Chitosan nanoparticles for non-viral gene therapy

The cationic polysaccharide chitosan has been widely used for non-viral transfection in vitro and in vivo and has many advantages over other polycations. Chitosan is biocompatible and biodegradable and protects DNA against DNase degradation. However following administration the ChitosanDNA polyplexes must overcome a series of barriers before DNA is delivered to the cell nucleus. This paper describes the most important parameters involved in the chitosan-DNA interaction and their effects of on the condensation, shape, size and protection of DNA. Strategies developed for chitosanDNA polyplexes to avoid non-specific interaction with blood components and to overcome intracellular obstacles as the crossing of die cell membrane, endosomal escape and nuclear import are presented. © 2006 American Chemical Society.

Orphan nuclear receptor NGFI-B forms dimers with nonclassical interface

The orphan receptor nerve growth factor-induced B (NGFI-B) is a member of the nuclear receptor's subfamily 4A (Nr4a). NGFI-B was shown to be capable of binding both as a monomer to an extended half-site containing a single AAAGGTCA motif and also as a homodimer to a widely separated everted repeat, as opposed to a large number of nuclear receptors that recognize and bind specific DNA sequences predominantly as homo- and/or heterodimers. To unveil the structural organization of NGFI-B in solution, we determined the quaternary structure of the NGFI-B LBD by a combination of ab initio procedures from small-angle X-ray scattering (SAXS) data and hydrogen-deuterium exchange followed by mass spectrometry. Here we report that the protein forms dimers in solution with a radius of gyration of 2.9 nm and maximum dimension of 9.0 nm. We also show that the NGFI-B LBD dimer is V-shaped, with the opening angle significantly larger than that of classical dimer's exemplified by estrogen receptor (ER) or retinoid X receptor (RXR). Surprisingly, NGFI-B dimers formation does not occur via the classical nuclear receptor dimerization interface exemplified by ER and RXR, but instead, involves an extended surface area composed of the loop between helices 3 and 4 and C-terminal fraction of the helix 3. Remarkably, the NGFI-B dimer interface is similar to the dimerization interface earlier revealed for glucocorticoid nuclear receptor (GR), which might be relevant to the recognition of cognate DNA response elements by NGFI-B and to antagonism of NGFI-B-dependent transcription exercised by GR in cells. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 The Protein Society.

Nuclear mitochondrial-like sequences in ants: Evidence from Atta cephalotes (Formicidae: Attini)

Nuclear mitochondrial-like sequences (numts) are copies of mitochondrial DNA that have migrated to the genomic DNA. We present the first characterization of numts in ants, these numts being homologues to a mitochondrial DNA fragment containing loci the 3′ portion of the cytochrome oxidase I gene, an intergenic spacer, the tRNA leucine gene and the 5′ portion of the cytochrome oxidase II gene. All 67 specimens of Atta cephalotes (Hymenoptera: Formicidae: Attini) investigated had these homologues, which are within two monophyletic groups that we called numt1 and numt2. Numt1 and numt2 sequences are less variable than mitochondrial sequences and released from the severe purifying selection constraining the evolution of mitochondrial genes. Their formation probably involved bottlenecks related to two distinct transfer events of ancient and fast evolving mitochondrial DNA fragments to comparative slowly evolving nuclear DNA regions. © 2007 The Authors.

Induction of chromosome aberrations in the Allium cepa test system caused by the exposure of seeds to industrial effluents contaminated with azo dyes

Numerous potentially mutagenic chemicals have been studied mainly because they can cause damaging and inheritable changes in the genetic material. Several tests are commonly used for biomonitoring pollution levels and to evaluate the effects of toxic and mutagenic agents present in the natural environment. This study aimed at assessing the potential of a textile effluent contaminated with azo dyes to induce chromosomal and nuclear aberrations in Allium cepa test systems. A continuous exposure of seeds in samples of the textile effluent in different concentrations was carried out (0.3%, 3%, 10%, and 100%). Cells in interphase and undergoing division were examined to assess the presence of chromosome aberrations, nuclear changes, and micronuclei. Our results revealed a mutagenic effect of the effluent at concentrations of 10% and 100%. At lower concentrations, the effluent (3% and 0.3%) did not induce mutagenic alterations in the test organism A. cepa. These findings are of concern, since cell damage may be transmitted to subsequent generations, possibly affecting the organism as a whole, as well as the local biota exposed to the effluent discharge. If the damage results in cell death, the development of the organism may be affected, which could also lead to its death. © 2008 Elsevier Ltd. All rights reserved.

