Expression of a bifunctional fusion of the Escherichia coli genes for trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase in transgenic rice plants increases trehalose accumulation and abiotic stress tolerance without stunting growth

Trehalose plays an important role in stress tolerance in plants. Trehalose-producing, transgenic rice (Oryza sativa) plants were generated by the introduction of a gene encoding a bifunctional fusion (TPSP) of the trehalose-6-phosphate (T-6-P) synthase (TPS) and T-6-P phosphatase (TPP) of Escherichia coli, under the control of the maize (Zea mays) ubiquitin promoter (Ubi1). The high catalytic efficiency (Seo et al., 2000) of the fusion enzyme and the single-gene engineering strategy make this an attractive candidate for high-level production of trehalose; it has the added advantage of reducing the accumulation of potentially deleterious T-6-P. The trehalose levels in leaf and seed extracts from Ubi1::TPSP plants were increased up to 1.076 mg g fresh weight(-1). This level was 200-fold higher than that of transgenic tobacco (Nicotiana tabacum) plants transformed independently with either TPS or TPP expression cassettes. The carbohydrate profiles were significantly altered in the seeds, but not in the leaves, of Ubi1::TPSP plants. It has been reported that transgenic plants with E. coli TPS and/or TPP were severely stunted and root morphology was altered. Interestingly, our Ubi1::TPSP plants showed no growth inhibition or visible phenotypic alterations despite the high-level production of trehalose. Moreover, trehalose accumulation in Ubi1::TPSP plants resulted in increased tolerance to drought, salt, and cold, as shown by chlorophyll fluorescence and growth inhibition analyses. Thus, our results suggest that trehalose acts as a global protectant against abiotic stress, and that rice is more tolerant to trehalose synthesis than dicots.     

Crystalline structure analysis of cellulose treated with sodium hydroxide and carbon dioxide by means of X-ray diffraction and FTIR spectroscopy

Crystalline structures of cellulose (named as Cell 1), NaOH-treated cellulose (Cell 2), and subsequent CO2-treated cellulose (Cell 2-C) were analyzed by wide-angle X-ray diffraction and FTIR spectroscopy. Transformation from cellulose I to cellulose II was observed by X-ray diffraction for Cell 2 treated with 15-20 wt% NaOH. Subsequent treatment with CO2 also transformed the Cell 2-C treated with 5-10 wt% NaOH. Many of the FTIR bands including 2901, 1431, 1282, 1236, 1202, 1165, 1032, and 897 cm(-1) were shifted to higher wave number (by 2-13 cm(-1)). However, the bands at 3352, 1373, and 983 cm(-1) were shifted to lower wave number (by 3-95 cm(-1)). In contrast to the bands at 1337, 1114, and 1058 cm(-1), the absorbances measured at 1263, 993, 897, and 668 cm(-1) were increased. The FTIR spectra of hydrogen-bonded OH stretching vibrations at around 3352 cm(-1) were resolved into three bands for cellulose I and four bands for cellulose II, assuming that all the vibration modes follow Gaussian distribution. The bands of 1 (3518 cm(-1)), 2 (3349 cm(-1)), and 3 (3195 cm(-1)) were related to the sum of valence vibration of an H-bonded OH group and an intramolecular hydrogen bond of 2-OH ...O-6, intramolecular hydrogen bond of 3-OH...O-5 and the intermolecular hydrogen bond of 6-O...HO-3', respectively. Compared with the bands of cellulose I, a new band of 4 (3115 cm(-1)) related to intermolecular hydrogen bond of 2-OH...O-2' and/or intermolecular hydrogen bond of 6-OH...O-2' in cellulose II appeared. The crystallinity index (CI) was obtained by X-ray diffraction [CI(XD)] and FTIR spectroscopy [CI(IR)]. Including absorbance ratios such as A1431,1419/A897,894 and A1263/A1202,1200, the CI(IR) was evaluated by the absorbance ratios using all the characteristic absorbances of cellulose. The CI(XD) was calculated by the method of Jayme and Knolle. In addition, X-ray diffraction curves, with and without amorphous halo correction, were resolved into portions of cellulose I and cellulose II lattice. From the ratio of the peak area, that is, peak area of cellulose I (or cellulose II)/total peak area, CI(XD) were divided into CI(XD-CI) for cellulose I and CI(XD-CII) for cellulose II. The correlation between CI(XD-CI) (or CI(XD-CII)) and CI(IR) was evaluated, and the bands at 2901 (2802), 1373 (1376), 897 (894), 1263, 668 cm(-1) were good for the internal standard (or denominator) of CI(IR), which increased the correlation coefficient. Both fraction of the absorbances showing peak shift were assigned as the alternate components of CI(IR). The crystallite size was decreased to constant value for Cell 2 treated at >or= 15 wt% NaOH. The crystallite size of Cell 2-C (cellulose II) was smaller than that of Cell 2 (cellulose I) treated at 5-10 wt% NaOH. But the crystallite size of Cell 2-C (cellulose II) was larger than that of Cell 2 (cellulose II) treated at 15-20 wt% NaOH.     

