Role of Ku80-dependent end-joining in delayed genomic instability in mammalian cells surviving ionizing radiation

Ionizing radiation induces delayed destabilization of the genome in the progenies of surviving cells. This phenomenon, which is called radiation-induced genomic instability, is manifested by delayed induction of radiation effects, such as cell death, chromosome aberration, and mutation in the progeny of cells surviving radiation exposure. Previously, there was a report showing that delayed cell death was absent in Ku80-deficient Chinese hamster ovary (CHO) cells, however, the mechanism of their defect has not been determined. We found that delayed induction of DNA double strand breaks and chromosomal breaks were intact in Ku80-deficient cells surviving X-irradiation, whereas there was no sign for the production of chromosome bridges between divided daughter cells. Moreover, delayed induction of dicentric chromosomes was significantly compromised in those cells compared to the wild-type CHO cells. Reintroduction of the human Ku86 gene complimented the defective DNA repair and recovered delayed induction of dicentric chromosomes and delayed cell death, indicating that defective Ku80-dependent dicentric induction was the cause of the absence of delayed cell death. Since DNA-PKcs-defective cells showed delayed phenotypes, Ku80-dependent illegitimate rejoining is involved in delayed impairment of the integrity of the genome in radiation-survived cells.     

A review of maternal and embryonic control of phase-dependent progeny characteristics in the desert locust

Hatchlings of the desert locust, Schistocerca gregaria, display phase polyphenism in body coloration and size. This phenomenon has been found to be maternally controlled and two different mechanisms have been proposed to explain the underlying process. One mechanism claims that a water-soluble pheromonal factor secreted by gregarious female adults into the foam plugs of egg pods induces darkening in their progeny. The other mechanism states that hatchling body coloration is pre-determined in the ovaries and that no foam factor is involved in this phenomenon. This mechanism was supported by the observation that hatchling melanization was not prevented by the early washing or separation of eggs, which should have removed the pheromonal factor from the eggs and produced green hatchlings according to the other mechanism. This paper reviews the latest findings related to this phenomenon with special reference to reproductive cycles and genetic differences. The close relationships between egg size and the degree of melanization in hatchlings may provide strong evidence against the possible involvement of the pheromonal factor, because egg size is determined in the ovarioles. Furthermore, the absence of “solitarizing” effects of early washing and separation on hatchling melanization was also confirmed in different genetic strains. A hypothesis proposed by others that such effects occur only in eggs from the first reproductive cycle was tested and rejected. Based on these and other results, a model to explain the mechanisms underlying the maternal control of progeny characteristics and embryonic control of melanization in the hatchling was proposed.     

Down-regulation of BCRP/ABCG2 in colorectal and cervical cancer

Expression of Breast Cancer Resistance Protein (BCRP/ABCG2) in tumor cells is associated with resistance to multiple chemotherapeutic agents. BCRP also protects against phototoxicity by mediating the efflux of protoporphyrins from cells. However, chemotherapy and photodynamic therapy are effective treatment options for cancer. Furthermore, protoporphyrins are essential, in the form of heme, for the synthesis of nitric oxide, over-production of which is associated with cancer. This raises the question as to whether the expression of this transporter is altered in cancer. To address this question, we investigated the expression of BCRP in colorectal cancer and cervical cancer. Paired normal and cancer tissues from colectomy specimens were used for the analysis of BCRP mRNA by RT-PCR and Northern blot. BCRP was analyzed by immunohistochemistry/immunofluorescence. Similar studies were also done with specimens of normal cervix and cancer cervix. A commercial dot blot was probed to quantify the expression of BCRP in paired normal and cancer cDNA samples from 154 patients with tumors in 19 different tissues. BCRP mRNA was present in normal colorectal tissue and showed a 6-fold decrease in cancer. BCRP was abundant in the normal colon and showed a decrease in colon cancer. The down-regulation of BCRP mRNA and protein was also evident in cervical cancer. There was also a decrease in BCRP mRNA in cancer in 12 of the 19 different tissues collected from 154 patients. These data show that cancer-associated down-regulation of BCRP is likely to be a common phenomenon in several tissues. Decreased expression of BCRP may have a role in tumorigenesis by allowing accumulation of genotoxins and over-production of nitric oxide.     

Active site analysis of fructosyl amine oxidase using homology modeling and site-directed mutagenesis

A three-dimensional structural model of fructosyl amine oxidase from the marine yeast Pichia N1-1 was generated using the crystal structure of monomeric sarcosine oxidase from Bacillus sp. B-0618 as template. The putative active site region was investigated by site-directed mutagenesis, identifying several amino acid residues likely playing important roles in the enzyme reaction. Asn354 was identified as a residue that plays an important role in substrate recognition and that can be substituted in order to change substrate specificity while maintaining high catalytic activity. While the Asn354Ala substitution had no effect on the V _max K _m⁻¹ value for fructosyl valine, the V _max K _m⁻¹ value for fructosyl-^ε N-lysine was decreased 3-fold, thus resulting in a 3-fold improvement in specificity for fructosyl valine over fructosyl-^ε N-lysine.     

