CHITIN — A promising biomaterial for tissue engineering and stem cell technologies

Chitin, after cellulose, is the second most abundant natural polymer. With a 200-year history of scientific research, chitin is beginning to see fruitful application in the fields of stem cell and tissue engineering. To date, however, research in chitin as a biomaterial appears to lag far behind that of its close relative, chitosan, due to the perceived difficulty in processing chitin. This review presents methods to improve the processability of chitin, and goes on further to discuss the unique physicochemical and biological characteristics of chitin that favor it as a biomaterial for regenerative medicine applications. Examples of the latter are presented, with special attention on the qualities of chitin that make it inherently suitable as scaffolds and matrices for tissue engineering, stem cell propagation and differentiation.     

Indinavir and nelfinavir inhibit proximal insulin receptor signaling and salicylate abrogates inhibition: Potential role of the NFkappa B pathway

The molecular basis of insulin resistance induced by HIV protease inhibitors (HPIs) remains unclear. In this study, Chinese hamster ovary cells transfected with high levels of human insulin receptor (CHO‐IR) and 3T3‐L1 adipocytes were used to elucidate the mechanism of this side effect. Indinavir and nelfinavir induced a significant decrease in tyrosine phosphorylation of the insulin receptor β‐subunit. Indinavir caused a significant increase in the phosphorylation of insulin receptor substrate‐1 (IRS‐1) on serine 307 (S307) in both CHO‐IR cells and 3T3‐L1 adipocytes. Nelfinavir also inhibited phosphorylation of Map/ERK kinase without affecting insulin‐stimulated Akt phosphorylation. Concomitantly, levels of protein tyrosine phosphatase 1B (PTP1B), suppressor of cytokines signaling‐1 and ‐3 (SOCS‐1 and ‐3), Src homology 2B (SH2B) and adapter protein with a pleckstrin homology domain and an SH2 domain (APS) were not altered significantly. When CHO‐IR cells were pre‐treated with sodium salicylate (NaSal), the effects of indinavir on tyrosine phosphorylation of the IR β‐subunit and phosphorylation of IRS‐1 at S307 were abrogated. These data suggest a potential role for the NFκB pathway in insulin resistance induced by HPIs. J. Cell. Biochem. 114: 1729–1737, 2013. © 2013 Wiley Periodicals, Inc.     

Variation in embolism occurrence and repair along the stem in drought‐stressed and re‐watered seedlings of a poplar clone

Root pressure and plasma membrane intrinsic protein (PIP) availability in the xylem have been recognized to participate in the refilling of embolized conduits, yet integration of the two mechanisms has not been reported in the same plant. In this study, 4‐month‐old seedlings of a hybrid poplar (Populus alba × Populus glandulosa) clone 84K were subjected to two contrasting soil‐water treatments, with the drought treatment involving withholding of water for 17 days to reduce the soil‐water content to 10% of the saturated field capacity, followed by a re‐watering cycle. The percentage loss of stem hydraulic conductance (PLC) sharply increased, and stomatal conductance and photosynthesis declined in response to drought stress; these processes were gradually restored following the subsequent re‐watering. Embolism was most severe in the middle portions of the stem, followed by the basal and top portions of the stems of seedlings subjected to drought stress and subsequent re‐watering. Although drought stress eliminated root pressure, re‐watering partially restored it in a short period of time. The expression of PIP genes in the xylem was activated by drought stress, and some PIP genes were further stimulated in the top portion after re‐watering. The dynamics of root pressure and differential expression of PIP genes along the stem coincided with changes in PLC, suggesting that root pressure and PIPs work together to refill the embolized vessels. On the basis of the recovery dynamics in PLC and g_smax (maximum stomatal conductance) after re‐watering, the stomatal closure and xylem cavitation exhibited fatigue due to drought stress.     

Time and litter species composition affect litter-mixing effects on decomposition rates

Aims

To assess whether the yew roots, which are able to provide a very constant environment due to their long life-span, can maintain the original arbuscular mycorrhizal (AM) fungal community during yew population decline.

