Extreme ocean acidification reduces the susceptibility of eastern oyster shells to a polydorid parasite.

Ocean acidification poses a threat to marine organisms. While the physiological and behavioural effects of ocean acidification have received much attention, the effects of acidification on the susceptibility of farmed shellfish to parasitic infections are poorly understood. Here we describe the effects of moderate (pH 7.5) and extreme (pH 7.0) ocean acidification on the susceptibility of Crassostrea virginica shells to infection by a parasitic polydorid, Polydora websteri. Under laboratory conditions, shells were exposed to three pH treatments (7.0, 7.5 and 8.0) for 3- and 5-week periods. Treated shells were subsequently transferred to an oyster aquaculture site (which had recently reported an outbreak of P. websteri) for 50 days to test for effects of pH and exposure time on P. websteri recruitment to oyster shells. Results indicated that pH and exposure time did not affect the length, width or weight of the shells. Interestingly, P. websteri counts were significantly lower under extreme (pH 7.0; ~50% reduction), but not moderate (pH 7.5; ~20% reduction) acidification levels; exposure time had no effect. This study suggests that extreme levels - but not current and projected near-future levels - of acidification (∆pH ~1 unit) can reduce the susceptibility of eastern oyster shells to P. websteri infections.

Maximizing production of Penicillium cyclopium partial acylglycerol lipase.

Penicillium cyclopium partial acylglycerol lipase production was maximized in shaken batch culture. The effect of inoculum size and substrate concentration on the lipase activity released in the culture medium was visualized using a surface response methodology based on a Doehlert experimental design. The main advantage of this approach is the low number of experiments required to construct a predictive model of the experimental domain. Substrate percentage (corn steep, w/v) ranged from 0.1% to 1.9% and inoculum from 100 spores/ml to 3,200 spores/ml. We determined that an optimal set of experimental conditions for high lipase production was 1.0% substrate and 3,200 spores/ml, with initial pH 5.0, temperature 25 degrees C and shaking speed 120 rpm. Between the conditions giving the minimum and the maximum lipase production, we observed a three-fold increase in both the predicted and the measured values.

Lipase-catalysed hydrolysis of short-chain substrates in solution and in emulsion: a kinetic study.

We have studied the enzymatic hydrolysis of solutions and emulsions of vinyl propionate, vinyl butyrate and tripropionin by lipases of various origin and specificity. Kinetic studies of the hydrolysis of short-chain substrates by microbial triacylglycerol lipases from Rhizopus oryzae, Mucor miehei, Candida rugosa, Candida antarctica A and by (phospho)lipase from guinea-pig pancreas show that these lipolytic enzymes follow the Michaelis-Menten model. Surprisingly, the activity against solutions of tripropionin and vinyl esters ranges from 70% to 90% of that determined against emulsions. In contrast, a non-hyperbolic (sigmoidal) dependence of enzyme activity on ester concentration is found with human pancreatic lipase, triacylglycerol lipase from Humicola lanuginosa (Thermomyces lanuginosa) and partial acylglycerol lipase from Penicillium camembertii and the same substrates. In all cases, no abrupt jump in activity (interfacial activation) is observed at substrate concentration corresponding to the solubility limit of the esters. Maximal lipolytic activity is always obtained in the presence of emulsified ester. Despite progress in the understanding of structure-function of lipases, interpretation of the mode of action of lipases active against solutions of short-chain substrates remains difficult. Actually, it is not known whether these enzymes, which possess a lid structure, are in open or/and closed conformation in the bulk phase and whether the opening of the lid that gives access to the catalytic triad is triggered by interaction of the enzyme molecule with monomeric substrates or/and multimolecular aggregates (micelles) both present in the bulk phase. From the comparison of the behaviour of lipases used in this study which, in some cases, follow the Michaelis-Menten model and, in others, deviate from classical kinetics, it appears that the activity of classical lipases against soluble short-chain vinyl esters and tripropionin depends not only on specific interaction with single substrate molecules at the catalytic site of the enzyme but also on physico-chemical parameters related to the state of association of the substrate dispersed in the aqueous phase. It is assumed that the interaction of lipase with soluble multimolecular aggregates of tripropionin or short-chain vinyl esters or the formation of enzyme-substrate mixed micelles with ester bound to lipase, might represent a crucial step that triggers the structural transition to the open enzyme conformation by displacement of the lid.

