The Taxonomic Status of Mico marcai (Alperin 1993) and Mico manicorensis (van Roosmalen et al. 2000) (Cebidae,Callitrichinae) from Southwestern Brazilian Amazonia

Species level taxonomic revisions of New World monkeys are scarce but necessary if we are to obtain a clear picture of this group’s diversity. In this work I revise taxonomically the little known marmoset Mico marcai¸ originally described as Callithrix argentata marcai Alperin, 1993, in light of more recently collected material. I show that this species is significantly different in facial pigmentation and pelage color from all other marmosets of the genus Mico in the Madeira–Tapajós interfluvium. The form first described as Callithrix manicorensis is treated as a junior synonym of Mico marcai, as there are no morphological differences or geographical barriers between the two putative taxa. Mico marcai occurs in the Aripuanã–Manicoré interfluvium, limited in the north by the Rio Madeira and in the south by enclaves of savannah vegetation. Mico marcai occurs in sympatry with Callibella humilis through at least part of its range, representing the only known case of sympatry of two species of marmoset. The northern range of Mico marcai is relatively undisturbed and has few human settlements, its southern limit, on the other hand, is crossed by a major Brazilian highway, and in the same region at least seven hydroelectric reservoirs are planned to be built.     

Purification,properties and substrate specificity of a digestive trypsin from Periplaneta americana (Dictyoptera) adults

A digestive trypsin from the American cockroach (Periplaneta americana, Dictyoptera) males was purified by a combination of anionic chromatographies in low and high pressure systems. The yield was 70% with a final specific activity of 2,000 units per mg protein (substrate: benzoyl-Arg-p-nitroanilide, BRpNA). Chemical modification with TLCK (k(obs)=3.3 M(-1) s(-1); stoichiometry 1:1) and PMSF (k(obs)=0.18 M(-1) s(-1); stoichiometry 1:1) confirmed that this peptidase is a trypsin. This enzyme has a molecular weight of 29 kDa (SDS-PAGE), a pI of 6.0 and a pH optimum of 8.9. Kinetic parameters using different colorimetric, fluorimetric and internally-quenched substrates indicated that P. americana trypsin prefers to hydrolyze synthetic substrates containing more than one amino acid residue and with an arginine residue at P1 position and a hydrophobic residue at P2. This enzyme presented a Km of 120 microM for BRpNA and is competitively inhibited by benzamidine (Ki=0.25 microM). Soybean trypsin inhibitor is a tight-binding inhibitor presenting a K(D) of 0.4 nM. Differences in substrate specificity and in the reactivity of the trypsin active site groups can be related to adaptation of insects to different hosts. P. americana trypsin is an excellent model for comparison as a basal group on evolutionary studies of insect trypsins.     

Adaptation of tobacco budworm Heliothis virescens to proteinase inhibitors may be mediated by the synthesis of new proteinases

The tobacco budworm Heliothis virescens is adapted to feed on tobacco leaves that have proteinase protein inhibitors (PIs). To study this adaptation, the midgut proteinases of Heliothis virescens larvae reared on artificial PI-free diet and on tobacco leaves were compared using ion exchange chromatography, hydrophobic chromatography, gel filtration and polyacrylamide gel electrophoresis at different conditions. SDS polyacrylamide-gradient gel electrophoresis (SDS-PAGE) and kinetic studies shown that leaf-fed larvae have a chymotrypsin (M(r) 26000) and four trypsins (T1-T4) with the following properties: T1, K(m) 0.3 microM, M(r) 70000; T2, K(m) 0.4 microM, M(r) 67000; T3, K(m) 2.4 microM, M(r) 29000; T4, K(m) 15 microM, M(r) 17000. Diet-fed larvae have a chymotrypsin (M(r) 26000) and a major trypsin (K(m) 2.9 microM, M(r) 29000). Native PAGE at different gel concentrations showed that in these conditions, only T1 and T2 occur in leaf-fed larvae, whereas gel filtration in the absence and presence of SDS revealed that T1 and T2 might arise by polymerization of T3 and T4, respectively. The data suggest that, in the presence of PI-containing food, H. virescens larvae express new trypsin molecules that form oligomers and are apparently less affected by PIs because of tighter binding to the substrate (lower K(m) values) and a putative decreased affinity for PIs.     

