Characterization of two isoenzymes of lipoxygenase from bush beans

Two isoenzymes of lipoxygenase, a and b, have been obtained from bush beans (Phaseolus vulgaris) as electrophoretically homogeneous proteins. Both proteins have a molecular weight of 100,000, contain 1 atom of iron, and appear to be composed of a single peptide chain. However, these enzymes appear to differ in some other respects. Thus, lipoxygenase a has an isoelectric point of 6.03 while lipoxygenase b has a value of 5.57. Their pH optima are 5 to 7 and 6.5 to 7, respectively. Both lipoxygenase a and b, when acting on linoleic acid plus the product hydroperoxide, generate what are presumably keto-dienes with an absorption maximum at 280 nm. Whereas lipoxygenase a can catalyze this secondary reaction in the presence of O₂, lipoxygenase b does so only under anaerobic conditions. Lipoxygenase a is stimulated by Ca²⁺ while lipoxygenase is not. An unexpected finding is the strong inhibition of lipoxygenase a by Mn²⁺ (50% inhibition at 12.5 μM under standard reaction conditions). Lipoxygenase b is inhibited by Mn²⁺ but only at concentrations about 250 times greater.     

Age‐specific mortality analysis of the dry forest kissing bug,Rhodnius neglectus

Age‐specific mortality patterns can be very different across insects with different life histories. Some holometabolous insects (like mosquitoes, fruit flies) show a pattern where mortality rate decelerates at older ages, whereas other holometabolous insects (bruchid beetles) and hemimetabolous insects (cotton stainers, milkweed bugs, and kissing bugs) show an age‐specific mortality pattern that increases through all ages. Kissing bugs are strictly hematophagous and are vectors of Trypanosoma cruzi Chagas, the etiologic agent of Chagas disease. Here, we tested whether cohort data from the dry forest kissing bug, Rhodnius neglectus Lent (Hemiptera: Reduviidae), supports an increase of mortality rate that decelerates with age. We analyzed the age‐specific mortality pattern of a cohort of 250 individuals of R. neglectus. We used a suite of seven models with different degrees of complexity, to model age‐dependent forms of change in mortality rate increase in R. neglectus in the laboratory. We used the Akaike model selection criterion to choose between models that consider absence or presence of mortality deceleration. Five of the seven models (logistic, Gavrilovs, Gompertz, DeMoivre, and exponential) showed a statistically significant fit to the mortality rate. Weak late‐age mortality deceleration in R. neglectus was supported by the best fit (logistic model), and this result is consistent with predictions of the disposable soma theory of senescence.     

Occurrence and biology of Rhodnius neglectus Lent, 1954 in palm trees of suburban areas of Belo Horizonte, Minas Gerais

Eighty-one "macaubeiras" palm-trees (Acrocomia sclerocarpa) from the periphery of Belo Horizonte city were cut down and dissected between April 1979 and July 1980. 60.5% of the trees were positive for R. neglectus and 463 specimens of this insect were collected, providing a mean rate of 9,45 individuals for positive palm-trees. R. neglectus seems to present a single annual generation in this region, but evidence was obtained that two generations may occur. Its spawn period is related to the hot months of the year, when younger insects prevail over the adults. This observation suggests that higher population densities are related to better feeding conditions and to minor densities of specific predators such as Telenomus sp., ants, spiders, hemipters, scorpions and pseudo scorpionidae. The Trypanosoma cruzi infection index was 15,9%, suggesting that R. neglectus is a very important vector of silvatic infections in the region. Marsupials are the main vertebrate source of T. cruzi infection. R. neglectus is strongly associated with palm-trees in the area, and with the various species of birds that haunt them. So far, there is no evidence suggesting R. neglectus to be an important vector of human Chagas' disease in the region.     