What's the signification of nuclear vacuoles in human sperm?

The aim of this study was to determine the extent of DNA fragmentation and the presence of single/denatured or double stranded of DNA in sperm with large nuclear vacuoles (LNV) selected by high-magnification. A total of 30 patients had fresh semen samples prepared by discontinuous concentration gradient. Sperm with normal nucleus (NN) and LNV were selected at 8400x magnification and placed in different slides. DNA fragmentation was determined by TUNEL assay. Denatured and double stranded DNA was identified by acridine orange fluorescence method. The percentage of DNA fragmentation in LNV sperm (29%) was significantly higher (P<0.001) than NN sperm (15.8%). Therefore, cleavage of genomic DNA in low molecular weight DNA fragments (mono and oligonucleosomes), and single strand breaks (nicks) in high molecular weight DNA occur more frequently in LNV. Identically, the percentage denatured stranded DNA in sperm with LNV (67.9%) was significantly higher (P <0.0001) than NN sperm (33%). The high level of denatured DNA in sperm with LNV suggests precocious decondensation and disaggregation of sperm chromatin fibers. Our results support an association between LNV sperm and DNA damage, and the routine selection and injection of morphological motile sperm at high magnification for ICSI. The adverse effect (DNA fragmentation or denaturation) leads to concern particularly about the possibility of iatrogenic transmission of genetic abnormalities. Copyright - SBRA - Sociedade Brasileira de Reprodução Assistida.

γ-Radiation induces apoptosis via sarcoplasmatic reticulum in guinea pig ileum smooth muscle cells

We investigated the effects of γ-radiation on cells isolated from the longitudinal smooth muscle layer of the guinea pig ileum, a relatively radioresistant tissue. Single doses (up to 50 Gy) reduced the amount of sarcoplasmatic reticulum and condensed the myofibrils, as shown by electron microscopy 3 days post-irradiation. After that, contractility of smooth muscle strips was reduced. Ca2+ handling was altered after irradiation, as shown in fura-2 loaded cells, with elevated basal intracellular Ca2+, reduced amount of intrareticular Ca2+, and reduced capacitive Ca2+ entry. Radiation also induced apoptosis, judged from flow cytometry of cells loaded with proprium iodide. Electron microscopy showed that radiation caused condensation of chromatin in dense masses around the nuclear envelope, the presence of apoptotic bodies, fragmentation of the nucleus, detachment of cells from their neighbors, and reductions in cell volume. Radiation also caused activation of caspase 12. Apoptosis was reduced by the administration of the caspase inhibitor Z-Val-Ala-Asp-fluoromethyl-ketone methyl ester (Z-VAD-FMK) during the 3 day period after irradiation, and by the chelator of intracellular Ca2+, 1,2-bis(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA), from 1 h before until 2 h after irradiation. BAPTA also reduced the effects of radiation on contractility, basal intracellular Ca2+, amount of intrareticular Ca2+, capacitative Ca2+ entry, and apoptosis. In conclusion, the effects of gamma radiation on contractility, Ca2+ handling, and apoptosis appear due to a toxic action of intracellular Ca2+. Ca2+-induced damage to the sarcoplasmatic reticulum seems a key event in impaired Ca2+ handling and apoptosis induced by γ-radiation. © 2008 Elsevier B.V. All rights reserved.

