Tyrosine-728 and glutamic acid-735 are essential for the metalloproteolytic activity of the lethal factor of Bacillus anthracis

The lethal factor (LF) of Bacillus anthracis is a Zn2+-endopeptidase specific for the MAPK-kinase family of proteins. The catalytic zinc atom is coordinated by a first shell of residues including the two histidines and the glutamate of the zinc-binding motif HExxH and by Glu-735. A characteristic feature of LF is the presence, within the second shell of residues, of a tyrosine (Tyr-728) in close proximity (3.3 A) to the zinc atom. To investigate the role of Tyr-728 and Glu-735, LF mutants with one or both of these two residues replaced by Ala were cloned, expressed, and purified from Escherichia coli. A fourth mutant was obtained by replacing Tyr-728 with Phe. Spectroscopic analysis of these mutants indicates that they fold in the same way as the parental molecule and that zinc stabilizes the structure of LF. These mutants have neither proteolytic activity nor in vivo toxicity. The possible role of Tyr-728 in catalysis is discussed.     

Birth of cloned pigs from zona-free nuclear transfer blastocysts developed in vitro before transfer

The objective of this work was to obtain cloned pig offspring by uterine transfer of blastocysts produced by zona-free manipulation. We started by defining the most suitable culture media for growing pig nuclear transfer embryos produced by zona-free micromanipulation comparing NCSU-23aa with Synthetic Oviduct Fluid (SOFaa) and with in vivo culture in the sheep oviduct. We found that parthenogenetic development to day 7 blastocyst in NCSU-23aa and sheep oviduct was significantly superior as compared to SOFaa (61.8%, 64% and 42.4 respectively) although blastocyst cell number was higher in the latter. Interestingly, when we compared the two media for the culture of nuclear transfer (NT) embryos derived from 3 different donor cell lines, we observed lower rates of development with NCSU-23aa (from 24.5% to 32.4%) while with SOFaa the development was significantly higher for two donor cell lines as compared to the third (44.4%, 48.9% and 20.6% respectively). A total of 244 blastocysts grown in SOFaa were transferred in four synchronized sows on day 5 or 6 of development. Two recipients farrowed 6 and 8 piglets corresponding to an efficiency of development to term of 8% and 16% of the transferred embryos respectively. Eleven pigs are now 10 month of age and those that have reached puberty have been proven to be fertile. Finally, this is the first report on the production of cloned pigs derived from the transfer of NT embryos at the blastocyst stage.     

Vertebral body innervation: Implications for pain

Vertebral fractures often cause intractable pain. To define the involvement of vertebral body innervation in pain, we collected specimens from male and female patients during percutaneous kyphoplasty, a procedure used for reconstruction of the vertebral body. Specimens were taken from 31 patients (9 men and 22 women) suffering high‐intensity pain before surgery. In total, 1,876 histological preparations were obtained and analysed. Immunohistochemical techniques were used to locate the nerves in the specimens. The nerve fibres were labelled by indirect immunofluorescence with the primary antibody directed against Protein Gene Product 9.5 (PGP 9.5), a pan‐neuronal marker; another primary antibody directed against type IV collagen (Col IV) was used to identify vessels and to determine their relationship with vertebral nerve fibres. The mean percentage of samples in which it was possible to identify nerve fibres was 35% in men and 29% in women. The percentages varied depending on the spinal level considered and the sex of the subject, nerve fibres being mostly present around vessels (95%). In conclusion, there is scarce innervation of the vertebral bodies, with a clear prevalence of fibres located around vessels. It seems unlikely that this pattern of vertebral body innervation is involved in vertebral pain or in pain relief following kyphoplasty. J. Cell. Physiol. 222: 488–491, 2010. © 2009 Wiley‐Liss, Inc.     

Neurogenesis during optic tectum regeneration in Xenopus laevis

The regenerative neurogenesis of the optic tectum of larval Xenopus laevis has been studied analyzing the proliferative and morphogenetic phases of the regeneration process after removal of one optic lobe. To this end, short‐term and long‐term pulses were carried out using the thymidine analog BrdU, selectively incorporated into cells during the S phase of the cell cycle. Results indicate that while in early larvae (stage 49/50, according to Nieuwkoop & Faber 1967 ) regeneration occurs mainly at the expense of the stem cells present in extensive proliferation zones (“matrix areas”) of the midbrain, in late larvae (stage 55/56) regeneration occurs at the expense of stem cells present in very limited matrix areas of the brain and of quiescent cells, which re‐enter the cell cycle following trauma. Moreover, in early larvae, morphogenesis of the optic tectum is carried out according to a precise spatio‐temporal order from rostro‐caudal to latero‐medial. By contrast, in late larvae, the topographical order of the regenerative morphogenesis of the optic lobe is completely altered. As a consequence, the regenerated optic tectum in early larvae has an apparently normal structure, while the regenerated optic tectum in late larvae lacks stratification.     

