Functional significance of the beta-hairpin in the insecticidal neurotoxin omega-atracotoxin-Hv1a

omega -Atracotoxin-Hv1a is an insect-specific neurotoxin whose phylogenetic specificity derives from its ability to antagonize insect, but not vertebrate, voltage-gated calcium channels. In order to help understand its mechanism of action and to enhance its utility as a lead compound for insecticide development, we used a combination of protein engineering and site-directed mutagenesis to probe the toxin for key functional regions. First, we constructed a Hairpinless mutant in which the C-terminal beta -hairpin, which is highly conserved in this family of neurotoxins, was excised without affecting the fold of the residual disulfide-rich core of the toxin. The Hairpinless mutant was devoid of insecticidal activity, indicating the functional importance of the hairpin. We subsequently developed a highly efficient system for production of recombinant toxin and then probed the hairpin for key functional residues using alanine-scanning mutagenesis followed by a second round of mutagenesis based on initial hits from the alanine scan. This revealed that two spatially proximal residues, Asn(27) and Arg(35), form a contiguous molecular surface that is essential for toxin activity. We propose that this surface of the beta -hairpin is a key site for interaction of the toxin with insect calcium channels.

Enzymology and molecular biology of prokaryotic sulfite oxidation

Despite its toxicity, sulfite plays a key role in oxidative sulfur metabolism and there are even some microorganisms which can use it as sole electron source. Sulfite is the main intermediate in the oxidation of sulfur compounds to sulfate, the major product of most dissimilatory sulfur-oxidizing prokaryotes. Two pathways of sulfite oxidation are known: (1) direct oxidation to sulfate catalyzed by a sulfite: acceptor oxidoreductase, which is thought to be a molybdenum-containing enzyme; (2) indirect oxidation under the involvement of the enzymes adenylylsulfate (APS) reductase and ATP sulfurylase and/or adenylylsulfate phosphate adenylyltransferase with APS as an intermediate. The latter pathway allows substrate phosphorylation and occurs in the bacterial cytoplasm. Direct oxidation appears to have a wider distribution; however, a redundancy of pathways has been described for diverse photo- or chemotrophic, sulfite-oxidizing prokaryotes. In many pro- and also eukaryotes sulfite is formed as a degradative product from molecules containing sulfur as a heteroatom. In these organisms detoxification of sulfite is generally achieved by direct oxidation to sulfate. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Myb-binding Protein 1a is a Nucleocytoplasmic Shuttling Protein that Utilizes CRM1-dependent and Independent Nuclear Export Pathways

Myb-binding protein 1a (Mybbp1a) is a novel nuclear protein localized predominantly, but not exclusively, in nucleoli. Although initially isolated as a c-Myb interacting protein, Mybbp1a is expressed ubiquitously, associates with a number of different transcription factors, and may play a role in both RNA polymerase I- and II-mediated transcriptional regulation. However, its precise function remains unclear. In this study we show that Mybbp1a is a nucleocytoplasmic shuttling protein and investigate the mechanisms responsible for both nuclear import and export. The carboxyl terminus of Mybbp1a, which contains seven short basic amino acid repeat sequences, is responsible for both nuclear and nucleolar localization, and this activity can be transferred to a heterologous protein. Deletion mapping demonstrated that these repeat sequences appear to act incrementally, with successive deletions resulting in a corresponding increase in the proportion of protein localized in the cytoplasm. Glutathione S-transferase pulldown experiments showed that the nuclear receptor importin-alpha/beta mediates Mybbp1a nuclear import. Interspecies heterokaryons were used to demonstrate that Mybbp1a was capable of shuttling between the nucleus and the cytoplasm. Deletion analysis and in vivo export studies using a heterologous assay system identified several nuclear export sequences which facilitate Mybbp1a nuclear export of Mybbp1a by CRM1-dependent and -independent pathways. (C) 2003 Elsevier Science (USA). All rights reserved.

