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The gradual accumulation and inadequate renewal of senescent cells over time drive organismal aging. Senescent cells undergo altered gene expression and release inflammatory mediators collectively termed the senescence-associated secretory phenotype (SASP), which significantly contributes to a spectrum of age-related disorders, including cancer. In the context of carcinogenesis, the SASP produced by senescent cells has been implicated in the promotion of epithelial cancers, including oral squamous cell carcinoma (OSCC), the most common form of oral cancer. Senescent cells within the tumor microenvironment release factors that amplify the growth and invasiveness of neighboring cancer cells. Senotherapeutics, including senolytics and senomorphics, emerge as promising modalities to target senescent cells and their associated inflammatory factors, thereby opening novel avenues for augmenting the efficacy of cancer treatments. Here, we review the general aspects of cellular senescence, focusing on the relation between senescence-related inflammation with cancer development. We also analyze the available evidence linking cellular senescence with OSCC, highlighting possible clinical applications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Niklander, Aránguiz, Faunes and Martínez-Flores.)

Prenatal ethanol exposure is associated with neurodevelopmental defects and long-lasting cognitive deficits, which are grouped as fetal alcohol spectrum disorders (FASD). The molecular mechanisms underlying FASD are incompletely characterized. Alternative splicing, including the insertion of microexons (exons of less than 30 nucleotides in length), is highly prevalent in the nervous system. However, whether ethanol exposure can have acute or chronic deleterious effects in this process is poorly understood. In this work, we used the bioinformatic tools VAST-TOOLS, rMATS, MAJIQ, and MicroExonator to predict alternative splicing events affected by ethanol from available RNA sequencing data. Experimental protocols of ethanol exposure included human cortical tissue development, human embryoid body differentiation, and mouse development. We found common genes with predicted differential alternative splicing using distinct bioinformatic tools in different experimental designs. Notably, Gene Ontology and KEGG analysis revealed that the alternative splicing of genes related to RNA processing and protein synthesis was commonly affected in the different ethanol exposure schemes. In addition, the inclusion of microexons was also affected by ethanol. This bioinformatic analysis provides a reliable list of candidate genes whose splicing is affected by ethanol during nervous system development. Furthermore, our results suggest that ethanol particularly modifies the alternative splicing of genes related to post-transcriptional regulation, which probably affects neuronal proteome complexity and brain function., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Fuentes-Beals et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: The thyroid hormones-thyroxine (T4) and 3,5,3'triiodothyronine (T3)-regulate the development of the central nervous system (CNS) in vertebrates by acting in different cell types. Although several T3 target genes have been identified in the brain, the changes in the transcriptome in response to T3 specifically in neural stem and progenitor cells (NSPCs) during the early steps of NSPCs activation and neurogenesis have not been studied in vivo. Here, we characterized the transcriptome of FACS-sorted NSPCs in response to T3 during Xenopus laevis metamorphosis., Results: We identified 1252 upregulated and 726 downregulated genes after 16 hours of T3 exposure. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that T3-upregulated genes were significantly enriched in rRNA processing and maturation, protein folding, ribosome biogenesis, translation, mitochondrial function, and proteasome. These results suggest that NSPCs activation induced by T3 is characterized by an early proteome remodeling through the synthesis of the translation machinery and the degradation of proteins by the proteasome., Conclusion: This work provides new insights into the dynamics of activation of NPSCs in vivo in response to T3 during a critical period of neurogenesis in the metamorphosis., (© 2022 American Association for Anatomy.)

The combination of molecular modeling methods to identify the putative binding site of inhibitors constitutes an important tool in drug discovery. In this work, we used these analyses to understand the potent inhibitory effect of naphthoquinone derivatives on heat shock protein 90 (Hsp90), one of the proteins involved in many types of cancer. Molecular docking results indicated that some favorable interactions of key amino acid residues at the binding site of Hsp90 with these quinones would be responsible for the inhibition of Hsp90 activity. Molecular docking and molecular dynamics simulation were carried out to further understand the binding modes and the interactions between the protein and these inhibitors. The main residues of the internal cavity were Val136, Phe138, Tyr139, Val150, Trp162 and Val186. The high concordance between the docking results and 3D-QSAR contour maps gives us helpful information about the environment of the binding site. Our results provide the bases for a rational modification of new molecules based in quinone scaffold, in order to design more potent Hsp90 inhibitors, which would exhibit highly potent antitumor activity.Communicated by Ramaswamy H. Sarma.

Lin28 is a highly conserved RNA binding protein that regulates stemness whose molecular role has been widely studied in vitro. However, the regulation and the molecular role of Lin28 during the development of the vertebrate central nervous system (CNS) in vivo are not completely understood. Here, the expression and the putative role of Lin28 in the development of the mammalian CNS are reviewed in the context of recent results showing the progressive cellular and molecular changes in neural progenitor cells. Downstream genes that may play a role during CNS development and the effect of misregulated expression of Lin28 are discussed. Evidence suggests that Lin28 promotes symmetric divisions over asymmetric divisions, increasing the number of progenitors during early neurogenesis. Future quantitative analysis of Lin28 isoforms levels and stabilities together with single cell transcriptomics data, cell cycle dynamics and cell fate analysis in Lin28 gain- and loss-of-function experiments will provide a better understanding of the molecular role of Lin28 during development., (© 2020. Springer Nature Switzerland AG.)

