Identification and kinetics analysis of a novel heparin-binding site (KEDK) in human tenascin-C

The interaction between tenascin-C (TN-C), a multi-subunit extracellular matrix protein, and heparin was examined using a surface plasmon resonance-based technique on a Biacore system. The aims of the present study were to examine the affinity of fibronectin type III repeats of TN-C fragments (TNIII) for heparin, to investigate the role of the TNIII4 domains in the binding of TN-C to heparin, and to delineate a sequence of amino acids within the TNIII4 domain, which mediates cooperative heparin binding. At a physiological salt concentration, and pH 7.4, TNIII3-5 binds to heparin with high affinity (K(D) = 30 nm). However, a major heparin-binding site in TNIII5 produces a modest affinity binding at a K(D) near 4 microm, and a second site in TNIII4 enhances the binding by several orders of magnitude, although it was far too weak to produce an observable binding of TNIII4 by itself. Moreover, mutagenesis of the KEDK sequence in the TNIII4 domain resulted in the significant reduction of heparin-binding affinity. In addition, residues in the KEDK sequences are conserved in TN-C throughout mammalian evolution. Thus the structure-based sequence alignment, mutagenesis, and sequence conservation data together reveal a KEDK sequence in TNIII4 suggestive of a minor heparin-binding site. Finally, we demonstrate that TNIII4 contains binding sites for heparin sulfate proteoglycan and enhances the heparin sulfate proteoglycan-dependent human gingival fibroblast adhesion to TNIII5, thus providing the biological significance of heparin-binding site of TNIII4. These results suggest that the heparin-binding sites may traverse TNIII4-5 and thus require KEDK in TNIII4 for optimal heparin-binding.     

Antioxidative effects of Cinnamomi cassiae and Rhodiola rosea extracts in liver of diabetic mice

Both Cinnamomi cassiae and Rhodiola rosea extracts are used as anti-diabetic folk medicines. Recently, increased oxidative stress was shown to play an important role in the etiology and pathogenesis of diabetes mellitus and its complications. This study was designed to examine the effects of Cinnamomi cassiae and Rhodiola rosea extracts on blood glucose, lipid peroxidation, the level of reduced glutathione and its related enzymes (glutathione reductase, glutathione S-transferase), and the activity of the antioxidant enzymes (catalase, superoxide dismutase and glutathione peroxidase) in the liver of db/db mice. Diabetic C57BL/Ks db/db mice were used as experimental models. Mice were divided into control (n=10), Cinnamomi cassiae (200 mg/kg/day, n=10), and Rhodiola rosea (200 mg/kg/day, n=10) treated groups for 12 weeks of treatment. These type II diabetic mice were used to investigate the effects of Cinnamomi cassiae and Rhodiola rosea on blood glucose, reduced glutathione, glutathione reductase, glutathione S-transferase, glutathione peroxidase, lipid peroxidation, catalase and superoxide dismutase. Cinnamomi cassiae and Rhodiola rosea extracts significantly decreased on blood glucose, increased levels of reduced glutathione and the activities of glutathione reductase, glutathione S-transferase, glutathione peroxidase, catalase and superoxide dismutase in the liver. Extract treatment also significantly decreased lipid peroxidation. Cinnamomi cassiae and Rhodiola rosea extracts may be effective for correcting hyperglycemia and preventing diabetic complications.     

Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG

Background

Receptors with a single transmembrane (TM) domain are essential for the signal transduction across the cell membrane. NMR spectroscopy is a powerful tool to study structure of the single TM domain. The expression and purification of a TM domain in Escherichia coli (E.coli) is challenging due to its small molecular weight. Although ketosteroid isomerase (KSI) is a commonly used affinity tag for expression and purification of short peptides, KSI tag needs to be removed with the toxic reagent cyanogen bromide (CNBr).

Result

The purification of the TM domain of p75 neurotrophin receptor using a KSI tag with the introduction of a thrombin cleavage site is described herein. The recombinant fusion protein was refolded into micelles and was cleaved with thrombin. Studies showed that purified protein could be used for structural study using NMR spectroscopy.

Conclusions

These results provide another strategy for obtaining a single TM domain for structural studies without using toxic chemical digestion or acid to remove the fusion tag. The purified TM domain of p75 neurotrophin receptor will be useful for structural studies.     

Effects of synthetic cohesin-containing scaffold protein architecture on binding dockerin-enzyme fusions on the surface of Lactococcus lactis

Background

In the last years, the biotechnological production of platform chemicals for fuel components has become a major focus of interest. Although ligno-cellulosic material is considered as suitable feedstock, the almost inevitable pretreatment of this recalcitrant material may interfere with the subsequent fermentation steps. In this study, the fungus Ustilago maydis was used to produce itaconic acid as platform chemical for the synthesis of potential biofuels such as 3-methyltetrahydrofuran. No studies, however, have investigated how pretreatment of ligno-cellulosic biomass precisely influences the subsequent fermentation by U. maydis. Thus, this current study aims to first characterize U. maydis in shake flasks and then to evaluate the influence of three exemplary pretreatment methods on the cultivation and itaconic acid production of this fungus. Cellulose enzymatically hydrolysed in seawater and salt-assisted organic-acid catalysed cellulose were investigated as substrates. Lastly, hydrolysed hemicellulose from fractionated beech wood was applied as substrate.

