Identification and characterisation of anti - Pseudomonas aeruginosa proteins in mucus of the brown garden snail, Cornu aspersum.

Background: Novel antimicrobial treatments are urgently needed. Previous work has shown that the mucus of the brown garden snail (Cornu aspersum) has antimicrobial properties, in particular against type culture collection strains of Pseudomonas aeruginosa. We hypothesised that it would also be effective against clinical isolates of the bacterium and that investigation of fractions of the mucus would identify one or more proteins with anti-pseudomonal properties, which could be further characterised. Materials and methods: Mucus was extracted from snails collected from the wild. Antimicrobial activity against laboratory and clinical isolates of Ps. aeruginosa was determined in disc diffusion assays. Mucus was purified using size exclusion chromatography and fractions containing anti-pseudomonal activity identified. Mass spectroscopy and high performance liquid chromatography analysis of these fractions yielded partial peptide sequences. These were used to interrogate an RNA transcriptome generated from whole snails. Results: Mucus from C. aspersum inhibited growth of type collection strains and clinical isolates of Ps. aeruginosa. Four novel C. aspersum proteins were identified; at least three are likely to have antimicrobial properties. The most interesting is a 37.4 kDa protein whilst smaller proteins, one 17.5 kDa and one 18.6 kDa also appear to have activity against Ps. aeruginosa. Conclusions: The study has identified novel proteins with antimicrobial properties which could be used to develop treatments for use in human medicine.

Subacute treatment with vascular endothelial growth factor after traumatic brain injury increases angiogenesis and gliogenesis.

Vascular endothelial growth factor (VEGF) is neuroprotective and induces neurogenesis and angiogenesis when given early after traumatic brain injury (TBI). However, the effects of VEGF administration in the subacute phase after TBI remain unknown. Mice were subjected to TBI and treated with vehicle or VEGF beginning 7 days later for an additional 7 days. The animals were injected with BrdU to label proliferating cells and examined with a motor-sensory scale at pre-determined time points. Mice were killed 90 days post injury and immunohistochemistry was used to study cell fates. Our results demonstrate that lesion volumes did not differ between the groups confirming the lack of neuroprotective effects in this paradigm. VEGF treatment led to significant increments in cell proliferation (1.9 fold increase vs. vehicle, P<0.0001) and angiogenesis in the lesioned cortex (1.7 fold increase vs. vehicle, P=0.0001) but most of the proliferating cells differentiated into glia and no mature newly-generated neurons were detected. In conclusion, VEGF induces gliogenesis and angiogenesis when given 7 days post TBI. However, treated mice had only insignificant motor improvements in this paradigm, suggesting that the bulk of the beneficial effects observed when VEGF is given early after TBI results from the neuroprotective effects.

Rapamycin is a neuroprotective treatment for traumatic brain injury.

The mammalian target of rapamycin, commonly known as mTOR, is a serine/threonine kinase that regulates translation and cell division. mTOR integrates input from multiple upstream signals, including growth factors and nutrients to regulate protein synthesis. Inhibition of mTOR leads to cell cycle arrest, inhibition of cell proliferation, immunosuppression and induction of autophagy. Autophagy, a bulk degradation of sub-cellular constituents, is a process that keeps the balance between protein synthesis and protein degradation and is induced upon amino acids deprivation. Rapamycin, mTOR signaling inhibitor, mimics amino acid and, to some extent, growth factor deprivation. In the present study we examined the effect of rapamycin, on the outcome of mice after brain injury. Our results demonstrate that rapamycin injection 4 h following closed head injury significantly improved functional recovery as manifested by changes in the Neurological Severity Score, a neurobehavioral testing. To verify the activity of the injected rapamycin, we demonstrated that it inhibits p70S6K phosphorylation, reduces microglia/macrophages activation and increases the number of surviving neurons at the site of injury. We therefore suggest that rapamycin is neuroprotective following traumatic brain injury and as a drug used in the clinic for other indications, we propose that further studies on rapamycin should be conducted in order to consider it as a novel therapy for traumatic brain injury.

Increase in peripheral benzodiazepine receptors and loss of glutamate NMDA receptors in a mouse model of closed head injury: a quantitative autoradiographic study.

