Functional and bioinformatics analyses reveal conservation of Cis-regulatory elements between sciaridae and Drosophilidae

The sciarid DNA puff C4 BhC4-1 gene is amplified and transcribed in salivary glands at the end of the larval stage. In transgenic Drosophila, the BhC4-1 promoter drives transcription in prepupal salivary glands and in the ring gland of late embryos. A bioinformatics analysis has identified 162 sequences similar to distinct regions of the BhC4-1 proximal promoter, which are predominantly located either in 5` or 3` regions or introns in the Drosophila melanogaster genome. A significant number of the identified sequences are found in the regulatory regions of Drosophila genes that are expressed in the salivary gland. Functional assays in Drosophila reveal that the BhC4-1 proximal promoter contains both a 129 bp (-186/-58) salivary gland enhancer and a 67 bp (-253/-187) ring gland enhancer that drive tissue specific patterns of developmentally regulated gene expression, irrespective of their orientation.

A DNA vaccine candidate encoding the structural prM/E proteins elicits a strong immune response and protects mice against dengue-4 virus infection

A DNA vaccine expressing dengue-4 virus premembrane (prM) and envelope (E) genes was produced by inserting these genes into a mammalian expression plasmid (pCI). Following a thorough screening, including confirmation of protein expression in vitro, a recombinant clone expressing these genes was selected and used to immunize BALB/c mice. After 3 immunizations all the animals produced detectable levels of neutralizing antibodies against dengue-4 virus. The cytokines levels and T cell proliferation, detected ex vivo from the spleen of the immunized mice, showed that our construction induced substantial immune stimulation after three doses. Even though the antibody levels, induced by our DNA vaccine, were lower than those obtained in mice immunized with dengue-4 virus the levels of protection were high with this vaccine. This observation is further supported by the fact that 80% of the vaccine immunized group was protected against lethal challenge. In conclusion, we developed a DNA vaccine employing the genes of the prM and E proteins from dengue-4 virus that protects mice against this virus. (C) 2010 Elsevier Ltd. All rights reserved.

Stability improvement of the fatty acid binding protein Sm14 from S. mansoni by Cys replacement: Structural and functional characterization of a vaccine candidate

The Schistosoma mansoni fatty acid binding protein (FABP), SmA, is a vaccine candidate against, S. mansoni and F hepatica. Previously, we demonstrated the importance of a correct fold to achieve protection in immunized animals after cercariae challenge [[10]. C.R.R. Ramos, R.C.R. Figueredo, T.A. Pertinhez, M.M. Vilar, A.L.T.O. Nascimento, M. Tendler, I. Raw, A. Spisni, P.L. Ho, Gene structure and M20T polymorphism of the Schistosoma mansoni Sm14 fatty acid-binding protein: structural, functional and immunoprotection analysis. J. Biol. Chem. 278 (2003) 12745-12751]. Here we show that the reduction of vaccine efficacy over time is due to protein dimerization and subsequent aggregation. We produced the mutants Sm14-M20(C62S) and Sm14M20(C62V) that, as expected, did not dimerize in SDS-PAGE. Molecular dynamics calculations and unfolding experiments highlighted a higher structural stability of these mutants with respect to the wild-type. In addition, we found that the mutated proteins, after thermal denaturation, refolded to their active native molecular architecture as proved by the recovery of the fatty acid binding ability. Sm14-M20(C62V) turned out to be the more stable form over time, providing the basis to determine the first 3D solution structure of a Sm14 protein in its apo-form. Overall, Sm14-M20(C62V) possesses an improved structural stability over time, an essential feature to preserve its immunization capability and, in experimentally immunized animals, it exhibits a protection effect against S. mansoni cercariae infections comparable to the one obtained with the wild-type protein. These facts indicate this protein as a good lead molecule for large-scale production and for developing an effective Sm14 based anti-helminthes vaccine. (C) 2008 Elsevier B.V. All rights reserved.

