Identification of a cysteine protease responsible for degradation of sperm histones during male pronucleus remodeling in sea urchins

We have identified a 60-kDa cysteine protease that is associated with chromatin in sea urchin zygotes. This enzyme was found to be present as a proenzyme in unfertilized eggs and was activated shortly after fertilization. At a pH of 7.8–8.0, found after fertilization, the enzyme degraded the five sperm-specific histones (SpH), while the native cleavage-stage (CS) histone variants remained unaffected. Based on its requirements for reducing agents, its inhibition by sulfhydryl blocking compounds and its sensitivity to the cysteine-type protease inhibitors (2S,3S)-translator-epoxysuccinyl-L-leucyl-amido-3-methylbutane-ethyl-ester (E-64 d), cystatin and leupeptin, this protease can be defined as a cysteine protease. Consistently, this protease was not affected by the serine-type protease inhibitors phenylmethylsulfonyl fluoride (PMSF) and pepstatin. The substrate selectivity and pH modulation of the protease activity strongly suggest its role in the removal of sperm-specific histones, which determines sperm chromatin remodeling after fertilization. This suggestion was further substantiated by the inhibition of sperm histones degradation in vivo by E-64 d. Based on these three lines of evidence, we postulate that this cysteine protease is responsible for the degradation of sperm-specific histones which occurs during male pronucleus formation. J. Cell. Biochem. 67:304–315, 1997. © 1997 Wiley-Liss, Inc.     

Particle bombardment and the genetic enhancement of crops: myths and realities

DNA transfer by particle bombardment makes use of physical processes to achieve the transformation of crop plants. There is no dependence on bacteria, so the limitations inherent in organisms such as Agrobacterium tumefaciens do not apply. The absence of biological constraints, at least until DNA has entered the plant cell, means that particle bombardment is a versatile and effective transformation method, not limited by cell type, species or genotype. There are no intrinsic vector requirements so transgenes of any size and arrangement can be introduced, and multiple gene cotransformation is straightforward. The perceived disadvantages of particle bombardment compared to Agrobacterium-mediated transformation, i.e. the tendency to generate large transgene arrays containing rearranged and broken transgene copies, are not borne out by the recent detailed structural analysis of transgene loci produced by each of the methods. There is also little evidence for major differences in the levels of transgene instability and silencing when these transformation methods are compared in agriculturally important cereals and legumes, and other non-model systems. Indeed, a major advantage of particle bombardment is that the delivered DNA can be manipulated to influence the quality and structure of the resultant transgene loci. This has been demonstrated in recently reported strategies that favor the recovery of transgenic plants containing intact, single-copy integration events, and demonstrating high-level transgene expression. At the current time, particle bombardment is the most efficient way to achieve plastid transformation in plants and is the only method so far used to achieve mitochondrial transformation. In this review, we discuss recent data highlighting the positive impact of particle bombardment on the genetic transformation of plants, focusing on the fate of exogenous DNA, its organization and its expression in the plant cell. We also discuss some of the most important applications of this technology including the deployment of transgenic plants under field conditions.     

Co-integration, co-expression and inheritance of unlinked minimal transgene expression cassettes in an apomictic turf and forage grass (Paspalum notatum Flugge)

Bahiagrass (Paspalum notatum Flugge) is an important turf and forage grass in the southeastern United States and other subtropical regions. Biolistic co-transfer of two unlinked, minimal, linear transgene expression cassettes (MCs) into the apomictic bahiagrass cv. Argentine was carried out to evaluate co-integration, quantify co-expression and analyze inheritance to apomictic seed progeny. Gold projectiles were coated with minimal unlinked nptII and bar expression cassettes in a 1:2 molar ratio. Complexity of transgene loci correlated with the amount of DNA used during gene transfer. Transgenic plants displayed a simple nptII integration pattern with 1–4 hybridization signals compared to the non-selected bar gene with 2 to more than 5 hybridization signals per transgenic line. Co-expression of unlinked nptII and bar genes occurred in 19 of the 20 co-transformed lines (95% co-expression frequency). Protein quantification revealed that several lines with complex integration patterns displayed a higher transgene expression than lines with simple transgene integration patterns. Several transgenic lines displayed hybridization signals indicative of concatemerization. Concatemers were confirmed following PCR amplification and sequence analysis of transgene loci. The obligate apomictic bahiagrass cv. Argentine produced uniform seed progeny without segregation of simple or complex transgene loci. NPTII- and PAT-ELISA, as well as herbicide application, confirmed stable expression of the nptII and bar gene at levels similar to the primary transformants. These results demonstrate that biolistic transfer of MCs support stable and high level co-expression of transgenes in bahiagrass.     

Morphological Characterization of Mycobacterium tuberculosis in a MODS Culture for an Automatic Diagnostics through Pattern Recognition

Tuberculosis control efforts are hampered by a mismatch in diagnostic technology: modern optimal diagnostic tests are least available in poor areas where they are needed most. Lack of adequate early diagnostics and MDR detection is a critical problem in control efforts.The Microscopic Observation Drug Susceptibility (MODS) assay uses visual recognition of cording patterns from Mycobacterium tuberculosis (MTB) to diagnose tuberculosis infection and drug susceptibility directly from a sputum sample in 7–10 days with a low cost.An important limitation that laboratories in the developing world face in MODS implementation is the presence of permanent technical staff with expertise in reading MODS.We developed a pattern recognition algorithm to automatically interpret MODS results from digital images. The algorithm using image processing, feature extraction and pattern recognition determined geometrical and illumination features used in an object-model and a photo-model to classify TB-positive images. 765 MODS digital photos were processed. The single-object model identified MTB (96.9% sensitivity and 96.3% specificity) and was able to discriminate non-tuberculous mycobacteria with a high specificity (97.1% M. avium, 99.1% M. chelonae, and 93.8% M. kansasii). The photo model identified TB-positive samples with 99.1% sensitivity and 99.7% specificity.This algorithm is a valuable tool that will enable automatic remote diagnosis using Internet or cellphone telephony. The use of this algorithm and its further implementation in a telediagnostics platform will contribute to both faster TB detection and MDR TB determination leading to an earlier initiation of appropriate treatment.     

