Soft X-Ray Microscopy Radiation Damage On Fixed Cells Investigated With Synchrotron Radiation FTIR Microscopy.

Radiation damage of biological samples remains a limiting factor in high resolution X-ray microscopy (XRM). Several studies have attempted to evaluate the extent and the effects of radiation damage, proposing strategies to minimise or prevent it. The present work aims to assess the impact of soft X-rays on formalin fixed cells on a systematic manner. The novelty of this approach resides on investigating the radiation damage not only with XRM, as often reported in relevant literature on the topic, but by coupling it with two additional independent non-destructive microscopy methods: Atomic Force Microscopy (AFM) and FTIR Microscopy (FTIRM). Human Embryonic Kidney 293 cells were exposed to different radiation doses at 1 keV. In order to reveal possible morphological and biochemical changes, the irradiated cells were systematically analysed with AFM and FTIRM before and after. Results reveal that while cell morphology is not substantially affected, cellular biochemical profile changes significantly and progressively when increasing dose, resulting in a severe breakdown of the covalent bonding network. This information impacts most soft XRM studies on fixed cells and adds an in-depth understanding of the radiation damage for developing better prevention strategies.

Structure of 1-aminocyclopropane-1-carboxylate synthase, a key enzyme in the biosynthesis of the plant hormone ethylene.

The 2.4 A crystal structure of the vitamin B6-dependent enzyme 1-aminocyclopropane-1-carboxylate (ACC) synthase is described. This enzyme catalyses the committed step in the biosynthesis of ethylene, a plant hormone that is responsible for the initiation of fruit ripening and for regulating many other developmental processes. ACC synthase has 15 % sequence identity with the well-studied aspartate aminotransferase, and a completely different catalytic activity yet the overall folds and the active sites are very similar. The new structure together with available biochemical data enables a comparative mechanistic analysis that largely explains the catalytic roles of the conserved and non-conserved active site residues. An external aldimine reaction intermediate (external aldimine with ACC, i.e. with the product) has been modeled. The new structure provides a basis for the rational design of inhibitors with broad agricultural applications.

Crystal structure of GABA-aminotransferase, a target for antiepileptic drug therapy.

gamma-Aminobutyrate aminotransferase (GABA-AT), a pyridoxal phosphate-dependent enzyme, is responsible for the degradation of the inhibitory neurotransmitter GABA and is a target for antiepileptic drugs because its selective inhibition raises GABA concentrations in brain. The X-ray structure of pig GABA-AT has been determined to 3.0 A resolution by molecular replacement with the distantly related enzyme ornithine aminotransferase. Both omega-aminotransferases have the same fold, but exhibit side chain replacements in the closely packed binding site that explain their respective specificities. The aldimines of GABA and the antiepileptic drug vinyl-GABA have been modeled into the active site.

Crystallization and preliminary crystallographic investigations of rhodanese from Azotobacter vinelandii.

The rhdA gene identified in Azotobacter vinelandii codes for a protein, RhdA, which displays rhodanese (thiosulfate-cyanide sulfurtransferase) activity. RhdA was overexpressed and purified to homogeneity. The protein crystallized in the orthorhombic space group P2(1)2(1)2 with unit-cell parameters a = 44.4, b = 150.8, c = 53.8 A; on a synchrotron source the diffraction patterns could be collected to a resolution limit of 1.8 A. Evaluation of the crystal density indicates that the crystal lattice accommodates one molecule per asymmetric unit and that the solvent content is 59% of the total volume.

Crystal structure of human ornithine aminotransferase complexed with the highly specific and potent inhibitor 5-fluoromethylornithine.

Ornithine aminotransferase (l-ornithine:2-oxoacid delta-aminotransferase; EC 2.6.1.13), a pyridoxal-5'-phosphate-dependent mitochondrial enzyme controls the l-ornithine level in tissues by catalyzing the transfer of the delta-amino group of l-ornithine to 2-oxoglutarate, producing l-glutamate- gamma-semialdehyde and l-glutamate. (2S, 5S)-5-Fluoromethylornithine is the only inhibitor exclusively specific for ornithine aminotransferase known to date. Both in vitro and in vivo, it blocks the enzyme by a suicide reaction leading to a covalent adduct with the cofactor. The crystal structure of the enzyme-inhibitor complex was solved at a resolution of 1.95 A. No significant conformational changes compared with the native enzyme structure were observed. The structure reveals the atomic details of the cofactor-inhibitor adduct and its interactions with the active site of the enzyme. The main residues responsible for specific binding of the inhibitor are Arg180, which forms a strong salt bridge with the alpha-carboxylate and Tyr55, which is involved in a short hydrogen bond with the alpha-amino group. The experimental observation that in the racemic mixture, (2S, 5S)-5-fluoromethylornithine is exclusively responsible for the enzyme inhibition can be explained on the basis of the active site topology. Model building studies strongly suggest that the natural substrate l-ornithine, in its external aldimine adduct with the enzyme, makes use of the same recognition site as the inhibitor. It is proposed that the neutralization of the active site Arg413 by a salt bridge with Glu235 also plays an important role in productive binding of both 5-fluoromethylornithine and l-ornithine. Arg180 and Arg413 are believed to be instrumental in recognition of l-glutamate, by binding its gamma and alpha-carboxylate groups, respectively. This requires a different side-chain conformation of Glu235. Lys292 is the only obvious candidate for catalyzing the rate-limiting proton transfer steps in the transamination reaction.