Hypertrophy and neuron loss: structural changes in sheep SCG induced by unilateral sympathectomy

Recently, superior cervical ganglionectomy has been performed to investigate a variety of scientific topics from regulation of intraocular pressure to suppression of lingual tumour growth. Despite these recent advances in our understanding of the functional mechanisms underlying superior cervical ganglion (SCG) growth and development after surgical ablation, there still exists a need for information concerning the quantitative nature of the relationships between the removed SCG and its remaining contralateral ganglion and between the remaining SCG and its modified innervation territory. To this end, using design-based stereological methods, we have investigated the structural changes induced by unilateral ganglionectomy in sheep at three distinct timepoints (2, 7 and 12 weeks) after surgery. The effects of time, and lateral (left-right) differences, were examined by two-way analyses of variance and paired t-tests. Following removal of the left SCG, the main findings were: (i) the remaining right SCG was bigger at shorter survival times, i.e. 74% at 2 weeks, 55% at 7 weeks and no increase by 12 weeks, (ii) by 7 weeks after surgery, the right SCG contained fewer neurons (no decrease at 2 weeks, 6% fewer by 7 weeks and 17% fewer by 12 weeks) and (iii) by 7 weeks, right SCG neurons were also larger and the magnitude of this increase grew substantially with time (no rise at 2 weeks, 77% by 7 weeks and 215% by 12 weeks). Interaction effects between time and ganglionectomy-induced changes were significant for SCG volume and mean perikaryal volume. These findings show that unilateral superior cervical ganglionectomy has profound effects on the contralateral ganglion. For future investigations, it would be interesting to examine the interaction between SCGs and their innervation targets after ganglionectomy. Is the ganglionectomy-induced imbalance between the sizes of innervation territories the milieu in which morphoquantitative changes, particularly changes in perikaryal volume and neuron number, occur? Mechanistically, how would those changes arise? Are there any grounds for believing in a ganglionectomy-triggered SCG cross-innervation and neuroplasticity? (C) 2011 ISDN. Published by Elsevier Ltd. All rights reserved.

Assessment of trace elements in fishes of Japanese foods marketed in Sao Paulo (Brazil)

In recent years, there has been increasing fish consumption in Brazil, largely due to the popularity of Japanese cuisine. No study, however, has previously assessed the presence of inorganic contaminants in species used in the preparation of Japanese food. In this paper, we determined total arsenic, cadmium, chromium, total mercury, and lead contents in 82 fish samples of Tuna (Thunnus thynnus), Porgy (Pagrus pagrus), Snook (Centropomus sp.), and Salmon (Salmo salar) species marketed in Sao Paulo (Brazil). Samples were mineralized in HNO(3)/H(2)O(2) for As, Cd, Cr and Pb, and in HNO(3)/H(2)SO(4)/V(2)O(5) for Hg. Inorganic contaminants were determined after the validation of the methodology using Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES); and for Hg, an ICP-coupled hydride generator was used. Concentration ranges for elements analyzed in mg kg(-1) (wet base) were as follows: Total As (0.11-10.82); Cd (0.005-0.047); Cr (0.008-0.259); Pb (0.026-0.481); and total Hg (0.0077-0.9681). As and Cr levels exceeded the maximum limits allowed by the Brazilian law (1 and 0.1 mg kg(-1)) in 51.2 and 7.3% of the total samples studied, respectively. The most contaminated species were porgy (As = 95% and Cr = 10%) and tuna (As 91% and Cr = 10%). An estimation of As, Cd, Pb, and Hg weekly intake was calculated considering a 60 kg adult person and a 350 g consumption of fish per week, with As and Hg elements presenting the highest contribution on diets reaching 222% of provisional tolerable weekly intake (PTWI) for As in porgy and 41% of PTWI for Hg in tuna. (C) 2010 Elsevier Ltd. All rights reserved.

