Alexandre Ferreira

Purification, molecular cloning, and properties of a β-glycosidase isolated from midgut lumen of Tenebrio molitor (Coleoptera) larvae

Two beta-glycosidases (M(r) 59k) were purified from midgut contents of larvae of the yellow mealworm, Tenebrio molitor (Coleoptera: Tenebrionidae). The two enzymes (betaGly1 and betaGly2) have identical kinetic properties, but differ in hydrophobicity. The two glycosidases were cloned and their sequences differ by only four amino acids. The T. molitor glycosidases are family 1 glycoside hydrolases and have the E379 (nucleophile) and E169 (proton donor) as catalytic amino acids based on sequence alignments. The enzymes share high homology and similarity with other insect, mammalian and plant beta-glycosidases. The two enzymes may hydrolyze several substrates, such as disaccharides, arylglucosides, natural occurring plant glucosides, alkylglucosides, oligocellodextrins and the polymer laminarin. The enzymes have only one catalytic site, as inferred from experiments of competition between substrates and sequence alignments. The observed inhibition by high concentrations of the plant glucoside amygdalin, used as substrate, is an artifact generated by transglucosylation. The active site of each purified beta-glycosidase has four subsites, of which subsites +1 and +2 bind glucose with more affinity. Subsite +2 has more affinity for hydrophobic groups, binding with increasing affinities: glucose, mandelonitrile and nitrophenyl moieties. Subsite +3 has more affinity for glucose than butylene moieties. The intrinsic catalytic constant calculated for hydrolysis of the glucose beta-1,4-glucosidic bond is 21.2 s(-1) x M(-1). The putative physiological role of these enzymes is the digestion of di- and oligosaccharides derived from hemicelluloses.

Characterization of a β-glycosidase highly active on disaccharides and of a β-galactosidase from Tenebrio molitor midgut lumen

The midgut of the yellow mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae) larvae has four β-glycosidases. The properties of two of these enzymes (βGly1 and βGly2) have been described elsewhere. In this paper, the characterization of the other two glycosidases (βGly3 and βGly4) is described. βGly3 has one active site, hydrolyzes disaccharides, cellodextrins, synthetic substrates and β-glucosides produced by plants. The enzyme is inhibited by amygdalin, cellotriose, cellotetraose and cellopentaose in high concentrations, probably due to transglycosylation. βGly3 hydrolyzes β 1,4-glycosidic linkages with a catalytic rate independent of the substrate polymerization degree (kint) of 11.9 s−1. Its active site is formed by four subsites, where subsites +1 and −1 bind glucose residues with higher affinity than subsite +2. The main role of βGly3 seems to be disaccharide hydrolysis. βGly4 is a β-galactosidase, since it has highest activity against β-galactosides. It can also hydrolyze fucosides, but not glucosides, and has Triton X-100 as a non-essential activator (Ka=15 μM, pH 4.5). βGly4 has two active sites that can hydrolyze p-nitrophenyl β-galactoside (NPβGal). The one hydrolyzing NPβGal with more efficiency is also active against methylumbellipheryl β-d-galactoside and lactose. The other active site hydrolyzes NPβFucoside and binds NPβGal weakly. βGly4 hydrolyzes hydrophobic substrates with high catalytical efficiency and is able to bind octyl-β-thiogalactoside in its active site with high affinity. The βGly4 physiological role is supposed to be the hydrolysis of galactolipids that are found in membranes from vegetal tissues. As the enzyme has a hydrophobic site where Triton X-100 can bind, it might be activated by membrane lipids, thus becoming fully active only at the surface of cell membranes.

Secretion of β-glycosidase by middle midgut cells and its recycling in the midgut of Tenebrio molitor larvae

There are four beta-glycosidases (betagly1, betagly2, betagly3, and betagly4) in Tenebrio molitor midgut larvae. betagly1 and betagly2 have identical kinetic properties, and differ in a few amino acid residues. Purified betagly1 was used to raise antibodies in a rabbit. The resulting antiserum recognizes in a Western blot only betagly1 and betagly2 in midgut tissue homogenates and contents. An immunocytochemical study carried out using confocal fluorescence and immunogold techniques showed that betagly1+betagly2 are secreted by exocytosis mainly from the distal part of the second third of T. molitor midguts. This is the first immunocytochemical study of an insect digestive enzyme that does not have polymers as substrates. Enzyme assays with 0.3 mM amygdalin, a condition that detects only betagly1+betagly2, revealed that most of those beta-glycosidases are found in the lumen of anterior and middle midgut. This supports the hypothesis that a countercurrent flux of fluid occurs in T. molitor midgut that is able to carry betagly1 and betagly2 to anterior midgut, in agreement with the enzyme recycling mechanism thought to occur in most insects.

