大肠杆菌热休克或冷休克启动子调控的基因表达载体

基因重组技术已经成为获得各种酶和生物活性蛋白的主要手段。虽然很多基因已在大肠杆菌中得到高效表达,但是当人们认定某种蛋白对科学研究或生产应用极为重要时,却常常因为其基因表达水平很低或产生包涵体而感到束手无策。表达载体pHsh和pEXC通过激活热休克或冷休克转录调控机制提高分子伴侣的表达水平,从而降低目标蛋白的细胞毒性并减少包涵体形成。应用于生物合成、分子修饰或生物降解的高温酶可以通过pHsh系统表达获得高产,而科研和诊疗所需要的来源于动植物和常温微生物的基因可以通过pEXC系统获得高效表达。这些新载体的发展为重组蛋白的小规模制备和大规模生产提供了新策略和有效途径。     

The role of lipid second messengers in aldosterone synthesis and secretion

Second messengers are small rapidly diffusing molecules or ions that relay signals between receptors and effector proteins to produce a physiological effect. Lipid messengers constitute one of the four major classes of second messengers. The hydrolysis of two main classes of lipids, glycerophospholipids and sphingolipids, generate parallel profiles of lipid second messengers: phosphatidic acid (PA), diacylglycerol (DAG), and lysophosphatidic acid versus ceramide, ceramide-1-phosphate, sphingosine, and sphingosine-1-phosphate, respectively. In this review, we examine the mechanisms by which these lipid second messengers modulate aldosterone production at multiple levels. Aldosterone is a mineralocorticoid hormone responsible for maintaining fluid volume, electrolyte balance, and blood pressure homeostasis. Primary aldosteronism is a frequent endocrine cause of secondary hypertension. A thorough understanding of the signaling events regulating aldosterone biosynthesis may lead to the identification of novel therapeutic targets. The cumulative evidence in this literature emphasizes the critical roles of PA, DAG, and sphingolipid metabolites in aldosterone synthesis and secretion. However, it also highlights the gaps in our knowledge, such as the preference for phospholipase D-generated PA or DAG, as well as the need for further investigation to elucidate the precise mechanisms by which these lipid second messengers regulate optimal aldosterone production.     

ω-O-Acylceramides but not ω-hydroxy ceramides are required for healthy lamellar phase architecture of skin barrier lipids

Epidermal omega-O-acylceramides (ω-O-acylCers) are essential components of a competent skin barrier. These unusual sphingolipids with ultralong N-acyl chains contain linoleic acid esterified to the terminal hydroxyl of the N-acyl, the formation of which requires the transacylase activity of patatin-like phospholipase domain containing 1 (PNPLA1). In ichthyosis with dysfunctional PNPLA1, ω-O-acylCer levels are significantly decreased, and ω-hydroxylated Cers (ω-OHCers) accumulate. Here, we explore the role of the linoleate moiety in ω-O-acylCers in the assembly of the skin lipid barrier. Ultrastructural studies of skin samples from neonatal Pnpla1^+/+ and Pnpla1^-/- mice showed that the linoleate moiety in ω-O-acylCers is essential for lamellar pairing in lamellar bodies, as well as for stratum corneum lipid assembly into the long periodicity lamellar phase. To further study the molecular details of ω-O-acylCer deficiency on skin barrier lipid assembly, we built in vitro lipid models composed of major stratum corneum lipid subclasses containing either ω-O-acylCer (healthy skin model), ω-OHCer (Pnpla1^-/- model), or combination of the two. X-ray diffraction, infrared spectroscopy, and permeability studies indicated that ω-OHCers could not substitute for ω-O-acylCers, although in favorable conditions, they form a medium lamellar phase with a 10.8 nm-repeat distance and permeability barrier properties similar to long periodicity lamellar phase. In the absence of ω-O-acylCers, skin lipids were prone to separation into two phases with diminished barrier properties. The models combining ω-OHCers with ω-O-acylCers indicated that accumulation of ω-OHCers does not prevent ω-O-acylCer-driven lamellar stacking. These data suggest that ω-O-acylCer supplementation may be a viable therapeutic option in patients with PNPLA1 deficiency.     