Comparative morphology and morphometry of alveolar macrophages from six species

Pulmonary alveolar macrophages (PAM) were collected from normal, healthy mice, rats, dogs, cynomolgus monkeys, chimpanzees, and humans and evaluated for morphologic and morphometric characteristics. The PAM of mice, rats, and dogs were morphologically similar and had statistically similar frequency distributions for size. The cell size distribution for these three species was relatively homogeneous. The PAM of nonhuman primates and humans were morphologically heterogenous with sometimes prominent cytoplasmic vacuolation, irregular cell outlines, and increased numbers of multinucleated cells as compared to the PAM of rodents and dogs. The mean size of human PAM was statistically greater than that for all other species evaluated, including nonhuman primates. These data indicate that significant differences in PAM morphology and size exist among species.     

Characterization of two glyceraldehyde-3-phosphate dehydrogenase isoenzymes from the pentalenolactone producer Streptomyces arenae.

Pentalenolactone (PL) irreversibly inactivates the enzyme glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating)] (EC 1.2.1.12) and thus is a potent inhibitor of glycolysis in both procaryotic and eucaryotic cells. We showed that PL-producing strain Streptomyces arenae TU469 contains a PL-insensitive glyceraldehyde-3-phosphate dehydrogenase under conditions of PL production. In complex media no PL production was observed, and a PL-sensitive glyceraldehyde-3-phosphate dehydrogenase, rather than the insensitive enzyme, could be detected. The enzymes had the same substrate specificity but different catalytic and molecular properties. The apparent Km values of the PL-insensitive and PL-sensitive enzymes for glyceraldehyde-3-phosphate were 100 and 250 microM, respectively, and the PL-sensitive enzyme was strongly inhibited by PL under conditions in which the PL-insensitive enzyme was not inhibited. The physical properties of the PL-insensitive enzyme suggest that the protein is an octamer, whereas the PL-sensitive enzyme, like other glyceraldehyde-3-phosphate dehydrogenases, appears to be a tetramer.     

Characterization of two cryptic species,Culicoides stigma and C.parroti (Diptera: Ceratopogonidae), based on barcode regions and morphology

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are insect vectors of economically important veterinary diseases such as African horse sickness, bluetongue, and Schmallenberg virus. The identification of Culicoides based on morphological features can be difficult. Three species of biting midges, Culicoides nubeculosus, C. stigma, and C. parroti have emerged in the laboratory from mud collected around watering troughs on a farm in northern France. Emerging Culicoides were characterized morphologically and molecularly using molecular markers. The closely related species C. stigma and C.parroti showed highly divergent sequences for both mitochondrial (cytochrome B and cytochrome oxidase I) and ribosomal DNA first internal transcribed spacer. A RFLP based on a single restriction using the same enzyme (HaeIII) for both cytochrome C oxidase I and cytochrome B is proposed to identify these species.     

When morphometry meets genetics: inferring the phylogeography of Carabus solieri using Fourier analyses of pronotum and male genitalia

Population differentiation is a crucial step in the speciation process and is therefore a central subject in studies of microevolution. Assessing divergence and inferring its dynamics in space and time generally require a wide array of markers. Until now however, most studies of population structure are based on molecular markers and those concerning morphological traits are more scarce. In the present work, we studied morphological differentiation among populations of the ground beetle Carabus solieri, and tested its congruence with genetic population structure. The shape of pronotum and aedeagus was assessed using Dual Axis Fourier Shape Analysis. manova on Fourier coefficients revealed highly significant morphological variation between populations and a similar geographical pattern of differentiation for both structures. On the whole, morphological and genetic patterns were also found to be congruent. Our analysis confirms the phylogeographical scenario proposing that two entities of C. solieri differentiated during the last glaciation events before recolonizing the actual range of the species. It also indicates a large introgression between the two differentiated entities in the centre of the range.     