Hyperammonemic coma caused by Providencia rettgeri infection in a child with prune belly syndrome.

OBJECTIVE: To report a case of hyperammonemic encephalopathy in the absence of liver disease caused by a urinary tract infection with a previously unreported organism. DESIGN: Case report. SETTING: Pediatric intensive care unit. Patient: Critically ill child who presented with hyperammonemic coma. INTERVENTION: Bladder drainage and antimicrobial therapy for the urinary tract infection. Measurements: Rapid resolution of the hyperammonemia. MAIN RESULTS: Rapid improvement in the patient's neurologic status and a return to his baseline function without neurologic deficit. CONCLUSIONS: Patients with abnormal urinary collecting systems who present with altered mental status should have a serum ammonia concentration sent as part of the initial evaluation. We have added Providencia species to the list of documented causative organisms in this rare association between urinary tract infections and hyperammonemia.

Optimal design for the maximization of Penicillium cyclopium lipase production.

Penicillium cyclopium triacylglycerol lipase production was maximized in stationary batch culture. We used a surface response methodology based on a Doehlert experimental design to study the effect on the lipase activity released in the culture medium of the three most important factors: substrate concentration, pH and inoculum. Besides reducing the number of experiments required for optimization, this technique allowed us to quantify the lipase activity in any part of the experimental domain.We determined an optimal set of conditions for high lipase production: 1% substrate (corn steep), pH 5.5 and an inoculum of 10(4) spores/ml. Between conditions giving the minimum and the maximum lipase production, we observed a nine-fold increase of both the predicted and measured values.

Kinetic properties of Penicillium cyclopium lipases studied with vinyl esters.

Penicillium cyclopium produces two lipases with different substrate specificities. Lipase I is predominantly active on triacylglycerols whereas lipase II hydrolyzes mono- and diacylglycerols but not triacylglycerols. In this study, we compared the kinetic properties of P. cyclopium lipases and human pancreatic lipase, a classical triacylglycerol lipase, by using vinyl esters as substrates. Results indicate that P. cyclopium lipases I and II and human pancreatic lipase hydrolyze solutions of vinyl propionate or vinyl butyrate at high relative rates compared with emulsions of the same esters, although, in all cases, maximal activity is reached in the presence of emulsified particles, at substrate concentrations above the solubility limit. It appears that partially water-soluble short-chain vinyl esters are suitable substrates for comparing the activity of lipolytic enzymes of different origin and specificity toward esters in solution and in emulsion.

Concentrates of DHA from fish oil by selective esterification of cholesterol by immobilized isoforms of lipase from Candida rugosa.

Two lipases (Lip A and Lip B), were purified from a commercial lipase preparation produced by Candida rugosa and partially characterized. The purified lipases were immobilized on Duolite A 568 and used in the selective esterification of cholesterol with free fatty acids from sardine fish oil. The results showed that Lip A and Lip B preferentially esterified saturated and monounsaturated fatty acids allowing a 3.4-fold (Lip B, 24 h) and 4-fold (Lip A, 10 h) enrichment of docosahexaenoic acid in the remaining free fatty acid fraction. Selectivity towards eicosapentaenoic acid was less pronounced. By this selective esterification docosahexaenoic acid was concentrated from 7.4 to 32% with a recovery of 95% of its initial content in sardine fish oil.

Production of extracellular lipases by Penicillium cyclopium purification and characterization of a partial acylglycerol lipase.

Penicillium cyclopium, grown in stationary culture, produces a type I lipase specific for triacylglycerols while, in shaken culture, it produces a type II lipase only active on partial acylglycerols. Lipase II has been purified by ammonium sulfate precipitation and chromatographies on Sephadex G-75 and DEAE-Sephadex. The enzyme exists in several glycosylated forms of 40-43 kDa, which can be converted to a single protein of 37 kDa by enzymatic deglycosylation. Activity of lipase II is maximal at pH 7.0 and 40 degrees C. The enzyme is stable from pH 4.5 to 7.0. Activity is rapidly lost at temperatures above 50 degrees C. The enzyme specifically hydrolyzes monoacylglycerols and diacylglycerols, especially of medium chain fatty acids. The sequence of the 20 first amino acid residues is similar to the N-terminal region of P. camembertii lipase and partially similar to lipases from Humicola lanuginosa and Aspergillus oryzae, but is different from Penicillium cyclopium lipase I. However, it can be observed that residues of valine and serine at positions 2 and 5 in Penicillium cyclopium lipase II are conserved in Penicillium expansum lipase, of which 16 out of the 20 first amino acid residues are similar to Penicillium cyclopium lipase I.

