Mycobacteriosis in zebrafish (Danio rerio) research facilities

The Zebrafish International Resource Center was established to support the zebrafish research community, and includes a diagnostic service. One of the most common diseases that we have diagnosed is mycobacteriosis, which represented 18% of the diagnostic cases submitted from November 1999 to June 2003. We describe here the severity of the disease and associated pathological changes of 24 diagnostic cases from 14 laboratories. Identifications of the bacteria are provided for seven of these cases. For two cases in which culture of the organism was not successful, these identifications were based on ribosomal DNA (rDNA) sequence analysis obtained directly from infected tissues. Biochemical characteristics and rDNA sequence analysis from cultures are reported for the other isolates. Two severe outbreaks from different facilities on different continents were associated with an organism identified as Mycobacterium haemophilum based on rDNA sequence from tissues. Another severe outbreak was associated with an organism most closely related to Mycobacterium peregrinum. These species are recognized pathogens of humans, but this is the first report of them from fish. Bacteria identified as Mycobacterium chelonae or M. abscessus were recovered from fish in cases categorized as moderate disease or as an incidental finding. These findings indicate that species of Mycobacterium previously undescribed from fish (i.e., M. haemophilum and M. peregrinum) may pose significant health problems in zebrafish research facilities, whereas species and strains that are already recognized as common in fish usually cause limited disease on a population basis in zebrafish.     

Growth Response of Coyote Willow (Salix exigua) Cuttings in Relation to Alluvial Soil Texture and Water Availability

A common approach to re‐establishing cottonwood–willow habitat along regulated rivers is through installing dormant, rootless cuttings, yet there is little published information exploring floodplain characteristics that optimize growth of southwestern riparian willows planted in this manner. The goal of this project was to evaluate relationships between growth attributes of Salix exigua and soil texture and soil water availability. Monitoring plots were established in five willow swales planted with dormant S. exigua cuttings along the banks of the Middle Rio Grande in central New Mexico. Data analysis revealed significantly higher aerial cover, height, and stem density for S. exigua plants installed in plots with intermediate levels (15–25%) of fine textured soils distributed through the soil profile. Similar relationships were found in relation to soil water availability. Regression analysis of percent fines and available water at different depth increments provided limited explanation of variability in willow growth attributes at different plots. Findings indicate that S. exigua plants established from cuttings can achieve heights and aerial cover values similar to naturally established willow bars if the floodplain soil profile contains intermediate levels of fine textured soils and the maximum depth to groundwater is within 1.5 m of the ground surface. Where sites are dominated by coarse sand, S. exigua growth may be improved if maximum depth to groundwater is within 1 m of the ground surface.     

Peptides of the liver stage antigen-1 (LSA-1) of Plasmodium falciparum bind to human hepatocytes

Synthetic peptides from the liver stage antigen-1 (LSA-1) antigen sequence were used in HepG2 cell and erythrocyte binding assays to identify regions that could be involved in parasite invasion. LSA-1 protein peptides 20630 ((21)INGKIIKNSEKDEIIKSNLRY(40)), 20637 ((157)KEKLQGQQSDSEQERRAY(173)), 20638 ((174)KEKLQEQQSDLEQERLAY(190)) and 20639 (191KEKLQEQQSDLEQERRAY(207)) had high binding activity in HepG2 assays. Were located in immunogenic regions; peptide cell binding was saturable. Peptide 20630 bound specifically to 48kDa HepG2 membrane surface protein. LSA-1 peptides 20630 ((21)INGKIIKNSEKDEIIKSNLRY(40)) and 20633 ((81)DKELTMSNVKNVSQTNFKSLY(100)) showed specific erythrocyte binding activity and inhibited merozoite invasion of erythrocytes in vitro. A monkey serum prepared against LSA-1 20630 peptide analog (CGINGKNIKNAEKPMIIKSNLRGC) inhibited merozoite invasion in vitro. The data suggest LSA-1 "High Activity Binding Peptides" could play a possible role in hepatic cell invasion as well as merozoite invasion of erythrocytes.     