Purification and characterization of two isoenzymes of lipoxygenase from soybeans

A chromatographic procedure for the purification of two lipoxygenase isoenzymes (linoleate: O₂ oxidoreductase, EC 1.13.11.12.) from soybean is described. The procedure for the purification of isoenzyme L-1 includes optimalized extraction, ammonium sulfate fractionation, heat treatment and gradient elution from a CM-Sephadex C-50 column. The purification of L-2 includes ammonium sulfate fractionation, gelfiltration on Sephadex G-150 and gradient elution from a DEAE-cellulose column. Both isoenzymes L-1 and L-2 appear homogeneous after Disc-PAGE. The isoelectric points are 5.6 for L-1 and 5.8 for L-2. Molecular weights are estimated as 100,000 for L-1 as well as L-2 applying three different methods. Both isoenzymes contain 0.9 mol iron per mol protien. The estimated turn over numbers are 8,200 mol linoleate per mol enzyme and min for L-1 and 3,100 for L-2. Amino acid compositions determined after acid hydrolysis show marked differences between L-1 and L-2, particularly with respect to the amino acids Lys, Phe, Ser, Gly and Leu. L-1 posesses a total of 9 cysteine molecules, 6 of which are present as disulfide bonds. L-2 posesses a total of 8 cysteine molecules with only one disulfide bond.These results have been presented in part at the 13th ISF Congress in Marseille on 2nd September 1976     

Separation and characterization of two chorismate-mutase isoenzymes from Nicotiana silvestris

Two isoenzymes of chorismate mutase (EC 5.4.99.5) were isolated and partially purified from leaves of diploid (2n=24) Nicotiana silvestris Speg. et Comes and from isogenic cells in a suspension culture originally established from haploid tissue. An isoenzyme denoted CM-1 (M _r=52,000) accounted for the major fraction of total activity recovered from suspension-cultured cells, while isoenzyme CM-2 (M _r=65,000) represented the major fraction of activity recovered from green leaf tissue. The ratio of isoenzyme levels from these two sources differed more than 20-fold. The subcellular location of isoenzyme CM-1 is known to be in the chloroplasts of green leaves or in proplastids of cultured cells, while isoenzyme CM-2 is located in the cytosol. Both isoenzymes were stable during partial purification, possessed broad pH optima for catalysis between 6.0 and 8.0, and were active without denaturation at temperatures at least as high as 45° C. Thiol reagents were unnecessary for either stability or activity of both isoenzymes. The affinity of isoenzyme CM-2 for substrate (K _m=0.24 mM) was almost an order of magnitude better than that of CM-1. The kinetic behavior of isoenzyme CM-1 was influenced by pH, while that of isoenzyme CM-2 was not. At pH 7.2, hyperbolic substrate-saturation curves (K _m=1.7 mM) were obtained for isoenzyme CM-1. At pH 6.1, however, isoenzyme CM-1 displayed relatively weak positive cooperativity, Hill plots yielding an n value of 1.2 At pH 6.1 the half-saturation ([S]_0.5) value was 2.5 mM.Abbreviations DEAE diethylaminoethyl - M _r molecular weight     

Biochemical and ecological characterization of two peroxidase isoenzymes from the mangrove, Rhizophora mangle

This study examines phenolic peroxidase (POX) in Rhizophora mangle L. leaves in order to assess its role in phenolic manipulation and H₂O₂ scavenging. Sun-exposed and understorey leaves experiencing varying degrees of nutrient stress were analysed from an oligotrophic cay off the coast of Belize. POX activity was unaffected by growth environment, but increased throughout leaf development and persisted through senescence and after abscission. Histochemical analyses indicated POX activity throughout leaf tissues, especially in the apoplast. Phenolics were similarly broadly distributed. Two isoenzymes of POX were partially characterized with pIs of 4.1 and 6.3 and masses of 65.5 and 54.3 kDa, respectively. The larger, more acidic isoenzyme showed especially high heat stability, showing no reduced activity after 24 h at 60 °C. Rhizophora mangle POX oxidized quercetin preferentially, and, to a lesser extent, coniferyl alcohol, caffeic acid, chlorogenic acid, and p-coumaric acid. It did not oxidize ascorbate, but ascorbate could act as a secondary electron donor in the presence of a phenolic substrate and H₂O₂. However, because quercetin and other aglycones were not present in R. mangle leaves, and because POX showed no activity with the most abundant leaf flavonoid, rutin, it was concluded that detoxification of H₂O₂ is secondary to the other roles of POX in manipulation of phenolics.     