Reproductive system of the male hermit crab Clibanarius sclopetarius: gonopore, spermatophore, and spermatozoal morphologies

In the present study, the morphology and biometry of the spermatophores of the western Atlantic hermit crab Clibanarius sclopetarius (Herbst, 1796) are described, and the results are placed in the context of the Paguroidea, in particular the Diogenidae. Individuals of C. sclopetarius were sampled from a human-impacted mangrove area of southern Brazil. The male reproductive system was removed, measured and analyzed using stereoscopic, light, transmission-electron and scanning-electron microscopy. This system is composed of lobular testes connected to the vas deferens, and gonopores with membranous coverage. The mature spermatophore consists of a spherical pack that stores sperm. These cells consist of a spherical acrosomal vesicle, an amorphous cytoplasm and a distal nucleus. The results revealed that the gonopores, testis and vas deferens have the expected characteristics of the family Diogenidae, while the non-tripartite morphology of the spermatophores and the sperm follow the patterns found only in the genus Clibanarius, and the presence of the dense perforatorial ring is, to date, unique in the species of the genus, being a possible apomorphic characteristic.

Structural and Biochemical Correlates of Na(+), K(+)-ATPase Driven Ion Uptake Across the Posterior Gill Epithelium of the True Freshwater Crab, Dilocarcinus pagei (Brachyura, Trichodactylidae)

To better comprehend the structural and biochemical underpinnings of ion uptake across the gills of true freshwater crabs, we performed an ultrastructural, ultracytochemical and morphometric investigation, and kinetically characterized the Na(+), K(+)-ATPase, in posterior gill lamellae of Dilocarcinus pagei. Ultrastructurally, the lamellar epithelia are markedly asymmetrical: the thick, mushroom-shaped, proximal ionocytes contain elongate mitochondria (41% cell volume) associated with numerous (approximate to 14 mu m(2) membrane per mu m(3) cytoplasm), deep invaginations that house the Na(+), K(+)-ATPase, revealed ultracytochemically. Their apical surface is amplified (7.5 mu m(2) mu m(-2)) by stubby evaginations whose bases adjoin mitochondria below the subcuticular space. The apical membrane of the thin, distal ionocytes shows few evaginations (1.6 mu m(2) mu m(-2)), each surrounding a mitochondrion, abundant in the cytoplasm below the subcuticular space; basolateral invaginations and mitochondria are few. Fine basal cytoplasmic bridges project across the hemolymph space, penetrating into the thick ionocytes, suggesting ion movement between the epithelia. Microsomal Na(+), K(+)-ATPase specific activity resembles marine crabs but is approximate to 5-fold less than in species from fluctuating salinities, and freshwater shrimps, suggesting ion loss compensation by strategies other than Na(+) uptake. Enzyme apparent K(+) affinity attains 14-fold that of marine crabs, emphasizing the relevance of elevated K(+) affinity to the conquest of fresh water. Western blotting and biphasic ouabain inhibition disclose two alpha-subunit isoforms comprising distinct functional isoenzymes. While enzyme activity is not synergistically stimulated by NH(4)(+) and K(+), each increases affinity for the other, possibly assuring appropriate intracellular K(+) concentrations. These findings reveal specific structural and biochemical adaptations that may have allowed the establishment of the Brachyura in fresh water. J. Exp. Zool. 313A:508-523, 2010. (C) 2010 Wiley-Liss, Inc.

Super-CHO - A cell line capable of autocrine growth under fully defined protein-free conditions

Chinese Hamster Ovary (CHO) cells are widely used for the large scale production of recombinant biopharmaceuticals. Growth of the CHO-K1 cell line has been demonstrated in serum-free medium containing insulin, transferrin and selenium. In an attempt to get autocrine growth in protein-free medium, DNA coding for insulin and transferrin production was transfected into CHO-K1 cells. Transferrin was expressed well, with clones secreting approximately 1000 ng/10(6)cells/24h. Insulin was poorly expressed, with rates peaking at 5 ng/10(6)cells/24h. Characterisation of the secreted insulin indicated that the CHO cells were incompletely processing the insulin molecule. Site-directed mutagenesis was used to introduce a furin (prohormone converting enzyme) recognition sequence into the insulin molecule, allowing the production of active insulin. However, the levels were still too low to support autocrine growth. Further investigations revealed insulin degrading activity (presumably due to the presence of insulin degrading enzymes) in the cytoplasm of CHO cells. To overcome these problems insulin-like growth factor I (instead of insulin) was transfected into the cells. IGF-1 was completely processed and expressed at rates greater than 500 ng/10(6)cells/24h. In this paper we report autonomous growth of the transfected CHO-K1 cell line expressing transferrin and IGF-1 in protein-free medium without the addition of exogenous growth factors. Growth rates and final cell densities of these cells were identical to that of the parent cell line CHO-K1 growing in insulin, transferrin, and selenium supplemented serum-free media.

