Geosmin and MIB events in a new reservoir in southern California.

Diamond Valley Lake is a large drinking water reservoir in western Riverside County, California near the city of Hemet. In almost 6 years since filling began in 1999, this reservoir has experienced five episodes involving either geosmin or 2-methylisoborneol (MIB). The first one was a short-lived but intense geosmin event in May 2000, associated with a planktonic cyanobacterium, Anabaena circinalis. Geosmin levels reached 750 ng/L at the surface. All the other episodes involved benthic proliferations in the littoral zone. In September 2002, an MIB-producing growth developed in a shallow area near the outlet tower, dominated by Oscillatoria cf. curviceps and O. limosa. A similar event occurred in October 2003. In March 2004, an extensive growth of cyanobacteria that included two geosmin producers developed at the east dam, but had minimal effect on geosmin levels in the water. Finally, there was a major MIB episode in October 2004, in which the primary causative organism was again Oscillatoria cf. curviceps. A band of benthic cyanobacteria developed all around the shoreline from 3-9 m deep, and surface MIB levels reached 63 ng/L. These events showed that a new reservoir in a mild climate can be colonised by benthic cyanobacteria that produce MIB and geosmin, within a relatively short time.

A guide to geosmin- and MIB-producing cyanobacteria in the United States.

A guide to confirmed geosmin- and MIB-producing cyanobacteria isolated in the United States is being prepared. This document will include 41 different species or morphologically distinct types from eight states and diverse aquatic sources isolated over a 22-year period. The organisms comprised by this guide demonstrate the importance of attached cyanobacteria as off-flavor agents, the strain specificity of MIB production, the existence of unicellular MIB producers, the occurrence of multiple geosmin and MIB producers in reservoirs, and the relationship of certain planktonic odor producers to species in other countries.

The cytoskeletal/non-muscle isoform of alpha-actinin is phosphorylated on its actin-binding domain by the focal adhesion kinase.

alpha-Actinin is tyrosine-phosphorylated in activated human platelets (Izaguirre, G., Aguirre, L., Ji, P., Aneskievich, B., and Haimovich, B. (1999) J. Biol. Chem. 274, 37012--37020). Analysis of platelet RNA by reverse transcription-polymerase chain reaction revealed that alpha-actinin expressed in platelets is identical to the cytoskeletal/non-muscle isoform. A construct of this isoform containing a His(6) tag at the amino terminus was generated. Robust tyrosine phosphorylation of the recombinant protein was detected in cells treated with the tyrosine phosphatase inhibitor vanadate. The tyrosine phosphorylation site was localized to the amino-terminal domain by proteolytic digestion. A recombinant alpha-actinin protein containing a Tyr --> Phe mutation at position 12 (Y12F) was no longer phosphorylated when expressed in vanadate-treated cells, indicating that tyrosine 12 is the site of phosphorylation. The wild type recombinant protein was not phosphorylated in cells lacking the focal adhesion kinase (FAK). Re-expression of FAK in these cells restored alpha-actinin phosphorylation. Purified wild type alpha-actinin, but not the Y12F mutant, was phosphorylated in vitro by wild type as well as a Phe-397 mutant of FAK. In contrast, no phosphorylation was detected in the presence of a kinase-dead FAK. Tyrosine phosphorylation reduced the amount of alpha-actinin that cosedimented with actin filaments. These results establish that alpha-actinin is a direct substrate for FAK and suggest that alpha-actinin mediates FAK-dependent signals that could impact the physical properties of the cytoskeleton.

Human aldehyde dehydrogenase catalytic activity and structural interactions with coenzyme analogs.

K(m) and V(max) values for 10 coenzyme analogs never previously studied with any aldehyde dehydrogenase and NADP(+) were compared with those for NAD(+) for three human aldehyde dehydrogenases (EC 1.2.1.3); the cytoplasmic E1 (the product of the aldh1 gene), the mitochondrial E2 (the product of the aldh2 gene) and the cytoplasmic E3 (the product of the aldh9 gene) isozymes. Structural information on changes in coenzyme-protein interactions were obtained via molecular dynamics (MD) studies with the E2 isozyme and quantum mechanical (QM) calculations were used to study changes in charge distribution of the pyridine ring and relative free energies of solvation of the purine ring in the analogs. E1 showed the broadest substrate specificity and was the only isozyme subject to substrate inhibition, both of which are suggested to be due to the two coenzyme conformations observed previously in the sheep crystal structure. NADP(+) selectivity is indicated to be influenced by Glu195 in E1 and E2. Substitutions in the purine ring affected K(m) but not V(max), with the changes in K(m) being dominated by the hydrophobicity of the purine ring as indicted by the QM calculations. Substitutions in the pyridine ring sometimes rendered the coenzymes inactive, with no consistent pattern observed for the three coenzymes. Structural analysis of the coenzyme analog-E2 MD simulations revealed different structural perturbations of the surrounding active site, though interactions with Asn169 and Glu399 were preserved in all cases.

