Clinical, hematological, total plasma protein and fibrinogen parameters of magellanic penguins ( Spheniscus magellanicus ) pre- and post-rehabilitation

Abstract: Magellanic penguins (Spheniscus magellanicus) usually arrive in poor body conditions at Brazilian beaches during the winter. Hematology provides valuable information about clinical and immunity status of the animals. The aims of this study were to determine the hematologic, total plasma protein (TPP) and fibrinogen profiles of young and adult magellanic penguins in PROAMAR and CETAS-SC, relating these results with the state of health and survival possibility of the animals. In Paraná 14 animals were evaluated in pre and eight in post-rehabilitation and 29 animals were evaluated in Santa Catarina after rehabilitation. Before rehabilitation, all animals showed weakness. In hematological exams of these animals, we found that anemia was present in 83% of the penguins that died and 50% of those which survived. The heterophils/lymphocytes (H/L) ratio was 3.87±0.57 in animals that died, significantly higher than the average of 2.20±0.30 for animals that survived. These two parameters are useful to assess the survival possibility of animals to rehabilitation. The body condition score was positively correlated with hematocrit and TPP, and negatively correlated with H/L ratio. After rehabilitation, the values were similar to other animals of the family Spheniscidae, with averages ranging from 1.64 to 1.90x106 erythrocytes/μL; 43.38 to 48.80% of hematocrit; 12.45 to 13.52g/dL of hemoglobin; 8,684 to 14,011 leukocytes/μL; 4,767 to 8,041 heterophils/μL; 3,215 to 4,951 lymphocytes/μL; 95 to 655 eosinophils/μl; 179.8 to 277.9 monocytes/μL; 141 to 184.9 basophils/μL; and 1.26 to 1.74 of H/L ratio. These parameters can therefore be used as reference values and release parameters for young and adult Magellanic penguins in captivity on the rehabilitation centers.

Probabilistic contact preferences in protein-ligand and protein-protein complexes

Modeller för intermolekulär växelvärkan utnyttjas brett inom biologin. Analys av kontakter mellan proteiner och läkemedelsforskning representerar typiska tillämpningsområden för dylika modeller. En modell som beskriver sådana molekylära växelverkningar kan utformas med hjälp av biofysisk teori, vilket tenderar att resultera i ytterst tung beräkningsbörda även för enkla tillämpningar. Ett alternativt sätt att formulera modeller är att utnyttja stora databaser som innehåller strukturmätningar gjorda med hjälp av till exempel röntgendiffraktion. Då man använder sig av empiriska mätdata direkt, möjliggör en statistisk modell att osäkerheten och inexaktheten i datat tas till hänsyn på ett adekvat sätt, samtidigt som beräkningsbördan håller sig på en rimligare nivå jämfört med kvantmekaniska metoder som i princip borde ge de optimala resultaten. I avhandlingen utvecklades en 3D modell för numerisk undersökning av intermolekulär växelverkan baserad på Bayesiansk statistik. Modellens syfte är att åstadkomma prognoser för det hurdana eller vilka molekylstrukturer prefereras i en given kontext, d.v.s. är mer sannolika inom ramen för interaktion. Modellen testades i essentiella molekyläromgivningar - en liten molekyl vid sin bindningsplats hos ett protein och en gränsyta mellan proteinerna i ett komplex. De erhållna numeriska resultaten motsvarar väl experimentella resultat som tidigare rapporterats i litteraturen, exempelvis kvalitativa bindningsaffiniteter och kemisk kännedom av vissa aminosyrors rumsliga förmågor att utgöra bindningar. I avhandlingen gjordes ytterligare preliminära tester av den statistiska ansatsen för modellering av den centrala molekylära strukturella anpassningsbarheten. I praktiken är den utvecklade modellen ämnad som ett led i en mer omfattande analysmetod, så som en s.k. farmakofor modell. Molekyylivuorovaikutusten mallintamista hyödynnetään laajasti biologisten kysymysten tarkastelussa. Tyypillisiä esimerkkejä sovelluskohteista ovat proteiinien väliset kontaktit ja lääkesuunnittelu. Vuorovaikutuksia kuvaavan mallin lähtökohta voi olla molekyyleihin liittyvä teoria, jolloin soveltamiseen liittyvä laskenta saattaa olla erityisen raskasta, tai suuri havaintojoukko joka on saatu aikaan esimerkiksi mittaamalla rakenteita röntgendiffraktio menetelmällä. Tilastollinen malli mahdollistaa havaintoaineistossa olevan epätarkkuuden ja epävarmuuden huomioimisen, samalla pitäen laskennallisen kuorman pienempänä verrattuna periaatteessa parhaan tuloksen antavaan kvanttimekaaniseen mallinnukseen. Väitöstyössä kehitettiin bayesiläiseen tilastotieteeseen perustuva 3D malli molekyylien välisten vuorovaikutusten laskennalliseen tarkasteluun. Mallin tehtävä on tuottaa ennusteita sen suhteen, minkä tai millaisten molekyylirakenteiden väliset kompleksit ovat etusijalla, toisin sanoen todennäköisempiä, vuorovaikutustilanteessa. Työssä kehitetyn menetelmän toimivuutta testattiin käyttötarkoituksen suhteen olennaisissa molekyyliympäristöissä - pieni molekyyli sitoutumiskohdassaan proteiinissa sekä rajapinta kahden proteiinin välilllä proteiinikompleksissa. Saadut laskennalliset tulokset vastasivat hyvin vertailuun käytettyjä kirjallisuudesta saatuja kokeellisia tuloksia, kuten laadullisia sitoutumisaffiniteetteja, sekä kemiallista tietoa esimerkiksi tiettyjen aminohappojen avaruudellisesta sidoksenmuodostuksesta. Väitöstyössä myös alustavasti testattiin tilastollista lähestymistapaa tärkeän molekyylien rakenteellisen mukautuvuuden mallintamiseen. Käytännössä malli on tarkoitettu osaksi jotakin laajempaa analyysimenetelmää, kuten farmakoforimallia.

