Recent advancements in the production and application of microbial pectinases: an overview

The wide utility and catalytic efficiency of microbial pectinase in various industries has greatly increased its global demand. Among the natural sources of pectinases, microbial pectinases are used frequently for its ease of production and unique physicochemical properties. Yet similar to other industrial enzymes, pectinases also face the constraint of thermo-tolerance and low yield in its economised production. The current review addresses the various strategies adopted to meet the high yield and thermo-tolerance of pectinases as well as the various attempts made in the field of pectinases to its improved production and better catalytic efficiency. The utilisation of natural as well as recombinant microbial sources, metagenomic approaches, metabolic engineering, site directed mutagenesis and media engineering techniques adopted in the field of pectinases have been discussed. The significance of pectinases in various industries is depicted by enlisting its applications. To the best our knowledge the current review is unique being the first attempt to compile the recent advancements in the field of pectinases.     

Tmod1 and CP49 Synergize to Control the Fiber Cell Geometry,Transparency, and Mechanical Stiffness of the Mouse Lens

The basis for mammalian lens fiber cell organization, transparency, and biomechanical properties has contributions from two specialized cytoskeletal systems: the spectrin-actin membrane skeleton and beaded filament cytoskeleton. The spectrin-actin membrane skeleton predominantly consists of α₂β₂-spectrin strands interconnecting short, tropomyosin-coated actin filaments, which are stabilized by pointed-end capping by tropomodulin 1 (Tmod1) and structurally disrupted in the absence of Tmod1. The beaded filament cytoskeleton consists of the intermediate filament proteins CP49 and filensin, which require CP49 for assembly and contribute to lens transparency and biomechanics. To assess the simultaneous physiological contributions of these cytoskeletal networks and uncover potential functional synergy between them, we subjected lenses from mice lacking Tmod1, CP49, or both to a battery of structural and physiological assays to analyze fiber cell disorder, light scattering, and compressive biomechanical properties. Findings show that deletion of Tmod1 and/or CP49 increases lens fiber cell disorder and light scattering while impairing compressive load-bearing, with the double mutant exhibiting a distinct phenotype compared to either single mutant. Moreover, Tmod1 is in a protein complex with CP49 and filensin, indicating that the spectrin-actin network and beaded filament cytoskeleton are biochemically linked. These experiments reveal that the spectrin-actin membrane skeleton and beaded filament cytoskeleton establish a novel functional synergy critical for regulating lens fiber cell geometry, transparency, and mechanical stiffness.     

Regulation of myocardial contractility and cell size by distinct PI3K-PTEN signaling pathways

The PTEN/PI3K signaling pathway regulates a vast array of fundamental cellular responses. We show that cardiomyocyte-specific inactivation of tumor suppressor PTEN results in hypertrophy, and unexpectedly, a dramatic decrease in cardiac contractility. Analysis of double-mutant mice revealed that the cardiac hypertrophy and the contractility defects could be genetically uncoupled. PI3Kalpha mediates the alteration in cell size while PI3Kgamma acts as a negative regulator of cardiac contractility. Mechanistically, PI3Kgamma inhibits cAMP production and hypercontractility can be reverted by blocking cAMP function. These data show that PTEN has an important in vivo role in cardiomyocyte hypertrophy and GPCR signaling and identify a function for the PTEN-PI3Kgamma pathway in the modulation of heart muscle contractility.     

Macroscopic assessment of cartilage shear: Effects of counter-surface roughness,synovial fluid lubricant,and compression offset

During joint articulation, cartilage is subjected to compression, shear, and sliding, mechanical factors that regulate and affect cartilage metabolism. The objective of this study was to use an in vitro material-on-cartilage shear test to elucidate the effects of counter-surface roughness (Polished, Mildly rough, and Rough), lubricants (phosphate buffered saline (PBS) and bovine synovial fluid (bSF)), and compression offset on the shearing and sliding of normal human talar cartilage under dynamic lateral displacement. Peak shear stress (σ_xz,m) and strain (E_xz,m) increased with increasing platen roughness and compression offset, and were 30% higher with PBS than with bSF. Compared to PBS, bSF was more effective as a lubricant for P than for M and R platens as indicated by the higher reduction in kinetic friction coefficient (?60% vs. ?20% and ?19%, respectively), σ_xz,m (?50% vs. ?14% and ?17%) and E_xz,m (?54% vs. ?19% and ?17%). Cartilage shear and sliding were evident for all counter-surfaces either at low compression offset (10%) or with high lateral displacement (70%), regardless of lubricant. An increase in tissue shear occurred with either increased compression offset or increased surface roughness. This material and biomechanical test system allow control of cartilage σ_xz,m and E_xz,m, and hence, sliding magnitude, for an imposed lateral displacement. It therefore can facilitate study of cartilage mechanobiological responses to distinct regimes of cartilage loading and articulation, such as shear with variable amounts of sliding.     

