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Strains and Culture Conditions. Strain CHO-K1 was obtained from the American Type Culture Collection. CHO-K1, mutant 29 11 ; , and the CHO strains constructed in this study were maintained in Ham's F-12 medium supplemented with 10% newborn calf serum, penicillin G 100 units ml ; , streptomycin sulfate 100 g ml ; , and NaHCO3 1.176 g liter ; under a 5% CO2 atmosphere of 100% humidity at 37C. An ouabain-resistant subclone of CHO-K1 cells was selected in the growth medium containing 1 mM ouabain, after mutagenesis with 400 g of ethyl methanesulfonate per ml of growth medium at 37C for 16 hr. For the isolation of an ouabain and 6-thioguanine-resistant clone, the ouabain-resistant cells were subjected to a second round of mutagenesis and then cultivated in the growth medium containing 30 M 6-thioguanine and 1 mM ouabain. A hybrid clone of the resultant thioguanine ouabain-resistant CHO-K1 and mutant 29 cells was selected and purified in the growth medium containing 5 g ml hypoxanthine, 0.02 g ml aminopterin, 5 g ml thymidine, and 1 mM ouabain after exposure of a mixed cell monolayer to 50% PEG 4000 in Ham's F-12 medium for 1 min. Metabolic Labeling of PtdSer with [14C]Serine. Approximately 5 105 cells were seeded into 60-mm-diameter dishes or the wells of a 24-well plate in Ham's F-12 medium suppleAbbreviations: CHO, Chinese hamster ovary; PtdSer, phosphatidylserine; PtdEtn, phosphatidylethanolamine; PSS, PtdSer synthase. * To whom reprint requests should be addressed. e-mail: kuge nih. go.jp. Present address: Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6, Asahi-machi, Machida-shi, Tokyo 194, Japan.
Induce extensive cell-to-cell fusion in MT-2 cultures, leading to complete lysis of the cells within three to four days after the initial appearance of syncitia data not shown ; . In contrast, MT-2 cultures infected with HIV-1 contained far fewer syncitia at comparable time points and multiplicities of infection. This difference in cytopathicity may explain why HIV-2 occasionally appears to be resistant to AZT in assays that measure virusmediated cell killing 18, 19 ; . Rapid syncitia formation by HIV-2 can potentially destroy cultures that are maintained at low drug concentrations, thereby saturating the assay and falsely inflating the resultant EC50. In contrast, assays that constrain viral infection to a single cycle are unaffected by strain-to-strain differences in replication capacity and cytopathic potential 12 ; , and therefore provide a more accurate comparison of the drug sensitivities of divergent HIV isolates.
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Ing blood or urine for past use of illegal drugs does not find impairment. After every accident, complete drug testing should be done. 7. Expand drug treatment availability and effectiveness by providing methadone, LAAM and other maintenance drugs through general practitioners, and to addicts in prisons and jails. Drug treatment health insurance should be on a parity with other coverage. Coordinate local drug treatment intake to maximize access to programs, and to match addicts to the most suitable treatment. Permit parents to keep and care for their young children while parents are in treatment. 8. Assure that anti-drug education is evaluated and effective. Americas largest teenage anti-drug program has been the ineffective D.A.R.E. curriculum. Like sex education, drug education should include abstinence and reality-based, harm reduction elements, and should encourage honest dialogue with parents and teachers. 9. Focus federal criminal justice resources only on the biggest, most violent and dangerous traffickers. Only ten percent of federal drug cases have been highlevel offenders. Repeal mandatory minimum sentences that give minor offenders kingpin sentences. 10. Medical patients with serious or terminal illness should never be denied the proper medication, whether opiates or marijuana. To carry out such a lifesaving, prevention and public health oriented plan, the next drug czar should have at least one quality: a background in public health. He or she should be unafraid of entrenched drug enforcement bureaucrats and anti-drug blow-hards, and not looking for a promotion. Someone like former Surgeon General C. Everett Koop comes to mind.
