A Comparison of Tomato Fruit Chloroplast and Chromoplast DNAs as Analyzed with Restriction Endonucleases

Chromoplast DNA was isolated from ripe-red tomato fruits, andits structure compared with that of chloroplast DNAs from maturegreen fruits and leaves. There was a good correspondence betweenthe structures analyzed by BamHI or EcoRI digestion and by hybridizationwith a probe for the gene of the ribulose 1,5-bisphosphate carboxylase/oxygenaselarge subunit. ¹ Present address: Koryo International College, Nisshin, Aichi470-01, Japan. (Received November 5, 1984; Accepted February 6, 1985)     

Chromosomal localization of the Epstein-Barr virus (EBV) genome in Bloom's syndrome B-lymphoblastoid cell lines transformed with EBV

The localization of the Epstein-Barr virus (EBV) genome in chromosomes of human B-lymphoblastoid cell lines (LCLs) transformed with EBV, and the effect of EBV DNA on the level of sister chromatid exchange (SCE) in Bloom's syndrome (BS) B-LCLs, were examined with chromosomal in situ hybridization techniques using a ³H-EBV DNA probe. EBV DNA was detected in chromosomes 1–5 and 13–15 at specific G band regions in BS as well as in normal B-LCLs, regardless of SCE. Several chromosomal sites (1p31, 1q31, 4q22–24, 5q21, 13q21, 14q21) carrying EBV DNA seemed to be very characteristic in normal as well as in BS B-LCLs. There was no statistically significant difference in silver grain counts due to EBV DNA and their distribution in different chromosomes or groups among normal and BS B-LCLs with normal and high SCE. These findings strongly indicate that EBV infection did not introduce a correcting factor for BS SCE.     

Biochemical studies on the mechanism of difference in the renal toxicity of 5-hydroxy-L-tryptophan between Sprague Dawley and Wistar rats.

The biochemical mechanisms of the renal toxicity of 5-hydroxy-L-tryptophan to rats were studied using Wistar and Sprague Dawley rats, which had different LD50 values. When the amino acid was injected intraperitoneally, Wistar rats, which had a low LD50 value of 5-hydroxy-L-tryptophan, excreted larger amonts of serotonin and smaller amounts of 5-hydroxyindole acetic acid into the urine than Spraque Dawley rats, which had a high LD50 value. The activity of renal aromatic L-amino acid decarboxylase was higher in Wistar rats than in Sprague Dawley rats, while the activity of renal aromatic amino acid transaminase was in an opposite relationship. The activity of renal monoamine oxidase was almost the same in both strains and the activity of renal UDP glucuronyltransferase in Wistar rats was higher than in Sprague Dawley rats. Since the renal damage caused in rats by 5-hydroxy-L-tryptophan was very similar to that caused by serotonin, the amine formed from the administered amino acid was thought to be an important factor for the renal necroses, and difference in serotonin formation from the administered precursor amino acid may be one of the important factors leading to the difference in LD50 values in the two strains of rats.     

Fast polyspermy block and activation potential : Correlated changes during oocyte maturation of a starfish

Sperm entry into the oocyte of the starfish, Asterina pectinifera, was prevented when the membrane potential of the oocyte was held more positive than −10 to −5 mV, and multiple sperm entries were induced when the potential was held more negative. Based on this potential-dependent fertilization block mechanism, it was demonstrated that an activation potential (AVP) which is induced immediately after the attachment of the first sperm to the egg surface plays the role of a fast polyspermy block. The AVP-mediated polyspermy block mechanism develops as the oocyte matures and deteriorates as it ages. AVPs of mature oocytes exceeded −5 mV (the critical potential level for fertilization block) within 1 sec, and the potential stayed at +12 mV even after the initiation of fertilization membrane elevation. Consequently, the entry of a second sperm is prevented. In contrast, AVPs of overripe oocytes took about 15 sec to attain −5 mV, or they did not attain −5 mV at all. In overripe oocytes multiple sperm entries were associated with “step depolarization(s)” in the rising phase of the AVPs before membrane elevation took place. Immature oocytes generated AVPs associated with sperm entries, but without membrane elevation. AVPs in immature oocytes were characterized by the step depolarization(s) in the rising phase, and an AVP could be evoked again by a second insemination 20 min after the first insemination. These findings indicate that immature oocytes lack both fast and slow polyspermy block mechanisms.     