Methods

The diversity of AM fungi (AMF) colonizing the roots of yew was analyzed by selecting the small subunit ribosomal RNA genes to construct a database of the overall community of AMF in the experimental area. A terminal restriction fragment length polymorphism (TRFLP) approach was used to identify the AMF communities present in yew roots. Physiological and environmental variables related to topology and soil and plant characteristics were determined as markers of habitat degradation.

Results

The AMF communities within yew roots were found to be dependent on soil, plant and topological variables indicative of habitat degradation surrounding the yew. The phylogenetic diversity of AMF associated to the yews was lower in habitats more exposed to degradation than in those better conserved.

Conclusions

The target yews can be grouped into two degradation levels. AMF communities were also affected by the degradation processes affecting their hosts. This finding rules out the role of these trees as refugia for their original AMF community, a fact that should be considered in plant reintroduction programs using AMF as bioenhancers.     

Molecular cloning and characterization of the mitogen-activated protein kinase kinase gene (MKK4) and its promoter sequence from oilseed rape (Brassica campestris L.)

Mitogen-activated protein kinase (MAPK) cascades are involved in various processes, including plant growth and development as well as biotic and abiotic stress responses. MAPK kinases (MKKs), which link MPKs and MAPKK kinases (MKKKs), are crucial in MAPK cascades because these kinases mediate various stress responses in plants. However, only few MKKs in Brassica campestris (rape) have been functionally characterized. In this study, a novel gene, MKK4 that belongs to a C MKK group, was isolated and characterized from rape. Bioinformatics analysis revealed that the length of cDNA was 1,317 bp with an open reading frame of 993 bp, which encodes a polypeptide containing 330 amino acids, including a putative signal peptide with 27 amino acid residues and a mature protein with 303 amino acids. The obtained MKK4 exhibited a predicted molecular mass of 36.5 kDa and an isoelectric point of 9.01. Quantitative real-time polymerase chain reaction analysis revealed that MKK4 expression could be induced by cold and salt. We also found that the MKK4 protein is localized in the nucleus. In addition, a 999 bp promoter fragment of MKK4 was cloned. Sequence analysis revealed that several putative regulatory elements were found in the MKK4 promoter. Transient expression assay showed that the MKK4 promoter fragments exhibited promoter activity and stimulated GFP expression. The effects of GFP gene expression at different temperatures and in different onion epidermis culture patterns were compared. Results showed that the MKK4 promoter could respond to low temperature and salt stress. These results suggested that MKK4 is possibly important for the regulation of cold- and salt-stress responses in plants.     

Genetic characterisation and heterologous expression of leucocin C, a class IIa bacteriocin from Leuconostoc carnosum 4010

Leuconostoc carnosum 4010 is a protective culture for meat products. It kills the foodborne pathogen Listeria monocytogenes by producing two class IIa (pediocin-like) bacteriocins, leucocin A and leucocin C. The genes for leucocin A production have previously been characterised from Leuconostoc gelidum UAL 187, whereas no genetic studies about leucocin C has been published. Here, we characterised the genes for the production of leucocins A and C in L. carnosum 4010. In this strain, leucocin A and leucocin C operons were localised in different plasmids. Unlike in L. gelidum, leucocin A operon in L. carnosum 4010 only contained the structural and the immunity genes lcaAB without transporter genes lcaECD. On the contrary, leucocin C cluster included two intact operons. Novel genes lecCI encode the leucocin C precursor and the 97-aa immunity protein LecI, respectively. LecI shares 48 % homology with the immunity proteins of sakacin P and listeriocin. Another leucocin C operon lecXTS, encoding an ABC transporter and an accessory protein, was 97 % identical with the leucocin A transporter operon lcaECD of L. gelidum. For heterologous expression of leucocin C in Lactococcus lactis, the mature part of the lecC gene was fused with the signal sequence of usp45 in the secretion vector pLEB690. L. lactis secreted leucocin C efficiently, as shown by large halos on lawns of L. monocytogenes and Leuconostoc mesenteroides indicators. The function of LecI was then demonstrated by expressing the gene lecI in L. monocytogenes. LecI-producing Listeria was less sensitive to leucocin C than the vector strain, thus corroborating the immunity function of LecI.     