A Modified phosphate-carrier protein theory is proposed as a non-target site mechanism For glyphosate resistance in weeds

Glyphosate is an herbicide that inhibits the enzyme 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPs) (EC 2.5.1.19). EPSPs is the sixth enzyme of the shikimate pathway, by which plants synthesize the aromatic amino acids phenylalanine, tyrosine, and tryptophan and many compounds used in secondary metabolism pathways. About fifteen years ago it was hypothesized that it was unlikely weeds would evolve resistance to this herbicide because of the limited degree of glyphosate metabolism observed in plants, the low resistance level attained to EPSPs gene overexpression, and because of the lower fitness in plants with an altered EPSPs enzyme. However, today 20 weed species have been described with glyphosate resistant biotypes that are found in all five continents of the world and exploit several different resistant mechanisms. The survival and adaptation of these glyphosate resistant weeds are related toresistance mechanisms that occur in plants selected through the intense selection pressure from repeated and exclusive use of glyphosate as the only control measure. In this paper the physiological, biochemical, and genetic basis of glyphosate resistance mechanisms in weed species are reviewed and a novel and innovative theory that integrates all the mechanisms of non-target site glyphosate resistance in plants is presented.

Effect of nitrate and ammonium on the growth and protein concentration of Microcystis viridis Lemmermann (Cyanobacteria)

Cyanobacteria are a very important group in aquatic systems, particularly in eutrophic waters. Therefore studies about their success in the environment are essential. Many hypotheses have tried to explain the dominance of Cyanobacteria, and several emphasized the importance of various nitrogen sources for the success of the group. In this study, we measured the effect of ammonium and nitrate on the growth and protein concentration of Microcystis viridis (Cyanobacteria). This species is well-known because bloom formation in eutrophic waters. The study was carried out, in experimental batch cultures, using the WC medium with different nitrogen sources: ammonium, nitrate, ammonium + nitrate (50% ammonium + 50% nitrate) and ammonium at different concentrations (to test for possible NH4+ toxicity). Protein, ammonium and nitrate concentrations were measured at end of each experiment, whereas samples for cell counts were taken daily. Results showed that Microcystis viridis grew faster with ammonium (µ = 0.393 day-1) than with nitrate (µ = 0.263 day-1) and ammonium + nitrate (µ = 0.325 day-1). This pattern is explained by the metabolism of ammonium that presents higher uptake and assimilation rates than nitrate. Maximum cell concentration, however, was higher in the ammonium + nitrate treatment, followed by nitrate treatment. Higher protein concentration were observed in the treatment with nitrate. In the ammonium toxicity test, no difference between the control and NH4+ at 50% was found. Thus, the ammonium concentrations used in these experiments were not toxic. Our results suggest that Cyanobacteria is able to grow on both nitrogen sources even if ammonium may allow faster growth and bloom development.

