The intraoperative impression and postoperative outcomes of gamma-irradiated corneas in corneal and glaucoma patch surgery.

PURPOSE: To report the short-term outcome of gamma-irradiated sterile cornea in corneal and glaucoma patch graft surgeries when viable endothelium is not necessary. METHODS: Surgeons who have used the irradiated corneas were asked to rate their impression from 1 (fair) to 4 (very good). Specific intraoperative questions included availability, ease of use, clarity, thickness, tensile strength, and suturing. Postoperative outcomes included epithelialization, biological incorporation, and clarity of tissue, as well as any adverse outcomes. RESULTS: Irradiated sterile cornea has been used in more than 150 surgeries so far. The major uses for irradiated cornea were as glaucoma patch graft (65.3%), as corneal patch graft (14%), in anterior lamellar keratoplasty (10.7%), and in keratoprosthesis (10%). The mean score for packaging of tissue was 3.75 (SD, 0.45), ease of use 3.83 (SD, 0.39), clarity 3.62 (SD, 0.51), thickness 3.67 (SD, 0.49), tensile strength 3.64 (SD, 0.50), and suturing 3.62 (SD, 0.65). Postoperative outcomes were also favorable, including epithelialization (mean ± SD, 3.46 ± 0.69), biological incorporation (mean ± SD, 3.46 ± 0.69), and clarity (mean ± SD, 3.43 ± 0.85). There were 4 episodes of corneal melt (all occurred in patients with previous corneal melt). There were no reported cases of tissue rejection or infection. CONCLUSIONS: Gamma-irradiated sterile cornea is a promising new development that may help increase the supply of donor cornea tissue. It virtually eliminates the risk of infection while providing a long shelf life. It is particularly helpful in emergency situations or in remote areas. However, longer follow-up and a prospective study comparing the outcomes of the irradiated corneas with those of the optisol-stored corneas are needed.

Hydrogenomics of the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus.

Caldicellulosiruptor saccharolyticus is an extremely thermophilic, gram-positive anaerobe which ferments cellulose-, hemicellulose- and pectin-containing biomass to acetate, CO(2), and hydrogen. Its broad substrate range, high hydrogen-producing capacity, and ability to coutilize glucose and xylose make this bacterium an attractive candidate for microbial bioenergy production. Here, the complete genome sequence of C. saccharolyticus, consisting of a 2,970,275-bp circular chromosome encoding 2,679 predicted proteins, is described. Analysis of the genome revealed that C. saccharolyticus has an extensive polysaccharide-hydrolyzing capacity for cellulose, hemicellulose, pectin, and starch, coupled to a large number of ABC transporters for monomeric and oligomeric sugar uptake. The components of the Embden-Meyerhof and nonoxidative pentose phosphate pathways are all present; however, there is no evidence that an Entner-Doudoroff pathway is present. Catabolic pathways for a range of sugars, including rhamnose, fucose, arabinose, glucuronate, fructose, and galactose, were identified. These pathways lead to the production of NADH and reduced ferredoxin. NADH and reduced ferredoxin are subsequently used by two distinct hydrogenases to generate hydrogen. Whole-genome transcriptome analysis revealed that there is significant upregulation of the glycolytic pathway and an ABC-type sugar transporter during growth on glucose and xylose, indicating that C. saccharolyticus coferments these sugars unimpeded by glucose-based catabolite repression. The capacity to simultaneously process and utilize a range of carbohydrates associated with biomass feedstocks is a highly desirable feature of this lignocellulose-utilizing, biofuel-producing bacterium.

Structure, function, and wavelength selection in blue-absorbing proteorhodopsin.

