A cytoplasmic RNA vector derived from nontransmissible Sendai virus with efficient gene transfer and expression

We have recovered a virion from defective cDNA of Sendai virus (SeV) that is capable of self-replication but incapable of transmissible-virion production. This virion delivers and expresses foreign genes in infected cells, and this is the first report of a gene expression vector derived from a defective viral genome of the Paramyxoviridae. First, functional ribonucleoprotein complexes (RNPs) were recovered from SeV cloned cDNA defective in the F (envelope fusion protein) gene, in the presence of plasmids expressing nucleocapsid protein and viral RNA polymerase. Then the RNPs were transfected to the cells inducibly expressing F protein. Virion-like particles thus obtained had a titer of 0.5 x 10(8) to 1. 0 x 10(8) cell infectious units/ml and contained F-defective RNA genome. This defective vector amplified specifically in an F-expressing packaging cell line in a trypsin-dependent manner but did not spread to F-nonexpressing cells. This vector infected and expressed an enhanced green fluorescent protein reporter gene in various types of animal and human cells, including nondividing cells, with high efficiency. These results suggest that this vector has great potential for use in human gene therapy and vaccine delivery systems.     

Cloning of a Sphingomonas paucimobilis SYK-6 Gene Encoding a Novel Oxygenase That Cleaves Lignin-Related Biphenyl and Characterization of the Enzyme

Sphingomonas paucimobilis SYK-6 transforms 2,2′-dihydroxy-3,3′-dimethoxy-5,5′-dicarboxybiphenyl (DDVA), a lignin-related biphenyl compound, to 5-carboxyvanillic acid via 2,2′,3-trihydroxy-3′-methoxy-5,5′-dicarboxybiphenyl (OH-DDVA) as an intermediate (15). The ring fission of OH-DDVA is an essential step in the DDVA degradative pathway. A 15-kb EcoRI fragment isolated from the cosmid library complemented the growth deficiency of a mutant on OH-DDVA. Subcloning and deletion analysis showed that a 1.4-kb DNA fragment included the gene responsible for the ring fission of OH-DDVA. An open reading frame encoding 334 amino acids was identified and designated ligZ. The deduced amino acid sequence of LigZ had 18 to 21% identity with the class III extradiol dioxygenase family, including the β subunit (LigB) of protocatechuate 4,5-dioxygenase of SYK-6 (Y. Noda, S. Nishikawa, K.-I. Shiozuka, H. Kadokura, H. Nakajima, K. Yano, Y. Katayama, N. Morohoshi, T. Haraguchi, and M. Yamasaki, J. Bacteriol. 172:2704–2709, 1990), catechol 2,3-dioxygenase I (MpcI) of Alcaligenes eutrophus JMP222 (M. Kabisch and P. Fortnagel, Nucleic Acids Res. 18:3405–3406, 1990), the catalytic subunit of the meta-cleavage enzyme (CarBb) for 2′-aminobiphenyl-2,3-diol from Pseudomonas sp. strain CA10 (S. I. Sato, N. Ouchiyama, T. Kimura, H. Nojiri, H. Yamane, and T. Omori, J. Bacteriol. 179:4841–4849, 1997), and 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) of Escherichia coli (E. L. Spence, M. Kawamukai, J. Sanvoisin, H. Braven, and T. D. H. Bugg, J. Bacteriol. 178:5249–5256, 1996). The ring fission product formed from OH-DDVA by LigZ developed a yellow color with an absorption maximum at 455 nm, suggesting meta cleavage. Thus, LigZ was concluded to be a ring cleavage extradiol dioxygenase. LigZ activity was detected only for OH-DDVA and 2,2′,3,3′-tetrahydroxy-5,5′-dicarboxybiphenyl and was dependent on the ferrous ion.Lignin is the most common aromatic compound in the biosphere, and the degradation of lignin is a significant step in the global carbon cycle. Lignin is composed of various intermolecular linkages between phenylpropanes and guaiacyl, syringyl, p-hydroxyphenyl, and biphenyl nuclei (5, 34). Lignin breakdown therefore involves multiple biochemical reactions involving the cleavage of intermonomeric linkages, demethylations, hydroxylations, side-chain modifications, and aromatic ring fission (10, 11, 19, 40).Soil bacteria are known to display ample metabolic versatility toward aromatic substrates. Sphingomonas paucimobilis SYK-6 (formerly Pseudomonas paucimobilis SYK-6) has been isolated with 2,2′-dihydroxy-3,3′-dimethoxy-5,5′-dicarboxybiphenyl (DDVA) as a sole carbon and energy source. This strain can also grow on syringate, 3-O-methylgallic acid (3OMGA), vanillate, and other dimeric lignin compounds, including β-aryl ether, diarylpropane (β-1), and phenylcoumaran (15). Analysis of the metabolic pathway has indicated that the dimeric lignin compounds are degraded to protocatechuate or 3OMGA (15) and that these compounds are cleaved by protocatechuate 4,5-dioxygenase encoded by ligAB (30). Among the dimeric lignin compounds, the degradation of β-aryl ether and the biphenyl structure is the most important, because β-aryl ether is most abundant in lignin (50%) and the biphenyl structure is so stable that its decomposition should be rate limiting in lignin degradation. We have already characterized the β-etherase and Cα-dehydrogenase genes (23–26) (ligFE and ligD, respectively) involved in the degradation of β-aryl ether. In this study, we focused on the genes responsible for the degradation of DDVA in SYK-6.In the proposed DDVA metabolic pathway of S. paucimobilis SYK-6 illustrated in Fig. ​Fig.1A,1A, DDVA is first demethylated to produce the diol compound 2,2′,3-trihydroxy-3′-methoxy-5,5′-dicarboxybiphenyl (OH-DDVA). OH-DDVA is then degraded to 5-carboxyvanillic acid (5-CVA), and this compound is converted to 3OMGA (15). The resulting product is cleaved by protocatechuate 4,5-dioxygenase. A ring cleavage enzyme for OH-DDVA has been thought to be involved in this pathway because the production of 5-CVA from OH-DDVA resembles the formation of benzoic acid from biphenyl by 2,3-dihydroxybiphenyl through the sequential action of a meta cleavage enzyme and a meta-cleavage compound hydrolase (Fig. ​(Fig.1B)1B) (1, 9, 13, 18, 21, 28). Open in a separate windowFIG. 1(A) Proposed metabolic pathway for DDVA by S. paucimobilis SYK-6. (B) Pathway for the conversion of 2,3-dihydroxybiphenyl (2,3-DHBP) to benzoate by the polychlorinated biphenyl-degrading bacteria. The proposed DDVA metabolic pathway follows the previous one (15). Enzymes: LigZ, OH-DDVA oxygenase; LigAB, protocatechuate 4,5-dioxygenase; BphC, 2,3-dihydroxybiphenyl 1,2-dioxygenase; BphD, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase. TCA, tricarboxylic acid.In this study, we isolated the ligZ gene encoding a ring cleavage enzyme for OH-DDVA. The nucleotide sequence of the gene was determined, and the ligZ gene product was characterized.     