A novel combinatorial biocatalytic approach for producing antibacterial compounds effective against Mycobacterium tuberculosis (TB)

Two bacterial hosts expressing cloned aromatic oxygenases were used to catalyze the oxidation and polymerization of indole and related substrates, creating mixtures of indigoid compounds comprised of novel dimers and trimers. Crude extracts and purified compounds were tested for their ability to inhibit the growth of Gram-positive organisms, in general, and Mycobacterium tuberculosis (TB), in particular. Of the 74 compounds tested against M. tuberculosis, ~66 % had minimum inhibitory concentrations (MIC) of 5 μg/ml or less. The most effective antibiotic found was designated SAB-P1, a heterodimer of indole and anthranil, which had a MIC of 0.16 μg/ml, and did not inhibit kidney cells (IC⁵⁰) at concentrations of >8 μg/ml. Combinatorial biocatalysis was used to create a series of halogenated derivatives of SAB-P1 with a wider therapeutic window. None of the derivatives had MIC values that were superior to SAB-P1, but some had a wider therapeutic window because of decreased kidney cell toxicity. Generally, the indigoid dimers that were effective against TB appeared to be specific for TB. Some of the trimers generated, however, had a broader spectrum of activity inhibiting not only TB (MIC?=?1.1 μg/ml) but also the growth of Mycobacterium smegmatis MC2 155, Bacillus cereus, Enterococcus faecalis, Staphylococcus epidermidis, Bacillus subtilis 168, and Clostridium acetobutylicum. The structure of two of the novel dimers (SAB-C4 and SAB-P1) and a trimer (SAB-R1) were solved using X-ray crystallography.     

Pipernonaline from Piper longum Linn. induces ROS-mediated apoptosis in human prostate cancer PC-3 cells

The antiproliferation effects of pipernonaline, a piperine derivative, were investigated on human prostate cancer PC-3 cells. It inhibited growth of androgen independent PC-3 and androgen dependent LNCaP prostate cells in a dose-dependent (30–90 μM) and time-dependent (24–48 h) manner. The growth inhibition of PC-3 cells was associated with sub-G₁ and G₀/G₁ accumulation, confirmed by the down-regulation of CDK2, CDK4, cyclin D1 and cyclin E, which are correlated with G₁ phase of cell cycle. Pipernonaline up-regulated cleavage of procaspase-3/PARP, but did not change expression of proapoptotic bax and antiapoptotic bcl-2 proteins. Its caspase-3 activation was confirmed by the caspase-3 assay kit. In addition, pipernonaline caused the production of reactive oxygen species (ROS), increase of intracellular Ca²⁺, and mitochondrial membrane depolarization, which these phenomena were reversed by N-acetylcysteine, a ROS scavenger. The results suggest that pipernonaline exhibits apoptotic properties through ROS production, which causes disruption of mitochondrial function and Ca²⁺ homeostasis and leads to its downstream events including activation of caspase-3 and cleavage of PARP in PC-3 cells. This is the first report of pipernonaline toward the anticancer activity of prostate cancer cells, which provides a role for candidate agent as well as the molecular basis for human prostate cancer.     

XBP-1u suppresses autophagy by promoting the degradation of FoxO1 in cancer cells

Autophagy is activated to maintain cellular energy homeostasis in response to nutrient starvation. However, autophagy is not persistently activated, which is poorly understood at a mechanistic level. Here, we report that turnover of FoxO1 is involved in the dynamic autophagic process caused by glutamine starvation. X-box-binding protein-1u (XBP-1u) has a critical role in FoxO1 degradation by recruiting FoxO1 to the 20S proteasome. In addition, the phosphorylation of XBP-1u by extracellular regulated protein kinases1/2 (ERK1/2) on Ser61 and Ser176 was found to be critical for the increased interaction between XBP-1u and FoxO1 upon glutamine starvation. Furthermore, knockdown of XBP-1u caused the sustained level of FoxO1 and the persistent activation of autophagy, leading to a significant decrease in cell viability. Finally, the inverse correlation between XBP-1u and FoxO1 expression agrees well with the expression profiles observed in many human cancer tissues. Thus, our findings link the dynamic process of autophagy to XBP-1u-induced FoxO1 degradation.