Haloarcula sebkhae sp. nov., an extremely halophilic archaeon from Algerian hypersaline environment.

A halophilic organism, SWO25T, was isolated from water sampled in Algeria at the salt lake (sebkha) of Ouargla. The novel strain stained Gram-negative, and cells were pleomorphic with a red pigmentation. Strain SWO25T grew optimally at 35-45 °C, at pH 6.0-8.0 and 0.05-0.25 M MgCl2 concentrations. Cells were extremely halophilic, with optimal growth at 4.3-5.1 M NaCl. The predominant membrane polar lipids were C20C20 glycerol diether derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate, phosphatidylglycerol sulfate, triglycosyl diether and diglycosyl diether. The major respiratory menaquinone component was MK-8. Cells were highly tolerant to the presence of decane and isooctane in the growth medium. Chemotaxonomic properties supported the assignment of strain SWO25T to the genus Haloarcula. The DNA G+C content was 61.1mol%. DNA-DNA hybridization and phylogenetic analyses of the 16S rRNA and rpoB' genes showed that strain SWO25T is distinct from known Haloarcula species. Based on phenotypic, chemotaxonomic, genotypic and phylogenetic data, we describe a novel species of the genus Haloarcula, for which the name Haloarculasebkhae sp. nov. is proposed. The type strain is SWO25T (=CIP 110583T=JCM 19018T).

A hemocyanin-derived antimicrobial peptide from the penaeid shrimp adopts an alpha-helical structure that specifically permeabilizes fungal membranes.

BACKGROUND: Hemocyanins are respiratory proteins with multiple functions. In diverse crustaceans hemocyanins can release histidine-rich antimicrobial peptides in response to microbial challenge. In penaeid shrimp, strictly antifungal peptides are released from the C-terminus of hemocyanins. METHODS: The three-dimensional structure of the antifungal peptide PvHCt from Litopenaeus vannamei was determined by NMR. Its mechanism of action against the shrimp pathogen Fusarium oxysporum was investigated using immunochemistry, fluorescence and transmission electron microscopy. RESULTS: PvHCt folded into an amphipathic α-helix in membrane-mimicking media and displayed a random conformation in aqueous environment. In contact with F. oxysporum, PvHCt bound massively to the surface of fungal hyphae without being imported into the cytoplasm. At minimal inhibitory concentrations, PvHCt made the fungal membrane permeable to SYTOX-green and fluorescent dextran beads of 4 kDa. Higher size beads could not enter the cytoplasm. Therefore, PvHCt likely creates local damages to the fungal membrane. While the fungal cell wall appeared preserved, gradual degeneration of the cytoplasm most often resulting in cell lysis was observed in fungal spores and hyphae. In the remaining fungal cells, PvHCt induced a protective response by the formation of daughter hyphae. CONCLUSION: The massive accumulation of PvHCt at the surface of fungal hyphae and subsequent insertion into the plasma membrane disrupt its integrity as a permeability barrier, leading to disruption of internal homeostasis and fungal death. GENERAL SIGNIFICANCE: The histidine-rich antimicrobial peptide PvHCt derived from shrimp hemocyanin is a strictly antifungal peptide, which adopts an amphipathic α-helical structure, and selectively binds to and permeabilizes fungal cells.

Halocin C8: an antimicrobial peptide distributed among four halophilic archaeal genera: Natrinema, Haloterrigena, Haloferax, and Halobacterium.

Halophilic archaea thrive in hypersaline ecosystems and produce antimicrobial peptides (AMPs) named halocins. AMPs are essential effectors of microbial interactions in natural ecosystems. Halocin C8 is a 7.4 kDa peptide produced by Natrinema sp. AS7092. Surrounded by genes involved in regulation and transport, the halC8 gene encodes a precursor, processed into the mature halocin and an immunity protein, protecting the producing strain against its halocin. This feature constitutes a unique property of halocin C8, as known AMPs and their immunity proteins are generally encoded on distinct ORFs in an operon. By complementary in silico and PCR-based approaches, the presence of halC8 in halophilic archaea collected from various parts of the world was evidenced. The full-length halC8 gene is restricted and consistently found in the genomes of strains belonging to the phylogenetically related genera Natrinema and Haloterrigena, along with transport and regulation genes. Functional expression of halC8 was demonstrated by RT-PCR and antimicrobial assays. Active halocin C8 was shown to contain five disulphide bridges, presumably conferring a compact structure resistant to harsh environmental conditions. In other archaeal genera, Haloferax and Halobacterium, genes encoding halocin C8 with diverging immunity protein moiety were evidenced. A phylogenetic analysis of halocin C8 sequences was conducted.