Macrophage colony-stimulating factor is produced by human T lymphoblastoid cell line, CEM-ON: identification by amino-terminal amino acid sequence analysis

A subclone, designated CEM-ON, derived from an azaguanine-resistant human leukemic T cell line, CEM-AG(R), constitutively secretes a colony-stimulating factor (CSF) which stimulates the production of macrophages from murine bone marrow progenitor cells. This CSF has been purified from serum-free conditioned medium. Highly purified CEM-ON CSF with a specific activity of 4.7 X 10(7) units/mg protein was obtained. Amino-terminal sequence analysis showed that the first 27 amino acids were identical to the amino-terminal sequence of the M-CSF (CSF-1) based on the cDNAs for human M-CSF. On SDS-PAGE analysis, CEM-ON CSF had an apparent molecular weight of 33,000-43,000; following reduction with 2-mercaptoethanol, this migrated as a 20,000-24,000 subunit, suggesting a homodimer structure. These results show that a human T cell line, CEM-ON, secretes M-CSF into its medium.     

Two Types of Alpha Satellite DNA in Distinct Chromosomal Locations in Azara's Owl Monkey

Alpha satellite DNA is a repetitive sequence known to be a major DNA component of centromeres in primates (order Primates). New World monkeys form one major taxon (parvorder Platyrrhini) of primates, and their alpha satellite DNA is known to comprise repeat units of around 340 bp. In one species (Azara''s owl monkey Aotus azarae) of this taxon, we identified two types of alpha satellite DNA consisting of 185- and 344-bp repeat units that we designated as OwlAlp1 and OwlAlp2, respectively. OwlAlp2 exhibits similarity throughout its entire sequence to the alpha satellite DNA of other New World monkeys. The chromosomal locations of the two types of sequence are markedly distinct: OwlAlp1 was observed at the centromeric constrictions, whereas OwlAlp2 was found in the pericentric regions. From these results, we inferred that OwlAlp1 was derived from OwlAlp2 and rapidly replaced OwlAlp2 as the principal alpha satellite DNA on a short time scale at the speciation level. A less likely alternative explanation is also discussed.     

Silencing Mutant Ataxin-3 Rescues Motor Deficits and Neuropathology in Machado-Joseph Disease Transgenic Mice

Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is an autosomal dominantly-inherited neurodegenerative disorder caused by the over-repetition of a CAG codon in the MJD1 gene. This expansion translates into a polyglutamine tract that confers a toxic gain-of-function to the mutant protein – ataxin-3, leading to neurodegeneration in specific brain regions, with particular severity in the cerebellum. No treatment able to modify the disease progression is available. However, gene silencing by RNA interference has shown promising results. Therefore, in this study we investigated whether lentiviral-mediated allele-specific silencing of the mutant ataxin-3 gene, after disease onset, would rescue the motor behavior deficits and neuropathological features in a severely impaired transgenic mouse model of MJD. For this purpose, we injected lentiviral vectors encoding allele-specific silencing-sequences (shAtx3) into the cerebellum of diseased transgenic mice expressing the targeted C-variant of mutant ataxin-3 present in 70% of MJD patients. This variation permits to discriminate between the wild-type and mutant forms, maintaining the normal function of the wild-type allele and silencing only the mutant form. Quantitative analysis of rotarod performance, footprint and activity patterns revealed significant and robust alleviation of gait, balance (average 3-fold increase of rotarod test time), locomotor and exploratory activity impairments in shAtx3-injected mice, as compared to control ones injected with shGFP. An important improvement of neuropathology was also observed, regarding the number of intranuclear inclusions, calbindin and DARPP-32 immunoreactivity, fluorojade B and Golgi staining and molecular and granular layers thickness. These data demonstrate for the first time the efficacy of gene silencing in blocking the MJD-associated motor-behavior and neuropathological abnormalities after the onset of the disease, supporting the use of this strategy for therapy of MJD.     

A novel,potent, and orally active VLA-4 antagonist with good aqueous solubility: trans-4-[1-[[2-(5-Fluoro-2-methylphenylamino)-7-fluoro-6-benzoxazolyl]acetyl]-(5S)-[methoxy(methyl)amino]methyl-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic acid

We have carried out the optimization of substituents at the C-3 or the C-5 position on the pyrrolidine ring of VLA-4 antagonist 3 with 2-(phenylamino)-7-fluorobenzoxazolyl moiety for the purpose of improving in vivo efficacy while maintaining good aqueous solubility. As a result, we successfully increased in vitro activity in the presence of 3% human serum albumin and achieved an exquisite lipophilic and hydrophilic balance of compounds suitable for oral administrative regimen. The modification resulted in the identification of zwitterionic compound 7n with (5S)-[methoxy(methyl)amino]methylpyrrolidine, which significantly alleviated bronchial hyper-responsiveness to acetylcholine chloride at 12.5 mg/kg, p.o. in a murine asthma model and showed favorable aqueous solubility (JP1, 89 μg/mL; JP2, 462 μg/mL). Furthermore, this compound showed good oral bioavailability (F = 54%) in monkeys.