Affinity purification of insoluble recombinant fusion proteins containing glutathione-S-transferase

Prokaryotic expression of polypeptides as fusion proteins with glutathione-S-transferase has recently been reported as a one-step means of purifying recombinant protein. The usefulness of the glutathione-S-transferase/glutathioneagarose system, however, is significantly limited by the frequent synthesis of recombinant proteins in insuluble form by Escherichia coli. We have found that for 5 separate fusion proteins containing glutathione-S-transferase and different domains of the large cystic fibrosis transmembrane conductance regulator, all were packaged in insoluble form by E. coli. Insolubility of these products made them inaccessible to one-step purification utilizing this scheme requires proper folding of recombinant glutathione-S-transferase to allow recognition on glutathione affinity agarose, we investigated the suitability of several alternative approaches for converting insoluble recombinant fusion proteins to a soluble form amenable to glutathione-agarose affinity purification. Low-temperature induction of fusion protein synthesis, but not incubation with anion-exchange resins, led to improved one-step purification of glutathione-S-transferase fusion proteins from E. coli cell lysate using mild, nondenaturing conditions. Solubilization in 8 mol/L urea, but not with other chaotropic agents or detergents, also allowed preparative yields of affinity-purified fusion protein. These techniques increase the usefulness of this recombinant protein purification scheme, and should be broadly applicable to diverse polypeptides synthesized as fusions with glutathione-S-transferase.     

Recombinant synthesis, purification, and nucleotide binding characteristics of the first nucleotide binding domain of the cystic fibrosis gene product.

The majority of mutations which lead to clinical cystic fibrosis are located within the two predicted nucleotide binding domains of the cystic fibrosis gene product. We have used a prokaryotic expression system to synthesize and purify the first nucleotide binding domain (NBD-1, amino acids 426-588) with and without the most common mutation associated with the disease (the deletion of phenylalanine at position 508, delta F508). Both wild type and delta F508 NBD-1 bind ATP-agarose in a quantitatively comparable manner; this binding was inhibited by excess Na2ATP, trinitrophenol-ATP, or 8-azido-ATP. Irreversible NBD-1 labeling by an ATP analog was demonstrated using [32P]8-azido-ATP. This covalent labeling was inhibited by preincubation with Na2ATP, with half-maximal inhibition for Na2ATP occurring at approximately 5 mM for both the wild type and delta F508 nucleotide binding domain. These experiments are among the first to confirm the expectation that the cystic fibrosis transmembrane conductance regulator NBD-1 binds nucleotide. Since, under the conditions used in our study, NBD-1 without phenylalanine 508 displays very similar nucleotide binding characteristics to the wild type protein, our results support previous structural models which predict that the delta F508 mutation should not cause an alteration in ATP binding.     

New Onset of Constipation during Long-Term Physical Inactivity: A Proof-of-Concept Study on the Immobility-Induced Bowel Changes

Background

The pathophysiological mechanisms underlining constipation are incompletely understood, but prolonged bed rest is commonly considered a relevant determinant.

Aims

Our primary aim was to study the effect of long-term physical inactivity on determining a new onset of constipation. Secondary aim were the evaluation of changes in stool frequency, bowel function and symptoms induced by this prolonged physical inactivity.

Methods

Ten healthy men underwent a 7-day run-in followed by 35-day study of experimentally-controlled bed rest. The study was sponsored by the Italian Space Agency. The onset of constipation was evaluated according to Rome III criteria for functional constipation. Abdominal bloating, flatulence, pain and urgency were assessed by a 100mm Visual Analog Scales and bowel function by adjectival scales (Bristol Stool Form Scale, ease of passage of stool and sense of incomplete evacuation). Daily measurements of bowel movements was summarized on a weekly score. Pre and post bed rest Quality of Life (SF-36), general health (Goldberg’s General Health) and depression mood (Zung scale) questionnaires were administered.

Results

New onset of functional constipation fulfilling Rome III criteria was found in 60% (6/10) of participants (p=0.03). The score of flatulence significantly increased whilst the stool frequency significantly decreased during the week-by-week comparisons period (repeated-measures ANOVA, p=0.02 and p=0.001, respectively). Stool consistency and bowel symptoms were not influenced by prolonged physical inactivity. In addition, no significant changes were observed in general health, in mood state and in quality of life at the end of bed rest

Conclusions

Our results provide evidence that prolonged physical inactivity is relevant etiology in functional constipation in healthy individuals. The common clinical suggestion of early mobilization in bedridden patients is supported as well.     

Matrix-M™ adjuvation broadens protection induced by seasonal trivalent virosomal influenza vaccine

Background

Influenza virus infections are responsible for significant morbidity worldwide and therefore it remains a high priority to develop more broadly protective vaccines. Adjuvation of current seasonal influenza vaccines has the potential to achieve this goal.

Methods

To assess the immune potentiating properties of Matrix-M?, mice were immunized with virosomal trivalent seasonal vaccine adjuvated with Matrix-M?. Serum samples were isolated to determine the hemagglutination inhibiting (HAI) antibody titers against vaccine homologous and heterologous strains. Furthermore, we assess whether adjuvation with Matrix-M? broadens the protective efficacy of the virosomal trivalent seasonal vaccine against vaccine homologous and heterologous influenza viruses.