Absence of galectin-3 does not affect the development of experimental tongue carcinomas in mice

Background: Galectin-3 is a lectin that presents pivotal roles in tumor biology and there are no studies evaluating their expression in dysplasias and carcinomas developed from tongue carcinogenesis models. Aims: To investigate the role of galectin-3 in the development of tongue carcinomas using a mouse model of oral carcinogenesis. Methods: Galectin-3-deficient (gal3(-/-)) and wild-type (gal3(+/+)) mice were challenged with 4-nitroquinoline-1-oxide in drinking water for 16 weeks and killed at different times. Tongues were removed and the number of dysplasias and carcinomas was counted. An immunohistochemical study for galectin-3 was performed only in the tongue from gal3(+/+) mice. Results: In both groups, a reduction of dysplasias and an increase of carcinomas from week 16 to week 32 (p > 0.05) were observed. A predominance of high cytoplasmic and nuclear galectin-3 expression was observed in carcinomas (64.7%) and dysplasias (55.5%), respectively (p > 0.05). The perilesional areas always presented a statistical cytoplasmic and nuclear galectin-3 overexpression. Conclusions: Absence of galectin-3 did not directly affect the process of carcinogenesis and a cytoplasm shift of galectin-3 seems to be associated with development of tongue carcinomas. (C) 2010 Elsevier Inc. All rights reserved.

Role of Fos-related antigen 1 in the progression and prognosis of ductal breast carcinoma

Aims: Fos-related antigen 1 (Fra-1) is a member of the activator protein 1 (AP-1) transcription factor family. Our objective was to evaluate the role of Fra-1 expression in breast carcinoma progression and prognosis. Methods and results: Fra-1 expression was investigated by immunohistochemistry in two tissue microarrays containing, respectively, 85 ductal carcinoma in situ (DCIS) and 771 invasive ductal carcinoma (IDC) samples. Staining was observed in the nucleus and cytoplasm of the carcinomas, but only nuclear staining was considered to be positive. Fibroblasts associated with IDC were also Fra-1-positive. The frequency of Fra-1 positivity in IDC (22.8%) was lower than that in DCIS (42.2%). No association was found between Fra-1 and clinico-pathological variables in DCIS. In IDC, Fra-1 expression correlated with aggressive phenotype markers, including: high grade, oestrogen receptor negativity and human epidermal growth factor receptor 2 (HER-2) positivity (P = 0.001, 0.015 and 0.004, respectively), and marginally with the presence of metastasis (P = 0.07). Fra-1 was more frequently positive in basal-like (34%) and in HER-2-positive (38.5%) subtypes than in luminal subtypes. Fra-1 presence did not correlate with survival. Conclusions: A high frequency of Fra-1 in DCIS tumours may be associated with early events in breast carcinogenesis. Although Fra-1 expression correlated with features of a more aggressive phenotype in IDC, no relationship with overall survival was found.

