Large scale production of phage antibody libraries using a bioreactor

One of the limitations of the use of phage antibody libraries in high throughput selections is the production of sufficient phage antibody library at the appropriate quality. Here, we successfully adapt a bioreactor-based protocol for the production of phage peptide libraries to the production of phage antibody libraries. The titers obtained in the stirred-tank bioreactor are 4 to 5 times higher than in a standard shake flask procedure, and the quality of the phage antibody library produced is indistinguishable to that produced using standard procedures as assessed by Western blotting and functional selections. Availability of this protocol will facilitate the use of phage antibody libraries in high-throughput scale selections.     

The PTH/PTHrP receptor can delay chondrocyte hypertrophy in vivo without activating phospholipase C

One G protein-coupled receptor (GPCR) can activate more than one G protein, but the physiologic importance of such activation has not been demonstrated in vivo. We have generated mice expressing exclusively a mutant form of the PTH/PTHrP receptor (DSEL) that activates adenylyl cyclase normally but not phospholipase C (PLC). DSEL mutant mice exhibit abnormalities in embryonic endochondral bone development, including delayed ossification and increased chondrocyte proliferation. Analysis of the differentiation of embryonic metatarsals in vitro shows that PTH(1-34) and forskolin inhibit, whereas active phorbol ester stimulates, hypertrophic differentiation. Thus, PLC signaling via the PTH/PTHrP receptor normally slows the proliferation and hastens the differentiation of chondrocytes, actions that oppose the dominant effects of PTH/PTHrP receptors and that involve cAMP-dependent signaling pathways.     

Sinorhizobium meliloti mutants lacking phosphotransferase system enzyme HPr or EIIA are altered in diverse processes, including carbon metabolism, cobalt requirements, and succinoglycan production

Sinorhizobium meliloti is a member of the Alphaproteobacteria that fixes nitrogen when it is in a symbiotic relationship. Genes for an incomplete phosphotransferase system (PTS) have been found in the genome of S. meliloti. The genes present code for Hpr and ManX (an EIIA(Man)-type enzyme). HPr and EIIA regulate carbon utilization in other bacteria. hpr and manX in-frame deletion mutants exhibited altered carbon metabolism and other phenotypes. Loss of HPr resulted in partial relief of succinate-mediated catabolite repression, extreme sensitivity to cobalt limitation, rapid die-off during stationary phase, and altered succinoglycan production. Loss of ManX decreased expression of melA-agp and lac, the operons needed for utilization of alpha- and beta-galactosides, slowed growth on diverse carbon sources, and enhanced accumulation of high-molecular-weight succinoglycan. A strain with both hpr and manX deletions exhibited phenotypes similar to those of the strain with a single hpr deletion. Despite these strong phenotypes, deletion mutants exhibited wild-type nodulation and nitrogen fixation when they were inoculated onto Medicago sativa. The results show that HPr and ManX (EIIA(Man)) are involved in more than carbon regulation in S. meliloti and suggest that the phenotypes observed occur due to activity of HPr or one of its phosphorylated forms.     

The Yo-Yo intermittent recovery test level 1 is reliable in young high-level soccer players

The aim of the study was to investigate test reliability of the Yo-Yo intermittent recovery test level 1 (YYIR1) in 36 high-level youth soccer players, aged between 13 and 18 years. Players were divided into three age groups (U15, U17 and U19) and completed three YYIR1 in three consecutive weeks. Pairwise comparisons were used to investigate test reliability (for distances and heart rate responses) using technical error (TE), coefficient of variation (CV), intra-class correlation (ICC) and limits of agreement (LOA) with Bland-Altman plots. The mean YYIR1 distances for the U15, U17 and U19 groups were 2024 ± 470 m, 2404 ± 347 m and 2547 ± 337 m, respectively. The results revealed that the TEs varied between 74 and 172 m, CVs between 3.0 and 7.5%, and ICCs between 0.87 and 0.95 across all age groups for the YYIR1 distance. For heart rate responses, the TEs varied between 1 and 6 bpm, CVs between 0.7 and 4.8%, and ICCs between 0.73 and 0.97. The small ratio LOA revealed that any two YYIR1 performances in one week will not differ by more than 9 to 28% due to measurement error. In summary, the YYIR1 performance and the physiological responses have proven to be highly reliable in a sample of Belgian high-level youth soccer players, aged between 13 and 18 years. The demonstrated high level of intermittent endurance capacity in all age groups may be used for comparison of other prospective young soccer players.     

Effects of GABA on acutely isolated neurons from the gustatory zone of the rat nucleus of the solitary tract

Responses of acutely isolated neurons from the rostral nucleus of the solitary tract (rNST) to GABA receptor agonists and antagonists were investigated using whole-cell recording in current clamp mode. The isolated neurons retain their morphology and can be divided into multipolar, elongate and ovoid cell types. Most rNST neurons (97%), including all three cell types, respond to GABA with membrane hyperpolarization and a reduction in input resistance. The GABA(A) receptor agonist muscimol reduces neuronal input resistance in a concentration-dependent manner, whereas the GABA(B) receptor agonist baclofen had no effect on any of the neurons tested. The GABA and muscimol reversal potentials were both found to be -75 mV Both the GABA competitive antagonist picrotoxin and the GABA(A) receptor antagonist bicuculline block the effect of GABA in a concentration-dependent manner. These results suggest that GABA activates all neurons in the rNST and that inhibitory synaptic activity is important in brainstem processing of gustatory and somatosensory information.      

Replacement of receptor cells in the hamster vomeronasal epithelium after nerve transection

Chemoreceptor cells in the vomeronasal and olfactory epithelium are replaced following experimentally induced degeneration. This study analyzes quantitatively the time course and degree of vomeronasal receptor cell replacement. Unilateral transection of the vomeronasal nerves in adult hamster was used to induce a retrograde degeneration of receptor cells in the vomeronasal organ. Histological measurement of both number of receptor cells and epithelial thickness were made for recovery times from 0 to 60 days. After nerve transection, there was a gradual degeneration of receptor cells, the number decreasing to 50% of control by day 2 and 16% by day 6. During days 7-15 maximum receptor cell replacement was observed. Cell number increased rapidly and reached a peak on day 15. At recovery times of 40-60 days, cell number returned to the control level. Epithelial thickness, however, decreased to 60-70% during the degeneration period (days 4-6) and did not return to control levels. After 40-60 days epithelial thickness remained at 70% of control. These results demonstrate that vomeronasal receptor cells are replaced following degeneration, but epithelial thickness does not return to control levels. These findings suggest that the number of replacement cells is not limited by the reduced thickness of the epithelium, and that recovery mechanisms may function to restore an optimum number of receptor cells.