Results

Matrix-M? adjuvation enhanced HAI antibody titers and protection against vaccine homologous strains. Interestingly, Matrix-M? adjuvation also resulted in HAI antibody titers against heterologous influenza B strains, but not against the tested influenza A strains. Even though the protection against heterologous influenza A was induced by the adjuvated vaccine, in the absence of HAI titers the protection was accompanied by severe clinical scores and body weight loss. In contrast, in the presence of heterologous HAI titers full protection against the heterologous influenza B strain without any disease symptoms was obtained.

Conclusion

The results of this study emphasize the promising potential of a Matrix-M?-adjuvated seasonal trivalent virosomal influenza vaccine. Adjuvation of trivalent virosomal vaccine does not only enhance homologous protection, but in addition induces protection against heterologous strains and thus provides overall more potent and broad protective immunity.

     

The Effect of a Spinal Cord Hemisection on Changes in Nitric Oxide Synthase Pools in the Site of Injury and in Regions Located Far Away from the Injured Site

1. The present study was designed to examine the nitric oxide synthase activities (constitutive and inducible) in the site of injury in response to Th10-Th11 spinal cord hemisection and, to determine whether unilateral disconnection of the spinal cord influences the NOS pools on the contra- and ipsilateral sides in segments located far away from the epicentre of injury.2. A radioassay detection was used to determine Ca²⁺-dependent and inducible nitric oxide synthase activities. Somal, axonal and neuropil neuronal nitric oxide synthase was assessed by immunocytochemical study. A quantitative assessment of neuronal nitric oxide synthase immunoreactivity was made by an image analyser. The level of neuronal nitric oxide synthase protein was measured by the Western blot analysis.3. Our data show the increase of inducible nitric oxide synthase activity and a decrease of Ca²⁺-dependent nitric oxide synthase activity in the injured site analysed 1 and 7 days after surgery. In segments remote from the epicentre of injury the inducible nitric oxide synthase activity was increased at both time points. Ca²⁺-dependent nitric oxide synthase activity had decreased in L5-S1 segments in a group of animals surviving for 7 days. A hemisection performed at thoracic level did not cause significant difference in the nitric oxide synthase activities and in the level of neuronal nitric oxide synthase protein between the contra- and ipsilateral sides in C6-Th1 and L5-S1 segments taken as a whole. Significant differences were observed, but only when the spinal cord was analysed segment by segment, and/or was divided into dorsal and ventral parts. The cell counts in the cervicothoracic (C7-Th1) and lumbosacral (L5-S1) enlargements revealed changes in neuronal nitric oxide synthase immunoreactivity on the ipsilateral side of the injury. The densitometric area measurements confirmed the reduction of somal, neuropil and axonal neuronal nitric oxide synthase immunoreactive staining in the ventral part of rostrally oriented segments.4. Our findings provide evidence that the changes in nitric oxide synthase pools are limited not only to impact zone, but spread outside the original lesion. The regional distribution of nitric oxide synthase activity and neuronal nitric oxide synthase immunoreactivity, measured segment by segment shows that nitric oxide may play a significant role in the stepping cycle in the quadrupeds.     

Up-regulation of myocardial connexin-43 in spontaneously hypertensive rats fed red palm oil is most likely implicated in its anti-arrhythmic effects

The purpose of this study was to test our hypothesis that red palm oil (RPO) intake may affect abnormalities of myocardial connexin-43 (Cx43) and protein kinase Cε (PKCε) signaling, and consequently the propensity of the spontaneously hypertensive rat heart (SHR) heart to arrhythmias. SHR and Wistar-Kyoto (WKY) rats fed a standard rat chow plus red palm oil (200?μL/day) for 5?weeks were compared with untreated rats. Cytosolic but not particulate PKCε expression as well as Cx43-mRNA, total Cx43 proteins, and its phoshorylated forms were increased, and disordered localization of Cx43 was attenuated in the left ventricle of RPO-fed SHR compared with untreated rats. These alterations were associated with suppression of early post-ischemic-reperfusion-related ventricular tachycardia and electrically inducible ventricular fibrillation. However, the treatment dose of RPO caused down-regulation of myocardial Cx43, but did not alter its cell membrane distribution or overall PKCε expression in WKY rats. It was, however, associated with poor arrhythmia protection, suggesting overdosing. Results indicate that SHR benefit from RPO intake, particularly because of its apparent anti-arrhythmic effects. This protection can be, in part, attributed to the preservation of cell-to-cell communication via up-regulation of myocardial Cx43, but not with PKCε activation.     

Seismic communication signals in the blind mole-rat (Spalax ehrenbergi ): electrophysiological and behavioral evidence for their processing by the auditory system

Based on morphological and behavioral findings we suggest that the seismic vibratory signals that blind mole-rats (Spalax ehrenbergi) use for intraspecific communication are picked up from the substrate by bone conduction and processed by the auditory system. An alternative hypothesis, raised by others, suggest that these signals are processed by the somatosensory system. We show here that brain stem and middle latency responses evoked by vibrations are similar to those evoked by high-intensity airborne clicks but are larger in their amplitudes, especially when the lower jaw is in close contact with the vibrating substrate. Bilateral deafening of the mole-rat or high-intensity masking noise almost completely eliminated these responses. Deafening also gradually reduced head-drumming behavior until its complete elimination about 4–6 weeks after surgery. Successive vibrations, at a rate of 0.5 vibrations/s, elicited prominent responses. At rates higher than 2 vibrations/s the amplitude of the brain stem response did not change, yet the middle latency response disappeared almost completely. It is concluded that the seismic signals that mole rats use for long distance communication are indeed processed primarily by the auditory system. Accepted: 11 May 1998