Comparison of exfoliative pap stain and AgNOR counts of the tongue in smokers and nonsmokers

Objective To compare exfoliative cytology from the oral mucosa of smokers and nonsmokers, with emphasis on proliferative activity. Methods Exfoliative cytology specimens were obtained from clinical normal mucosa from the lateral border of the tongue in 30 nonsmokers and 30 smokers ranging in age from 40 to 70 years of age, who were seen at the Heart Institute's Patient Center and the Smoking Cessation Program of the University Hospital, University of São Paulo Medical School (InCor-HCFMUSP). The cytologic specimens were evaluated by Papanicolaou staining and AgNOR quantification in order to evaluate the presence of cytological alterations suggestive of inflammation, dysplasia, keratinization, and proliferative activity of epithelial cells. Results Only Papanicolaou Class I and Class II smears were observed. Inflammatory alterations were found in 90% of smokers and in 87% of nonsmokers. The number of AgNORs/nucleus differed significantly between smokers and nonsmokers (3.372 ± 0.375 versus 2.732 ± 0.236). Conclusions Within the limitations of this research, the results indicate higher proliferative activity in smoking patients compared to nonsmoking patients, even in the absence of clinical lesions. © The Author(s) 2008.

Seleção de espermatozoides para ICSI

The application of assisted reproduction techniques has provided help to many men seeking to father a child, although the current success of these procedures remains suboptimal. Today some protocols allow sperm to be selected according to their ultrastructural morphology or surface molecular characteristics. On the other hand, successful human reproduction relies partly on the inherent integrity of sperm DNA. Therefore, it is now necessary to improve the safety of the sperm selection method. It is urgent to optimize procedures to isolate spermatozoa for ICSI with low risk of DNA damage. In recent years, two technologies have attracted the attention of specialists as methods capable of identifying a spermatozoon with low risk of DNA damage: Ultrastructural morphology sperm selection at high magnification and sperm head birefringence selection. This review analyses these two technologies. © Todos os direitos reservados a SBRA - Sociedade Brasileira de Reprodução Assistida.

EIF5A dimerizes not only in vitro but also in vivo and its molecular envelope is similar to the EF-P monomer

The protein eukaryotic initiation factor 5A (eIF5A) is highly conserved among archaea and eukaryotes, but not in bacteria. Bacteria have the elongation factor P (EF-P), which is structurally and functionally related to eIF5A. eIF5A is essential for cell viability and the only protein known to contain the amino acid residue hypusine, formed by post-translational modification of a specific lysine residue. Although eIF5A was initially identified as a translation initiation factor, recent studies strongly support a function for eIF5A in the elongation step of translation. However, the mode of action of eIF5A is still unknown. Here, we analyzed the oligomeric state of yeast eIF5A. First, by using size-exclusion chromatography, we showed that this protein exists as a dimer in vitro, independent of the hypusine residue or electrostatic interactions. Protein-protein interaction assays demonstrated that eIF5A can form oligomers in vitro and in vivo, in an RNA-dependent manner, but independent of the hypusine residue or the ribosome. Finally, small-angle X-ray scattering (SAXS) experiments confirmed that eIF5A behaves as a stable dimer in solution. Moreover, the molecular envelope determined from the SAXS data shows that the eIF5A dimer is L-shaped and superimposable on the tRNAPhe tertiary structure, analogously to the EF-P monomer. © 2012 Springer-Verlag.

Evaluation epidermal p53 immunostaining by digital image analysis

Digital techniques have been developed and validated to assess semiquantitatively immunohistochemical nuclear staining. Currently visual classification is the standard for qualitative nuclear evaluation. Analysis of pixels that represents the immunohistochemical labeling can be more sensitive, reproducible and objective than visual grading. This study compared two semiquantitative techniques of digital image analysis with three techniques of visual analysis imaging to estimate the p53 nuclear immunostaining. Methods: Sixty-three sun-exposed forearm-skin biopsies were photographed and submitted to three visual analyses of images: the qualitative visual evaluation method (0 to 4 +), the percentage of labeled nuclei and HSCORE. Digital image analysis was performed using ImageJ 1.45p; the density of nuclei was scored per ephitelial area (DensNU) and the pixel density was established in marked suprabasal epithelium (DensPSB). Results: Statistical significance was found in: the agreement and correlation among the visual estimates of evaluators, correlation among the median visual score of the evaluators, the HSCORE and the percentage of marked nuclei with the DensNU and DensPSB estimates. DensNU was strongly correlated to the percentage of p53-marked nuclei in the epidermis, and DensPSB with the HSCORE. Conclusion: The parameters presented herein can be applied in routine analysis of immunohistochemical nuclear staining of epidermis. © 2012 John Wiley & Sons A/S.

