Esterase patterns and phylogenetic relationships of Drosophila species in the saltans subgroup (saltans group)

The esterase patterns of sixteen strains from four species in the saltans subgroup were analyzed using polyacrylamide gel electrophoresis. Thirty-four esterase bands were detected. By using alpha and beta naphthyl acetates as substrates, they were classified in 18 alpha-esterases (they hydrolyse the alpha-naphtyl substrate), 15 beta-esterases (they hydrolyse the beta-naphtyl substrate) and 1 alpha/beta-esterase (it hydrolyses the alpha and beta-naphtyl substrates). Among the alpha-esterases, three were detected exclusively in males. Malathion, Eserine and pCMB were used as inhibitors in order to characterize biochemically the esterases. The results indicated the presence of cholinesterases, carboxylesterases and acetylesterases. The degree of mobility of the bands in the gels, their specificity to alpha and beta naphthyl acetates and the results of the inhibition tests allowed us to recognize tentatively nine genetic loci. Phylogenetic relationships among species inferred on the basis of the esterase patterns by PAUP 4.0 b8, with neighbor-joining search and a bootstrap analysis showed that, although the four species are closely related, D. septentriosaltans, D. saltans and D. austrosaltans are closer to each other than to D. prosaltans. These results showed to be consistent with phylogenetic relationships previously inferred from inversion polymorphism.

The effects of copper ions on the synthesis of periplasmic and membrane proteins in Acidithiobaeillus ferrooxidans as analyzed by SDS-PAGE and 2D-PAGE

The effects of 200 mM copper ions on the synthesis of membrane and periplasmic proteins were investigated in iron-grown cells of Acidithiobacillus ferrooxidans (At. ferrooxidans). Total membrane protein profiles of cells grown in the absence of copper ions (unadapted cells) and in the presence of copper ions (copper-adapted cells) were compared by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Crude preparations of outer membrane and periplasmic proteins were analyzed by SDS-PAGE. The synthesis of proteins was diminished or increased in the presence of copper ions. Low molecular weight proteins (< 14 kDa) were significantly repressed by copper. These proteins are probably acidic proteins located in the outer membrane. An over-expression of a periplasmic protein of about 17 kDa was detected in the copper-adapted cells and was assumed to be rusticyanin, a 16.5-kDa periplasmic copper protein present in At. ferrooxidans cells and involved in the electron-transport chain of the iron oxidation pathway. To our knowledge, this is the first report of a possible involvement of the rusticyanin and outer membrane proteins in the mechanism of copper resistance in At. ferrooxidans. (C) 2003 Elsevier B.V. All rights reserved.

PURIFICATION AND PROPERTIES OF SHIKIMATE DEHYDROGENASE FROM CUCUMBER (CUCUMIS-SATIVUS L)

Shikimate dehydrogenase (SDH, EC 1.1.1.25) extracted from cucumber pulp (Cucumis sativus L.) was purified 7-fold by precipitation with ammonium sulfate and elution from columns of Sephadex G-25, DEAE-cellulose, and hydroxyapatite. Two activity bands were detected on polyacrylamide gel electrophoresis at the last purification step. pH optimum was 8.7, and molecular weight of 45 000 was estimated on a Sephadex G-100 column. SDH was inhibited competitively by protocatechuic acid with a K(i) value of 2 x 10-4 M. K(m) values of 6 x 10-5 and 1 x 10-5 M were determined for shikimic acid and NADP+, respectively. The enzyme was completely inhibited by HgCl2 and p-(chloromercuri)benzoate (PCMB). NaCl and KCl showed partial protection against inhibition by PCMB. Heat inactivation between 50 and 55-degrees-C was biphasic, and the enzyme was completely inactivated after 10 min at 60-degrees-C. Incubation of SDH with either NADP+ or shikimic acid protected the enzyme against heat inactivation.

Protein profile of Acidithiobacillus ferrooxidans strains exhibiting different levels of tolerance to metal sulfates

Strains of Acidithiobacillus ferrooxidans exhibited differences in the inhibition of Fe(2+) oxidation in the presence of 250 mm of cadmium, zinc, and manganese sulfates in respirometric assays. Strains LR and I35 were practically not inhibited, whereas strains SSP and V3 showed significant inhibition (30-70%). Analysis by SDS-PAGE of total proteins from cells grown in the absence of metal sulfates showed different profiles between the more tolerant strains (LR and 135) and the more susceptible ones (SSP and V3). Total proteins of strains LR and V3 were also resolved by two-dimensional polyacrylamide gel electrophoresis (2-DE). A set of major proteins (40, 32, 22, and 20 kDa) could be identified only in the more tolerant strain LR. Our results show that protein profiles analysis could differentiate A. ferrooxidans strains that considerably differ in the tolerance to metal sulfates and present low genomic similarity as revealed by Random Amplified Polymorphic DNA (RAPD) data obtained previously in our laboratory.