The absorption maximum of blue proteorhodopsin (BPR) is the most blue-shifted of all retinal proteins found in archaea or bacteria, with the exception of sensory rhodopsin II (SRII). The absorption spectrum also exhibits a pH dependence larger than any other retinal protein. We examine the structural origins of these two properties of BPR by using optical spectroscopy, homology modeling, and molecular orbital theory. Bacteriorhodopsin (BR) and SRII are used as homology parents for comparative purposes. We find that the tertiary structure of BPR based on SRII is more realistic with respect to free energy, dynamic stability, and spectroscopic properties. Molecular orbital calculations including full single- and double-configuration interaction within the chromophore pi-electron system provide perspectives on the wavelength regulation in this protein and indicate that Arg-95, Gln-106, Glu-143, and Asp-229 play important, and in some cases pH-dependent roles. A possible model for the 0.22 eV red shift of BPR at low pH is examined, in which Glu-143 becomes protonated and releases Arg-95 to rotate up into the binding site, altering the electrostatic environment of the chromophore. At high pH, BPR has spectroscopic properties similar to SRII, but at low pH, BPR has spectroscopic properties more similar to BR. Nevertheless, SRII is a significantly better homology model for BPR and opens up the question of whether this protein serves as a proton pump, as commonly believed, or is a light sensor with structure-function properties more comparable to those of SRII. The function of BPR in the native organism is discussed with reference to the results of the homology model.

Discovery and characterization of a Coenzyme A disulfide reductase from Pyrococcus horikoshii. Implications for this disulfide metabolism of anaerobic hyperthermophiles.

We have cloned NADH oxidase homologues from Pyrococcus horikoshii and P. furiosus, and purified the recombinant form of the P. horikoshii enzyme to homogeneity from Escherichia coli. Both enzymes (previously referred to as NOX2) have been shown to act as a coenzyme A disulfide reductases (CoADR: CoA-S-S-CoA + NAD(P)H + H+-->2CoA-SH + NAD(P)+). The P. horikoshii enzyme shows a kcat app of 7.2 s(-1) with NADPH at 75 degrees C. While the enzyme shows a preference for NADPH, it is able to use both NADPH and NADH efficiently, with both giving roughly equal kcats, while the Km for NADPH is roughly eightfold lower than that for NADH. The enzyme is specific for the CoA disulfide, and does not show significant reductase activity with other disulfides, including dephospho-CoA. Anaerobic reductive titration of the enzyme with NAD(P)H proceeds in two stages, with an apparent initial reduction of a nonflavin redox center with the first reduction resulting in what appears to be an EH2 form of the enzyme. Addition of a second of NADPH results in the formation of an apparent FAD-NAD(P)H complex. The behavior of this enzyme is quite different from the mesophilic staphylococcal version of the enzyme. This is only the second enzyme with this activity discovered, and the first from a strict anaerobe, an Archaea, or hyperthermophilic source. P. furiosus cells were assayed for small molecular mass thiols and found to contain 0.64 micromol CoA.g dry weight(-1) (corresponding to 210 microM CoA in the cell) consistent with CoA acting as a pool of disulfide reducing equivalents.

Transcriptional analysis of biofilm formation processes in the anaerobic, hyperthermophilic bacterium Thermotoga maritima.

Thermotoga maritima, a fermentative, anaerobic, hyperthermophilic bacterium, was found to attach to bioreactor glass walls, nylon mesh, and polycarbonate filters during chemostat cultivation on maltose-based media at 80 degrees C. A whole-genome cDNA microarray was used to examine differential expression patterns between biofilm and planktonic populations. Mixed-model statistical analysis revealed differential expression (twofold or more) of 114 open reading frames in sessile cells (6% of the genome), over a third of which were initially annotated as hypothetical proteins in the T. maritima genome. Among the previously annotated genes in the T. maritima genome, which showed expression changes during biofilm growth, were several that corresponded to biofilm formation genes identified in mesophilic bacteria (i.e., Pseudomonas species, Escherichia coli, and Staphylococcus epidermidis). Most notably, T. maritima biofilm-bound cells exhibited increased transcription of genes involved in iron and sulfur transport, as well as in biosynthesis of cysteine, thiamine, NAD, and isoprenoid side chains of quinones. These findings were all consistent with the up-regulation of iron-sulfur cluster assembly and repair functions in biofilm cells. Significant up-regulation of several beta-specific glycosidases was also noted in biofilm cells, despite the fact that maltose was the primary carbon source fed to the chemostat. The reasons for increased beta-glycosidase levels are unclear but are likely related to the processing of biofilm-based polysaccharides. In addition to revealing insights into the phenotype of sessile T. maritima communities, the methodology developed here can be extended to study other anaerobic biofilm formation processes as well as to examine aspects of microbial ecology in hydrothermal environments.

