Antonio G. de Bianchi

Cathepsin B and acid phosphatase activities during Musca domestica embryogenesis

Abstract During Musca domestica embryogenesis, we detected a proteolytic activity that is probably involved in yolk protein degradation. This was characterized as a cathepsin B-like proteinase, with a pH optimum of 4.6 and a molecular weight of 25,000 ± 5000. An acid phosphatase activity was also found during M. domestica embryogenesis. This enzyme has a molecular weight equal to 84,300 ± 3000, is inhibited by fluoride and tartrate and therefore may be of lysosomal origin. The activity patterns of both enzymes are similar during embryogenesis. Acid phosphatase inhibition does not have any effect on the cathepsin proteolytic action against yolk protein, indicating that the two enzymes do not have a cooperative action for initiation of vitellin hydrolysis.

Formation of Nitrosyl Hemoglobin and Nitrotyrosine during Murine Leishmaniasis

Peroxynitrite, the potent oxidant formed by the fast reaction between nitric oxide and superoxide anion, has been suggested to be the reactive intermediate responsible for some of the pathologies associated with an overproduction of nitric oxide. In this report, we demonstrate that both nitric oxide and peroxynitrite are formed during infection of the susceptible mouse strain, BALBk, with Leishmania amazonensis. Nitric oxide was detected as the nitrosyl hemoglobin complex by EPR analysis of blood drawn from mice at35, 64 and 148 days of infection. The levels of nitrosyl hemoglobin complex increased with disease evolution, which in the murine model used is characterized by skin lesions, ulceration and visceral‐ization of the parasites. Peroxynitrite formation was inferred from immunoreaction of homogenates obtained from footpad lesions in the late stages of the infection with anti‐nitrotyrosine antibody; homogenates from parasites drawn from the lesions were also immunoreactive, although to a lesser extent. Analysis of protein homogenates by gel electrophoresis and western blots suggests that peroxynitrite may degrade proteins in vivo, in addition to nitrating them. The results demonstrate that peroxynitrite is formed during murine leishmaniasis and may play a role in the aggravation of the disease.

MUSCA DOMESTICA HEMOLYMPH FERRITIN

We describe a method for the purification of ferritin from Musca domestica larval hemolymph. Musca ferritin occurs in hemolymph predominantly as a native protein with molecular weight equal to 550,000 and subunits of 26,000. The average iron content of purified ferritin was determined to be 3,000 +/ 600 iron atoms per molecule. The iron contents of ferritin was heterogeneous; both fully iron loaded molecules and apoferritin are probably present in the Musca hemolymph. The anti-ferritin serum raised in rabbit was able to recognize native ferritin but was not reactive with the protein subunits isolated by SDS-PAGE. The ferritin concentration in hemolymph attains a maximum of 0.28 mg/ml in the wandering stage larvae decreasing to 0.13 mg/ml at the middle of pupal stadium. The ferritin contents of midgut and fat bodies were also determined. Fat body ferritin content is greatly reduced when the feeding larva passes into wandering stage.

Processing of procathepsin from Musca domestica eggs

The major source of amino acids for insect embryos are yolk proteins which accumulate in developing oocytes and are hydrolyzed during embryogenesis. Studies on Musca domestica embryogenesis indicated that a cathepsin B-like proteinase is responsible for yolk protein degradation (Ribolla et al., 1993). In this study, we report the purification of mature cathepsin and show that it is made up of a single 41 kDa polypeptide chain. The Musca domestica cathepsin NH2-terminal 11-residue sequence was determined (Ala-Pro-Lys-Tyr-Val-Asp-Tyr-Gly-Glu-Asn-Glu) and reveals homology with other cathepsins of the papain family. Experiments using serum anti-cathepsin show that the enzyme is stored in oocytes as a 55 kDa zymogen. The activation of the zymogen occurs in vitro only at low pH. In vitro activation in the presence of cysteine protease inhibitors is blocked at an intermediary polypeptide of 48 kDa. Kinetic studies of this activation process at pH 3.5 and 4.6 show that the zymogen is processed in a manner similar to that of pepsin (Foltmann, 1986) and papain (Vernet et al., 1991). We propose that Musca domestica cathepsin zymogen activation occurs in two steps. First, an intramolecular cleavage of the procathepsin polypeptide chain (55,000), induced by low pH gives rise to an intermediary polypeptide (48,000) which then undergoes autolysis to produce the mature enzyme (41,000).

A transcriptome analysis of the Aedes aegypti vitellogenic fat body

Abstract Aedes (Stegomyia) aegypti is an important dengue vector in tropical and subtropical zones throughout the world. A transcriptome of Ae. aegypti vitellogenic fat bodies is described here. The fat body is a dynamic tissue that participates in multiple biochemical functions of intermediate metabolism. A total of 589 randomly selected cDNAs were assembled into 262 clusters based on their primary sequence similarities. The putative translated proteins were classified into categories based on their function in accordance with significant similarity using the BlastX at NCBI FTP site and Pfam (Bateman et al. 2000) and SMART (Schultz et al. 2000) databases. The characterization of transcripts expressed in the fat body of Ae. aegypti at 24 hours post blood meal provides a basic tool for understanding the processes occurring in this organ and could identify putative new genes whose promoters can be used to specifically express transgenes in the fat bodies of Ae. aegypti.