A rapid procedure for the isolation of the Lys-49 myotoxin II from Bothrops moojeni (caissaca) venom: Biochemical characterization, crystallization, myotoxic and edematogenic activity

A, M. Soares, V, M, Rodrigues, M. I. Homsi-Brandeburgo, M. H. Toyama, F, R, Lombardi, K. Arni and J. R, Giglio. A rapid procedure for the isolation of the Lys-49 myotoxin II from Bothrops moojeni (caissaca) venom: Biochemical characterization, crystallization, myotoxic and edematogenic activity. Toxicon 36, 503-514, 1998.-Bothrops moojeni snake venom was fractionated on a CM-Sepharose column which was previously equilibrated with 0.05 M ammonium bicarbonate buffer at pH 8.0 and subsequently eluted with an ammonium bicarbonate concentration gradient from 0.05 to 0.5 M at constant pH (8.0) and temperature (25 degrees C). The fraction which eluted last (M-VI) showed, after direct lyophilization, a single band by polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE, indicating an approximate M,. of 14 000 and 77 000, in the presence and absence of dithiothreitol, respectively. Its amino acid composition revealed a high level of hydrophobic and basic amino acids as well as 13 half-cystine residues. Its isoelectric point and extinction coefficient (E-1.0cm(1.0mg/ml) at 278 nm and pH 7.0) were 8.2 and 1.170, respectively. M-VI was devoid of phospholipase A(2) (PLA(2)) activity on egg yolk, as well as of hemorrhagic, anticoagulant and coagulant activities, but could induce drastic necrosis on skeletal muscle fibres as well as rapid and transient edema on the rat paw. Its N-terminal sequence: SLFELGKMILQETGKNPAKSYGVYGCNCGVGGRGKPKDATDRCCYVHKCCYK.... revealed high homology with other Lys 49 PLA(2)-like myotoxins from other bothropic venoms. Orthorhombic crystals of M-VI? which diffracted to a maximal resolution of 1.6 Angstrom. were obtained and indicated the presence of a dimer in the asymmetrical unit. (C) 1998 Elsevier B.V. Ltd. All rights reserved.

Sulfation of intrinsic glycoproteins of the rabbit vitreous

The experiments reported here were designed to characterize the intrinsic vitreous glycoproteins and to understand the process of their sulfation. Rabbits were injected intravitreally with S-35-sodium sulfate and killed at several time intervals after injection. In another series of experiments, rabbits were injected either with S-35-sodium sulfate, H-3-fucose or H-3-tyrosine, associated or not associated with tunicamycin administration. Vitreous from the control eyes was also digested with N-glycosidase.. Furthermore, ciliary bodies, the putative source of the intrinsic vitreous glycoproteins, were incubated with S-35-sodium sulfate in the presence or absence of the protein synthesis inhibitor cycloheximide, and the culture media recovered for analysis. These and the vitreous samples of the other experiments were processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. Except for serum albumin, practically all polypeptide bands of the vitreous and culture media were labeled with radioactive sulfate and were shown to undergo renewal. The experiments using tunicamycin or enzyme treatment suggest that radioactive sulfate was incorporated not only into the carbohydrate side chains of the glycoproteins but also into the amino acid tyrosine of the polypeptide backbone of these glycoproteins. (C) 1998 Academic Press.

Esterase patterns and phylogenetic relationships of species and strains included in the Drosophila buzzatii cluster

Ten strains of two species in the Drosophila buzzatii cluster (D. serido and D. seriema) were examined as to esterase patterns using polyacrylamide gel electrophoresis. The migration rate of esterases, and their substrate specificity to alpha and beta naphthyl acetates, were analysed. Other esterase features such as inhibition behaviour, presence in males and females and location in the head, thorax or abdomen of flies, were also examined. The present data,together with results obtained by others for eight strains of D. koepferae, D. serido, D. seriema and D. buzzatii, show that 69 bands have been detected in the eighteen strains studied. This total number of bands was used for comparison of strains and species by similarity index, analysis of dependence and cluster analysis. The comparisons confirmed the existence of a high degree of similarity among D. seriema strains and among D. koepferae strains, but indicated differentiation among the D. serido strains. Two strains (D69R2 and D69R5) which differed from the others of the latter species, showed closer affinities with D. buzzatii, which indicates the need for further work on those strains classified as D. serido.

