In vitro splicing analysis showed that availability of a cryptic splice site is not a determinant for alternative splicing patterns caused by +1G-->A mutations in introns of the dystrophin gene.

BACKGROUND: Splicing patterns are critical for assessing clinical phenotype of mutations in the dystrophin gene. However, it is still unclear how to predict alternative splicing pathways in such cases of splice-site mutation in the dystrophin gene. OBJECTIVE: To identify elements determining alternative splicing pathways in intron +1G-->A mutations of the dystrophin gene. RESULTS: We found that exon 25 is spliced out in the +1G-->A mutation in intron 25, resulting in mild Becker muscular dystrophy, and that a cryptic splice site within exon 45 was activated in severe Duchenne muscular dystrophy with a mutation of +1G-->A mutation in 45. Furthermore, in vitro splicing analysis using a pre-constructed expression vector showed that the mutant intron 25 produced one transcript that lacked exon 25. In contrast, the same splice-site mutation in intron 45 produced three splicing products. One product used the same cryptic donor splice site within exon 45 as the in vivo donor site and another product used a cryptic splice site within the vector sequence. Notably, the available cryptic splice site was not activated by the same G-->A mutation of intron 25. CONCLUSION: It was concluded that sequences inserted into the in vitro splicing assay minigene contain cis-elements that determine splicing pathways. By taking other +1G-->A mutations in the introns of the dystrophin gene reported in the literature into consideration, it seems that cryptic splice-site activation is seen only in strong exons. This finding will help to elucidate the molecular pathogenesis of dystrophinopathy and to predict efficiency of induction of exon skipping with antisense oligonucleotides for treatment of Duchenne muscular dystrophy.

Large-scale manufacturing of all four 2'-deoxynucleosides via novel strategies including a chemo-enzymatic process.

A chemical synthesis of 2-deoxyribose-1-phosphate 2 and its enzymatic conversion into purine 2'-deoxynucleosides (dNus) are shown. Besides the chemo-enzymatic process for purine dNus, a modified process for practical dC preparation is also established. Consequently, a series of practical manufacturing processes of all four dNus have been realized via novel strategies.

Development of chemo-enzymatic process and manufacture of deoxynucleosides.

Manufacturing method for 2'-deoxynucleosides (dNus) has been developed using novel chemo-enzymatic process. The method consists of the chemical synthesis of 2'-deoxyribose 1-phosphate (dRP) and the enzymatic conversions of dRP into 2'-deoxyadenosine (dA), 2'-deoxyguanosine (dG) and 2'-deoxycytidine (dC). Large-scale preparation is successfully done, which demonstrates an applicability of this strategy to practical use. As thymidine is already under commercial-scale production, all four dNus are now ready for scale-up.

Antitumor activity of 5'-O-dipalmitoylphosphatidyl 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine is enhanced by long-circulating liposomalization.

We previously synthesized the 5'-O-diacylphosphatidyl derivative of 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine (CNDAC), a novel antitumor nucleoside, and observed it to have a high antitumor activity. Since this compound is readily incorporated into liposomal membranes, we liposomalized the compound using a formulation for conventional and long-circulating liposomes, and investigated the antitumor activity of liposomal 5'-O-dipalmitoylphosphatidyl CNDAC (DPP-CNDAC). Long-circulating liposomes composed of DPP-CNDAC, dipalmitoylphosphatidylcholine, cholesterol and palmityl-D-glucuronide (PGlcUA) (2:2:2:1 as a molar ratio), as well as liposomes containing dipalmitoylphosphatidylglycerol (DPPG) instead of palmityl-D-glucuronide and those composed of only DPP-CNDAC, were injected intravenously into Meth A sarcoma-bearing mice. DPP-CNDAC showed suppression of tumor growth, whereas CNDAC did not at the same concentration, suggesting that 5'-phosphatidylation is useful to enhance therapeutic efficacy. Furthermore, liposomal DPP-CNDAC reduced the acute toxicity, and liposomes containing PGlcUA showed more enhanced activities of reducing tumor growth and increasing the lifetime of the mice than liposomes containing DPPG. To obtain a higher therapeutic efficacy, we injected long-circulating liposomal DPP-CNDAC 5 times. The tumor growth was suppressed to 13.2% (86.8% inhibition), and the survival time of the tumor-bearing mice increased to 128.5% with one completely cured mouse out of five. Next, the effect of DPP-CNDAC incorporation on the in vivo behavior of PGlcUA and DPPG liposomes was examined by a non-invasive method using positron emission tomography (PET). Liposomes were labeled with [2-(18)F]-2-fluoro-2-deoxy-D-glucose, and administered to tumor-bearing mice. PET images and time-activity curves indicated that DPP-CNDAC/PGlcUA-liposomes tended to accumulate in tumor tissues a little bit more than DPP-CNDAC/DPPG-liposomes, although the difference between the two kinds of liposomal distribution was not as marked as between PGlcUA and DPPG liposomes, suggesting that DPP-CNDAC incorporation partly affected the liposomal behavior in vivo but that the long-circulating character of PGlcUA-liposomes might not be fully abolished. Thus, the enhanced therapeutic efficacy of long circulating liposomalized DPP-CNDAC observed here may be due to passive targeting of DPP-CNDAC to the tumor tissue, making this formulation of DPP-CNDAC useful for cancer chemotherapy.

