The habitat disruption induces immune-suppression and oxidative stress in honey bees

The honey bee is a major insect used for pollination of many commercial crops worldwide. Although the use of honey bees for pollination can disrupt the habitat, the effects on their physiology have never been determined. Recently, honey bee colonies have often collapsed when introduced in greenhouses for pollination in Japan. Thus, suppressing colony collapses and maintaining the number of worker bees in the colonies is essential for successful long-term pollination in greenhouses and recycling of honey bee colonies. To understand the physiological states of honey bees used for long-term pollination in greenhouses, we characterized their gene expression profiles by microarray. We found that the greenhouse environment changes the gene expression profiles and induces immune-suppression and oxidative stress in honey bees. In fact, the increase of the number of Nosema microsporidia and protein carbonyl content was observed in honey bees during pollination in greenhouses. Thus, honey bee colonies are likely to collapse during pollination in greenhouses when heavily infested with pathogens. Degradation of honey bee habitat by changing the outside environment of the colony, during pollination services for example, imposes negative impacts on honey bees. Thus, worldwide use of honey bees for crop pollination in general could be one of reasons for the decline of managed honey bee colonies.     

Production of tropane alkaloids by transformed root cultures of Atropa belladonna in stirred bioreactors with a stainless steel net

A scale up of transformed root cultures of Atropa belladonna from a 300-ml flask to a 30-l tank was accomplished without any reduction in alkaloid productivity. Cutting treatment of seed cultures showed no distinct effect on root growth, morphology, and alkaloid content in conical flasks during 1 month of culture. Randomly cut roots thus grown were further cultivated in 3-l and 30-l modified stirred bioreactors for a scale-up culture. After 1 month of culture, 1490 mg of tropane alkaloids was produced by a 30-l culture of A. belladonna transformed roots. These roots contained the same level of atropine (5.4 mg/ g dw) as the roots of this plant grown in the field for 12 months and still contained a considerable amount of other alkaloids including 1.6 mg/g dw of 6-β-hydroxyhyoscyamine, 0.9 mg/g dw of scopolamine, and 2.0 mg/g dw of littorine. Received: 12 June 1998 / Revision received: 31 August 1998 / Accepted: 27 October 1998     

Mechanistic insights into tRNA cleavage by a contact-dependent growth inhibitor protein and translation factors

Contact-dependent growth inhibition is a mechanism of interbacterial competition mediated by delivery of the C-terminal toxin domain of CdiA protein (CdiA–CT) into neighboring bacteria. The CdiA–CT of enterohemorrhagic Escherichia coli EC869 (CdiA–CT^EC869) cleaves the 3′-acceptor regions of specific tRNAs in a reaction that requires the translation factors Tu/Ts and GTP. Here, we show that CdiA–CT^EC869 has an intrinsic ability to recognize a specific sequence in substrate tRNAs, and Tu:Ts complex promotes tRNA cleavage by CdiA–CT^EC869. Uncharged and aminoacylated tRNAs (aa-tRNAs) were cleaved by CdiA–CT^EC869 to the same extent in the presence of Tu/Ts, and the CdiA–CT^EC869:Tu:Ts:tRNA(aa-tRNA) complex formed in the presence of GTP. CdiA–CT^EC869 interacts with domain II of Tu, thereby preventing the 3′-moiety of tRNA to bind to Tu as in canonical Tu:GTP:aa-tRNA complexes. Superimposition of the Tu:GTP:aa-tRNA structure onto the CdiA–CT^EC869:Tu structure suggests that the 3′-portion of tRNA relocates into the CdiA–CT^EC869 active site, located on the opposite side to the CdiA–CT^EC869 :Tu interface, for tRNA cleavage. Thus, CdiA–CT^EC869 is recruited to Tu:GTP:Ts, and CdiA–CT:Tu:GTP:Ts recognizes substrate tRNAs and cleaves them. Tu:GTP:Ts serves as a reaction scaffold that increases the affinity of CdiA–CT^EC869 for substrate tRNAs and induces a structural change of tRNAs for efficient cleavage by CdiA–CT^EC869.     

