The effect of pressure on leucine and thymidine incorporation by free-living bacteria and by bacteria attached to sinking oceanic particles

The effect of pressure on upper ocean free-living bacteria and bacteria attached to rapidly sinking particles was investigated through studying their ability to synthesize DNA and protein by measuring their rate of 3H-thymidine and 3H-leucine incorporation. Studies were carried out on samples from the NE Atlantic under the range of pressures (1–430 atm) encountered by sinking aggregates during their journey to the deep-sea bed. Thymidine and leucine incorporation rates per bacterium attached to sinking particles from 200 m were about six and ten times higher, respectively, than the free-living bacterial assemblage. The ratio of leucine incorporation rate per cell to thymidine incorporation rate per cell was significantly different between the larger attached (18.9:1) and smaller free-living (10.4:1) assemblages. The rates of leucine and thymidine incorporation decreased exponentially with increasing pressure for the free-living and linearly for attached bacteria, while there was no significant influence of pressure on cell numbers. At 100 atm leucine and thymidine incorporation rate per free-living bacterium was reduced to 73 and 20%, respectively, relative to that measured at 1 atm. Pressure of 100 atm reduced leucine and thymidine incorporation per attached bacterium to 94 and 70%, and at 200 atm these rates were reduced to 34 and 51%, respectively, relative to those measured at 1 atm. There was no significant uncoupling of thymidine and leucine incorporation for either the free-living or attached bacterial assemblages with increasing pressure, indicating that the processess of DNA and protein synthesis may be equally affected by increasing pressure. It is therefore unlikely that bacteria, originating from surface waters, attached to rapidly sinking particles play a role in particle remineralization below approximately 1000–2000 m. These results may help to explain the occurrence of relatively fresh aggregates on the deep-sea bed that still contain sufficient organic carbon to fuel the rapid growth of benthic micro-organisms; they also indicate that the effect of pressure on microbial processes may be important in oceanic biogeochemical cycles.

Prognostic significance of thymidylate synthase, dihydropyrimidine dehydrogenase and thymidine phosphorylase protein expression in colorectal cancer patients treated with or without 5-fluorouracil-based chemotherapy

Background: Low tumour expression levels of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and thymidine phosphorylase (TP) have been linked with improved outcome for colorectal cancer (CRC) patients treated with 5-fluorouracil (5-FU). It is unclear whether this occurs because such tumours have better prognosis or they are more sensitive to 5-FU treatment.

Synthesis and Phosphorus Sulfur Bond-Cleavage Of 3'- Thiothymidylyl(3'5')Thymidine

The title compound is readily prepared from 5'-O-monomethoxytrityl-3'-thiothymidine (5); cleavage of the P–S bond can be accomplished by mild oxidative hydrolysis.

Kinetic Parameters of Thymidine Kinase and DNA Synthesis During Liver Regeneration: Role Ofthyroid Hormones

The role of thyroid hormones in DNA synthesis and in the activity of Thymidille kinase (TK), a key regulatory enzyme of DNA synthesis was studied in proliferating hepatocytes in vivo. Liver regeneration after partial hepatectomy was used as a model for controlled cell division in rats having different thyroid status - euthyroid, hypothyroid and 3,3',5'-triiodo-L-thyronine (T))-heated hypothyroid. Partial hepatectomy caused a significant elevation of DNA synthesis (p<0.01) in all the three groups compared to their sham-operated counterparts. Hypothyroid liepatectomised animals showed significantly lower (p<0.01) level of DNA synthesis than euthyroid hepatectomised animals. A single subcutaneous close of 1'3 to hypothyroid shamoperated animals resulted in a significant increase (p<0.01) of DNA synthesis in the intact liver. 17tis was comparable to the level of DNA synthesis occurring in regenerating liver of euthyroid animals. In hypothyroid hepatectomised animals, "1'3 showed an additive effect on l)NA synthesis and this group exhibited maximum level of DNA synthesis (p<0.0I ). Studies of the kinetic parameters of TK show that the Michelis-Menten constant, (K111) of TK for thymidine was altered by the thyroid status. K11 increased significantly (p<0.01) in untreated hypothyroid animals when compared to the euthyroid rats. '13 treatment of hypothyroid animals reversed this effect and this group showed the lowest value for K111 (p<0.01). Thus our results indicate that thyroid hormones can influence DNA synthesis during liver regeneration and they may regulate the activity of enzymes such as 17rymidine kinase which are important for DNA synthesis and hence cell division.