Coordinated expression of galectin-3 and galectin-3-binding sites in malignant mammary tumors: implications for tumor metastasis

Galectin-3 is a glycan-binding protein that mediates cell-cell and/or cell-extracellular matrix (ECM) interactions. Although galectin-3 is implicated in the progression of various types of cancers, the mechanisms by which galectin-3 enhances metastasis remain unclear. In order to elucidate the role of galectin-3 in the complex multistage process of cancer metastasis, we examined galectin-3 and galectin-3-binding site expression in a series of 82 spontaneous canine mammary tumors (CMT) and two CMT cell lines. Benign CMT tumors exhibited strong nuclear/cytoplasmic galectin-3 immunostaining, whereas malignant CMT tumors and metastases exhibited dramatically decreased galectin-3 expression with the majority of the immunostaining confined to the cytoplasm. Interestingly, intravascular tumor cells overexpressed galectin-3 regardless of their location. CMT-U27 xenografts displayed the same pattern of galectin-3 expression found in spontaneous malignant CMT. In parallel with the downregulation of galectin-3, malignant CMT displayed an overall loss of galectin-3-binding sites in the ECM and focal expression of galectin-3-binding sites mainly detected in intravascular tumor cells and endothelium. Furthermore, loss of galectin-3-binding sites was correlated with the downregulation of GLT25D1, a beta (1-O) galactosyltransferase that modifies collagen, and upregulation of stromal galectin-1. Finally, GLT25D1 mRNA expression was strikingly downregulated in malignant CMT-U27 compared with the benign cell line, and its expression was further de-creased in a galectin-3 knockdown CMT-U27 cell line. We therefore hypothesized that the loss of galectin-3-binding sites in the ECM in conjunction with the overexpression of galectin-3 in specific tumor cell subpopulations are crucial events for the development of mammary tumor metastases.

Ultrastructural characterization of CD133(+) stem cells bound to superparamagnetic nanoparticles: possible biotechnological applications

CD133 antigen is an integral membrane glycoprotein that can bind with different cells. Originally, however. this cellular surface antigen was expressed in human stem cells and in various cellular progenitors of the haematopoietic system. Human cord blood has been described as an excellent source of CD133(+) haematopoietic progenitor cells with a large application potential. One of the main objectives of the present study is to describe for the first time the ultrastructural characteristics of CD133(+) stem cells using transmission electronic microscopy. Another objective of the manuscript is to demonstrate through transmission electronic microscopy the molecular image of magnetic nanoparticles connected to the stein cells of great biotechnological importance, as well as demonstrating the value of this finding for electronic paramagnetic resonance and its related nanobioscientific value. Ultrastructural results showed the monoclonal antibody anti-CD133 bound to the superparamagnetic nanoparticles by the presence of electrondense granules in cell membrane, as well as in the cytoplasm, revealing the ultrastructural characteristics of CD133(+) cells, exhibiting a round morphology with discrete cytoplasmic projections, having an active nucleus that follows this morphology. The cellular cytoplasm was filled up with mitochondrias, as well as microtubules and vesicles pinocitic. characterizing the process as being related to internalization of the magnetic nanoparticles that were endocyted by the cells in question. Electronic paramagnetic resonance analysis of the CD133(+) stem cells detected that the small (spectrum) generated by the labelled cells comes from the superparamagnetic nanoparticles that are bound to them. These results strongly suggest that these CD133(+) cells can be used in nanobiotechnology applications, with benefits in different biomedical areas.

Long-Term Astroglial Reaction and Neuronal Plasticity in the Subcortical Visual Pathways After a Complete Ablation of Telencephalon in Pigeons (Columba livia)

This paper analyzes the astroglial and neuronal responses in subtelencephalic structures, following a bilateral ablation of the telencephalon in the Columba livia pigeons. Control birds received a sham operation. Four months later the birds were sacrificed and their brains processed for glial fribillary acid protein (GFAP) and neurofilament immunohistochemistry, markers for astrocytes and neurons, respectively. Computer-assisted image analysis was employed for quantification of the immunoreactive labeling in the nucleus rotundus (N.Rt) and the optic tectum (OT) of the birds. An increased number of GFAP immunoreactive astrocytes were found in several subregions of the N.Rt (p .001), as well as in layers 1, 2cd, 3, and 6 of the OT (p .001) of the lesioned animals. Neurofilament immunoreactivity decreased massively in the entire N.Rt of the lesioned birds; however, remaining neurons with healthy aspect showing large cytoplasm and ramified branches were detected mainly in the periphery of the nucleus. In view of the recently described paracrine neurotrophic properties of the activated astrocytes, the data of the present study may suggest a long-lasting neuroglial interaction in regions of the lesioned bird brain far from injury. Such events may trigger neuronal plasticity in remaining brain structures that may lead spontaneous behavior recovery as the one promoted here even after a massive injury.

