Proteolytic activity in the adult and larval stages of the human roundworm parasite Angiostrongylus costaricensis

Angiostrongylus costaricensis is a nematode that causes abdominal angiostrongyliasis, a widespread human parasitism in Latin America. This study aimed to characterize the protease profiles of different developmental stages of this helminth. First-stage larvae (L1) were obtained from the faeces of infected Sigmodon hispidus rodents and third-stage larvae (L3) were collected from mollusks Biomphalaria glabrata previously infected with L1. Adult worms were recovered from rodent mesenteric arteries. Protein extraction was performed after repeated freeze-thaw cycles followed by maceration of the nematodes in 40 mM Tris base. Proteolysis of gelatin was observed by zymography and found only in the larval stages. In L3, the gelatinolytic activity was effectively inhibited by orthophenanthroline, indicating the involvement of metalloproteases. The mechanistic class of the gelatinases from L1 could not be precisely determined using traditional class-specific inhibitors. Adult worm extracts were able to hydrolyze haemoglobin in solution, although no activity was observed by zymography. This haemoglobinolytic activity was ascribed to aspartic proteases following its effective inhibition by pepstatin, which also inhibited the haemoglobinolytic activity of L1 and L3 extracts. The characterization of protease expression throughout the A. costaricensis life cycle may reveal key factors influencing the process of parasitic infection and thus foster our understanding of the disease pathogenesis.

Detection of the B"-GWGR variant in the southernmost region of Brazil: unveiling the complexity of the human immunodeficiency virus-1 subtype B epidemic

Typical human immunodeficiency virus-1 subtype B (HIV-1B) sequences present a GPGR signature at the tip of the variable region 3 (V3) loop; however, unusual motifs harbouring a GWGR signature have also been isolated. Although epidemiological studies have detected this variant in approximately 17-50% of the total infections in Brazil, the prevalence of B"-GWGR in the southernmost region of Brazil is not yet clear. This study aimed to investigate the C2-V3 molecular diversity of the HIV-1B epidemic in southernmost Brazil. HIV-1 seropositive patients were ana-lysed at two distinct time points in the state of Rio Grande do Sul (RS98 and RS08) and at one time point in the state of Santa Catarina (SC08). Phylogenetic analysis classified 46 individuals in the RS98 group as HIV-1B and their molecular signatures were as follows: 26% B"-GWGR, 54% B-GPGR and 20% other motifs. In the RS08 group, HIV-1B was present in 32 samples: 22% B"-GWGR, 59% B-GPGR and 19% other motifs. In the SC08 group, 32 HIV-1B samples were found: 28% B"-GWGR, 59% B-GPGR and 13% other motifs. No association could be established between the HIV-1B V3 signatures and exposure categories in the HIV-1B epidemic in RS. However, B-GPGR seemed to be related to heterosexual individuals in the SC08 group. Our results suggest that the established B"-GWGR epidemics in both cities have similar patterns, which is likely due to their geographical proximity and cultural relationship.

A nuclear ribosomal DNA pseudogene in triatomines opens a new research field of fundamental and applied implications in Chagas disease

A pseudogene, designated as "ps(5.8S+ITS-2)", paralogous to the 5.8S gene and internal transcribed spacer (ITS)-2 of the nuclear ribosomal DNA (rDNA), has been recently found in many triatomine species distributed throughout North America, Central America and northern South America. Among characteristics used as criteria for pseudogene verification, secondary structures and free energy are highlighted, showing a lower fit between minimum free energy, partition function and centroid structures, although in given cases the fit only appeared to be slightly lower. The unique characteristics of "ps(5.8S+ITS-2)" as a processed or retrotransposed pseudogenic unit of the ghost type are reviewed, with emphasis on its potential functionality compared to the functionality of genes and spacers of the normal rDNA operon. Besides the technical problem of the risk for erroneous sequence results, the usefulness of "ps(5.8S+ITS-2)" for specimen classification, phylogenetic analyses and systematic/taxonomic studies should be highlighted, based on consistence and retention index values, which in pseudogenic sequence trees were higher than in functional sequence trees. Additionally, intraindividual, interpopulational and interspecific differences in pseudogene amount and the fact that it is a pseudogene in the nuclear rDNA suggests a potential relationships with fitness, behaviour and adaptability of triatomine vectors and consequently its potential utility in Chagas disease epidemiology and control.

A brief introduction to molecular orbital theory of simple polyatomic molecules for undergraduate chemistry students

A simple, four-step method for better introducing undergraduate students to the fundamentals of molecular orbital (MO) theory of the polyatomic molecules H2O, NH3, BH3 and SiH4 using group theory is reported. These molecules serve to illustrate the concept of ligand group orbitals (LGOs) and subsequent construction of MO energy diagrams on the basis of molecular symmetry requirements.