Baclofen Solution for Low-Volume Therapeutic Delivery.

OBJECTIVES: Baclofen is a zwitterion molecule where increased ions in the excipient increase the solubility. We developed baclofen in a stable solution similar to cerebrospinal fluid (CSF) without bicarbonate and proteins to improve the solubility of the baclofen and to reduce the potential toxicity to the central nervous system (CNS) and subarachnoid space. The objective is to develop a solution of baclofen wherein baclofen is solubilized in a multivalent physiological ion solution such as artificial cerebrospinal fluid (aCSF) at a concentration from 2 mg/cc to 10 mg/cc. METHODS: First, to determine the solubility of Baclofen in aCSF, solubility was determined at six different pH levels at 37°C, by the addition of aCSF to a known amount of Baclofen. The final concentrations were confirmed by high performance liquid chromatography (HPLC) analysis. Second, the stability of Baclofen at 4 mg/cc investigated in a test manufacturing batch utilizing standard methods of production of 1500 20 cc vials inverted for 18 months at 25°C at 60% humidity. The stability and purity of the baclofen was verified at 18 months by HPLC analysis. RESULTS: Baclofen was initially soluble between pH of 6-8 above 7 mg/cc but fell back to 6.3-5.8 mg/cc level with time. Baclofen produced in vials with inversion were noted to be stable at 4 mg/cc at 18 months with less than 2% breakdown of the baclofen in solution. CONCLUSION: Baclofen is much more soluble in artificial CSF than normal saline. The artificial CSF may also be less toxic to the subarachnoid space than saline.

Antagonistic interactions and production of halocin antimicrobial peptides among extremely halophilic prokaryotes isolated from the solar saltern of Sfax, Tunisia.

Thirty-five extremely halophilic microbial strains isolated from crystallizer (TS18) and non-crystallizer (M1) ponds in the Sfax solar saltern in Tunisia were examined for their ability to exert antimicrobial activity. Antagonistic assays resulted in the selection of eleven strains that displayed such antimicrobial activity and they were further characterized. Three cases of cross-domain inhibition (archaea/bacteria or bacteria/archaea) were observed. Four archaeal strains exerted antimicrobial activity against several other strains. Three strains, for which several lines of evidence suggested the antimicrobial activity was, at least in part, due to peptide/protein agents (Halobacterium salinarum ETD5, Hbt. salinarum ETD8, and Haloterrigena thermotolerans SS1R12), were studied further. Optimal culture conditions for growth and antimicrobial production were determined. Using DNA amplification with specific primers, sequencing and RT-PCR analysis, Hbt. salinarum ETD5 and Hbt. salinarum ETD8 were shown to encode and express halocin S8, a hydrophobic antimicrobial peptide targeting halophilic archaea. Although the gene encoding halocin H4 was amplified from the genome of Htg. thermotolerans SS1R12, no transcript could be detected and the antimicrobial activity was most likely due to multiple antimicrobial compounds. This is also the first report that points to four different strains isolated from different geographical locations with the capacity to produce identical halocin S8 proteins.

Isolation and characterization of environmental bacteria capable of extracellular biosorption of mercury.