Purification and structural characterization of bovine cathelicidins, precursors of antimicrobial peptides.

Cathelicidins are a novel family of antimicrobial peptide precursors from mammalian myeloid cells. They are characterized by a conserved N-terminal region while the C-terminal antimicrobial domain can vary considerably in both primary sequence and length. Four cathelicidins, proBac5, proBac7, prododecapeptide and proBMAP-28, have been concurrently purified from bovine neutrophils, using simple and rapid methodologies. The correlation of ES-MS data from the purified proteins with their cDNA-deduced sequences has revealed several common features of their primary sequence, such as the presence of N-terminal 5-oxoproline (pyroglutamate) residues and two disulfide bridges in a 1-2, 3-4 arrangement. The N-terminal domains of the cathelicidins present one or two Asp-Pro bonds, which are particularly acid-labile in proBac5 and proBac7, but stable in prododecapeptide. This suggests that the spatial organization around these bonds may vary in different cathelicidins, and favour hydrolysis in some cases. An unexpected feature of the prododecapeptide is that it exists as dimers formed by three possible combinations of its two isoforms. The isolation of a truncated, monomeric form of this protein, lacking the cysteine-containing antimicrobial dodecapeptide, indicates that dimerization occurs via disulfide bridge formation at the level of the C-terminal domain and that the dodecapeptide is likely released as a dimer from its precursor. Sequence-based secondary structure predictions and CD results indicate for cathelicidins a 30-50% content of extended conformation and <20% content of alpha-helical conformation, with the alpha-helical segment placed near the N-terminus. Finally, similarity searching and topology-based structure prediction underline a significant sequential and structural similarity between the conserved N-terminal domain of cathelicidins and cystatin-like domains, placing this family within the cystatin superfamily. When assayed against cathepsin L, unlike the potent cystatin inhibitors, three of the four cathelicidins show only a poor inhibitory activity (Ki = 0.6-3 microM).

PMAP-37, a novel antibacterial peptide from pig myeloid cells. cDNA cloning, chemical synthesis and activity.

A molecular biological approach, based on preproregion homology in the precursors of several diverse antibacterial peptides, was used to clone a pig bone marrow cDNA encoding a novel 167-residue polypeptide. The preproregion of this polypeptide is highly similar to corresponding regions in congeners from pig, cattle and rabbit. It is followed by a unique, cationic, 37-residue sequence, which was predicted to have a high propensity for an alpha-helical conformation. A peptide, termed PMAP-37, corresponding to this sequence, was chemically synthesized and shown to undergo a transition from a random coil to an ordered, mainly helical, conformation on addition of trifluoroethanol. This behaviour is typical of an amphipathic alpha helix, a structure common to several membrane-active, antimicrobial peptides. In vitro experiments showed that PMAP-37 strongly inhibits the growth of several strains of Gram-negative and Gram-positive bacteria, with minimal inhibitory concentrations ranging over 1-4 microM, and permeabilizes the inner membrane of Escherichia coli. Interestingly, the 15-32 stretch of PMAP-37 show a remarkable similarity to N-terminal stretches in cecropins B and A from Drosophila melanogaster and Cecropia hyalophora, respectively. This affords an uncommon example of sequence convergence.

Molecular cloning and chemical synthesis of a novel antibacterial peptide derived from pig myeloid cells.

A group of myeloid precursors of defense peptides has recently been shown to have highly homologous N-terminal regions. Using a strategy based on this homology, a novel cDNA was cloned from pig bone marrow RNA and found to encode a 153-residue polypeptide. This comprises a highly conserved region encompassing a 29-residue signal peptide and a 101-residue prosequence, followed by a unique, 23-residue, cationic, C-terminal sequence. A peptide corresponding to this C-terminal sequence was chemically synthesized and shown to exert antimicrobial activity against both Gram positive and negative bacteria at concentrations of 2-16 microM. The activity of this potent and structurally novel antibacterial peptide appears to be mediated by its ability to damage bacterial membranes, as shown by the rapid permeabilization of the inner membrane of Escherichia coli.

Chemical synthesis and biological activity of a novel antibacterial peptide deduced from a pig myeloid cDNA.