Synthesis and antiviral activity of 5-substituted (2'S)-2'-deoxy-2'-C-methylcytidines and -uridines.

Synthesis of several 5-substituted (2'S)-2'-deoxy-2'-C-methylcytidines (8) and -uridines (6, 11) has been accomplished using radical deoxygenation of the 2'-tert-alcohols via their methyl oxalyl esters as a key reaction. Anti-herpes simplex virus type-1 and -2, and anti-varicella-zoster virus activities of the newly synthesized nucleosides were evaluated. Among them, the 5-iodouracil derivative 6e showed the most potent activity against herpes simplex virus type-1, with an EC50 of 0.14 micrograms/mL without showing cytotoxicity up to 100 micrograms/mL, but had a weak activity against herpes simplex virus type-2 and no activity against varicella-zoster virus up to 50 micrograms/mL in vitro. Although the 5-fluorocytosine derivative 8b had a potent anti-herpes simplex virus type-1 activity (EC50 = 0.22 micrograms/mL), it was rather cytotoxic to the CCRF-HSB-2 human T-cell line (IC50 > or = 1.0 microgram/mL).

Effects of nifedipine on physical dependence on barbital or diazepam in rats.

The effects of nifedipine on the development of physical dependence on barbital and diazepam in rats were examined using the drug-admixed food method. Rats were chronically treated with either barbital- or barbital in combination with nifedipine-admixed food for 28 days, and with either diazepam- or diazepam in combination with nifedipine-admixed food for 26 days, on schedules of gradually increasing doses of barbital or diazepam. Withdrawal was conducted by substituting normal food for drug-admixed food on the last day of the treatment. Co-administration of nifedipine with barbital potentiated weight loss and withdrawal scores after the termination of barbital treatment. However, the withdrawal signs after the termination of diazepam treatment were not affected by co-administration of nifedipine with diazepam. These results suggest that nifedipine potentiates the development of physical dependence on barbital but not diazepam. It is known that co-administration of dihydropyridine derivative nitrendipine suppresses the development of physical dependence on ethanol. Basing on the differences in sensitivity of central depressants, barbiturates, benzodiazepines and ethanol, to three types of voltage-dependent Ca2+ channels, such as L-, N- and T-types studied so far, the development of physical dependence on central depressants may be modified differently by L-type Ca2+ channel blockers, corresponding to respective depressants.

[A case of inclusion body myositis (IBM) associated with lipid-storage vacuoles].

A 62-year-old woman, who has had slowly progressive muscle weakness and wasting of the four limbs for past 10 years, was evaluated by muscle biopsies; i.e. the deltoid, a moderately atrophic muscle, and the quadriceps femoris, a muscle that was only mildly involved. The specimen from the deltoid disclosed the basic muscle structures having been collapsed, containing many fibers undergoing degeneration. There were rimmed vacuoles and infiltration of macrophages and round cells into the muscle fibers and perimysial space. Inclusion bodies containing fibrillary structures were identified in sarcolemmal nuclei by electron microscopy. When the quadriceps femoris muscle was examined, however, a large number of vacuoles were interspersed between myofibrils that contained numerous lipid droplets. There were no pathological changes suggestive of IBM in this specimen. The case described here is unique in that the muscle biopsies from two separate muscle revealed a distinctly different pathology. Although we were unable to determine the cause of lipid-storage in this case, it is our impression that inclusion body myositis is not a uniform disorder but the characteristic pathology to this particular myositis might be an ultimate feature of several diverse degenerative and/or inflammatory muscle disorders.

Ultrastructural localization of anti-Purkinje cell antibody-binding sites in paraneoplastic cerebellar degeneration.