<Emphasis Type="Italic">Phellinus</Emphasis> extracts inhibit migration and matrix metalloproteinase secretion in porcine coronary artery endothelial cells

Phellinus linteus is a fungus which is found primarily in tropical regions of the Americas, Africa, and Asia.P. linteus has been used in traditional medical practice for the treatment of arthritis, liver damage and cancer. Angiogenesis is a process that involves migration, proliferation and cell differentiation, as well as the formation of new capillary structures. The anti-angiogenic activities evidenced by natural compounds may actually be a critical effect for the inhibition of angiogenesis-dependent disease by these agents via the blockage of vascular development. This study assessed the effects of water extracts fromP. linteus (Phellinus extracts) on primary cultured porcine coronary artery endothelial cells (PCAECs).Phellinus extracts induced no changes in DNA synthesis or cell numbers, but inhibited the migration of PCAECs.Phellinus extracts also induced a reduction in the secretion of matrix metalloproteinase-2 (MMP-2) and MMP-9. Our results show that, in endothelial cells,Phellinus extracts may inhibit angiogenesis by reducing levels of MMP-2 and MMP-9 secretion.     

Human Embryonic Stem Cell Technology: Large Scale Cell Amplification and Differentiation

Embryonic stem cells (ESC) hold the promise of overcoming many diseases as potential sources of, for example, dopaminergic neural cells for Parkinson’s Disease to pancreatic islets to relieve diabetic patients of their daily insulin injections. While an embryo has the innate capacity to develop fully functional differentiated tissues; biologists are finding that it is much more complex to derive singular, pure populations of primary cells from the highly versatile ESC from this embryonic parent. Thus, a substantial investment in developing the technologies to expand and differentiate these cells is required in the next decade to move this promise into reality. In this review we document the current standard assays for characterising human ESC (hESC), the status of ‘defined’ feeder-free culture conditions for undifferentiated hESC growth, examine the quality controls that will be required to be established for monitoring their growth, review current methods for expansion and differentiation, and speculate on the possible routes of scaling up the differentiation of hESC to therapeutic quantities.     

Serine palmitoyltransferase inhibitor myriocin induces growth inhibition of B16F10 melanoma cells through G(2) /M phase arrest

Objectives: Melanoma is the most aggressive form of skin cancer, and it resists chemotherapy. Candidate drugs for effective anti‐cancer treatment have been sought from natural resources. Here, we have investigated anti‐proliferative activity of myriocin, serine palmitoyltransferase inhibitor, in the de novo sphingolipid pathway, and its mechanism in B16F10 melanoma cells. Material and methods: We assessed cell population growth by measuring cell numbers, DNA synthesis, cell cycle progression, and expression of cell cycle regulatory proteins. Ceramide, sphingomyelin, sphingosine and sphingosine‐1‐phosphate levels were analysed by HPLC. Results: Myriocin inhibited proliferation of melanoma cells and induced cell cycle arrest in the G₂/M phase. Expressions of cdc25C, cyclin B1 and cdc2 were decreased in the cells after exposure to myriocin, while expression of p53 and p21^waf1/cip1 was increased. Levels of ceramide, sphingomyelin, sphingosine and sphingosine‐1‐phosphate in myriocin‐treated cells after 24 h were reduced by approximately 86%, 57%, 75% and 38%, respectively, compared to levels in control cells. Conclusions: Our results suggest that inhibition of sphingolipid synthesis by myriocin in melanoma cells may inhibit expression of cdc25C or activate expression of p53 and p21^waf1/cip1, followed by inhibition of cyclin B1 and cdc2, resulting in G₂/M arrest of the cell cycle and cell population growth inhibition. Thus, modulation of sphingolipid metabolism by myriocin may be a potential target of mechanism‐based therapy for this type of skin cancer.     

Relationship Between β-Amyloid and Mitochondrial Dynamics

Mitochondria as dynamic organelles undergo morphological changes through the processes of fission and fusion which are major factors regulating their functions. A disruption in the balance of mitochondrial dynamics induces functional disorders in mitochondria such as failed energy production and the generation of reactive oxygen species, which are closely related to pathophysiological changes associated with Alzheimer’s disease (AD). Recent studies have demonstrated a relationship between abnormalities in mitochondrial dynamics and impaired mitochondrial function, clarifying the effects of morphofunctional aberrations which promote neuronal cell death in AD. Several possible signaling pathways have been suggested for a better understanding of the mechanism behind the key molecules regulating mitochondrial morphologies. However, the exact machinery involved in mitochondrial dynamics still has yet to be elucidated. This paper reviews the current knowledge on signaling mechanisms involved in mitochondrial dynamics and the significance of mitochondrial dynamics in controlling associated functions in neurodegenerative diseases, particularly in AD.     