Several myeloid precursors of antibacterial peptides have recently been shown to share homologous pre- and pro-regions. Taking advantage of this homology, a novel cDNA was cloned from pig bone marrow RNA. This encodes a 166-residue polypeptide with highly conserved pre- (29 residues) and pro- (101 residues) sequences, followed by a unique, 36-residue C-terminal sequence. Structure analyses of this C-terminal region have identified a highly cationic sequence predicted to adopt an amphipathic alpha-helical conformation. A peptide corresponding to this sequence was chemically synthesized and shown to arrest the growth of both Gram-positive and Gram-negative bacteria. At least for Escherichia coli, the activity of this peptide appears to be mediated by its ability to permeabilize the bacterial membranes.

A cDNA derived from pig bone marrow cells predicts a sequence identical to the intestinal antibacterial peptide PR-39.

We have recently shown that a group of antimicrobial peptides of bovine neutrophils share highly identical pro-sequences. In this paper we report the cDNA sequence of a 172 amino acid residue pig myeloid protein showing a similar pro-sequence of 101 residues. The carboxyl-moiety of the predicted protein is identical to the mature form of the proline- and arginine-rich antibacterial peptide named PR-39, isolated from pig intestine.

A novel cDNA sequence encoding a pig leukocyte antimicrobial peptide with a cathelin-like pro-sequence.

It has recently been shown that the precursors of various structurally unrelated leukocyte antimicrobial peptides share similar pro-regions. These, in turn, are highly identical to a cysteine proteinase inhibitor named cathelin, or PLCPI. In this paper we report a novel cDNA sequence of porcine bone marrow origin, encoding a protein characterized by a cathelin-like domain. The putative protein is 147 amino acid residue long, with a calculated mass of 16479 Da and appears to be the precursor of a recently isolated antimicrobial peptide named protegrin PG-2. The unique sequence of the mature PG-2 is located at the C-terminus of the precursor. Similar to the previously reported precursors, both the signal peptide and the pro-sequence of pre-proPG-2 appear highly conserved.

The cDNA of the neutrophil antibiotic Bac5 predicts a pro-sequence homologous to a cysteine proteinase inhibitor that is common to other neutrophil antibiotics.

Bac5 is a 5-kDa proline- and arginine-rich antibiotic, stored as inactive precursor (proBac5) in the large granules of bovine neutrophils. A full-length cDNA encoding the precursor form of Bac5 has been cloned. The encoded protein (pre-proBac5) has a calculated mass of 20,031 Da and a pI of 9.21. This comprises a putative signal peptide of 29 amino acid residues and a 101-residue pro-sequence that precede the mature antibiotic. The pro-sequence is acidic and may neutralize the highly cationic Bac5, thus accounting for the inactivation of the antibiotic activity observed in in vitro experiments. The structure of mature Bac5 agrees closely with the amino acid sequence previously determined, with an additional tripeptide tail predicting carboxyl-terminal amidation. A valyl residue is deduced at the cleavage site for the proteolytic maturation of proBac5, consistent with a previous observation showing elastase as the enzyme involved in this processing step. The region upstream of Bac5 reveals high identity to corresponding regions of two neutrophil antimicrobial polypeptides, CAP18 from rabbit and bovine indolicidin. The COOH-terminal sequences of these antibiotics are completely unrelated. The proregion also exhibits remarkable similarity to pig cathelin, an inhibitor of cathepsin L, indicating a common evolutionary origin.

cDNA sequence analysis of an antibiotic dodecapeptide from neutrophils.

The full-length cDNA of a neutrophil antibiotic dodecapeptide has been cloned by reverse transcription/PCR from bovine bone marrow RNA. This peptide was originally isolated from bovine neutrophils, and shown to exert a potent antimicrobial activity in vitro on both Escherichia coli and Staphylococcus aureus. The cDNA codes for a polypeptide of 155 amino acid residues with a predicted mass of 17,629 Da and a pI of 8.03. The deduced sequence comprises a putative signal peptide of 29 amino acids, a 114 residue pro-region, and a carboxy-terminal dodecapeptide corresponding to the mature antibiotic. The pro-sequence displays extensive identity to corresponding regions of other structurally unrelated antibiotic peptides of bovine neutrophils recently cloned.

cDNA cloning of the neutrophil bactericidal peptide indolicidin.

A structurally novel, tryptophan-rich antimicrobial tridecapeptide amide, named indolicidin, has recently been purified from bovine neutrophils (Selsted et al. (1992) J. Biol. Chem. 267, 4292-4295). Here we describe the molecular cloning of this endoantibiotic, which is synthesised in bone marrow cells as a 144 amino acid residue precursor. The encoded protein has a predicted mass of 16479 Da and a pI of 6.51. A putative signal peptide of 29 amino acids precedes a 101 residue pro-region. The mature peptide is at the 3' end of the open reading frame. A glycine, not found in purified indolicidin, is present at the carboxyl terminus of the deduced sequence and is very likely involved in post-translational peptide amidation.