Structure, processing and midgut secretion of putative peritrophic membrane ancillary protein (PMAP) from Tenebrio molitor larvae

A cDNA coding for a Tenebrio molitor midgut protein named peritrophic membrane ancillary protein (PMAP) was cloned and sequenced. The complete cDNA codes for a protein of 595 amino acids with six insect-allergen-related-repeats that may be grouped in A (predicted globular)- and B (predicted nonglobular)-types forming an ABABAB structure. The PMAP-cDNA was expressed in Pichia pastoris and the recombinant protein (64kDa) was purified to homogeneity and used to raise antibodies in rabbits. The specific antibody detected PMAP peptides (22kDa) in the anterior and middle midgut tissue, luminal contents, peritrophic membrane and feces. These peptides derive from PMAP, as supported by mass spectrometry, and resemble those formed by the in vitro action of trypsin on recombinant PMAP. Both in vitro and in vivo PMAP processing seem to occur by attack of trypsin to susceptible bonds in the coils predicted to link AB pairs, thus releasing the putative functional AB structures. The AB-domain structure of PMAP is found in homologous proteins from several insect orders, except lepidopterans that have the apparently derived protein known as nitrile-specifier protein. Immunocytolocalization shows that PMAP is secreted by exocytosis and becomes entrapped in the glycocalyx, before being released into midgut contents. Circumstantial evidence suggests that PMAP-like proteins have a role in peritrophic membrane type 2 formation.

Simultaneous Isolation of Three Different Stem Cell Populations from Murine Skin

The skin is a rich source of readily accessible stem cells. The level of plasticity afforded by these cells is becoming increasingly important as the potential of stem cells in Cell Therapy and Regenerative Medicine continues to be explored. Several protocols described single type stem cell isolation from skin; however, none of them afforded simultaneous isolation of more than one population. Herein, we describe the simultaneous isolation and characterization of three stem cell populations from the dermis and epidermis of murine skin, namely Epidermal Stem Cells (EpiSCs), Skin-derived Precursors (SKPs) and Mesenchymal Stem Cells (MSCs). The simultaneous isolation was possible through a simple protocol based on culture selection techniques. These cell populations are shown to be capable of generating chondrocytes, adipocytes, osteocytes, terminally differentiated keratinocytes, neurons and glia, rendering this protocol suitable for the isolation of cells for tissue replenishment and cell based therapies. The advantages of this procedure are far-reaching since the skin is not only the largest organ in the body, but also provides an easily accessible source of stem cells for autologous graft.

Organophosphate and carbamate poisonings in the northwest of Paraná state, Brazil from 1994 to 2005: clinical and epidemiological aspects

In the present study, clinical and epidemiological aspects of 529 intoxication cases of organophosphate or carbamate pesticides in the northwest of the state of Parana, Brazil, over a twelve-year period (1994-2005), are presented. One hundred-five of 257 patients (40.8%) who attempted suicide were admitted to Intensive Care Units (ICUs), with an average hospital stay of two days (range 1-40 days). Men corresponded to 56.4% of the cases of suicide attempts and sixteen individuals died. One hundred-forty patients intoxicated due to occupational exposure were all young adults and nine of them were admitted to ICU, with average hospital stays of eight days (range 1-16 days). Of these cases, two patients died. One hundred twenty-four patients intoxicated due to accidental exposure were mainly children and had a hospital average stay of four days. Twenty patients were admitted to the ICU, and one of them died. Overall complications included respiratory failure, convulsions, and aspiration pneumonia. Deliberate ingestion of organophosphates and carbamates was much more toxic than occupational and accidental exposure. Men aged 15-39 years were the most likely to attempt suicide with these agents and had more prolonged ICU with significant complications and mortality.

Exposição a hidroquinona e ao fenol sobre a resposta inflamatória pulmonar induzida por bactéria

A gravidade dos efeitos causados pela exposicao ambiental e ocupacional ao benzeno determinou o controle de sua utilizacao. No entanto, mesmo nestas condicoes, toxicidade ao sistema imune e nervoso tem sido descrita. A toxicidade do benzeno e determinada pelos seus produtos de biotransformacao, em que fenol (FE) e hidroquinona (HQ) tem papel relevante na imunotoxicidade. Neste contexto, o presente trabalho mostra que a exposicao de ratos Wistar, machos, a doses de 5 ou 10 mg/kg de HQ (via i.p., uma vez ao dia, 13 doses consecutivas, com intervalos de 2 dias a cada 5 doses) provocou reducoes acentuadas no influxo de leucocitos polimorfonucleares (PMN) e mononucleares (MN) para o pulmao 24 horas apos inalacao de Lipopolissacarideo (LPS) de Salmonella abortus. Diferentemente, a migracao de leucocitos em animais expostos ao FE nao foi alterada. A exposicao a ambos os agentes quimicos simultaneamente (dose de 5 mg/kg cada) manteve a reducao na migracao de MN detectada em animais expostos a HQ e potencializou o efeito inibitorio da HQ sobre a migracao de leucocitos PMN. Os prejuizos nas migracoes de leucocitos nao foram decorrentes de modificacoes no numero destas celulas na circulacao. E importante ressaltar que os efeitos foram induzidos por doses dos agentes quimicos que nao causaram prejuizo a funcao hepatica ou renal, determinados pela atividade das transaminases hepaticas e a concentracao de creatinina no soro. Em conjunto, os dados obtidos mostram a exposicao a baixas doses de HQ nao provoca alteracoes nos parâmetros empregados como indicadores de toxicidade. No entanto, os efeitos toxicos sao manifestados resposta do organismo ao trauma.