Complete mitochondrial genome of freshwater pearl mussel Lamellidens marginalis (Lamarck, 1819) and its phylogenetic relation within unionidae family

Molecular Biology Reports - Freshwater mussels play a key role in ecology and are often considered as ecological indicators. Conversely, these molluscs are one of the most threatened groups due to...     

Dental pulp–derived stem cells inhibit osteoclast differentiation by secreting osteoprotegerin and deactivating AKT signalling in myeloid cells

Osteoclasts (OCs) differentiate from the monocyte/macrophage lineage, critically regulate bone resorption and remodelling in both homeostasis and pathology. Various immune and non-immune cells help initiating activation of myeloid cells for differentiation, whereas hyper-activation leads to pathogenesis, and mechanisms are yet to be completely understood. Herein, we show the efficacy of dental pulp–derived stem cells (DPSCs) in limiting RAW 264.7 cell differentiation and underlying molecular mechanism, which has the potential for future therapeutic application in bone-related disorders. We found that DPSCs inhibit induced OC differentiation of RAW 264.7 cells when co-cultured in a contact-free system. DPSCs reduced expression of key OC markers, such as NFATc1, cathepsin K, TRAP, RANK and MMP-9 assessed by quantitative RT-PCR, Western blotting and immunofluorescence detection methods. Furthermore, quantitative RT-PCR analysis revealed that DPSCs mediated M2 polarization of RAW 264.7 cells. To define molecular mechanisms, we found that osteoprotegerin (OPG), an OC inhibitory factor, was up-regulated in RAW 264.7 cells in the presence of DPSCs. Moreover, DPSCs also constitutively secrete OPG that contributed in limiting OC differentiation. Finally, the addition of recombinant OPG inhibited OC differentiation in a dose-dependent manner by reducing the expression of OC differentiation markers, NFATc1, cathepsin K, TRAP, RANK and MMP9 in RAW 264.7 cells. RNAKL and M-CSF phosphorylate AKT and activate PI3K-AKT signalling pathway during osteoclast differentiation. We further confirmed that OPG-mediated inhibition of the downstream activation of PI3K-AKT signalling pathway was similar to the DPSC co-culture–mediated inhibition of OC differentiation. This study provides novel evidence of DPSC-mediated inhibition of osteoclastogenesis mechanisms.     

Significantly Enhanced Heme Retention Ability of Myoglobin Engineered to Mimic the Third Covalent Linkage by Nonaxial Histidine to Heme (Vinyl) in Synechocystis Hemoglobin

Heme proteins, which reversibly bind oxygen and display a particular fold originally identified in myoglobin (Mb), characterize the “hemoglobin (Hb) superfamily.” The long known and widely investigated Hb superfamily, however, has been enriched by the discovery and investigation of new classes and members. Truncated Hbs typify such novel classes and exhibit a distinct two-on-two α-helical fold. The truncated Hb from the freshwater cyanobacterium Synechocystis exhibits hexacoordinate heme chemistry and bears an unusual covalent bond between the nonaxial His¹¹⁷ and a heme porphyrin 2-vinyl atom, which remains tightly associated with the globin unlike any other. It seems to be the most stable Hb known to date, and His¹¹⁷ is the dominant force holding the heme. Mutations of amino acid residues in the vicinity did not influence this covalent linkage. Introduction of a nonaxial His into sperm whale Mb at the topologically equivalent position and in close proximity to vinyl group significantly increased the heme stability of this prototype globin. Reversed phase chromatography, electrospray ionization-MS, and MALDI-TOF analyses confirmed the presence of covalent linkage in Mb I107H. The Mb mutant with the engineered covalent linkage was stable to denaturants and exhibited ligand binding and auto-oxidation rates similar to the wild type protein. This indeed is a novel finding and provides a new perspective to the evolution of Hbs. The successful attempt at engineering heme stability holds promise for the production of stable Hb-based blood substitute.     

Isozymes of <Emphasis Type="Italic">α</Emphasis>-amylases from newly isolated <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> CKB19: Production from immobilized cells

The aim of this study was to produce two isozymes of α-amylase by immobilization of a newly isolated soil bacterium. The bacterium was identified as Bacillus thuringiensis CKB19 on the basis of its 16S rRNA profile. Enzyme production by free cells increased linearly with cell growth up to 34 h in starch containing enriched liquid media. The active bacterial cells were immobilized in Caalginate beads, and operational stability of the entrapped cell was optimized for amylase production. Enzyme production was optimal at an alginate concentration of 2 g% (w/v), calcium chloride concentration of 1 M, and with 300 beads (each bead contained 2 × 10⁷ cells)/250 mL flask. Amylase production by the immobilized cells was about 3 times higher than free cell fermentation after 34 h of incubation. It was observed that the immobilized bacterium secreted two different amylases (Am-I and Am-II) into the culture fluid. The molecular masses of Am-I and Am-II were 59.6 and 44.7 kd, respectively, and showed optimum activity at pH 5.0 and 9.0. Both amylases showed optimum activity at 40°C and were stable at the same temperature, with losses of only 10 and 20% (for Am I and Am II, respectively) of their original activities after 24 h of incubation. Further, both amylases were salt tolerant (up to 4 M NaCl) and hydrolyzed raw starchy foods into glucose. All these characteristics make this enzyme mixture suitable for use as a digestive aid and for the improvement of digestibility of animal feed ingredients.     

