Specific localization of scallop gill epithelial calmodulin in cilia

Calmodulin has been isolated and characterized from the gill of the bay scallop aequipecten irradians. Quantitative electrophoretic analysis of epithelial cell fractions show most of the calmodulin to be localized in the cilia, specifically in the detergent- solubilized membrane-matrix fraction. Calmodulin represents 2.2 +/- 0.3 percent of the membrane-matrix protein or 0.41 +/- 0.5 percent of the total ciliary protein. Its concentration is at least 10(-4) M if distributed uniformly within the matrix. Extraction in the presence of calcium suggests that the calmodulin is not bound to the axoneme proper. The ciliary protein is identified as a calmodulin on the basis of its calcium- dependent binding to a fluphenazine-sepharose affinity column and its comigration with bovine brain calmodulin on alkaline-urea and SDS polyacrylamide gels in both the presence and absence of calcium. Scallop ciliary calmodulin activates bovine brain phosphodiesterase to the same extent as bovine brain and chicken gizzard calmodulins. Containing trimethyllysine and lacking cysteine and tryptophan, the amino acid composition of gill calmodulin is typical of known calmodulins, except that it is relatively high in serine and low in methionine. Its composition is less acidic than other calmodulins, in agreement with an observed isoelectric point approximately 0.2 units higher than that of bovine brain. Comparative tryptic peptide mapping of scallop gill ciliary and bovine brain calmodulins indicates coincidence of over 75 percent of the major peptides, but at least two major peptides in each show no near-equivalency. Preliminary results using ATP-reactivated gill cell models show no effect of calcium at micromolar levels on ciliary beat or directionality of the lateral cilia, the cilia which constitute the vast majority of those isolated. However, ciliary arrest will occur at calcium levels more than 150 muM. Because calmodulin usually functions in the micromolar range, its role in this system is unclear. Scallop gill ciliary calmodulin may be involved in the direct regulation of dyneintubule sliding, or it may serve some coupled calcium transport function. At the concentration in which it is found, it must also at least act as a calcium buffer.     

In vitro insulin-like actions of human growth hormone: a study with an insulin antibody

When polyclonal insulin antibodies were preincubated with either adipose tissue from hypophysectomized rats or adipocytes from normal rats, human growth hormone failed to stimulate glucose oxidation. Removal of insulin from adipocytes through incubation with pyruvate at pH 7.0, followed by washing three times, also abolished subsequent in vitro insulin-like action of hGH. Administration of the same insulin antibody to hypophysectomized rats 30 minutes prior to injection of hGH did not inhibit the insulin-like activity of the hGH as measured by its ability to decrease serum glucose and non-esterified fatty acid levels. It is concluded that the in vitro promotion of glucose oxidation by hGH requires insulin. Because of the uncertainty of complete removal of insulin in intact animals, such a conclusion cannot be made regarding in vivo insulin-like action of hGH.     

Comparison of the acute metabolic effects of 22,000-dalton and 20,000-dalton growth hormone in human subjects

The acute metabolic effects of 20,000-dalton human growth hormone (hGH20K) in man have not previously been tested. We compared changes in concentrations of free fatty acids (FFA), glucose, and insulin in nine growth hormone deficient children following injection of 22,000-dalton intact human growth hormone (hGH22K) and the smaller variant, hGH20K. There was a significant decline (37%) in the mean FFA concentration from baseline to 1/2 hour post-injection and from baseline to 1 hour post-injection (36%) in the children given hGH22K, but no such decline was seen after injection of hGH20K. No significant differences in mean insulin or glucose concentrations were noted between the two treatment groups, and glucose and insulin concentrations did not acutely change after injection of either hormone. The results of this study indicate that hGH20K has a diminished activity for suppression of FFA as compared to hGH22K. This suggests that GH residues 32-46, missing in hGH20K, constitute all or part of the region of hGH22K producing this response, or that the different primary structures of the two hormones result in tertiary structural differences and altered biological activity.     

Diabetogenic response to streptozotocin varies among obese yellow and among lean agouti (BALB/c x VY)F1 hybrid mice

To test the hypothesis that the elevated insulin levels in obese neoplasia-susceptible yellow Avy/- mice might be a major factor stimulating tumor formation, it is necessary to use normoinsulinemic yellow mice. Although our attempt to obtain normoinsulinemic, euglycemic mice by streptozotocin treatment was unsuccessful, we did observe significant differences in the responsiveness to this treatment among mice of identical genotype. These differences were observed among female yellow Avy/A and agouti A/a (BALB/c x VY)F1 hybrid mice in the responses of body weight gain, plasma glucose, and plasma insulin levels to a single intraperitoneal injection of either 150 or 200 mg/kg streptozotocin (STZ) at 4 weeks of age followed by a 22-week observation period. Among animals treated with the high streptozotocin dose, 80% of the yellow mice gained almost no weight and became grossly hyperglycemic and hypoinsulinemic; however, only 55% of the agouti mice exhibited such a strong response. In the low dose group, 25% of the yellow mice responded with reduced body weight gain, decreased insulin, and elevated glucose levels whereas none of the agouti mice exhibited such responses. More pancreatic islet tissue mass was present in the untreated yellow control mice than among the comparable agouti mice by the end of the study. In both streptozotocin dose groups and in both genotypes, islet tissue mass was reduced to a much greater extent in the more responsive mice than in the less responsive mice. There appeared to be no correlation between islet tissue mass and insulin level. The phenotypic variation in responsiveness to an exogenous agent among test animals of a single inbred or F1 hybrid genotype reported here is not unique to this F1 hybrid since it is seen in most chronic bioassays when relatively low levels of agent are used.     

