Impact of serine protease inhibitor alpha1-antitrypsin on expression of endoplasmic reticulum stress-induced proinflammatory factors in adipocytes

Obesity-induced endoplasmic reticulum (ER) stress contributes to low-grade chronic inflammation in adipose tissue and may cause metabolic disorders such as diabetes mellitus and dyslipidemia. Identification of high serpina A1 (alpha-1 antitrypsin, A1AT) expression in mouse adipose tissue and adipocytes prompted us to explore the role of A1AT in the inflammatory response of adipocytes under ER stress. We aimed to determine the role of A1AT expression in adipocytes with ER stress during regulation of adipocyte homeostasis and inflammation. To this end, we chemically induced ER stress in A1AT small interfering RNA-transfected differentiating adipocytes using thapsigargin. Induction of CCAAT-enhancer-binding protein homologous protein (CHOP), an ER stress marker, by thapsigargin was lower in A1AT-deficient SW872 adipocytes. Thapsigargin or the proinflammatory cytokine tumor necrosis factor (TNF)α increased basal expression of cytokines such as interleukin (IL)-1β and IL-8 in both SW872 and primary omental adipocytes. This thapsigargin- or TNFα-induced expression of proinflammatory genes was increased by A1AT deficiency. These findings indicate that adipose A1AT may suppress the ER stress response to block excessive expression of proinflammatory factors, which suggests that A1AT protects against adipose tissue dysfunction associated with ER stress activation.     

Cauliflower mosaic virus 35 S promoter directs S phase specific expression in plant cells

Summary An experimental system to study cell cycle specific gene expression in plant cells was developed using protoplasts from tobacco cells synchronized by aphidicolin treatment. Chimeric plasmids consisting either of the chloramphenicol acetyltransferase (CAT) gene downstream of the cauliflower mosaic virus (CaMV) 35 S promoter or the nopaline synthase (nos) promoter were introduced into synchronized protoplasts of four cell cycle stages by electroporation. In the case of the CaMV 35 S promoter cyclic oscillation of CAT activity was observed which paralleled the cell cycle of the recipient cells. The peak of CAT activity was found in the S phase, while no such cyclic change was observed in the case of the nos promoter. This system clearly shows that it is feasible to search for a cell cycle specific promoter. The significance of these observations is discussed in relation to the study of plant cells.     

Experession and integration of genes introduced into highly synchronized plant protoplasts

Summary Chimaeric genes containing the chloramphenicol acetyltransferase (CAT) coding sequence were introduced into protoplasts of suspension-cultured tobacco cells using improved conditions of electroporation (Okada et al. 1986). CAT activity became detectable in the protoplasts within 3 h, was maximal during a period of 18–36 h after electroporation, and then declined gradually. Alpha-amanitin added to the medium abolished the transient expression of the CAT gene. The closed circular form of input DNA was as effective as the linear form for the transient expression. The suspension culture was treated with aphidicolin, and S, G₂, M and G₁ phases were identified in the highly synchronized cell cycle obtained by releasing the cells from the inhibition of DNA synthesis. When a chimacric CAT gene was introduced into M phase protoplasts prepared from the synchronized culture, the transient expression of the CAT gene was 3–4 times higher than when it was introduced into protoplasts of other cell cycle phases. The frequency of stable transformation with a chimaeric neomycin phosphotransferase II gene was studied using the same system. G-418-resistant transformants were obtained from M phase protoplasts at frequencies 2–8 times those obtained from protoplasts at other cell cycle phases. The results indicate that the absence of the nuclear membrane in mitotic cells favours delivery to the nucleus of exogenous DNA introduced into the cytoplasm.     