Biophysical characterization of the recombinant importin-α from neurospora crassa

Neurospora crassa has been widely used as a model organism and contributed to the development of biochemistry and molecular biology by allowing the identification of many metabolic pathways and mechanisms responsible for gene regulation. Nuclear proteins are synthesized in the cytoplasm and need to be translocated to the nucleus to exert their functions which the importin-α receptor has a key role for the classical nuclear import pathway. In an attempt to get structural information of the nuclear transport process in N. crassa, we present herein the cloning, expression, purification and structural studies with N-terminally truncated IMPα from N. crassa (IMPα-Nc). Circular dichroism analysis revealed that the IMPα-Nc obtained is correctly folded and presents a high structural conservation compared to other importins-α. Dynamic light scattering, analytical size-exclusion chromatography experiments and molecular dynamics simulations indicated that the IMPα-Nc unbound to any ligand may present low stability in solution. The IMPα-Nc theoretical model displayed high similarity of its inner concave surface, which binds the cargo proteins containing the nuclear localization sequences, among IMPα from different species. However, the presence of non-conserved amino acids relatively close to the NLS binding region may influence the binding specificity of IMPα-Nc to cargo proteins. Copyright © 2012 Bentham Science Publishers. All Rights Reserved.

In vitro assessment of the cytotoxic, apoptotic, and mutagenic potentials of Isatin

Isatin (1H-indole-2,3-dione) is a chemical found in various medicinal plant species and responsible for a broad spectrum of pharmacological and biological properties that may be beneficial to human health, as an anticonvulsant, antibacterial, antifungal, antiviral, and anticancer agent. The aim of the present study was to determine in vitro the cytotoxic, mutagenic, and apoptotic effects of isatin on CHO-K1 and HeLa cells using the MTT viability assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), micronucleus (MN) test, apoptosis index, and nuclear division index (NDI). The 5 isatin concentrations evaluated in the mutagenicity and apoptosis tests were 0.5, 1, 5, 10, and 50 μM, selected through a preliminary MTT assay. Positive (doxorubicin, DXR) and negative (phosphate buffered saline, PBS) control groups were also included in the analysis. Isatin did not exert a mutagenic effect on CHO-K1 after 3 and 24 h of treatment or on HeLa cells after 24 h. However, 10 and 50 μM concentrations inhibited cell proliferation and promoted apoptosis in both CHO-K1 and HeLa cells. Data indicate that the cytotoxic, apoptotic, and antiproliferative effects of isatin were concentration independent and cell line independent. The authors thank Profa Dra Eiko Nakagawa Itano for the use the spectrophotometer and the Conselho Nacional para o Desenvolvimento Científico e Tecnológico for master's scholarships to P. M. Cândido-Bacani and grants to T. R. Calvo, W. Vilegas, E. A. Varanda and I. M. S. Cólus. The Conselho Nacional para o Desenvolvimento Científico e Tecnológico provided funding for this study. © 2013 Taylor & Francis Group, LLC.

