Trafficking and localization studies of recombinant alpha1, 3- fucosyltransferase VI stably expressed in CHO cells

Peripheral alpha1,3-fucosylation of glycans occurs by the action of either one of five different alpha1,3-fucosyltransferases (Fuc-Ts) cloned to date. Fuc-TVI is one of the alpha1,3-fucosyltransferases which is capable to synthesize selectin ligands. The major alpha1, 3- fucosyltransferase activity in human plasma is encoded by the gene for fucosyltransferase VI, which presumably originates from liver cells. While the sequence, chromosomal localization, and kinetic properties of Fuc-TVI are known, immunocytochemical localization and trafficking studies have been impossible because of the lack of specific antibodies. Here we report on the development and characterization of a peptide-specific polyclonal antiserum monospecific to Fuc-TVI and an antiserum to purified soluble recombinant Fuc-TVI crossreactive with Fuc-TIII and Fuc-TV. Both antisera were applied for immunodetection in stably transfected CHO cells expressing the full-length form of this enzyme (CHO clone 61/11). Fuc-TVI was found to be a resident protein of the Golgi apparatus. In addition, more than 30% of cell-associated and released enzyme activity was found in the medium. Maturation and release of Fuc-TVI was analyzed in metabolically labeled CHO 61/11 cells followed by immunoprecipitation. Fuc-TVI occurred in two forms of 47 kDa and 43 kDa bands, while the secreted form was detected as a 43 kDa. These two different intracellular forms arose by posttranslational modification, as shown by pulse-chase experiments. Fuc-TVI was released to the supernatant by proteolytic cleavage as a partially endo-H resistant glycoform.      

Fluorescence properties of actin and analysis of the content of native actin in its preparations]

Changes of properties of actin preparations from rabbit skeletal muscles in the course of purification were studied. It is shown that independent on initial properties of actin preparations at successive polimerization, sedimentation and depolimerization cycles: 1) the quantity of protein in supernatant diminishes progressively, 2) intrinsic viscosity of F-actin increases, 3) the value of spectral parameter A = (I320/I365)296., which in characteristic of the fluorescence spectrum position of tryptophan residues, increases and approaches the extremal value, 4) the effect of short wave shift of the spectrum at actin polimerazation becomes more pronounced. The actin preparation with the extremal value of A = 2,6 (native actin) has [eta] = 8,8; deltaAg leads to f approximately 0,25; lambdamax=325 nm. Inactivation of actin results in the long-wave shift of fluorescence spectrum (lambdamax=337 nm, A = 1,30) suggesting the disturbance of exclusively compact globular structure of native protein macromolecular. The ratio is described which enables to use parameter A for the quick estimation of the content of native actin in its preparations. The technical simplicity of the measurement of parameter A enables to use it for the characterization of individual fractions in gelfiltration of actin preparations.     

Effect of cooling to low temperatures on viability of human skin keratinocytes at different stages of differentiation

The goal of this study was to conduct a comparative analysis of the degree of resistance to low temperatures of human epidermal cells found at different stages of differentiation. The action of liquid-nitrogen vapors was analyzed in experiments in vitro at various temperature regimes on fragments of the human integral skin and on isolated from them and cultivated keratinocytes. The degree of resistance of keratinocytes to the action of cooling to low temperatures was evaluated by their ability to form a multilayer stratum in culture, which indicates the preservation of the viability of the cells treated with cold. This approach allowed one to reveal the diapason of optimal regimes of the action of low temperatures on cells in the composition of tissue and after their conversion into culture. The quantitative ratios of the epidermal stem, transitory, and differentiated cells in a population of viable cells before and after exposure to low temperatures were determined with antibodies that correspond to their different stages of differentiation. The degree of resistance of keratinocytes to action of cooling to low temperatures was evaluated by their ability to form a multilayer stratum in culture, which indicates preservation of viability of the cells treated with cold. The results of this study show that the resistance of human epidermal cells to low temperature differs depending on their stage of differentiation both in situ and in vitro. The epidermal stem and transitory cells are more stable than the differentiated cells.     

Isolation of human basal keratinocytes by selective adhesion to extracellular matrix proteins

Epidermal human cells (keratinocytes) differently interact with extracellular matrix proteins of the skin basal membrane depending on the stages of their differentiation. The pool of basal keratinocytes commonly includes stem cells and transient amplifying cells. They directly attach to the skin basal membrane. Keratinocytes change their adhesive properties during differentiation, lose direct interaction with the basal membrane and move to suprabasal epidermal strata. From this, it is suggested that basal and primarily stem cells can be isolated from a heterogenous keratinocyte population due to their selective adhesion to the extracellular matrix proteins. In the current study, we analysed the specificity of interaction between primary keratinocytes and extracellular matrix proteins (collagens of I and IV types, laminin-2/4, fibronectin and matrigel). We have demonstrated that the basal keratinocytes extracted from the skin have different adhesive abilities. The rapidly spreading cells usually interacted with collagen and fibronectin rather that with laminin-2/4 or matrigel. The majority of these cells being represented by basal keratinocytes. Our data demonstrate that the applied method of keratinocyte selection may be directed for precise isolation of skin stem from a common cell population.     

