The N-acylethanolamine-hydrolyzing acid amidase (NAAA)

Bioactive N-acylethanolamines, including the endocannabinoid anandamide and anti-inflammatory N-palmitoylethanolamine, are hydrolyzed to fatty acids and ethanolamine in animal tissues by the catalysis of fatty acid amide hydrolase (FAAH). We recently cloned cDNA of N-acylethanolamine-hydrolyzing acid amidase (NAAA), another enzyme catalyzing the same reaction, from human, rat, and mouse. NAAA reveals no sequence homology with FAAH and belongs to the choloylglycine hydrolase family. The most striking catalytic property of NAAA is pH optimum at 4.5-5, which is consistent with its immunocytochemical localization in lysosomes. In rat, NAAA is highly expressed in lung, spleen, thymus, and intestine. Notably, the expression level of NAAA is exceptionally high in rat alveolar macrophages. The primary structure of NAAA exhibits 33-35% amino acid identity to that of acid ceramidase, a lysosomal enzyme hydrolyzing ceramide to fatty acid and sphingosine. NAAA actually showed a low, but detectable ceramide-hydrolyzing activity, while acid ceramidase hydrolyzed N-lauroylethanolamine. Thus, NAAA is a novel lysosomal hydrolase, which is structurally and functionally similar to acid ceramidase. These results suggest a unique role of NAAA in the degradation of N-acylethanolamines.     

Diversity and Antimicrobial Activity of Endophytic Actinomycetes Isolated from Plant Roots in Thailand

Microbiology - In this study, 37 endophytic actinomycetes were isolated from roots of 14 plant species obtained in Thailand, where biological diversity is known to be high. The isolates were...     

Sequence- and seed-structure-dependent polymorphic fibrils of alpha-synuclein

Synucleinopathies comprise a diverse group of neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. These share a common pathological feature, the deposition of alpha-synuclein (a-syn) in neurons or oligodendroglia. A-syn is highly conserved in vertebrates, but the primary sequence of mouse a-syn differs from that of human at seven positions. However, structural differences of their aggregates remain to be fully characterized. In this study, we found that human and mouse a-syn aggregated in vitro formed morphologically distinct amyloid fibrils exhibiting twisted and straight structures, respectively. Furthermore, we identified different protease-resistant core regions, long and short, in human and mouse a-syn aggregates. Interestingly, among the seven unconserved amino acids, only A53T substitution, one of the familial PD mutations, was responsible for structural conversion to the straight-type. Finally, we checked whether the structural differences are transmissible by seeding and found that human a-syn seeded with A53T aggregates formed straight-type fibrils with short protease-resistant cores. These results suggest that a-syn aggregates form sequence-dependent polymorphic fibrils upon spontaneous aggregation but become seed structure-dependent upon seeding.     

Heterozygous loss of Zbtb38 leads to early embryonic lethality via the suppression of Nanog and Sox2 expression

ObjectivesMammalian DNA methyltransferases are essential to re‐establish global DNA methylation patterns during implantation, which is critical for transmitting epigenetic information to the next generation. In contrast, the significance of methyl‐CpG binding proteins (MBPs) that bind methylated CpG remains almost unknown at this stage. We previously demonstrated that Zbtb38 (also known as CIBZ)—a zinc finger type of MBP—is required for mouse embryonic stem (ES) cell proliferation by positively regulating Nanog expression. However, the physiological function of Zbtb38 in vivo remains unclear.Materials and MethodsThis study used the Cre‐loxP system to generate conditional Zbtb38 knockout mice. Cell proliferation and apoptosis were studied by immunofluorescence staining. Quantitative real‐time PCR, immunoblotting and immunofluorescence were performed to investigate the molecular mechanisms.ResultsGermline loss of the Zbtb38 single allele resulted in decreased epiblast cell proliferation and increased apoptosis shortly after implantation, leading to early embryonic lethality. Heterozygous loss of Zbtb38 reduced the expression of Nanog, Sox2, and the genes responsible for epiblast proliferation, differentiation, and cell viability. Although this early lethal phenotype, Zbtb38 is dispensable for ES cell establishment and identity.ConclusionsThese findings indicate that Zbtb38 is essential for early embryonic development via the suppression of Nanog and Sox2 expression.

Heterozygous loss of Zbtb38 leads to aberrant epiblast cell proliferation and apoptosis shortly after implantation. Heterozygous loss of Zbtb38 reduced the expression of Nanog and Sox2 in ICM and epiblast.     