Electrochemical Gating of Tricarboxylic Acid Cycle in Electricity-Producing Bacterial Cells of Shewanella

Energy-conversion systems mediated by bacterial metabolism have recently attracted much attention, and therefore, demands for tuning of bacterial metabolism are increasing. It is widely recognized that intracellular redox atmosphere which is generally tuned by dissolved oxygen concentration or by appropriate selection of an electron acceptor for respiration is one of the important factors determining the bacterial metabolism. In general, electrochemical approaches are valuable for regulation of redox-active objects. However, the intracellular redox conditions are extremely difficult to control electrochemically because of the presence of insulative phospholipid bilayer membranes. In the present work, the limitation can be overcome by use of the bacterial genus Shewanella , which consists of species that are able to respire via cytochromes abundantly expressed in their outer-membrane with solid-state electron acceptors, including anodes. The electrochemical characterization and the gene expression analysis revealed that the activity of tricarboxylic acid (TCA) cycle in Shewanella cells can be reversibly gated simply by changing the anode potential. Importantly, our present results for Shewanella cells cultured in an electrochemical system under poised potential conditions showed the opposite relationship between the current and electron acceptor energy level, and indicate that this unique behavior originates from deactivation of the TCA cycle in the (over-)oxidative region. Our result obtained in this study is the first demonstration of the electrochemical gating of TCA cycle of living cells. And we believe that our findings will contribute to a deeper understanding of redox-dependent regulation systems in living cells, in which the intracellular redox atmosphere is a critical factor determining the regulation of various metabolic and genetic processes.     

Design and synthesis of biotin- or alkyne-conjugated photoaffinity probes for studying the target molecules of PD 404182

To investigate the mechanism of action of the potent antiviral compound PD 404182, three novel photoaffinity probes equipped with a biotin or alkyne indicator were designed and synthesized based on previous structure–activity relationship studies. These probes retained the potent anti-HIV activity of the original pyrimidobenzothiazine derivatives. In photoaffinity labeling studies using HIV-1-infected H9 cells (H9IIIB), eight potential proteins were observed to bind PD 404182.     

DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

Genome editing can introduce designed mutations into a target genomic site, but also into unintended off-target sites. DAJIN, a novel nanopore sequencing data analysis tool, identifies and quantifies allele numbers and their mutation patterns, reporting consensus sequences and visualizing mutations in alleles at single-nucleotide resolution.     

Differential cell affinity and sorting of anterior and posterior cells during outgrowth of recombinant avian limb buds

To examine the role of position-specific differences in cell-cell affinity, recombinant limb buds composed of dissociated and reaggregated cells derived from anterior (A) and posterior (P) limb bud fragments were analyzed. Dissociated anterior and/or posterior cells were differentially labeled, and their behavior was analyzed during recombinant limb bud outgrowth. We find that anterior and posterior cells sort out from one another to form alternating anterior and posterior stripes of cells that extend distally along the proximal-distal axis. These alternating stripes are prominent across the A/P axis in whole-mount preparations of recombinant limb buds after 48 h of outgrowth when the presumptive autopod is dorsal-ventrally flattened and digit rudiments are not evident. After 96 h, when digital and interdigital regions are clearly defined, we find evidence that A/P stripes do not follow obvious anatomical boundaries. The formation of A/P stripes is not inhibited by grafts of ZPA tissue, suggesting that polarizing activity does not influence cell-cell affinity early in limb outgrowth. In vitro studies provide evidence that cell sorting is not dependent on the limb bud ectoderm or the AER; however, cells sort out without organizing into stripes. Gene expression studies using anterior-specific (Alx-4) and posterior-specific (Shh, Bmp-2, and Hoxd-13) marker genes failed to reveal expression domains that corresponded to stripe formation. Control recombinant limb buds composed of anterior, central, or posterior mesenchyme formed digits in a position-specific manner. A/P recombinant limb buds that develop to later stages form digits that are characteristic of central recombinant limbs. These data provide the first definitive evidence of A/P cell sorting during limb outgrowth in vivo and suggest that differential cell affinities play a role in modulating cell behavior during distal outgrowth.     

siRNA-mediated inhibition of endogenous Huntington disease gene expression induces an aberrant configuration of the ER network in vitro