Tyrosine phosphorylation of alpha-actinin in activated platelets.

The integrin alpha(IIb)beta(3) mediates tyrosine phosphorylation of a 105-kDa protein (pp105) in activated platelets. We have partially purified a 105-kDa tyrosine-phosphorylated protein from platelets stimulated with phorbol 12-myristate 13-acetate and obtained the sequence of an internal 12-mer peptide derived from this protein. The sequence was identical to human alpha-actinin sequences deposited in the Swiss Protein Database. alpha-Actinin, a 105-kDa protein in platelets, was subsequently purified from activated platelets by four sequential chromatographic steps. Fractions were analyzed by Western blotting and probed with alpha-actinin and anti-phosphotyrosine antibodies. The distribution of alpha-actinin and pp105 overlapped throughout the purification. Furthermore, in the course of this purification, a 105-kDa tyrosine-phosphorylated protein was only detected in fractions that contained alpha-actinin. The purified alpha-actinin protein was immunoprecipitated with antibodies to phosphotyrosine in the absence but not in the presence of phenyl phosphate. alpha-Actinin resolved by two-dimensional gel electrophoresis of activated platelet lysates was recognized by the antibodies to phosphotyrosine, whereas pretreatment of the platelets with bisindolylmaleimide, a protein kinase C inhibitor that prevents tyrosine phosphorylation of pp105, inhibited the reactivity of the antibodies to phosphotyrosine with alpha-actinin. Taken together, these data demonstrate that a fraction of alpha-actinin is tyrosine-phosphorylated in activated platelets.

Metabolism of retinaldehyde and other aldehydes in soluble extracts of human liver and kidney.

Purification and characterization of enzymes metabolizing retinaldehyde, propionaldehyde, and octanaldehyde from four human livers and three kidneys were done to identify enzymes metabolizing retinaldehyde and their relationship to enzymes metabolizing other aldehydes. The tissue fractionation patterns from human liver and kidney were the same, indicating presence of the same enzymes in human liver and kidney. Moreover, in both organs the major NAD(+)-dependent retinaldehyde activity copurified with the propionaldehyde and octanaldehyde activities; in both organs the major NAD(+)-dependent retinaldehyde activity was associated with the E1 isozyme (coded for by aldh1 gene) of human aldehyde dehydrogenase. A small amount of NAD(+)-dependent retinaldehyde activity was associated with the E2 isozyme (product of aldh2 gene) of aldehyde dehydrogenase. Some NAD(+)-independent retinaldehyde activity in both organs was associated with aldehyde oxidase, which could be easily separated from dehydrogenases. Employing cellular retinoid-binding protein (CRBP), purified from human liver, demonstrated that E1 isozyme (but not E2 isozyme) could utilize CRBP-bound retinaldehyde as substrate, a feature thought to be specific to retinaldehyde dehydrogenases. This is the first report of CRBP-bound retinaldehyde functioning as substrate for aldehyde dehydrogenase of broad substrate specificity. Thus, it is concluded that in the human organism, retinaldehyde dehydrogenase (coded for by raldH1 gene) and broad substrate specificity E1 (a member of EC 1. 2.1.3 aldehyde dehydrogenase family) are the same enzyme. These results suggest that the E1 isozyme may be more important to alcoholism than the acetaldehyde-metabolizing enzyme, E2, because competition between acetaldehyde and retinaldehyde could result in abnormalities associated with vitamin A metabolism and alcoholism.

Studies of the subtilin leader peptide as a translocation signal in Escherichia coli K12.

