Terrestrial mollusks (Mollusca: Gastropoda) of Costa Rica: classification, distribution and conservation

Terrestrial mollusks are poorly known worldwide. The country has 183 reported species, 30% endemic and 7% are probably extinct. About 400 species are expected to inhabit the country. Biology, ecology, distribution, genetics and other areas of research are unknown for more than 95% of the species. The most diverse families are Spiraxidae, Orthalicidae and Subulinidae. However, the family that may have more species is Euconulidae. Euconulids inhabit the highlands, where less work has been done. The study of species of highlands will also rise the endemism rate. Future taxonomic, biological and ecological work should consider their low vagility, tendency to produce new taxa in sympatry, specific microhabitat requirements, hermaphroditism, high evolutionary rate (10% per million years), and divergence between species (2 to 30%). Urgent studies to protect the Costa Rican malacofauna include: distribution, abundance, effect of land use and climate changes on populations.     

Metabolism of N-alkylated spermine analogues by polyamine and spermine oxidases

N-alkylated polyamine analogues have potential as anticancer and antiparasitic drugs. However, their metabolism in the host has remained incompletely defined thus potentially limiting their utility. Here, we have studied the degradation of three different spermine analogues N,N′-bis-(3-ethylaminopropyl)butane-1,4-diamine (DESPM), N-(3-benzyl-aminopropyl)-N′-(3-ethylaminopropyl)butane-1,4-diamine (BnEtSPM) and N,N′-bis-(3-benzylaminopropyl)butane-1,4-diamine (DBSPM) and related mono-alkylated derivatives as substrates of recombinant human polyamine oxidase (APAO) and spermine oxidase (SMO). APAO and SMO metabolized DESPM to EtSPD [K _m(APAO) = 10 μM, k _cat(APAO) = 1.1 s⁻¹ and K _m(SMO) = 28 μM, k _cat(SMO) = 0.8 s⁻¹, respectively], metabolized BnEtSPM to EtSPD [K _m(APAO) = 0.9 μM, k _cat(APAO) = 1.1 s⁻¹ and K _m(SMO) = 51 μM, k _cat(SMO) = 0.4 s⁻¹, respectively], and metabolized DBSPM to BnSPD [K _m(APAO) = 5.4 μM, k _cat(APAO) = 2.0 s⁻¹ and K _m(SMO) = 33 μM, k _cat(SMO) = 0.3 s⁻¹, respectively]. Interestingly, mono-alkylated spermine derivatives were metabolized by APAO and SMO to SPD [EtSPM K _m(APAO) = 16 μM, k _cat(APAO) = 1.5 s⁻¹; K _m(SMO) = 25 μM, k _cat(SMO) = 8.2 s⁻¹; BnSPM K _m(APAO) = 6.0 μM, k _cat(APAO) = 2.8 s⁻¹; K _m(SMO) = 19 μM, k _cat(SMO) = 0.8 s⁻¹, respectively]. Surprisingly, EtSPD [K _m(APAO) = 37 μM, k _cat(APAO) = 0.1 s⁻¹; K _m(SMO) = 48 μM, k _cat(SMO) = 0.05 s⁻¹] and BnSPD [K _m(APAO) = 2.5 μM, k _cat(APAO) = 3.5 s⁻¹; K _m(SMO) = 60 μM, k _cat(SMO) = 0.54 s⁻¹] were metabolized to SPD by both the oxidases. Furthermore, we studied the degradation of DESPM, BnEtSPM or DBSPM in the DU145 prostate carcinoma cell line. The same major metabolites EtSPD and/or BnSPD were detected both in the culture medium and intracellularly after 48 h of culture. Moreover, EtSPM and BnSPM were detected from cell samples. Present data shows that inducible SMO parallel with APAO could play an important role in polyamine based drug action, i.e. degradation of parent drug and its metabolites, having significant impact on efficiency of these drugs, and hence for the development of novel N-alkylated polyamine analogues.     

Mss51 and Ssc1 Facilitate Translational Regulation of Cytochrome c Oxidase Biogenesis

