Can recombinant tree shrew (Tupaia belangeri chinensis) chymosin coagulate cow (Bos taurus) milk?

Genetically engineered chymosin from the tree shrew (Tupaia belangeri chinensis) has been obtained and partially characterized for the first time. The target enzyme was produced in Escherichia coli, strain BL21(DE3). It was shown that tree shrew recombinant chymosin coagulates cow milk (Bos taurus). The total and specific milk-clotting activity of the obtained enzyme was 0.7–5.3 IMCU/mL and 8.8–16.6 IMCU/mg. The nonspecific proteolytic activity of tree shrew recombinant chymosin in relation to total bovine casein was 30 and 117% higher than that of recombinant chymosin of cow and of single-humped camel respectively. It was found that in comparison with most of the known genetically engineered chymosins, the tree shrew enzyme showed exceptionally low thermal stability. After heating at 45°C, the coagulation ability of tree shrew recombinant chymosin decreased by more than 40%, and at 50°C the enzyme lost more than 90% of the initial milk-clotting activity. The Michaelis constant (Km), enzyme turnover number (kcat), and catalytic efficiency (kcat/Km) for genetically engineered chymosin from the tree shrew were 6.3 ± 0.1 µM, 11 927 ± 3169 s–1 and 1968 ± 620 µM–1 s–1, respectively. Comparative analysis showed that the primary structure of the chymosin-sensitive site of cow kappa-casein and the supposed similar sequence of tree shrew kappa-casein differed by 75%. The ability of tree shrew recombinant chymosin to coagulate cow’s milk, along with a low thermal stability and high catalytic efficiency with respect to the substrate, imitating the chymosin-sensitive site of cow kappa-casein, suggests that this enzyme is of potential interest for cheese making

The effect of thioredoxin and prochymosin coexpression on the refolding of recombinant alpaca chymosin

The milk-clotting enzyme chymosin is a member of the group of aspartate proteinases. Chymosin is the main component of rennet traditionally obtained from the stomachs of dairy calves and widely used to coagulate milk in the production of various types of cheese. Another source of chymosin, which does not require the killing of animals, is based on recombinant DNA technology. Recombinant alpaca chymosin has a number of valuable technological properties that make it attractive for use in cheese-making as an alternative to recombinant bovine chymosin. The purpose of this work is to study the effect of coexpression of thioredoxin and prochymosin on the refolding of the recombinant zymogen and the activity of alpaca chymosin. To achieve this goal, on the basis of the pET32a plasmid, an expression vector was constructed containing the thioredoxin A gene fused to the N-terminal sequence of the marker enzyme zymogen, alpaca prochymosin. Using the constructed vector, pETTrxProChn, a strain-producer of the recombinant chimeric protein thioredoxin-prochymosin was obtained. The choice of prochymosin as a model protein is due to the ability of autocatalytic activation of this zymogen, in which the pro-fragment is removed, together with the thioredoxin sequence attached to it, with the formation of active chymosin. It is shown that Escherichia coli strain BL21 transformed with the pET-TrxProChn plasmid provides an efficient synthesis of the thioredoxin-prochymosin chimeric molecule. However, the chimeric protein accumulates in inclusion bodies in an insoluble form. Therefore, a renaturation procedure was used to obtain the active target enzyme. Fusion of thioredoxin capable of disulfide-reductase activity to the N-terminal sequence of prochymosin provides optimal conditions for zymogen refolding and increases the yield of recombinant alpaca chymosin immediately after activation and during long-term storage by 13 and 15 %, respectively. The inclusion of thioredoxin in the composition of the chimeric protein, apparently, contributes to the process of correct reduction of disulfide bonds in the prochymosin molecule, which is reflected in the dynamics of the increase in the milk-clotting activity of alpaca chymosin during long-term storage

MODERN CONCEPTS OF THE PATHOGENESIS OF POLYCYSTIC OVARY SYNDROME (LITERATURE REVIEW)

Polycystic ovarian syndrome (PCOS) is one of the most common gynecological disorders, which affects 5-20 % of young women all over the world in different ethnic groups and races, and is a frequent cause of infertility, a miscarriage, complicated pregnancy and childbirth. PCOS is a polysymptomatic disease characterized by hyperandrogenism, menstrual dysfunction and multifollicular structure of the ovaries with ultrasound examination. The main manifestations of PCOS lie in the basis of N1H and Rotterdam diagnostic criteria. There are several different phenotypes of polycystic ovary syndrome in accordance with these criteria. At the same time, PCOS is a metabolic disorder, and the role of insulin resistance has been proven in development of this condition. 1n consequence of which pathogenesis, diagnosis and treatment are of interest not only for gynecologists, but also for endocrinologists, cardiologists, and others clinicians, as PCOS is a serious problem associated with obesity, increased risks of endometrial adenocarcinoma, hypertension and cardiovascular complications, type 2 diabetes and etc. Moreover, economic costs are billions of dollars ayear around the world for the treatment of complications of PCOS. To date, many researchers have been studying the formation of PCOS, and our article discusses the theories explaining pathogenesis and etiology of this disease, clinical manifestations and current approaches to diagnosis of PCOS

SNX3 controls Wingless/Wnt secretion through regulating retromer-dependent recycling of Wntless