Subtilin is an antimicrobial peptide of the lantibiotic family that is produced by Gram-positive Bacillus subtilis, and its biosynthesis involves expression of presubtilin which consists of a leader segment and a mature segment. The leader segment is unlike a typical sec-type general secretion signal, and its ability to mediate translocation through a non-sec pathway has been previously studied by fusing the subtilin leader to an alkaline phosphatase reporter and expressing it in B. subtilis 168 [Izaguirre, G. and Hansen, J. N. (1997) Appl. Environ. Microbiol. 63, 3965-3971]. In this work, we have expressed the same subtilin leader-AP fusion in Gram-negative Escherichia coli, and found that the AP polypeptide is translocated into the periplasmic compartment and assembles into an enzymatically active form. The subtilin leader segment was not cleaved from this enzymatically active AP, which remained associated with the membrane. Conversion of the cells to spheroplasts followed by treatment with proteinase K showed that about 50% of the bound AP was sufficiently exposed on the surface of the spheroplasts to be inactivated by proteolytic cleavage.

Methylglyoxal as substrate and inhibitor of human aldehyde dehydrogenase: comparison of kinetic properties among the three isozymes.

Methylglyoxal was demonstrated to be a substrate for the isozymes E1, E2 and E3 of human aldehyde dehydrogenase. Pyruvate was the product from the oxidation of methylglyoxal by the three isozymes. At pH 7.4 and 25 degrees C, the major and minor components of the E3 isozyme catalyzed the reaction with Vmax of 1.1 and 0.8 mumol NADH min-1 mg-1 protein, respectively, compared to 0.067 and 0.060 mumol NADH min-1 mg-1 protein for the E1 and E2 isozymes, respectively. The E2 isozyme had a K(m) for methylglyoxal of 8.6 microM, the lowest compared to 46 microM for E1 and 586 and 552 microM for the major and minor components of the E3 isozyme, respectively. Both components of the E3 isozyme showed substrate inhibition by methylglyoxal, with Ki values of 2.0 mM for the major component and 12 mM for the minor component at pH 9.0. Substrate inhibition by methylglyoxal was not observed with the E1 and E2 isozymes. Methylglyoxal strongly inhibited the glycolaldehyde activity of the E1 and E2 isozymes. Mixed-type models of inhibition were employed as an approach to calculate the inhibition constants, 44 and 10.6 microM for E1 and E2 isozymes, respectively.

Use of alkaline phosphatase as a reporter polypeptide to study the role of the subtilin leader segment and the SpaT transporter in the posttranslational modifications and secretion of subtilin in Bacillus subtilis 168.

The subtilin leader segment of presubtilin was fused to alkaline phosphatase (AP), which was used as a reporter polypeptide to study the role of the subtilin leader segment in posttranslational modifications during the conversion of presubtilin to subtilin and in the translocation of presubtilin from the cytoplasm of Bacillus subtilis 168 to the extracellular medium. It was observed that the subtilin leader segment could be utilized by a wild-type transporter, but secretion was enhanced if the subtilin transporter was available. The subtilin leader was not cleaved away from the AP component of the precursor until the precursor had been transported to the cell wall, and none of the AP was released into the medium until after cleavage had occurred. The role of SpaT, which is an ABC transporter that has been implicated in subtilin secretion, was explored by making a large in-frame deletion from the central region of SpaT and observing the effect on translocation of the AP reporter. Instead of having an effect on translocation, the deletion disrupted proteolytic cleavage of the subtilin leader segment and release of the AP reporter into the medium. The AP that was secreted by means of the subtilin leader segment had not undergone any posttranslational modifications, as assessed by amino acid composition analysis and enzymatic activity analysis.

Tissue distribution of human aldehyde dehydrogenase E3 (ALDH9): comparison of enzyme activity with E3 protein and mRNA distribution.

The tissue distribution of the E3 isozyme of human aldehyde dehydrogenase has been investigated by three methods: enzyme activity assay employing betaine aldehyde as substrate, Western blotting employing E3 isozyme-specific antibodies, and Northern blotting using a human liver E3 cDNA as probe. All three methods showed that E3 isozyme was universally distributed among all tissues tested. The highest levels of the E3 isozyme activity were found in liver, adrenal gland, and kidney. These same tissues also showed highest levels of the E3 protein via the Western blot. This distribution is consistent with the possible physiological role of E3 isozyme in the synthesis of the osmolyte, betaine, and the neurotransmitter, GABA. Northern blot analysis, however, differed from that of enzyme assay and the Western blot in that it showed highest mRNA levels in skeletal and heart muscles, which had low enzyme activities and E3 protein levels.

Ammonia-oxidizing bacteria in a chloraminated distribution system: seasonal occurrence, distribution and disinfection resistance.