The intricate biogenesis of multimeric organellar enzymes of dual genetic origin entails several levels of regulation. In Saccharomyces cerevisiae, mitochondrial cytochrome c oxidase (COX) assembly is regulated translationally. Synthesis of subunit 1 (Cox1) is contingent on the availability of its assembly partners, thereby acting as a negative feedback loop that coordinates COX1 mRNA translation with Cox1 utilization during COX assembly. The COX1 mRNA-specific translational activator Mss51 plays a fundamental role in this process. Here, we report that Mss51 successively interacts with the COX1 mRNA translational apparatus, newly synthesized Cox1, and other COX assembly factors during Cox1 maturation/assembly. Notably, the mitochondrial Hsp70 chaperone Ssc1 is shown to be an Mss51 partner throughout its metabolic cycle. We conclude that Ssc1, by interacting with Mss51 and Mss51-containing complexes, plays a critical role in Cox1 biogenesis, COX assembly, and the translational regulation of these processes.Translational regulation is a fundamental mechanism used to control the accumulation of key proteins in a large variety of biogenetic and physiological processes in both prokaryotic and eukaryotic cells (20, 23). Translational autoregulation is a particular form of regulation exerted by the protein being translated. It is a well-established control mechanism for bacteriophage and prokaryotic systems (15), and it has also been reported in eukaryotes (4). Usually, the newly synthesized protein binds to its own mRNA to repress translation (20). However, repression can also be exerted by nascent chains interacting with the ribosome (49).Translational autoregulation also occurs in semiautonomous eukaryotic organelles of ancestral bacterial origin, namely, mitochondria and chloroplasts. During evolution, these organelles have retained a few genes in their own genomes, which are transcribed within the organelle, and the mRNAs are translated on organellar ribosomes. Most proteins synthesized within the organelles are part of large multimeric enzyme complexes devoted to energy production. These complexes are formed by subunits of dual genetic origin, nuclear and organellar, and assemble in the organellar membranes. Interestingly, intraorganellar translation of certain subunits has been proposed to be regulated by the availability of their assembly partners (1, 39, 54, 55). A distinctive characteristic of these systems is the involvement of ternary factors, mRNA-specific translational activators whose availability would be regulated by the specific gene products. The players and mechanisms involved remain largely unknown.We have focused on the characterization, in the yeast Saccharomyces cerevisiae, of an assembly-controlled translational regulatory system that operates during the biogenesis of cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain. The three subunits forming the COX catalytic core (1, 2, and 3) are encoded in the mitochondrial DNA (mtDNA), and the remaining eight subunits are encoded in the nuclear DNA. Subunits 1 and 2 coordinate the heme A and copper prosthetic groups of the enzyme. COX biogenesis requires the assistance of a large number of ancillary factors acting at all the levels of the process (11). COX assembly is thought to be linear, consisting of the sequential addition of subunits to an initial seed formed by the mtDNA-encoded subunit 1 (Cox1) in both mammalian and yeast cells (11).The concerted accumulation of COX subunits is regulated by posttranslational degradation of most unassembled Cox1 and the other highly hydrophobic core subunits (27). Recently, we along with others have proposed an additional level of regulation, namely, an assembly-controlled synthesis of Cox1 (1, 2, 39, 56). In S. cerevisiae, COX1 mRNA translation is under the control of Mss51 and Pet309 (8, 30). Mss51 is a key element of the regulatory system. Mss51 acts on the 5′ untranslated region (UTR) of COX1 mRNA to promote translation initiation (39, 56) and additionally acts on a target in the protein coding sequence of COX1 mRNA, perhaps to promote elongation (39). Mss51 and newly synthesized Cox1 form a transient complex (2, 39) that is stabilized by Cox14 (2). We have postulated that these interactions downregulate Cox1 synthesis when COX assembly is impaired by trapping Mss51 and limiting its availability for COX1 mRNA translation (2). According to this model, the release of Mss51 from the ternary complex and its availability for Cox1 synthesis probably occur when Cox1 acquires its prosthetic groups or interacts with other COX subunits, a step possibly catalyzed by Shy1, a protein involved in maturation and/or assembly of Cox1 (2, 10, 34). Coa1 could also participate in Cox1 maturation and stabilize the ternary Cox1/Mss51/Cox14 complex until it interacts with Shy1 (34, 40). Further studies are required to understand how Mss51 is recycled from its posttranslational function to become available for COX1 mRNA translation and to fully clarify how this regulatory mechanism operates.In this study, we have analyzed protein-interacting partners of Mss51 in the wild type and a collection of COX assembly mutants. We found that the native molecular weight (MW) of Mss51 is dependent on both the status of COX assembly and the synthesis of Cox1. The mitochondrial Hsp70 (mtHsp70) chaperone Ssc1 interacts with Mss51 and with several high-molecular weight Mss51-containing complexes involving the COX1 mRNA translational apparatus, Cox1, and several Cox1 assembly factors. Mutants defective in Cox1 maturation or in other aspects of COX biogenesis accumulate distinct ratios of these complexes. In this way, Cox1 regulates its own translation through the action of Mss51 and Ssc1.     

Heat-induced programmed cell death in Leishmania infantum is reverted by Bcl-XL expression

An increasing number of reports indicate that single-celled organisms are able to die following what seems to be an ordered program of cell death with strong similarities to apoptosis from higher eukaryotes. DNA degradation and several other apoptotic-like processes have also been described in the parasitic protozoa Leishmania. However, the existence of an apoptotic death in this parasite is still a matter of controversy. Our results indicate that most of the processes of macromolecular degradation and organelle dysfunction observed in mammalian cells during apoptosis can also be reproduced in promastigotes of the genus Leishmania when incubated at temperatures above 38°C. These processes can be partially reversed by the expression of the anti-apoptotic mammalian gene Bcl-X_L, which suggests that this family of apoptosis-regulating proteins was present very early in the evolution of eukaryotic cells.     

