Neurotoxic activity and ultrastructural changes in muscles caused by the brown widow spider Latrodectus geometricus venom

Brown widow spider (Latrodectus geometricus) venom (BrWSV) produces few local lesions and intense systemic reactions such as cramps, harsh muscle pains, nausea, vomiting and hypertension. Approximately 16 protein bands under reducing conditions and ~ 14 bands under non-reducing conditions on a 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis were observed. Neurotoxic clinical manifestations were confirmed in vivo, while proteolytic activity was demonstrated on gelatine film. Severe ultrastructural damages in mice skeletal muscles were observed at 3, 6, 12 and 24 h postinjection with at total of 45 µg of venom protein. Infiltration of eosinophils and ruptures of the cellular membranes were observed in the muscles along with swelling of the nuclear cover and interruption of the collagen periodicity. Altered mitochondrias and autophage vacuoles, nuclear indentation and mitochondria without cristae, slight increment of intermyofibrillar and subsarcolemic spaces and myelinic figures formation were also observed. In the capillary, endothelial membrane unfolding into the lumen was noticed; along with myelinic figures compatible with a toxic myopathy. Swollen sarcotubular systems with lysis of membrane, intense mitochondria autophagia and areas without pinocytic vesicles were observed. Swollen mitochondria surrounded by necrotic areas, myofibrillar disorganization and big vacuolas of the sarcotubular system, degenerated mitochondrium with formation of myelinic figure was seen. Glycogenosomes with small particulate, muscle type glycogen was noticed. Autophagic vacuole (autophagolysosomes) and necrotic areas were also noticed. These damages may be due to interactive effects of the multifactorial action of venom components. However, Latrodectus geometricus venom molecules may also be utilized as neuro therapeutic tools, as they affect neuronal activities with high affinity and selectivity. To our knowledge, the present study is the first ultrastructural report in the literature of muscle injuries and neurological and proteolytic activities caused by BrWSV.

Cucumber seedling dependence on cotyledonary leaves for early growth

The objective of this work was to evaluate the dependence of cucumber (Cucumis sativus L.) seedlings on cotyledonary leaves for early growth and establishment. Sets of two uniform emerging seedlings were used to quantify the initial growth and dry matter accumulation, as well as the intensity and stage of cotyledon damage in seedling establishment and to determine cotyledon protein, amino acid and carbohydrate contributions to the growing seedling. Cucumber seedling establishment was found to be highly dependent on cotyledonary leaves. Root system establishment was highly dependent on the health of the aerial part. One cotyledon was enough to maintain aerial growth of seedlings after unfolding the first true leaf. Cucumber seedlings depended on both cotyledons to keep root system growth at least until leaf area was equivalent to cotyledon area. Covering one or both cotyledons of seedlings with one unfolded leaf increased carbohydrate content of uncovered cotyledon and leaves compared with control seedlings. Cucumber seedlings are highly dependent on cotyledonary leaves and aerial parts are less dependent than root system. Cotyledon damage at early stages of plant establishment would adversely impact crop yield by reducing plant density, an important yield component, or slowing down seedling growth and establishment.

Estudo microcalorimétrico da interação de tensoativos n-alquil-sulfato de sódio com tripsina a 298 k

Systematic study of the interactions of ionic surfactants with protein trypsin in buffer solution pH 3.5, 7.0 and 9.0, ionic strength 10 mM at 298 K was done using the microcalorimetric technique. In this study, anionic surfactant solutions of the sodium n-alkyl sulfates series (C8, C10, C12 and C14) were used. The enthalpy of interaction (ΔintHº) shows that the interaction of the surfactants C8, C10, C12 and C14 with trypsin in the solution pH 3.5 is an endothermic process with the value of ΔintHº decreasing linearly with increasing carbon chain length, which is attributed to the unfolding of the polypeptide chain. In the solution pH 7.0, we observed the same trend except for C14. In the solution pH 9.0, from C10 the enthapy of interaction didn't change with the increasing of the carbon chain length due to unfolding of the polypeptide. We concluded that when trypsin is folded, the enthalpy of interaction shows a linear relationship with the surfactant's hydrophobicity, in agreement with Traube's rule.