Results

U. maydis was characterized on shake flask level regarding its itaconic acid production on glucose. Nitrogen limitation was shown to be a crucial condition for the production of itaconic acid. For itaconic acid concentrations above 25 g/L, a significant product inhibition was observed. Performing experiments that simulated influences of possible pretreatment methods, U. maydis was only slightly affected by high osmolarities up to 3.5 osmol/L as well as of 0.1 M oxalic acid. The production of itaconic acid was achieved on pretreated cellulose in seawater and on the hydrolysed hemicellulosic fraction of pretreated beech wood.

Conclusion

The fungus U. maydis is a promising producer of itaconic acid, since it grows as single cells (yeast-like) in submerged cultivations and it is extremely robust in high osmotic media and real seawater. Moreover, U. maydis can grow on the hemicellulosic fraction of pretreated beech wood. Thereby, this fungus combines important advantages of yeasts and filamentous fungi. Nevertheless, the biomass pretreatment does indeed affect the subsequent itaconic acid production. Although U. maydis is insusceptible to most possible impurities from pretreatment, high amounts of salts or residues of organic acids can slow microbial growth and decrease the production. Consequently, the pretreatment step needs to fit the prerequisites defined by the actual microorganisms applied for fermentation.     

Suppression of inflammation by recombinant Salmonella typhimurium harboring CCL22 microRNA

Atopic dermatitis (AD) is an inflammatory, chronically relapsing, puritic skin disorder. These syndromes result from multifactorial inheritance, with interaction between genetic and environmental factors. In particular, the macrophage-derived chemokine CCL22 is directly implicated in skin inflammatory reactions and its levels are significantly elevated in serum and correlated with disease severity in AD. We tested the suppression of the CCL22 gene by microRNA (miRNA) and observed the effects in mice with inflammation similar to AD. We used Salmonella as a vector to deliver miRNA. The recombinant strain of Salmonella typhimurium expressing CCL22 miRNA (ST-miRCCL22) was prepared for in vivo knockdown of CCL22. ST-miRCCL22 was orally inoculated into mice and the CCL22 gene suppressed with CCL22 miRNA in the activated lymphocytes. IgE and interleukin-4 were inhibited and interferon-γ was induced after treatments with ST-miRCCL22 and CCL22 was suppressed. Further, Th17 cells were suppressed in the atopic mice treated with ST-miRCCL22. These results suggested that suppression of the CCL22 gene using Salmonella induced anti-inflammatory effects.     

Clinical features and predictors for disease natural progression in adults with Pompe disease: a nationwide prospective observational study

Background

Due partly to physicians’ unawareness, many adults with Pompe disease are diagnosed with great delay. Besides, it is not well known which factors influence the rate of disease progression, and thus disease outcome. We delineated the specific clinical features of Pompe disease in adults, and mapped out the distribution and severity of muscle weakness, and the sequence of involvement of the individual muscle groups. Furthermore, we defined the natural disease course and identified prognostic factors for disease progression.

Methods

We conducted a single-center, prospective, observational study. Muscle strength (manual muscle testing, and hand-held dynamometry), muscle function (quick motor function test), and pulmonary function (forced vital capacity in sitting and supine positions) were assessed every 3–6 months and analyzed using repeated-measures ANOVA.

Results

Between October 2004 and August 2009, 94 patients aged between 25 and 75 years were included in the study. Although skeletal muscle weakness was typically distributed in a limb-girdle pattern, many patients had unfamiliar features such as ptosis (23%), bulbar weakness (28%), and scapular winging (33%). During follow-up (average 1.6 years, range 0.5-4.2 years), skeletal muscle strength deteriorated significantly (mean declines of ?1.3% point/year for manual muscle testing and of ?2.6% points/year for hand-held dynamometry; both p<0.001). Longer disease duration (>15 years) and pulmonary involvement (forced vital capacity in sitting position <80%) at study entry predicted faster decline. On average, forced vital capacity in supine position deteriorated by 1.3% points per year (p=0.02). Decline in pulmonary function was consistent across subgroups. Ten percent of patients declined unexpectedly fast.

Conclusions

Recognizing patterns of common and less familiar characteristics in adults with Pompe disease facilitates timely diagnosis. Longer disease duration and reduced pulmonary function stand out as predictors of rapid disease progression, and aid in deciding whether to initiate enzyme replacement therapy, or when.

     

MicroRNA miR-199a* regulates the MET proto-oncogene and the downstream extracellular signal-regulated kinase 2 (ERK2)

MicroRNAs (miRNAs) constitute a class of small noncoding RNAs that play important roles in a variety of biological processes including development, apoptosis, proliferation, and differentiation. Here we show that the expression of miR-199a and miR-199a* (miR-199a/a*), which are processed from the same precursor, is confined to fibroblast cells among cultured cell lines. The fibroblast-specific expression pattern correlated well with methylation patterns: gene loci on chromosome 1 and 19 were fully methylated in all examined cell lines but unmethylated in fibroblasts. Transfection of miR-199a and/or -199a* mimetics into several cancer cell lines caused prominent apoptosis with miR-199a* being more pro-apoptotic. The mechanism underlying apoptosis induced by miR-199a was caspase-dependent, whereas a caspase-independent pathway was involved in apoptosis induced by miR-199a* in A549 cells. By employing microarray and immunoblotting analyses, we identified the MET proto-oncogene as a target of miR-199a*. Studies with a luciferase reporter fused to the 3'-untranslated region of the MET gene demonstrated miR-199a*-mediated down-regulation of luciferase activity through a binding site of miR-199a*. Interestingly, extracellular signal-regulated kinase 2 (ERK2) was also down-regulated by miR-199a*. Coordinated down-regulation of both MET and its downstream effector ERK2 by miR-199a* may be effective in inhibiting not only cell proliferation but also motility and invasive capabilities of tumor cells.