Silenciamiento génico en insectos plaga que afectan la industria agrícola usando ARN de interferencia / Gene silencing in pest insects that affect the agricultural industry using interference RNA

Resumen Los insectos plaga, son especies de organismos vivos que en forma constante se encuentran en poblaciones altas, ocasionando daños económicos en los cultivos. Generalmente, suele tratarse de especies puntuales, por lo general, sólo una o dos, que pueden causar gran afectación económica en el sector de la agricultura. En las últimas 3 décadas se ha venido desarrollando el concepto de un proceso biológico, detectado en eucariotas ampliamente, mediante el que se pueden silenciar genes, a partir de ARN de doble cadena (ARNdc). Esta maquinaria se ha investigado para conocer su funcionamiento y buscar potenciales aplicaciones que podrían tener en el campo de la biotecnología. En varios estudios se encontró que el silenciamiento de genes se debe a las interacciones enzimáticas intracelulares citoplasmáticas con moléculas de ARN pequeñas (ARNsi), que actúan sobre el ARN mensajero (ARNm) intracelular, impidiendo que este se traduzca a proteína. Mediante este mecanismo se busca silenciar genes específicos en insectos plaga, que sean esenciales para que el insecto pueda vivir y de esa manera evitar la proliferación de la plaga. Este artículo recopila los estudios realizados acerca del ARN de interferencia, referidos al mecanismo genético de los insectos, como alternativa para su control.

AbstractPest insects are species of living organisms that are constantly found in high populations,causing economic crops damage. Generally, it tends to be specific species, usually onlyone or two, which can cause great economic damage in the agricultural sector. In thelast 3 decades, the concept of a biological process has been developed, widely detected in eukaryotes, by which genes can be silenced, from double-stranded RNA (dsRNA). This machinery has been investigated to understand its operation and to look for potential applications that it could have in the field of biotechnology. In several studies it was found that gene silencing is due to cytoplasmic intracellular enzymatic interactions with small RNA molecules (siRNA), which act on intracellular messenger RNA (mRNA), preventing it from translating a protein. Through this mechanism, the aim is to silence specific genes in pest insects, which are essential for the insect to live and thus prevent the proliferation of the pest. This article compiles the studies carried out on RNA interference, referring to the genetic mechanism of insects, as an alternative for its control.

New multienzymatic complex formed between human cathepsin D and snake venom phospholipase A2

Background Cathepsin D (CatD) is a lysosomal proteolytic enzyme expressed in almost all tissues and organs. This protease is a multifunctional enzyme responsible for essential biological processes such as cell cycle regulation, differentiation, migration, tissue remodeling, neuronal growth, ovulation, and apoptosis. The overexpression and hypersecretion of CatD have been correlated with cancer aggressiveness and tumor progression, stimulating cancer cell proliferation, fibroblast growth, and angiogenesis. In addition, some studies report its participation in neurodegenerative diseases and inflammatory processes. In this regard, the search for new inhibitors from natural products could be an alternative against the harmful effects of this enzyme. Methods An investigation was carried out to analyze CatD interaction with snake venom toxins in an attempt to find inhibitory molecules. Interestingly, human CatD shows the ability to bind strongly to snake venom phospholipases A2 (svPLA2), forming a stable muti-enzymatic complex that maintains the catalytic activity of both CatD and PLA2. In addition, this complex remains active even under exposure to the specific inhibitor pepstatin A. Furthermore, the complex formation between CatD and svPLA2 was evidenced by surface plasmon resonance (SPR), two-dimensional electrophoresis, enzymatic assays, and extensive molecular docking and dynamics techniques. Conclusion The present study suggests the versatility of human CatD and svPLA2, showing that these enzymes can form a fully functional new enzymatic complex.

In vitro multi-enzyme molecular machines - a review / 生物工程学报

In vitro multi-enzyme molecular machines that follow the designed multi-enzyme pathways, require the rational optimization and adaptation of several purified or partially purified enzyme components, in order to convert certain substrates into target compounds in vitro in an efficient manner. This type of molecular machine is component-based and modularized, so that its design, assembly, and regulation processes are highly flexible. Recently, the advantages of in vitro multi-enzyme molecular machines on the precise control of reaction process and the enhancement of product yield have suggested their great application potential in biomanufacturing. Studies on in vitro multi-enzyme molecular machines have become an important branch of synthetic biology, and are gaining increasing attentions. This article systematically reviews the enzyme component-/module-based construction strategy of in vitro multi-enzyme molecular machines, as well as the research progress on the improvement of compatibility among enzyme components/modules. The current challenges and future prospects of in vitro multi-enzyme molecular machines are also discussed.