OBJECTIVE: Anti-Purkinje cell antibodies have been found specifically in patients with paraneoplastic cerebellar degeneration. We investigated the ultrastructural localization of antigen-antibody reaction products by means of immune electron microscopy, using an antibody identical to anti-Yo antibody, which occurs most frequently and which immunostains the cytoplasm of Purkinje cells. DESIGN: The antibody used here (anti-Us) was obtained from a patient with paraneoplastic cerebellar degeneration. Based on results indicating 94% homology between the amino acid sequences of the antigens recognized by anti-Us and anti-Yo antibodies, these two antigens are considered to be the same. After immunostaining of the specimen from the rat cerebellum by the avidin-biotin peroxidase complex method, ultrathin sections were examined with an electron microscope without counterstaining. RESULTS: Immunoreaction products were found to be localized on membrane-bound ribosomes and free ribosomes. There were no reaction products in the endoplasmic reticulum lumen, smooth endoplasmic reticulum, Golgi complex, mitochondria, or nucleus. CONCLUSION: Although it has been noted that there is a leucine-zipper motif in the amino acid sequence of the antigenic protein that can bind to DNA, our findings indicate that this protein exists on the ribosome and may regulate protein synthesis rather than gene expression in the nucleus.

Idiopathic AA amyloidosis manifested by autonomic neuropathy, vestibulocochleopathy, and lattice corneal dystrophy.

A 69-year-old Japanese woman with non-familial amyloidosis had polyneuropathy and profound autonomic neuropathy, and kappa chain monoclonal gammopathy. Immunohistopathological examination showed protein AA and protein AP in the amyloid deposits. She showed involvement of the vestibulocochlear nerve and lattice dystrophy of the cornea. Vestibulocochleopathy and corneal lattice dystrophy have been reported in familial amyloid polyneuropathy type IV, Finnish type, but never in non-familial amyloidosis.

Oxygen consumption stimulated by increases in permeability of Na+ of mouse diaphragm muscle in vitro.

1. The effects on oxygen consumption of agents that modify Na(+)-permeability were examined in mouse diaphragm muscle perfused with a bathing solution that contained (+)-tubocurarine in a flow-through mode. Twitch tension and levels of Na+ and K+ in the muscle were also measured. 2. Unstimulated preparations decreased the concentration of oxygen in the bathing solution, indicating a basal level of oxygen consumption. Electrical stimulation of the muscle further decreased the concentration of oxygen. Potassium cyanide eliminated both the basal and the stimulated consumption of oxygen. 3. Veratridine facilitated the effect of stimulation at 0.1 Hz on both the consumption of oxygen and the twitch tension. Ouabain antagonized those effects. 4. Twitch contractions were blocked in the presence of dantrolene sodium or by pretreatment with ethylene glycol. Electrical stimulation of such preparations still caused a residual but considerably decreased consumption of oxygen. Ouabain and tetrodotoxin reduced the residual consumption of oxygen. 5. Ouabain significantly increased the levels of Na+ in the tissue, while veratridine alone did not. The effect of ouabain was further potentiated by the simultaneous presence of veratridine. 6. These results indicate that the enhancement of the sarcolemmal permeability to Na+ increases the rate oxygen consumption. This concept supports the hypothesis that Na+ homeostasis depends on energy consumption.

Sodium salicylate facilitates calcium-dependent release of transmitter at mouse neuromuscular junctions.

1. The effects of sodium salicylate on the frequency of miniature endplate potentials (m.e.p.ps) and on the quantal content of endplate potentials (e.p.ps) in mouse diaphragm muscles at 36 degrees C and 24 degrees C were studied by conventional microelectrode techniques. 2. At 36 degrees C, salicylate (10 mM) elevated the frequency of m.e.p.ps in a manner which was insensitive to [Mg2+]0 and independent of the presence of [Ca2+]0. Lowering the temperature to 24 degrees C abolished the stimulating effect. 2,4-Dinitrophenol (10 microM) had similar effects at 36 degrees C and these disappeared at 24 degrees C. All subsequent experiments were performed at 24 degrees C. 3. Salicylate (10 mM) further increased the frequency (F) of m.e.p.ps stimulated by [Ca2+]0 in a depolarizing solution. When [Ca2+]0 was varied in the absence of salicylate, a linear relationship between ln(F) and ln([ CA2+]0) was obtained. Salicylate shifted this relationship to the left, with respect to the control, without altering the slope. 4. Salicylate (5 mM) also increased the quantal content (m) of e.p.ps in a solution that contained 5 mM Mg2+, in a concentration-dependent fashion. As [Ca2+]0 was varied in the absence of salicylate, a linear relationship between ln(m) and ln[(Ca2+]0) was observed. Salicylate shifted this linear relationship to the left, with respect to the control, without altering the slope. Doubling the concentration of [Mg2+]0 antagonized this effect of salicylate on the quantal content of e.p.ps. 5. These results indicate that salicylate enhances the effect of changing [Ca2+]0. Salicylate probably facilitates the entry of Ca2+ into the nerve terminal or sensitizes the process that is regulated by Ca2 , thereby stimulating the release of transmitter. Surface negative charges may have an important role in the effect of [Ca22]0.