Corneal Perforation Repair Using a Novel Lyophilized Amniotic Membrane Graft Technique: Plug and Patch.

BACKGROUND The use of amniotic membranes for corneal perforations using different surgical techniques has been widely described in the literature. This case report is a novel variation in the technique that can be useful for incorporating in clinical practice when the need arises. CASE REPORT A 36-year-old male patient presented to our clinic with a corneal ulcer in his left eye caused by herpetic keratitis, treated with a topical non-steroidal anti-inflammatory (indomethacin 0.1% solution). Examination revealed a paracentral 2-mm wide corneal perforation on the site of the corneal ulcer. The patient was admitted to the hospital. He was treated with intravenous piperacillin-ofloxacine, and an emergency surgical intervention using a lyophilized amniotic membrane was performed using a "plug and patch" technique. Postoperatively, the patient received 48 h of intravenous antibiotics and was discharged on topical antibiotic/corticosteroid eyedrops along with a 10-day course of oral antibiotics (ofloxacin) and antiviral therapy (valaciclovir). Three months after surgery, the anterior chamber was formed, the corneal defect was closed, and visual acuity improved. One year after initial presentation, anterior segment optical coherence tomography showed a large scarred but healed cornea. CONCLUSIONS We report the successful use of combination of a single round-shaped rolled amniotic membrane with a multilayered amniotic membrane transplantation for the treatment of a 2-mm-wide perforated corneal ulcer. This technique allowed for preservation of the globe integrity without the need for a keratoplasty, stopped further tissue loss, and was associated with a rapid visual recovery.

[Behçet's disease : Description and analysis of a French single-center retrospective study of 51 patients]. / Maladie de Behçet : description et analyse d'une série rétrospective monocentrique française de 51 patients.

PURPOSE: To evaluate if the presence of uveitis in Behçet's disease (BD) is associated with a particular clinical phenotype and to analyze the prognostic impact of a missed diagnosis of BD at the time the uveitis is diagnosed. MATERIEL AND METHODS: Ophthalmologic and systemic clinical features of 51 patients with BD were recorded retrospectively. We compared the clinical phenotype of patients with ocular manifestations with those without ocular manifestations. The patients were divided into two groups depending on the progression of their visual acuity: "decreased visual acuity" versus "stable or improved visual acuity." RESULTS: In the group of patients with ocular involvement, there was a mean 2.3 systemic manifestations, vs. 3.2 in the group without ocular manifestations (P=0.004). When BD was diagnosed prior to the onset of uveitis, we counted fewer patients in the "decreased visual acuity" group in comparison with the patients who had no prior diagnosis of BD at the onset of the uveitis (91.3% in the "decreased visual acuity" group, P=0.04). The time before initiation of immunosuppressive treatment or a biological agent was shorter for these patients (4.4 vs. 39.3 months, P=0.007). CONCLUSION: It appears that different phenotypes exist according to whether or not the BD patient has ocular involvement. Moreover, the visual prognosis is better if the uveitis occurs in patients who have already been diagnosed with BD, due to earlier initiation of immunosuppressive therapy.

Secondary Corneal Myxoma After DMEK (Descemet Membrane Endothelial Keratoplasty).

BACKGROUND This is a clinical case of secondary corneal myxoma, which developed 18 months after Descemet membrane endothelial keratoplasty (DMEK). The DMEK was performed to treat viral endotheliitis and, postoperatively, a diagnosis of Crohn's disease was made. CASE REPORT A 52-year-old male, with no prior clinical history, presented with an endotheliitis in the left eye. The hypothesis of an undetected herpes infection was favored, and an antiviral treatment was prescribed using valacyclovir (1 g orally, 3 times daily). After 3 months of antiviral treatment, the endotheliitis was successfully controlled and a combined intervention of DMEK endothelial graft and phacoemulsification was performed. A corneal tumor was found 18 months after a successful DMEK procedure and was surgically removed. A pathological examination revealed a secondary corneal myxoma. CONCLUSIONS Corneal myxomas are rare lesions, often secondary to trauma in the Bowman's membrane, which is why it is often called a myxomatous corneal degeneration. In this instance, there is no link with Carney complex. However, myxomas under the eyelid or within the orbit are often associated with cardiac myxomas. Treatment is strictly surgical, either by simple excision or by surgical excision followed by graft. To the best of our knowledge, this is the first time that such an association between DMEK and secondary corneal myxoma has been described in the literature.

Draft Genome Sequence of Acetobacter aceti Strain 1023, a Vinegar Factory Isolate.