In vivo and in vitro metacyclogenesis tests of two strains of Trypanosoma cruzi in the triatomine vectors Triatoma pseudomaculata and Rhodnius neglectus: short/long-term and comparative study

Metacyclogenesis of Trypanosoma cruzi of the Y and Berenice strains was studied in Triatoma pseudomaculata and Rhodnius neglectus. Results in vivo showed a higher production of metacyclic trypomastigotes in R. neglectus' digestive tube than in T. pseudomaculata. In vitro experiments were also carried out in order to compare the behavior of culture forms of T. cruzi incubated in extracts of different compartments (stomach, intestine, and rectum) of the digestive tract of both species of triatomines. A higher percentage of metacyclic trypomastigotes for both parasite strains, Y and Berenice, was detected in the rectum extract of R. neglectus in comparison to that from T. pseudomaculata. The same results were obtained with in vitro experiments, using parasites incubated in urine from each of those vectors. The adhesion of parasites to the incubated rectum epithelial cells was also compared. In incubations with the Y strain no significant differences were detected between the two triatomine species but, however, with the Berenice strain the mean percentage of cells with adhered parasites was higher in R. neglectus than in T. pseudomaculata.     

Rapid purification of two lipase isoenzymes from Candida rugosa

Summary We describe a two-step method for the purification of two lipases (lipases A and B) from C. rugosa. The purification procedure includes Phenyl-Sepharose and Sephacryl HR 100 chromatographies. The enzymes obtained were pure according to criteria of specific activity and neutral sugar content.     

Characterization of Toxoplasma gondii from domestic animals from Minas Gerais, Brazil

In an attempt to isolate and characterize Toxoplasma gondii from the State of Minas Gerais, Brazil, musculature samples from 72 pigs, 25 dogs, 28 free-range chickens and 50 chickens produced in industrialized farms were collected. Antibodies to T. gondii have not been detected in pigs, but were found in nine (40.9 %) out of 22 dogs, and in 15 (53.6 %) of 28 free range chickens. T. gondii was not isolated from pigs and industrialized chickens, but from eight dogs and 11 free range chickens. In order to determine T. gondii virulence, female BALB/c mice were inoculated with 10(3), 10(2), 10(1) and 10(0) tachyzoites of the 19 isolates. The strains RH (virulent) and ME49 (non-virulent) were used as references. Isolates were divided into three groups according to the virulence phenotype: five isolates were classified into virulent in mice, one into non-virulent and 13 into intermediate virulent. Nested-PCR of T. gondii SAG2 locus amplified DNA from 21 out of 22 DNA samples directly extracted from heart of free range chickens. These samples were genotyped through a PCR-RFLP assay. Seventeen (80.9 %) were classified into type I; one (4.8 %) into type III and three (14.3 %) into type I or II.     

Genetic profile of the arylamine N-acetyltransferase 2 coding gene among individuals from two different regions of Brazil

Arylamine N-acetyltranferase 2 is the main enzyme responsible for the isoniazid metabolization into hepatotoxic intermediates and the degree of hepatotoxicity severity has been attributed to genetic variability in the NAT2 gene. The main goal of this study was to describe the genetic profile of the NAT2 gene in individuals from two different regions of Brazil: Rio de Janeiro and Goiás States. Therefore, after preparation of DNA samples from 404 individuals, genotyping of the coding region of NAT2 was performed by direct PCR sequencing. Thirteen previously described SNPs were detected in these Brazilian populations, from which seven: 191 G>A; 282 C>T; 341 T>C; 481 C>T; 590 G>A; 803 A>G and 857 G>A are the most frequent in other populations. The presence of so-called ethnic-specific SNPs in our population is in accordance with the Brazilians' multiple ancestry. Upon allele and genotype analysis, the most frequent NAT2 alleles were respectively NAT2*5B (33%), NAT2*6A (26%) and NAT2*4 (20%) being NAT2*5/*5 the more prevalent genotype (31.7%). These results clearly demonstrate the predominance in the studied Brazilian groups of NAT2 alleles associated with slow over the fast and intermediate acetylator genotypes. Additionally, in Rio de Janeiro, a significantly higher frequency of intermediate acetylation status was found when compared to Goiás (42.5% versus 25%) (p=0.05), demonstrating that different regions of a country with a population characterized by a multi-ethnic ancestry may present a large degree of variability in NAT2 allelic frequencies. This finding has implications in the determination of nationwide policies for use of appropriate anti-TB drugs.     