Deciphering the Antimicrobial Potential of Taxus wallichiana Zucc: Identification and Characterization Using Bioassay-Guided Fractionation

Taxus wallichiana Zucc. is a high valued medicinal plant and has been mainly studied for its anti-cancer properties. However, research on its other important biological activities, such as its antimicrobial potential, still needs attention. The focus of the present study is to investigate the antimicrobial activity of secondary metabolites of T. wallichiana needles against 3 different groups of microorganisms, i. e., bacteria, actinobacteria, and fungi. Bioactive compounds from T. wallichiana needles were separated through column chromatography, and, TLC-bioautography. Mobile phases were optimized using Snyder's selectivity triangle. Antimicrobial spots were fractionated and compounds were identified by gas chromatography-mass spectroscopy (GC/MS) and liquid chromatography-mass spectrometry (LC/MS). Functional groups were characterized using Fourier transform infrared spectrometry (FTIR) and nuclear magnetic resonance (NMR) was used to identify the molecular structures. GC/MS and LC/MS data analysis confirm the presence of fatty acids (arachidic acid, behenic acid, palmitic acid, and stearic acid), vitamins (nicotinamide), and alkaloids (cinchonine, timolol), aminobenzamides (procainamide), carbocyclic sugar (myoinositol), and alkane hydrocarbon (hexadecane), having antimicrobial activity in the needles of T. wallichiana. To the best of our knowledge, this is the first report on the isolation and characterization of antimicrobial compounds from the needles of Taxus wallichiana (Himalayan yew). The data obtained from the present study will be supportive to the new drug discoveries in modern medicine with various combinations of medicinal plant's active constituents that can be used for curing many diseases.     

Penta- and hexa-coordinate ferric hemoglobins display distinct pH titration profiles measured by Soret peak shifts

Hemoglobins with diverse characteristics have been identified in all kingdoms of life. Their ubiquitous presence indicates that these proteins play important roles in physiology, though function for all hemoglobins are not yet established with certainty. Their physiological role may depend on their ability to bind ligands, which in turn is dictated by their heme chemistry. However, we have an incomplete understanding of the mechanism of ligand binding for these newly discovered hemoglobins and the measurement of their kinetic parameters depend on their coordination at the heme iron. To gain insights into their functional role, it is important to categorize the new hemoglobins into either penta- or hexa-coordinated varieties. We demonstrate that simple pH titration and absorbance measurements can determine the coordination state of heme iron atom in ferric hemoglobins, thus providing unambiguous information about the classification of new globins. This method is rapid, sensitive and requires low concentration of protein. Penta- and hexa-coordinate hemoglobins displayed distinct pH titration profiles as observed in a variety of hemoglobins. The pentacoordinate distal histidine mutant proteins of hexacoordinate hemoglobins and ligand-bound hexacoordinate forms of pentacoordinate hemoglobins reverse the pH titration profiles, thus validating the sensitivity of this spectroscopic technique.     

Ecological variations in adult life table attributes of Aedes aegypti (L.) from the desert and coastal regions of India

The ecological variation in biological and adult life-table attributes of two populations of Aedes aegypti (Diptera: Culicidae) from the desert (Jodhpur) and coastal (Kolkata) regions of India are assessed to understand the reproductive and survival strategies. The results showed that females lived longer than males in both strains. The desert strain was more r-strategist because of its higher intrinsic rate of increase (r_m = 0.23), finite rate of increase (λ = 1.25), lower life expectancy of males (7.9 days) and females (14.4 days), mean generation time (T = 19.2 days) and doubling time (DT = 3.0 days). However, there was no difference in net reproductive rate (R₀) between the desert and coastal strains. The coastal strain showed a longer female life expectancy (22.0 days) than the desert strain (14.4 days). However, the fecundity (eggs/female/day) was lower in the coastal strain (11.4) than in the desert strain (15.1). Conclusively, the desert (Jodhpur) strain is adapted to a better r-strategy than the coastal (Kolkata) strain of Ae. aegypti, which might be helpful to flourish in harsh environmental conditions. This study may provide accurate predictions of Ae. aegypti population dynamics for vector management.     

Comparison of innate immune activation after prolonged feeding of milk fermented with three species of Lactobacilli

The present investigation aimed at identifying the abilities of three different species of probiotic lactobacilli to modulate cellular immune responses in mouse neutrophils and macrophages in vivo over a study period of 60 days. Neutrophil respiratory burst enzymes (cytochrome c reductase and MPO) showed remarkable increased activity (P ≤ 0.01) after consumption of milks fermented by different species of probiotics over 30 and 60 days of feeding trials. Enzyme activities (β‐galactosidase and β‐glucuronidase) and nitric oxide production also increased considerably (P ≤ 0.01) in macrophages, both in peritoneal fluid and in enriched cell cultures. The effects of enhanced enzyme activities were corroborated by simultaneous increases in the phagocytic activities of neutrophils and macrophages. The increases in cellular functions were invariably maximal during the first 30 days of study and were maintained, but did not increase, over the next 30 days. Further, Lactobacillus helveticus‐fed groups were most effective at modulating neutrophil functions whereas Lactobacillus paracasei‐fed groups were more potent at enhancing macrophage functions. Together, our results indicate that probiotics have strain specific effects on stimulating cellular functions while not causing excessive stimulation of the immune system over longer feeding periods, thereby resulting in maximum and stable health benefits.