A Novel Method for the Synthesis of ddA and F-ddA Via Regioselective 2′-O-Deacetylation of 9-(2,5-DI-O-Acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)adenine

Abstract

Regioselective 2′-O-deacetylation of 9-(2,5-di-O-acetyl-3-bromo-3-deoxy-β-D-xylofuranosyl)adenine (1) is achieved by treatment of 1 with β-cyclodextrin (β-CyD) / aq. NaHCO₃ or N₂H₄·H₂O / EtOH. The 9-(5-O-Acetyl-3-bromo-3-deoxy-β-D-xylo-furanosyl)adenine (2) obtained is a common intermediate for the synthesis of 2′,3′-dideoxy-adenosine (ddA) (7) and 9-(2-fluoro-2,3-dideoxy-β-D-threo-pentofuranosyl)-adenine (F-ddA) (9).     

Activation of histidine decarboxylase through post-translational cleavage by caspase-9 in a mouse mastocytoma P-815

L-Histidine decarboxylase (HDC) is the rate-limiting enzyme for histamine synthesis in mammals. Although accumulating evidence has indicated the post-translational processing of HDC, it remains unknown what kinds of proteases are involved. We investigated the processing of HDC in a mouse mastocytoma, P-815, using a lentiviral expression system. HDC was expressed as a 74-kDa precursor form, which is cleaved to yield the 55- and 60-kDa forms upon treatment with butyrate. Alanine-scanning mutations revealed that two tandem aspartate residues (Asp(517)-Asp(518), Asp(550)-Asp(551)) are critical for the processing. Treatment with butyrate caused an increase in the enzyme activity of the cells expressing the wild type HDC, but not in the cells expressing the processing-incompetent mutant. An increase in histamine synthesis by butyrate was accompanied by formation of the 55- and 60-kDa form of HDC. In addition, the in vitro translated 74-kDa form of HDC was found to undergo a limited cleavage by purified human caspase-9, whereas the alanine-substituted mutants were not. Processing and enzymatic activation of HDC in P-815 cells was enhanced in the presence of a Zn(2+) chelator, TPEN. Although treatment with butyrate and TPEN drastically augmented the protease activity of caspase-3, and -9, no apoptotic cell death was observed. Both enzymatic activation and processing of HDC were completely suppressed by a pan-caspase inhibitor, partially but significantly by a specific inhibitor for caspase-9, but not by a caspase-3 inhibitor. These results suggest that, in P-815 cells, histamine synthesis is augmented through the post-translational cleavage of HDC, which is mediated by caspase-9.     

Ceiling culture-derived proliferative adipocytes retain high adipogenic potential suitable for use as a vehicle for gene transduction therapy

Adipose tissue is expected to provide a source of proliferative cells for regenerative medicine and cell-transplantation therapies using gene transfer manipulation. We have recently identified ceiling culture-derived proliferative adipocytes (ccdPAs) from the mature adipocyte fraction as cells suitable as a therapeutic gene vehicle because of their stable proliferative capacity. In this study, we examined the capability of adipogenic differentiation of the ccdPAs compared with stromal vascular fraction (SVF)-derived progenitor cells (adipose-derived stem cells, ASCs) with regard to their multipotential ability to be converted to another lineage and therefore their potential to be used for regenerative medicine research. After in vitro passaging, the surface antigen profile and the basal levels of adipogenic marker genes of the ccdPAs were not obviously different from those of the ASCs. However, the ccdPAs showed increased lipid-droplet accumulation accompanied with higher adipogenic marker gene expression after stimulation of differentiation compared with the ASCs. The higher adipogenic potential of the ccdPAs than the ASCs from the SVF was maintained for 42 days in culture. Furthermore, the difference in the adipogenic response was enhanced after partial stimulation without indomethacin. These results indicate that the ccdPAs retain a high adipogenic potential even after in vitro passaging, thus suggesting the commitment of ccdPAs to stable mature adipocytes after autotransplantation, indicating that they may have potential for use in regenerative and gene-manipulated medicine.     

