Isolation and characterization of the plasma membrane from the yeast Pichia pastoris

Despite similarities of cellular membranes in all eukaryotes, every compartment displays characteristic and often unique features which are important for the functions of the specific organelles. In the present study, we biochemically characterized the plasma membrane of the methylotrophic yeast Pichia pastoris with emphasis on the lipids which form the matrix of this compartment. Prerequisite for this effort was the design of a standardized and reliable isolation protocol of the plasma membrane at high purity. Analysis of isolated plasma membrane samples from P. pastoris revealed an increase of phosphatidylserine and a decrease of phosphatidylcholine compared to bulk membranes. The amount of saturated fatty acids in the plasma membrane was higher than in total cell extracts. Ergosterol, the final product of the yeast sterol biosynthetic pathway, was found to be enriched in plasma membrane fractions, although markedly lower than in Saccharomyces cerevisiae. A further characteristic feature of the plasma membrane from P. pastoris was the enrichment of inositol phosphorylceramides over neutral sphingolipids, which accumulated in internal membranes. The detailed analysis of the P. pastoris plasma membrane is discussed in the light of cell biological features of this microorganism especially as a microbial cell factory for heterologous protein production.     

HMG CoA reductase inhibitors

Beta-hydroxy-beta-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (HMG CoA RIs) are now being prescribed in many different countries, and the number of patients treated with these compounds is estimated to be over 1.5 million. The spectrum of indications for these drugs is enlarging. Thus, not only patients with primary hypercholesterolemia are suitable candidates for therapy with HMG CoA RIs, but also patients with other forms of familial and non-familial (secondary) dyslipidemias. The safety and tolerance profile is remarkably good and adverse effects tend to be mild and rare. Currently, several primary and secondary intervention trials are ongoing or in preparation to investigate the influence of HMG CoA RIs on cardiovascular morbidity and mortality.     

Human TLR8 senses UR/URR motifs in bacterial and mitochondrial RNA

Toll‐like receptor (TLR) 13 and TLR2 are the major sensors of Gram‐positive bacteria in mice. TLR13 recognizes Sa19, a specific 23S ribosomal (r) RNA‐derived fragment and bacterial modification of Sa19 ablates binding to TLR13, and to antibiotics such as erythromycin. Similarly, RNase A‐treated Staphylococcus aureus activate human peripheral blood mononuclear cells (PBMCs) only via TLR2, implying single‐stranded (ss) RNA as major stimulant. Here, we identify human TLR8 as functional TLR13 equivalent that promiscuously senses ssRNA. Accordingly, Sa19 and mitochondrial (mt) 16S rRNA sequence‐derived oligoribonucleotides (ORNs) stimulate PBMCs in a MyD88‐dependent manner. These ORNs, as well as S. aureus‐, Escherichia coli‐, and mt‐RNA, also activate differentiated human monocytoid THP‐1 cells, provided they express TLR8. Moreover, Unc93b1 ^−/−‐ and Tlr8 ^−/−‐THP‐1 cells are refractory, while endogenous and ectopically expressed TLR8 confers responsiveness in a UR/URR RNA ligand consensus motif‐dependent manner. If TLR8 function is inhibited by suppression of lysosomal function, antibiotic treatment efficiently blocks bacteria‐driven inflammatory responses in infected human whole blood cultures. Sepsis therapy might thus benefit from interfering with TLR8 function.     

Oxylipins in fungi

In nearly every living organism, metabolites derived from lipid peroxidation, the so-called oxylipins, are involved in regulating developmental processes as well as environmental responses. Among these bioactive lipids, the mammalian and plant oxylipins are the best characterized, and much information about their physiological role and biosynthetic pathways has accumulated during recent years. Although the occurrence of oxylipins and enzymes involved in their biosynthesis has been studied for nearly three decades, knowledge about fungal oxylipins is still scarce as compared with the situation in plants and mammals. However, the research performed so far has shown that the structural diversity of oxylipins produced by fungi is high and, furthermore, that the enzymes involved in oxylipin metabolism are diverse and often exhibit unusual catalytic activities. The aim of this review is to present a synopsis of the oxylipins identified so far in fungi and the enzymes involved in their biosynthesis.     

