Differential subcellular localization of functionally divergent survivin splice variants

Survivin is an inhibitor of apoptosis protein (IAP) that is markedly overexpressed in most cancers. We identified two novel functionally divergent splice variants, i.e. non-antiapoptotic survivin-2B and antiapoptotic survivin-deltaEx3. Because survivin-2B might be a naturally occurring antagonist of antiapoptotic survivin variants, we analyzed the subcellular distribution of these proteins. PSORT II analysis predicted a preferential cytoplasmic localization of survivin and survivin-2B, but a preferential nuclear localization of survivin-deltaEx3. GFP-tagged survivin variants confirmed the predicted subcellular localization and additionally revealed a cell cycle-dependent nuclear accumulation of survivin-deltaEx3. Moreover, a bipartite nuclear localization signal found exclusively in survivin-deltaEx3 may support cytoplasmic clearance of survivin-deltaEx3. In contrast to the known association between survivin and microtubules or centromeres during mitosis, no corresponding co-localization became evident for survivin-deltaEx3 or survivin-2B. In conclusion, our study provided data on a differential subcellular localization of functionally divergent survivin variants, suggesting that survivin isoforms may perform different functions in distinct subcellular compartments and distinct phases of the cell cycle.     

Cholesterol transfer via endoplasmic reticulum contacts mediates lysosome damage repair

Lysosome integrity is essential for cell viability, and lesions in lysosome membranes are repaired by the ESCRT machinery. Here, we describe an additional mechanism for lysosome repair that is activated independently of ESCRT recruitment. Lipidomic analyses showed increases in lysosomal phosphatidylserine and cholesterol after damage. Electron microscopy demonstrated that lysosomal membrane damage is rapidly followed by the formation of contacts with the endoplasmic reticulum (ER), which depends on the ER proteins VAPA/B. The cholesterol‐binding protein ORP1L was recruited to damaged lysosomes, accompanied by cholesterol accumulation by a mechanism that required VAP–ORP1L interactions. The PtdIns 4‐kinase PI4K2A rapidly produced PtdIns4P on lysosomes upon damage, and knockout of PI4K2A inhibited damage‐induced accumulation of ORP1L and cholesterol and led to the failure of lysosomal membrane repair. The cholesterol–PtdIns4P transporter OSBP was also recruited upon damage, and its depletion caused lysosomal accumulation of PtdIns4P and resulted in cell death. We conclude that ER contacts are activated on damaged lysosomes in parallel to ESCRTs to provide lipids for membrane repair, and that PtdIns4P generation and removal are central in this response.     

Terminally differentiated postmitotic tumor cells in a rat rhabdomyosarcoma cell line

A permanent rat rhabdomyosarcoma cell line (BA-HAN-1C) has been established, the phenotype of which is characterized by the coexistence of undifferentiated mononuclear cells and differentiated multinuclear myotube-like giant cells. The failure of attempts to separate these two cell types by repeated recloning procedures indicates their close histogenetic relationship and suggests that differentiation in this tumor proceeds in a similar manner to that in normal striated muscle where postmitotic myotubes arise from mononuclear myoblasts by fusion. The morphologically undifferentiated mononuclear tumor cells were shown to be actively proliferating and to incorporate thymidine methyl-3H(3H-TdR). The myotube-like giant cells neither incorporated 3H-TdR nor underwent mitosis or exhibited any clonogenic potential. After retransplantation into syngenic rats, tumor growth was markedly retarded when the tumor cell inoculum contained a high percentage of myotube-like giant cells. These data show that proliferative activity in this rhabdomyosarcoma cell line is confined to the mononuclear tumor cell compartment, the multinuclear myotube-like giant cells having withdrawn from the cell cycle and represent terminally differentiated postmitotic cells. This cell line should provide a valuable tool for further investigation of coherent aspects of proliferation and differentiation using various differentiation inducers.     

Study of human allergic milk whey protein from different mammalian species using computational method

Nowadays, safety and quality assessment of food used for human consumption have to consider by its possible contribution to the maintenance or improvement of the consumer''s health. Milk is an important food with many nutrients. Cow milk is an important source of energy, protein, vitamins and minerals for the growing child as well as adults. But, numerous cow milk proteins have been implicated in allergic responses and most of these have been shown to contain multiple allergic epitopes. The present study disclosed best alternatives to cow milk, which are not allergic and as good as cow milk in nutritional value. The in silico analysis of casein (alpha s1, alpha s2, beta and kappa) and beta-lactoglobulin, unveils that sheep milk is a more suitable alternate to cow milk for allergic infants and buffalo milk for allergic adult humans.     

