Hansenula polymorpha: An attractive model organism for molecular studies of peroxisome biogenesis and function

In wild-type Hansenula polymorpha the proliferation of peroxisomes is induced by various unconventional carbon- and nitrogen sources. Highest induction levels, up to 80% of the cytoplasmic volume, are observed in cells grown in methanol-limited chemostat cultures. Based on our accumulated experience, we are now able to precisely adjust both the level of peroxisome induction as well as their protein composition by specific adaptations in growth conditions. During the last few years a series of peroxisome-deficient (per) mutants of H. polymorpha have been isolated and characterized. Phenotypically these mutants are characterized by the fact that they are not able to grow on methanol. Three mutant phenotypes were defined on the basis of morphological criteria, namely: (a) mutants completely lacking peroxisomes (Per-; 13 complementation groups); (b) mutants containing few small peroxisomes which are partly impaired in the peroxisomal import of matrix proteins (Pim-; five complementation groups); and (c) mutants with aberrations in the peroxisomal substructure (Pss-; two complementation groups). In addition, several conditional Per-, Pim- and Pss- mutants have been obtained. In all cases the mutant phenotype was shown to be caused by a recessive mutation in one gene. However, we observed that different mutations in one gene may cause different morphological mutant phenotypes. A detailed genetic analysis revealed that several PER genes, essential for peroxisome biogenesis, are tightly linked and organized in a hierarchical fashion. The use of both constitual and conditional per mutants in current and future studies of the molecular mechanisms controlling peroxisome biogenesis and function is discussed.

Hansenula polymorpha Swi1p and Snf2p are essential for methanol utilisation

We have cloned the Hansenula polymorpha SWI1 and SNF2 genes by functional complementation of mutants that are defective in methanol utilisation. These genes encode proteins similar to Saccharomyces cerevisiae Swi1p and Snf2p, which are subunits of the SWI/SNF complex. This complex belongs to the family of nucleosome-remodeling complexes that play a role in transcriptional control of gene expression. Analysis of the phenotypes of constructed H. polymorpha SWI1 and SNF2 disruption strains indicated that these genes are not necessary for growth of cells on glucose, sucrose, or various organic nitrogen sources which involve the activity of peroxisomal oxidases. Both disruption strains showed a moderate growth defect on glycerol and ethanol, but were fully blocked in methanol utilisation. In methanol-induced cells of both disruption strains, two peroxisomal enzymes involved in methanol metabolism, alcohol oxidase and dihydroxyacetone synthase, were hardly detectable, whereas in wild-type cells these proteins were present at very high levels. We show that the reduction in alcohol oxidase protein levels in H. polymorpha SWI1 and SNF2 disruption strains is due to strongly reduced expression of the alcohol oxidase gene. The level of Pex5p, the receptor involved in import of alcohol oxidase and dihydroxyacetone synthase into peroxisomes, was also reduced in both disruption strains compared to that in wild-type cells.

Метаболический синдром и присоединение бактериальной инфекции как факторы риска фатального исхода при гриппе А / H1N1, осложненном пневмонией

Metabolic syndrome and bacterial infection as risk factors of death in influenza А / H1N1 complicated by pneumonia.Метаболический синдром и присоединение бактериальной инфекции как факторы риска фатального исхода при гриппе А / H1N1, осложненном пневмонией

ЛИПОПОЛИСАХАРИД-СВЯЗЫВАЮЩИЙ БЕЛОК, НЕОПТЕРИН И ИНТЕРФЕРОН-Г КАК ПОКАЗАТЕЛИ АКТИВНОСТИ ВОСПАЛЕНИЯ У БОЛЬНЫХ ОСТРЫМ БРУЦЕЛЛЁЗОМ

Brucellosis is characterized by nonspecific clinical manifestations, the possibility of subclinical flow, the development of relapses and chronic course. Currently, there are no laboratory criteria to assess the activity of inflammation in brucellosis, the effectiveness of the therapy, predict the outcome of the disease and the risks of recurrence. Available in clinical practice, laboratory tests to assess inflammation, in particular, erythrocyte sedimentation rate, C-reactive protein, leukocyte level, with brucellosis infection are almost not informative. An important role in the development of the cellular immune response against brucella is played by interferon-γ, lipopolysaccharide-binding protein and neopterin. The aim of the study was to determine the level of lipopolysaccharide-binding protein, neopterin and interferon-γ, in the serum of patients with acute form of brucellosis before and after antibacterial treatment. When studying the blood of patients with acute brucellosis before and after therapy, the indices of neopterin, lipopolysaccharide-binding protein and interferon-γ were significantly higher than normal values. The obtained results testify to the persisting active inflammation and the formation of a chronic brucellosis. Determination of the level of lipopolysaccharide-binding protein, neopterin and interferon-γ in the blood of patients with brucellosis can be used as markers of inflammation and in monitoring the effectiveness of antibacterial therapy.Цель: определение уровня ЛПС-белка, неоптерина и ИФН- γ в сыворотке крови больных с острой формой бруцеллеза до и после лечения антибактериальными препаратами. Материалы и методы: в исследование включены 65 больных острым бруцеллезом. Для определения уровня ЛПС-белка в сыворотке крови использовали тестсистемы «Hycultbiotech, Netherlands», ELISA. Уровень неоптерина в сыворотке крови определяли с помощью тест-систем Neopterin ELISA «IBL, Hamburg». Определение уровня ИФН- γ в сыворотке крови проводили тестсистемами Вектор Бест А-8752 гамма-ИнтерферонИФА-БЕСТ производства «Вектор-БЕСТ», Россия. Группу сравнения составили 32 здоровых донора, сопоставимые по полу и возрасту с больными бруцеллезом, не болевшие этой инфекцией, не вакцинированные против этой инфекции. Специфические лабораторные исследования крови, определение уровня ЛПС-белка, уровня ИФН-γ и неоптерина в образцах сыворотки крови проводились в лабораториях Ставропольского научно-исследовательского противочумного института Роспотребнадзора Результаты: при исследовании крови больных острым бруцеллезом до и после лечения определены показатели неоптерина, липополисахарид-связывающего белка и интерферона- γ, значительно превышающие нормальные значения. Полученные результаты свидетельствуют о сохраняющемся активном воспалении и формировании хронического бруцеллеза. Заключение: патогенетической основой практически закономерной трансформации острой стадии инфекции в хроническую является несостоятельность врожденного и адаптивного иммунитета в отношении бруцелл с созданием условий для незавершенного фагоцитоза и долгосрочного внутриклеточного паразитирования. ИФН- γ, неоптерин и ЛПС-белок относятся к иммуномодулирующим факторам реакции иммунитета на возбудителя, и определение их уровня в крови больных бруцеллезом возможно использовать в качестве маркеров воспаления и в мониторинге эффективности антибактериальной терапии

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field