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.

Identification of midgut microvillar proteins from Tenebrio molitor and Spodoptera frugiperda by cDNA library screenings with antibodies.

The objective of this study was to identify midgut microvillar proteins in insects appearing earlier (Coleoptera) and later (Lepidoptera) in evolution. For this, cytoskeleton-free midgut microvillar membrane from Spodoptera frugiperda (Lepidoptera) and Tenebrio molitor (Coleoptera) were used to raise antibodies. These were used for screening midgut cDNA expression libraries. Positive clones were sequenced, assembled and searched for similarities with gene/protein databases. The predicted midgut microvillar proteins from T. molitor were: cockroach allergens (unknown function), peritrophins (peritrophic membrane proteins), digestive enzymes (aminopeptidase, alpha-mannosidase) and unknown proteins. Predicted S. frugiperda midgut proteins may be grouped into six classes: (a) proteins involved in protection of midgut (thioredoxin peroxidase, aldehyde dehydrogenase, serpin and juvenile hormone epoxide hydrolase); (b) digestive enzymes (astacin, transporter-like amylase, aminopeptidase, and carboxypeptidase); (c) peritrophins; (d) proteins associated with microapocrine secretion (gelsolin, annexin); (e) membrane-tightly bound-cytoskeleton proteins (fimbrin, calmodulin) and (f) unidentified proteins. The novel approach is compared with others and microvillar function is discussed in the light of the predicted proteins.

Secretion of beta-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.

Purification, molecular cloning, and properties of a beta-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.

[Rabies epizootic in the urban area of Ribeirão Preto, São Paulo, Brazil]. / Epizootia de raiva na área urbana de Ribeirão Preto, SP, Brasil.

This report describes some epidemiological aspects of a rabies epizootic that started in 1995 in the urban area of Ribeirão Preto, SP, Brazil, and discusses its main causes. All laboratory confirmed cases were described according to a set of epidemiological variables. Simultaneously, information was raised concerning rabies vaccine coverage and epidemiological surveillance activities. In addition to one human case, 58 rabid animals were confirmed in 1995 (54 dogs, 3 cats. and 1 bat). There were 20 cases in 1996 (18 dogs and 2 cats). Geographical distribution was uneven in the city, with higher concentrations observed in the Western, Northern, and Southwestern sections, corresponding to the poorest areas. No seasonal variation was observed. The main reasons for the epizootic were low rabies vaccine coverage in animals and severe failures in epidemiological surveillance activities in the years immediately prior to 1995. This epizootic illustrates the risk of neglecting such activities, even in a city with a reasonably good health system, located in one of the most economically developed areas of the country. Vigorous preventive measures markedly reduced the number of cases.

A case of human rabies in the urban area of Ribeirã Preto, SP, Brazil.

A 39-year old male patient was admitted to the University Hospital of the Faculty of Medicine of Ribeirão, Preto with signs and symptoms of sudden dyspnea, generalized myalgia and behavioral disorders. The initial suspicion was alcohol abstinence syndrome and the patient was referred for psychiatric and neurologic care. The evolution of the patient with a worsening of signs and symptoms, presence of crises of tachypnea, agitation, difficulty to swallow, irritability and hydrophobia, and his report of having been bitten by a suspected dog raised the hypothesis of rabies. The diagnosis was confirmed by examination of a corneal impression, biological tests in the cerebrospinal fluid (CSF) and saliva and visualization of Negri bodies in nervous tissue (direct immunofluorescence). The patient evolved with agitation, aggressiveness, and worsening tachypnea intercalating with apnea, and died on the 4th day after admission.