A Multicenter,Randomized, Double-Blind,Placebo-Controlled Trial of High-Dose Rebamipide Treatment for Low-Dose Aspirin-Induced Moderate-to-Severe Small Intestinal Damage

Background

Low-dose aspirin (LDA) frequently causes small bowel injury. While some drugs have been reported to be effective in treating LDA-induced small intestinal damage, most studies did not exclude patients with mild damage thought to be clinically insignificant.

Aim

We conducted a multicenter, randomized, double-blind, placebo-controlled trial to assess the efficacy of a high dose of rebamipide, a gastroprotective drug, for LDA-induced moderate-to-severe enteropathy.

Methods

We enrolled patients who received 100 mg of enteric-coated aspirin daily for more than 3 months and were found to have more than 3 mucosal breaks (i.e., erosions or ulcers) in the small intestine by capsule endoscopy. Eligible patients were assigned to receive either rebamipide 300 mg (triple dose) 3 times daily or placebo for 8 weeks in a 2:1 ratio. Capsule endoscopy was then repeated. The primary endpoint was the change in the number of mucosal breaks from baseline to 8 weeks. Secondary endpoints included the complete healing of mucosal breaks at 8 weeks and the change in Lewis score (an endoscopic score assessing damage severity) from baseline to 8 weeks.

Results

The study was completed by 38 patients (rebamipide group: n = 25, placebo group: n = 13). After 8 weeks of treatment, rebamipide, but not placebo, significantly decreased the number of mucosal breaks (p = 0.046). While the difference was not significant (p = 0.13), the rate of complete mucosal break healing in the rebamipide group (32%, 8 of 25) tended to be higher than that in the placebo group (7.7%, 1 of 13). Rebamipide treatment significantly improved intestinal damage severity as assessed by the Lewis score (p = 0.02), whereas placebo did not. The triple dose of rebamipide was well tolerated.