HnRNP D activates production of HPV16 E1 and E6 mRNAs by promoting intron retention

Human papillomavirus type 16 (HPV16) E1 and E6 proteins are produced from mRNAs with retained introns, but it has been unclear how these mRNAs are generated. Here, we report that hnRNP D act as a splicing inhibitor of HPV16 E1/E2- and E6/E7-mRNAs thereby generating intron-containing E1- and E6-mRNAs, respectively. N- and C-termini of hnRNP D contributed to HPV16 mRNA splicing control differently. HnRNP D interacted with the components of splicing machinery and with HPV16 RNA to exert its inhibitory function. As a result, the cytoplasmic levels of intron-retained HPV16 mRNAs were increased in the presence of hnRNP D. Association of hnRNP D with HPV16 mRNAs in the cytoplasm was observed, and this may correlate with unexpected inhibition of HPV16 E1- and E6-mRNA translation. Notably, hnRNP D40 interacted with HPV16 mRNAs in an HPV16-driven tonsillar cancer cell line and in HPV16-immortalized human keratinocytes. Furthermore, knockdown of hnRNP D in HPV16-driven cervical cancer cells enhanced production of the HPV16 E7 oncoprotein. Our results suggest that hnRNP D plays significant roles in the regulation of HPV gene expression and HPV-associated cancer development.     

Severely reduced production of klotho in human chronic renal failure kidney

We recently identified a novel gene, termed klotho (kl) that is involved in the development of a syndrome in mice resembling human aging. A defect of the kl gene expression in mice leads to multiple disorders including arteriosclerosis, osteoporosis, ectopic calcification, and skin atrophy together with short life-span and infertility. Patients with chronic renal failure (CRF), develop multiple complications that are reminiscent of phenotypes observed in kl mutant mice. Furthermore, the kl gene is mainly expressed in kidney and brain. These evidences above suggest the possible involvement of Klotho function in the complications arising in CRF patients. To investigate the above possibility, we examined the kidneys of 10 clinically or histologically diagnosed CRF cases. The level of kl gene expression was measured by utilizing RNase protection assay. The expression of Klotho protein was assayed by utilizing Western blot analysis and by immunohistochemistry. The levels of kl mRNA expression were greatly reduced in all CRF kidneys. Moreover, the production of Klotho protein was also severely reduced in all CRF kidneys. These results suggest that the decrease in kl gene expression in CRF patients may underlie the deteriorating process of multiple complications in the CRF patients.     

Accumulation and degradation of thiamin-binding protein and level of thiamin in wheat seeds during seed maturation and germination

Changes in the levels of thiamin-binding globulin and thiamin in wheat seeds during maturation and germination were studied. The thiamin-binding activity of the seed proteins increased with seed development after flowering. The thiamin content of the seeds also increased with development. Thiamin-binding activity decreased during seed germination. On the other hand, immunological analysis using an antibody directed against the thiamin-binding protein isolated from wheat seeds showed that the thiamin-binding globulin accumulated in the aleurone layer of the seeds during maturation, and then the protein was degraded and disappeared during seed germination. These results suggested that the thiamin-binding globulin of wheat seeds was synthesized and accumulated in the aleurone layer of the seeds with seed development, similar to the thiamin-binding albumin in sesame seeds, and that thiamin bound to the thiamin-binding globulin in the dormant wheat seeds for germ growth during germination.     

Comparative effect of repeated ingestion of difructose anhydride III and palatinose on the induction of gastrointestinal symptoms in humans

We evaluated the safety and change in fermentability from repeated ingestion of difructose anhydride III (DFAIII) in humans. A randomized controlled single-blind crossover study with thirteen subjects was conducted. Each subject ingested 5 g of DFAIII or palatinose daily for 12 days, before and after which the subject was loaded with 10 g of DFAIII and had breath hydrogen measured from 0 to 9 h (DL test) to evaluate the fermentability of DFAIII. The defecation frequency and abdominal symptom score were the same between each ingestion period. Moreover, DFAIII ingestion had no influence on blood test results. Only the breath hydrogen excretion in post-DFAIII ingestion was slightly higher at h 8 than the pre-ingestion. Consequently, repeated ingestion of DFAIII for 12 days was as safe as palatinose ingestion, especially with respect to abdominal symptoms and blood test results, and its high resistance to enterobacterial fermentation in humans was not impaired.