Chaperone protein 14-3-3 and protein kinase A increase the relative abundance of low agonist sensitivity human alpha 4 beta 2 nicotinic acetylcholine receptors in Xenopus oocytes

Alpha4 and beta2 nicotinic acetylcholine (nACh) receptor subunits expressed heterologously in Xenopus oocytes assemble into a mixture of receptors with high and low agonist sensitivity whose relative abundance is influenced by the heteropentamer subunit ratio. We have found that inhibition of protein kinase A by KT5720 decreased maximal [3H]cytisine binding and acetylcholine (ACh)-induced current responses, and increased the relative proportion of alpha4beta2 receptors with high agonist sensitivity. Mutation of serine 467, a putative protein kinase A substrate in a chaperone protein binding motif within the large cytoplasmic domain of the alpha4 subunit, to alanine or asparate decreased or increased, respectively, maximal [3H]cytisine binding and ACh response amplitude. Expression of alpha4S467A mutant subunits decreased steady levels of alpha4 and the relative proportion of alpha4beta2 receptors with low agonist sensitivity, whilst expression of alpha4S467D increased steady levels of alpha4 and alpha4beta2 receptors with low agonist sensitivity. Difopein, an inhibitor of chaperone 14-3-3 proteins, decreased [3H]cytisine binding and ACh responses and increased the proportion of alpha4beta2 with high sensitivity to activation by ACh. Thus, post-translational modification affecting steady-state levels of alpha4 subunits provides a possible means for physiologically relevant, chaperone-mediated variation in the relative proportion of high and low agonist sensitivity alpha4beta2 nACh receptors.     

In vivo light-driven DNA binding and cellular uptake of nucleic acid stains

Chemical derivatization of nucleic stains such as ethidium bromide or DAPI with tailored, photoresponsive caging groups, allows for "on demand" spatiotemporal control of their in vivo nucleic acid binding, as well as for improving their cellular uptake. This effect was particularly noteworthy for a nitro-veratryloxycarbonyl-caged derivative of ethidium bromide that, in contrast with the parent stain, is effectively internalized into living cells. The activation strategy works in light-accessible, therapeutically relevant settings, such as human retinas, and can even be applied for the release of active compounds in the eyes of living mice.     

Preparation, stability, and in vitro performance of vesicles made with diblock copolymers

Vesicles made completely from diblock copolymers-polymersomes-can be stably prepared by a wide range of techniques common to liposomes. Processes such as film rehydration, sonication, and extrusion can generate many-micron giants as well as monodisperse, approximately 100 nm vesicles of PEO-PEE (polyethyleneoxide-polyethylethylene) or PEO-PBD (polyethyleneoxide-polybutadiene). These thick-walled vesicles of polymer can encapsulate macromolecules just as liposomes can but, unlike many pure liposome systems, these polymersomes exhibit no in-surface thermal transitions and a subpopulation even survive autoclaving. Suspension in blood plasma has no immediate ill-effect on vesicle stability, and neither adhesion nor stimulation of phagocytes are apparent when giant polymersomes are held in direct, protracted contact. Proliferating cells, in addition, are unaffected when cultured for an extended time with an excess of polymersomes. The effects are consistent with the steric stabilization that PEG-lipid can impart to liposomes, but the present single-component polymersomes are far more stable mechanically and are not limited by PEG-driven micellization. The results potentiate a broad new class of technologically useful, polymer-based vesicles.     

Genotype and allele frequencies of polymorphic cytochromes P450 CYP1A2 and CYP2E1 in Mexicans

CYP1A2 and CYP2E1 are two of the main cytochrome P450 isoforms involved in the metabolism of commonly used drugs and xenobiotic compounds considered to be responsible for or possible participants in the development of several human diseases. Individual susceptibility to developing these pathologies relies, among other factors, on genetic polymorphism which depends on ethnic differences, as the frequency of mutant genotypes varies in different human populations. Thus the aim of this study was to investigate the frequency of CYP1A2 5'-flanking region and CYP2E1 Rsa I/Pst I polymorphisms in Mexicans by PCR-RFLP methods. The DNA of 159 subjects was analysed and mutant allele frequencies of 30% for CYP2E1 Rsa I/Pst I sites and 43% for CYP1A2 5'-flanking region were found. These frequencies are higher than those previously reported for other human populations.     

Decontamination of surfaces by lysozyme encapsulated in reverse micelles

Cells and enzymes can be used to decontaminate soil, water supplies, personal equipment, weapons and hospital equipment that have been exposed to bacteria, toxins or viruses. One of the problems associated with the use of microorganisms and enzymes for decontamination purposes is that the presence of water is not acceptable for some applications such as electronic equipment. One way of circumventing this problem is to allow the enzyme to distribute between a water phase and an organic phase-containing surfactant and then use the encapsulated enzyme in reverse micelles directly into the device to be clean. Reverse micelles were used to deliver the enzyme (lysozyme) to the cell-surface interface. They serve as a way to increase the local concentration of lysozyme and decrease the amount of water delivered. Specifically, we explored the lysis by free lysozyme and lysozyme encapsulated in reverse micelles of Klebsiella pneumoniae and Staphylococcus epidermidis attached to steel, glass, and hydroxyapatite. These two bacteria have been selected because they are known to be pathogenic and because of their differences in cell wall structure. Lysozyme was added to the surfaces in either reverse micelles or as a free solution and was tested under conditions of stirring and no stirring. Stirring was implemented to study the interplay between mass transfer limitations and surface roughness. We have shown that free lysozyme or lysozyme encapsulated in reverse micelles is capable of decontaminating surfaces of different texture. Lysis of the cells is slower when the encapsulated enzyme is used but lysis is more complete.     