Leibniz's theory of bodies: monadic aggregates, phenomena, or both?

Leibniz's conception of bodies seems to be a puzzling theory. Bodies are seen as aggregates of monads and as wellfounded phenomena. This has initiated controversy and unending discussions. The paper attempts to resolve the apparent inconsistencies by a new and formally spirited reconstruction of Leibniz's theory of monads and perception, on the one hand, and a (re-)formulation and precisation of his concept of preestablished harmony, on the other hand. Preestablished harmony is modelled basically as a covariation between the monadic and the ideal realm.

Analysis and forecast of the storage needs of soybeans in Brazil

The existence of a minimum storage capacity of grains as a condition for the maintenance of regulator physical stocks has been used as a strategic factor in the agribusiness expansion. However, in Brazil the storage infrastructure has not followed the growth of the agricultural sector. This fact is evident in the case of soybeans that currently represent 49% of grain production in the country, whose volume production has been increasing significantly over the years. This study aimed to predict the futureneeds of static storage capacity of soybeans from historical data to estimate the investment needed to install storage units in Brazil for the next five years. A statistic analysis of collected data allowed a forecast and identification of the number of storage units that should be installed to meet the storage needs of soybeans in the next five years. It was concluded that by 2015 the soybean storage capacity should be 87 million tons, and to store 49% of soybeans produced, 1,104 storage units should be installed at a cost of R$ 442 million.

The theory of constraints in manufacturing

In this paper, a systematic and quantitative view is presented for the application of the theory of constraints in manufacturing. This is done employing the operational research technique of mathematical programming. The potential of the theory of constraints in automated manufacturing is demonstrated.

A 37-kb restriction map of the human immunoglobulin lambda variable locus, VB cluster, harboring four functional genes and two non-coding Vl sequences

The human immunoglobulin lambda variable locus (IGLV) is mapped at chromosome 22 band q11.1-q11.2. The 30 functional germline v-lambda genes sequenced untill now have been subgrouped into 10 families (Vl1 to Vl10). The number of Vl genes has been estimated at approximately 70. This locus is formed by three gene clusters (VA, VB and VC) that encompass the variable coding genes (V) responsible for the synthesis of lambda-type Ig light chains, and the Jl-Cl cluster with the joining segments and the constant genes. Recently the entire variable lambda gene locus was mapped by contig methodology and its one- megabase DNA totally sequenced. All the known functional V-lambda genes and pseudogenes were located. We screened a human genomic DNA cosmid library and isolated a clone with an insert of 37 kb (cosmid 8.3) encompassing four functional genes (IGLV7S1, IGLV1S1, IGLV1S2 and IGLV5a), a pseudogene (VlA) and a vestigial sequence (vg1) to study in detail the positions of the restriction sites surrounding the Vl genes. We generated a high resolution restriction map, locating 31 restriction sites in 37 kb of the VB cluster, a region rich in functional Vl genes. This mapping information opens the perspective for further RFLP studies and sequencing

The use of non-human primates as animal models for the study of hepatitis viruses

Hepatitis viruses belong to different families and have in common a striking hepatotropism and restrictions for propagation in cell culture. The transmissibility of hepatitis is in great part limited to non-human primates. Enterically transmitted hepatitis viruses (hepatitis A virus and hepatitis E virus) can induce hepatitis in a number of Old World and New World monkey species, while the host range of non-human primates susceptible to hepatitis viruses transmitted by the parenteral route (hepatitis B virus, hepatitis C virus and hepatitis delta virus) is restricted to few species of Old World monkeys, especially the chimpanzee. Experimental studies on non-human primates have provided an invaluable source of information regarding the biology and pathogenesis of these viruses, and represent a still indispensable tool for vaccine and drug testing.

Involvement of the actin cytoskeleton and p21rho-family GTPases in the pathogenesis of the human protozoan parasite Entamoeba histolytica

It has been estimated that infection with the enteric protozoan parasite Entamoeba histolytica kills more than 50,000 people a year. Central to the pathogenesis of this organism is its ability to directly lyse host cells and cause tissue destruction. Amebic lesions show evidence of cell lysis, tissue necrosis, and damage to the extracellular matrix. The specific molecular mechanisms by which these events are initiated, transmitted, and effected are just beginning to be uncovered. In this article we review what is known about host cell adherence and contact-dependent cytolysis. We cover the involvement of the actin cytoskeleton and small GTP-binding proteins of the p21rho-family in the process of cell killing and phagocytosis, and also look at how amebic interactions with molecules of the extracellular matrix contribute to its cytopathic effects.

