Glutathione S-transferase and S-crystallins of cephalopods: Evolution from active enzyme to lens-refractive proteins

Our previous studies have shown that the S-crystallins of cephalopod (Ommastrephes sloani pacificus) eye lenses comprise a family of at least ten members which are evolutionarily related to glutathione S-transferase (GST, EC 2.5.1.18). Here we show by cDNA cloning that there are at least 24 different S-crystallins that are 46–99% identical to each other by amino acid sequence in the squid Loligo opalescens. In each species, all but one S-crystallin (SL11 in O. pacificus and Lops4 in L. opalescens) examined has an inserted central peptide of variable length and sequence. cDNA expression studies conducted in Escherichia coli showed that squid GST (which is expressed little in the lens) has very high enzymatic activity using 1-chloro-2, 4-dinitrobenzene (CDNB) as a substrate; by contrast, SL20-1 of O. pacificus and Lops 12 of L. opalescens (which are encoded by abundant lens mRNAs) have no GST activity. Interestingly, SL11 and Lops4 have some enzymatic activity with the CDNB substrate. Site-specific mutations at Y7 or W38, both residues essential for activity of vertebrate GSTs, or insertion of the central peptide present in the inactive SL20-1, reduced the specific activity of squid GST by 30- to 100-fold. These data indicate that the S-crystallins consist of a family of enzymatically inactive proteins (when using CDNB as a substrate) which is considerably larger than previously believed and that GST activity was lost by gradual drift in sequence as well as by insertion of an extra peptide by exon shuffling. The results are also consistent with the idea that SL11 and Lops4 are orthologous crystallins representing the first descendants of the ancestral GST gene in the pathway which gave rise to the extensive S-crystallin family of lens proteins. Correspondence to: S.I. Tomarev     

Prostaglandin H Synthetase-Mediated Metabolism of Dopamine: Implication for Parkinson's Disease

Abstract: Differences in prostaglandin H synthetase (PHS) activity in the substantia nigra of age- and post-mortem interval-matched parkinsonian, Alzheimer's, and normal control brain tissue were assessed. Prostaglandin E₂ (PGE₂, an index of PHS activity) was higher in substantia nigra of parkinsonian brain tissue than Alzheimer's or control tissue. Incubation of substantia nigra slices with arachidonic acid (AA) increased PGE₂ synthesis. Dopamine stimulated PHS synthesis of PGE₂. [³H]Dopamine was activated by PHS to electrophilic intermediate(s) that covalently bound to DNA, microtubulin protein, bovine serum albumin, and sulfhydryl reagents. When AA was replaced by hydrogen peroxide, PHS/H₂O₂-supported binding proceeded at rates similar to those observed with PHS/AA. Indomethacin and aspirin inhibited AA-mediated cooxidation of dopamine but not H₂O₂-mediated metabolism. PHS-mediated metabolism of dopamine was not affected by monoamine oxidase inhibitors. Substrate requirements and effects of specific inhibitors suggest cooxidation of dopamine is mediated by the hydroperoxidase activity of PHS. ³²P-postlabeling was used to detect dopamine-DNA adducts. PHS/AA activation of dopamine in the presence of DNA resulted in the formation of five dopamine-DNA adducts, i.e., 23, 43, 114, 70, and 270 amol/µg DNA. DNA adduct formation was PHS, AA, and dopamine dependent. PHS catalyzed cooxidation of dopamine in dopaminergic neuronal degeneration is discussed.     

Polymorphism, mesomorphism, and metastability of monoelaidin in excess water.

The polymorphic and metastable phase behavior of monoelaidin dry and in excess water was studied by using high-sensitivity differential scanning calorimetry and time-resolved x-ray diffraction in the temperature range of 4 degrees C to 60 degrees C. To overcome problems associated with a pronounced thermal history-dependent phase behavior, simultaneous calorimetry and time-resolved x-ray diffraction measurements were performed on individual samples. Monoelaidin/water samples were prepared at room temperature and stored at 4 degrees C for up to 1 week before measurement. The initial heating scan from 4 degrees C to 60 degrees C showed complex phase behavior with the sample in the lamellar crystalline (Lc0) and cubic (Im3m, Q229) phases at low and high temperatures, respectively. The Lc0 phase transforms to the lamellar liquid crystalline (L alpha) phase at 38 degrees C. At 45 degrees C, multiple unresolved lines appeared that coexisted with those from the L alpha phase in the low-angle region of the diffraction pattern that have been assigned previously to the so-called X phase (Caffrey, 1987, 1989). With further heating the X phase converts to the Im3m cubic phase. Regardless of previous thermal history, cooling calorimetric scans revealed a single exotherm at 22 degrees C, which was assigned to an L alpha+cubic (Im3m, Q229)-to-lamellar gel (L beta) phase transition. The response of the sample to a cooling followed by a reheating or isothermal protocol depended on the length of time the sample was incubated at 4 degrees C. A model is proposed that reconciles the complex polymorphic, mesomorphic, and metastability interrelationships observed with this lipid/water system. Dry monoelaidin exists in the lamellar crystalline (beta) phase in the 4 degrees C to 45 degrees C range. The beta phase transforms to a second lamellar crystalline polymorph identified as beta* at 45 degrees C that subsequently melts at 57 degrees C. The beta phase observed with dry monoelaidin is identical to the LcO phase formed by monoelaidin that was dispersed in excess water and that had not been previously heated.     