Accumulation of toxic metals in the environment represents a public health and wildlife concern. Bacteria resistant to toxic metals constitute an attractive biomass for the development of systems to decontaminate soils, sediments, or waters. In particular, biosorption of metals within the bacterial cell wall or secreted extracellular polymeric substances (EPS) is an emerging process for the bioremediation of contaminated water. Here the isolation of bacteria from soil, effluents, and river sediments contaminated with toxic metals permitted the selection of seven bacterial isolates tolerant to mercury and associated with a mucoid phenotype indicative of the production of EPS. Inductively coupled plasma-optical emission spectroscopy and transmission electron microscopy in conjunction with X-ray energy dispersive spectrometry revealed that bacteria incubated in the presence of HgCl2 sequestered mercury extracellularly as spherical or amorphous deposits. Killed bacterial biomass incubated in the presence of HgCl2 also generated spherical extracellular mercury deposits, with a sequestration capacity (40 to 120 mg mercury per g [dry weight] of biomass) superior to that of live bacteria (1 to 2 mg mercury per g [dry weight] of biomass). The seven strains were shown to produce EPS, which were characterized by Fourier transform-infrared (FT-IR) spectroscopy and chemical analysis of neutral-carbohydrate, uronic acid, and protein contents. The results highlight the high potential of Hg-tolerant bacteria for applications in the bioremediation of mercury through biosorption onto the biomass surface or secreted EPS.

Intranasally applied human olfactory mucosa neural progenitor cells migrate to damaged brain regions.

Aim: To determine if intranasally administered olfactory mucosa progenitor cells (OMPCs) migrate to damaged areas of brain. Materials & methods: Rowett Nude (RNU) adult rats were injured using the Marmarou model then 2 weeks later received intranasally-delivered human OMPC. After 3 weeks, rats were sacrificed and brain sectioned. The mean distances from the human OMPCs to markers for degenerative neuronal cell bodies (p-c-Jun+), axonal swellings on damaged axons (ß-APP+) and random points in immunostained sections were quantified. One-way ANOVA was used to analyze data. Results: The human OMPCs were seen in specific areas of the brain near degenerating cell bodies and damaged axons. Conclusion: Intranasally delivered human OMPC selectively migrate to brain injury sites suggesting a possible noninvasive stem cell delivery for brain injury.

Phase IIB Randomized Trial on the Use of 4-Aminopyridine in Guillain-Barré Syndrome.

OBJECTIVE: To determine the safety and efficacy of orally delivered 4-aminopyridine (4-AP) in persons with Guillain-Barré Syndrome (GBS) >6 months from initial diagnosis. DESIGN: A randomized, double-blind, placebo-controlled, crossover study. SETTING: Tertiary care clinical outpatient program. PARTICIPANTS: Nineteen participants enrolled (14 male, 5 female; N=19), neurologic impairment secondary to GBS and functional loss on the FIM motor score (stable for ≥12mo) and >3.0 but <5.0 on the American Spinal Injury motor scale. Twelve participants (mean age, 59y; range, 23-77y) completed the study. INTERVENTIONS: A 4-AP dose-escalation study with 8 weeks in each period with a 3-week washout period, followed by 3 months open-label follow-up. MAIN OUTCOME MEASURES: FIM motor score was the primary outcome measure; also evaluated were the American Spinal Injury motor strength score (all limbs), handheld dynamometer, 6-minute walk test, Medical Outcomes Study 12-Item Short Form, Center for Epidemiological Studies Depression scale, Positive and Negative Affect Schedule, pain, GBS disability scale, Jepsen-Taylor Hand Function Test, Minnesota Manual Dexterity Test and Minnesota Rate of Manipulation Test, Get Up and Go Test, McGill Pain Inventory, Craig Handicap Assessment and Reporting Technique, and participant self-evaluation. RESULTS: Seven participants discontinued the study prematurely: 3 because of adverse events, 3 because of travel difficulties or relocation, and 1 because of pretreatment laboratory abnormalities. After removing 3 participants with maximum FIM scores, 4-AP arm trended superior to placebo (P=.065). Patients subjectively could always tell when they were on the active agent usually by tingling sensations or a sense of wellness. No statistically significant differences were found for other outcome measures although there were strong trends. CONCLUSIONS: This study demonstrates the safety of 4-AP in the patient population with GBS as the predominate goal of the study. A trend toward improved function after treatment was noted with most patients electing to stay on the medication after the trial.

Isolation and characterization of two members of the siderophore-microcin family, microcins M and H47.