Effect of sigma factor S (sigmaS) on the stability of penicillin-binding protein 3 (PBP3) of Escherichia colt K12

The stability of penicillin-binding protein 3 (PBP3), a cell septum synthesizing protein, was analyzed at different incubation temperatures in three Escherichia coli K12 strains carrying a PBP3-overproducing plasmid. The stability of PBP3 was significantly reduced in stationary phase cells shifted to 42°C for 4 h, compared to samples incubated at 28 or 37°C. The half-life of PBP3 in the C600 strain was 60 min at 42°C, while samples incubated at 28 or 37°C had PBP3 half-lives greater than 4 h. Analysis of the PBP3 content in mutants deficient in rpoS (coding for the stationary phase sigma factor, sigmaS) and rpoH (coding for the heat shock sigma factor, sigma32) genes after shift to 42°C showed that stability of the protein was controlled by sigmaS but not by sigma32. These results suggest that control of the PBP3 levels in E. coli K12 is through a post-transcriptional mechanism regulated by the stationary phase regulon. We demonstrated that stability of PBP3 in E. coli K12 involves degradation of the protein. Moreover, we observed that incubation of cells at 42°C significantly reduces the stability of PBP3 in early stationary phase cells in a process controlled by sigmaS.

A comparative study of eleven protein systems in tamarins, genus Saguinus (Platyrrhini, Callitrichinae)

The genetic variability of six tamarin taxa, genus Saguinus, was analyzed comparatively using protein data from eleven systems coded by 15 loci. S. fuscicollis weddelli and S. midas midas were the most polymorphic taxa, and S. bicolor the least. The results of the phylogenetic analyses (UPGMA and neighbor-joining) and the genetic distances between taxa were generally consistent with their geographic and probable phylogenetic relationships. Analyses of the S. bicolor and S. midas populations suggested that they represent no more than three subspecies of a single species, S. midas, with the bicolor forms belonging to a single subspecies, S. midas bicolor. If supported by additional studies, this would have important implications for the conservation of the bicolor form, which is endangered with extinction. The genetic similarity of S. fuscicollis and S. mystax was also consistent with their geographical and morphological proximity, although more data from a larger number of taxa will be required before the taxonomic relationships within the genus can be defined.

Milk protein polymorphisms in Brazilian Zebu cattle

Five bovine milk protein polymorphisms were studied in Zebuine cattle raised in Brazil, through horizontal electrophoresis on starch gel containing urea and 2-mercaptoethanol, using basic and acidic buffer systems. Allelic frequencies for a-La, b-Lg, aS1-Cn, b-Cn and k-Cn loci were estimated in six Gyr herds (N = 283), six Guzerat herds (N = 205), one Nelore herd (N = 17) and one Sindi herd (N = 22), all from São Paulo or Minas Gerais State, Brazil. Genotypic frequencies observed for each locus and breed studied are in accordance with the assumption of genetic equilibrium, demonstrating absence of high inbreeding levels for the breeds tested. The FST value found indicated significant genetic differentiation among breeds; however, the Gyr and Guzerat herds showed significantly different gene frequencies. Genetic distance estimates among zebuine breeds studied and the Holstein breed, taken as a reference for a taurine breed, showed strong differences between these two racial groups

Thyroid function in post-weaning rats whose dams were fed a low-protein diet during suckling

This study was designed to evaluate the thyroid and pituitary hormone levels in post-weaning rats whose dams were fed a low-protein diet during suckling (21 days). The dams and pups were divided into 2 groups: a control group fed a diet containing 22% protein that supplies the necessary amount of protein for the rat and is the usual content of protein in most commercial rat chow, and a diet group fed a low-protein (8%) diet in which the protein was substituted by an isocaloric amount of starch. After weaning all dams and pups received the 22% protein diet. Two hours before sacrifice of pups aged 21, 30 and 60 days, a tracer dose (0.6 µCi) of 125I was injected (ip) into each animal. Blood and thyroid glands of pups were collected for the determination of serum T4, T3 and TSH and radioiodine uptake. Low protein diet caused a slight decrease in radioiodine uptake at 21 days, and a significant decrease in T3 levels (128 ± 14 vs 74 ± 9 ng/dl, P<0.05), while T4 levels did not change and TSH was increased slightly. At 30 days, T3 and TSH did not change while there was a significant increase in both T4 levels (4.8 ± 0.3 vs 6.1 ± 0.2 µg/dl, P<0.05) and in radioiodine uptake levels (0.34 ± 0.02 vs 0.50 ± 0.03%/mg thyroid, P<0.05). At 60 days serum T3, T4 and TSH levels were normal, but radioiodine uptake was still significantly increased (0.33 ± 0.02 vs 0.41 ± 0.03%/mg thyroid, P<0.05). Thus, it seems that protein malnutrition of the dams during suckling causes hypothyroidism in the pups at 21 days that has a compensatory mechanism increasing thyroid function after refeeding with a 22% protein diet. The radioiodine uptake still remained altered at 60 days, when all the hormonal serum levels returned to the normal values, suggesting a permanent change in the thyroid function

