Antiprotozoal and cysteine proteases inhibitory activity of dipeptidyl enoates

A family of dipeptidyl enoates has been prepared and tested against the parasitic cysteine proteases rhodesain, cruzain and falcipain-2 related to sleeping sickness, Chagas disease and malaria, respectively. They have also been tested against human cathepsins B and L1 for selectivity. Dipeptidyl enoates resulted to be irreversible inhibitors of these enzymes. Some of the members of the family are very potent inhibitors of parasitic cysteine proteases displaying k_2nd (M^?1s^?1) values of seven orders of magnitude. In vivo antiprotozoal testing was also performed. Inhibitors exhibited IC₅₀ values in the micromolar range against Plasmodium falciparum, Trypanosoma brucei, Trypanosoma cruzi and even more promising lower values against Leishmania donovanii.     

Budding yeast Rif1 binds to replication origins and protects DNA at blocked replication forks

Despite its evolutionarily conserved function in controlling DNA replication, the chromosomal binding sites of the budding yeast Rif1 protein are not well understood. Here, we analyse genome‐wide binding of budding yeast Rif1 by chromatin immunoprecipitation, during G1 phase and in S phase with replication progressing normally or blocked by hydroxyurea. Rif1 associates strongly with telomeres through interaction with Rap1. By comparing genomic binding of wild‐type Rif1 and truncated Rif1 lacking the Rap1‐interaction domain, we identify hundreds of Rap1‐dependent and Rap1‐independent chromosome interaction sites. Rif1 binds to centromeres, highly transcribed genes and replication origins in a Rap1‐independent manner, associating with both early and late‐initiating origins. Interestingly, Rif1 also binds around activated origins when replication progression is blocked by hydroxyurea, suggesting association with blocked forks. Using nascent DNA labelling and DNA combing techniques, we find that in cells treated with hydroxyurea, yeast Rif1 stabilises recently synthesised DNA. Our results indicate that, in addition to controlling DNA replication initiation, budding yeast Rif1 plays an ongoing role after initiation and controls events at blocked replication forks.     

A New Glucose-6-Phosphate Dehydrogenase Variant, G6PD Orissa (44 Ala→Gly), is the Major Polymorphic Variant in Tribal Populations in India

Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is usually found at high frequencies in areas of the world where malaria has been endemic. The frequency and genetic basis of G6PD deficiency have been studied in Africa, around the Mediterranean, and in the Far East, but little such information is available about the situation in India. To determine the extent of heterogeneity of G6PD, we have studied several different Indian populations by screening for G6PD deficiency, followed by molecular analysis of deficient alleles. The frequency of G6PD deficiency varies between 3% and 15% in different tribal and urban groups. Remarkably, a previously unreported deficient variant, G6PD Orissa (44 Ala→Gly), is responsible for most of the G6PD deficiency in tribal Indian populations but is not found in urban populations, where most of the G6PD deficiency is due to the G6PD Mediterranean (188 Ser→Phe) variant. The K of G6PD Orissa is fivefold higher than that of the normal enzyme. This may be due to the fact that the alanine residue that is replaced by glycine is part of a putative coenzyme-binding site.     

Effect of insectary rearing on host preference and oviposition behavior of the fruit fly parasitoid Diachasmimorpha longicaudata

Diachasmimorpha longicaudata (Ashmead) has been produced in the laboratory for >160 generations on the larvae of oriental fruit fly, Bactrocera dorsalis (Hendel), the propagation hosts raised routinely on a semi-synthetic wheat diet formulation. Choice tests using modified stinging units were conducted in the laboratory to investigate whether insectary rearing had altered the host seeking and oviposition behavior of female parasitoids. Results showed that fruit fly larvae that developed in papaya, Carica papaya L. var. solo, were less preferred for oviposition than fruit fly larvae that developed on wheat diet when both were exposed concurrently to naive D. longicaudata females (= females without prior oviposition experience). The substrates (pureed papaya or wheat diet) in which treatment larvae were exposed to parasitoids did not affect oviposition preference of gravid D. longicaudata for wheat diet-reared fruit fly larvae. Our study demonstrated the possibility that rearing in an insectary system may have modified the parasitization behavior of female D. longicaudata.     

Evolution of eutherian cytochrome c oxidase subunit II: heterogeneous rates of protein evolution and altered interaction with cytochrome c

Cytochrome c oxidase subunit II (COII), encoded by the mitochondrial genome, exhibits one of the most heterogeneous rates of amino acid replacement among placental mammals. Moreover, it has been demonstrated that cytochrome c oxidase has undergone a structural change in higher primates which has altered its physical interaction with cytochrome c. We collected a large data set of COII sequences from several orders of mammals with emphasis on primates, rodents, and artiodactyls. Using phylogenetic hypotheses based on data independent of the COII gene, we demonstrated that an increased number of amino acid replacements are concentrated among higher primates. Incorporating approximate divergence dates derived from the fossil record, we find that most of the change occurred independently along the New World monkey lineage and in a rapid burst before apes and Old World monkeys diverged. There is some evidence that Old World monkeys have undergone a faster rate of nonsynonymous substitution than have apes. Rates of substitution at four-fold degenerate sites in primates are relatively homogeneous, indicating that the rate heterogeneity is restricted to nondegenerate sites. Excluding the rate acceleration mentioned above, primates, rodents, and artiodactyls have remarkably similar nonsynonymous replacement rates. A different pattern is observed for transversions at four-fold degenerate sites, for which rodents exhibit a higher rate of replacement than do primates and artiodactyls. Finally, we hypothesize specific amino acid replacements which may account for much of the structural difference in cytochrome c oxidase between higher primates and other mammals.      