Mammalian telomeres end in a large duplex loop.

Mammalian telomeres contain a duplex array of telomeric repeats bound to the telomeric repeat-binding factors TRF1 and TRF2. Inhibition of TRF2 results in immediate deprotection of chromosome ends, manifested by loss of the telomeric 3' overhang, activation of p53, and end-to-end chromosome fusions. Electron microscopy reported here demonstrated that TRF2 can remodel linear telomeric DNA into large duplex loops (t loops) in vitro. Electron microscopy analysis of psoralen cross-linked telomeric DNA purified from human and mouse cells revealed abundant large t loops with a size distribution consistent with their telomeric origin. Binding of TRF1 and single strand binding protein suggested that t loops are formed by invasion of the 3' telomeric overhang into the duplex telomeric repeat array. T loops may provide a general mechanism for the protection and replication of telomeres.

Biochemical and structural characterization of triacylglycerol lipase from Penicillium cyclopium.

An extracellular lipase, active on water-insoluble triacylglycerols, has been isolated from Penicillium cyclopium. The purified enzyme has a molecular mass of 29 kDa by gel filtration and SDS-polyacrylamide gel electrophoresis. It hydrolyzes emulsions of tributyrin, trioctanoin, and olive oil at the same rate as pancreatic lipase and shows very low activity against partial acylglycerols (monooctanoin and dioctanoin) and methyl esters. It is stable at 35 degrees C for 60 min and has maximal activity in a pH range of 8-10. Hydrolysis of triacylglycerols by P. cyclopium lipase is inhibited by detergents such as Triton X-100. Comparison of the sequence of the 20 first amino acid residues of P. cyclopium triacylglycerol lipase with other Penicillium lipases indicates a high homology with previously characterized lipases produced by P. expansum and P. solitum which are enzymes of comparable size and substrate specificity. Conversely, homology between P. cyclopium lipase and P. simplicissimum lipase, a nonspecific lipolytic enzyme, is low. Penicillium cyclopium triacylglycerol lipase shows no homology with P. camembertii lipase which is specific to monoacylglycerol and diacylglycerol.

Comparative topical anaesthesia of EMLA and liposome-encapsulated tetracaine.

BACKGROUND: The eutectic mixture of local anaesthetics (EMLA) provides effective topical anaesthesia after a minimum of 60 to 90 min application. Since liposome-encapsulated tetracaine (LET) can provide rapid dermal penetration, the goal of this study was to compare the local anaesthetic effects of EMLA and LET in human volunteers after 60 min application. METHODS: After obtaining institutional approval and informed consent, healthy volunteers were recruited in a double blind, crossover, randomized trial. The study creams (0.5 ml EMLA and 0.5 ml LET 5%) were applied randomly to opposite arms for 60 min. The discomfort of i.v. catheterization was assessed using a visual analogue pain score (VAS). Cutaneous side effects of the creams were recorded. RESULTS: Sixty-one subjects were studied. Twenty-one were excluded because of technical difficulties. Forty subjects completed the study and were included in the data analysis. The mean ( +/- SD) VAS was lower for LET than for EMLA (10.9 +/- 9.0 mm vs 22.7 +/- 17.1 mm, P < 0.001). Erythema secondary to vasodilatation occurred more frequent in the LET group than in the EMLA group (33 vs 3, P < 0.001). One subject with a history of atopy developed a rash at the LET application site. CONCLUSION: Liposome-encapsulated tetracaine can provide a more effective topical anaesthesia than EMLA for intravenous catheterization after 60 min application. Clinical evaluations are necessary to determine the efficacy and safety of LET in providing topical anaesthesia for various invasive percutaneous procedures in other patient populations.

Association of putative origins of replication with the nuclear matrix in normal human fibroblasts.