Conclusions

High-dose rebamipide is effective for the treatment of LDA-induced moderate-to-severe enteropathy.

Trial Registration

UMIN Clinical Trials Registry UMIN000003463     

Comparison of conventional and liquid-based cytology, and human papillomavirus testing using SurePath preparation in Japan

We compared the detection rate of cervical neoplasias between a liquid-based cytology (LBC) method using SurePath and the conventional method. We also studied the feasibility of human papillomavirus (HPV) typing by linear array assay. Cytological specimens from 1551 Japanese women were prepared using the conventional and SurePath methods; the cytological and histological results from biopsy samples were compared. HPV typing using an HPV linear array assay was carried out on residual specimens using the SurePath method. The cytodiagnostic results showed a concordance rate of 85.3% (Κ= 0.46) between the two methods. The sensitivity of lesions histopathologically diagnosed as CIN1 or above was not significantly different between the two methods (P = 0.575-1.000). The receiver operating characteristic curve analysis of the detectability in CIN2 or above revealed no significant difference between the two methods (P = 0.096). Among the 44 patients who underwent HPV typing using a linear array assay, 33 samples were eligible for HPV testing and were stored at ambient temperature. In conclusion, the SurePath and conventional methods have equivalent abilities for detecting cervical lesions. After preparation for cytological diagnosis, use of the remaining cells from the SurePath specimens to perform HPV typing using the linear array method could be feasible.     

Preparation and characterization of novel 4-bromo-3,4-dimethyl-1-phenyl-2-phospholene 1-oxide and the analogous phosphorus heterocycles or phospha sugars

4-Bromo-3,4-dimethyl-1-phenyl-2-phospholene 1-oxide (3c) was first synthesized from 3,4-dimethyl-1-phenyl-2-phospholene 1-oxide (2c) by a bromo-radical substitution reaction occurred at C-4 position by N-bromosuccinimide and 2,2′-azobisisobutyronitrile. The novel phospha sugar analogue 3c exerted high anti-proliferative effect on U937 cells evaluated by MTT in vitro methods and was much more efficient than that of Gleevec®, which is known as a molecule targeting chemotherapeutical agent. The substitution of 2-phospholenes at C-3 and C-4 position with methyl groups as well as 4-bromo substituent suggests a good anti-proliferative effect.     

Insecticidal Constituents from the Argentine Liverwort Plagiochila bursata

The new 2,3‐secoaromadendrane 1 , together with the known compounds plagiochilines A and M ( 2 and 3 , resp.), fusicogigantone A ( 4 ), and 1,4‐dimethylazulene ( 5 ) were isolated from an Argentine collection of the liverwort Plagiochila bursata. Structures were elucidated by extensive 1D‐ and 2D‐NMR studies. Compounds 2 and 4 , incorporated to the larval diet at 100 μg per g of diet, reduced the larval growth of Spodoptera frugiperda (Lepidoptera: Noctuidae) by 66±29% and 25±8% and produced 55 and 75% larval mortality at early instars and 20 and 25% pupal mortality, respectively. Treatment with compound 2 also produced abdomen and wing malformation in adults leading to impossibility to mate.     

Zierane sesquiterpene lactone,cembrane and fusicoccane diterpenoids,from the Tahitian liverwort Chandonanthus hirtellus

Cembrane-type diterpenoids, 13,18,20-epi-iso-chandonanthone (1) and (8E)-4α-acetoxy-12α,13α-epoxycembra-1(15),8-diene (2), two fusicoccane-type diterpenoids, fusicoauritone 6α-methyl ether (3) and 6β,10β-epoxy-5β-hydroxyfusicocc-2-ene (4) and a zierane sesquiterpene γ-lactone, chandolide (5) were isolated from the Tahitian liverwort Chandonanthus hirtellus (Web.) Mitt., together with eight known diterpenoids, chandonanthine (6), fusicogigantone A (7), fusicogigantone B (8), fusicogigantepoxide (9), anadensin (10), fusicoauritone (11), ent-verticillol (12) and ent-epi-verticillol (13). Their structures were established by a combination of extensive NMR spectroscopy and/or X-ray crystallographic analyses. Compounds 1, 5 and 10 showed weak cytotoxic activity against HL-60. Compound 3 also indicated weak cytotoxic activity against KB cell lines.