Purification and characterization of two aldose reductase isoenzymes from rabbit muscle

Using a modification of the procedure of Kormann et al. (Kormann, A. W., Hurst, R. O., and Flynn, T. G. (1972) Biochim. Biophys. Acta 258, 40-55) for the purification of glycerol dehydrogenase, two enzymes have been purified from the skeletal muscle of male rabbits. From a consideration of their properties these enzymes have been named aldose reductase 1 and aldose reductase 2, respectively. Both enzymes are monomeric by the criteria of gel filtration and polyacrylamide gel electrophoresis in sodium dodecyl sulfate and both reductases are immunologically identical as shown by double immunodiffusion and rocket immunoelectrophoresis. Aldose reductases 1 and 2 have almost identical amino acid compositions, their NH2 termini are blocked and the COOH termini of both enzymes are apparently identical. The enzymes differ, however, in molecular weight with aldose reductase 2 having Mr = 41,500 and aldose reductase 1 Mr 40,200. Both enzymes have the broad substrate specificity typical of the aldehyde reductase family of enzymes; Km values of aldose reductase 1 for aldo sugars were similar to those reported for rabbit lens aldose reductase, and both aldose reductase 1 and 2 were inhibited by the commercial aldose reductase inhibitors Alrestatin and Sorbinil. Two aldose reductases, immunologically and electrophoretically identical to the muscle enzymes, were found in rabbit lens. Two aldose reductases were also detected in the skeletal muscle of male rats and pigs and in pig and bovine lens. The presence of relatively large amounts of aldose reductase in muscle identifies a new and rich source of the enzyme.     

Purification and characterization of two glutamate dehydrogenase isoenzymes from Brassica napus

Glutamate dehydrogenase (L-glutamate: NAD⁺ oxidoreductase, EC 1.4.1.2) was purified from Brassica napus leaves. Isoenzyme 1 (GDH1), with the lowest, and isoenzyme 7 (GDH7) with the highest electrophoretic mobility were characterized. The native GDH was estimated to have a molecular mass of about 239 kDa and consisted of six identical 41.4-kDa subunits for GDH1 and 42.4-kDa subunits for GDH7. The pH optima of both isoenzymes in amination and deamination reactions were 9.0 and 9.5, respectively. At optimum pH, the K_m values for ammonium, 2-oxoglutarate, NADH, NAD and glutamate did not differ between the two isoenzymes. Addition of 10 mM EGTA inhibited the amination activity of GDH1, but that of GDH7 remained at about 30 %. Cellular fractionation experiments showed that both GDH1 and GDH7 localized in mitochondria with a loose association with the mitochondrial membrane.     

Characterization of neuropeptide F-like immunoreactivity in the blood-feeding hemipteran, Rhodnius prolixus

The invertebrate neuropeptide Y (NPY) homolog, neuropeptide F (NPF), has been characterized for a wide range of invertebrate phyla, including platyhelminthes, molluscs, and arthropods. Current hypotheses suggest that NPF may be capable of regulating responses to diverse external cues related to nutritional status and feeding. The qualitative and quantitative distribution of an NPF-like peptide in fifth instar Rhodnius prolixus was undertaken using an antiserum raised against Drosophila NPF. Immunohistochemistry reveals NPF-like immunoreactive neurons and processes in the central nervous system, stomatogastric nervous system and peripheral nervous system. The distribution of NPF-like immunoreactivity within the medial neurosecretory cells of the brain and neurohemal areas of the corpus cardiacum and dorsal vessel, suggests NPF may act as a neurohormone. Immunoreactive processes are present over the surface of the hindgut and the immunoreactivity in these processes is greatly reduced in intensity 24h post-feeding. The quantification of partially purified NPF-like material in the CNS of R. prolixus was conducted by HPLC fractionation and radioimmunoassay. The results suggest that NPF-like material is present in fifth instar R. prolixus and likely released into the hemolymph following a blood meal.