The promoters of two carboxylases in a C4 plant (maize) direct cell-specific, light-regulated expression in a C3 plant (rice)

C₄ plants have two carboxylases which function in photosynthesis. One, phosphoenolpyruvate carboxylase (PEPC) is localized in mesophyll cells, and the other, ribulose bisphosphate carboxylase (RuBPC) is found in bundle sheath cells. In contrast, C₃ plants have only one photosynthetic carboxylase, RuBPC, which is localized in mesophyll cells. The expression of PEPC in C₃ mesophyll cells is quite low relative to PEPC expression in C₄ mesophyll cells. Two chimeric genes have been constructed consisting of the structural gene encoding β-glucuronidase (GUS) controlled by two promoters from C₄ (maize) photosynthetic genes: (i) the PEPC gene (pepc) and (ii) the small subunit of RuBPC (rbcS). These constructs were introduced into a C₃ cereal, rice. Both chimeric genes were expressed almost exclusively in mesophyll cells in the leaf blades and leaf sheaths at high levels, and no or very little activity was observed in other cells. The expression of both genes was also regulated by light. These observations indicate that the regulation systems which direct cell-specific and light-inducible expression of pepc and rbcS in C₄ plants are also present in C₃ plants. Nevertheless, expression of endogenous pepc in C₃ plants is very low in C₃ mesophyll cells, and the cell specificity of rbcS expression in C₃ plants differs from that in C₄ plants. Rice nuclear extracts were assayed for DNA-binding protein(s) which interact with a cis-regulatory element in the pepc promoter. Gel-retardation assays indicate that a nuclear protein with similar DNA-binding specificity to a maize nuclear protein is present in rice. The possibility that differences in pepc expression in a C₃ plant (rice) and C₄ plant (maize) may be the result of changes in cis-acting elements between pepc in rice and maize is discussed. It also appears that differences in the cellular localization of rbcS expression are probably due to changes in a trans-acting factor(s) required for rbcS expression.     

Identification of proteins whose synthesis is preferentially enhanced by polyamines at the level of translation in mammalian cells

In Escherichia coli, several proteins whose synthesis is enhanced by polyamines at the level of translation have been identified. We looked for proteins that are similarly regulated in eukaryotes using a mouse mammary carcinoma FM3A cell culture system. Polyamine deficiency was induced by adding an inhibitor of ornithine decarboxylase, α-difluoromethylornithine, to the medium. Proteins enhanced by polyamines were determined by comparison of protein levels in control and polyamine-deficient cells using two-dimensional gel electrophoresis, and were identified by Edman degradation and/or LC/MALDI-TOF/TOF tandem mass spectrometry. Polyamine stimulation of the synthesis of these proteins at the level of translation was confirmed by measuring levels of the corresponding mRNAs and proteins, and levels of the [³⁵S]methionine pulse-labeled proteins. The proteins identified in this way were T-complex protein 1, β subunit (Cct2); heterogenous nuclear ribonucleoprotein L (Hnrpl); and phosphoglycerate mutase 1 (Pgam1). Since Cct2 was most strongly enhanced by polyamines among three proteins, the mechanism of polyamine stimulation of Cct2 synthesis was studied using NIH3T3 cells transiently transfected with genes encoding Cct2-EGFP fusion mRNA with normal or mutated 5′-untranslated region (5′-UTR) of Cct2 mRNA. Polyamines most likely enhanced ribosome shunting on the 5′-UTR of Cct2 mRNA.     

Enhanced plasma ghrelin levels in rats with streptozotocin-induced diabetes

Human saliva, which contains nitrite, is normally mixed with gastric juice, which contains ascorbic acid (AA). When saliva was mixed with an acidic buffer in the presence of 0.1 mM AA, rapid nitric oxide formation and oxygen uptake were observed. The oxygen uptake was due to the oxidation of nitric oxide, which was formed by AA-dependent reduction of nitrite under acidic conditions, by molecular oxygen. A salivary component SCN⁻ enhanced the nitric oxide formation and oxygen uptake by the AA/nitrite system. The oxygen uptake by the AA/nitrite/SCN⁻ system was also observed in an acidic buffer solution. These results suggest that oxygen is normally taken up in the stomach when saliva and gastric juice are mixed.     

