Expression of N-linked sialyl Le(x) determinants and O-glycans in the carbohydrate moiety of human amniotic fluid transferrin during pregnancy

Transferrin, a glycoprotein involved in iron transport in body fluids, was isolated from amniotic fluid of a hydramniospatient by sequential anion-exchange chromatography and gel filtration. The N-glycans of human amniotic fluid transferrin (hAFT) were enzymatically liberated by PNGase-F digestion, isolated by gel filtration and fractionated by (high-pH) anion-exchange chromatography. After alkaline borohydride treatment of native hAFT, the released O-glycans were isolated by gel filtration and fractionated by anion-exchange chroma-tography. Structure elucidation of 14 N- and 2 O-glycans was performed by 500 or 600 MHz1H-NMR spectroscopy. Besides conventional N-glycans established earlier for human serum transferrin (hST), new (alpha1-3)-fucosylated N- glycans were found, representing sialyl Le(x) elements. Furthermore, as compared to hST, a higher degree of (alpha1-6)-fucosylation and an increase in branching from di- to triantennary compounds has been detected. The presence of O-glycans is demonstrated for the first time in transferrin.      

Microbial signaling and systems biology

A report of the symposium on Signaling and Systems Biology held during the Society for General Microbiology Spring Meeting, 29-30 March 2010, Edinburgh, UK.     

Genetic instabilities of triplet repeat sequences by recombination

The expansion of triplet repeat sequences is an initial step in the disease etiology of a number of hereditary neurological disorders in humans. Diseases such as myotonic dystrophy, Huntington's, several spinocerebellar ataxias, fragile X syndrome, and Friedreich's ataxia are caused by the expansions of CTG.CAG, CGG.CCG, or GAA.TTC repeats. The mechanisms of the expansion process have been investigated intensely in E. coli, yeast, transgenic mice, mammalian cell culture, and in human clinical cases. Whereas studies from 1994-1999 have implicated DNA replication and repair at the paused synthesis sites due to the unusual conformations of the triplet repeat sequences, recent work has shown that homologous recombination (gene conversion) is a powerful mechanism for generating massive expansions, in addition to, or in concert with, replication and repair.     

Consequences of concurrent Ascaridia galli and Escherichia coli infections in chickens

   

The principal aim of this study was to evaluate dissociative anaesthesia for castration of colts during field conditions. Three dissociative anaesthetic protocols were evaluated during castration of colts in an animal hospital. The protocol considered to be the most suitable was thereafter evaluated during castration of colts under field conditions. Respiratory and haemodynamic parameters and the response to surgery were determined during anaesthesia. All horses breathed air spontaneously during anaesthesia. Under hospital conditions 26 colts were randomised to receive one of three anaesthetic protocols: Romifidine and tiletamine-zolazepam (RZ); acepromazine, romifidine and tiletamine-zolazepam (ARZ); or acepromazine, romifidine, butorphanol and tiletamine-zolazepam (ARBZ). The surgeon was blinded to the anaesthetic protocol used and decided whether supplemental anaesthesia was needed to complete surgery. Under field conditions 31 colts were castrated during anaesthesia with the ARBZ protocol. All inductions, anaesthesia and recoveries were calm and without excitation under both hospital and field conditions. Surgery was performed within 5–20 minutes after the horses had assumed lateral recumbency during both hospital and field castrations. Under hospital conditions some horses needed supplemental anaesthesia with all three anaesthetic protocols to complete surgery. Interestingly, none of the horses castrated with protocol ARBZ under field conditions needed additional anaesthesia. Cardiorespiratory changes were within acceptable limits in these clinically healthy colts.     

Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: II. Effects on skeletal muscle atrophy

Background

Chronic obstructive pulmonary disease (COPD) is accompanied by pulmonary inflammation and associated with extra-pulmonary manifestations, including skeletal muscle atrophy. Glycogen synthase kinase-3 (GSK-3) has been implicated in the regulation of muscle protein- and myonuclear turnover; two crucial processes that determine muscle mass. In the present study we investigated the effect of the selective GSK-3 inhibitor SB216763 on muscle mass in a guinea pig model of lipopolysaccharide (LPS)-induced pulmonary inflammation-associated muscle atrophy.

Methods

Guinea pigs were pretreated with either intranasally instilled SB216763 or corresponding vehicle prior to each LPS/saline challenge twice weekly. Pulmonary inflammation was confirmed and indices of muscle mass were determined after 12 weeks. Additionally, cultured skeletal muscle cells were incubated with tumor necrosis factor α (TNF-α) or glucocorticoids (GCs) to model the systemic effects of pulmonary inflammation on myogenesis, in the presence or absence of GSK-3 inhibitors.

Results

Repeated LPS instillation induced muscle atrophy based on muscle weight and muscle fiber cross sectional area. Intriguingly, GSK-3 inhibition using SB216763 prevented the LPS-induced muscle mass decreases and myofiber atrophy. Indices of protein turnover signaling were unaltered in guinea pig muscle. Interestingly, inhibition of myogenesis of cultured muscle cells by TNF-α or synthetic GCs was prevented by GSK-3 inhibitors.

Conclusions

In a guinea pig model of LPS-induced pulmonary inflammation, GSK-3 inhibition prevents skeletal muscle atrophy without affecting pulmonary inflammation. Resistance to inflammation- or GC-induced impairment of myogenic differentiation, imposed by GSK-3 inhibition, suggests that sustained myogenesis may contribute to muscle mass maintenance despite persistent pulmonary inflammation. Collectively, these results warrant further exploration of GSK-3 as a potential novel drug target to prevent or reverse muscle wasting in COPD.     

Study of permeation and blocker binding in TMEM16A calcium-activated chloride channels

We studied the effects of mutations of positively charged amino acid residues in the pore of X. tropicalis TMEM16A calcium-activated chloride channels: K613E, K628E, K630E; R646E and R761E. The activation and deactivation kinetics were not affected, and only K613E showed a lower current density. K628E and R761E affect anion selectivity without affecting Na⁺ permeation, whereas K613E, R646E and the double mutant K613E + R646E affect anion selectivity and permeability to Na⁺. Furthermore, altered blockade by the chloride channel blockers anthracene-9-carboxylic acid (A-9-C), 4, 4''-Diisothiocyano-2,2''-stilbenedisulfonic acid (DIDS) and T16inh-A01 was observed. These results suggest the existence of 2 binding sites for anions within the pore at electrical distances of 0.3 and 0.5. These sites are also relevant for anion permeation and blockade.