Hemes férricos pentacoordenados e hexacoordenados dos monômeros d nativo e reconstituído da hemoglobina extracelular de Glossoscolex paulistus: estudos espectroscópicos em meio ácido

UV-Vis and fluorescence spectroscopic studies of the native and reconstituted d monomers of Glossoscolex paulistus were performed in acid medium. The coexistence of distinct species shows the complexity of the equilibria. Besides the hexacoordinate low spin hemichrome, with bands at 535 and 565 nm, a pentacoordinate high spin hemichrome is identified by the blue-shifted low intensity Soret band (371 nm) and the LMCT band (643 nm). The pentacoordinate hemichrome must be related to the partial unfolding of the polypeptide.

Estudo espectroscópico do equilíbrio entre espécies hexacoordenada e pentacoordenada dos monômero d nativo e reconstituído da hemoglobina extracelular gigante de Glossoscolex paulistus em meio alcalino

UV-Vis and fluorescence spectroscopic studies of native and reconstituted d monomers from Glossoscolex paulistus were performed in alkaline pH. In addition to the presence of aquomet and hemichrome hexacoordinated species, a pentacoordinated species occurs. This latter species, which presents an imidazolate group of axial histidine as fifth ligand, is identified by the blue-shifted low-intensity Soret band and LMCT band. The pentacoordinated species must occur due to partial polypeptide unfolding.

Developmental and behavioral effects of postnatal amitraz exposure in rats

The effects of postnatal amitraz exposure on physical and behavioral parameters were studied in Wistar rats, whose lactating dams received the pesticide (10 mg/kg) orally on days 1, 4, 7, 10, 13, 16 and 19 of lactation; control dams received distilled water (1 ml/kg) on the same days. A total of 18 different litters (9 of them control and 9 experimental) born after a 21-day gestation were used. The results showed that the median effective time (ET50) for fur development, eye opening, testis descent and onset of the startle response were increased in rats postnatally exposed to amitraz (2.7, 15.1, 21.6 and 15.3 days, respectively) compared to those of the control pups (1.8, 14.0, 19.9 and 12.9 days, respectively). The ages of incisor eruption, total unfolding of the external ears, vaginal and ear opening and the time taken to perform the grasping hindlimb reflex were not affected by amitraz exposure. Pups from dams treated with amitraz during lactation took more time (in seconds) to perform the surface righting reflex on postnatal days (PND) 3 (25.0 ± 2.0), 4 (12.3 ± 1.2) and 5 (8.7 ± 0.9) in relation to controls (10.6 ± 1.2; 4.5 ± 0.6 and 3.4 ± 0.4, respectively); the climbing response was not changed by amitraz. Postnatal amitraz exposure increased spontaneous motor activity of male and female pups in the open-field on PND 16 (140 ± 11) and 17 (124 ± 12), and 16 (104 ± 9), 17 (137 ± 9) and 18 (106 ± 8), respectively. Data on spontaneous motor activity of the control male and female pups were 59 ± 11 and 69 ± 10 for days 16 and 17 and 49 ± 9, 48 ± 7 and 56 ± 7 for days 16, 17 and 18, respectively. Some qualitative differences were also observed in spontaneous motor behavior; thus, raising the head, shoulder and pelvis matured one or two days later in the amitraz-treated offspring. Postnatal amitraz exposure did not change locomotion and rearing frequencies or immobility time in the open-field on PND 30, 60 and 90. The present findings indicate that postnatal exposure to amitraz caused transient developmental and behavioral changes in the exposed offspring and suggest that further investigation of the potential health risk of amitraz exposure to developing human and animal offsprings may be warranted.