Modulation of Depth-dependent Properties in Tissue-engineered Cartilage with a Semi-permeable Membrane and Perfusion: A Continuum Model of Matrix Metabolism and Transport

The functional properties of cartilaginous tissues are determined predominantly by the content, distribution, and organization of proteoglycan and collagen in the extracellular matrix. Extracellular matrix accumulates in tissue-engineered cartilage constructs by metabolism and transport of matrix molecules, processes that are modulated by physical and chemical factors. Constructs incubated under free-swelling conditions with freely permeable or highly permeable membranes exhibit symmetric surface regions of soft tissue. The variation in tissue properties with depth from the surfaces suggests the hypothesis that the transport processes mediated by the boundary conditions govern the distribution of proteoglycan in such constructs. A continuum model (DiMicco and Sah in Transport Porus Med 50:57–73, 2003) was extended to test the effects of membrane permeability and perfusion on proteoglycan accumulation in tissue- engineered cartilage. The concentrations of soluble, bound, and degraded proteoglycan were analyzed as functions of time, space, and non-dimensional parameters for several experimental configurations. The results of the model suggest that the boundary condition at the membrane surface and the rate of perfusion, described by non-dimensional parameters, are important determinants of the pattern of proteoglycan accumulation. With perfusion, the proteoglycan profile is skewed, and decreases or increases in magnitude depending on the level of flow-based stimulation. Utilization of a semi-permeable membrane with or without unidirectional flow may lead to tissues with depth-increasing proteoglycan content, resembling native articular cartilage.     

Evaluation of fungal culture filtrate containing chitinase as a biocontrol agent against Helicoverpa armigera

AIMS: To evaluate the biocontrol efficacy of culture filtrate containing chitinase from Trichoderma harzianum against Heliothis. METHODS AND RESULTS: T. harzianum was cultured by submerged fermentation using colloidal chitin as sole carbon source. The ability of the culture filtrate to hydrolyse colloidal chitin indicated the presence of chitinase as one of its components. Biocontrol assay on Heliothis showed that the culture filtrate is a potent antifeedant as it reduced the feeding rate and body weight of the larvae. It reduced the successful pupation and increased larval and pupal mortality in a dosage-dependent manner when applied topically. The highest mortalities (70%) were recorded for groups treated with 2000 U ml(-1) chitinase activity. The percentage of adult emergence was zero for the highest chitinase concentration (2000 U ml(-1)) tried. CONCLUSIONS: The studies showed that the culture filtrate containing chitinase from T. harzianum is capable of negatively affecting the growth and metamorphosis of Heliothis larvae. SIGNIFICANCE AND IMPACT OF THE STUDY: In view of the need for safer and environmentally friendly pest management tools, the present study could help in the development of enzyme-based biopesticides against Heliothis.     

Antistress activity of ethanolic extract of Asparagus racemosus Willd roots in mice

Ethanolic extract of the roots of A. racemosus improved the stress tolerance in chemical writhing test and swimming endurance test at all the doses as compared to stress control group. Restraint stress induced elevation of blood glucose, triglyceride and cholesterol levels were significantly lowered by pretreatment with extract. Moreover, stress induced variations in levels of lipid peroxidation, nitric oxide, protein and glutathione content in mouse brain were significantly ameliorated by pretreatment with extract. The extract attenuated the elevated weight of adrenal glands and increased the reduced weight of the spleen during stress. In conclusion, the results suggest antistress property of Asparagus racemosus in different model of stress.     