Intravesical treatment of bladder cancer with recombinant human interferon-β

Summary In order to examine its clinical efficacy, recombinant human interferon- (rIFN-) was instilled intravesically into 51 patients with superficial bladder cancer. Ten patients, who received intermittent intravesical instillation at a dose of (3–36) × 10⁶ U rIFN- on days 1–3 every week, showed no response. Thirty-two patients received intravesical instillation at a dose of (3–36) × 10⁶ U every day for 10–20 days. Eight patients showed partial response, indicating an efficacy rate of 25%. Nine patients received divided doses of 18 × 10⁶ U twice a day every day for 10–20 days. Six patients showed partial response, indicating an efficacy rate of 67%. This value was significantly higher than that obtained by administering divided doses. The response to intravesical instillation therapy with rIFN- varies with treatment protocol. Frequent and longer exposure to rIFN- may induce better regression of superficial bladder cancer. Six incidences of side-effects were found in five cases (9.8%): pollakiuria in one, pain on micturition in two, fever in two, and eruption in one case. All of these side-effects were slight and reversible after drug withdrawal. Laboratory tests showed only a few changes with low severity. Thus, rIFN- is potentially a new drug for instillation therapy of superficial bladder cancer, in view of the absence of adverse effects.     

Decrease in ovarian platelet-activating factor during ovulation in the gonadotropin-primed immature rat

Platelet-activating factor (PAF) is a biologically active phospholipid that is released locally during acute inflammatory reactions and tissue injury. Since there is evidence that the biochemical events of mammalian ovulation resemble an inflammatory reaction, the objective of this study was to determine whether ovarian levels of PAF change during ovulation. At 2-h intervals during the ovulatory process in gonadotropin-primed 25-day-old Wistar rats, the ovaries were extirpated, homogenized, and extracted for lipids. The extracts were subjected to thin-layer chromatography (TLC), and the portion of the silica gel that comigrated with PAF was re-extracted and assayed for PAF activity. The PAF was measured (in fmole equivalents of synthetic PAF) by a bioassay based on the capacity of aliquots of the extracts to release [3H]-serotonin from platelets isolated from whole blood of rabbits and prelabeled with [3H]-serotonin. The ovarian level of PAF decreased (p less than 0.01) by 36% from 6.67 +/- 0.77 to 4.27 +/- 0.45 fmoles/mg ovary by 2 h after treatment with human chorionic gonadotropin (hCG), and it declined another 14% by 4 h after hCG. The ovarian PAF remained at this reduced level for up to 24 h after hCG. The administration of indomethacin (5 mg/rat, s.c.) or epostane (5 mg/rat, s.c.) at 1 h after hCG prevented ovulation, but neither drug affected the decline in ovarian PAF. Preliminary tests showed that the lipid extracts from the ovaries also contained PAF inhibitor(s) that comigrated with PAF on the TLC plates. Similar to PAF, the lipid-soluble inhibitor(s) decreased (p less than 0.05) in the ovaries within 4 h after hCG treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

A cAMP-dependent protein kinase inhibitor modulates the blocking action of ATP and 5-hydroxydecanoate on the ATP-sensitive K+ channel.

We studied the blocking mechanism of 5-hydroxydecanoate, a novel antiarrhythmic agent, on the ATP-sensitive K+ channel in the single ventricular myocytes using the inside-out patch clamp technique. The channel activity in response to 5-hydroxydecanoate varied with each membrane patch corresponding to the sensitivity to ATP. In this condition the exogenous application of cAMP or cAMP-dependent protein kinase (PKA) obviously recovered the ATP-sensitive K+ channel activity after channel deactivation. By contrast, in membrane patches exhibited low sensitivity to ATP, endogenous cAMP-dependent protein kinase inhibitor (PKI) depressed the channel activity and restored the inhibitory action of 5-hydroxydecanoate and ATP on the channel. These results suggest that PKA-PKI system is involved in the regulatory mechanism of gating activity of the ATP-sensitive K+ channel and the blocking action of 5-hydroxydecanoate and ATP appears to be exerted by potentiating the inhibitory action of PKI on the channel.     

Crystallization and preliminary X-ray structure analysis of pigeon egg-white lysozyme.

Calcium binding lysozyme from pigeon egg-white was crystallized by the hanging drop vapor diffusion technique using ammonium sulphate as a precipitant. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), and have unit cell dimensions of a = 34.2 A, b = 34.8 A, and c = 99.4 A. One asymmetric unit contains one molecule of the pigeon lysozyme. The crystals diffract X-rays at least to 2.0 A resolution and are suitable for high resolution structure analysis. The diffraction data up to 3.0 A resolution were collected with a diffraction image processor, DIP100, using a Fuji imaging plate as an area detector. The structure was solved by the molecular replacement technique and refined to an R factor of 0.216. Least-squares fitting of the main-chains of pigeon egg-white lysozyme with those of chicken egg-white lysozyme and baboon alpha-lactalbumin showed that the main-chain folding of pigeon lysozyme is more similar to that of chicken lysozyme than that of alpha-lactalbumin. The largest differences between the pigeon and chicken lysozymes are in the surface loop regions.     