Sedentary life style of Neotropical sedge wrens promotes song imitation

To what extent has the style of song development among songbirds coevolved with other life history strategies? Among Cistothorus wrens in North America, it seems that sedentary or site-faithful habits of marsh wrens, C. palustris, favour song imitation, but seminomadic habits of sedge wrens, C. platensis, favour song improvisation, whereby each male generates a large but unique song repertoire. In this study, we tested whether more sedentary populations of sedge wrens in the Neotropics would imitate songs. At our primary study site near Cartago, Costa Rica, breeding birds were colour-banded during 1995 and 1996, and follow-up surveys revealed that the birds remained at this site the year round. Extensive tape recording and analysis of songs showed that males had large song repertoires (200-300+ songs), and that many songs were shared among neighbouring males. In addition, males only 27 km distant, at La Pastora, used different songs. Furthermore, matched countersinging, in which two males answer each other with identical song types, was recorded near Brasilia, in Brazil. The sharing of songs among permanent neighbours, microgeographical variation in song, and matched countersinging can be achieved only through song imitation, thus revealing a striking difference in the style of song development among different populations of the sedge wren. In the Neotropics, having predictable neighbours throughout life appears to have favoured song imitation, so that individuals can interact using a common, learned code typical of the local population; among more mobile populations in North America, however, individuals improvise large repertoires of species-typical songs, thereby enabling singing males to communicate with any individual, no matter what the population of origin. Strategies of song development must correlate with life history features, and further surveys are needed to make sense of the great diversity of singing behaviours among songbirds. Copyright 1999 The Association for the Study of Animal Behaviour.     

Properties of purified recombinant human polyamine oxidase,PAOh1/SMO

The discovery of an inducible oxidase whose apparent substrate preference is spermine indicates that polyamine catabolism is more complex than that originally proposed. To facilitate the study of this enzyme, the purification and characterization of the recombinant human PAOh1/SMO polyamine oxidase are reported. Purified PAOh1/SMO oxidizes both spermine (K(m)=1.6 microM) and N(1)-acetylspermine (K(m)=51 microM), but does not oxidize spermidine. The purified human enzyme also does not oxidize eight representative antitumor polyamine analogues; however, specific oligamine analogues were found to be potent inhibitors of the oxidation of spermine by PAOh1/SMO. The results of these studies are consistent with the hypothesis that PAOh1/SMO represents a new addition to the polyamine metabolic pathway that may represent a new target for antineoplastic drug development.     

Functional homologs of cyanovirin-N amenable to mass production in prokaryotic and eukaryotic hosts

Cyanovirin-N (CV-N) is under development as a topical (vaginal or rectal) microbicide to prevent sexual transmission of human immunodeficiency virus (HIV), and an economically feasible means for very large-scale production of the protein is an urgent priority. We observed that N-glycosylation of CV-N in yeast eliminated the anti-HIV activity, and that dimeric forms and aggregates of CV-N occurred under certain conditions, potentially complicating the efficient, large-scale manufacture of pure monomeric CV-N. We therefore expressed and tested CV-N homologs in which the glycosylation-susceptible Asn residue at position 30 was replaced with Ala, Gln, or Val, and/or the Pro at position 51 was replaced by Gly to eliminate potential conformational heterogeneity. All homologs exhibited anti-HIV activity comparable to wild-type CV-N, and the Pro51Gly homologs were significantly more stable proteins. These glycosylation-resistant, functional cyanovirins should be amenable to large-scale production either in bacteria or in eukaryotic hosts.     

Cytochrome c release and mitochondria involvement in programmed cell death induced by acetic acid in Saccharomyces cerevisiae

Evidence is presented that mitochondria are implicated in the previously described programmed cell death (PCD) process induced by acetic acid in Saccharomyces cerevisiae. In yeast cells undergoing a PCD process induced by acetic acid, translocation of cytochrome c (CytC) to the cytosol and reactive oxygen species production, two events known to be proapoptotic in mammals, were observed. Associated with these events, reduction in oxygen consumption and in mitochondrial membrane potential was found. Enzymatic assays showed that the activity of complex bc(1) was normal, whereas that of cytochrome c oxidase (COX) was strongly decreased. This decrease is in accordance with the observed reduction in the amounts of COX II subunit and of cytochromes a+a(3). The acetic acid-induced PCD process was found to be independent of oxidative phosphorylation because it was not inhibited by oligomycin treatment. The inability of S. cerevisiae mutant strains (lacking mitochondrial DNA, heme lyase, or ATPase) to undergo acetic acid-induced PCD and in the ATPase mutant (knockout in ATP10) the absence of CytC release provides further evidence that the process is mediated by a mitochondria-dependent apoptotic pathway. The understanding of the involvement of a mitochondria-dependent apoptotic pathway in S. cerevisiae PCD process will be most useful in the further elucidation of an ancestral pathway common to PCD in metazoans.