Transcriptional analysis of dynamic heat-shock response by the hyperthermophilic bacterium Thermotoga maritima.

The thermal stress response of the hyperthermophilic bacterium Thermotoga maritima was characterized using a 407-open reading frame-targeted cDNA microarray. Transient gene expression was followed for 90 min, following a shift from 80 degrees C to 90 degrees C. While some aspects of mesophilic heat-shock response were conserved in T. maritima, genome content suggested differentiating features that were borne out by transcriptional analysis. Early induction of predicted heat-shock operons hrcA-grpE-dnaJ (TM0851-TM0850-TM0849), groES-groEL (TM0505-TM0506), and dnaK-sHSP (TM0373-TM0374) was consistent with conserved CIRCE elements upstream of hrcA and groES. Induction of the T. maritima rpoE/ sigW and rpoD/ sigA homologs suggests a mechanism for global heat-shock response in the absence of an identifiable ortholog to a major heat-shock sigma factor. In contrast to heat-shock response in Escherichia coli, the majority of genes encoding ATP-dependent proteases were downregulated, including clpP (TM0695), clpQ (TM0521), clpY (TM0522), lonA (TM1633), and lonB (TM1869). Notably, T. maritima showed indications of a late heat-shock response with the induction of a marR homolog (TM0816), several other putative transcriptional regulators (TM1023, TM1069), and two alpha-glucosidases (TM0434 and TM1068). Taken together, the results reported here indicate that, while T. maritima shares core elements of the bacterial heat-shock response with mesophiles, the thermal stress regulatory strategies of this organism differ significantly. However, it remains to be elucidated whether these differences are related to thermophilicity or phylogenetic placement.

Heat shock response by the hyperthermophilic archaeon Pyrococcus furiosus.

Collective transcriptional analysis of heat shock response in the hyperthermophilic archaeon Pyrococcus furiosus was examined by using a targeted cDNA microarray in conjunction with Northern analyses. Differential gene expression suggests that P. furiosus relies on a cooperative strategy of rescue (thermosome [Hsp60], small heat shock protein [Hsp20], and two VAT-related chaperones), proteolysis (proteasome), and stabilization (compatible solute formation) to cope with polypeptide processing during thermal stress.

Characterization of plasmid pRT1 from Pyrococcus sp. strain JT1.

We discovered a 3,373-bp plasmid (pRT1) in the hyperthermophilic archaeon Pyrococcus sp. strain JT1. Two major open reading frames were identified, and analysis of the sequence revealed some resemblance to motifs typically found in plasmids that replicate via a rolling-circle mechanism. The presence of single-stranded DNA replication intermediates of pRT1 was detected, confirming this mode of replication.

Regulation of endo-acting glycosyl hydrolases in the hyperthermophilic bacterium Thermotoga maritima grown on glucan- and mannan-based polysaccharides.