Procathepsin and acid phosphatase are stored in Musca domestica yolk spheres

Yolk spheres present in mature invertebrate oocytes are composed of yolk proteins and proteolytic enzymes. In the fly Musca domestica, yolk proteins are degraded during embryogenesis by a cathepsin-like proteinase that is stored as a zymogen. An acid phosphatase is also active in the yolk spheres during Musca embryogenesis. In this paper we show that procathepsin and acid phosphatase are initially stored by a different pathway from the one followed by yolk protein precursors. Both enzymes are taken up by the oocytes and transitorily stored into small vesicles (lysosomes) surrounding the early yolk spheres. Fusion of both structures, the early yolk spheres and lysosomes, creates the mature yolk spheres.

Protein synthesis in the salivary glands of Rhynchosciara americana

Abstract During the fourth larval instar the salivary gland of Rhynchosciara americana is engaged in the synthesis of a silk-like protein secretion. This secretion is composed of a small number of polypeptides whose molecular weights vary between 20,300 and 152,000. The polypeptide composition of this secretion changes as the larvae pass through different periods in the fourth instar toward pupation, and the size of its constituent polypeptides generally decreases. When the larvae stop feeding at the beginning of communal cocoon spinning (third period), an exponential increase in the production of secretion occurs until it represents 75% of the protein synthesis in the gland. In vitro experiments with [ 3 H]leucine incorporation show that all the major secreted polypeptides are produced in the posterior region of the gland, with the anterior region making only a small contribution. Each polypeptide is produced during a specific stage of larval development. Thus the salivary gland follows a precise developmental pattern of protein synthesis which is typical of highly specialized organs.

Morphological aspects of Culex quinquefasciatus salivary glands

The salivary glands of Culex quinquefasciatus female mosquitoes are paired organs composed of two lateral lobes with proximal and distal secretory portions, and a medial lobe. All portions comprise a simple epithelium that surrounds a salivary duct. In the apical portion of the medial lobe, non-secretory cells strongly resemble cells involved in ion and water transport. The general architecture of the secretory portions is similar between lobes. The appearance of the secretory material and the morphological aspect of the apical cell membrane are the most distinctive features among the three secretory portions. Cells in the lateral proximal lobe display thin membrane projections extending into a translucent and finely filamentous secretory product. At the lateral distal portion, the apical cell membrane forms an intricate meshwork that encloses a dark secretory product. Medial lobe secretory cells also contain secretory cavities surrounded by intracytoplasmic vesicles, all containing a very dark and uniform product. Scattered cells holding numerous vacuoles, some of them containing a small and electron-dense granule eccentrically located and resembling those of the diffuse endocrine system, are frequently observed in the periphery of all secretory portions. Immunofluorescence assays revealed that the distal portion of the lateral lobes contains apyrase, an enzyme putatively responsible for platelet aggregation inhibition, diffusely distributed in the cell cytoplasm.

Chemical composition and rate of synthesis of the larval salivary secretion of the fly Rhynchosciara americana

Abstract The salivary secretion of Rhynchosciara americana was chemically analysed. The secretion shows a yellow colour, with a pH of 7·5 and protein as its major component (94·5 per cent of the secretion dry weight). Carbohydrates are minor components of the secretion which amount to 3·4 per cent of the secretion dry weight, of which 2·3 per cent are neutral carbohydrates and 1·1 per cent are galactosamine. The major amino acids present in the secretion proteins are aspartic acid, glycine, serine, and glutamic acid. The salivary secretion proteins can be separated into eleven protein fractions by urea-acrylamide gel electrophoresis from which nine fractions are PAS positive. The salivary pigment moves together with the protein fraction No. 8, which is quantitatively the most important one, and has spectral characteristics identical to a haemolymph pigment. The higher rate of gland protein labelling by 14 C-phenylalanine determined in vivo and in vitro occurs around the middle of the spinning stage at the same time as the appearance of the large chromosomal puffs. The role of the salivary secretion in cocoon production is discussed.

Structural properties of Musca domestica storage protein

The storage protein of Musca domestica is a hexameric protein with an apparent molecular weight of 500,000. The hexamers are assembled by at least three types of polypeptides: p1 and p2 with apparent molecular weights of 83,000 each, and p3 with an apparent molecular weight of 89,000. The storage protein contains 26.1% of tyrosine plus phenylalanine and 0.68% of carbohydrates, which were identified as mannose and/or glucose and possibly N-acetylglucosamine by means of their interaction with lectins. About 50% of the storage protein molecules bind to a Concanavalin A affinity column. The storage protein hexamers begin to dissociate into monomers near pH 7.0 and become totally dissociated at pH 8.5. The hexamers of Musca storage protein are partially dissociated by iodination and are not affected by treatment with Triton X-100.