Antimalarial activity of 1-aryl-3,3-dialkyltriazenes

The antimalarial activity of 1-aryl-3,3-dialkyltriazenes to Plasmodium berghei NK-65 in infected mice was evaluated at an intraperitoneal dose of 100mg/kgbw. Some of these compounds were found to possess potent antimalarial activity.     

Gene silencing by RNA interference in the koji mold Aspergillus oryzae

We found the orthologous genes required for RNA interference (RNAi) in the Aspergillus oryzae genome database, and constructed a set of tools for gene silencing using RNAi in A. oryzae. This system utilizes compatible restriction enzyme sites so that only a single target gene fragment is required to create the hairpin RNA cassette. For ease of handling, we also separated the construction of the hairpin RNA cassette for the target gene from its subsequent introduction into the expression vector. Using the brlA gene as a target for RNAi, we detected decreased mRNA levels and a delayed conidiation phenotype in the transformants. Furthermore, even though A. oryzae possesses three copies of the alpha-amylase gene, a single copy of an alpha-amylase RNAi construct was sufficient to downregulate the mRNA levels and decrease the enzymatic activity to 10% of control levels. Gene silencing by RNAi should provide a powerful genetic tool for post-genomic studies of the industrially important fungus A. oryzae.     

Mek1/2 MAPK kinases are essential for Mammalian development, homeostasis, and Raf-induced hyperplasia

The p42/p44 mitogen-activated protein kinase (MAPK) cascade includes Ras, Raf, Mek, and Erk MAPK. To determine the effect of a full knockout at a single level of this signaling pathway in mammals, and to investigate functional redundancy between Mek1 and Mek2, we disrupted these genes in murine and human epidermis. Loss of either protein alone produced no phenotype, whereas combined Mek1/2 deletion in development or adulthood abolished Erk1/2 phosphorylation and led to hypoproliferation, apoptosis, skin barrier defects, and death. Conversely, a single copy of either allele was sufficient for normal development. Combined Mek1/2 loss also abolished Raf-induced hyperproliferation. Human tissue deficient in either Mek isoform was normal, whereas loss of both proteins led to hypoplasia, which was rescued by active Erk2 expression. These data indicate that Mek1/2 are functionally redundant in the epidermis, where they act as a linear relay in the MAPK pathway to mediate development and homeostasis.     

Natural dental caries in molars of osteogenic disorder Shionogi rats

Osteogenic disorder Shionogi (ODS) rats are genetically defective in ascorbic acid biosynthesis. They exhibit a gait abnormality due to dysfunctional bone formation and display various dental abnormalities. Conditions of the oral cavity and tooth quality both influence the development of dental caries. This study was designed to determine the characteristics of dental caries in ODS/ ShiJclod/od rats. Caries were scored and compared among ODS/ShiJclod/od, ODS/ShiJcl+/+, and Jcl:Wistar retired breeders. Among male rats, the caries scores of the ODS/ShiJclod/od and ODS/ShiJcl+/+ groups were similar to each other but greater than those in Jcl:Wistar rats, whereas among female rats, caries scores in ODS/ShiJclod/od animals were equivalent to or somewhat greater than those in ODS/ShiJcl+/+ rats, whose scores were markedly greater than those of Jcl:Wistar rats. The results suggest that ODS/ShiJcl rats were more susceptible to dental caries than were Jcl:Wistar rats. Under the conditions of the study, caries scores between ODS/ ShiJclod/od and ODS/ShiJcl+/+ rats differed only among parous females.     

Characterization of the Rv3378c gene product, a new diterpene synthase for producing tuberculosinol and (13R, S)-isotuberculosinol (nosyberkol), from the Mycobacterium tuberculosis H37Rv genome

The Rv3377c and Rv3378c genes from Mycobacterium tuberculosis are specifically found in the virulent Mycobacterium species, but not in the avirulent species. The Rv3378c-encoded enzyme produced tuberculosinol 2 (5(6), 13(14)-halimadiene-15-ol), 13R-5a and 13S-isotuberculosinol 5b (5(6), 14(15)-halimadiene-13-ol) as its enzymatic products from tuberculosinyl diphosphate 3, indicating that the Rv3378c enzyme catalyzed the nucleophilic addition of a water molecule after the release of a diphosphate moiety. The three enzymatic products 2, 5a, and 5b were produced irrespective of the N- and C-terminal His-tagged Rv3378c enzymes, and of the maltose-binding protein fusion enzyme; the product distribution ratio was identical between the enzymes as 1:1 for 2:5, and 1:3 for 5a:5b. The successful separation of 5a and 5b by a chiral HPLC column provided the first complete assignments of 1H- and 13C-NMR data for 5a and 5b. The enzymatic mechanism for producing 2, 5a, and 5b is proposed here, and the optimal catalytic conditions and kinetic parameters, in addition to the divalent metal effects, are described. Site-directed mutagenesis of Asp into Asn, targeted at the DDXXD motif, resulted in significantly decreased enzymatic activity.     

Molecular characterization of a Cryptosporidium isolate from a banded mongoose Mungos mungo

Cryptosporidium spp. has been found in more than 150 species of mammals, but there has been no report in mongooses. In this study, we report the isolation of Cryptosporidium sp. in a banded mongoose Mungos mungo, which was brought from Tanzania to Japan; the isolate was analyzed genetically to validate the occurrence of a new, host-adapted genotype. Cryptosporidium diagnostic fragments of 18S ribosomal RNA and 70-kDa heat shock protein genes were amplified from this isolate and compared with the other Cryptosporidium species and genotypes reported previously. Analyses showed that the mongoose isolate represents a new genotype, closely related to that of bears.     

Neoculin, a taste-modifying sweet protein, accumulates in ripening fruits of cultivated Curculigo latifolia

Neoculin is a sweet protein with a taste-modifying activity of converting sourness to sweetness. It occurs in the fruits of Curculigo latifolia, a wild plant found in tropical Asia. We successfully cultivated the plant and evaluated the production of neoculin. The neoculin content of the fruit was high for 10 weeks after flowering, following which the yield decreased gradually. The optimal period for harvesting the fruits with sensory activity coincided with this 10-week peak period during which the amount of neoculin was 1-3mg in the whole fruit and 1.3mg/g of pulp. Immunohistochemical staining showed that neoculin occurred in the whole fruit, especially at the basal portion. Although it is known that neoculin comprises an acidic subunit (NAS) with an N-glycosylated moiety and a basic subunit (NBS), protein gel blot analysis revealed the presence of a non-glycosylated NAS species. This suggests the presence of multiple NAS-NBS heterodimers in our cultivar.     

Degradation of caspase-activated DNase by the ubiquitin-proteasome system

DNA fragmentation is one of the most characteristic features of apoptotic cells and caspase-activated DNase (CAD) is considered to be a major nuclease responsible for DNA fragmentation. CAD forms a complex with its inhibitor (ICAD), which is also required for the functional folding of CAD, leading to CAD stabilization in cells. In this paper, we investigated the involvement of the ubiquitin-proteasome system in CAD stability. The expression and ubiquitination of CAD was remarkably increased by MG132 treatment in the absence of ICAD. These results suggest that CAD protein may be preferentially degraded by the ubiquitin-proteasome system in the absence of ICAD to maintain protein quality control.