Increases in peripheral type benzodiazepine receptors (PTBR) have been utilized for the detection of neuroinflammation and neurotoxicity in the brain. We have investigated the relationship between PTBR and NMDA receptor binding density in mice with closed head injury (CHI) using quantitative autoradiography. CHI was induced by a weight drop in nine mice, four of which received a single injection of the rat sarcoma (Ras) inhibitor famesyl thiosalicylate (FTS) 1 h after the insult. Sham controls received anesthesia but no contusion. The neurological status of the mice was evaluated at 1 h, and hence up to 7 days using a neurological severity score (NSS). Animals were killed 7 days after CHI and consecutive brain sections were incubated with [3H]PK11195, a PTBR antagonist, or [3H]MK801, an n-methyl-d-aspartate receptor (NMDAR) use-dependent antagonist. CHI produced large (two- to threefold), widespread increases in PK11195 binding in the traumatized hemisphere and a significant decrease (20%-40%) in NMDAR binding limited to regions at close proximity to the lesion. Histologically, these regions were characterized by glial proliferation and neuronal loss. Significant increases in PTBR binding, but no concomitant decrease in NMDAR, were identified in several regions remote from the lesion, including the contralateral ventrolateral striatum and the ipsilateral ventral thalamus. Drug treatment significantly improved the neurological deficits but had only a marginal effect on PTBR. These results support a complex role for glial activation and PTBR increases in the context of CHI.

Solution structure, hydrodynamics and thermodynamics of the UvrB C-terminal domain.

The solution structure, thermodynamic stability and hydrodynamic properties of the 55-residue C-terminal domain of UvrB that interacts with UvrC during excision repair in E. coli have been determined using a combination of high resolution NMR, ultracentrifugation, 15N NMR relaxation, gel permeation, NMR diffusion, circular dichroism and differential scanning calorimetry. The subunit molecular weight is 7,438 kDa., compared with 14.5+/-1.0 kDa. determined by equilibrium sedimentation, indicating a dimeric structure. The structure determined from NMR showed a stable dimer of anti-parallel helical hairpins that associate in an unusual manner, with a small and hydrophobic interface. The Stokes radius of the protein decreases from a high plateau value (ca. 22 A) at protein concentrations greater than 4 microM to about 18 A at concentrations less than 0.1 microM. The concentration and temperature-dependence of the far UV circular dichroism show that the protein is thermally stable (Tm ca. 71.5 degrees C at 36 microM). The simplest model consistent with these data was a dimer dissociating into folded monomers that then unfolds co-operatively. The van't Hoff enthalpy and dissociation constant for both transition was derived by fitting, with deltaH1=23 kJ mol(-1). K1(298)=0.4 microM and deltaH2= 184 kJ mol(-1). This is in good agreement with direct calorimetric analysis of the thermal unfolding of the protein, which gave a calorimetric enthalpy change of 181 kJ mol(-1) and a van't Hoff enthalpy change of 354 kJ mol(-1), confirming the dimer to monomer unfolding. The thermodynamic data can be reconciled with the observed mode of dimerisation. 15N NMR relaxation measurements at 14.1 T and 11.75 T confirmed that the protein behaves as an asymmetric dimer at mM concentrations, with a flexible N-terminal linker for attachment to the remainder of the UvrB protein. The role of dimerisation of this domain in the excision repair mechanism is discussed.

Crystal structure of Escherichia coli UvrB C-terminal domain, and a model for UvrB-uvrC interaction.

A crystal structure of the C-terminal domain of Escherichia coli UvrB (UvrB') has been solved to 3.0 A resolution. The domain adopts a helix-loop-helix fold which is stabilised by the packing of hydrophobic side-chains between helices. From the UvrB' fold, a model for a domain of UvrC (UvrC') that has high sequence homology with UvrB' has been made. In the crystal, a dimerisation of UvrB domains is seen involving specific hydrophobic and salt bridge interactions between residues in and close to the loop region of the domain. It is proposed that a homologous mode of interaction may occur between UvrB and UvrC. This interaction is likely to be flexible, potentially spanning > 50 A.

NMR assignments and secondary structure of the UvrC binding domain of UvrB.

The 55 residue C-terminal domain of UvrB that interacts with UvrC during excision repair in Escherichia coli has been expressed and purified as a (His)6 fusion construct. The fragment forms a stable folded domain in solution. Heteronuclear NMR experiments were used to obtain extensive 15N, 13C and 1H NMR assignments. NOESY and chemical shift data showed that the protein comprises two helices from residues 630 to 648 and from 652 to 670. 15N relaxation data also show that the first 11 and last three residues are unstructured. The effective rotational correlation time within the structured region is not consistent with a monomer. This oligomerisation may be relevant to the mode of dimerisation of UvrB with the homologous domain of UvrC.