Cellulase and xylanase activity in relation to biofilm formation by two intertidal filamentous fungi in a novel polymethylmethacrylate conico-cylindrical flask

A polymethylmethacrylate (PMMA) conico-cylindrical flask (CCF) with an inner arrangement consisting of eight equidistantly spaced rectangular strips mounted radially on a circular disk to provide additional surface area for fungal attachment was employed for production of cellulase by Chaetomium crispatum and xylanase by Gliocladium viride. The design allowed comparison of production between CCFs with hydrophobic surface (PMMA-CCF), hydrophilic glass surface (GS-CCF) and 500-ml Erlenmeyer flask (EF). Compared with the EF, endo-β-1,4-glucanase and FPase (filter paper degradation) activities increased from 0.044 to 0.156 and from 0.008 to 0.021 IU/ml, respectively, in the PMMA-CCF, while growth of C. crispatum was higher by at most 1.38-fold compared with the other vessels. Xylanase production in the EF was at most 5.08-fold higher and growth of G. viride was at most 1.52-fold higher compared with the other vessels. Temporal pattern of biofilm development based on two-channel fluorescence detection of extracellular polymeric substances (EPSs) and whole cells in a confocal laser scanning microscope demonstrated increase by 100% in biovolume, 25% in thickness and 62.5% both in substratum coverage and total spreading of C. crispatum biofilm in PMMA-CCF over 6 days. Biovolume of G. viride biofilm in GS-CCF increased by 150% over 4 days while that in PMMA-CCF enhanced by 200% over 2 days. Biofilm thickness in PMMA-CCF was 44% higher compared with GS-CCF and increased by 175% over 2 days. Substratum coverage was 38% higher in GS-CCF compared with PMMA-CCF. Thus, reactor surface area and property, shear forces and biofilm formation influenced enzyme production.     

TC-PTP dephosphorylates the guanine nucleotide exchange factor C3G (RapGEF1) and negatively regulates differentiation of human neuroblastoma cells

The guanine nucleotide exchange factor, C3G (RapGEF1), functions in multiple signaling pathways involved in cell adhesion, proliferation, apoptosis and actin reorganization. C3G is regulated by tyrosine phosphorylation on Y504, known to be mediated by c-Abl and Src family kinases. In the present study we explored the possibility of cellular phospho-C3G (pC3G) being a substrate of the intracellular T-cell protein tyrosine phosphatase TC-PTP (PTPN2) using the human neuroblastoma cell line, IMR-32. In vivo and in vitro binding assays demonstrated interaction between C3G and TC-PTP. Interaction is mediated through the Crk-binding region of C3G and C-terminal noncatalytic residues of TC-PTP. C3G interacted better with a substrate trap mutant of TC48 and this complex formation was inhibited by vanadate. Endogenous pC3G colocalized with catalytically inactive mutant TC48 in the Golgi. Expression of TC48 abrogated pervanadate and c-Src induced phosphorylation of C3G without affecting total cellular phospho-tyrosine. Insulin-like growth factor treatment of c-Src expressing cells resulted in dephosphorylation of C3G dependent on the activity of endogenous TC48. TC48 expression inhibited forskolin induced tyrosine phosphorylation of C3G and neurite outgrowth in IMR-32 cells. Our results identify a novel Golgi localized substrate of TC48 and delineate a role for TC48 in dephosphorylation of substrates required during differentiation of human neuroblastoma cells.     

Production of embryonic and fetal-like red blood cells from human induced pluripotent stem cells

We have previously shown that human embryonic stem cells can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. We report here that we have produced iPS from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells. We show that regardless of the age of the donor cells, the iPS produced are fully reprogrammed into a pluripotent state that is undistinguishable from that of hESCs by low and high-throughput expression and detailed analysis of globin expression patterns by HPLC. This suggests that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with erythroid differentiation regardless of the age or the tissue type of the donor cells, at least as detected in these assays. The ability to produce large number of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications.     