Effect of lead acetate on neurobehavioral development of rats

We investigated the effects of lead exposure during the pre- and postnatal period on the neurobehavioral development of female Wistar rats (70-75 days of age, 120-150 g) using a protocol of lead intoxication that does not affect weight gain. Wistar rats were submitted to lead acetate intoxication by giving their dams 1.0 mM lead acetate. Control dams received deionized water. Growth and neuromotor development were assessed by monitoring daily the following parameters in 20 litters: body weight, ear unfolding, incisor eruption, eye opening, righting, palmar grasp, negative geotaxis, cliff avoidance and startle reflex. Spontaneous alternation was assessed on postnatal day 17 using a T maze. The animals' ability to equilibrate on a beaker rim was measured on postnatal day 19. Lead intoxication was confirmed by measuring renal, hepatic and cerebral lead concentration in dams and litters. Lead treatment hastened the day of appearance of the following parameters: eye opening (control: 13.5 ± 0.6, N = 88; lead: 12.9 ± 0.6, N = 72; P<0.05), startle reflex (control: 13.0 ± 0.8, N = 88; lead: 12.0 ± 0.7, N = 72; P<0.05) and negative geotaxis. On the other hand, spontaneous alternation performance was hindered in lead-exposed animals (control: 37.6 ± 19.7; lead: 57.5 ± 28.3% of alternating animals; P<0.05). These results suggest that lead exposure without concomitant undernutrition alters rat development, affecting specific subsets of motor skills.

Partially folded intermediates during trypsinogen denaturation

The equilibrium unfolding of bovine trypsinogen was studied by circular dichroism, differential spectra and size exclusion HPLC. The change in free energy of denaturation was = 6.99 ± 1.40 kcal/mol for guanidine hydrochloride and = 6.37 ± 0.57 kcal/mol for urea. Satisfactory fits of equilibrium unfolding transitions required a three-state model involving an intermediate in addition to the native and unfolded forms. Size exclusion HPLC allowed the detection of an intermediate population of trypsinogen whose Stokes radii varied from 24.1 ± 0.4 Å to 26.0 ± 0.3 Å for 1.5 M and 2.5 M guanidine hydrochloride, respectively. During urea denaturation, the range of Stokes radii varied from 23.9 ± 0.3 Å to 25.7 ± 0.6 Å for 4.0 M and 6.0 M urea, respectively. Maximal intrinsic fluorescence was observed at about 3.8 M urea with 8-aniline-1-naphthalene sulfonate (ANS) binding. These experimental data indicate that the unfolding of bovine trypsinogen is not a simple transition and suggest that the equilibrium intermediate population comprises one intermediate that may be characterized as a molten globule. To obtain further insight by studying intermediates representing different stages of unfolding, we hope to gain a better understanding of the complex interrelations between protein conformation and energetics.

Neonatal administration of citalopram delays somatic maturation in rats

We investigated the somatic maturation of neonate rats treated during the suckling period with citalopram, a selective serotonin reuptake inhibitor. Groups with 6 male neonates were randomly assigned to different treatments 24 h after birth. Each litter was suckled by one of the dams until the 21st postnatal day. Body weight, head axis and tail length were measured daily from the 1st to the 21st postnatal day. Time of ear unfolding, auditory conduit opening, incisor eruption, and eye opening was determined. Pups received 5 mg (Cit5), 10 mg (Cit10) or 20 mg/kg (Cit20) citalopram sc, or saline (0.9% NaCl, w/v, sc). Compared to saline, body weight was lower (24.04%, P < 0.01) for Cit10 from the 10th to the 21st day and for Cit20 from the 6th to the 21st day (38.19%, P < 0.01). Tail length was reduced in the Cit20 group (15.48%, P < 0.001) from the 8th to the 21st day. A reduction in mediolateral head axis (10.53%, P < 0.05) was observed from the 11th to the 21st day in Cit10 and from the 6th to the 21st day in Cit20 (13.16%, P < 0.001). A reduction in anteroposterior head axis was also observed in the Cit20 group (5.28%, P < 0.05) from the 13th to the 21stday. Conversely, this axis showed accelerated growth from the 12th to the 21stday in the Cit5 group (13.05%, P < 0.05). Auditory conduit opening was delayed in the Cit5 and Cit20 groups and incisor eruption was delayed in all citalopram groups. These findings show that citalopram injected during suckling to rats induces body alterations and suggest that the activity of the serotoninergic system participates in growth mechanisms.

Protein dynamics: hydration and cavities

The temperature-pressure behavior of proteins seems to be unique among the biological macromolecules. Thermodynamic as well as kinetic data show the typical elliptical stability diagram. This may be extended by assuming that the unfolded state gives rise to volume and enthalpy-driven liquid-liquid transitions. A molecular interpretation follows from the temperature and the pressure dependence of the hydration and cavities. We suggest that positron annihilation spectroscopy can provide additional quantitative evidence for the contributions of cavities to the dynamics of proteins. Only mature amyloid fibrils that form from unfolded proteins are very resistant to pressure treatment.

