Conotoxin GI: disulfide bridges, synthesis, and preparation of iodinated derivatives

The 13 amino acid toxic peptide from the marine snail Conus geographus, conotoxin GI, blocks the acetylcholine receptor at the neuromuscular junction. In this report, we describe a method for analyzing disulfide bonding in nanomole amounts of small cystine-rich peptides. The procedure involves partial reduction and a double-label alkylation of cysteine residues. Using this method, we show that the natural conotoxin GI has a (2-7, 3-13) disulfide configuration. The structure of conotoxin GI has been confirmed by chemical synthesis. The preparation and purification of molecularly homogeneous, iodinated derivatives of this toxin are also described. All derivatives, including the [diiodohistidine,diiodotyrosine]conotoxin GI, retained at least half of the biological activity of unmodified toxin. Since the tetraiodinated toxin, which is greater than 25% by weight iodine, retains considerable toxicity, unmodified histidine and tyrosine residues in conotoxin GI are not crucial for biological activity.     

Removal of trace metal ions from industrial waters, nuclear effluents and drinking water, with the aid of cross-linked N-carboxymethyl chitosan

Chitoplex (cross-linked N-carboxymethyl chitosan) is particularly effective in removing trace metal ions (0·01 mg/liter) from brines (50 g/liter), carrier-free radioisotopes such as cobalt-60 from nuclear effluents, and toxic contaminants such as lead and cadmium (0·01 mg/liter) from drinking waters. Chitoplex is indifferent to large concentrations of magnesium.     

Foraging for patchily-distributed leaf miners by the parasitic wasp,Dacnusa sibirica

We studied the rules used by the female parasitoid, Dacnusa sibirica Telenga (Hymenoptera: Braconidae), for deciding when to leave a tomato leaflet on which she is searching for larvae of the leafminer, Liriomyza bryoniae Kalt. (Diptera: Agromyzidae). Females would deposit a marking pheromone on the leaflet and would leave the leaflet when the amount of the pheromone accumulated to the threshold L, which is proportional to the amount of search effort on the leaflet. L appears to increase with host density since it rises after every encounter with a host (or mine).D. sibirica would employ an area-concentrated search, which is advantageous in foraging for hosts showing a clumped distribution.     

Suppression of humoral response during the course of Candida albicans infection in mice

This paper aims at demonstrating the non-specific immunosuppression as regards thyme-dependent antigens sheep erythrocytes (SRBC) during the course of Candida albicans systemic infection.Three lots of syngeneic /BALB/c mice, 8–12 weeks of age, were used. The first normal lot was inoculated via the intraperitoneal route with a (SRBC) suspension (4×10⁸ cells ml) in a Hank's balanced saline solution. The primary response of antibodies formed by splenic cells was measured from 4 to 8 days after inoculation using the direct plaque forming cells technique. The second lot was infected by the same route with a suspension of Candida albicans (1×10⁷ cells). Positive retrocultures from the blood and kidneys of these infected mice were obtained. These yeasts cultivated in a Sabouraud medium were harvested after 20 h at 37 °C. Following the same methodology the immune response to SRBC was determined. The serum obtained from infected mice was transferred to a third lot of mice at different intervals during the course of the infection. The immune response to SRBC was done by the direct plaque-forming cells technique. Controls were carried out using normal donors and recipients.A suppression of the immune response was obtained as from the 2nd day of inoculation up to the 28th day. It was not possible to transfer such suppression passively by means of the serum.These results suggest that the systemic infection by Candida albicans induce a non-specific immunosuppression in the organism, already demonstrated in viral infections, bacteria, protozoaria and metazoaria in mammals.In some way, this will contribute to explain the mechanisms of immune response to Candida albicans.     

In vitro synthesis of myelin proteolipid protein from rat brain using a total homogenate system

In vitro synthesis of myelin proteolipid protein (PLP) was explored at different ages using rat brain total homogenates, incubated for 30 min with [³H]glycine. Total proteolipids, extracted from the incubated samples, were separated by SDSPAGE and the radioactivity was measured in the band corresponding to myelin PLP. The incorporation into PLP in relation to the incorporation into brain total proteins increased from 0.04% at 10 days of age to 0.63% at 20 days, and declined slowly thereafter. Time course experiments were carried out using brain homogenates obtained from rats of 20 days of age (i.e. at the period of maximal synthesis of PLP). Labeled PLP molecules were already found at 2.5 min of incubation and the incorporation of the label into this protein, relative to the incorporation into total proteins, did not vary throughout the entire incubation time (30 min). Pulsechase experiments using a similar system and adding cycloheximide at different incubation times showed that the appearance of label into mature PLP was immediately blocked by the inhibitor of protein synthesis. These data suggest that PLP is synthesized as such and not as a pre-protein which is subsequently processed to render mature PLP.     

