF and other growth factors. Biological features of EPCs were observed at different time points, and EPCs were identified by morphology, fluorescence double-staining and flow cytometry. Indirect immunofluorescence was performed to analyze the expression of alpha-SM-actin, calponin of VSMCs special antigen. Co-culture system of EPCs and VSMCs was established by transwell permeable support. FBS (20%) was used to stimulate the proliferation of VSMCs. In a VSMCs/EPCs co-culture system, the DNA synthesis ability, total protein level and cell cycle of VSMCs were determined by BrdU marking method,protein quantitation and flow cytometry after co-culture for 6, 12, 24,48 and 72 h. After co-culture for 12, 24, 48, and 72 h, the DNA synthesis ability and total protein level of VSMCs significantly decreased compared with the control group (P<0.05). Flow cytometry showed that the percentage of S phase of VSMCs in VSMCs/EPCs co-cultured group significantly decreased and the percentage of G1 phase increased markedly compared with the control group (P<0.05). The maximal inhibitory effect was observed at 48 h. Early EPCs could inhibit the proliferation of VSMCs.

Mifepristone abrogates repopulation of ovarian cancer cells in between courses of cisplatin treatment

Repopulation of cancer cells escaping lethal chemotherapy is a critical factor hindering treatment success. One strategy to inhibit tumor cell repopulation is the use of cytostatic compounds between courses of lethal chemotherapy. In this study, we tested the hypothesis that mifepristone (MF), a steroid compound with demonstrated growth inhibition activity in ovarian cancer, should be efficacious in inducing cytostasis and preventing repopulation of ovarian cancer cells if given among rounds of cisplatin (CDDP) treatment. We established an in vitro approach wherein ovarian cancer cells with high (OV2008) or low (SK-OV-3) sensitivity to CDDP were exposed to 3 (OV2008) or 2 (SK-OV-3) rounds of lethal doses of CDDP for 1 h, 12 (OV2008) or 24 (SK-OV-3) days apart. Every 4 or 8 days cell number, cell viability, cell cycle traverse, and colony-forming capacity of viable cells was analyzed. Although CDDP killed the vast majority of cells, there were remnant cells escaping CDDP lethality and repopulating the culture, as evidenced by increased cell number, improved clonogenic capacity of viable cells, and normalization of DNA synthesis. Conversely, when cells were exposed to CDDP for 1 h, and 5, 10 or 20 microM MF was present in the culture medium after CDDP removal, the number, clonogenic capacity, and DNA synthesis ability of the cells were reduced in a dose-dependent manner. The blockage by MF of post-CDDP repopulation was accompanied by a remarkable increase in the percentage of cells expressing the cell death marker cleaved poly(ADP-ribose) polymerase and the mitotic marker phospho-histone H3, suggesting that MF also potentiated CDDP lethality and that the cells likely die due to mitotic failure. In summary, this is the first study reporting that presence of cytostatic concentrations of MF after courses of lethal doses of CDDP prevents repopulation of remnant ovarian cancer cells surviving CDDP treatment.

Inhibition of DNA synthesis by downregulation of cyclin A but not Skp 2 overexpression in human hepatocellular carcinoma cells

Cyclin A is an S and G2/M phase regulatory protein and associates with Skp 2 in many transformed cells. Our previous results showed that 12 (39%) and 17 (55%) out of 31 hepatocellular carcinoma (HCC) patients exhibited higher protein expression levels of cyclin A and Skp 2, respectively, in their tumorous compared to non-tumorous tissues. In the present study, we used Western blot analysis to show that 3 out of 6 HCC cell lines, HA59T, HA22T and HCC36, exhibited overexpression of cyclin A, among which the HCC36 cell line also expressed a higher Skp 2 protein level. Moreover, we used the antisense oligonucleotide phosphorothioates to down regulate the overexpression of cyclin A and Skp 2 proteins to determine whether or not these two proteins are involved in the mitogenesis of HCC36 cells. After treatment with antisense oligonucleotide phosphorothioates, the gene product of cyclin A or Skp 2 was suppressed dose-dependently as revealed by Western blot analyses. By [3H]thymidine incorporation assay, we found that downregulation of cyclin A but not Skp 2 overexpression could inhibit the DNA synthesis ability of HCC36 cells, suggesting that abnormal Skp 2 expression is not directly correlated with the HCC proliferation.

培養成長軟骨細胞に対するパルス電磁場刺激の影響

Recently, pulsing electromagnetic field (PEMF) stimulation has been used for the treatment of delayed unions and non-unions clinically, but the mechanism is not clearly known. As a result, we investigated the effect of PEMFs on costal growth-cartilage cells of rabbits in culture. Cell growth and the ability of DNA synthesis and glycosaminoglycan (GAG) synthesis in the cells were measured.A PEMF generator was made in this department by using Helmholtz coil. Repetitive pulse burst (RPB) with a burst width of 76ms, a pulse width of 230μs and 6.4Hz was passed through this coil. The magnetic field strength reached 4 Gauss at the point where culture dishes were positioned.Growth-cartilage cells were isolated from New Zealand White rabbits (_??_ 300-600g). Culture medium was HamF-12 with 10% FCS. The ability of DNA synthesis was measured by 3H-thymidine incorporation. The ability of GAG synthesis was measured by 35S-sulfuric acid incorporation.We concluded that PEMF stimulation tended to promote cell growth and metabolism, especially immediately after stimulation.

Temperature sensitivity of human spermatogonia and spermatocytes in vitro.

To study the effect of temperature on human spermatogenesis, both the number and DNA synthesis of germ cells were investigated in tissue fragments of human testes cultured for 22 h at 31 degrees C and 37 degrees C. The number of differentiated germ cells such as spermatids and spermatozoa cultured at 37 degrees C was significantly smaller than that cultured at 31 degrees C. The number of spermatogonia and resting primary spermatocytes was not significantly different between these two temperatures, but the functional ability of DNA synthesis in these cells was significantly lower at 37 degrees C than at 31 degrees C. It seems that in normal body temperature (37 degrees C) differentiated germ cells such as spermatids and spermatozoa are fragile and the DNA synthesis of spermatogonia and resting primary spermatocytes is retarded.

Semi-conservative synthesis of DNA in UV-sensitive mutant cells of Chinese hamster after UV-irradiation

A study was made of the rate of semi-conservative DNA synthesis in asynchronous UV-resistant (clone V79) and UV-sensitive clones (VII and XII) of Chinese hamster cells after UV-irradiation. In all 3 clones studied, UV-irradiation (5–30 J/m 2) induced a decrease in the rate of DNA synthesis during the subsequent 1–2 h. In the resistant clone (V79) recovery of DNA synthesis rate started after the first 2 h post-irradiation (5 J/m 2) and by the 3rd hour reached its maximum value, which constituted 70% of that observed in control, non-irradiated cells. The UV-sensitive mutant clones VII and XII showed no recovery in the rate of DNA synthesis during 6–7 h post-irradiation. The results obtained show that the survival of cells is correlated with the ability of DNA synthesis to recover after UV-irradiation in 3 clones studied. The observed recovery of UV-inhibited DNA synthesis in mutant clones may be due to certain defects in DNA repair.