Effect of a Decoction to Nourish Qi and Invigorate the Spleen on the Activity of Mitochondrial Respiratory Chain Enzyme Complexes in Cardiomyocytes of Rats with Spleen Qi Deficiency Syndrome / 中国医科大学学报

Objective To investigate the effect of a decoction to nourish qi and invigorate the spleen on mitochondrial respiratory chain enzyme complex activity in cardiomyocytes of rats with spleen qi deficiency syndrome. Methods Rats were randomly divided into normal,model,and treatment groups. The model and treatment groups were treated by diet intervention combined with the limit swim method. The general condition and spleen qi deficiency syndrome were assessed on day 15. After the success of the model,the normal and model groups were treated with a con?ventional feeding method combined with normal saline ,and the treatment group was treated by diet intervention combined with a decoction to nour?ish qi and invigorate the spleen for 9 weeks. The activity of two mitochondrial respiratory chain enzyme complexes was observed. Results The ac?tivity of mitochondrial respiratory chain enzyme complexⅡand complexⅣin the model group was significantly lower than the activity in the nor?mal and treatment groups(P<0.05). The activity levels of complexⅡand complexⅣwere significantly different between the model group and the treatment group(P<0.05). Conclusion Spleen qi deficiency can cause decreased activity of mitochondrial respiratory chain enzyme com?plexes in myocardial cells. The decoction to nourish qi and invigorate the spleen can modulate the activity of myocardial mitochondrial respiratory chain enzyme complexesⅡandⅣ.

Mitochondrial function and its correlation with age in articular chondrocytes of Kashin-Beck disease / 西安交通大学学报(医学版)

Objective To evaluate the mitochondrial function of chondrocytes in Kashin-Beck disease (KBD) patients and its correlation with age.Methods Mitochondrial function was evaluated by analyzing the activities of respiratory chain enzyme complexes and citrate synthase (CS),intracellular adenosine triphosphate (ATP)contents, changes in mitochondrial membrane potential (ΔΨm),as well as the intracellular reactive oxygen species (ROS) contents.Correlation of mitodhondrial function and age was analyzed with linear regression.Results Activities of complexes Ⅱ (P = 0.001 ),Ⅲ (P = 0.005 )and Ⅳ (P = 0.005 )were reduced in KBD articular chondrocytes compared with cells from normal controls.However,the mitochondrial mass was increased in KBD samples. Cultured KBD chondrocytes had reduced cellular ATP level (P = 0.001 )and mitochondrial membrane potential (P =0.001),but increased citrate synthase activity (P =0.04)and intracellular ROS level (P <0.001).We found no correlation between mitochondrial function and donor age either in normal or in KBD chondrocytes. Conclusion Mitochondrial function is altered in articular chondrocytes of KBD,and no correlation was found between mitochondrial function and donor age.

Co-expression of human malaria parasite Plasmodium falciparum orotate phosphoribosyltransferase and orotidine 5?-monophosphate decarboxylase as enzyme complex in Escherichia coli: a novel strategy for drug development

Background: Human malaria parasite Plasmodium falciparum operates de novo pyrimidine biosynthetic pathway. The fifth and sixth enzymes of the pathway form a heterotetrameric complex, containing two molecules each of orotate phosphoribosyltransferase (OPRT) and orotidine 5’-monophosphate decarboxylase (OMPDC). Objective: Define the function of OPRT-OMPDC enzyme complex of P. falciparum by co-expressing the enzymes in Escherichia coli. Methods: The constructed plasmids containing either P. falciparum OPRT or OMPDC were cloned in E. coli by co-transformation. Both genes were co-expressed as OPRT-OMPDC enzyme complex and the complex was purified by chromatographic techniques, including N2+-NTA affinity, Hi Trap Q HP anion-exchange, uridine 5’- monophosphate affinity, and Superose 12 gel-filtration columns. Physical and kinetic properties of the enzyme complex were analyzed for its molecular mass. Results: Co-transformation of PfOPRT and PfOMPDC plasmids in E. coli were achieved with a clone containing DNA ratio of 1:2, respectively. Both plasmids remained stable and were functionally expressed in the E. coli cell for at least 20 weeks. The P. falciparum OPRT-OMPDC enzyme complex were co-expressed and the complex was co-eluted in all chromatographic columns during purification and physical analysis. The molecular mass of the complex was 130 kDa, whereas the PfOPRT and PfOMPDC component were 35.6 and 41.5 kDa, respectively. The enzymatic activities of the complex were competitively inhibited by their products of each enzyme component. Conclusion: P. falciparum OPRT and OMPDC in E. coli as an enzyme complex were co-transformed and functionally co-expressed. These have similar properties to the native enzyme purified directly from P. falciparum, and this character is different from that of the human host organism. The enzyme complex would be suitable as new target to research selective inhibitors as suitable drugs to better control this disease.