Biosynthesis of intestinal microvillar proteins. Pulse-chase labelling studies on maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV

The biogenesis of three intestinal microvillar enzymes, maltase-glucoamylase (EC 3.2.1.20), aminopeptidase A (aspartate aminopeptidase, EC 3.4.11.7) and dipeptidyl peptidase IV (EC 3.4.14.5), was studied by pulse-chase labelling of pig small-intestinal explants kept in organ culture. The earliest detectable forms of the enzymes were polypeptides of Mr 225000, 140000 and 115000 respectively. These were found to represent the enzymes in a 'high-mannose' state of glycosylation, as judged by their susceptibility to treatment with endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96). After about 40-60 min of chase, maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV were further modified to yield the mature polypeptides of Mr 245000, 170000 and 137000 respectively, which were expressed at the microvillar membrane after 60-90 min of chase. The fact that the enzymes before reaching the microvillar membrane were found in a Ca2+-precipitated membrane fraction (intracellular and basolateral membranes), but not in soluble form, indicates that during biogenesis maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV are transported and assembled in a membrane-bound state.     

Reproductive isolation and patterns of genetic differentiation in a cryptic butterfly species complex

Molecular studies of natural populations are often designed to detect and categorize hidden layers of cryptic diversity, and an emerging pattern suggests that cryptic species are more common and more widely distributed than previously thought. However, these studies are often decoupled from ecological and behavioural studies of species divergence. Thus, the mechanisms by which the cryptic diversity is distributed and maintained across large spatial scales are often unknown. In 1988, it was discovered that the common Eurasian Wood White butterfly consisted of two species (Leptidea sinapis and Leptidea reali), and the pair became an emerging model for the study of speciation and chromosomal evolution. In 2011, the existence of a third cryptic species (Leptidea juvernica) was proposed. This unexpected discovery raises questions about the mechanisms preventing gene flow and about the potential existence of additional species hidden in the complex. Here, we compare patterns of genetic divergence across western Eurasia in an extensive data set of mitochondrial and nuclear DNA sequences with behavioural data on inter‐ and intraspecific reproductive isolation in courtship experiments. We show that three species exist in accordance with both the phylogenetic and biological species concepts and that additional hidden diversity is unlikely to occur in Europe. The Leptidea species are now the best studied cryptic complex of butterflies in Europe and a promising model system for understanding the formation of cryptic species and the roles of local processes, colonization patterns and heterospecific interactions for ecological and evolutionary divergence.     

Diabetes alters the mRNA expression of insulin-like growth factors and their receptors in the mouse fallopian tube and uterus during the preimplantation stages

The possible role of insulin-like growth factors (IGFs) and their receptors (IGFRs) in the pathogenesis of diabetic embryopathy was investigated. Sexually mature female ICR mice of 6-8 weeks old were made diabetic by a single intraperitoneal injection with 200 mg/kg streptozotocin ten days prior to mating. Fallopian tubes and uterine tissues were obtained from the superovulated diabetic and normal mice 48, 72 and 96 hours following human chorionic gonadotropin (hCG) injection. The mRNA expression of IGF-1 and IGF-2 as well as their receptors was determined in the tissues using Real-time Polymerase Chain Reaction (Real-time PCR). The mRNA expression of IGF-1 in the fallopian tube and uterus of the diabetic mice was significantly lower 72 and 96 hours after hCG treatment, respectively, as compared to the controls. The mRNA expression of IGF-1R at 96 hours post-hCG treatment was significantly higher in the fallopian tube and lower in the uterus of the diabetic mice as compared to the controls. The mRNA expression IGF-2 in the fallopian tube was significantly higher 48 and 96 hours after hCG treatment, but was lower in the uterus of diabetic mice 96 hours after hCG treatment as compared to controls. The mRNA expression of IGF-2R in the diabetic mice was significantly higher 48 and 96 hours (the fallopian tube) and 48 hours (uterus) after hCG treatments as compared to the controls. In conclusion, an alteration in mRNA expression of IGFs and their receptors in the diabetic mice as observed in this study could possibly result in diabetic embryopathy.     

Analysis of simple sequence repeat markers linked with blast disease resistance genes in a segregating population of rice (Oryza sativa)

Among 120 simple sequence repeat (SSR) markers, 23 polymorphic markers were used to identify the segregation ratio in 320 individuals of an F(2) rice population derived from Pongsu Seribu 2, a resistant variety, and Mahsuri, a susceptible rice cultivar. For phenotypic study, the most virulent blast (Magnaporthe oryzae) pathotype, P7.2, was used in screening of F(2) population in order to understand the inheritance of blast resistance as well as linkage with SSR markers. Only 11 markers showed a good fit to the expected segregation ratio (1:2:1) for the single gene model (d.f. = 1.0, P < 0.05) in chi-square (χ(2)) analyses. In the phenotypic data analysis, the F(2) population segregated in a 3:1 (R:S) ratio for resistant and susceptible plants, respectively. Therefore, resistance to blast pathotype P7.2 in Pongsu Seribu 2 is most likely controlled by a single nuclear gene. The plants from F(2) lines that showed resistance to blast pathotype P7.2 were linked to six alleles of SSR markers, RM168 (116 bp), RM8225 (221 bp), RM1233 (175 bp), RM6836 (240 bp), RM5961 (129 bp), and RM413 (79 bp). These diagnostic markers could be used in marker assisted selection programs to develop a durable blast resistant variety.     