Molecular cloning and biochemical characterization of the first Na-channel α-type toxin peptide (Acra4) from Androctonus crassicauda scorpion venom

Due to the medical importance played in Turkey by stings of the scorpion Androctonus crassicauda, its venom has been studied with more attention. In this communication we report a new toxic peptide, named Acra4, because it is the fourth peptide completely characterized from venom of this scorpion. The peptide contains 64 amino acid residues stabilized by four disulfide bridges, with a molecular weight of 6937 Da. Purification of the lethal peptide was performed by three steps of high performance liquid chromatography (HPLC) separations, and the molecular weight was determined by mass spectrometry analysis and the full amino acid sequence was obtained by direct Edman degradation in conjunction with gene cloning. The LD₅₀ of Acra4 was 50.5 ng/20 g mouse body weight (95% confidence intervals from 48.8 to 52.2 ng/20 g mouse body weight). Additionally, from a sample of cDNA of A. crassicauda four genes were cloned displaying sequence similarities to known scorpion toxins, and are reported here as potentially toxic peptides, named Acra5 to Acra8. Electrophysiological studies of Acra4 were performed using Na⁺-channels expressed in F11 cell culture, by patch-clamp recordings. This is the first time that such peptide from A. crassicauda having a specific Na⁺-channel α-type effect is reported. Its affinity toward Na⁺-channels in F11 cell line is in the order of 1 μM concentration.     

Animal Research Ethics in Africa: Is Tanzania Making Progress?

The significance of animals in research cannot be over‐emphasized. The use of animals for research and training in research centres, hospitals and schools is progressively increasing. Advances in biotechnology to improve animal productivity require animal research. Drugs being developed and new interventions or therapies being invented for cure and palliation of all sorts of animal diseases and conditions need to be tested in animals for their safety and efficacy at some stages of their development. Drugs and interventions for human use pass through a similar development process and must be tested pre‐clinically in laboratory animals before clinical trials in humans can be conducted. Therefore, animals are important players in research processes which directly and indirectly benefit animals and humans. However, questions remain as to whether these uses of animals consider the best interests of animals themselves. Various research and training institutions in Tanzania have established some guidelines on animal use, including establishing animal ethics committees. However, most institutions have not established oversight committees. In institutions where there may be guidelines and policies, there are no responsible committees or units to directly oversee if and how these guidelines and policies are enforced; thus, implementation becomes difficult or impossible. This paper endeavours to raise some issues associated with the responsible use of animals in research and training in Tanzania and highlights suggestions for improvement of deficiencies that exist in order to bridge the gap between what ought to be practised and what is practised.     

Sandflies (Diptera: Psychodidae) associated with opossum nests at urban sites in southeastern Brazil: a risk factor for urban and periurban zoonotic Leishmania transmission?

Sandflies associated with opossum nests are reported for the first time in the yards of residences located in the urban area of the municipality of Monte Mor, situated in the metropolitan region of Campinas, state of São Paulo, Brazil. Eleven specimens of Evandromyia cortelezzii and one of Evandromyia lenti were captured in two Didelphis albiventris nests. Ev. cortelezzii is considered a secondary vector species for the transmission of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum in the Neotropics. This association may contribute to the introduction, establishment and maintenance of urban and periurban zoonotic transmission outbreaks of Leishmania and should therefore be investigated further.     

The Dominance of Introduced Plant Species in the Diets of Migratory Galapagos Tortoises Increases with Elevation on a Human‐Occupied Island

The distribution of resources and food selection are fundamental to the ecology, life history, physiology, population dynamics, and conservation of animals. Introduced plants are changing foraging dynamics of herbivores in many ecosystems often with unknown consequences. Galapagos tortoises, like many herbivores, undertake migrations along elevation gradients driven by variability in vegetation productivity which take them into upland areas dominated by introduced plants. We sought to characterize diet composition of two species of Galapagos tortoises, focussing on how the role of introduced forage species changes over space and the implications for tortoise conservation. We quantified the distribution of tortoises with elevation using GPS telemetry. Along the elevation gradient, we quantified the abundance of introduced and native plant species, estimated diet composition by recording foods consumed by tortoises, and assessed tortoise physical condition from body weights and blood parameter values. Tortoises ranged between 0 and 429 m in elevation over which they consumed at least 64 plant species from 26 families, 44 percent of which were introduced species. Cover of introduced species and the proportion of introduced species in tortoise diets increased with elevation. Introduced species were positively selected for by tortoises at all elevations. Tortoise physical condition was either consistent or increased with elevation at the least biologically productive season on Galapagos. Santa Cruz tortoises are generalist herbivores that have adapted their feeding behavior to consume many introduced plant species that has likely made a positive contribution to tortoise nutrition. Some transformed habitats that contain an abundance of introduced forage species are compatible with tortoise conservation.