Drug delivery of anti-restenosis agent by 40 - 60°C heating to porcine aortic smooth muscle cells in vitro.

We investigated the uptake of anti-restenosis agent in vascular smooth muscle cells with heating observing the fluorescence intensity of Oregon green labeled paclitaxel in vitro. The heating temperature to porcine aortic smooth muscle cells was varied from 40 to 60°C in 5 s in order to simulate laser-mediated short-duration heating balloon. The cells were contacted with the agent from 1 to 30 min in 37°C after the heating. We measured the agent uptake characteristics on agent concentration and duration in 37°C as a reference. The uptake of the agent in the cells increased with increasing of both the concentration around the cells and contact duration in the case of 37°C. When the cells were heated with 40°C in 5 s and then contacted with the agent in 30 minutes, the uptake of the agent in the cells significantly increased. The uptake of the agent with 50°C or 60°C in 5 s did not show any increasing. We prospected that 40°C heating to the smooth muscle cells would promote the agent uptake ability of the cells because of homeostasis of the cells.

Novel short-duration heating balloon dilatation with uniform temperature distribution: the heating conditions to suppress neo-intimal hyperplasia.

We investigate the relation between the influences on smooth muscle cells and the chronic performances of our novel short-duration heating balloon dilatation to reveal the heating conditions which can suppress the neo-intimal hyperplasia after our heating dilatations. The temperature of prototype balloon catheter surface was measured during short-duration heating balloon dilatation ex vivo. There existed 2 °C temperature variations in the long direction of prototype balloon catheter at a maximum. The neo-intimal hyperplasia occupancy rate after our short-duration heating dilatations were measured in vivo porcine study. The neo-intimal hyperplasia was suppressed most at 75 °C in balloon peak temperature in vivo. The estimated dead rate of smooth muscle cells at this condition was about 13% by the Arrhenius equation. We think that the suppression of neo-intimal hyperplasia was obtained after our short-duration heating dilatation due to the proper decrease of smooth muscle cells by heating and no thermal damages to the adventitia and surrounding tissues.

Clinical factors affecting the efficacy of vancomycin in methicillin-resistant Staphylococcus aureus pneumonia.

OBJECTIVE: Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen. Patients with risk factors such as long hospital admission and compromised hosts are prone to MRSA infection, particularly MRSA pneumonia that is not resolved effectively and causes significant mortality. To identify the factors affecting the efficacy of vancomycin (VCM) therapy on MRSA pneumonia, a retrospective investigation was carried out. METHODS: Severity rating of pneumonia, pharmacokinetic parameters of VCM, clinical improvement following VCM therapy, clinical data and patient's background were investigated in 40 patients from January 2003 to March 2008. The outcome was evaluated 30 days after the initiation of VCM therapy, and multivariate logistic regression analysis was performed. RESULTS: The 30 day mortality rate was 15.0% (6 patients). As a result of multivariate logistic regression analysis, underlying malignancy and parenteral nutrition as the route of feeding during VCM therapy (odds ratio (OR) = 22.3, 95% confidence interval (CI) = 1.55-322; OR = 12.6, 95% CI = 1.15-139, respectively) were found to be significant factors affecting the survival. No pharmacokinetic parameters of VCM and severity of pneumonia were significant. CONCLUSION: Underlying malignancy and parenteral nutrition, which were associated with nutrition and the immune system, were found to affect the survival after 30 days of VCM therapy.

Terminal deoxynucleotidyltransferase is negatively regulated by direct interaction with proliferating cell nuclear antigen.