Altered histology of the thymus and spleen in contaminant-exposed juvenile American alligators

Morphological differences in spleen and thymus are closely related to functional immune differences. Hormonal regulation of the immune system has been demonstrated in reptilian splenic and thymic tissue. Spleens and thymus were obtained from juvenile alligators at two reference sites in Florida, USA: Orange Lake and a National Wildlife Refuge, Lake Woodruff, as well as from a contaminated lake, Lake Apopka. Lake Apopka has been extensively polluted with agricultural pesticides. Tissues were prepared for histological analysis to determine if previously detected endocrine abnormalities associated with contaminant exposure might also be reflected in morphological differences in splenic and thymic structures important for immunological response. Similar tissues were taken from captive-raised juvenile female alligators (3 years old) that were hatched from eggs collected on Lake Woodruff and Lake Apopka. Differences in thymic ratios (medulla/cortex) were found among alligators collected from the two lakes (P = 0.0051). Alligators from Lake Apopka had smaller thymic ratios than animals from either reference lake. Males from Lake Woodruff had significantly smaller lymphocyte sheaths in the spleen than females (P = 0.0009), indicative of a normal sexual dimorphism. Lymphocyte sheath width differed among females obtained from the three lakes, with females from Lake Apopka having the smallest sheath width and those from Orange Lake having the largest. Malpighian body area was largest in alligators from Orange Lake, intermediate in Lake Woodruff, and smallest in Lake Apopka. In contrast to that observed for wild-caught animals, no difference was found in the thymic medulla/cortex ratio of captive-raised female alligators (P = 0.378). Captive-raised female alligators from Lake Apopka and Lake Woodruff displayed lake-associated differences in lymphocyte sheath width as observed in wild animals; Lake Apopka alligators had smaller lymphocyte sheath width compared to Woodruff alligators (P = 0.0396). In contrast to wild-caught animals, area of the Malpighian bodies did not differ by lake in the captive-raised female alligators (P = 0.066). The enlarged thymic cortex suggests a change in T-lymphocyte maturation within the thymus of alligators from a contaminated environment, Lake Apopka. The results point to alterations in the histology of the thymus and spleen. Further studies are required to examine the functional significance of these observations.     

Evolutionary potential of (beta/alpha)8-barrels: functional promiscuity produced by single substitutions in the enolase superfamily

The members of the mechanistically diverse, (beta/alpha)(8)-barrel fold-containing enolase superfamily evolved from a common progenitor but catalyze different reactions using a conserved partial reaction. The molecular pathway for natural divergent evolution of function in the superfamily is unknown. We have identified single-site mutants of the (beta/alpha)(8)-barrel domains in both the l-Ala-d/l-Glu epimerase from Escherichia coli (AEE) and the muconate lactonizing enzyme II from Pseudomonas sp. P51 (MLE II) that catalyze the o-succinylbenzoate synthase (OSBS) reaction as well as the wild-type reaction. These enzymes are members of the MLE subgroup of the superfamily, share conserved lysines on opposite sides of their active sites, but catalyze acid- and base-mediated reactions with different mechanisms. A comparison of the structures of AEE and the OSBS from E. coli was used to design the D297G mutant of AEE; the E323G mutant of MLE II was isolated from directed evolution experiments. Although neither wild-type enzyme catalyzes the OSBS reaction, both mutants complement an E. coli OSBS auxotroph and have measurable levels of OSBS activity. The analogous mutations in the D297G mutant of AEE and the E323G mutant of MLE II are each located at the end of the eighth beta-strand of the (beta/alpha)(8)-barrel and alter the ability of AEE and MLE II to bind the substrate of the OSBS reaction. The substitutions relax the substrate specificity, thereby allowing catalysis of the mechanistically diverse OSBS reaction with the assistance of the active site lysines. The generation of functionally promiscuous and mechanistically diverse enzymes via single-amino acid substitutions likely mimics the natural divergent evolution of enzymatic activities and also highlights the utility of the (beta/alpha)(8)-barrel as a scaffold for new function.