Coexistence of cardiac-type and fast skeletal-type myosin light chains in embryonic chicken cardiac muscle

Myosin from embryonic chicken ventricle contained a light chain component which comigrated with fast skeletal myosin light chain 1 (Lf1) on two dimensional electrophoresis in addition to cardiac type light chains (Lc1 and Lc2). Immunoblot analysis showed that this minor light chain band reacted with anti-Lf1 antibody. Antigens binding with anti-Lc1 and anti-Lf1 antibodies were located on myofibrils in embryonic cardiac muscle cells in vivo and in vitro. From these observations, we conclude that a small amount of Lf1 exists in embryonic chicken cardiac muscle.     

Genomic organization and mutational analysis of HERG, a gene responsible for familial long QT syndrome

Familial long QT syndrome (LQTS) is characterized by prolonged ventricular repolarization. Clinical symptoms include recurrent syncopal attacks, and sudden death may occur as a result of ventricular tachyarrhythmias. Three genes responsible for this syndrome (KVLQT1, HERG, and SCN5A) have been identified so far, and mutations have been reported on the basis of partially characterized genomic organization. To optimize the search for HERG mutations, we have determined the genomic structure of HERG and investigated mutations in LQTS families. Human genomic clones containing the HERG gene were isolated from a human genomic library by using reverse-transcribed polymerase chain reaction (RT-PCR) products from this gene as probes. We determined exon/intron boundaries and flanking intronic sequences by using primers synthesized on the basis of the HERG cDNA sequence available in the DNA database. HERG was shown to consist of 15 exons spanning approximately 19 kb on chromosome 7q35. Subsequently, we synthesized oligonucleotide primers to cover the entire coding region and searched for mutations in 36 Japanese LQTS families. When genomic DNA from each proband was examined by the PCR/single-strand conformation polymorphism technique followed by direct DNA sequencing, five novel mutations were detected. Each mutation was present in affected relatives of the respective proband. This work should increase the efficiency of screening mutations associated with HERG. Received: 4 November 1997 / Accepted: 5 January 1998     

Functional gamma delta T-lymphocyte defect associated with human immunodeficiency virus infections.

BACKGROUND: Antiviral cellular immune responses may influence immunological homeostasis in HIV-infected persons. Recent data indicate that V gamma 9/V delta 2 T lymphocytes display potent cytotoxic activities against human cells infected with certain viruses including HIV. Understanding the role of gamma delta T cells in the course of HIV infection may be helpful for designing novel treatment strategies for HIV-associated disorders. MATERIALS AND METHODS: The constitutive recognition of Daudi cells and monoethyl pyrophosphate (Etpp) by peripheral blood V gamma 9/V delta 2 T cells was assessed using a proliferation assay. The cytotoxicity of Daudi-stimulated lymphocyte populations was measured by chromium release assays. The HIV infectivity for gamma delta T cell clones was determined by measuring the levels of HIV p24 in cell supernatants. The effect of in vitro HIV-infection on cytokine mRNA production by gamma delta T cell clones was assessed by PCR. RESULTS: The constitutive proliferative responses of peripheral blood V gamma 9/V delta 2 T cells and the lytic functions of Daudi-expanded lymphoid cells from HIV+ persons were substantially diminished in comparison with those of HIV-seronegative persons. These alterations were present in asymptomatic HIV+ persons prior to substantial alpha beta CD4+ T cell loss. Productive HIV infection of gamma delta T cells in vitro had no measurable effect either on their proliferative response to Daudi stimuli or on the expression of cytokine mRNAs for IFN-gamma, IL-2, IL-4, IL-5, IL-6, IL-10, and IL-13. CONCLUSIONS: The constitutive responsiveness of V gamma 9/V delta 2 T lymphocytes to Daudi and Etpp is severely altered in HIV+ persons. HIV infection of gamma delta T cells in vitro does not substantially change their cytokine expression or antigenic response.