Nitrification in chloraminated drinking water can have a number of adverse effects on water quality, including a loss of total chlorine and ammonia-N and an increase in the concentration of heterotrophic plate count bacteria and nitrite. To understand how nitrification develops, a study was conducted to examine the factors that influence the occurrence of ammonia-oxidizing bacteria (AOB) in a chloraminated distribution system. Samples were collected over an 18-month period from a raw-water source, a conventional treatment plant effluent, and two covered, finished-water reservoirs that previously experienced nitrification episodes. Sediment and biofilm samples were collected from the interior wall surfaces of two finished-water pipelines and one of the covered reservoirs. The AOB were enumerated by a most-probable-number technique, and isolates were isolated and identified. The resistance of naturally occurring AOB to chloramines and free chlorine was also examined. The results of the monitoring program indicated that the levels of AOB, identified as members of the genus Nitrosomonas, were seasonally dependent in both source and finished waters, with the highest levels observed in the warm summer months. The concentrations of AOB in the two reservoirs, both of which have floating covers made of synthetic rubber (Hypalon; E.I. du Pont de Nemours & Co., Inc., Wilmington, Del.), had most probable numbers that ranged from less than 0.2 to greater than 300/ml and correlated significantly with temperature and levels of heterotrophic plate count bacteria. No AOB were detected in the chloraminated reservoirs when the water temperature was below 16 to 18 degrees C. The study indicated that nitrifiers occur throughout the chloraminated distribution system. Higher concentrations of AOB were found in the reservoir and pipe sediment materials than in the pipe biofilm samples. The AOB were approximately 13 times more resistant to monochloramine than to free chlorine. After 33 min of exposure to 1.0 mg of monochloramine per liter (pH 8.2, 23 degrees C), 99% of an AOB culture was inactivated. The amounts of this disinfectant that are currently used (1.5 mg/liter at a 3:1 ratio of chlorine to ammonia-N) may be inadequate to control the growth of these organisms in the distribution system.

Degradation of 2-methylisoborneol by aquatic bacteria.

2-Methylisoborneol (MIB) is a musty- or muddy-smelling compound which occurs in some natural waters and which is difficult to remove by conventional water treatment methods. Bacterial degradation of MIB was examined in batch culture experiments. Cultures able to metabolize MIB were enriched in a mineral salts medium supplemented with milligram-per-liter levels of the compound and were inoculated with water and sediment samples from reservoirs where MIB is seasonally produced. Bacteria from degrading cultures were isolated on R2A agar and identified as predominantly Pseudomonas spp. Degradation occurred only in cultures consisting of three or more different bacteria. MIB supported growth as the sole added carbon source at 1 to 6.7 mg/liter. MIB was also degraded at microgram-per-liter levels in sterile filtered lake water inoculated with washed bacteria and in synthetic medium supplemented with various sugars or acetate. Complete degradation of MIB took from 5 days to more than 2 weeks. Enrichment with isoborneol, a structural analog of MIB, failed as a preenrichment for MIB degraders. Isoborneol at 20 to 40 mg/liter readily supported bacterial growth, whereas MIB at 12 to 20 mg/liter took months to degrade. The relative recalcitrance of MIB compared with isoborneol may be a result of the additional methyl group in MIB.

Geosmin and 2-methylisoborneol from cyanobacteria in three water supply systems.

Three Oscillatoria strains and one Anabaena species were isolated from three different water supply systems in California that experienced earthy-musty taste and odor problems in their drinking water. Unialgal cultures, free of actinomycetes, were purged using the Grob closed-loop stripping analysis method, and the resulting methylene chloride extracts were analyzed on a gas chromatograph/mass spectrometer. Geosmin was produced by Oscillatoria simplicissima and Anabaena scheremetievi, and 2-methylisoborneol was produced by O. curviceps and O. tenuis. These compounds are the two major causes of earthy-musty tastes and odors in water. In three instances, the major odorant found in culture was previously identified in the water or sediment sample from which the respective organism was isolated. O. curviceps was implicated in a taste and odor episode involving 2-methylisoborneol in a major reservoir. Geosmin and 2-methylisoborneol were easily detected with culture samples of only 4 to 25 ml.

Melanoma maligno de la coroides en un niño de 13 meses. Informe de un caso / Choroid malignant melanoma in a 13-month old infant. Report of one case

Se presenta un caso de melanoma maligno en la coroides de crecimiento plano con signos de buftalmía e hipertensión ocular en un paciente de dos meses de nacido, con comprobación histopatológica a los trece meses de edad, con invasión al cuerpo ciliar, iris y cámara anterior. Se hace breve revisión de la literatura médica extranjera (AU)