Insights into the role of hydration in protein structure and stability obtained through hydrostatic pressure studies

A thorough understanding of protein structure and stability requires that we elucidate the molecular basis for the effects of both temperature and pressure on protein conformational transitions. While temperature effects are relatively well understood and the change in heat capacity upon unfolding has been reasonably well parameterized, the state of understanding of pressure effects is much less advanced. Ultimately, a quantitative parameterization of the volume changes (at the basis of pressure effects) accompanying protein conformational transitions will be required. The present report introduces a qualitative hypothesis based on available model compound data for the molecular basis of volume change upon protein unfolding and its dependence on temperature.

The powerful high pressure tool for protein conformational studies

The pressure behavior of proteins may be summarized as a the pressure-induced disordering of their structures. This thermodynamic parameter has effects on proteins that are similar but not identical to those induced by temperature, the other thermodynamic parameter. Of particular importance are the intermolecular interactions that follow partial protein unfolding and that give rise to the formation of fibrils. Because some proteins do not form fibrils under pressure, these observations can be related to the shape of the stability diagram. Weak interactions which are differently affected by hydrostatic pressure or temperature play a determinant role in protein stability. Pressure acts on the 2º, 3º and 4º structures of proteins which are maintained by electrostatic and hydrophobic interactions and by hydrogen bonds. We present some typical examples of how pressure affects the tertiary structure of proteins (the case of prion proteins), induces unfolding (ataxin), is a convenient tool to study enzyme dissociation (enolase), and provides arguments to understand the role of the partial volume of an enzyme (butyrylcholinesterase). This approach may have important implications for the understanding of the basic mechanism of protein diseases and for the development of preventive and therapeutic measures.

Volume and energy folding landscape of prion protein revealed by pressure

The main hypothesis for prion diseases proposes that the cellular protein (PrP C) can be altered into a misfolded, ß-sheet-rich isoform, the PrP Sc (from scrapie). The formation of this abnormal isoform then triggers the transmissible spongiform encephalopathies. Here, we discuss the use of high pressure as a tool to investigate this structural transition and to populate possible intermediates in the folding/unfolding pathway of the prion protein. The latest findings on the application of high pressure to the cellular prion protein and to the scrapie PrP forms will be summarized in this review, which focuses on the energetic and volumetric properties of prion folding and conversion.

A aventura da crítica

The adventure of the critic. The aim of this paper is to reply the critical observations made by Fernandes, Rego and Gala in this number of Revista de Economia Política about a paper of mine, also published in this same journal in January 2006, which deals with the relationship between Economics and Rethoric and its unfolding in Brazil. Answering these critical observations I have tried to show that: a) it is not easy, as they do, to associate Habermas’s project to the defense of the approach of Rethoric in Economics; Habermas himself has a lot of objections to the association of his project with Rorty’s pragmatism which seems to be the strongest McCloskey’s influence; b) it is not true that my considerations have a kind of epistemological immunity and that they are not liable to contestations; if it seems so it is because the nature of the materialistic approach itself. At the end I observe that my carpers didn’t reply my observations about the unfolding of the rethorical project in Brazil and that this is, at some measure, surprising, because they are central personages in it.

Ciência econômica e modelos de explicação científica: retomando a questão

Economic science and scientific explanation models. Economics was born under the sign of methodological vagueness. The first author who tried to solve the question (John Stuart Mill) asserted that it is a kind of knowledge that uses the abstract (direct deductive) method but also that it is an exception because for all other phenomena in the field of moral science the correct method is the inverse deductive or historical method. On the other side, functionalist explanations that are condemned by the scientific precepts of economics are present in the science since its beginning with Adam Smith. Economics went ahead without worriment about these methodological issues but this indefiniteness has never gone off stage. Since the start of XXI century new facts are emerging. Developments in Psychology are giving a new breath to Friedman's point of view and seem to fortify the mainstream (Rogebert e Nordberg, 2005), whereas unfolding in complexity science promises to throw it down and put in its place another kind of explanation borrowed from Biology (Beinhocker, 2006). All this stimulates to retake the question. We do this here, under a critical vision, taking the taxonomy of scientific explanations framed by modern positivism.