Quadro seroproteico como auxílio diagnóstico na anemia hemolítica imunomediada em cães

No presente protocolo experimental, determinaram-se os proteinogramas séricos, por intermédio da eletroforese em gel de poliacrilamida contendo duodecil sulfato de sódio (SDS-PAGE), de 120 cães com raças e idades variadas e atendidos junto ao Hospital Veterinário Governador Laudo Natel da FCAV/Unesp, com o objetivo principal de comparar diferentes frações seroproteicas em estados anêmicos regenerativos, arregenerativos, imunomediados primários e secundários. Os referidos animais foram distribuídos em cinco grupos experimentais: grupo 1: 20 cães de controle; grupo 2: 28 cães com anemia regenerativa não imune; grupo 3: 27 cães com anemia arregenerativa não imune; grupo 4: 10 cães com anemia hemolítica imunomediada primária; grupo 5: 35 cães com anemia hemolítica imunomediada secundária. A técnica SDS-PAGE permitiu o fracionamento de 24 proteínas, cujos pesos moleculares (PM) variaram de 18.000 a 165.000 daltons (Da). Os cães com AHIM primária e secundária apresentaram 24 frações proteicas em seus traçados eletroforéticos, enquanto que cães de controle (1) e portadores de anemia regenerativa (2) e arregenerativa (3) de natureza não imune apresentaram 23 frações de proteínas, cuja proteína de peso molecular 68.000Da não foi encontrada. Dessa forma, 23 frações proteicas foram detectadas e revelaram-se comuns aos proteinogramas dos cães de controle e daqueles dos quatro grupos experimentais. Destas, identificaram-se nominalmente 11 frações proteicas, e as demais foram estudadas com base nos seus respectivos pesos moleculares. em relação aos cães de controle, os anêmicos (grupos 2, 3, 4 e 5) apresentaram maiores concentrações de transferrina sérica e entre estes os animais portadores da AHIM primária. Todos os cães anêmicos apresentaram teores séricos de haptoglobina e fosforilase significativamente maiores que os controles, enquanto que a concentração sérica de ceruloplasmina foi significativamente maior nestes. Tais achados analisados em conjunto agregam informações adicionais úteis à elucidação das AHIMs em cães.

Comparative cytogenetic and allozyme analysis of Mugil rubrioculus and M. curema (Teleostei : Mugilidae) from Venezuela

A comparative cytogenetic and allozyme analysis of sympatric specimens of Mugil rubrioculus and M. curema from Venezuela is reported. Specimens of M. rubrioculus exhibit a 2n=48 karyotype with exclusively acrocentric (NF=48) chromosomes, one pair of NORs interstitially located on chromosome pair number 8 and constitutive heterochromatin distributed in pericentromeric position of all chromosomes. Specimens of M. curema show cytogenetic features significantly different in comparison to M. rubrioculus in terms of chromosome number and morphology (2n=24 biarmed chromosomes, NF=48) and NORs location (telomeric region of the largest metacentric pair). Starch gel electrophoresis analysis at 20 presumptive loci reveals a reduced genetic difterentiation between the two species. In fact, though a total of ten private alleles are identified; all loci share alleles between the two species and the obtained Nei's genetic distance (D= 0.060) is lower than the values obtained between other congeneric mullet species. Thus, the cytogenetic and allozyme data sets indicate quite different degrees of genetic divergence between M. rubrioculus and M. curema. This could either reflect an underestimate of molecular divergence owing to cryptic variation or different rates of molecular/chromosomal evolution. Whatever the explanation, this study confirms the power of karyological data in discriminating species of Mugilidae.

AN IMPROVED ELECTROPHORETIC METHOD FOR THE DETERMINATION OF SERUM MILK PROTEIN VARIANTS IN GYR-HOLSTEIN COWS

Milk serum proteins such as alpha-lactalbumin (ALA) and beta-lactoglobulin (BLG) present biochemical polymorphism which is under the control of codominant autosomal alleles. In the present report, we propose modifications of traditional electrophoretic techniques such as increasing the running gel concentration from 5 to 10% and the addition of 5 M urea to the stacking gel, which permitted the detection of two variants (A and B) at the ALA and BLG loci. About 8 mul of milk serum (6 mg/ml protein) and 10 pl of total fresh milk were applied. Bovine serum albumin (BSA) and immunolactoglobulins (ILG) could also be discriminated. Total fresh milk was as useful as the purified serum milk proteins for the discrimination of ALA and BLG serum milk protein polymorphism by alkaline vertical slab polyacrylamide gel electrophoresis. However, BSA and ILG ran with caseins, which prevented their characterization in this system.

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.

Electrophoretic variants of intracellular catalase of different Candida species

Intracellular and extracellular catalases of different species of Candida were investigated using different culture media. All the Candida strains produced intracellular catalase, whose enzymatic activity was detected by non-denaturating polyacrylamide gradient (4-30%) gel electrophoresis. The cell extracts presented a major 230 kDa catalase band and in some strains variants of catalase with different molecular weights were detected. Candida catalase activity was not affected by heating at 50degreesC and incubation with beta-mercaptoethanol, but treatment with sodium dodecyl sulphate inhibited or reduced enzymatic activity. Extracellular enzyme activity was not detected in any of the culture filtrate extracts tested.

Chondroitin sulfate proteoglycans are structural renewable constituents of the rabbit vitreous body

Purpose: To characterize the vitreous intrinsic proteoglycans, investigate their dynamics, and examine their role in the supramolecular organization of the vitreous. Methods: Vitreous from normal rabbits was collected and processed for observation with the transmission electron microscope after treatment with glycosidases. Also, rabbits were injected intravitreally with [S-35]-sodium sulfate and sacrificed at several time intervals after the injection. Proteoglycans (PGs) were assayed in the vitreous supernatant or in whole samples extracted with guanidine hydrochloride by polyacrylamide or agarose gel electrophoresis, followed respectively by fluorography or autoradiography, and ion-exchange chromatography and gel-filtration chromatography, combined with glycolytic treatment of the samples. The sulfated glycosaminoglycans (GAGs) were characterized by agarose gel electrophoresis after treating vitreous samples with protease and specific glycosidases. Results: the electron microscopic study revealed a network with hyaluronic acid ( HA) as thin threads coating and connecting collagen fibrils. The elimination of the HA coat showed chondroitin sulfate granules (8-25 nm) arranged at regular intervals on the fibril surface. The chondroitinase ABC digestion, besides removing the granules, also caused the formation of thicker bundles of the collagen fibrils. The PG and GAG analysis indicated that there are three renewable PGs in the vitreous ( e. g., one heparan-and two chondroitin-sulfate ones). Conclusions: At least one of the chondroitin sulfate PGs is involved in the interactions that occur in the vitreous structure, mainly by providing adequate spacing between the collagen fibrils, a condition that is probably required for the transparency of the vitreous.

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.