A chromatographic method for the production of a human immunoglobulin G solution for intravenous use

Immunoglobulin G (IgG) of excellent quality for intravenous use was obtained from the cryosupernatant of human plasma by a chromatographic method based on a mixture of ion-exchange, DEAE-Sepharose FF and arginine Sepharose 4B affinity chromatography and a final purification step by Sephacryl S-300 HR gel filtration. The yield of 10 experimental batches produced was 3.5 g IgG per liter of plasma. A solvent/detergent combination of 1% Tri (n-butyl) phosphate and 1% Triton X-100 was used to inactivate lipid-coated viruses. Analysis of the final product (5% liquid IgG) based on the mean for 10 batches showed 94% monomers, 5.5% dimers and 0.5% polymers and aggregates. Anticomplementary activity was 0.3 CH50/mg IgG and prekallikrein activator levels were less than 5 IU/ml. Stability at 37ºC for 30 days in the liquid state was satisfactory. IgG was stored in flasks (2.5 g/flask) at 4 to 8ºC. All the characteristics of the product were consistent with the requirements of the 1997 Pharmacopée Européenne.

Adhesion of the human pathogen Sporothrix schenckii to several extracellular matrix proteins

The pathogenic fungus Sporothrix schenckii is the causative agent of sporotrichosis. This subcutaneous mycosis may disseminate in immunocompromised individuals and also affect several internal organs and tissues, most commonly the bone, joints and lung. Since adhesion is the first step involved with the dissemination of pathogens in the host, we have studied the interaction between S. schenckii and several extracellular matrix (ECM) proteins. The binding of two morphological phases of S. schenckii, yeast cells and conidia, to immobilized type II collagen, laminin, fibronectin, fibrinogen and thrombospondin was investigated. Poly (2-hydroxyethyl methacrylate) (poly-HEMA) was used as the negative control. Cell adhesion was assessed by ELISA with a rabbit anti-S. schenckii antiserum. The results indicate that both morphological phases of this fungus can bind significantly to type II collagen, fibronectin and laminin in comparison to the binding observed with BSA (used as blocking agent). The adhesion rate observed with the ECM proteins (type II collagen, fibronectin and laminin) was statistically significant (P<0.05) when compared to the adhesion obtained with BSA. No significant binding of conidia was observed to either fibrinogen or thrombospondin, but yeast cells did bind to the fibrinogen. Our results indicate that S. schenckii can bind to fibronectin, laminin and type II collagen and also show differences in binding capacity according to the morphological form of the fungus.

A study of the interaction between Helicobacter pylori and components of the human fibrinolytic system

The interaction of plasminogen, tissue plasminogen activator (t-PA) and urokinase with a clinical strain of Helicobacter pylori was studied. Plasminogen bound to the surface of H. pylori cells in a concentration-dependent manner and could be activated to the enzymatic form, plasmin, by t-PA. Affinity chromatography assays revealed a plasminogen-binding protein of 58.9 kDa in water extracts of surface proteins. Surface-associated plasmin activity, detected with the chromogenic substrate CBS 00.65, was observed only when plasminogen and an exogenous activator were added to the cell suspension. The two physiologic plasminogen activators, t-PA and urokinase, were also shown to bind to and remain active on the surface of bacterial cells. epsilon-Aminocaproic acid caused partial inhibition of t-PA binding, suggesting that the kringle 2 structure of this activator is involved in the interaction with surface receptors. The activation of plasminogen by t-PA, but not urokinase, strongly depended on the presence of cells and a 25-fold enhancer effect on the initial velocity of activation by t-PA compared to urokinase was established. Furthermore, a relationship between cell concentration and the initial velocity of activation was demonstrated. These findings support the concept that plasminogen activation by t-PA on the bacterial surface is a surface-dependent reaction which offers catalytic advantages.

Measuring higher order optical aberrations of the human eye: techniques and applications

In the present paper we discuss the development of "wave-front", an instrument for determining the lower and higher optical aberrations of the human eye. We also discuss the advantages that such instrumentation and techniques might bring to the ophthalmology professional of the 21st century. By shining a small light spot on the retina of subjects and observing the light that is reflected back from within the eye, we are able to quantitatively determine the amount of lower order aberrations (astigmatism, myopia, hyperopia) and higher order aberrations (coma, spherical aberration, etc.). We have measured artificial eyes with calibrated ametropia ranging from +5 to -5 D, with and without 2 D astigmatism with axis at 45º and 90º. We used a device known as the Hartmann-Shack (HS) sensor, originally developed for measuring the optical aberrations of optical instruments and general refracting surfaces in astronomical telescopes. The HS sensor sends information to a computer software for decomposition of wave-front aberrations into a set of Zernike polynomials. These polynomials have special mathematical properties and are more suitable in this case than the traditional Seidel polynomials. We have demonstrated that this technique is more precise than conventional autorefraction, with a root mean square error (RMSE) of less than 0.1 µm for a 4-mm diameter pupil. In terms of dioptric power this represents an RMSE error of less than 0.04 D and 5º for the axis. This precision is sufficient for customized corneal ablations, among other applications.