Coiled-coil binding of the leucine zipper domains of APOL1 is necessary for the open cation channel conformation

Apolipoprotein L-I (APOL1) is a channel-forming effector of innate immunity. The common human APOL1 variant G0 provides protection against infection with certain Trypanosoma and Leishmania parasite species, but it cannot protect against the trypanosomes responsible for human African trypanosomiasis. Human APOL1 variants G1 and G2 protect against human-infective trypanosomes but also confer a higher risk of developing chronic kidney disease. Trypanosome-killing activity is dependent on the ability of APOL1 to insert into membranes at acidic pH and form pH-gated cation channels. We previously mapped the channel’s pore-lining region to the C-terminal domain (residues 332–398) and identified a membrane-insertion domain (MID, residues 177–228) that facilitates acidic pH-dependent membrane insertion. In this article, we further investigate structural determinants of cation channel formation by APOL1. Using a combination of site-directed mutagenesis and targeted chemical modification, our data indicate that the C-terminal heptad-repeat sequence (residues 368–395) is a bona fide leucine zipper domain (ZIP) that is required for cation channel formation as well as lysis of trypanosomes and mammalian cells. Using protein-wide cysteine-scanning mutagenesis, coupled with the substituted cysteine accessibility method, we determined that, in the open channel state, both the N-terminal domain and the C-terminal ZIP domain are exposed on the intralumenal/extracellular side of the membrane and provide evidence that each APOL1 monomer contributes four transmembrane domains to the open cation channel conformation. Based on these data, we propose an oligomeric topology model in which the open APOL1 cation channel is assembled from the coiled-coil association of C-terminal ZIP domains.     

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.     

Origin of Embryo-Like Structures in Soybean Anther Culture Investigated Using SSR Marker

The Satt418 microsatellite locus was examined in order to investigate the origin of embryo-like structures (ELS) obtained from soybean anther culture. Four heterozygous plants were used as anther donors. A total of 7000 anthers were placed on the induction medium under culture conditions known to trigger androgenic response. After 60 days of culture, upper portion of 216 ELS were carefully removed and transferred to a proliferation medium, in order to obtain sufficient tissue for DNA extraction. Callogenic masses originated from 114 ELS were screened for the Satt418 microsatellite locus. Heterozygous and homozygous ELS were identified, suggesting occurrence of somatic embryogenesis and androgenesis in the same system. This unexpected morphogenic response seems to be genotype-dependent.     

Distribution of nutrient reserves during spinning in tissues of the larva of the fly, Rhynchosciara americana

The amount of protein, carbohydrate, lipids, and free amino acids were examined in the spinning stage in the fat body, haemolymph, skeletal muscle, and gut of Rhynchosciara americana. Protein and lipids increase in the fat body soon after the animal stopped feeding, probably at the expense of the digestion of the gut contents and of the reserves of the gut wall. Afterwards there is a fall in protein and lipids in the fat body. Haemolymph protein rises a little at the beginning of spinning and then decreases steadily during cocoon production. Carbohydrate and free amino acids decrease from the beginning of spinning in all tissues studied. Quantitatively, the most important decrease of carbohydrate during spinning occurs in the fat body whereas that of free amino acids occurs in the haemolymph. Lipid increases during spinning in the skeletal muscle, probably due to enlargement of the lateral fat body which occurs as a contaminant in the skeletal muscle preparation. The Malpighian tubules contain a large amount of calcium carbonate, which is eliminated during spinning. A correlation of our chemical data with histochemical data recently published is presented and the physiological implications of our findings are discussed in comparison to other insects.     

The role of cortical pattern in timing of cell division and morphogenesis in Stentor.

This paper describes experiments involving reciprocal exchange of the oral apparatus between a large (L) and a small (S) strain of Stentor coeruleus. Mature L-strain stentors given and S-strain oral apparatus formed an oral primordium in response to the presence of abnormally small oral structures and mostly divided although a few reorganized instead. Conversely, when S-strain stentors in early division were given a large L-strain oral apparatus, they resorbed the developing primordium and returned to interphase. When combined with previous results, these findings provide evidence for the hypothesis that the time of cell division in Stentor is determined by a "critical" ratio between the size of the oral apparatus and the size of the cell body (somatic) cortex. This ratio develops because the somatic cortex grows during interphase and the oral apparatus does not. Cell division in Stentor therefore appears to be initiated by a cortical pattern change resulting from cell surface growth during interphase.