Thematic Review Series: Recent Advances in the Treatment of Lysosomal Storage Diseases: Lysosomal storage diseases and the heat shock response: convergences and therapeutic opportunities

Lysosomes play a vital role in the maintenance of cellular homeostasis through the recycling of cell constituents, a key metabolic function which is highly dependent on the correct function of the lysosomal hydrolases and membrane proteins, as well as correct membrane lipid stoichiometry and composition. The critical role of lysosomal functionality is evident from the severity of the diseases in which the primary lesion is a genetically defined loss-of-function of lysosomal hydrolases or membrane proteins. This group of diseases, known as lysosomal storage diseases (LSDs), number more than 50 and are associated with severe neurodegeneration, systemic disease, and early death, with only a handful of the diseases having a therapeutic option. Another key homeostatic system is the metabolic stress response or heat shock response (HSR), which is induced in response to a number of physiological and pathological stresses, such as protein misfolding and aggregation, endoplasmic reticulum stress, oxidative stress, nutrient deprivation, elevated temperature, viral infections, and various acute traumas. Importantly, the HSR and its cardinal members of the heat shock protein 70 family has been shown to protect against a number of degenerative diseases, including severe diseases of the nervous system. The cytoprotective actions of the HSR also include processes involving the lysosomal system, such as cell death, autophagy, and protection against lysosomal membrane permeabilization, and have shown promise in a number of LSDs. This review seeks to describe the emerging understanding of the interplay between these two essential metabolic systems, the lysosomes and the HSR, with a particular focus on their potential as a therapeutic target for LSDs.     

An overview of Bacillus proteases: from production to application

Proteases have a broad range of applications in industrial processes and products and are representative of most worldwide enzyme sales. The genus Bacillus is probably the most important bacterial source of proteases and is capable of producing high yields of neutral and alkaline proteolytic enzymes with remarkable properties, such as high stability towards extreme temperatures, pH, organic solvents, detergents and oxidizing compounds. Therefore, several strategies have been developed for the cost-effective production of Bacillus proteases, including optimization of the fermentation parameters. Moreover, there are many studies on the use of low-cost substrates for submerged and solid state fermentation. Other alternatives include genetic tools such as protein engineering in order to obtain more active and stable proteases and strain engineering to better secrete recombinant proteases from Bacillus through homologous and heterologous protein expression. There has been extensive research on proteases because of the broad number of applications for these enzymes, such as in detergent formulations for the removal of blood stains from fabrics, production of bioactive peptides, food processing, enantioselective reactions, and dehairing of skins. Moreover, many commercial proteases have been characterized and purified from different Bacillus species. Therefore, this review highlights the production, purification, characterization, and application of proteases from a number of Bacillus species.     

Cleistanthane diterpenoids from the resin of Vellozia pyrantha A.A.Conc and their chemotaxonomic significance

Vellozia pyrantha A.A.Conc. is endemic to the rupestrian fields in northern Chapada Diamantina National Park (Brazil). This species has the peculiar characteristic of only producing flowers after a massive fire event occurs in the surrounding areas. This is the first report on the assessment of the chemical composition of Vellozia pyrantha resin, where five cleistanthane diterpenoids (1–5) were identified. Their structures were proposed after careful analysis of ¹H and ¹³C NMR, DEPT 135, HMBC, HMQC, and COSY spectra as well as comparison with the literature. Cleistanthane diterpenoids show great the potential to be used as chemosystematics biomarkers for the genus Vellozia and, therefore, their chemotaxonomic significance is discussed.     

Biotransformation of geranylated- and acetylated-phloroglucinols by Gibberella fujikuroi into molecules with increased antifungal activity against Botrytis cinerea

Terpenylated phenols possess interesting biological activities. These properties vary mainly according to the type of terpene associated and the degree of oxidation of the molecule. The search for new active molecules for application in different areas of knowledge includes the structural modification of these through ecological methodologies, such as biotransformation. The aims of this study were the biotransformation of geranylated- and acetylated-phloroglucinol by the fungus Gibberella fujikuroi and the evaluation of the antifungal activity of the derivatives. Five major derivatives were identified after biotransformation, highlighting the formation of specific monoacetylated products. In vitro antifungal activity assays against the phytopathogenic fungus Botrytis cinerea indicated that deacetylated derivatives possess higher activity compared to the precursor molecule. In other biotransformation reactions, a relationship between the release of the alkyl chain from the aromatic ring with a decrease of the antifungal activity, was observed. The in vivo tests in infected tomato plants with B. cinerea confirmed the antifungal activity of the derivatives observed in in vitro experiments.