Thiobacillus ferrooxidans response to copper and other heavy metals: growth, protein synthesis and protein phosphorylation

Respirometric experiments demonstrated that the oxygen uptake by Thiobacillus ferrooxidans strain LR was not inhibited in the presence of 200 mM copper. Copper-treated and untreated cells from this T. ferrooxidans strain were used in growth experiments in the presence of cadmium, copper, nickel and zinc. Growth in the presence of copper was improved by the copper-treated cells. However, no growth was observed for these cells, within 190 h of culture, when cadmium, nickel and zinc were added to the media. Changes in the total protein synthesis pattern were detected by two-dimensional polyacrylamide gel electrophoresis for T. ferrooxidans LR cells grown in the presence of different heavy metals. Specific proteins were induced by copper (16, 28 and 42 kDa) and cadmium (66 kDa), whereas proteins that had their synthesis repressed were observed for all the heavy metals tested. Protein induction was also observed in the cytosolic and membrane fractions from T. ferrooxidans LR cells grown in the presence of copper. The level of protein phosphorylation was increased in the presence of this metal.

AMINO-ACID-SEQUENCE OF TSTX-V, AN ALPHA-TOXIN FROM TITYUS-SERRULATUS SCORPION-VENOM, AND ITS EFFECT ON K+ PERMEABILITY OF BETA-CELLS FROM ISOLATED RAT ISLETS OF LANGERHANS

Highly purified Tityustoxin V (TsTX-V), an alpha-toxin isolated from the venom of the Brazilian scorpion Tityus serrulatus, was obtained by ion exchange chromatography on carboxymethylcellulose-52. It was shown to be homogeneous by reverse phase high performance liquid chromatography, N-terminal sequencing (first 39 residues) of the reduced and alkylated protein and by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and tricine. Following enzymatic digestion, the complete amino acid sequence (64 residues) was determined. The sequence showed higher homology with the toxins from the venoms of the North African than with those of the North and South American scorpions. Using the rate of Rb-86(+) release from depolarized rat pancreatic beta-cells as a measure of K+ permeability changes, TsTX-V (5.6 mu g/ml) was found to increase by 2.0-2.4-fold the rate of marker outflow in the presence of 8.3 mM glucose. This effect was persistent and slowly reversible, showing similarity to that induced by 100 mu-M veratridine, an agent that increases the open period of Na+ channels, delaying their inactivation. It is suggested that, by extending the depolarized period, TsTX-V indirectly affects beta-cell voltage-dependent K+ channels, thus increasing K+ permeability.

Purification and characterization of a cyclomaltodextrin glucanotransferase from Paenibacillus campinasensis strain H69-3

A cyclomaltodextrin glucanotransferase (E.C. 2.4.1.19) from a newly isolated alkalophilic and moderately thermophilic Paenibacillus campinasensis strain H69-3 was purified as a homogeneous protein from culture supernatant. Cyclomaltodextrin glucanotransferase was produced during submerged fermentation at 45 degrees C and purified by gel filtration on Sephadex G50 ion exchange using a Q-Sepharose column and ion exchange using a Mono-Q column. The molecular weight of the purified enzyme was 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the pI was 5.3. The optimum pH for enzyme activity was 6.5, and it was stable in the pH range 6.0-11.5. The optimum temperature was 65 degrees C at pH 6.5, and it was thermally stable up to 60 degrees C without substrate during 1 h in the presence of 10 mm CaCl2. The enzyme activity increased in the presence of Co2+, Ba2+, and Mn2+. Using maltodextrin as substrate, the K-m and K-cat were 1.65 mg/mL and 347.9 mu mol/mg.min, respectively.

Changes in plasma protein concentrations in ponies with experimentally induced alimentary laminitis

Objective-To determine whether plasma protein concentrations were altered in ponies with alimentary laminitis.Animals-12 adult ponies.Procedure-Acute laminitis was induced in 6 ponies by oral administration of carbohydrate (85% corn starch, 15% wood flour); the other 6 ponies were used as controls. A physical examination was performed and blood samples were collected immediately before and 4, 8, 12, 24, and 28 hours after administration of carbohydrate. Plasma protein concentrations were determined by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis.Results-19 plasma proteins ranging from a molecular weight of 24,000 to a molecular weight of 350,000 were identified in all 12 ponies. Plasma concentrations of proteins with molecular weights of 350,000 (fibrinogen), 130,000 (ceruloplasmin), 118,000 (c-reactive protein), 67,000 (alpha(1)-antitrypsin I), 65,000 (alpha(1)-antitrypsin II), 50,000 (haptoglobulin), and 45,000 (acid glycoprotein) were significantly increased in ponies with laminitis, compared with concentrations in control ponies.Conclusion-Changes in plasma protein concentrations are detectable within 4 hours after the onset of alimentary laminitis in ponies.Clinical Relevance-Measurement of plasma protein concentrations may be useful in monitoring the progression of laminitis in ponies.