The genome sequence of the hyperthermophilic bacterium Thermotoga maritima encodes a number of glycosyl hydrolases. Many of these enzymes have been shown in vitro to degrade specific glycosides that presumably serve as carbon and energy sources for the organism. However, because of the broad substrate specificity of many glycosyl hydrolases, it is difficult to determine the physiological substrate preferences for specific enzymes from biochemical information. In this study, T. maritima was grown on a range of polysaccharides, including barley beta-glucan, carboxymethyl cellulose, carob galactomannan, konjac glucomannan, and potato starch. In all cases, significant growth was observed, and cell densities reached 10(9) cells/ml. Northern blot analyses revealed different substrate-dependent expression patterns for genes encoding the various endo-acting beta-glycosidases; these patterns ranged from strong expression to no expression under the conditions tested. For example, cel74 (TM0305), a gene encoding a putative beta-specific endoglucananse, was strongly expressed on all substrates tested, including starch, while no evidence of expression was observed on any substrate for lam16 (TM0024), xyl10A (TM0061), xyl10B (TM0070), and cel12A (TM1524), which are genes that encode a laminarinase, two xylanases, and an endoglucanase, respectively. The cel12B (TM1525) gene, which encodes an endoglucanase, was expressed only on carboxymethyl cellulose. An extracellular mannanase encoded by man5 (TM1227) was expressed on carob galactomannan and konjac glucomannan and to a lesser extent on carboxymethyl cellulose. An unexpected result was the finding that the cel5A (TM1751) and cel5B (TM1752) genes, which encode putative intracellular beta-specific endoglucanases, were induced only when T. maritima was grown on konjac glucomannan. To investigate the biochemical basis of this finding, the recombinant forms of Man5 (M(r), 76,900) and Cel5A (M(r), 37,400) were expressed in Escherichia coli and characterized. Man5, a T. maritima extracellular enzyme, had a melting temperature of 99 degrees C and an optimun temperature of 90 degrees C, compared to 90 and 80 degrees C, respectively, for the intracellular enzyme Cel5A. While Man5 hydrolyzed both galactomannan and glucomannan, no activity was detected on glucans or xylans. Cel5A, however, not only hydrolyzed barley beta-glucan, carboxymethyl cellulose, xyloglucan, and lichenin but also had activity comparable to that of Man5 on galactomannan and higher activity than Man5 on glucomannan. The biochemical characteristics of Cel5A, the fact that Cel5A was induced only when T. maritima was grown on glucomannan, and the intracellular localization of Cel5A suggest that the physiological role of this enzyme includes hydrolysis of glucomannan oligosaccharides that are transported following initial hydrolysis by extracellular glycosidases, such as Man5.

Molecular analysis of the role of two aromatic aminotransferases and a broad-specificity aspartate aminotransferase in the aromatic amino acid metabolism of Pyrococcus furiosus.

The genes encoding aromatic aminotransferase II (AroAT II) and aspartate aminotransferase (AspAT) from Pyrococcus furiosus have been identified, expressed in Escherichia coli and the recombinant proteins characterized. The AroAT II enzyme was specific for the transamination reaction of the aromatic amino acids, and uses a-ketoglutarate as the amino acceptor. Like the previously characterized AroAT I, AroAT II has highest efficiency for phenylalanine (k(cat)/Km = 923 s(-1) mM(-1)). Northern blot analyses revealed that AroAT I was mainly expressed when tryptone was the primary carbon and energy source. Although the expression was significantly lower, a similar trend was observed for AroAT II. These observations suggest that both AroATs are involved in amino acid degradation. Although AspAT exhibited highest activity with aspartate and alpha-ketoglutarate (k(cat) approximately 105 s(-1)), it also showed significant activity with alanine, glutamate and the aromatic amino acids. With aspartate as the amino donor, AspAT catalyzed the amination of alpha-ketoglutarate, pyruvate and phenyl-pyruvate. No activity was detected with either branched-chain amino acids or alpha-keto acids. The AspAT gene (aspC) was expressed as a polycistronic message as part of the aro operon, with expression observed only when the aromatic amino acids were absent from the growth medium, indicating a role in the biosynthesis of the aromatic amino acids.

Proteolysis in hyperthermophilic microorganisms.

Proteases are found in every cell, where they recognize and break down unneeded or abnormal polypeptides or peptide-based nutrients within or outside the cell. Genome sequence data can be used to compare proteolytic enzyme inventories of different organisms as they relate to physiological needs for protein modification and hydrolysis. In this review, we exploit genome sequence data to compare hyperthermophilic microorganisms from the euryarchaeotal genus Pyrococcus, the crenarchaeote Sulfolobus solfataricus, and the bacterium Thermotoga maritima. An overview of the proteases in these organisms is given based on those proteases that have been characterized and on putative proteases that have been identified from genomic sequences, but have yet to be characterized. The analysis revealed both similarities and differences in the mechanisms utilized for proteolysis by each of these hyperthermophiles and indicated how these mechanisms relate to proteolysis in less thermophilic cells and organisms.