Huntingtin is a ubiquitously expressed cytoplasmic protein encoded by the Huntington disease (HD) gene, in which a CAG expansion induces an autosomal dominant progressive neurodegenerative disorder; however, its biological function has not been completely elucidated. Here, we report for the first time that short interfering RNA (siRNA)-mediated inhibition of endogenous Hdh (a mouse homologue of huntingtin) gene expression induced an aberrant configuration of the endoplasmic reticulum (ER) network in vitro. Studies using immunofluorescence microscopy with several ER markers revealed that the ER network appeared to be congregated in various types of cell lines transfected with siRNA directed against Hdh, but not with other siRNAs so far tested. Other subcellular organelles and structures, including the nucleus, Golgi apparatus, mitochondria, lysosomes, microtubules, actin cytoskeletons, cytoplasm, lipid rafts, and plasma membrane, exhibited normal configurations. Western blot analysis of cellular prion protein (PrP(C)) revealed normal glycosylation, which is a simple marker of post-translational modification in the ER and Golgi compartments, and immunofluorescence microscopy detected no altered subcellular distribution of PrP(C) in the post-ER compartments. Further investigation is required to determine whether the distorted ER network, i.e., loss of the huntingtin function, participates in the development of HD.     

Isolation and characterization of Streptomyces, Actinoplanes, and Methylibium strains that are involved in degradation of natural rubber and synthetic poly(cis-1,4-isoprene)

Rubber-degrading bacteria were screened for the production of clearing zones around their colonies on latex overlay agar plates. Novel three bacteria, Streptomyces sp. strain LCIC4, Actinoplanes sp. strain OR16, and Methylibium sp. strain NS21, were isolated. To the best of our knowledge, this is the first report on the isolation of a Gram-negative rubber-degrading bacterium other than γ-proteobacteria. Gel permeation chromatography analysis revealed that these strains degraded poly(cis-1,4-isoprene) to low-molecular-weight products. The occurrence of aldehyde groups in the degradation products by NS21 was suggested by staining with Schiff's reagent and 1H-nuclear magnetic resonance spectroscopy. The lcp gene of LCIC4, which showed 99% amino acid sequence identity with that of Streptomyces sp. strain K30, was cloned, and contained a putative twin-arginine motif at its N terminus. It is located next to oxiB, which is estimated to be responsible for oxidation of degradation intermediate of rubber in K30. Southern hybridization analysis using LCIC4 lcp probe revealed the presence of a lcp-homolog in OR16. These results suggest that the lcp-homologs are involved in rubber degradation in LCIC4 and OR16.     

Differences in resource storage pattern between Laminaria longissima and Laminaria diabolica (Laminariaceae; Phaeophyta) reflecting their morphological characteristics

The kelps Laminaria longissima and L. diabolica, belonging to the groups of L. angustata and L. japonica, respectively, differ greatly in their morphological characteristics although their geographical distributions overlap widely along the eastern coast of Hokkaido. To clarify the interaction between the morphological and physiological characteristics of the two species, and their link with environmental variables, hatchery-raised young sporophytes of L. longissima and L. diabolica collected from Hokkaido were cultivated simultaneously under similar conditions in Matsushima Bay, Miyagi, from January to July 2004. Seasonal morphological characteristics, gross photosynthetic rate, nutrient uptake rates, and resource contents were examined. The blade lengths of L. longissima and L. diabolica reached a maximum of 329.9 cm and 256.7 cm, respectively, in April to May, and decreased to 284.4 cm and 68.6 cm, respectively, in July. The total elongation length of L. longissima (412.5 cm) was similar to that of L. diabolica (373.8 cm). However, the total erosion length of L. longissima (145.9 cm) was approximately half that of L. diabolica (302.9 cm). The gross photosynthetic rate and uptake rates of NH₄-N, NO₃-N, and PO₄-P of the two species were similar. However, the carbon, nitrogen, and phosphorus contents were transferred and stored in the whole blade tissues in the case of L. longissima, but in the meristem of L. diabolica from May to June. These results suggest that morphological differences are a response to different resource storage patterns. The storage patterns of L. longissima and L. diabolica are likely to be genetically fixed characteristics, which have evolved in adaptation to the specific habitat environments of the groups of L. angustata and L. japonica. The low water temperature and rich nutrients provided by the Oyashio Current are conducive to storage of resources in the whole blade tissues and a large surface area retained for photosynthesis and nutrient uptake in the L. angustata group. Conversely, high temperature and poor nutrients, or large fluctuations in these parameters, provided by the Tsushima Warm Current are more conducive to intensive storage of resources in the meristem for maturation and further growth in the L. japonica group. L. diabolica retains the storage pattern of the L. japonica group but grows in regions affected by the Oyashio Current, allowing it to become the widest distributed Laminaria species.