Lipoproteins of Actinomyces viscosus induce inflammatory responses through TLR2 in human gingival epithelial cells and macrophages

Actinomyces viscosus has been suggested to be associated with periodontal disease. However, the pathogenicity of this bacterium is not known. In this study, we examined inflammation-inducing activity by A. viscosus. Whole cells and a lipophilic fraction of A. viscosus ATCC19246 induced production of interleukin-8 and tumor necrosis factor alpha from both human oral epithelial cells and human monocytoid cells. This cytokine production was blocked by lipoprotein lipase treatment of the lipophilic fraction. In addition, anti-Toll-like receptor 2 antibody blocked the cytokine production. These results suggest that lipoprotein of A. viscosus triggers inflammatory responses in periodontitis by activation of Toll-like receptor 2.     

Excess of de novo deleterious mutations in genes associated with glutamatergic systems in nonsyndromic intellectual disability

Little is known about the genetics of nonsyndromic intellectual disability (NSID). We hypothesized that de novo mutations (DNMs) in synaptic genes explain an important fraction of sporadic NSID cases. In order to investigate this possibility, we sequenced 197 genes encoding glutamate receptors and a large subset of their known interacting proteins in 95 sporadic cases of NSID. We found 11 DNMs, including ten potentially deleterious mutations (three nonsense, two splicing, one frameshift, four missense) and one neutral mutation (silent) in eight different genes. Calculation of point-substitution DNM rates per functional and neutral site showed significant excess of functional DNMs compared to neutral ones. De novo truncating and/or splicing mutations in SYNGAP1, STXBP1, and SHANK3 were found in six patients and are likely to be pathogenic. De novo missense mutations were found in KIF1A, GRIN1, CACNG2, and EPB41L1. Functional studies showed that all these missense mutations affect protein function in cell culture systems, suggesting that they may be pathogenic. Sequencing these four genes in 50 additional sporadic cases of NSID identified a second DNM in GRIN1 (c.1679_1681dup/p.Ser560dup). This mutation also affects protein function, consistent with structural predictions. None of these mutations or any other DNMs were identified in these genes in 285 healthy controls. This study highlights the importance of the glutamate receptor complexes in NSID and further supports the role of DNMs in this disorder.     

Development of Neospora caninum cultured with human serum in vitro and in vivo

Because there has been no report of symptomatic Neospora caninum infection in humans, we examined the effect of human serum on the parasite's growth in either a bovine angioendothelial cell or Caco-2 cell culture in vitro and in immunocompromised mice in vivo. There was no difference in intracellular parasite numbers between cells incubated with human serum at 24 hr after challenge and those incubated with fetal bovine serum (FBS), which has no titer for the anti-N. caninum agglutination antibody test. Serum of sheep infected with N. caninum, which has the anti-N. caninum antibody, reduced the numbers of the intracellular parasite significantly. We also showed that there was no inhibitory effect on the intracellular multiplication of the parasite in cells incubated with human serum through incorporation of 3H-uracil. CB-17 scid mice administered human serum daily and challenged with N. caninum died on day 20 or 22 after challenge, when large numbers of parasite clusters were found in the brain, oviduct, adrenal gland, lung, stomach, spleen, skeletal muscle, pancreas, and mesenteric lymph nodes. Scid mice administered FBS survived until the end of the experiment. These results suggest that adult human serum may have no inhibitory effect on the development of N. caninum in vitro and in vivo.     

Amino acid sequence of cytochrome c-552 from a thermophilic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus.

The complete amino acid sequence of cytochrome c-552 from an extremely thermophilic hydrogen bacterium, Hydrogenobacter thermophilus TK-6 (IAM 12695), was determined. It is a single polypeptide chain of 80 residues, and its molecular weight, including heme, was calculated to be 7,599. The sequence of cytochrome c-552 from H. thermophilus TK-6 closely resembles that of cytochromes c-551 from Pseudomonas species. Moreover, the tertiary structure of Hydrogenobacter cytochrome c-552 is suggested to be similar to that of cytochrome c-551 from Pseudomonas aeruginosa. The sequence similarity between Hydrogenobacter cytochrome c-552 and that of other bacteria physiologically related to H. thermophilus is not high.