Nonglucosylated oligosaccharides are transferred to protein in MI8-5 Chinese hamster ovary cells

A CHO mutant MI8-5 was found to synthesize Man9-GlcNAc2-P-P-dolichol rather than Glc3Man9GlcNAc2-P-P-dolichol as the oligosaccharide-lipid intermediate in N-glycosylation of proteins. MI8-5 cells were incubated with labeled mevalonate, and the prenol was found to be dolichol. The mannose-labeled oligosaccharide released from oligosaccharide-lipid of MI8-5 cells was analyzed by HPLC and alpha-mannosidase treatment, and the data were consistent with a structure of Man9GlcNAc2. In addition, MI8-5 cells did not incorporate radioactivity into oligosaccharide- lipid during an incubation with tritiated galactose, again consistent with MI8-5 cells synthesizing an unglucosylated oligosaccharide-lipid. MI8-5 cells had parental levels of glucosylphosphoryldolichol synthase activity. However, in two different assays, MI8-5 cells lacked dolichol- P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase activity. MI8-5 cells were found to synthesize glucosylated oligosaccharide after they were transfected with Saccharomyces cerevisiae ALG 6, the gene for dolichol-P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase. MI8-5 cells were found to incorporate mannose into protein 2-fold slower than parental cells and to approximately a 2-fold lesser extent.      

FIGENIX: Intelligent automation of genomic annotation: expertise integration in a new software platform

Background  

Two of the main objectives of the genomic and post-genomic era are to structurally and functionally annotate genomes which consists of detecting genes' position and structure, and inferring their function (as well as of other features of genomes). Structural and functional annotation both require the complex chaining of numerous different software, algorithms and methods under the supervision of a biologist. The automation of these pipelines is necessary to manage huge amounts of data released by sequencing projects. Several pipelines already automate some of these complex chaining but still necessitate an important contribution of biologists for supervising and controlling the results at various steps.     

Single amino acid supplementation in aminoacidopathies: a systematic review

Aminoacidopathies are a group of rare and diverse disorders, caused by the deficiency of an enzyme or transporter involved in amino acid metabolism. For most aminoacidopathies, dietary management is the mainstay of treatment. Such treatment includes severe natural protein restriction, combined with protein substitution with all amino acids except the amino acids prior to the metabolic block and enriched with the amino acid that has become essential by the enzymatic defect. For some aminoacidopathies, supplementation of one or two amino acids, that have not become essential by the enzymatic defect, has been suggested. This so-called single amino acid supplementation can serve different treatment objectives, but evidence is limited. The aim of the present article is to provide a systematic review on the reasons for applications of single amino acid supplementation in aminoacidopathies treated with natural protein restriction and synthetic amino acid mixtures.     

Regenerative potential of human muscle stem cells in chronic inflammation

Introduction

Chronic inflammation is a profound systemic modification of the cellular microenvironment which could affect survival, repair and maintenance of muscle stem cells. The aim of this study was to define the role of chronic inflammation on the regenerative potential of satellite cells in human muscle.

Methods

As a model for chronic inflammation, 11 patients suffering from rheumatoid arthritis (RA) were included together with 16 patients with osteoarthritis (OA) as controls. The mean age of both groups was 64 years, with more females in the RA group compared to the OA group. During elective knee replacement surgery, a muscle biopsy was taken from the distal musculus vastus medialis. Cell populations from four RA and eight OA patients were used for extensive phenotyping because these cell populations showed no spontaneous differentiation and myogenic purity greater than 75% after explantation.

Results

After mononuclear cell explantation, myogenic purity, viability, proliferation index, number of colonies, myogenic colonies, growth speed, maximum number of population doublings and fusion index were not different between RA and OA patients. Furthermore, the expression of proteins involved in replicative and stress-induced premature senescence and apoptosis, including p16, p21, p53, hTERT and cleaved caspase-3, was not different between RA and OA patients. Mean telomere length was shorter in the RA group compared to the OA group.

Conclusions

In the present study we found evidence that chronic inflammation in RA does not affect the in vitro regenerative potential of human satellite cells. Identification of mechanisms influencing muscle regeneration by modulation of its microenvironment may, therefore, be more appropriate.     