Multiple genes encoding 2,3-dihydroxybiphenyl 1,2-dioxygenase in the gram-positive polychlorinated biphenyl-degrading bacterium Rhodococcus erythropolis TA421, isolated from a termite ecosystem.

Rhodococcus erythropolis TA421 was isolated from a termite ecosystem and is able to degrade a wide range of polychlorinated biphenyl (PCB) congeners. Genetic and biochemical analyses of the PCB catabolic pathway of this organism revealed that there are four different bphC genes (bphC1, bphC2, bphC3, and bphC4) which encode 2,3-dihydroxybiphenyl dioxygenases. As determined by Southern hybridization, none of the bphC genes exhibits homology to any other bphC gene. bphC1, bphC2, and bphC4 encode enzymes that have narrow substrate specificities and cleave the first aromatic ring in the meta position. In contrast, bphC3 encodes a meta cleavage dioxygenase with broad substrate specificity. Asturias et al. have shown that the closely related organism Rhodococcus globerulus P6 contains three different bphC genes (bphC1, bphC2, and bpHC3) which encode meta cleavage dioxygenases. The data suggest that there is a diverse family of bphC genes which encode PCB meta cleavage dioxygenases in members of the genus Rhodococcus.     

A serotype-specific epitope of dengue virus 1 identified by phage displayed random peptide library

Abstract From a panel of monoclonal antibodies of dengue viruses, a serotype-specific epitope of dengue virus 1 was screened from a random peptide library displayed on phage. The epitope was the determinant reactive with monoclonal antibody 15F3-1 that was specific to dengue 1. The screening was monitored by a dot blotting procedure, and after three rounds of screening a consensus motif, HRYSWK, was found. This sequence matches the sequence HKYSWK, corresponding to the amino acid residues 885–890 of polyprotein or residues 111–116 of the non-structural protein 1 of dengue virus serotype 1. The linear epitope was confirmed by testing the antigenicity of chemically synthesized 8-branched peptide.     

Prostaglandin F2 alpha increases responsiveness of pulmonary airways in dogs

We studied the effect of prostaglandin F2 alpha (PGF2 alpha) on the responsiveness of pulmonary airways in dogs. Airway responsiveness was assessed by determining the bronchoconstrictor response to increasing concentrations of acetylcholine aerosol delivered to the airways. In each of five dogs, we determined responsiveness during treatment with physiologic saline, histamine, or PGF2 alpha aerosols. The doses of histamine and PGF2 alpha were determined by establishing the largest dose of each which could be given to the dog without causing bronchoconstriction (subthreshold doses). We found that airway responsiveness was not significantly different during histamine treatment than after saline, however, responsiveness increased during treatment with PGF2 alpha. In addition, the hyperresponsiveness induced by PGF2 alpha was prevented by pretreatment with the ganglion blocking drug hexamethonium (5 mg/kg given intravenously). The results show that PGF2 alpha specifically increases the responsiveness of pulmonary airways in doses that do not cause bronchoconstriction, and suggest that the hyperresponsiveness involves a neural mechanism such as increased responsiveness of airway sensory nerves.     

Ultrastructure and enzyme digestion of nucleoli and associated structures in hypothalamic nerve cells viewed in resinless sections

Estrogen has been shown to affect ventromedial hypothalamic (VMH) nerve cell nucleoli in ovariectomized rats, by causing an increase in the number of electron-dense aggregates associated with nucleoli. In order to characterize these nucleolus-associated structures and other nuclear components, we examined the ultrastructure of ventromedial hypothalamic nucleoli and nuclei revealed by enzyme digestions (pepsin, RNase and DNase) in resinless thin sections. Digestion by pepsin did not cause obvious alterations in the morphology of the nucleolus or its related structures. Pepsin treatment followed by RNase, however, reduced the density of the nucleolus, while that of the nucleolus-associated structure and other related structures remained unchanged. Conversely pepsin treatment followed by DNase, reduced the density of nucleolus-associated and other chromatin structures, but had no effect on the density of the nucleolus. Pepsin treatment followed by RNase and then DNase treatment, reduced the density of the nucleolus and nucleolus-associated structures. A residual nucleolus and nucleolus-associated structure remained after this treatment. Stereo viewing of resinless sections shows that the nucleolus, its associated structures, and other related structures, are associated with fine filaments that may comprise the nuclear matrix. The nucleolus-associated structure containing DNA may direct RNA synthesis at an increased rate in estrogen-treated hypothalamic cells.     

Crosslinking of uteroglobin by transglutaminase

Uteroglobin, a progesterone induced, pregnancy related protein, can be incorporated into higher molecular weight proteins by human placental Factor XIIIa. This process is time dependent, requires CaCl2 and can be inhibited by the addition of polylysine, dansylcadavarine or histamine. Crosslinking of uteroglobin into higher molecular weight proteins can also be brought about by guinea pig liver transglutaminase. Such a process may be involved in the modification of epididymal spermatozoa to suppress their antigenicity.