Effect Insulin on DNA Synthesis and Kinetic Parameters of Thymidine Kinase During Liver Regenaration

The effect of insulin on cell proliferation in vivo has been studied in hepatectomised streptozotocin- diabetic rats. The extent of cell proliferation in sham and hepatectomized- control, diabetic and insulin treated rats were monitored by determining DNA content and [3H]thymidine incorporation into DNA. The kinetic parameters of thymidine kinase a regulatory enzyme for DNA synthesis was also studied in these groups. The rate of DNA synthesis in liver of streptozotocin -diabetic rats was significantly higher 24 hrs post-hepatectomy compared to control and insulin treated diabetic groups. Kinetic studies of thymidine kinase revealed that there was no change in the Michaelis -Menten constant (Km) whereas maximum velocity (Vmax) was elevated in the diabetic hepatectomized groups compared to control and insulin treated hepatectomized groups. Thus our study elucidates the role of insulin in thymidine kinase activity and DNA synthesis.

Synthesis, binding and cellular uptake properties of oligodeoxynucleotides containing cationic bicyclo-thymidine residues

The synthesis and incorporation into oligodeoxynucleotides of two novel derivatives of bicyclothymidine carrying a cationic diaminopropyl or lysine unit in the C(6′)-β position is described. Compared to unmodified DNA these oligonucleotides show Tm-neutral behavior when paired against complementary DNA and are destabilizing when paired against RNA. Unaided uptake experiments of a decamer containing five lys-bcT units into HeLa and HEK293T cells showed substantial internalization with mostly cytosolic distribution which was not observed in the case of an unmodified control oligonucleotide.

Gene therapy of cancer: Mechanisms of ``bystander effect'' killing in cells expressing the herpes simplex virus-thymidine kinase gene and its potential clinical applications

At the fore-front of cancer research, gene therapy offers the potential to either promote cell death or alter the behavior of tumor-cells. One example makes use of a toxic phenotype generated by the prodrug metabolizing gene, thymidine kinase (HSVtk) from the Herpes Simplex Virus. This gene confers selective toxicity to a relatively nontoxic prodrug, ganciclovir (GCV). Tumor cells transduced with the HSVtk gene are sensitive to 1-50 $\mu$M GCV; normal tissue is insensitive up to 150-250 $\mu$M GCV. Utilizing these different sensitivities, it is possible to selectively ablate tumor cells expressing this gene. Interestingly, if a HSVtk$\sp+$ expressing population is mixed with a HSVtk$\sp-$ population at high density, all the cells are killed after GCV administration. This phenomenon for killing all neighboring cells is termed the "bystander effect", which is well documented in HSVtk$\sp-$ GCV systems, though its exact mechanism of action is unclear.^ Using the mouse colon carcinoma cell line CT26, data are presented supporting possible mechanisms of "bystander effect" killing of neighboring CT26-tk$\sp-$cells. A major requirement for bystander killing is the prodrug GCV: as dead or dying CT26tk$\sp+$ cells have no toxic effect on neighboring cells in its absence. In vitro, it appears the bystander effect is due to transfer of toxic GCV-metabolites, through verapamil sensitive intracellular-junctions. Additionally, possible transfer of the HSVtk enzyme to bystander cells after GCV addition, may play a role in bystander killing. A nude mouse model suggests that in a 50/50 (tk$\sp+$/tk$\sp-$) mixture of CT26 cells the bystander eradication of tumors does not involve an immune component. Additionally in a possible clinical application, the "bystander effect" can be directly exploited to eradicate preexisting CT26 colon carcinomas in mice by intratumoral implantation of viable or lethally irradiated CT26tk$\sp+$ cells and subsequent GCV administration. Lastly, an application of this toxic phenotype gene to a clinical marking protocol utilizing a recombinant adenoviral vector carrying the bifunctional protein GAL-TEK to eradicate spontaneously-arisen or vaccine-induced fibrosarcomas in cats is demonstrated. ^