Relationships among msx gene structure and function in zebrafish and other vertebrates

The zebrafish genome contains at least five msx homeobox genes, msxA, msxB, msxC, msxD, and the newly isolated msxE. Although these genes share structural features common to all Msx genes, phylogenetic analyses of protein sequences indicate that the msx genes from zebrafish are not orthologous to the Msx1 and Msx2 genes of mammals, birds, and amphibians. The zebrafish msxB and msxC are more closely related to each other and to the mouse Msx3. Similarly, although the combinatorial expression of the zebrafish msx genes in the embryonic dorsal neuroectoderm, visceral arches, fins, and sensory organs suggests functional similarities with the Msx genes of other vertebrates, differences in the expression patterns preclude precise assignment of orthological relationships. Distinct duplication events may have given rise to the msx genes of modern fish and other vertebrate lineages whereas many aspects of msx gene functions during embryonic development have been preserved.      

SEXUAL REPRODUCTION AND GENETIC VARIATION IN THE SUBAERIAL ALGA CEPHALEUROS VIRESCENS (ULVOPHYCEAE,CHLOROPHYTA)

Cephaleuros virescens is a pantropical subaerial green alga with no known long‐range dispersal mechanisms. Sexual reproduction is relatively rare and may involve intragametangial fusion of identical, mitotically produced gametes. This situation may be a consequence of adaptation to the subaerial habitat. Genetic variation among populations of C. virescens may be very low and might be positively correlated to the distance (hence, time) separating populations. Thus, assessing the global biogeography of C. virescens requires analysis of what might be low levels of variation. Because C. virescens occurs on literally hundreds of different host species, the question of host‐races must also be considered. Preliminary analysis of local populations of C. virescens, originally obtained as field collections from three different host species and subsequently raised in culture, is the first step in addressing the biogeography of this alga. We are using the AFLP plant mapping protocol by PE Applied Biosystems to detect genetic variability in the three isolates of C. virescens. AFLP is a PCR‐based DNA fingerprinting technique that detects the presence or absence of restriction fragments rather than fragment length differences. Because the number of restriction fragments that can be detected with the AFLP technique is “virtually unlimited,” it is a very powerful tool for assessing the degree of relatedness or variability among cultivars or isolates. AFLP techniques have been used successfully to distinguish morphologically identical bacteria, determine relatedness among soybean accessions, reveal genetic variability within bee samples, and identifyfall armyworm strains and hybrids.     

Fuzzy rule‐based decision support system for evaluation of long‐established forest restoration projects

Although the importance of monitoring and evaluation of restoration actions is increasingly acknowledged, availability of accurate, quantitative monitoring data is very rare for most restoration areas, particularly for long‐established restoration projects. We propose using fuzzy rule‐based expert systems to evaluate the degree of success of restoration actions when available information on project results and impacts largely relies on expert‐based qualitative assessments and rough estimates of quantitative values. These systems use fuzzy logic to manage the uncertainty present in the data and to integrate qualitative and quantitative information. To illustrate and demonstrate the potential of fuzzy rule‐based systems for restoration evaluation, we applied this approach to seven forest restoration projects implemented in Spain between 1897 and 1952, using information compiled in the REACTION database on Mediterranean forest restoration projects. The information available includes both quantitative and expert‐based qualitative data, and covers a wide variety of indicators grouped into technical, structural, functional, and socioeconomic criteria. The fuzzy rule‐based system translates expert knowledge of restoration specialists and forest managers into a set of simple logic rules that integrate information on individual indicators into more general evaluation criteria. The rule‐based approach proposed here can be readily applicable to any kind of restoration project, provided that some information, even if vague and uncertain, is available for a variety of assessment indicators. The evaluation of long‐established forest restoration projects implemented in Spain revealed important asymmetries in the degree of restoration success between technical, structural, functional, and socioeconomic criteria.     

Surface structure changes of rat adipocytes during lypolysis stimulated by various lypolysis stimulated by various lypolytic agents

A qualitative and quantitative electron microscopic study was performed on rat adipocytes during stimulation of lipolysis by various agents. Scanning electron microscopy of control cells revealed a spherical cell with a textured glycocalyx surface exhibiting small irregular projections. Globular surface evaginations or protrusions measuring 8-18 μM in diameter were seen on cell hemispheres, and there was an average of one protrusion for every two hemispheres examined. Distribution analysis showed that 60 percent of the hemispheres had no protrusions, and 25, 10, and 5 percent of the hemispheres had one, two or three protrusions, respectively. Thin-section and freeze- fracture electron microscopy of the protrusions showed a small triglyceride droplet surrounded by a thin cytoplasmic rim that was continuous with the main cytoplasmic matrix. The glycocalyx coating and plasma membrane extended from the cell surface onto, and over, the protrusion. Scanning microscopy of cells stimulated by lipolytic agents, including epinephrine, adrenocorticotropic hormone, theophylline, and dibutyryl cyclic AMP, revealed a dose-dependent increase in the number of protrusions per cell hemisphere. Maximal concentrations of lipolytic hormones cuase an average 2.5-fold increase in the number of protrusions per hemisphere without changing the average size of the protrusions. Only 40 percent of the stimulated cell hemispheres exhibited no protrusions; over 15 percent of the cells contained three or more; and a number of the protrusions were multilobulate. Insulin prevented the increase in the number of protrusions and the change in distribution caused by the lipolytic hormones but did not prevent the increase caused by theophylline and dibutryl cyclic AMP. The data suggest that the protrusions are a structural feature of the cell and may be related to the lypolytic pathway. These observations may help explain some of the discrepant biochemical data relating to hormonal stimulation of lipolysis.