Surface masking shapes the traffic of the neuropeptide Y Y2 receptor

The neuropeptide Y (NPY) Y2 receptor shows a large masked surface population in adherent CHO cells or in forebrain cell aggregates, but not in dispersed cells or in particulates from these sources. This is related to adhesion via acidic motifs in the extracellular N-terminal domain. Masking of the Y2 receptor is lifted by non-permeabilizing mechanical dispersion of cells, which also increases internalization of Y2 agonists. Mechanical dispersion and detachment by EDTA expose the same number of surface sites. As we have already shown, phenylarsine oxide (PAO), a cysteine-bridging agent, and to a lesser extent also the cysteine alkylator N-ethylmaleimide, unmask the surface Y2 sites without cell detachment or permeabilization. We now demonstrate that unmasking by permeabilizing but non-detaching treatment with cholesterol-binding detergents digitonin and edelfosine compares with and overlaps that of PAO. The caveolar/raft cholesterol-targeting macrolide filipin III however produces only partial unmasking. Depletion of the surface sites by N-terminally clipped Y2 agonists indicates larger accessibility for a short highly helical peptide. These findings indicate presence of a dynamic masked pool including majority of the cell surface Y2 receptors in adherent CHO cells. This compartmentalization is obviously involved in the low internalization of Y2 receptors in these cells.     

Osteopontin is an endogenous modulator of the constitutively activated phenotype of pulmonary adventitial fibroblasts in hypoxic pulmonary hypertension

Increased cell proliferation and migration, of several cell types are key components of vascular remodeling observed in pulmonary hypertension (PH). Our previous data demonstrate that adventitial fibroblasts isolated from pulmonary arteries of chronically hypoxic hypertensive calves (termed PH-Fibs) exhibit a "constitutively activated" phenotype characterized by high proliferative and migratory potential. Osteopontin (OPN) has been shown to promote several cellular activities including growth and migration in cancer cells. We thus tested the hypothesis that elevated OPN expression confers the "activated" highly proproliferative and promigratory/invasive phenotype of PH-Fibs. Our results demonstrate that, both in vivo and ex vivo, PH-Fibs exhibited increased expression of OPN, as well as its cognate receptors, α(V)β(3) and CD44, compared with control fibroblasts (CO-Fibs). Augmented OPN expression in PH-Fibs corresponded to their high proliferative, migratory, and invasive properties and constitutive activation of ERK1/2 and AKT signaling. OPN silencing via small interfering RNA or sequestering OPN production by specific antibodies led to decreased proliferation, migration, invasion, and attenuated ERK1/2, AKT phosphorylation in PH-Fibs. Furthermore, increasing OPN levels in CO-Fibs via recombinant OPN resulted in significant increases in their proliferative, migratory, and invasive capabilities to the levels resembling those of PH-Fibs. Thus our data suggest OPN as an essential contributor to the activated (highly proliferative, migratory, and proinvasive) phenotype of pulmonary adventitial fibroblasts in hypoxic PH.     

Biomechanical assessment of tissue retrieved after in vivo cartilage defect repair: tensile modulus of repair tissue and integration with host cartilage

Failure to restore the mechanical properties of tissue at the repair site and its interface with host cartilage is a common problem in tissue engineering procedures to repair cartilage defects. Quantitative in vitro studies have helped elucidate mechanisms underlying processes leading to functional biomechanical changes. However, biomechanical assessment of tissue retrieved from in vivo studies of cartilage defect repair has been limited to compressive tests. Analysis of integration following in vivo repair has relied on qualitative histological methods. The objectives of this study were to develop a quantitative biomechanical method to assess (1) the tensile modulus of repair tissue and (2) its integration in vivo, as well as determine whether supplementation of transplanted chondrocytes with IGF-I affected these mechanical properties. Osteochondral blocks were obtained from a previous 8 month study on the effects of IGF-I on chondrocyte transplantation in the equine model. Tapered test specimens were prepared from osteochondral blocks containing the repair/native tissue interface and adjacently located blocks of intact native tissue. Specimens were then tested in uniaxial tension. The tensile modulus of repair tissue averaged 0.65 MPa, compared to the average of 5.2 MPa measured in intact control samples. Integration strength averaged 1.2 MPa, nearly half the failure strength of intact cartilage samples, 2.7 MPa. IGF-I treatment had no detectable effects on these mechanical properties. This represents the first quantitative biomechanical investigation of the tensile properties of repair tissue and its integration strength in an in vivo joint defect environment.