Effects of the utilization of homeopathic elements in commercial diluent on swine sperm viability

It has been speculated that the homeopathic treatment of sperm cells in order to improve semen quality could be promising. However, few data is available and its use in spermatozoa requires investigation. It is well established that mitochondrial membrane potential is an important viability parameter of spermatozoa and it is intimately related to reproductive efficiency. In this manner, new technologies in order to improve the activity of sperm cells and, finally, the fecundity of swine herds are of extremely importance. Due to the lack of knowledge of homeopathic treatment effect on spermatozoa, the aim of the present study was to verify the effect of three different homeopathic treatments on viability of boar sperm cells. Three homeopathic treatments composed by Pulsatila CH6, Pulsatila and Avena CH6, Avena CH6 and one control treatment (sucrose) were added to diluted boar semen, which were cooled for 24 or 48 h. Interestingly, no positive effect of homeopathic treatments was observed over semen viability. However, it was demonstrated that the 24 h of cooling storage provided more viable sperm cells when compared to the 48-h period. This effect of storage period on sperm viability was assessed by intact plasmatic membrane, intact acrosome and mitochondrial membrane potential evaluation.

Coordinated regulation of follicle development by germ and somatic cells

The continuum of folliculogenesis begins in the fetal ovary with the differentiation of the oogonia and their isolation within the primordial follicles. Primordial follicle activation is an enigmatic process, whereby some follicles enter the growing pool to become primary follicles, thereby embarking on an irreversible progression towards ovulation or atresia. This process is under the coordinated regulation of factors from the oocyte itself, as well as from the somatic cells of the ovary, in particular the theca and granulosa cells, which are structural components of the follicle. These two influences provide the principal stimuli for the growth of the follicle to the late preantral or early antral stage of development. The endocrine effects of the gonadotrophins FSH and LH are essential to the continued progression of the follicle and most atresia can be attributed to the failure to receive or process the gonadotrophin signals. The peri-ovulatory state has received intensive investigation recently, demonstrating a coordinated role for gonadotrophins, steroids, epidermal growth factor family proteins and prostaglandins. Thus, a complex programme of coordinated interaction of governing elements from both germ and somatic cell sources is required for successful follicle development.

Saethre-Chotzen Syndrome, Pro136His TWIST Mutation, Hearing Loss, and External and Middle Ear Structural Anomalies: Report on a Brazilian Family

Objective: To describe the clinical, speech, hearing, and imaging findings in three members of a Brazilian family with Saethre-Chotzen syndrome (SCS) who presented some unusual characteristics within the spectrum of the syndrome. Design: Clinical evaluation was performed by a multidisciplinary team. Direct sequencing of the polymerase chain reaction amplified coding region of the TWIST1 gene, routine and electrophysiological hearing evaluation, speech evaluation, and imaging studies through computed tomography (CT) scan and magnetic resonance imaging (MRI) were performed. Results: TWIST1 gene analysis revealed a Pro136His mutation in all patients. Hearing evaluation showed peripherial and mixed hearing loss in two of the patients, one of them with severe unilateral microtia. Computed tomography scan showed structural middle ear anomalies, and MRI showed distortion of the skull contour as well as some of the brain structures. Conclusions: We report a previously undescribed TWIST1 gene mutation in patients with SCS. There is evidence that indicates hearing loss (conductive and mixed) can be related both with middle ear (microtia, high jugular bulb, and enlarged vestibules) as well as with brain stem anomalies. Here we discuss the relationship between the gene mutation and the clinical, imaging, speech, and hearing findings.

Cell compartmentalisation in planctomycetes: novel types of structural organisation for the bacterial cell