Control of the phosphorylation of the astrocyte marker glial fibrillary acidic protein (GFAP) in the immature rat hippocampus by glutamate and calcium ions: possible key factor in astrocytic plasticity

The present review describes recent research on the regulation by glutamate and Ca2+ of the phosphorylation state of the intermediate filament protein of the astrocytic cytoskeleton, glial fibrillary acidic protein (GFAP), in immature hippocampal slices. The results of this research are discussed against a background of modern knowledge of the functional importance of astrocytes in the brain and of the structure and dynamic properties of intermediate filament proteins. Astrocytes are now recognized as partners with neurons in many aspects of brain function with important roles in neural plasticity. Site-specific phosphorylation of intermediate filament proteins, including GFAP, has been shown to regulate the dynamic equilibrium between the polymerized and depolymerized state of the filaments and to play a fundamental role in mitosis. Glutamate was found to increase the phosphorylation state of GFAP in hippocampal slices from rats in the post-natal age range of 12-16 days in a reaction that was dependent on external Ca2+. The lack of external Ca2+ in the absence of glutamate also increased GFAP phosphorylation to the same extent. These effects of glutamate and Ca2+ were absent in adult hippocampal slices, where the phosphorylation of GFAP was completely Ca2+-dependent. Studies using specific agonists of glutamate receptors showed that the glutamate response was mediated by a G protein-linked group II metabotropic glutamate receptor (mGluR). Since group II mGluRs do not act by liberating Ca2+ from internal stores, it is proposed that activation of the receptor by glutamate inhibits Ca2+ entry into the astrocytes and consequently down-regulates a Ca2+-dependent dephosphorylation cascade regulating the phosphorylation state of GFAP. The functional significance of these results may be related to the narrow developmental window when the glutamate response is present. In the rat brain this window corresponds to the period of massive synaptogenesis during which astrocytes are known to proliferate. Possibly, glutamate liberated from developing synapses during this period may signal an increase in the phosphorylation

Diclofenac plasma protein binding: PK-PD modelling in cardiac patients submitted to cardiopulmonary bypass

Twenty-four surgical patients of both sexes without cardiac, hepatic, renal or endocrine dysfunctions were divided into two groups: 10 cardiac surgical patients submitted to myocardial revascularization and cardiopulmonary bypass (CPB), 3 females and 7 males aged 65 ± 11 years, 74 ± 16 kg body weight, 166 ± 9 cm height and 1.80 ± 0.21 m2 body surface area (BSA), and control, 14 surgical patients not submitted to CPB, 11 female and 3 males aged 41 ± 14 years, 66 ± 14 kg body weight, 159 ± 9 cm height and 1.65 ± 0.16 m2 BSA (mean ± SD). Sodium diclofenac (1 mg/kg, im Voltaren 75® twice a day) was administered to patients in the Recovery Unit 48 h after surgery. Venous blood samples were collected during a period of 0-12 h and analgesia was measured by the visual analogue scale (VAS) during the same period. Plasma diclofenac levels were measured by high performance liquid chromatography. A two-compartment open model was applied to obtain the plasma decay curve and to estimate kinetic parameters. Plasma diclofenac protein binding decreased whereas free plasma diclofenac levels were increased five-fold in CPB patients. Data obtained for analgesia reported as the maximum effect (EMAX) were: 25% VAS (CPB) vs 10% VAS (control), P<0.05, median measured by the visual analogue scale where 100% is equivalent to the highest level of pain. To correlate the effect versus plasma diclofenac levels, the EMAX sigmoid model was applied. A prolongation of the mean residence time for maximum effect (MRTEMAX) was observed without any change in lag-time in CPB in spite of the reduced analgesia reported for these patients, during the time-dose interval. In conclusion, the extent of plasma diclofenac protein binding was influenced by CPB with clinically relevant kinetic-dynamic consequences