In this paper we provide the first biochemical evidence of the existence of a family of structure-related antimicrobial peptides, the siderophore-microcins, in the Enterobacteriaceae family. We isolated and characterized two novel siderophore-microcins, MccM and MccH47, previously characterized through genetic studies. MccM and MccH47 were expressed from several Escherichia coli strains containing the microcin gene clusters. The spectra of their bactericidal activities were found to be restricted to some species of the Enterobacteriaceae. MccM and MccH47 were unable to inhibit the growth of strains carrying mutations in the fepA, cir, and fiu genes, which showed the requirement of the iron-catecholate receptors for their recognition. The MccM and MccH47 peptide moieties contain 77 and 60 residues, respectively, and are derived from the microcin precursors McmA and MchB, respectively. In addition, both peptides carried a C-terminal posttranslational modification containing a salmochelin-like siderophore moiety also found in MccE492 (X. Thomas et al., J. Biol. Chem., 279:28233-28242, 2004). Interestingly, when MccM was isolated from E. coli Nissle 1917, which lacks the two genes necessary for modification biosynthesis, it was devoid of posttranslational modification. Those two genes could be complemented by their homologues from the MccH47 gene cluster, thereby showing their functional interchangeability between at least two members of the siderophore-microcin family. Finally, from the sequence analysis of the MccE492 gene cluster, we hypothesized the existence of an additional member of the siderophore-microcin family. Therefore, we propose that the siderophore-microcin family contains five representatives.

Targeted ablation of mesenteric projecting sympathetic neurons reduces the hemodynamic response to pain in conscious, spinal cord-transected rats.

Individuals with spinal cord injuries above thoracic level 6 (T(6)) experience episodic bouts of life-threatening hypertension as part of a condition termed autonomic dysreflexia. The paroxysmal hypertension can be caused by a painful stimulus below the level of the injury. Targeted ablation of mesenteric projecting sympathetic neurons may reduce the severity of autonomic dysreflexia by reducing sympathetic activity. Therefore, cholera toxin B subunit (CTB) conjugated to saporin (SAP; a ribosomal inactivating protein that binds to and inactivates ribosomes) was injected into the celiac ganglion to test the hypothesis that targeted ablation of mesenteric projecting sympathetic neurons reduces the pressor response to pain in conscious, spinal cord-transected rats. Nine Sprague-Dawley male rats underwent a spinal cord transection between thoracic vertebrae 4 and 5. Following recovery (5 wk), all rats were instrumented with a radio telemetry device for recording arterial pressure and bilateral catheters in the gluteus maximus muscles for the infusion of hypertonic saline (hNa(+)Cl(-)). Subsequently, the hemodynamic responses to intramuscular injection of hNa(+)Cl(-) (100 microl and 250 microl, in random order) were determined. Following the experiments in the no celiac ganglia injected condition (NGI), rats received injections of CTB-SAP (n = 5) or CTB (n = 3) into the celiac ganglia. CTB-SAP rats, compared with NGI and CTB rats, had reduced pressor responses to hNa(+)Cl(-). Furthermore, the number of stained neurons in the celiac ganglia and spinal cord (segments T(6)-T(12)), was reduced in CTB-SAP rats. Thus, CTB-SAP retrogradely transported from the celiac ganglia is effective at ablating mesenteric projecting sympathetic neurons and reducing the pressor response to pain in spinal cord-transected rats.

Mechanical, topographical and chemical cues combined with physical therapy for peripheral nerve injuries.

Aim: The aim of this paper is to evaluate biomaterial cues combined with physical therapy (PT) on functional recovery in a rat sciatic nerve injury model. Materials & methods: Nerve growth conduits were filled with longitudinally aligned hyaluronic acid fibers and microspheres containing neurotrophic factor (growth factor [GF]). All animals received behavior and functional testing throughout the study, which concluded with measurement of compound muscle action potentials and contractile force of the gastrocnemius muscle. Results & conclusion: Including GF improved recovery of gross motor function and increased sensory pain sensation. During the 4 weeks that animals participated in PT, these groups showed higher static sciatic index scores. Including GF and PT has the potential to improve clinical outcomes following peripheral nerve injury.

The extremely halophilic archaeon Halobacterium salinarum ETD5 from the solar saltern of Sfax (Tunisia) produces multiple halocins.