The genome sequence of Acetobacter aceti 1023, an acetic acid bacterium adapted to traditional vinegar fermentation, comprises 3.0 Mb (chromosome plus plasmids). A. aceti 1023 is closely related to the cocoa fermenter Acetobacter pasteurianus 386B but possesses many additional insertion sequence elements.

Formyl-coenzyme A (CoA):oxalate CoA-transferase from the acidophile Acetobacter aceti has a distinctive electrostatic surface and inherent acid stability.

Bacterial formyl-CoA:oxalate CoA-transferase (FCOCT) and oxalyl-CoA decarboxylase work in tandem to perform a proton-consuming decarboxylation that has been suggested to have a role in generalized acid resistance. FCOCT is the product of uctB in the acidophilic acetic acid bacterium Acetobacter aceti. As expected for an acid-resistance factor, UctB remains folded at the low pH values encountered in the A. aceti cytoplasm. A comparison of crystal structures of FCOCTs and related proteins revealed few features in UctB that would distinguish it from nonacidophilic proteins and thereby account for its acid stability properties, other than a strikingly featureless electrostatic surface. The apparently neutral surface is a result of a "speckled" charge decoration, in which charged surface residues are surrounded by compensating charges but do not form salt bridges. A quantitative comparison among orthologs identified a pattern of residue substitution in UctB that may be a consequence of selection for protein stability by constant exposure to acetic acid. We suggest that this surface charge pattern, which is a distinctive feature of A. aceti proteins, creates a stabilizing electrostatic network without stiffening the protein or compromising protein-solvent interactions.

Structural basis and evolution of redox regulation in plant adenosine-5'-phosphosulfate kinase.

Adenosine-5'-phosphosulfate (APS) kinase (APSK) catalyzes the phosphorylation of APS to 3'-phospho-APS (PAPS). In Arabidopsis thaliana, APSK is essential for reproductive viability and competes with APS reductase to partition sulfate between the primary and secondary branches of the sulfur assimilatory pathway; however, the biochemical regulation of APSK is poorly understood. The 1.8-Å resolution crystal structure of APSR from A. thaliana (AtAPSK) in complex with ß,γ-imidoadenosine-5'-triphosphate, Mg(2+), and APS provides a view of the Michaelis complex for this enzyme and reveals the presence of an intersubunit disulfide bond between Cys86 and Cys119. Functional analysis of AtAPSK demonstrates that reduction of Cys86-Cys119 resulted in a 17-fold higher k(cat)/K(m) and a 15-fold increase in K(i) for substrate inhibition by APS compared with the oxidized enzyme. The C86A/C119A mutant was kinetically similar to the reduced WT enzyme. Gel- and activity-based titrations indicate that the midpoint potential of the disulfide in AtAPSK is comparable to that observed in APS reductase. Both cysteines are invariant among the APSK from plants, but not other organisms, which suggests redox-control as a unique regulatory feature of the plant APSK. Based on structural, functional, and sequence analyses, we propose that the redox-sensitive APSK evolved after bifurcation of the sulfur assimilatory pathway in the green plant lineage and that changes in redox environment resulting from oxidative stresses may affect partitioning of APS into the primary and secondary thiol metabolic routes by having opposing effects on APSK and APS reductase in plants.

A specialized citric acid cycle requiring succinyl-coenzyme A (CoA):acetate CoA-transferase (AarC) confers acetic acid resistance on the acidophile Acetobacter aceti.

Microbes tailor macromolecules and metabolism to overcome specific environmental challenges. Acetic acid bacteria perform the aerobic oxidation of ethanol to acetic acid and are generally resistant to high levels of these two membrane-permeable poisons. The citric acid cycle (CAC) is linked to acetic acid resistance in Acetobacter aceti by several observations, among them the oxidation of acetate to CO2 by highly resistant acetic acid bacteria and the previously unexplained role of A. aceti citrate synthase (AarA) in acetic acid resistance at a low pH. Here we assign specific biochemical roles to the other components of the A. aceti strain 1023 aarABC region. AarC is succinyl-coenzyme A (CoA):acetate CoA-transferase, which replaces succinyl-CoA synthetase in a variant CAC. This new bypass appears to reduce metabolic demand for free CoA, reliance upon nucleotide pools, and the likely effect of variable cytoplasmic pH upon CAC flux. The putative aarB gene is reassigned to SixA, a known activator of CAC flux. Carbon overflow pathways are triggered in many bacteria during metabolic limitation, which typically leads to the production and diffusive loss of acetate. Since acetate overflow is not feasible for A. aceti, a CO(2) loss strategy that allows acetic acid removal without substrate-level (de)phosphorylation may instead be employed. All three aar genes, therefore, support flux through a complete but unorthodox CAC that is needed to lower cytoplasmic acetate levels.