Response threshold to aversive stimuli in stimulated early protein-malnourished rats

Two animal models of pain were used to study the effects of short-term protein malnutrition and environmental stimulation on the response threshold to aversive stimuli. Eighty male Wistar rats were used. Half of the pups were submitted to malnutrition by feeding their mothers a 6% protein diet from 0 to 21 days of age while the mothers of the other half (controls) were well nourished, receiving 16% protein. From 22 to 70 days all rats were fed commercial lab chow. Half of the animals in the malnourished and control groups were maintained under stimulating conditions, including a 3-min daily handling from 0 to 70 days and an enriched living cage after weaning. The other half was reared in a standard living cage. At 70 days, independent groups of rats were exposed to the shock threshold or to the tail-flick test. The results showed lower body and brain weights in malnourished rats when compared with controls at weaning and testing. In the shock threshold test the malnourished animals were more sensitive to electric shock and environmental stimulation increased the shock threshold. No differences due to diet or environmental stimulation were found in the tail-flick procedure. These results demonstrate that protein malnutrition imposed only during the lactation period is efficient in inducing hyperreactivity to electric shock and that environmental stimulation attenuates the differences in shock threshold produced by protein malnutrition

A 28-fold increase in secretory protein synthesis is associated with DNA puff activity in the salivary gland of Bradysia hygida (Diptera, Sciaridae)

When the first group of DNA puffs is active in the salivary gland regions S1 and S3 of Bradysia hygida larvae, there is a large increase in the production and secretion of new salivary proteins demonstrable by [3H]-Leu incorporation. The present study shows that protein separation by SDS-PAGE and detection by fluorography demonstrated that these polypeptides range in molecular mass from about 23 to 100 kDa. Furthermore, these proteins were synthesized mainly in the S1 and S3 salivary gland regions where the DNA puffs C7, C5, C4 and B10 are conspicuous, while in the S2 region protein synthesis was very low. Others have shown that the extent of amplification for DNA sequences that code for mRNA in the DNA puffs C4 and B10 was about 22 and 10 times, respectively. The present data for this group of DNA puffs are consistent with the proposition that gene amplification is necessary to provide some cells with additional gene copies for the production of massive amounts of proteins within a short period of time (Spradling AC and Mahowald AP (1980) Proceedings of the National Academy of Sciences, USA, 77: 1096-1100).

Changes in protein profile during coagulation of latex from Carica papaya

We describe the changes in peptide composition by SDS-PAGE analysis of latex from Carica papaya collected at various times after incision of the unripe fruit. The data show that during latex coagulation several peptides are processed in an orderly fashion.

Effect of protein malnutrition on the glycolytic and glutaminolytic enzyme activity of rat thymus and mesenteric lymph nodes

The activity of important glycolytic enzymes (hexokinase, phosphofructokinase, aldolase, phosphohexoseisomerase, pyruvate kinase and lactate dehydrogenase) and glutaminolytic enzymes (phosphate-dependent glutaminase) was determined in the thymus and mesenteric lymph nodes of Wistar rats submitted to protein malnutrition (6% protein in the diet rather than 20%) from conception to 12 weeks after birth. The wet weight (g) of the thymus and mesenteric lymph nodes decreased due to protein malnutrition by 87% (from 0.30 ± 0.05 to 0.04 ± 0.01) and 75% (0.40 ± 0.04 to 0.10 ± 0.02), respectively. The protein content was reduced only in the thymus from 102.3 ± 4.4 (control rats) to 72.6 ± 6.6 (malnourished rats). The glycolytic enzymes were not affected by protein malnutrition, but the glutaminase activity of the thymus and lymph nodes was reduced by half in protein-malnourished rats as compared to controls. This fact may lead to a decrease in the cellularity of the organ and thus in its size, weight and protein content.

Large-scale production and purification of recombinant protein from an insect cell/baculovirus system in Erlenmeyer flasks: application to the chicken poly(ADP-ribose) polymerase catalytic domain

A simple and inexpensive shaker/Erlenmeyer flask system for large-scale cultivation of insect cells is described and compared to a commercial spinner system. On the basis of maximum cell density, average population doubling time and overproduction of recombinant protein, a better result was obtained with a simpler and less expensive bioreactor consisting of Erlenmeyer flasks and an ordinary shaker waterbath. Routinely, about 90 mg of pure poly(ADP-ribose) polymerase catalytic domain was obtained for a total of 3 x 109 infected cells in three liters of culture