Purification and properties of buffalo (Bubalus bubalis) erythrocyte hexokinase

Buffalo erythrocytes contain one isozyme of hexokinase that apparently lacks microheterogeneity as shown by chromatographic properties. A single protein band was detected by means of Western blotting using an antibody raised in rabbits against homogeneous rat brain hexokinase I. The native protein has a molecular weight of 200,000 +/- 2880 by gel filtration. Partial purification of erythrocyte hexokinase by a combination of several procedures, including affinity chromatography, which was previously applied successfully to the purifica tion of other mammalian type I hexokinases, produced a partially purified enzyme that showed several contami nants after SDS-polyacrylamide gel electrophoresis. The affinity of buffalo erythrocyte hexokinase for glucose (K-m = 0.012 +/- 0.001 mM) is lower than most other mammal hexokinases type I. It phosphorylates other sugars, with considerably higher K-m values. This isozyme is able to use MgATP but does not use MgGTP, MgCTP or MgUTP. We used inhibition patterns, obtained with products to elucidate enzyme sequential mechanisms. Our results are clearly in agreement with a random sequential mechanism and in disagreement with an ordered sequential mechanism with either glucose or ATP as the obligatory first substrates. The ADP inhibition was of mixed type with both ATP and glucose as substrates. (C) 1997 Elsevier B.V.

Extracellular matrix of porcine pericardium: Biochemistry and collagen architecture

Pericardial tissue has been used to construct bioprostheses employed in the repair of different kinds of injuries, mostly cardiac. However, calcification and mechanical failure have been the main causes of the limited durability of cardiac bioprostheses constructed with bovine pericardium. In the course of this work, a study was conducted on porcine fibrous pericardium, its microscopic structure and biochemical nature. The general morphology and architecture of collagen were studied under conventional light and polarized light microscopy. The biochemical study of the pericardial matrix was conducted according to the following procedures: swelling test, hydroxyproline and collagen dosage, quantification of amino acids in soluble collagen, component extraction of the extracellular matrix of the right and left ventral regions of pericardium with different molarities of guanidine chloride, protein and glycosaminoglycan (GAG) dosage, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and total GAG analysis. Microscopic analysis showed collagen fibers arranged in multidirectionally oriented layers forming a closely knit web, with a larger number of fibers obliquely oriented, initiating at the lower central region toward the upper left lateral relative to the heart. No qualitative differences were found between proteins extracted from the right and left regions. Likewise, no differences were found between fresh and frozen material. Protein dosages from left frontal and right frontal pericardium regions showed no significant differences. The quantities of extracted GAGs were too small for detection by the method used. Enzymatic digestion and electrophoretic analysis showed that the GAG found is possibly dermatan sulfate. The proteoglycan showed a running standard very similar to the small proteoglycan decorin.

Variability of esterase patterns in adult flies of the saltans species group of Drosophila (subgenus Sophophora)

Esterases are known for their involvement in several physiological processes and high degree of polymorphism, in many organisms. Such polymorphism has been used to characterize species and species groups and to study genetic changes occurred in their evolutionary history. In the present study, the esterase patterns of 19 strains from 10 species representative of the five subgroups of the saltans species group were analyzed using polyacrylamide gel electrophoresis and alpha- and beta- naphthyl acetates as substrates. Fifty-one esterase bands were detected and classified as 31 alpha-esterases, 18 beta-esterases and two alpha/beta-esterases. on the basis of the inhibition patterns using Malathion and eserine sulfate, 34 bands were classified as carboxylesterases, 14 as acethylesterases and three as cholinesterases. Ten gene loci were tentatively established on the basis of data on band position in the gel, substrate preference and inhibition pattern. Twenty bands were species-specific, the remaining being shared by species from the same or different subgroups. Bands detected exclusively in males and bands with a different frequency or degree of expression between sexes were also detected. In the gels prepared for analysis of gene expression in the body parts (head, thorax and abdomen), the degree of expression of the beta-esterases was higher in the thorax, while the alpha-esterases were expressed predominantly in the abdomen and thorax. A global view of the data available at present on the esterases of the species from the saltans group and their degree of polymorphism are presented, as well as the possibility of using some beta-esterases, because of their characteristics in the gels, as markers for species identification.

A Preliminary and Qualitative Metallomics Study of Mercury in the Muscle of Fish from Amazonas, Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)