Preparation and purification of Cry1Ah protein candidate reference material / 生物工程学报

With the rapid development of transgenic technology, the safety of genetically modified products has received extensive attention. Certified reference materials for the detection of genetically modified organisms play important roles in ensuring comparability and traceability of the qualitative and quantitative detection of genetically modified products. However, the development of protein reference materials is relatively slow, and one of the difficulties is the preparation of protein candidates with high purity. The cry1Ah1 gene of Bacillus thuringiensis has been used for the development of transgenic insect-resistant crops because of its excellent insecticidal activity against lepidopteran pests such as Asian corn borer, and has obtained transgenic lines with good insect resistance traits. In order to develop Cry1Ah protein certified reference material, it is urgent to establish a preparation and purification system. In this study, a system for preparing Cry1Ah protein by Bt expression system was optimized, and a high-purity Cry1Ah protein (size exclusion chromatography purity: 99.6%) was obtained by ion-exchange chromatography and size exclusion chromatography stepwise purification. The results of biological activity assay showed that there was no significant difference in the insecticidal activity of purified Cry1Ah protein and protoxin against diamondback moths (Plutella xylostella). Finally, the amino acid sequence of the activated Cry1Ah protein was determined using Edman degradation and mass spectrometry. In summary, the obtained Cry1Ah pure protein can be used for the development of protein reference materials.

Historia de los terrenos del Hospital Clínico y la Facultad de Medicina de la Universidad de Chile / History of the University of Chile Faculty of Medicine and clinical hospital location

The history of the location of the University of Chile Faculty of Medicine North Campus is derived from a farm of Pedro de Valdivia founder of the city of Santiago de la Nueva Extremadura and governor of the “Reyno de Chile”. This work narrates succinctly the history of this particular location from the Spanish Conquest period to present days.

Expression, purification of recombinant human cryptochrome I and its application in preparation of protective agent for radiotherapy / 生物工程学报

Radiotherapy is a treatment for cancer with undesired by-effects. In order to develop a new radiation protective agent that could reduce the by-effects, we tried to express and purify human cryptochrome 1 (hCRY1). The coding sequence of hCRY1 was inserted into prokaryotic expression plasmid pET28a(+), and this protein was purified from Escherichia coli BL21(DE3) after IPTG induction, ultrasonication, inclusion body dissolution, gradient dialysis, nickel column purification and ultrafiltration. The yield of hCRY1 in 1 L E. coli culture (LB medium) was about 10-15 mg. The radiation protective efficiency of hCRY1 was monitored by detecting X-ray-induced H2A.X foci in HaCaT cells. The results of immunofluorescence show that hCRY1 significantly reduces X-ray stimulated DNA damage response. The apoptosis of HaCaT cell was also detected, and the repression of H2A.X foci formation was not due to hCRY1's cytotoxity. All these data suggest a potential application of recombinant hCRY1 as a protective agent for radiotherapy.

AMP-activated protein kinase as a key molecular link between metabolism and clockwork

Circadian clocks regulate behavioral, physiological and biochemical processes in a day/night cycle. Circadian oscillators have an essential role in the coordination of physiological processes with the cyclic changes in the physical environment. Such mammalian circadian clocks composed of the positive components (BMAL1 and CLOCK) and the negative components (CRY and PERIOD (PER)) are regulated by a negative transcriptional feedback loop in which PER is rate-limiting for feedback inhibition. In addition, posttranslational modification of these components is critical for setting or resetting the circadian oscillation. Circadian regulation of metabolism is mediated through reciprocal signaling between the clock and metabolic regulatory networks. AMP-activated protein kinase (AMPK) in the brain and peripheral tissue is a crucial cellular energy sensor that has a role in metabolic control. AMPK-mediated phosphorylation of CRY and Casein kinases I regulates the negative feedback control of circadian clock by proteolytic degradation. AMPK can also modulate the circadian rhythms through nicotinamide adenine dinucleotide-dependent regulation of silent information regulator 1. Growing evidence elucidates the AMPK-mediated controls of circadian clock in metabolic diseases such as obesity and diabetes. In this review, we summarize the current comprehension of AMPK-mediated regulation of the circadian rhythms. This will provide insight into understanding how their components regulate the metabolism.