Effect of self-association on the structural organization of partially folded proteins: inactivated actin

The propensity to associate or aggregate is one of the characteristic properties of many nonnative proteins. The aggregation of proteins is responsible for a number of human diseases and is a significant problem in biotechnology. Despite this, little is currently known about the effect of self-association on the structural properties and conformational stability of partially folded protein molecules. G-actin is shown to form equilibrium unfolding intermediate in the vicinity of 1.5 M guanidinium chloride (GdmCl). Refolding from the GdmCl unfolded state is terminated at the stage of formation of the same intermediate state. An analogous form, known as inactivated actin, can be obtained by heat treatment, or at moderate urea concentration, or by the release of Ca(2+). In all cases actin forms specific associates comprising partially folded protein molecules. The structural properties and conformational stability of inactivated actin were studied over a wide range of protein concentrations, and it was established that the process of self-association is rather specific. We have also shown that inactivated actin, being denatured, is characterized by a relatively rigid microenvironment of aromatic residues and exhibits a considerable limitation in the internal mobility of tryptophans. This means that specific self-association can play an important structure-forming role for the partially folded protein molecules.     

Effects of retinoic acid metabolites on proliferation and differentiation of the clonal rhabdomyosarcoma cell line BA-HAN-1C

Summary— The clonal rat rhabdomyosarcoma cell line BA-HAN-IC is composed of proliferating mononuclear cells, some of which spontaneously fuse to terminally differentiated myotube-like giant cells. This cell line has been shown to be susceptible to differentiation induction with all-trans retinoic acid (RA). Since it is still unknown whether exclusively all-trans RA itself or also its metabolites can act as inductive compounds in our cell line, we exposed BA-HAN-1C cells to the metabolites 4-hydroxy RA, 4-oxo RA and 5,6epoxy RA. Exposure to these RA metabolites resulted in a significant inhibition of proliferation (P < 0.001) and induction of cellular differentiation, as evidenced by a significant increase in the number of myotube-like giant cells (P < 0.05) and a significant increase in creatine kinase activity (P < 0.05). However, differences in the inductive potency of these RA metabolites became apparent. Furthermore, RA metabolites exhibited a significantly weaker (P < 0.05) inductive activity when compared to all-trans RA. Summarizing our results we could demonstrate that the endogenous metabolites 4-hydroxy RA, 4-oxo RA and 5,6-epoxy RA are not merely deactivated cellular excretion products of all-trans RA, but potent inducers of differentiation and inhibitors of proliferation, possibly contributing to the complex physiological actions of retinoic acid.     

Health-related quality of life and functional ability in patients with early arthritis during remission steered treatment: results of the IMPROVED study

Introduction

The aim of this study was to investigate patient reported outcomes (PROs) of functional ability and health related quality of life (HRQoL) in patients with early (rheumatoid) arthritis during one year of remission steered treatment.

Methods

In this study, 610 patients with early rheumatoid arthritis (RA) or undifferentiated arthritis (UA) were treated with methotrexate (MTX) and tapered high dose of prednisone. Patients in early remission (Disease Activity Score (DAS) <1.6 after 4 months) tapered prednisone to zero and when in persistent remission, also tapered MTX. Patients not in early remission were randomized to either MTX + hydroxychloroquine + sulphasalazine + prednisone (arm 1) or to MTX + adalimumab (arm 2). Every 4 months, patients filled out the Health Assessment Questionnaire (HAQ) and the McMaster Toronto Arthritis Patient Preference Questionnaire (MACTAR), the Short Form 36 (SF-36) and visual analogue scales (VAS). Change scores were compared between treatment groups. The association with achieving remission was analyzed using linear mixed models.

Results

During year 1, patients who achieved early remission had the most improvement in PROs with scores comparable to the general population. Patients in the randomization arms showed less improvement. Scores were comparable between the arms. There was a significant association between achieving remission and scores of HAQ, MACTAR and physical HRQoL.

Conclusions

In early arthritis, PROs of functional ability and HRQoL after one year of remission steered treatment reach normal values in patients who achieved early remission. In patients not in early remission, who were randomized to two strategy arms, PROs improved less, with similar scores in both treatment arms.

Trial registrations

ISRCTN11916566 and EudraCT2006-006186-16     

High throughput screening to investigate the interaction of stem cells with their extracellular microenvironment

Stem cells in vivo are housed within a functional microenvironment termed the “stem cell niche.” As the niche components can modulate stem cell behaviors like proliferation, migration and differentiation, evaluating these components would be important to determine the most optimal platform for their maintenance or differentiation. In this review, we have discussed methods and technologies that have aided in the development of high throughput screening assays for stem cell research, including enabling technologies such as the well-established multiwell/microwell plates and robotic spotting, and emerging technologies like microfluidics, micro-contact printing and lithography. We also discuss the studies that utilized high throughput screening platform to investigate stem cell response to extracellular matrix, topography, biomaterials and stiffness gradients in the stem cell niche. The combination of the aforementioned techniques could lay the foundation for new perspectives in further development of high throughput technology and stem cell research.