Overexpression of the VvLTP1 gene interferes with somatic embryo development in grapevine.

Grapevine (Vitis vinifera L.) embryos have an early developmental pattern which differs from the one observed in model angiosperms such as Arabidopsis, in that the plane of divisions show variations from one individual to another. Furthermore, the protoderm (the first tissue to differentiate) does not form in one step but rather, gradually with time during globule formation. In Arabidopsis, expression pattern of a particular lipid transfer protein (LTP) isoform, AtLTP1, appears to be related to protoderm establishment, and is considered as a molecular marker of its differentiation. To investigate whether a similar role for LTPs in the development of grapevine embryos, we investigated the expression pattern of VvLTP1, a Vitis homologue of AtLTP1, in somatic embryo development. Expression of the GUS reporter gene under the control of the VvLTP1 promoter demonstrated that this LTP isoform is a marker of protoderm formation, and confirmed that this tissue forms sequentially over time. Ectopic expression of VvLTP1 under the control of the 35S promoter led to grossly misshapen embryos, which failed to acquire bilateral symmetry and displayed an abnormal epidermal layer. These results indicate that a correct spatial or temporal expression, or both, of this gene is essential for grapevine embryo development.

Atomic-resolution crystal structure of thioredoxin from the acidophilic bacterium Acetobacter aceti.

The crystal structure of thioredoxin (AaTrx) from the acetic acid bacterium Acetobacter aceti was determined at 1 A resolution. This is currently the highest resolution crystal structure available for any thioredoxin. Thioredoxins facilitate thiol-disulfide exchange, a process that is expected to be slow at the low pH values encountered in the A. aceti cytoplasm. Despite the apparent need to function at low pH, neither the active site nor the surface charge distribution of AaTrx is notably different from that of Escherichia coli thioredoxin. Apparently the ancestral thioredoxin was sufficiently stable for use in A. aceti or the need to interact with multiple targets constrained the variation of surface residues. The AaTrx structure presented here provides a clear view of all ionizable protein moieties and waters, a first step in understanding how thiol-disulfide exchange might occur in a low pH cytoplasm, and is a basis for biophysical studies of the mechanism of acid-mediated unfolding. The high resolution of this structure should be useful for computational studies of thioredoxin function, protein structure and dynamics, and side-chain ionization.

Alanine racemase from the acidophile Acetobacter aceti.

Acetobacter aceti converts ethanol to acetic acid, and survives acetic acid exposure by tolerating cytoplasmic acidification. Alanine racemase (Alr) is a pyridoxal 5' phosphate (PLP) -dependent enzyme that catalyzes the interconversion of the d- and l-isomers of alanine and has a basic pH optimum. Since d-alanine is essential for peptidoglycan biosynthesis, Alr must somehow function in the acidic cytoplasm of A. aceti. We report the partial purification of native A. aceti Alr (AaAlr) and evidence that it is a rather stable enzyme. The C-terminus of AaAlr has a strong resemblance to the ssrA-encoded protein degradation signal, which thwarted initial protein expression experiments. High-activity AaAlr forms lacking a protease recognition sequence were expressed in Escherichia coli and purified. Biophysical and enzymological experiments confirm that AaAlr is intrinsically acid-resistant, yet has the catalytic properties of an ordinary Alr.

Structural basis for interaction of O-acetylserine sulfhydrylase and serine acetyltransferase in the Arabidopsis cysteine synthase complex.

In plants, association of O-acetylserine sulfhydrylase (OASS) and Ser acetyltransferase (SAT) into the Cys synthase complex plays a regulatory role in sulfur assimilation and Cys biosynthesis. We determined the crystal structure of Arabidopsis thaliana OASS (At-OASS) bound with a peptide corresponding to the C-terminal 10 residues of Arabidopsis SAT (C10 peptide) at 2.9-A resolution. Hydrogen bonding interactions with key active site residues (Thr-74, Ser-75, and Gln-147) lock the C10 peptide in the binding site. C10 peptide binding blocks access to OASS catalytic residues, explaining how complex formation downregulates OASS activity. Comparison with bacterial OASS suggests that structural plasticity in the active site allows binding of SAT C termini with dissimilar sequences at structurally similar OASS active sites. Calorimetric analysis of the effect of active site mutations (T74S, S75A, S75T, and Q147A) demonstrates that these residues are important for C10 peptide binding and that changes at these positions disrupt communication between active sites in the homodimeric enzyme. We also demonstrate that the C-terminal Ile of the C10 peptide is required for molecular recognition by At-OASS. These results provide new insights into the molecular mechanism underlying formation of the Cys synthase complex and provide a structural basis for the biochemical regulation of Cys biosynthesis in plants.