The fatty acyl-CoA reductase Waterproof mediates airway clearance in Drosophila

The transition from a liquid to a gas filled tubular network is the prerequisite for normal function of vertebrate lungs and invertebrate tracheal systems. However, the mechanisms underlying the process of gas filling remain obscure. Here we show that waterproof, encoding a fatty acyl-CoA reductase (FAR), is essential for the gas filling of the tracheal tubes during Drosophila embryogenesis, and does not affect branch network formation or key tracheal maturation processes. However, electron microscopic analysis reveals that in waterproof mutant embryos the formation of the outermost tracheal cuticle sublayer, the envelope, is disrupted and the hydrophobic tracheal coating is damaged. Genetic and gain-of-function experiments indicate a non-cell-autonomous waterproof function for the beginning of the tracheal gas filling process. Interestingly, Waterproof reduces very long chain fatty acids of 24 and 26 carbon atoms to fatty alcohols. Thus, we propose that Waterproof plays a key role in tracheal gas filling by providing very long chain fatty alcohols that serve as potential substrates for wax ester synthesis or related hydrophobic substances that ultimately coat the inner lining of the trachea. The hydrophobicity in turn reduces the tensile strength of the liquid inside the trachea, leading to the formation of a gas bubble, the focal point for subsequent gas filling. Waterproof represents the first enzyme described to date that is necessary for tracheal gas filling without affecting branch morphology. Considering its conservation throughout evolution, Waterproof orthologues may play a similar role in the vertebrate lung.     

The lipidome and proteome of microsomes from the methylotrophic yeast Pichia pastoris

The methylotrophic yeast Pichia pastoris is a popular yeast expression system for the production of heterologous proteins in biotechnology. Interestingly, cell organelles which play an important role in this process have so far been insufficiently investigated. For this reason, we started a systematic approach to isolate and characterize organelles from P. pastoris. In this study, we present a procedure to isolate microsomal membranes at high purity. These samples represent endoplasmic reticulum (ER) fractions which were subjected to molecular analysis of lipids and proteins. Organelle lipidomics included a detailed analysis of glycerophospholipids, fatty acids, sterols and sphingolipids. The microsomal proteome analyzed by mass spectrometry identified typical proteins of the ER known from other cell types, especially Saccharomyces cerevisiae, but also a number of unassigned gene products. The lipidome and proteome analysis of P. pastoris microsomes are prerequisite for a better understanding of functions of this organelle and for modifying this compartment for biotechnological applications.     

Infection,inflammation and exercise in cystic fibrosis

Regular exercise is positively associated with health. It has also been suggested to exert anti-inflammatory effects. In healthy subjects, a single exercise session results in immune cell activation, which is characterized by production of immune modulatory peptides (e.g. IL-6, IL-8), a leukocytosis and enhanced immune cell functions. Upon cessation of exercise, immune activation is followed by a tolerizing phase, characterized by a reduced responsiveness of immune cells. Regular exercise of moderate intensity and duration has been shown to exert anti-inflammatory effects and is associated with a reduced disease incidence and viral infection susceptibility. Specific exercise programs may therefore be used to modify the course of chronic inflammatory and infectious diseases such as cystic fibrosis (CF).Patients with CF suffer from severe and chronic pulmonary infections and inflammation, leading to obstructive and restrictive pulmonary disease, exercise intolerance and muscle cachexia. Inflammation is characterized by a hyper-inflammatory phenotype. Patients are encouraged to engage in exercise programs to maintain physical fitness, quality of life, pulmonary function and health.In this review, we present an overview of available literature describing the association between regular exercise, inflammation and infection susceptibility and discuss the implications of these observations for prevention and treatment of inflammation and infection susceptibility in patients with CF.