7th SOSORT consensus paper: conservative treatment of idiopathic & Scheuermann's kyphosis

Abstract

Thoracic hyperkyphosis is a frequent problem and can impact greatly on patient's quality of life during adolescence. This condition can be idiopathic or secondary to Scheuermann disease, a disease disturbing vertebral growth. To date, there is no sound scientific data available on the management of this condition. Some studies discuss the effects of bracing, however no guidelines, protocols or indication's of treatment for this condition were found. The aim of this paper was to develop and verify the consensus on managing thoracic hyperkyphosis patients treated with braces and/or physiotherapy.

Methods

The Delphi process was utilised in four steps gradually modified according to the results of a set of recommendations: we involved the SOSORT Board twice, then all SOSORT members twice, with a Pre-Meeting Questionnaire (PMQ), and during a Consensus Session at the SOSORT Lyon Meeting with a Meeting Questionnaire (MQ).

Results

There was an unanimous agreement on the general efficacy of bracing and physiotherapy for this condition. Most experts suggested the use of 4-5 point bracing systems, however there was some controversy with regards to physiotherapeutic aims and modalities.

Conclusion

The SOSORT panel of experts suggest the use of rigid braces and physiotherapy to correct thoracic hyperkyphosis during adolescence. The evaluation of specific braces and physiotherapy techniques has been recommended.     

Differentiation of naive CD4<Superscript>+</Superscript> T cells towards T helper 2 cells is not impaired in rheumatoid arthritis patients

An impaired differentiation of naive CD4+ T cells towards Th2 cells may contribute to the chronic tissue-destructive T-cell activity in rheumatoid arthritis (RA). The differentiation of naive CD4+ T cells into memory Th2 cells by IL-7 in comparison with that by IL-4 was studied in RA patients and in healthy controls. Naive CD4+ T cells from peripheral blood were differentiated by CD3/CD28 costimulation in the absence of or in the presence of IL-7 and/or IL-4. The production of IFN-gamma and IL-4 was measured by ELISA and by single-cell FACS analysis to indicate Th1 and Th2 cell activity. CD3/CD28 costimulation and IL-7 were early inducers of IL-4 production, but primarily stimulated IFN-gamma production. In contrast, in short-term cultures exogenously added IL-4 did not prime for IL-4 production but suppressed IL-7-induced IFN-gamma production. Upon long-term stimulation of naive CD4+ T cells, IFN-gamma production was differentially regulated by IL-7 and IL-4, but IL-4 production was increased by both IL-7 and IL-4. IL-7 and IL-4 additively induced polarization towards a Th2 phenotype. This susceptibility of naive CD4+ T cells to become Th2 cells upon culture with IL-7 and IL-4 was increased in RA patients compared with that in healthy controls. These findings demonstrate that, in RA patients, differentiation of naive CD4+ T cells towards a Th2 phenotype by CD3/CD28 costimulation, IL-7 and IL-4 is not impaired. The perpetuation of arthritogenic T-cell activity in RA therefore seems not to be the result of intrinsic defects of naive CD4+ T cells to develop towards suppressive memory Th2 cells.     

Disentangling Woodland Caribou Movements in Response to Clearcuts and Roads across Temporal Scales

Although prey species typically respond to the most limiting factors at coarse spatiotemporal scales while addressing biological requirements at finer scales, such behaviour may become challenging for species inhabiting human altered landscapes. We investigated how woodland caribou, a threatened species inhabiting North-American boreal forests, modified their fine-scale movements when confronted with forest management features (i.e. clearcuts and roads). We used GPS telemetry data collected between 2004 and 2010 on 49 female caribou in a managed area in Québec, Canada. Movements were studied using a use – availability design contrasting observed steps (i.e. line connecting two consecutive locations) with random steps (i.e. proxy of immediate habitat availability). Although caribou mostly avoided disturbances, individuals nonetheless modulated their fine-scale response to disturbances on a daily and annual basis, potentially compromising between risk avoidance in periods of higher vulnerability (i.e. calving, early and late winter) during the day and foraging activities in periods of higher energy requirements (i.e. spring, summer and rut) during dusk/dawn and at night. The local context in which females moved was shown to influence their decision to cross clearcut edges and roads. Indeed, although females typically avoided crossing clearcut edges and roads at low densities, crossing rates were found to rapidly increase in greater disturbance densities. In some instance, however, females were less likely to cross edges and roads as densities increased. Females may then be trapped and forced to use disturbed habitats, known to be associated with higher predation risk. We believe that further increases in anthropogenic disturbances could exacerbate such behavioural responses and ultimately lead to population level consequences.