Involvement of Astroglial Fibroblast Growth Factor-2 and Microglia in the Nigral 6-OHDA Parkinsonism and a Possible Role of Glucocorticoid Hormone on the Glial Mediated Local Trophism and Wound Repair

We have observed in previous studies that 6-hydroxydopamine (6-OHDA)-induced lesions in the nigrostriatal dopamine (DA) system promote increases of the astroglial basic fibroblast growth factor (FGF-2, bFGF) synthesis in the ascending DA pathways, event that could be modified by adrenosteroid hormones. Here, we first evaluated the changes of microglial reactivity in relation to the FGF-2-mediated trophic responses in the lesioned nigrostriatal DA system. 6-OHDA was injected into the left side of the rat substantia nigra. The OX42 immunohistochemistry combined with stereology showed the time course of the microglial activation. The OX42 immunoreactivity (IR) was already increased in the pars compacta of the substantia nigra (SNc) and ventral tegmental area (VTA) 2 h after the 6-OHDA injection, peaked on day 7, and remained increased on the 14th day time-interval. In the neostriatum, OX42 immunoreactive (ir) microglial profiles increased at 24 h, peaked at 72 h, was still increased at 7 days but not 14 days after the 6-OHDA injection. Two-colour immunofluorescence analysis of the tyrosine hydroxylase (TH) and OX42 IRs revealed the presence of small patches of TH IR within the activated microglia. A decreased FGF-2 IR was seen in the cytoplasm of DA neurons of the SNc and VTA as soon as 2 h after 6-OHDA injection. The majority of the DA FGF-2 ir cells of these regions had disappeared 72 h after neurotoxin. The astroglial FGF-2 IR increased in the SNc and VTA, which peaked on day 7. Two-colour immunofluorescence and immunoperoxidase analyses of the FGF-2 and OX42 IRs revealed no FGF-2 IR within the reactive or resting microglia. Second, we have evaluated in a series of biochemical experiments whether adrenocortical manipulation can interfere with the nigral lesion and the state of local astroglial reaction, looking at the TH and GFAP levels respectively. Rats were adrenalectomized (ADX) and received a nigral 6-OHDA stereotaxical injection 2 days later and sacrificed up to 3 weeks after the DA lesion. Western blot analysis showed time-dependent decrease and elevation of TH and GFAP levels, respectively, in the lesioned versus contralateral midbrain sides, events potentiated by ADX and worsened by corticosterone replacement. ADX decreased the levels of FGF-2 protein (23 kDa isoform) in the lesioned side of the ventral midbrain compared contralaterally. The results indicate that reactive astroglia, but not reactive microglia, showed an increased FGF-2 IR in the process of DA cell degeneration induced by 6-OHDA. However, interactions between these glial cells may be relevant to the mechanisms which trigger the increased astroglial FGF-2 synthesis and thus may be related to the trophic state of DA neurons and the repair processes following DA lesion. The findings also gave further evidence that adrenocortical hormones may regulate astroglial-mediated trophic mechanisms and wound repair events in the lesioned DA system that may be relevant to the progression of Parkinson`s disease.

Molecular and ultrastructural studies of the fibrotic lesions of bovine focal proliferative fibrogranulomatous panniculitis (Lechiguana)

This work aimed to investigate some aspects related to the pathogenicity of Lechiguana, a bovine fibroproliferative lesion characterized by rapid collagen accumulation. Light and transmission electron microscopy and in situ hybridization studies were performed in order to elucidate the fibrogenic activity of this lesion. The characterization of fibroblastic plasticity in the lesion was done by immunohistochemical study for alpha-smooth-muscle cell actin. The ovoid-shaped cells presented positive reaction for alpha-smooth-muscle cell actin in their cytoplasm and, at the electron-microscopic level demonstrated basal lamina-like material adjacent to the external surface and collagen fibrils that corresponded to a cell population phenotypically similar to the myofibroblast. We also investigated alpha 1 collagen type I mRNA at different times of evolution of Lechiguana lesions, using isotopic and non-isotopic in situ hybridization. The results strongly suggest the involvement of a myofibroblast-like cell population that expresses mRNA for type I collagen and is probably associated with the increase of collagen deposition.

