Functional expression of adrenoreceptors in mesenchymal stromal cells derived from the human adipose tissue

Cultured mesenchymal stromal cells (MSCs) from different sources represent a heterogeneous population of proliferating non-differentiated cells that contains multipotent stem cells capable of originating a variety of mesenchymal cell lineages. Despite tremendous progress in MSC biology spurred by their therapeutic potential, current knowledge on receptor and signaling systems of MSCs is mediocre. Here we isolated MSCs from the human adipose tissue and assayed their responsivity to GPCR agonists with Ca^2 + imaging. As a whole, a MSC population exhibited functional heterogeneity. Although a variety of first messengers was capable of stimulating Ca²⁺ signaling in MSCs, only a relatively small group of cells was specifically responsive to the particular GPCR agonist, including noradrenaline. RT-PCR and immunocytochemistry revealed expression of α1B-, α2A-, and β2-adrenoreceptors in MSCs. Their sensitivity to subtype-specific adrenergic agonists/antagonists and certain inhibitors of Ca²⁺ signaling indicated that largely the α2A-isoform coupled to PLC endowed MSCs with sensitivity to noradrenaline. The all-or-nothing dose-dependence was characteristic of responsivity of robust adrenergic MSCs. Noradrenaline never elicited small or intermediate responses but initiated large and quite similar Ca²⁺ transients at all concentrations above the threshold. The inhibitory analysis and Ca²⁺ uncaging implicated Ca²⁺-induced Ca²⁺ release (CICR) in shaping Ca²⁺ signals elicited by noradrenaline. Evidence favored IP₃ receptors as predominantly responsible for CICR. Based on the overall findings, we inferred that adrenergic transduction in MSCs includes two fundamentally different stages: noradrenaline initially triggers a local and relatively small Ca²⁺ signal, which next stimulates CICR, thereby being converted into a global Ca²⁺ signal.     

Xylanase of the micromycete <Emphasis Type="Italic">Rhizopus</Emphasis> var. <Emphasis Type="Italic">microsporus</Emphasis> 595: Preparation,structural and functional characteristics,and application

Procedures for the production of endo-1,4-β-xylanase have been developed. An active producer—Rhizopus var. microsporus BKMF-595—has been chosen, and the conditions of surface and submerged cultivation, as well as the composition of the culture medium for this strain, have been optimized to ensure maximum yield of the target enzyme. Activity of xylomicrosporin Px equaled 123 U/g, while the activity of xylomicrosporin Gx equaled 25 U/cm³. Homogeneous enzyme preparations, purified 59.44-fold and 72.6-fold, have been obtained. The dependence of endo-1,4-β-xylanase catalytic activity on temperature and pH of the reaction medium has been studied. The enzyme has been shown to be most stable in the pH range 5.0–6.0 and to be thermostable. Amino acid composition and subunit structure of the enzyme were determined; the molecular masses of the subunits equaled 50 and 56 kDa. Carboxyl groups of glutamic and aspartic acid residues of the active center were experimentally shown to play an important role in catalysis. The potential of this enzyme for beer production has been demonstrated.     

Respiratory allergy to Blomia tropicalis: Immune response in four syngeneic mouse strains and assessment of a low allergen-dose,short-term experimental model

Background

The dust mite Blomia tropicalis is an important source of aeroallergens in tropical areas. Although a mouse model for B. tropicalis extract (BtE)-induced asthma has been described, no study comparing different mouse strains in this asthma model has been reported. The relevance and reproducibility of experimental animal models of allergy depends on the genetic background of the animal, the molecular composition of the allergen and the experimental protocol.

Objectives

This work had two objectives. The first was to study the anti-B. tropicalis allergic responses in different mouse strains using a short-term model of respiratory allergy to BtE. This study included the comparison of the allergic responses elicited by BtE with those elicited by ovalbumin in mice of the strain that responded better to BtE sensitization. The second objective was to investigate whether the best responder mouse strain could be used in an experimental model of allergy employing relatively low BtE doses.