BACKGROUND: The repertoires of Ig and TcR are generated by a combinatorial rearrangement of variable (V), diversity (D), and joining (J) segments (V(D)J recombination) in B- and T-cells. Terminal deoxynucleotidyltransferase (TdT) adds extra nucleotides (N nucleotides) at the junctions of the gene segments to enhance the Ig and TcR genes diversity. Using an anti-TdT antibody column, TdT has been purified as a member of a megadalton protein complex from rat thymus. The N region would be synthesized with the large protein complex. RESULTS: The cDNAs for proliferating cell nuclear antigen (PCNA) were isolated by yeast two-hybrid screening as the gene products which directly interacted with TdT. The interaction between PCNA and TdT was confirmed by co-immunoprecipitation, both in vitro and in vivo. TdT binds directly to a PCNA trimer, as shown by gel filtration. TdT interacts with PCNA in its DNA polymerization domain (DPD), but not in its BRCA-1 C-terminal (BRCT) domain. TdT activity was reduced to 17% of the maximum value by TdT/PCNA complex formation. CONCLUSION: TdT interacts directly with PCNA through its DPD. A functional consequence of this interaction is the negative regulation of TdT activity. These findings suggest that TdT catalyses the addition of N nucleotides under the negative control of PCNA during V(D)J recombination.

Terminal deoxynucleotidyltransferase directly interacts with a novel nuclear protein that is homologous to p65.

BACKGROUND: Terminal deoxynucleotidyltransferase (TdT) is a DNA polymerase that enhances Ig and TcR gene diversity in the N region in B- and T-cells. TdT is found as a member of a large protein complex in the lysate of the thymocytes. To elucidate the molecular mechanism of the synthesis of the N region, we first attempted to isolate the genes with products that are interacting directly with TdT. RESULTS: Using a yeast two-hybrid system, we isolated a cDNA clone encoding a novel nuclear protein that interacts with TdT. This protein was designated as TdT interacting factor 1 (TdIF1). TdIF1 has a high degree of homology to the transcription factor p65, which belongs to the nuclear receptor superfamily. TdIF1 contains HMG-I and HMG-Y DNA binding domains (AT-hooks) and can bind to single- and double-stranded DNA. TdT and TdIF1 were co-eluted at position 232 kDa by gel filtration of MOLT4 lysate. TdIF1 can enhance TdT activity fourfold in vitro assay system using oligo(dT)16 as primers. CONCLUSIONS: TdIF1 binds directly to TdT, both in vitro and in vivo. TdIF1 and TdT exist as the members of a 232 kDa protein complex. TdIF1 can enhance TdT activity maximum fourfold in vitro assay system, suggesting that it positively regulates the synthesis of the N region during V(D)J recombination in the Ig and TcR genes.

PAF inhibitory activity of diketopiperazines: structure-activity relationships.

FR900452, a natural product isolated from the culture broth of Streptomyces phaeofaciens No. 7739, was found to inhibit PAF-induced rabbit platelet aggregation with an IC50 of 3.7 x 10(-7)M. FR900452, 1-methyl-3-[1-[5- methylthiomethyl-6-oxo-3-(2-oxo-3-cyclopenten-1-yli- dene)-2-piperazinyl]ethyl]-2-indoline, has an oxocylopentylidene group incorporated as a vinylogous amide in a diketopiperazine skeleton. This unique structure led us to synthesize diketopiperazine derivatives, 3-arylalkyl- 6-substituted-piperazine-2,5-diones. Their observed PAF inhibitory activity suggest that the D-D configuration of diketopiperazine is an important factor for anti-PAF activity and that the hydrophobic aromatic portion may play a specific role in the binding of the diketopiperazine to the PAF receptor.

Studies on macrocyclic lactone antibiotics. XIII. Anti-tubulin activity and cytotoxicity of rhizoxin derivatives: synthesis of a photoaffinity derivative.

Chemical modification of the side chain in rhizoxin, a potent antimitotic agent, was attempted in order to study structure-activity relationships and also to devise a probe for photoaffinity labeling of tubulin. An OsO4/NaIO4 oxidation gave a nor-rhizoxin 20-al (5) which was converted to 20-ol (6) by a NaBH3CN reduction. Starting from these two compounds as key intermediates, a series of Wittig reaction products 7-2, and of 20-O-acylates 13-21 were prepared and their anti-tubulin activity and cytotoxicity were determined. An aryl azide derivative 23 was synthesized as a photoaffinity analogue.