Mutational analysis of 6-aminohexanoate-dimer hydrolase: relationship between nylon oligomer hydrolytic and esterolytic activities

Carboxylesterase (EII') from Arthrobacter sp. KI72 has 88% homology to 6-aminohexanoate-dimer hydrolase (EII) and possesses ca. 0.5% of the level of 6-aminohexanoate-linear dimer (Ald)-hydrolytic activity of EII. To study relationship between Ald-hydrolytic and esterolytic activities, random mutations were introduced into the gene for Hyb-24 (an EII/EII' hybrid with the majority of the sequence deriving for EII' and possessing an EII'-like level of Ald-hydrolytic activity). Either a G181D or a D370Y substitution in Hyb-24 increased the Ald-hydrolytic activity ca. 10-fold, and a G181D/D370Y double substitution increased activity ca. 100-fold. On the basis of kinetic studies and the three-dimensional structure of the enzyme, we suggest that binding of Ald is improved by these mutations. D370Y increased esterolytic activity for glycerylbutyrate ca. 30-50-fold, whereas G181D decreased the activity to 30% of the parental enzyme.     

In vitro assays for adhesion and migration of osteoblastic cells (Saos-2) on titanium surfaces

The first event occurring at the boundary between a metal implant and living tissue is the attachment of cells onto the metal surface of the implant. The attachment characteristics of the metal in this situation are critical in determining its biocompatibility and usefulness as artificial bone and tooth implants. Using the human osteosarcoma cell line Saos-2, we attempted to establish simple and reliable methods for evaluating the attachment of cultured osteoblastic cells onto titanium samples that had been subjected to various surface treatments. Fluorescence actin imaging showed that cells cultured on titanium with hydrofluoric acid etching (HF-Ti) exhibited delayed spreading of their cytoplasm, as compared to cells cultured for the same length of time on nitrided titanium or physically polished titanium. The HF-Ti-cultured cells also exhibited poor assembly of focal contacts, as visualized by vinculin immunofluorescence. Furthermore, in motility assays based on an in vitro wound model, cells cultured on HF-Ti migrated more slowly than cells cultured on other titanium surfaces. These data suggest that Saos-2 cells attach less effectively to the HF-Ti surface. The methods described in this study should be useful for assessing the initial interactions of cultured cells with various materials, including metals.S.G. is the recipient of a grant awarded to foreign students by the government of Japan. This study was supported by the Integrated Center for Science (INCS) at Ehime University.     

A model of retinal ischemia-reperfusion injury in rats by subconjunctival injection of endothelin-1

The retinal ischemia-reperfusion model is used in the study of transient ischemia-related diseases, such as central retinal artery occlusion, angle-closure glaucoma, and others. There are two methods for experimentally producing an ischemia-reperfusion model in the rat retina: (i) the intraocular pressure is greatly raised by increasing the height of the infusion bottle connected with the needle in the anterior chamber; or (ii) the blood vessel that accompanies the optic nerve in retina is ligated. However, each method has some drawbacks. For example, in the first method, the needle must be fixed in the anterior chamber for 1 hr, thus, the technique is not stable and mechanical damage to ocular structures sometimes occurs. In the second method, because of the unavoidable involvement of the optic nerve, damage to the nerve induces retinal changes unrelated to ischemia. In this study, we injected endothelin (ET)-1 under the conjunctiva of the eyeball (subconjunctival injection), and evaluated whether a retinal ischemia-reperfusion model could be generated by this method, simply and noninvasively. We injected 4 x 10(-5) M ET-1 solution into the right eye of the rat and injected a control vehicle (artificial tears) into the left eye. From 5-60 mins after the injection, 50 mg/ml fluorescein isothiocyanate (FITC)-dextran was injected to the left ventricle of heart. Then, the retina was removed and flat mounted. We compared the perfusion conditions of the FITC-dextran to each retina in the right and left eye. There was a complete perfusion of FITC-dextran in the retinal main artery, vein, and the capillary vessels in all of the control eyes. However, perfusion could not be completely observed in the ET-1 injected eye from 5-35 mins after injection; afterwards, the flow was returned. This method of subconjunctival injection of ET-1 is, thus, a feasible technical option for producing a retinal ischemia-reperfusion model in rat.     

Altered expression of endothelin, vascular endothelial growth factor, and its receptor in hepatic tissue in endotoxemic rat

Sepsis involves a heterogeneous class of syndromes, and septic shock, a severe form of sepsis, is associated with the development of progressive damage in multiple organs. The present study examined the time-dependent alterations of endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF) levels in liver tissue in a septic rat model. Healthy male Wistar rats aged 15 weeks received 15 mg/kg lipopolysaccharide (LPS) and were sacrificed at different time points (1, 3, 6, and 10 hrs after treatment). Rats that did not receive LPS were considered to be controls. A 28-fold increase in the ET-1 level was observed in liver tissue 10 hrs after LPS administration. VEGF was also altered in hepatic tissue in a time-dependent manner. A gradual increase of VEGF expression in liver tissue after LPS administration was observed. Expression of Flt-1, the vascular permeability receptor of VEGF, was also increased in liver tissue after LPS administration. ET-1 is a potent vasoconstrictor and, therefore, may play a role in the regulation of hepatic perfusion in a sepsis model. On the other hand, VEGF may be involved in capillary leakage in liver tissue after LPS administration. The present findings suggest that there might be a loss of balance between the ET-1 and VEGF levels in the septic liver at different time points, which could contribute to the pathogenesis of acute liver injury in endotoxemia.