Methods

Groups of mice of four different syngeneic strains were sensitized subcutaneously with 100 μg of BtE on days 0 and 7 and challenged four times intranasally, at days 8, 10, 12, and 14, with 10 μg of BtE. A/J mice, that were the best responders to BtE sensitization, were used to compare the B. tropicalis-specific asthma experimental model with the conventional experimental model of ovalbumin (OVA)-specific asthma. A/J mice were also sensitized with a lower dose of BtE.

Results

Mice of all strains had lung inflammatory-cell infiltration and increased levels of anti-BtE IgE antibodies, but these responses were significantly more intense in A/J mice than in CBA/J, BALB/c or C57BL/6J mice. Immunization of A/J mice with BtE induced a more intense airway eosinophil influx, higher levels of total IgE, similar airway hyperreactivity to methacholine but less intense mucous production, and lower levels of specific IgE, IgG1 and IgG2 antibodies than sensitization with OVA. Finally, immunization with a relatively low BtE dose (10 μg per subcutaneous injection per mouse) was able to sensitize A/J mice, which were the best responders to high-dose BtE immunization, for the development of allergy-associated immune and lung inflammatory responses.

Conclusions

The described short-term model of BtE-induced allergic lung disease is reproducible in different syngeneic mouse strains, and mice of the A/J strain was the most responsive to it. In addition, it was shown that OVA and BtE induce quantitatively different immune responses in A/J mice and that the experimental model can be set up with low amounts of BtE.     

Rats go genomic

A report on the meeting 'Rat Genomics and Models', Cold Spring Harbor, USA, 8-11 December 2005.     

A Dutch guideline for the treatment of scoliosis in neuromuscular disorders

Background

Children with neuromuscular disorders with a progressive muscle weakness such as Duchenne Muscular Dystrophy and Spinal Muscular Atrophy frequently develop a progressive scoliosis. A severe scoliosis compromises respiratory function and makes sitting more difficult. Spinal surgery is considered the primary treatment option for correcting severe scoliosis in neuromuscular disorders. Surgery in this population requires a multidisciplinary approach, careful planning, dedicated surgical procedures, and specialized after care.

Methods

The guideline is based on scientific evidence and expert opinions. A multidisciplinary working group representing experts from all relevant specialties performed the research. A literature search was conducted to collect scientific evidence in answer to specific questions posed by the working group. Literature was classified according to the level of evidence.

Results

For most aspects of the treatment scientific evidence is scarce and only low level cohort studies were found. Nevertheless, a high degree of consensus was reached about the management of patients with scoliosis in neuromuscular disorders. This was translated into a set of recommendations, which are now officially accepted as a general guideline in the Netherlands.

Conclusion

In order to optimize the treatment for scoliosis in neuromuscular disorders a Dutch guideline has been composed. This evidence-based, multidisciplinary guideline addresses conservative treatment, the preoperative, perioperative, and postoperative care of scoliosis in neuromuscular disorders.     

The effect of daily exposure to low hardening temperature on plant vital activity

Phenomenological responses of plants to daily short-term exposure to low hardening temperature was studied under chamber and field conditions. Experiments were carried out on cucumber (Cucumis sativus L.), barley (Hordeum vulgare L.), marigolds (Tagetes L.), and petunia (Petunia × hybrida) plants. The obtained data demonstrated a similar pattern of response in all studied plant species to different variants of exposure to low hardening temperature. The main features of plant response to daily short-term exposure to low hardening temperature include: a higher increment in cold tolerance (cf. two-or threefold increase relative to constant low hardening temperature) that peaked on day 5 (cf. day 2 at constant low hardening temperature) and was maintained for 2 weeks (cf. 3–4 days at constant low hardening temperature); a simultaneous increase in heat tolerance (cf. twofold relative to constant low hardening temperature) maintained over a long period (cf. only in the beginning of the exposure to constant low hardening temperature); a sharp drop in the subsequent cold tolerance after plant incubation in the dark (cf. a very low decrease in cold tolerance following the exposure to constant low hardening temperature); a combination of high cold tolerance and high photochemical activity of the photosynthetic apparatus (cf. a low non-photochemical quenching at constant low hardening temperature); and the capacity to increase cold tolerance in response to repeated short-term exposures to low hardening temperature in plants grown outdoors (cf. a gradual increase after repeated exposure to constant low hardening temperature). Possible mechanisms underlying the plant response to daily short-term exposure to low temperature are proposed.