Several metabolic processes, such as DNA organization and replication, transcription, and RNA processing are closely associated with the nuclear matrix. Nuclear matrix attachment regions are nucleotide sequences holding DNA tightly complexed with the nuclear scaffold and are resistant to extractions with detergents and high salt solutions. The role of matrix attachment regions in DNA replication has not been completely clarified, but they have been identified in close association with origins of replication in mammalian cells. We isolated nuclear matrix-associated DNA from normal human fibroblasts synchronized to different phases of the cell cycle and cloned compatible fragments into pUC19. We tested the homology of a fraction of the available clones to DNA replicated at the beginning of the S phase in human fibroblasts. We confirmed that nuclear matrix-associated DNA isolated from cells in G0 and G1 phases of the cell cycle contains sequences that are among the earliest replicated regions in the human genome. This finding supports the hypothesis that matrix attachment regions in human DNA are located in close proximity to origins of DNA replication.

Differential induction and regulation of peroxisomal enzymes: predictive value of peroxisome proliferation in identifying certain nonmutagenic carcinogens.

Hypolipidemic drugs and certain plasticizers markedly increase the number of peroxisomes in liver parenchymal cells. Continued exposure to peroxisome proliferators has been shown to produce essentially similar pleiotropic responses leading eventually to the development of liver tumors in rats and mice. These agents are not mutagenic in short-term test systems and do not appear to interact with or damage DNA. Accordingly, the events leading to or associated with the induction of peroxisome proliferation have been postulated to play a role in the development of liver tumors. Recent evidence indicates that persistent peroxisome proliferation leads to the formation of 8-hydroxyguanosine in rat liver DNA, which supports the role for oxidative stress. The mRNAs of the three peroxisomal beta-oxidation genes are induced over 20-fold in the livers of rats treated with nafenopin, Wy-14643, BR-931, and other structurally diverse peroxisome proliferators. This increase in beta-oxidation mRNAs is evident within 30 min to 1 hr and was maximal 8 to 16 hr after the administration of a single dose of these agents by gavage. The peroxisomal catalase and urate oxidase mRNAs increase about 2-fold in the livers of rats treated chronically with peroxisome proliferators. These results indicate that peroxisome proliferators differentially regulate different peroxisomal enzymes. The tissue specificity of peroxisomal beta-oxidation gene regulation by xenobiotics supports the contention that the development of liver tumors following exposure to peroxisome proliferators correlates well with the inducibility of peroxisome proliferation and the beta-oxidation genes. Although these agents are known to exert mitogenic response in liver, it is unlikely that stimulation of DNA synthesis alone is responsible for tumor development. Cell proliferation may, however, play a secondary role. The morphological phenomenon of peroxisome proliferation should serve as a simple, sensitive, and valuable biological indicator for the identification of nongenotoxic or nonmutagenic chemicals that may be carcinogenic. An understanding of the cellular and molecular basis of peroxisome proliferation is a prerequisite for the evaluation of toxicological implications of this phenomenon.

Inhibition of viral mRNA translation in interferon-treated L cells infected with reovirus.

Murine L cells were treated with interferon (IFN) concentrations which reduced by 75 to 80% the synthesis of viral mRNA after infection with reovirus. Protein synthesis was not inhibited in these cells up to 6 h after infection, but a large fraction of the viral mRNA was not associated with polyribosomes and sedimented at about 50S. In contrast, most of the reovirus mRNA was associated with polyribosomes in control infected cells. This mRNA was of similar size to non-polyribosomal mRNA from IFN-treated cells when analyzed by Northern blot hybridization with a cloned cDNA for the s2 reovirus mRNA, indicating that the non-polyribosomal mRNA was not appreciably degraded. Viral mRNA was labeled with [3H]uridine and the non-polyribosomal mRNA was isolated from IFN-treated cells. This mRNA could quantitatively bind to 80S initiation complexes when incubated in a rabbit reticulocyte cell-free system. These findings indicated that the non-polyribosomal RNA was translatable, but that its binding to functional initiation complexes was inhibited in IFN-treated cells by a discriminatory mechanism, which did not affect translation of cellular mRNA. Previous experiments showed that mRNA is blocked in 48S complexes when the alpha subunit of initiation factor eIF-2 is phosphorylated by the double-stranded RNA-dependent protein kinase induced by IFN. A localized activation of this kinase could explain the block of viral mRNA in 48S complexes. By labeling the phosphoproteins of IFN-treated cells with 32P, eIF-2 (alpha P) was shown to cosediment with non-polyribosomal mRNA, presumably in 48S complexes.