The extremely halophilic archaeon Halobacterium salinarum strain ETD5 was previously isolated from the solar saltern of Sfax (Tunisia) and shown to encode and express halocin S8. The Hbt. salinarum ETD5 culture supernatant was shown here to exhibit high antimicrobial activity against several halophilic archaea and bacteria of different genera, showing a cross-domain inhibition. The antimicrobial activity was destroyed by proteases, thus pointing to halocins. A bioguided purification procedure was applied using two chromatography steps and antimicrobial assays directed against Halorubrum chaoviator ETR14. In-gel screening assay showed the presence of two antimicrobial bands of approximately 8 and 14 kDa, for which characterization was investigated by N-terminal sequencing and mass spectrometry. The full-length form of halocin S8 that contains 81 amino acids and differs from the 36 amino acid short-length halocin S8 previously described from an uncharacterized haloarchaeon S8a, was identified in the 8 kDa halocin band. A novel halocin that we termed halocin S14 was found in the 14 kDa band. It exhibits amino acid sequence identities with the N-terminally truncated region of the archaeal Mn-superoxide dismutase. These results show that Hbt. salinarum ETD5 produces multiple halocins, a feature that had not been described until now in the domain Archaea.

Two enzymes catalyze the maturation of a lasso peptide in Escherichia coli.

Microcin J25 (MccJ25) is a gene-encoded lasso peptide secreted by Escherichia coli which exerts a potent antibacterial activity by blocking RNA polymerase. Here we demonstrate that McjB and McjC, encoded by genes in the MccJ25 gene cluster, catalyze the maturation of MccJ25. Requirement for both McjB and McjC was shown by gene inactivation and complementation assays. Furthermore, the conversion of the linear precursor McjA into mature MccJ25 was obtained in vitro in the presence of McjB and McjC, all proteins being produced by recombinant expression in E. coli. Analysis of the amino acid sequences revealed that McjB could possess proteolytic activity, whereas McjC would be the ATP/Mg(2+)-dependent enzyme responsible for the formation of the Gly1-Glu8 amide bond. Finally, we show that putative lasso peptides are widespread among Proteobacteria and Actinobacteria.

Proteomics analysis of the nacre soluble and insoluble proteins from the oyster Pinctada margaritifera.

Shell nacre is laid upon an organic cell-free matrix, part of which, paradoxically, is water soluble and displays biological activities. Proteins in the native shell also constitute an insoluble network and offer a model for studying supramolecular organization as a means of self-ordering. Consequently, difficulties are encountered in extraction and purification strategies for protein characterization. In this work, water-soluble proteins and the insoluble conhiolin residue of the nacre of Pinctada margaritifera matrix were analyzed via a proteomics approach. Two sequences homologous to nacre matrix proteins of other Pinctada species were identified in the water-soluble extract. One of them is known as a fundamental component of the insoluble organic matrix of nacre. In the conchiolin, the insoluble residue, four homologs of Pinctada nacre matrix proteins were found. Two of them were the same as the molecules characterized in the water-soluble extract. Results established that soluble and insoluble proteins of the nacre organic matrix share constitutive material. Surprisingly, a peptide in the conchiolin residue was found homologous to a prismatic matrix protein of Pinctada fucata, suggesting that prismatic and nacre matrices may share common proteins. The insoluble properties of shell matrix proteins appear to arise from structural organization via multimerization. The oxidative activity, found in the water-soluble fraction of the nacre matrix, is proposed as a leading process in the transformation of transient soluble proteins into the insoluble network of conchiolin during nacre growth.

Functional recovery in rats with chronic spinal cord injuries after exposure to an enriched environment.

BACKGROUND/OBJECTIVE: The objective of this study was to determine the effect of environmental enrichment on the sensorimotor function of rats with chronic spinal cord injuries. DESIGN: Adult Sprague-Dawley rats received a contusive injury of moderate severity at vertebral level T8 using a weight-drop device. Three months after injury, 1 randomized group (n = 16) of rats was placed in an enriched environment, whereas the control group (n = 16) remained housed in standard laboratory cages (2/cage). METHODS: Animals were placed in an enriched environment for 4 weeks beginning at 3 months after injury. The enriched environment consisted of a large cage (5-6 rats/cage) with access to items such as tubes, ramps, and running wheel, with items changed daily. MAIN OUTCOME MEASURES: Functional evaluation consisted of the open field Basso, Beattie and Bresnahan (BBB) locomotor test and the tests that form the combined behavioral score (CBS). The CBS includes motor score, toe spread, placing, withdrawal, righting, inclined plane, hot plate, and swim tests. Behavioral testing was repeated 7 times before and after the period of intervention. RESULTS: The group placed in the enriched environment scored significantly better on the BBB (ANOVA repeated-measures, P < 0.01) test and CBS (ANOVA repeated-measures, P < 0.01). CONCLUSIONS: Environmental enrichment results in significant functional improvement in animals with spinal cord injury even with a substantial delay in initiating treatment after injury. The features of an enriched environment that may be responsible for the improvement include social interactions, exercise, and novel items in an interesting environment. These findings suggest a continued plasticity of the chronically injured rat spinal cord and a possible therapeutic intervention for people with spinal cord injury.