Cleavage of Bax is mediated by caspase-dependent or -independent calpain activation in dopaminergic neuronal cells: protective role of Bcl-2.

Two cysteine protease families, caspase and calpain, are known to participate in cell death. We investigated whether a stress-specific protease activation pathway exists, and to what extent Bcl-2 plays a role in preventing drug-induced protease activity and cell death in a dopaminergic neuronal cell line, MN9D. Staurosporine (STS) induced caspase-dependent apoptosis while a dopaminergic neurotoxin, MPP(+) largely induced caspase-independent necrotic cell death as determined by morphological and biochemical criteria including cytochrome c release and fluorogenic caspase cleavage assay. At the late stage of both STS- and MPP(+)-induced cell death, Bax was cleaved into an 18-kDa fragment. This 18-kDa fragment appeared only in the mitochondria-enriched heavy membrane fraction of STS-treated cells, whereas it was detected exclusively in the cytosolic fraction of MPP(+)-treated cells. This proteolytic cleavage of Bax appeared to be mediated by calpain as determined by incubation with [(35)S]methionine-labelled Bax. Thus, cotreatment of cells with calpain inhibitor blocked both MPP(+)- and STS-induced Bax cleavage. Intriguingly, overexpression of baculovirus-derived inhibiting protein of caspase, p35 or cotreatment of cells with caspase inhibitor blocked STS- but not MPP(+)-induced Bax cleavage. This appears to indicate that calpain activation may be either dependent or independent of caspase activation within the same cells. However, cotreatment with calpain inhibitor rescued cells from MPP(+)-induced but not from STS-induced neuronal cell death. In these paradigms of dopaminergic cell death, overexpression of Bcl-2 prevented both STS- and MPP(+)-induced cell death and its associated cleavage of Bax. Thus, our results suggest that Bcl-2 may play a protective role by primarily blocking drug-induced caspase or calpain activity in dopaminergic neuronal cells.     

Astrocyte ethanol exposure reveals persistent and defined calcium response subtypes and associated gene signatures

Astrocytes play a critical role in brain function, but their contribution during ethanol (EtOH) consumption remains largely understudied. In light of recent findings on the heterogeneity of astrocyte physiology and gene expression, an approach with the ability to identify subtypes and capture this heterogeneity is necessary. Here, we combined measurements of calcium signaling and gene expression to define EtOH-induced astrocyte subtypes. In the absence of a demonstrated EtOH receptor, EtOH is believed to have effects on the function of many receptors and downstream biological cascades that underlie calcium responsiveness. This mechanism of EtOH-induced calcium signaling is unknown and this study provides the first step in understanding the characteristics of cells displaying these observed responses. To characterize underlying astrocyte subtypes, we assessed the correlation between calcium signaling and astrocyte gene expression signature in response to EtOH. We found that various EtOH doses increased intracellular calcium levels in a subset of astrocytes, distinguishing three cellular response types and one nonresponsive subtype as categorized by response waveform properties. Furthermore, single-cell RNA-seq analysis of astrocytes from the different response types identified type-enriched discriminatory gene expression signatures. Combining single-cell calcium responses and gene expression analysis identified specific astrocyte subgroups among astrocyte populations defined by their response to EtOH. This result provides a basis for identifying the relationship between astrocyte susceptibility to EtOH and corresponding measurable markers of calcium signaling and gene expression, which will be useful to investigate potential subgroup-specific influences of astrocytes on the physiology and pathology of EtOH exposure in the brain.     

Accumulation of pTreg cells is detrimental in late‐onset (aged) mouse model of multiple sclerosis

Although typically associated with onset in young adults, multiple sclerosis (MS) also attacks the elderly, which is termed late‐onset MS. The disease can be recapitulated and studied in a mouse model, experimental autoimmune encephalomyelitis (EAE). The onset of induced EAE is delayed in aged mice, but disease severity is increased relative to young EAE mice. Given that CD4⁺FoxP3⁺ regulatory T (Treg) cells play an ameliorative role in MS/EAE severity, and the aged immune system accumulates peripheral Treg (pTreg) cells, failure of these cells to prevent or ameliorate EAE disease is enigmatic. When analyzing the distribution of Treg cells in EAE mice, the aged mice exhibited a higher proportion of polyclonal (pan‐) pTreg cells and a lower proportion of antigen‐specific pTreg cells in the periphery but lower proportions of both pan‐ and antigen‐specific Treg cells in the central nervous system (CNS). Furthermore, in the aged inflamed CNS, CNS‐Treg cells exhibited a higher plasticity, and T effector (CNS‐Teff) cells exhibited greater clonal expansion, disrupting the Treg/Teff balance. Transiently inhibiting FoxP3 or depleting pTreg cells partially corrected Treg distribution and restored the Treg/Teff balance in the aged inflamed CNS, thereby ameliorating the disease in the aged EAE mice. These results provide evidence and mechanism that accumulated aged pTreg cells play a detrimental role in neuronal inflammation of aged MS.