Comparative nutritional and mycochemical contents,biological activities and LC/MS screening of tuber from new recipe cultivation technique with wild type tuber of tiger’s milk mushroom of species <Emphasis Type="Italic">Lignosus rhinocerus</Emphasis>

Tiger’s milk mushroom is known for its valuable medicinal properties, especially the tuber part. However, wild tuber is very hard to obtain as it grows underground. This study first aimed to cultivate tiger’s milk mushroom tuber through a cultivation technique, and second to compare nutritional and mycochemical contents, antioxidant and cytotoxic activities and compound screening of the cultivated tuber with the wild tuber. Results showed an increase in carbohydrate content by 45.81% and protein content by 123.68% in the cultivated tuber while fat content reduced by 13.04%. Cultivated tuber also showed an increase of up to 64.21% for total flavonoid-like compounds and 62.51% of total β-d-glucan compared to the wild tuber. The antioxidant activity of cultivated tuber and wild tuber was 760 and 840 µg mL^?1, respectively. The cytotoxic activity of boiled water extract of cultivated tuber against a human lung cancer cell line (A549) was 65.50?±?2.12 µg mL^?1 and against a human breast cancer cell line (MCF7) was 19.35?±?0.11 µg mL^?1. β-d-glucan extract from the purification of boiled water extract of cultivated tuber showed cytotoxic activity at 57.78?±?2.29 µg mL^?1 against A549 and 33.50?±?1.41 µg mL^?1 against MCF7. However, the β-glucan extract from wild tuber did not show a cytotoxic effect against either the A549 or MCF7 cell lines. Also, neither of the extracts from cultivated tuber and wild tuber showed an effect against a normal cell line (MRC5). Compound profiling through by liquid chromatography mass spectrometry (LC/MS) showed the appearance of new compounds in the cultivated tuber. In conclusion, our cultivated tuber of tiger’s milk mushroom using a new recipe cultivation technique showed improved nutrient and bioactive compound contents, and antioxidant and cytotoxic activities compared to the wild tuber. Further investigations are required to obtain a better quality of cultivated tuber.     

A New Cold-Adapted,Organic Solvent Stable Lipase from Mesophilic Staphylococcus epidermidis AT2

The gene encoding a cold-adapted, organic solvent stable lipase from a local soil-isolate, mesophilic Staphylococcus epidermidis AT2 was expressed in a prokaryotic system. A two-step purification of AT2 lipase was achieved using butyl sepharose and DEAE sepharose column chromatography. The final recovery and purification fold were 47.09 % and 3.45, respectively. The molecular mass of the purified lipase was estimated to be 43 kDa. AT2 lipase was found to be optimally active at pH 8 and stable at pH 6–9. Interestingly, this enzyme demonstrated remarkable stability at cold temperature (<30 °C) and exhibited optimal activity at a temperature of 25 °C. A significant enhancement of the lipolytic activity was observed in the presence of Ca²⁺, Tween 60 and Tween 80. Phenylmethylsulfonylfluoride, a well known serine inhibitor did not cause complete inhibition of the enzymatic activity. AT2 lipase exhibited excellent preferences towards long chain triglycerides and natural oils. The lipolytic activity was stimulated by dimethylsulfoxide and diethyl ether, while more than 50 % of its activity was retained in methanol, ethanol, acetone, toluene, and n-hexane. Taken together, AT2 lipase revealed highly attractive biochemical properties especially because of its stability at low temperature and in organic solvents.     

Combination mode of antimalarial drug mefloquine and human serum albumin: Insights from spectroscopic and docking approaches

The interaction between mefloquine (MEF), the antimalarial drug, and human serum albumin (HSA), the main carrier protein in blood circulation, was explored using fluorescence, absorption, and circular dichroism spectroscopic techniques. Quenching of HSA fluorescence with MEF was characterized as static quenching and thus confirmed the complex formation between MEF and HSA. Association constant values for MEF-HSA interaction were found to fall within the range of 3.79-5.73 × 10⁴ M^˗1 at various temperatures (288, 298, and 308 K), which revealed moderate binding affinity. Hydrogen bonds and hydrophobic interactions were predicted to connect MEF and HSA together in the MEF-HSA complex, as deduced from the thermodynamic data (ΔS = +133.52 J mol⁻¹ K⁻¹ and ΔH = +13.09 kJ mol⁻¹) of the binding reaction and molecular docking analysis. Three-dimensional fluorescence spectral analysis pointed out alterations in the microenvironment around aromatic amino acid (tryptophan and tyrosine) residues of HSA consequent to the addition of MEF. Circular dichroic spectra of HSA in the wavelength ranges of 200-250 and 250-300 nm hinted smaller changes in the protein's secondary and tertiary structures, respectively, induced by MEF binding. Noncovalent conjugation of MEF to HSA bettered protein thermostability. Site marker competitive drug displacement results suggested HSA Sudlow's site I as the MEF binding site, which was also supported by molecular docking analysis.     

Pretreatment of empty palm fruit bunch for production of chemicals via catalytic pyrolysis

The effect of chemical pretreatments using NaOH, H₂O₂, and Ca(OH)₂ on Empty Palm Fruit Bunches (EPFB) to degrade EPFB lignin before pyrolyis was investigated. Spectrophotometer analysis proved consecutive addition of NaOH and H₂O₂ decomposed almost 100% of EPFB lignin compared to 44% for the Ca(OH)₂, H₂O₂ system while NaOH and Ca(OH)₂ used exclusively could not alter lignin much. Next, the pretreated EPFB was catalytically pyrolyzed. Experimental results indicated the phenolic yields over Al-MCM-41 and HZSM-5 catalysts were 90 wt% and 80 wt%, respectively compared to 67 wt% yield for the untreated sample under the same set of conditions. Meanwhile, the experiments with HY zeolite yielded 70 wt% phenols.