Penicillin amidohydrolases in fungal autolysis

The production of penicillin G and penicillin V amidohydrolases or acylases (E.C.3.5.1.11) was studied during the autolysis of filamentous fungi in a mineral medium, and in the same medium with phenoxyacetic acid as inducer. In all the studied fungi, enzymes showing penicillin G and penicillin V amidohydrolase activities were found. Generally, an increase of these activities during fungal autolysis was observed. The presence of phenoxyacetic acid in the medium did not increase these activities. The activities found in the culture fluids were generally higher than that found in the mycelial extracts. Under these conditions, beta-lactamases (penicillinases) were not found. The fungi Alternaria alternata, Fusarium culmorum, Penicillium oxalicum, and the species Penicillium 222 were chosen to study penicillin G and penicillin V acylases. The enzymes were precipitated with tannic acid from the culture fluid of their autolyzed cultures. Some kinetic constants of these activities were determined.     

Limb development in the gekkonid lizard gonatodes albogularis: A reconsideration of homology in the lizard carpus and tarsus

Despite the attention squamate lizards have received in the study of digit and limb loss, little is known about limb morphogenesis in pentadactyl lizards. Recent developmental studies have provided a basis for understanding lizard autopodial element homology based on developmental and comparative anatomy. In addition, the composition and identity of some carpal and tarsal elements of lizard limbs, and reptiles in general, have been the theme of discussions about their homology compared to non‐squamate Lepidosauromorpha and basal Amniota. The study of additional embryonic material from different lizard families may improve our understanding of squamate limb evolution. Here, we analyze limb morphogenesis in the gekkonid lizard Gonatodes albogularis describing patterns of chondrogenesis and ossification from early stages of embryonic development to hatchlings. Our results are in general agreement with previous developmental studies, but we also show that limb development in squamates probably involves more chondrogenic elements for carpal and tarsal morphogenesis, as previously recognized on the grounds of comparative anatomy. We provide evidence for the transitory presence of distal carpale 1 and intermedium in the carpus and tibiale, intermedium, distal centralia, and distal tarsale 2 in the tarsus. Hence, we demonstrate that some elements that were believed to be lost in squamate evolution are conserved as transitory elements during limb development. However, these elements do not represent just phylogenetic burden but may be important for the morphogenesis of the lizard autopodium. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Coumarin A/AA induces apoptosis‐like cell death in HeLa cells mediated by the release of apoptosis‐inducing factor

It has been demonstrated that naturally occurring coumarins have strong biological activity against many cancer cell lines. In this study, we assessed the cytotoxicity induced by the naturally isolated coumarin A/AA in different cancer cell lines (HeLa, Calo, SW480, and SW620) and in normal peripheral‐blood mononuclear cells (PBMCs). Cytotoxicity was evaluated using the MTT assay. The results demonstrate that coumarin A/AA was cytotoxic in the four cancer cell lines tested and importantly was significantly less toxic in PBMCs isolated from healthy donors. The most sensitive cancer cell line to coumarin A/AA treatment was Hela. Thus, the programmed cell death (PCD) mechanism induced by this coumarin was further studied in this cell line. DNA fragmentation, histomorphology, cell cycle phases, and subcellular distribution of PCD proteins were assessed. The results demonstrated that DNA fragmentation, but not significant cell cycle disruptions, was part of the PCD activated by coumarin A/AA. Interestingly, it was found that apoptosis‐inducing factor (AIF), a proapoptotic protein of the mitochondrial intermembrane space, was released to the cytoplasm in treated cells as detected by the western blot analysis in subcellular fractions. Nevertheless, the active form of caspase‐3 was not detected. The overall results indicate that coumarin A/AA induces a caspase‐independent apoptotic‐like cell death program in HeLa cells, mediated by the early release of AIF and suggest that this compound may be helpful in clinical oncology. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:263–272, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20288     

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