Identification of low molecular weight molecules as new components of the nacre organic matrix.

Nacre of Pinctada margaritifera displays a number of interesting biological activities on bone, mainly concentrated in a water-soluble organic matrix representing 0.24% of the nacre weight. Dialysis of that matrix through 8 kDa and 1 kDa cut-off membranes showed that 60% of it is made of small molecules of molecular masses below 1 kDa. Reversed-phase high-performance liquid chromatography of the small molecule fractions and subsequent electrospray ionization mass spectrometric analysis of 19 fractions thereof indicated the presence of at least 110 different molecules, in the range 100 Da-700 Da. Evidence for aggregate-forming properties of the small molecules was given. Amino acid analysis revealed that most of the small molecules were not peptides and tandem mass spectrometric gas-phase fragmentations clearly indicated a structural relationship between several molecules. Intriguingly, differences of a single Dalton between mono-charged ions peaks were observed. Further, approximately 40 analytes could be arranged in a ladder-like manner with mass spaces of 57 Da. Some of the water-soluble peptide sequences obtained after MS/MS fragmentation revealed that the 57 Da shift corresponds to the repetition of glycine residues. Furthermore, the exchange of glycine against alanine explains the 14 Da shift observed between some peptides. These data show for the first time that small molecules, especially peptides, are prevalent components of nacre. The molecular species described in this report might have a functional role in nacre.

The iron-siderophore transporter FhuA is the receptor for the antimicrobial peptide microcin J25: role of the microcin Val11-Pro16 beta-hairpin region in the recognition mechanism.

The role of the outer-membrane iron transporter FhuA as a potential receptor for the antimicrobial peptide MccJ25 (microcin J25) was studied through a series of in vivo and in vitro experiments. The requirement for both FhuA and the inner-membrane TonB-ExbB-ExbD complex was demonstrated by antibacterial assays using complementation of an fhuA(-) strain and by using isogenic strains mutated in genes encoding the protein complex respectively. In addition, MccJ25 was shown to block phage T5 infection of Escherichia coli, in vivo, by inhibiting phage adhesion, which suggested that MccJ25 prevents the interaction between the phage and its receptor FhuA. This in vivo activity was confirmed in vitro, as MccJ25 inhibited phage T5 DNA ejection triggered by purified FhuA. Direct interaction of MccJ25 with FhuA was demonstrated for the first time by size-exclusion chromatography and isothermal titration calorimetry. MccJ25 bound to FhuA with a 2:1 stoichiometry and a K(d) of 1.2 microM. Taken together, our results demonstrate that FhuA is the receptor for MccJ25 and that the ligand-receptor interaction may occur in the absence of other components of the bacterial membrane. Finally, both differential scanning calorimetry and antimicrobial assays showed that MccJ25 binding involves external loops of FhuA. Unlike native MccJ25, a thermolysin-cleaved MccJ25 variant was unable to bind to FhuA and failed to prevent phage T5 infection of E. coli. Therefore the Val11-Pro16 beta-hairpin region of MccJ25, which is disrupted upon cleavage by thermolysin, is required for microcin recognition.

Olfactory mucosa autografts in human spinal cord injury: a pilot clinical study.