Decreased immunoexpression of survivin could be a potential marker in human non-alcoholic fatty liver disease progression?

Background/aim Regulation of apoptosis in non-alcoholic fatty liver disease (NAFLD) has been a theme of growing debate. Although no other study assessed the role of survivin in NAFLD, its expression has been reported in hepatic carcinogenesis because of other aetiological factors with relevant discrepancies. The aim of this study was to assess the pattern of survivin immunoexpression by tissue microarray along the whole spectrum of NAFLD, including non-alcoholic steatohepatitis (NASH)-related hepatocelular carcinoma (HCC). Methods Liver biopsies from 56 patients with NAFLD were evaluated: 18 with steatosis, 21 non-cirrhotic NASH, 10 NASH-related cirrhosis, seven NASH-related HCC, as compared with 71 HCC related to other causes and with 12 normal livers. Results Survivin immunoexpression in NAFLD was restricted to cytoplasm and was found to be progressively lower in advanced stages, including cirrhosis and HCC: steatosis vs NASH-related cirrhosis (P=0.0243); steatosis vs NASH-related HCC (P=0.0010); NASH vs NASH-related cirrhosis (P=0.0318); and NASH vs NASH-related HCC (P=0.0007), thus suggesting a deregulation of apoptosis from NAFLD towards HCC. Interestingly, survivin immunoreactivity in NASH-related HCC was also found to be significantly lower than in HCC related to other causes (P < 0.05). Remarkably, nuclear staining for survivin was not detected in any case of NAFLD, contrasting to its presence in all other cases of HCC. Conclusions Survivin immunoexpression in NASH-related HCC is herein originally found substantially different than in HCC related to other causes, thus requiring further studies to elucidate the role of survivin in human NAFLD progression.

Congenital Hyperinsulinism in Brazilian Neonates: A Study of Histology, KATP Channel Genes, and Proliferation of beta Cells

Congenital hyperinsulinism (CHI) is a rare pancreatic beta-cell disease of neonates, characterized by inappropriate insulin secretion with severe persistent hypoglycemia, with regard to which many questions remain to be answered, despite the important acquisition of its molecular mechanisms in the last decade. The aim of this study was to examine pancreatic histology, beta-cell proliferation (immunohistochemistry with double staining for Ki-67/insulin), and beta-cell adenosine triphosphate-sensitive potassium channels genes from 11 Brazilian patients with severe medically unresponsive CHI who underwent pancreatectomy. Pancreatic histology and beta-cell proliferation in CHI patients were compared to pancreatic samples from 19 age-matched controls. Ten cases were classified as diffuse form (D-CHI) and 1 as focal form (F-CHI). beta-cell nucleomegaly and abundant cytoplasm were absent in controls and were observed only in D-CHI patients. The Ki-67 labeling index (Ki-67-LI) was used to differentiate the adenomatous areas of the F-CHI case (10.15%) from the ""loose cluster of islets`` found in 2 D-CHI samples (2.29% and 2.43%) and 1 control (1.54%) sample. The Ki-67-LI was higher in the F-CHI adenomatous areas, but D-CHI patients also had significantly greater Ki-67-LI (mean value = 2.41%) than age-matched controls (mean value = 1.87%) (P = 0.009). In this 1st genetic study of CHI patients in Brazil, no mutations or new polymorphisms were found in the 33-37 exons of the ABCC8 gene (SUR1) or in the entire exon of the KCNJ11 gene (Kir 6.2) in 4 of 4 patients evaluated. On the other hand, enhanced beta-cell proliferation seems to be a constant feature in CHI patients, both in diffuse and focal forms.