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Allow for subsequent colonization of recipient testes. Recently it was reported that short-term survival of isolated mouse, rat, and pig type A spermatogonia in culture was improved in the presence of stem cell factor and granulocyte-macrophage-colony stimulating factor whereas LIF was not effective [34]. Appropriate long-term culture systems for germ cells from domestic animals will have to be determined empirically. Availability of these culture systems will be of utmost importance for the genetic modification of male germ cells in vitro. The different results of spermatogonial transplantation from rabbits and dogs to mouse recipients compared to transplantation of rodent germ cells supports the hypothesis that increased phylogenetic distance between donor and recipient leads to incompatibilities between transplanted germ cells and Sertoli cells of the recipient or the microenvironment in the recipient seminiferous tubule. Hamster spermatogenesis in mouse testes was less efficient, and more defective spermatozoa were observed compared to rat spermatogenesis in the mouse [12, 14]. Although classification is still controversial, rabbits and rodents as well as carnivora and rodents were separated at least as early as the late paleocene epoch approximately 60 million yr ago [35] ; , whereas rats and mice diverged 1011 million yr ago, and the mouse-hamster separation was approximately 16 million yr ago [36]. It appears therefore, that the extent of xenogeneic spermatogenesis decreases with increasing phylogenetic distance between donor and recipient. Compatibility of cell surface molecule configurations [37] and the presence of growth factors, cytokines, or other paracrine signals in the recipient testis are likely to be important in determining whether donor stem cells can survive and undergo differentiation. Rat spermatogenesis in mouse testis is supported by mouse Sertoli cells [12], and in the present study there was no indication that donor Sertoli cells were present in the recipient seminiferous tubules. Transplanted donor cell populations contain Sertoli cells, but these cells have stopped dividing [38] and have little opportunity to colonize the mouse basement membrane already occupied by endogenous Sertoli cells. Furthermore, the round germ cells of rabbit and dog in recipient seminiferous tubules were found in association with nonstained mouse ; somatic cells on the basement membrane. Thus, the mouse seminiferous tubule provides a suitable environment for germ cells from distant species, such as rabbit and dog, to interact with supporting cells and associate with the basement membrane. Proliferation of these undifferentiated germ cells occurs for more than 1 yr in both rabbit and dog, with characteristic intercellular bridges. Therefore, the important first steps of xenogeneic colonization can occur across a wide phylogenetic gap. The conditions necessary to support differentiation steps of xenogeneic spermatogenesis now must be determined. Interspecies spermatogonial transplantation provides a unique system for studying the cellular and molecular events that regulate the sequential steps of spermatogenesis. Increasing knowledge of the factors controlling spermatogonial proliferation and differentiation will aid in understanding disturbances of spermatogenesis and will enable us to manipulate the microenvironment of recipient seminiferous tubules to support spermatogenesis of phylogenetically distant donor species. In combination with cryopreservation of germ cells and improved culture systems, spermatogonial transplantation then can be used to preserve valuable genetic material and could ultimately serve as an.
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ABSTRACT The role of DNA repair in mutagenesis was studied in normal, repair-proficient Chinese hamster ovary cells and in two mutant strains that are deficient in excision repair. By using the mutagen 7-bromomethylbenz[a]anthracene 7-BrMeBA ; and the technique of alkaline elution of DNA, the mutants were found to be defective at or before the incision step of excision repair. Dose-responses were determined for cell killing, mutation induction at three loci, and sister chromatid exchanges over a survival range of.1.O-O.1 after 7-BrMeBA treatment. The mutants were 5-fold more sensitive to killing than were the normal cells, but the degree of hypersensitivity to mutation induction varied depending on the mutant strain, the genetic marker, and the dose of mutagen. In each instance, the dose-response curve for mutations was essentially linear in the repair-deficient cells. In the normal cells, however, the curves for induced resistance to thioguanine and azaadenine were complex and were curvilinear with increasing slope at low doses. This behavior may be attributable to saturation of the excision repair system. No difference was seen in the efficiency of inducing ouabain-resistant mutations in the repair-deficient cells compared to the normal cells, indicating a qualitatively different behavior of this marker. These results are consistent with excision repair of 7-BrMeBA damage being error-free in Chinese hamster ovary cells. Sister chromatid exchange, another manifestation of DNA damage, also was induced with greater efficiency in the repair-deficient cells. An understanding of how cellular DNA repair processes influence mutation frequencies is of paramount importance in learning how environmental insults to genetic material may lead to heritable mutations or cancer. Several human genetic diseases suggest causal relationships between defects in repairing DNA damage and increased probabilities of developing neoplasia 1 ; . The best known syndrome is xeroderma pigmentosum XP ; , which has well-documented deficiencies in excision repair in response to UV radiation or certain chemicals 2-6 ; . In culture, fibroblasts from XP individuals show increased sensitivity to killing and mutation induction by various mutagens 4, 6-9 ; , establishing a direct link between mutagenesis and carcinogenesis. The role of DNA repair in mutagenesis in diverse organisms has been studied with respect to the particular repair pathway acting on the DNA lesions. In bacteria the uvr excision repair pathway appears to be an error-free process, and mutagenesis is a consequence mainly of an induced error-prone system, termed "SOS" repair 10, 11 ; . In yeast also, chemical and UV mutagenesis are dependent on the integrity of certain repair systems 12, 13 ; . In mammalian cells the genetic control of mutagenesis is not well understood. Most studies with fibroblasts from XP and normal persons have indicated that the UV excision repair system is essentially error-free 8, 9, 14 ; although and tiagabine.