Interchain heterogeneity in 2-hydroxyethylglutamine-l-valine random copolymers

Solid state circular dichroism (c.d.) and infrared (i.r.) studies of water soluble and insoluble fractions of poly(hydroxyethylglutamine-valine) random copolymers, prepared from parent γ-benzyl l-glutamate valine copolymers, show that interchain conformational heterogeneity with interchain compositional heterogeneity is present when the respective N-carboxyanhydrides are copolymerized in dioxan or benzene/methylene chloride. Use of previously determined reactivity ratios for the aforementioned copolymer systems permits the determination of the variation of the average copolymer composition, $\text{f}$_G, with conversion. The experimentally determined average copolymer composition.$\text{f}$_G for the use of the respective reactivity ratios and the copolymer hydroxyethylglutamine, valine are predicted by the use of the respective reactivity ratios and the copolymer composition equation. As the valine content of the copolymer chains in the fractions increases, the expected increase in β-sheet contribution is seen. Comparison of the experimentally determined solid state c.d. spectra with Greenfield and Fasman's computer generated c.d. spectra for varying amounts of α-helix, β-sheet and random structures, shows that the water insoluble fractions with their increased valine contents have a greater contribution of β-sheet structure than the respective soluble fractions.     

Integrin alpha4beta1 promotes focal adhesion kinase-independent cell motility via alpha4 cytoplasmic domain-specific activation of c-Src

The fibronectin binding integrins alpha5beta1 and alpha4beta1 generate signals pivotal for cell migration through distinct yet undefined mechanisms. For alpha5beta1, beta1-mediated activation of focal adhesion kinase (FAK) promotes c-Src recruitment to FAK and the formation of a FAK-Src signaling complex. Herein, we show that FAK expression is essential for alpha5beta1-stimulated cell motility and that exogenous expression of human alpha4 in FAK-null fibroblasts forms a functional alpha4beta1 receptor that promotes robust cell motility equal to the alpha5beta1 stimulation of wild-type and FAK-reconstituted fibroblasts. alpha4beta1-stimulated FAK-null cell spreading and motility were dependent on the integrity of the alpha4 cytoplasmic domain, independent of direct paxillin binding to alpha4, and were not affected by PRNK expression, a dominant-negative inhibitor of Pyk2. alpha4 cytoplasmic domain-initiated signaling led to a approximately 4-fold activation of c-Src which did not require paxillin binding to alpha4. Notably, alpha4-stimulated cell motility was inhibited by catalytically inactive receptor protein-tyrosine phosphatase alpha overexpression and blocked by the p50Csk phosphorylation of c-Src at Tyr-529. alpha4beta1-stimulated cell motility of triple-null Src(-/-), c-Yes(-/-), and Fyn(-/-) fibroblasts was dependent on c-Src reexpression that resulted in p130Cas tyrosine phosphorylation and Rac GTPase loading. As p130Cas phosphorylation and Rac activation are common downstream targets for alpha5beta1-stimulated FAK activation, our results support the existence of a novel alpha4 cytoplasmic domain connection leading to c-Src activation which functions as a FAK-independent linkage to a common motility-promoting signaling pathway.     

Analysis of nuclear transport signals in the human apurinic/apyrimidinic endonuclease (APE1/Ref1)

The mammalian abasic-endonuclease1/redox-factor1 (APE1/Ref1) is an essential protein whose subcellular distribution depends on the cellular physiological status. However, its nuclear localization signals have not been studied in detail. We examined nuclear translocation of APE1, by monitoring enhanced green fluorescent protein (EGFP) fused to APE1. APE1's nuclear localization was significantly decreased by deleting 20 amino acid residues from its N-terminus. Fusion of APE1's N-terminal 20 residues directed nuclear localization of EGFP. An APE1 mutant lacking the seven N-terminal residues (ND7 APE1) showed nearly normal nuclear localization, which was drastically reduced when the deletion was combined with the E12A/D13A double mutation. On the other hand, nearly normal nuclear localization of the full-length E12A/D13A mutant suggests that the first 7 residues and residues 8–13 can independently promote nuclear import. Both far-western analyses and immuno-pull-down assays indicate interaction of APE1 with karyopherin alpha 1 and 2, which requires the 20 N-terminal residues and implicates nuclear importins in APE1's nuclear translocation. Nuclear accumulation of the ND7 APE1(E12A/D13A) mutant after treatment with the nuclear export inhibitor leptomycin B suggests the presence of a previously unidentified nuclear export signal, and the subcellular distribution of APE1 may be regulated by both nuclear import and export.     