Synthesis, chiral HPLC resolution and configuration assignment of 1-phenylglyceryl trinitrate stereomers

As an introductory study of in vitro vasodilating activity, the access to the four stereomers of 1-phenylglyceryl trinitrate is described using achiral and chiral chromatography. For semi-preparative separation of the enantiomers, a Chiralcel OD (250 x 10 mm, 10 microm) was used. Catalytic reduction leading to the corresponding stereomers of 1-phenylglycerol allowed absolute configuration assignments. The same methods were used for the separation and configuration assignment of the enantiomers of 3-phenylpropane-1,2-diyl dinitrate.     

Annexin2 coating the surface of enlargeosomes is needed for their regulated exocytosis

Enlargeosomes are small cytoplasmic vesicles that undergo rapid, Ca2+-dependent exo/endocytosis. The role of the cytoskeleton in these processes was unknown. In PC12-27 cells, microtubule disassembly had little effect on enlargeosomes, whereas microfilament disassembly increased markedly both their resting and stimulated exocytosis, and inhibited their endocytosis. Even at rest enlargeosomes are coated at their cytosolic surface by an actin-associated protein, annexin2, bound by a dual, Ca2+-dependent and Ca2+-independent mechanism. In contrast, the other enlargeosome marker, desmoyokin/Ahnak, is transported across the organelle membrane, apparently by an ABC transporter, and binds to its lumenal face. Annexin2-GFP expression revealed that, upon stimulation, the slow and random enlargeosome movement increases markedly and becomes oriented toward the plasma membrane. After annexin2 downregulation enlargeosome exocytosis induced by both [Ca2+]i rise and cytoskeleton disruption is inhibited, and the NGF-induced differentiation is blocked. Binding of annexin2 to the enlargeosome membrane, the most extensive ever reported (>50% annexin2 bound to approximately 3% of total membrane area), seems therefore to participate in the regulation of their exocytosis.     

Identification and biochemical characterization of Rap2C, a new member of the Rap family of small GTP-binding proteins

The Rap family of small GTP-binding proteins is composed by four different members: Rap1A, Rap1B, Rap2A and Rap2B. In this work we report the identification and characterization of a fifth member of this family of small GTPases. This new protein is highly homologous to Rap2A and Rap2B, binds labeled GTP on nitrocellulose, and is recognized by a specific anti-Rap2 antibody, but not by an anti-Rap1 antibody. The protein has thus been named Rap2C. Binding of GTP to recombinant purified Rap2C was Mg(2+)-dependent. However, accurate comparison of the kinetics of nucleotide binding and release revealed that Rap2C bound GTP less efficiently and possessed slower rate of GDP release compared to the highly homologous Rap2B. Moreover, in the presence of Mg(2+), the relative affinity of Rap2C for GTP was only about twofold higher than that for GDP, while, under the same conditions, Rap2B was able to bind GTP with about sevenfold higher affinity than GDP. When expressed in eukaryotic cells, Rap2C localized at the plasma membrane, as dictated by the presence of a CAAX motif at the C-terminus. We found that Rap2C represented the predominant Rap2 protein expressed in circulating mononuclear leukocytes, but was not present in platelets. Importantly, Rap2C was found to be expressed in human megakaryocytes, suggesting that the protein may be down-regulated during platelets generation. This work demonstrates that Rap2C is a new member of the Rap2 subfamily of proteins, able to bind guanine nucleotides with peculiar properties, and differently expressed by various hematopoietic subsets. This new protein may therefore contribute to the still poorly clarified cellular events regulated by this subfamily of GTP-binding proteins.     

Reversible skeletal neuromuscular paralysis induced by different lysophospholipids

Lysophosphatidylcholine rapidly paralyses the neuromuscular junction (NMJ), similarly to snake phospholipase A2 neurotoxins, implicating a lipid hemifusion-pore transition in neuroexocytosis. The mode and kinetics of NMJ paralysis of different lysophospholipids (lysoPLs) in high or low [Mg2+] was investigated. The following order of potency was found: lysophosphatidylcholine>lysophosphatidylethanolamine>lysophosphatidic acid>lysophosphatidylserine>lysophosphatidylglycerol. The latter two lysoPLs closely mimic the profile of paralysis caused by the toxins in high [Mg2+]. This paralysis is fully reversed by albumin washing. These findings provide novel insights on the mode of action of snake neurotoxins and qualify lysoPLs as novel agents to study neuroexocytosis.     

Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase

FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl(2), as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8+/-1.3nmol of FAD synthesized/min/mg protein and exhibited a K(M) value for FMN of 1.5+/-0.3microM. This is the first report on characterization of human FADS, and the first cloning and over-expression of FADS from an organism higher than yeast.