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Fresh clam meat 100 g ; was measured and added to 100 ml of distilled water. The mixture was blended three times for 30 s at maximum speed, incubated 1 h at and then centrifuged at 5000 g. The supernatant 100 l ; was used for hemagglutination. The titer was determined by serial 2-fold dilution and defined as the dilution factor. The blood type did not affect the hemagglutination titer.
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Type I disease, including bone problems and organ enlargement, can be successfully relieved through administration of mannose-terminated placental or recombinant glucocerebrosidase. Types II and III, the acute neuronopathic or infantile and subacute neuronopathic or juvenile varieties, respectively, are more severe forms of Gaucher's disease. These forms of Gaucher's disease typically involve neurologic signs, which currently available enzyme replacement therapies are unable to reverse. The first data demonstrating the clinical effectiveness of weekly i.v. infusions of placental derived glucocerebrosidase for the treatment of Gaucher's disease were reported in 1990 by Barton et al. Proc. Natl. Acad. Sci. USA 87, 1913 1916 ; . In April of 1991, placental derived glucocerebrosidase Ceredase ; was approved by the FDA. By this time research into a recombinant means to express the protein was in progress. The driving force behind this effort was the anticipated lack of supply of placental derived material when full marketing would be achieved. A recombinant production system utilizing a dihydrofolate reductase DHFR ; deficient Chinese hamster ovary CHO ; cell line was developed. The cDNA for human b-glucocerebrosidase!
Group of 405 primary care patients with uncomplicated dysthymia, major depression, or both Figure 1 ; . This cohort consisted of middle-aged patients, the majority of whom were female, white, employed, earning an income exceeding the poverty level, married, and living with others Table 1 ; . Depressive symptoms of the 405 protocol-eligible patients as measured by the HSCL-2016 were moderate to severe; primary care physicians already had prescribed an antidepressant medication for 90% of them. A substantial minority of patients screened positive for symptoms of generalized anxiety, panic, or both. On the WHODAS18 subscales, patients acknowledged only mild limitations in self-care but moderate to severe limitations in performing life activities and societal participation. The relationship of patient characteristics with and ting
Fig ure 3 Positron emission tomography imaging using [11 C]raclopride ; reveals that the human brain striatum ; occupancy of D2 by quetiapine and clozapine rapidly falls off within 24 hours, in contrast to that for ha loperi dol, which maintains its D2 occupancy constant over 24 hours adapted from refs. 38, 53, 63 and thioguanine.
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Thiopurine methyltransferase polymorphisms that affect mercaptopurine therapy. a | The leukaemia drug mercaptopurine MP ; is converted by the enzyme hypoxanthine phosphoribosyl transferase HPRT ; to its active metabolites, thioguanine nucleotides TGNs ; . TGNs are incorporated into DNA and result in antileukaemic effects, but also in myelosuppression.The enzyme thiopurine methyltransferase TPMT ; competes for the MP substrate, catabolizing it to methylmercaptopurine MeMP ; , an inactive metabolite. b | The population frequency distribution of TPMT activity displays a trimodal pattern. The three modes of TPMT activity indicated here in erythrocytes ; correspond to 0.3% of the population being homozygous mut mut ; for mutations in TPMT, 10% being heterozygous wt mut ; for mutations in TPMT, and 90% being wild-type wt wt ; TPMT. c | TPMT genotype is related to phenotype. Mut mut homozygotes have very little TPMT activity, and therefore accumulate excess amounts of intracellular TGNs, leading to myelosuppression and an increased risk of secondary cancers. Wt wt homozygotes express high levels of this enzyme and produce low levels of active TGNs, which decreases the risk of myelosuppression, but might also increase the risk of relapse of the leukaemia. Heterozygotes wt mut ; have intermediate TGN levels. NATURE REVIEWS | CANCER VOLUME 1 | NOVEMBER 2001 | 99 DH Insight Briefing October 2005 Oncology - Page 40 Defined Health, 2005.
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