Structure of a NADH-insensitive hexameric citrate synthase that resists acid inactivation.

Acetobacter aceti converts ethanol to acetic acid, and strains highly resistant to both are used to make vinegar. A. aceti survives acetic acid exposure by tolerating cytoplasmic acidification, which implies an unusual adaptation of cytoplasmic components to acidic conditions. A. aceti citrate synthase (AaCS), a hexameric type II citrate synthase, is required for acetic acid resistance and, therefore, would be expected to function at low pH. Recombinant AaCS has intrinsic acid stability that may be a consequence of strong selective pressure to function at low pH, and unexpectedly high thermal stability for a protein that has evolved to function at approximately 30 degrees C. The crystal structure of AaCS, complexed with oxaloacetate (OAA) and the inhibitor carboxymethyldethia-coenzyme A (CMX), was determined to 1.85 A resolution using protein purified by a tandem affinity purification procedure. This is the first crystal structure of a "closed" type II CS, and its active site residues interact with OAA and CMX in the same manner observed in the corresponding type I chicken CS.OAA.CMX complex. While AaCS is not regulated by NADH, it retains many of the residues used by Escherichia coli CS (EcCS) for NADH binding. The surface of AaCS is abundantly decorated with basic side chains and has many fewer uncompensated acidic charges than EcCS; this constellation of charged residues is stable in varied pH environments and may be advantageous in the A. aceti cytoplasm.

Molecular basis of ergosterol-induced protection of grape against botrytis cinerea: induction of type I LTP promoter activity, WRKY, and stilbene synthase gene expression.

Type I lipid transfer proteins (LTPs) are basic, 9-kDa cystein-rich proteins believed to be involved in plant defense mechanisms. A 2,100-bp fragment containing the coding region of Vitis vinifera lipid transfer protein 1 (VvLTP1) and 1,420-bp of its promoter region was isolated by screening a grape genomic library. In silico analysis revealed several putative, defense-related, cis-regulatory elements such as W- and MYB-boxes, involved in the binding of WRKY and MYB transcription factors, respectively. The 5'-truncated versions of the VvLTP1 promoter were generated, cloned in front of the beta-glucuronidase (GUS) reporter gene, and introduced in tobacco plants and grapevine cell suspensions using Agrobacterium spp. Single MYB- and the W-boxes identified on the 0.250-kbp fragment were sufficient to induce GUS activity in transgenic tobacco plants after transient expression of MYB and WRKY. Ergosterol, a nonspecific fungal elicitor, induced GUS activity in transgenic grapevine cell suspensions transformed with the 1,420- and 750-bp promoter containing a palindromic arrangement of two W-boxes but not the 650- or 250-bp fragment, where only one W-box was present. Moreover, ergosterol triggered WRKY, VvLTP1, and stilbene synthase gene expression in grape plantlets and enhanced protection against Botrytis cinerea. The molecular basis of ergosterol-induced protection is discussed.

Biochemical and structural studies of N5-carboxyaminoimidazole ribonucleotide mutase from the acidophilic bacterium Acetobacter aceti.

N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) mutase (PurE) catalyzes the reversible interconversion of acid-labile compounds N5-CAIR and 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). We have examined PurE from the acidophilic bacterium Acetobacter aceti (AaPurE), focusing on its adaptation to acid pH and the roles of conserved residues His59 and His89. Both AaPurE and Escherichia coli PurE showed quasi-reversible acid-mediated inactivation, but wt AaPurE was much more stable at pH 3.5, with a > or = 20 degrees C higher thermal unfolding temperature at all pHs. His89 is not essential and does not function as part of a proton relay system. The kcat pH-rate profile was consistent with the assignment of pK1 to unproductive protonation of bound nucleotide and pK2 to deprotonation of His59. A 1.85 A resolution crystal structure of the inactive mutant H59N-AaPurE soaked in CAIR showed that protonation of CAIR C4 can occur in the absence of His59. The resulting species, modeled as isoCAIR [4(R)-carboxy-5-iminoimidazoline ribonucleotide], is strongly stabilized by extensive interactions with the enzyme and a water molecule. The carboxylate moiety is positioned in a small pocket proposed to facilitate nucleotide decarboxylation in the forward direction (N5-CAIR --> CAIR) [Meyer, E., Kappock, T. J., Osuji, C., and Stubbe, J. (1999) Biochemistry 38, 3012-3018]. Comparisons with model studies suggest that in the reverse (nonbiosynthetic) direction PurE favors protonation of CAIR C4. We suggest that the essential role of protonated His59 is to lower the barrier to decarboxylation by stabilizing a CO2-azaenolate intermediate.