The potato virus X TGBp2 movement protein associates with endoplasmic reticulum-derived vesicles during virus infection

The green fluorescent protein (GFP) gene was fused to the potato virus X (PVX) TGBp2 gene, inserted into either the PVX infectious clone or pRTL2 plasmids, and used to study protein subcellular targeting. In protoplasts and plants inoculated with PVX-GFP:TGBp2 or transfected with pRTL2-GFP:TGBp2, fluorescence was mainly in vesicles and the endoplasmic reticulum (ER). During late stages of virus infection, fluorescence became increasingly cytosolic and nuclear. Protoplasts transfected with PVX-GFP:TGBp2 or pRTL2-GFP:TGBp2 were treated with cycloheximide and the decline of GFP fluorescence was greater in virus-infected protoplasts than in pRTL2-GFP:TGBp2-transfected protoplasts. Thus, protein instability is enhanced in virus-infected protoplasts, which may account for the cytosolic and nuclear fluorescence during late stages of infection. Immunogold labeling and electron microscopy were used to further characterize the GFP:TGBp2-induced vesicles. Label was associated with the ER and vesicles, but not the Golgi apparatus. The TGBp2-induced vesicles appeared to be ER derived. For comparison, plasmids expressing GFP fused to TGBp3 were transfected to protoplasts, bombarded to tobacco leaves, and studied in transgenic leaves. The GFP:TGBp3 proteins were associated mainly with the ER and did not cause obvious changes in the endomembrane architecture, suggesting that the vesicles reported in GFP:TGBp2 studies were induced by the PVX TGBp2 protein. In double-labeling studies using confocal microscopy, fluorescence was associated with actin filaments, but not with Golgi vesicles. We propose a model in which reorganization of the ER and increased protein degradation is linked to plasmodesmata gating.     

Phytoestrogenic effects of black tea extract (Camellia sinensis) in an oophorectomized rat (Rattus norvegicus) model of osteoporosis

The adverse side effects of currently available anti-osteoporotic agents warrant the search for compounds with less toxic effects. In this study, we assessed the phytoestrogenic potentiality of whole aqueous extract of black tea (BTE) in a bilaterally oophorectomized rat model (2.5%, 1 ml/100 g body weight/day for 28 days). Although the supplementation was given for 28 days but, sign of revival of copulation period (estrous stage) from non-receptive diestrous stage was first noticed after 21 days of BTE supplementation in bilaterally oophorectomized rats. This was accompanied by a significant increase in serum estradiol level. To test whether this increase in serum estradiol level could have an influence upon the oophorectomy-induced damage of bone, we assessed marker parameters of bone resorption and osteoclastic activity (tartrate-resistant acid phosphatase), collagen degradation (urinary hydroxyproline), bone loss (bone ash mineral content) and bone breaking strength (bone density). Results indicated that increase in serum estradiol level after BTE supplementation could significantly diminish oophorectomy-induced decaying changes in bone. This study proposes that aqueous BTE may be assessed as a phytoestrogenic compound for prevention against estrogen deficiency-related osteoporotic damages.     

HMGB1 contributes to the development of acute lung injury after hemorrhage

High mobility group box 1 (HMGB1) is a novel late mediator of inflammatory responses that contributes to endotoxin-induced acute lung injury and sepsis-associated lethality. Although acute lung injury is a frequent complication of severe blood loss, the contribution of HMGB1 to organ system dysfunction in this setting has not been investigated. In this study, HMGB1 was detected in pulmonary endothelial cells and macrophages under baseline conditions. After hemorrhage, in addition to positively staining endothelial cells and macrophages, neutrophils expressing HMGB1 were present in the lungs. HMGB1 expression in the lung was found to be increased within 4 h of hemorrhage and then remained elevated for more than 72 h after blood loss. Neutrophils appeared to contribute to the increase in posthemorrhage pulmonary HMGB1 expression since no change in lung HMGB1 levels was found after hemorrhage in mice made neutropenic with cyclophosphamide. Plasma concentrations of HMGB1 also increased after hemorrhage. Blockade of HMGB1 by administration of anti-HMGB1 antibodies prevented hemorrhage-induced increases in nuclear translocation of NF-kappa B in the lungs and pulmonary levels of proinflammatory cytokines, including keratinocyte-derived chemokine, IL-6, and IL-1 beta. Similarly, both the accumulation of neutrophils in the lung as well as enhanced lung permeability were reduced when anti-HMGB1 antibodies were injected after hemorrhage. These results demonstrate that hemorrhage results in increased HMGB1 expression in the lungs, primarily through neutrophil sources, and that HMGB1 participates in hemorrhage-induced acute lung injury.