BACKGROUND/OBJECTIVE: Olfactory mucosa is a readily accessible source of olfactory ensheathing and stem-like progenitor cells for neural repair. To determine the safety and feasibility of transplanting olfactory mucosa autografts into patients with traumatically injured spinal cords, a human pilot clinical study was conducted. METHODS: Seven patients ranging from 18 to 32 years of age (American Spinal Injury Association [ASIA] class A) were treated at 6 months to 6.5 years after injury. Olfactory mucosa autografts were transplanted into lesions ranging from 1 to 6 cm that were present at C4-T6 neurological levels. Operations were performed from July 2001 through March 2003. Magnetic resonance imaging (MRI), electromyography (EMG), and ASIA neurological and otolaryngological evaluations were performed before and after surgery. RESULTS: MRI studies revealed moderate to complete filling of the lesion sites. Two patients reported return of sensation in their bladders, and one of these patients regained voluntary contraction of anal sphincter. Two of the 7 ASIA A patients became ASIA C. Every patient had improvement in ASIA motor scores. The mean increase for the 3 subjects with tetraplegia in the upper extremities was 6.3 +/- 1.2 (SEM), and the mean increase for the 4 subjects with paraplegia in the lower extremities was 3.9 +/- 1.0. Among the patients who improved in their ASIA sensory neurological scores (all except one patient), the mean increase was 20.3 +/- 5.0 for light touch and 19.7 +/- 4.6 for pinprick. Most of the recovered sensation below the initial level of injury was impaired. Adverse events included sensory decrease in one patient that was most likely caused by difficulty in locating the lesion, and there were a few instances of transient pain that was relieved by medication. EMG revealed motor unit potential when the patient was asked to perform movement. CONCLUSION: This study shows that olfactory mucosa autograft transplantation into the human injured spinal cord is feasible, relatively safe, and potentially beneficial. The procedure involves risks generally associated with any surgical procedure. Long-term patient monitoring is necessary to rule out any delayed side effects and assess any further improvements.

Antimicrobial peptides and proteins in the face of extremes: Lessons from archaeocins.

Archaeocins are ribosomally-synthesized antimicrobial peptides or proteins produced by archaea. Halocins and sulfolobicins are produced by archaea belonging to the order Halobacteriales (Euryarchaeota) and Sulfolobales (Crenarchaeota), respectively. These weapons contribute helping the producer to prosper in spite of the microbial warfare. Given the fact that many archaea thrive in various extreme environments, archaeocins are challenged with inhospitable and destructive environmental conditions. Their structural features and mechanisms of action, which could be original, mostly remain to be deciphered. This review summarizes the present knowledge on halocins and sulfolobicins, the two classes of archaeocins that have been evidenced until now, and brings light on aspects of emerging research such as their ecological role or potential applications. Other antimicrobial compounds produced by archaea are also considered.

Microcin J25, from the macrocyclic to the lasso structure: implications for biosynthetic, evolutionary and biotechnological perspectives.

Microcin J25 (MccJ25) is a cyclic antibacterial peptide secreted by a fecal isolate of Escherichia coli. It exerts highly potent activity on Salmonella and Escherichia species. The microcin is recognized at the outer membrane of sensitive strains by the FhuA multifunctional protein, which belongs to the iron/siderophore receptor family, and inhibits bacterial transcription through binding to the RNA-polymerase beta' subunit. The mcjABCD genetic system carried by the wild type 50-kb pTUC100 plasmid contains four genes involved in MccJ25 production and immunity. MccJ25 results from the proteolytic cleavage of a 58-residue precursor at a specific Lys-Gly bond. The resulting mature peptide consists of 21 unmodified amino acids, mostly hydrophobic and includes a single dehydration. The initially described macrocyclic structure of MccJ25, which mostly relied on manual Edman sequencing of the thermolysin-cleaved form (t-MccJ25), involved a head-to-tail cyclisation of the 21-residue precursor. This structure did not prove to be consistent with recent IT-MS CID experiments conducted either on the native microcin or on peptides resulting from acidic or enzymatic cleavages, which are in favour of an 8-residue ring followed by a 13-residue tail. Cyclisation thus occurs between the N-terminus (Gly1) and the Glu8 side chain carboxyl group. The solution three-dimensional structure shows threading of the tail into the ring, thus forming a highly stable lasso type structure. Such a structure was described previously for enzyme inhibitors from Actinobacteria and is consistent with the ability of MccJ25 to inhibit RNA polymerase. The lasso structure is discussed in terms of phylogenetical and biotechnological perspectives.