Drosophila Wingless (Wg) acts as a morphogen during development. Wg secretion is controlled by a seven-pass transmembrane cargo Wntless (Wls). We have recently identified retromer as a key regulator involved in Wls trafficking. As sorting nexin (SNX) molecules are essential components of the retromer complex, we hypothesized that specific SNX(s) is required for retromer-mediated Wnt secretion. Here, we generated Drosophila mutants for all of the eight snx members, and identified Drosophila SNX3 (DSNX3) as an essential molecule required for Wg secretion. We show that Wg secretion and its signaling activity are defective in Dsnx3 mutant clones in wing discs. Wg levels in the culture medium of Dsnx3-depleted S2 cells are also markedly reduced. Importantly, Wls levels are strikingly reduced in Dsnx3 mutant cells, and overexpression of Wls can rescue the Wg secretion defect observed in Dsnx3 mutant cells. Moreover, DSNX3 can interact with the retromer component Vps35, and co-localize with Vps35 in early endosomes. These data indicate that DSNX3 regulates Wg secretion via retromer-dependent Wls recycling. In contrast, we found that Wg secretion is not defective in cells mutant for Drosophila snx1 and snx6, two components of the classical retromer complex. Ectopic expression of DSNX1 or DSNX6 fails to rescue the Wg secretion defect in Dsnx3 mutant wing discs and in Dsnx3 dsRNA-treated S2 cells. These data demonstrate the specificity of the DSNX3-retromer complex in Wls recycling. Together, our findings suggest that DSNX3 acts as a cargo-specific component of retromer, which is required for endocytic recycling of Wls and Wg/Wnt secretion

Dynamic auroral storms on Saturn as observed by the Hubble Space Telescope

We present observations of significant dynamics within two UV auroral storms observed on Saturn using the Hubble Space Telescope in April/May 2013. Specifically, we discuss bursts of auroral emission observed at the poleward boundary of a solar wind-induced auroral storm, propagating at ∼330% rigid corotation from near ∼01 h LT toward ∼08 h LT. We suggest that these are indicative of ongoing, bursty reconnection of lobe flux in the magnetotail, providing strong evidence that Saturn’s auroral storms are caused by large-scale flux closure. We also discuss the later evolution of a similar storm and show that the emission maps to the trailing region of an energetic neutral atom enhancement. We thus identify the auroral form with the upward field-aligned continuity currents flowing into the associated partial ring current

Sharp boundaries of Dpp signalling trigger local cell death required for Drosophila leg morphogenesis

Article available at http://dx.doi.org/10.1038/ncb1518Morphogens are secreted signalling molecules that govern many developmental processes1. In the Drosophila wing disc, the transforming growth factor (TGF) homologue Decapentaplegic (Dpp) forms a smooth gradient and specifies cell fate by conferring a defined value of morphogen activity. Thus, neighbouring cells have similar amounts of Dpp protein, and if a sharp discontinuity in Dpp activity is generated between these cells, Jun kinase (JNK)-dependent apoptosis is triggered to restore graded positional information2, 3. To date, it has been assumed that this apoptotic process is only activated when normal signalling is distorted. However, we now show that a similar process occurs during normal development: rupture in Dpp activity occurs during normal segmentation of the distal legs of Drosophila. This sharp boundary of Dpp signalling, independently of the absolute level of Dpp activity, induces a JNK—reaper-dependent apoptosis required for the morphogenesis of a particular structure of the leg, the joint. Our results show that Dpp could induce a developmental programme not only in a concentration dependent manner, but also by the creation of a sharp boundary of Dpp activity. Furthermore, the same process could be used either to restore a normal pattern in response to artificial disturbance or to direct a morphogenetic process.This work has been supported by grants from the Dirección General de Investigación Científica y Técnica (BMC 2002-00300), the Comunidad Autónoma de Madrid (08.1/0031/2001.1 and GR/SAL/0147/2004) and an Institutional Grant from the Fundación Ramón Areces. C.M. is a recipient of a Formación del Personal Universitario (F.P.U.) fellowship from the Ministerio de Educación y Ciencia.Peer reviewe

MESSENGER observations of Mercury's magnetic field structure

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/96229/1/jgre3136.pd

The Sno Oncogene Antagonizes Wingless Signaling during Wing Development in Drosophila

The Sno oncogene (Snoo or dSno in Drosophila) is a highly conserved protein and a well-established antagonist of Transforming Growth Factor-β signaling in overexpression assays. However, analyses of Sno mutants in flies and mice have proven enigmatic in revealing developmental roles for Sno proteins. Thus, to identify developmental roles for dSno we first reconciled conflicting data on the lethality of dSno mutations. Then we conducted analyses of wing development in dSno loss of function genotypes. These studies revealed ectopic margin bristles and ectopic campaniform sensilla in the anterior compartment of the wing blade suggesting that dSno functions to antagonize Wingless (Wg) signaling. A subsequent series of gain of function analyses yielded the opposite phenotype (loss of bristles and sensilla) and further suggested that dSno antagonizes Wg signal transduction in target cells. To date Sno family proteins have not been reported to influence the Wg pathway during development in any species. Overall our data suggest that dSno functions as a tissue-specific component of the Wg signaling pathway with modest antagonistic activity under normal conditions but capable of blocking significant levels of extraneous Wg, a role that may be conserved in vertebrates

FIP1/RCP Binding to Golgin-97 Regulates Retrograde Transport from Recycling Endosomes to the trans-Golgi Network

This study shows that Rab11 and its binding protein FIP1 are required for retrograde delivery of TGN38 and Shiga toxin from early/recycling endosomes to the TGN. We also demonstrate that Golgin-97 as a FIP1-binding protein and that this binding regulates the targeting of retrograde transport vesicles to the TGN