The organisation of cells of the planctomycete species Pirellula marina, Isosphaera pallida, Gemmata obscuriglobus, Planctomyces mat-is and Candidatus Brocadia anammoxidans was investigated based on ultrastructure derived from thin-sections of cryosubstituted cells, freeze-fracture replicas, and in the case of Gemmata obscuriglobus and Pirellllla marina, computer-aided 3-D reconstructions from serial sections of cryosubstituted cells. All planctomycete cells display a peripheral ribosome-free region, termed here the paryphoplasm, surrounding the perimeter of the cell, and an interior region including any nucleoid regions as well as ribosome-like particles, bounded by a single intracytoplasmic membrane (ICM), and termed the pirellulosome in Pirellula species. Immunogold labelling and RNase-gold cytochemistry indicates that in planctomycetes all the cell DNA is contained wholly within the interior region bounded by the ICM, and the paryphoplasm contains no DNA but at least some of the cell's RNA. The ICM in Isosphaera pallida and Planctomyces mat-is is invaginated such that the paryphoplasm forms a major portion of the cell interior in sections, but in other planctomycetes it remains as a peripheral zone. In the anaerobic ammonium-oxidising (anammox process) chemoautotroph Candidatus Brocadia anammoxidans the interior region bounded by ICM contains a further internal single-membrane-bounded region, the anam-moxosome. In Gemmata obscuriglobus. the interior ICM-bounded region contains the nuclear body, a double-membrane-bounded region containing the cell's nucleoid and all genomic DNA in addition to some RNA. Shared features of cell compartmentalisation in different planctomycetes are consistent with the monophyletic nature of the planctomycetes as a distinct division of the Bacteria. The shared organisational plan for the planctomycete cell constitutes a new type not known in cells of other bacteria.

Structural and morphological anomalies in magnetosomes: possible biogenic origin for magnetite in ALH84001

We report biogenic magnetite whiskers, with axial ratios of 6: 1, elongated in the [1 1 1]. [1 1 2] and [1 0 0] directions, resembling the magnetite whiskers detected in the Martian meteorite ALH84001 by Bradley ct nl., and interpreted by those authors as evidence of vapour-phase (abiogenic) growth. Magnetosomal whiskers with extended defects consistent with screw dislocations and magnetosomes resembling flattened twinned platelets, as well as other twinning phenomena and other structural defects, are also reported here. Magnetosomes with teardrop-shaped. cuboidal. irregular and jagged structures similar to those detected in ALH84001 by McKay et al.. coprecipitation of magnetite possibly with amorphous calcium carbonate, coprecipitation of magnetite possibly with amorphous silica, the incorporation of titanium in volutin inclusions and disoriented arrays of magnetosomes are also described. These observations demonstrate that the structures of the magnetite particles in ALH84001. their spatial arrange ment and coprecipitation with carbonates and proximity to silicates are consistent with being biogenic. Electron-beam-induced flash-melting of magnetosomes produced numerous screw dislocations in the (1 1 1). (1 0 0) and (1 1 0) lattice planes and induced fusion of platelets. From this, the lack of screw dislocations reported in the magnetite particles in ALH84001 (McKay et al.. and Bradley et al.) indicates that they have a low-temperature origin.

A nanometre-scale non-periodic structural variation in high temperature superconducting ceramics and the implications for properties

Non-periodic structural variation has been found in the high T-c cuprates, YBa2Cu3O7-x and Hg0.67Pb0.33Ba2Ca2Cu3O8+delta, by image analysis of high resolution transmission electron microscope (HRTEM) images. We use two methods for analysis of the HRTEM images. The first method is a means for measuring the bending of lattice fringes at twin planes. The second method is a low-pass filter technique which enhances information contained by diffuse-scattered electrons and reveals what appears to be an interference effect between domains of differing lattice parameter in the top and bottom of the thin foil. We believe that these methods of image analysis could be usefully applied to the many thousands of HRTEM images that have been collected by other workers in the high temperature superconductor field. This work provides direct structural evidence for phase separation in high T-c cuprates, and gives support to recent stripes models that have been proposed to explain various angle resolved photoelectron spectroscopy and nuclear magnetic resonance data. We believe that the structural variation is a response to an opening of an electronic solubility gap where holes are not uniformly distributed in the material but are confined to metallic stripes. Optimum doping may occur as a consequence of the diffuse boundaries between stripes which arise from spinodal decomposition. Theoretical ideas about the high T-c cuprates which treat the cuprates as homogeneous may need to be modified in order to take account of this type of structural variation.