Classification of videocapsule endoscopy image patterns: comparative analysis between patients with celiac disease and normal individuals

Background  

Quantitative disease markers were developed to assess videocapsule images acquired from celiac disease patients with villous atrophy, and from control patients.     

Expression of biologically active recombinant rat IgE-binding protein in Escherichia coli

IgE-binding protein (epsilon BP) is a protein which has affinity for IgE and was originally identified in rat basophilic leukemia (RBL) cells. Subsequently, it was found to be the rat homolog of CBP35, a murine beta-galactoside-specific lectin. This protein is also designated as L-34 and RL-29 and studied independently by several laboratories. More recently, CBP35 (epsilon BP) was found to be equivalent to Mac-2, a surface marker on activated macrophages. Using rat epsilon BP cDNA, we have succeeded in expressing recombinant epsilon BP in Escherichia coli. Milligram quantities of homogeneous epsilon BP could be obtained from bacterial lysate in a one-step affinity purification procedure utilizing lactosyl-Sepharose 4B and elution with a lactose gradient. The recombinant epsilon BP (r epsilon BP) binds mouse IgE and retains reactivity to anti-peptide antibodies specific for a sequence within rat epsilon BP. The purified r epsilon BP exhibits binding activity to various saccharides, with affinity for N-acetyllactosamine greater than thiodigalactoside greater than lactose much greater than D-galactose greater than L-arabinose, an order identical to that exhibited by native epsilon BP isolated from RBL cells. The recombinant lectin displayed hemagglutination activity when tested with rabbit erythrocytes. Although epsilon BP shares sequence homology to other lectins containing S-type (thiol-dependent) carbohydrate-recognition domains, r epsilon BP is resistant to air oxidation and does not require reducing agents for maintaining its activity. Furthermore, the single cysteine residue appears to be unexposed and can be alkylated only when the protein is denatured in 5.6 M guanidinium hydrochloride. The availability of a source for a large quantity of epsilon BP should facilitate the analysis of biological function(s) and structure-activity relationships of this lectin.     

A new gating site in human aquaporin-4: Insights from molecular dynamics simulations

Aquaporin-4 (AQP4) is the predominant water channel in different organs and tissues. An alteration of its physiological functioning is responsible for several disorders of water regulation and, thus, is considered an attractive target with a promising therapeutic and diagnostic potential. Molecular dynamics (MD) simulations performed on the AQP4 tetramer embedded in a bilayer of lipid molecules allowed us to analyze the role of spontaneous fluctuations occurring inside the pore. Following the approach by Hashido et al. [Hashido M, Kidera A, Ikeguchi M (2007) Biophys J 93: 373–385], our analysis on 200 ns trajectory discloses three domains inside the pore as key elements for water permeation. Herein, we describe the gating mechanism associated with the well-known selectivity filter on the extracellular side of the pore and the crucial regulation ensured by the NPA motifs (asparagine, proline, alanine). Notably, on the cytoplasmic side, we find a putative gate formed by two residues, namely, a cysteine belonging to the loop D (C178) and a histidine from loop B (H95). We observed that the spontaneous reorientation of the imidazole ring of H95 acts as a molecular switch enabling H-bond interaction with C178. The occurrence of such local interaction seems to be responsible for the narrowing of the pore and thus of a remarkable decrease in water flux rate. Our results are in agreement with recent experimental observations and may represent a promising starting point to pave the way for the discovery of chemical modulators of AQP4 water permeability.     

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.     

AQP4-Dependent Water Transport Plays a Functional Role in Exercise-Induced Skeletal Muscle Adaptations

In this study we assess the functional role of Aquaporin-4 (AQP4) in the skeletal muscle by analyzing whether physical activity modulates AQP4 expression and whether the absence of AQP4 has an effect on osmotic behavior, muscle contractile properties, and physical activity. To this purpose, rats and mice were trained on the treadmill for 10 (D10) and 30 (D30) days and tested with exercise to exhaustion, and muscles were used for immunoblotting, RT-PCR, and fiber-type distribution analysis. Taking advantage of the AQP4 KO murine model, functional analysis of AQP4 was performed on dissected muscle fibers and sarcolemma vesicles. Moreover, WT and AQP4 KO mice were subjected to both voluntary and forced activity. Rat fast-twitch muscles showed a twofold increase in AQP4 protein in D10 and D30 rats compared to sedentary rats. Such increase positively correlated with the animal performance, since highest level of AQP4 protein was found in high runner rats. Interestingly, no shift in muscle fiber composition nor an increase in AQP4-positive fibers was found. Furthermore, no changes in AQP4 mRNA after exercise were detected, suggesting that post-translational events are likely to be responsible for AQP4 modulation. Experiments performed on AQP4 KO mice revealed a strong impairment in osmotic responses as well as in forced and voluntary activities compared to WT mice, even though force development amplitude and contractile properties were unvaried. Our findings definitively demonstrate the physiological role of AQP4 in supporting muscle contractile activity and metabolic changes that occur in fast-twitch skeletal muscle during prolonged exercise.