High chromosome variability and the presence of multivalent associations in buthid scorpions

In this study, we investigated the mitotic and meiotic chromosomes of 11 Buthidae scorpion species, belonging to three genera (Ananteris, Rhopalurus and Tityus), to obtain detailed knowledge regarding the mechanisms underlying the intraspecific and/or interspecific diversity of chromosome number and the origin of the complex chromosome associations observed during meiosis. The chromosomes of all species did not exhibit a localised centromere region and presented synaptic and achiasmatic behaviour during meiosis I. Spermatogonial and/or oogonial metaphase cells of these buthids showed diploid numbers range from 2n = 6 to 2n = 28. In most species, multivalent chromosome associations were observed in pachytene and postpachytene nuclei. Moreover, intraspecific variability associated with the presence or absence of chromosome chains and the number of chromosomes in the complex meiotic configurations was observed in some species of these three genera. Silver-impregnated cells revealed that the number and location of nucleolar organiser regions (NORs) remained unchanged despite extensive chromosome variation; notably, two NORs located on the terminal or subterminal chromosome regions were commonly observed for all species. C-banded and fluorochrome-stained cells showed that species with conspicuous blocks of heterochromatin exhibited the lowest rate of chromosomal rearrangement. Based on the investigation of mitotic and meiotic cells, we determined that the intraspecific variability occurred as a consequence of fission/fusion-type chromosomal rearrangements in Ananteris and Tityus species and reciprocal translocation in Rhopalurus species. Furthermore, we verified that individuals presenting the same diploid number differ in structural chromosome organisation, giving rise to intraspecific differences of chromosome association in meiotic cells (bivalent-like elements or chromosome chains). © 2013 Springer Science+Business Media Dordrecht.

Fetal-Maternal Interactions in the Synepitheliochorial Placenta Using the eGFP Cloned Cattle Model

Background: To investigate mechanisms of fetal-maternal cell interactions in the bovine placenta, we developed a model of transgenic enhanced Green Fluorescent Protein (t-eGFP) expressing bovine embryos produced by nuclear transfer (NT) to assess the distribution of fetal-derived products in the bovine placenta. In addition, we searched for male specific DNA in the blood of females carrying in vitro produced male embryos. Our hypothesis is that the bovine placenta is more permeable to fetal-derived products than described elsewhere. Methodology/Principal Findings: Samples of placentomes, chorion, endometrium, maternal peripheral blood leukocytes and blood plasma were collected during early gestation and processed for nested-PCR for eGFP and testis-specific Y-encoded protein (TSPY), western blotting and immunohistochemistry for eGFP detection, as well as transmission electron microscopy to verify the level of interaction between maternal and fetal cells. TSPY and eGFP DNA were present in the blood of cows carrying male pregnancies at day 60 of pregnancy. Protein and mRNA of eGFP were observed in the trophoblast and uterine tissues. In the placentomes, the protein expression was weak in the syncytial regions, but intense in neighboring cells on both sides of the fetal-maternal interface. Ultrastructurally, our samples from t-eGFP expressing NT pregnancies showed to be normal, such as the presence of interdigitating structures between fetal and maternal cells. In addition, channels-like structures were present in the trophoblast cells. Conclusions/Significance: Data suggested that there is a delivery of fetal contents to the maternal system on both systemic and local levels that involved nuclear acids and proteins. It not clear the mechanisms involved in the transfer of fetal-derived molecules to the maternal system. This delivery may occur through nonclassical protein secretion; throughout transtrophoblastic-like channels and/or by apoptotic processes previously described. In conclusion, the bovine synepitheliochorial placenta displays an intimate fetal-maternal interaction, similar to other placental types for instance human and mouse. © 2013 Pereira et al.

Action of andiroba oil and permethrin on the central nervous and reproductive systems of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) ticks females. A confocal study

Research for acaricides with lower toxicity and impact on the environment has been intensified. An alternative would be the use of natural compounds or of synthetic products in lower concentrations than the ones sold commercially. Thus, this study describes the action of andiroba seed oil on the nuclei of the ovary and synganglion cells of Rhipicephalus sanguineus, and presents an analysis of the nuclear morphology of the nervous system cells of this tick species when exposed to permethrin. The results obtained showed that, although no changes have been observed in the genetic material of the ovary cells exposed to the andiroba oil, this compound, as well as permethrin, has neurotoxic action on the females of this species. The damages caused to the physiology of the synganglion, due to the loss of integrity of the genetic material, would result in the impairment of the metabolism of other systems of R. sanguineus ticks. © 2013 Elsevier Ltd.