An approach for differentiating uniform glutamatergic neurons from mouse embryonic stem cells

Much effort is being marshaled to generate uniform neuronal populations from embryonic stem (ES) cells, but a completely reliable method has yet to be developed. Here we modified and established a method that brings us closer to this goal. By examining many parameters, we found that the optimal timing of applying a freshly made trypsin/EDTA (ethylenediaminetetraacetic acid) solution to dissociate embryoid bodies determines the success of the outcome. Analyses demonstrated that with this approach, more than 87% of cells differentiated into glutamatergic neurons. Hence, these uniform neurons that were differentiated from ES cells provide an ideal cellular model for many aspects of research.     

Neuroprotective effects of granulocyte-colony stimulating factor in a novel transgenic mouse model of SCA17

Spinocerebellar ataxia type 17 (SCA17) is an autosomal dominant inherited disorder characterized by degeneration of spinocerebellar tracts and selected brainstem neurons owing to the expansion of a CAG repeat of the human TATA-binding protein (hTBP) gene. To gain insight into the pathogenesis of this hTBP mutation, we generated transgenic mice with the mutant hTBP gene driven by the Purkinje specific protein (Pcp2/L7) gene promoter. Mice with the expanded hTBP allele developed ataxia within 2-5 months. Behavioral analysis of L7-hTBP transgenic mice showed reduced fall latency in a rotarod assay. Purkinje cell degeneration was identified by immunostaining of calbindin and IP3R1. Reactive gliosis and neuroinflammation occurred in the transgenic cerebellum, accompanied by up-regulation of GFAP and Iba1. The L7-hTBP transgenic mice were thus confirmed to recapitulate the SCA17 phenotype and were used as a disease model to explore the potential of granulocyte-colony stimulating factor in SCA17 treatment. Our results suggest that granulocyte-colony stimulating factor has a neuroprotective effect in these transgenic mice, ameliorating their neurological and behavioral deficits. These data indicate that the expression of the mutant hTBP in Purkinje cells is sufficient to produce cell degeneration and an ataxia phenotype, and constitutes a good model for better analysis of the neurodegeneration in SCA17.     

Field site selection: getting it right first time around

The selection of suitable field sites for integrated control of Anopheles mosquitoes using the sterile insect technique (SIT) requires consideration of the full gamut of factors facing most proposed control strategies, but four criteria identify an ideal site: 1) a single malaria vector, 2) an unstructured, relatively low density target population, 3) isolation of the target population and 4) actual or potential malaria incidence. Such a site can exist in a diverse range of situations or can be created. Two contrasting SIT field sites are examined here: the desert-flanked Dongola Reach of the Nile River in Northern State, Sudan, where malaria is endemic, and the island of La Reunion, where autochthonous malaria is rare but risk is persistent. The single malaria-transmitting vector at both sites is Anopheles arabiensis. In Sudan, the target area is a narrow 500 km corridor stretching from the rocky terrain at the Fourth Cataract - just above the new Merowe Dam, to the northernmost edge of the species range, close to Egypt. Vector distribution and temporal changes in density depend on the Nile level, ambient temperature and human activities. On La Reunion, the An. arabiensis population is coastal, limited and divided into three areas by altitude and exposure to the trade winds on the east coast. Mosquito vectors for other diseases are an issue at both sites, but of primary importance on La Reunion due to the recent chikungunya epidemic. The similarities and differences between these two sites in terms of suitability are discussed in the context of area-wide integrated vector management incorporating the SIT.     

Ethical,legal and social aspects of the approach in Sudan

The global malaria situation, especially in Africa, and the problems frequently encountered in chemical control of vectors such as insecticide resistance, emphasize the urgency of research, development and implementation of new vector control technologies that are applicable at regional and local levels. The successful application of the sterile insect technique (SIT) for the control of the New World screwworm Cochliomyia hominivorax and several species of fruit flies has given impetus to the use of this method for suppression or elimination of malaria vectors in some areas of Africa including Northern State of Sudan. The research and development phase of the Northern State feasibility study has been started. Sudanese stakeholders are working side-by-side with the International Atomic Energy Agency in the activities of this important phase. Several ethical, legal and social issues associated with this approach arose during this phase of the project. They need to be seriously considered and handled with care. In this paper, these issues are described, and the current and proposed activities to overcome potential hurdles to ensure success of the project are listed.