Background: Lin28 regulates stem cell biology and developmental timing. At the molecular level Lin28 inhibits the biogenesis of the micro RNA let-7 and directly controls the transcription and translation of several genes. In Xenopus, Lin28 overexpression delays metamorphosis and affects the expression of genes of the thyroid hormone (TH) axis. The TH carrier albumin, synthesized by the liver, is down-regulated in limbs and tail after Lin28 overexpression. The molecular mechanisms underlying the interaction between Lin28, let-7, and the hypothalamus-pituitary-thyroid gland (HPT) axis are unknown., Results: We found that precursor and mature forms of let-7 increase during Xenopus metamorphosis. In the liver, lin28b is down-regulated and albumin is up-regulated during metamorphosis. Overexpression of a truncated form of Lin28a (Lin28aΔC), which has been shown not to interact with RNA helicase A to regulate translation, delays metamorphosis, indicating that the translational regulation domain is not required to inhibit the HPT axis. Importantly, both full length Lin28a and Lin28aΔC block the increase of albumin mRNA in the liver independently of changes in TH signaling., Conclusions: These results suggest that Lin28 delays metamorphosis through regulation of let-7 and that the decrease of the TH carrier albumin is one of the early changes after Lin28 overexpression., (© 2019 Wiley Periodicals, Inc.)

Sequences derived from parvoviruses (family Parvoviridae ) are relatively common in animal genomes, but the functional significance of these endogenous parvoviral element (EPV) sequences remains unclear. In this study, we used a combination of in silico and molecular biological approaches to investigate a fusion gene carried by guinea pigs (genus Cavia ) that is partially derived from an EPV. This gene, named enRep-M9l , encodes a predicted polypeptide gene product comprising a partial myosin 9-like ( M9l ) gene fused to a 3' truncated, EPV-encoded replicase. We examined the genomic and phylogenetic characteristics of the EPV locus ( enRep ) that encodes the viral portions of enRep-M9l , revealing that it derives from an ancient dependoparvovirus (genus Dependoparvovirus ) that was incorporated into the guinea pig germ line between approximately 22 and 35 million years ago (MYA). Despite these ancient origins, the regions of the enRep locus that are expressed in the enRep-M9l gene are conserved across multiple species in the family Caviidae (guinea pigs and cavies), consistent with a potential function at the amino acid level. Using molecular biological approaches, we further demonstrated that (i) enRep-M9l mRNA is broadly transcribed in guinea pig cells, (ii) the cloned enRep-M9l transcript can express a protein of the expected size in guinea pig cells in vitro , and (iii) the expressed protein localizes to the cytosol. Our findings demonstrate that, consistent with a functional role, the enRep-M9l fusion gene is evolutionarily conserved, broadly transcribed, and capable of expressing protein. IMPORTANCE DNA from viruses has been "horizontally transferred" to mammalian genomes during evolution, but the impact of this process on mammalian biology remains poorly understood. The findings of our study indicate that a novel gene has evolved in guinea pigs through fusion of host and virus genes., (Copyright © 2019 American Society for Microbiology.)

Insect metamorphosis has been a classic model to understand the role of hormones in growth and timing of developmental transitions. In addition to hormones, transitions in some species are regulated by genetic programs, such as the heterochronic gene network discovered in C. elegans. However, the functional link between hormones and heterochronic genes is not clear. The heterochronic gene lin-28 is involved in the maintenance of stem cells, growth and developmental timing in vertebrates. In this work, we used gain-of-function and loss-of-function experiments to study the role of Lin-28 in larval growth and the timing of metamorphosis of Drosophila melanogaster. During the late third instar stage, Lin-28 is mainly expressed in neurons of the central nervous system and in the intestine. Loss-of-function lin-28 mutant larvae are smaller and the larval-to-pupal transition is accelerated. This faster transition correlates with increased levels of ecdysone direct target genes such as Broad-Complex (BR-C) and Ecdysone Receptor (EcR). Overexpression of Lin-28 does not affect the timing of pupariation but most animals are not able to eclose, suggesting defects in metamorphosis. Overexpression of human Lin-28 results in delayed pupariation and the death of animals during metamorphosis. Altogether, these results suggest that Lin-28 is involved in the control of growth during larval development and in the timing and progression of metamorphosis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Metamorphosis is a classic example of developmental transition, which involves important morphological and physiological changes that prepare the organism for the adult life. It has been very well established that amphibian metamorphosis is mainly controlled by Thyroid Hormone (TH). Here, we show that the heterochronic gene Lin28 is downregulated during Xenopus laevis metamorphosis. Lin28 overexpression before activation of TH signaling delays metamorphosis and inhibits the expression of TH target genes. The delay in metamorphosis is rescued by incubation with exogenous TH, indicating that Lin28 works upstream or parallel to TH. High-throughput analyses performed before any delay on metamorphosis or change in TH signaling showed that overexpression of Lin28 reduces transcript levels of several hormones secreted by the pituitary, including the Thyroid-Stimulating Hormone (TSH), and regulates the expression of proteins involved in TH transport, metabolism and signaling, showing that Lin28 disrupts TH function at different levels. Our data demonstrates that the role of Lin28 in controlling developmental transitions is evolutionary conserved and establishes a functional interaction between Lin28 and thyroid hormone function introducing a new regulatory step in perinatal development with implications for our understanding of endocrine disorders., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Developmental transitions include molting in some invertebrates and the metamorphosis of insects and amphibians. While the study of Caenorhabditis elegans larval transitions was crucial to determine the genetic control of these transitions, Drosophila melanogaster and Xenopus laevis have been classic models to study the role of hormones in metamorphosis. Here we review how heterochronic genes (lin-4, let-7, lin-28, lin-41), hormones (dafachronic acid, ecdysone, thyroid hormone) and the environment regulate developmental transitions. Recent evidence suggests that some heterochronic genes also regulate transitions in higher organisms that they are controlled by hormones involved in metamorphosis. We also discuss evidence demonstrating that heterochronic genes and hormones regulate the proliferation and differentiation of embryonic and neural stem cells. We propose the hypothesis that developmental transitions are regulated by an evolutionary conserved mechanism in which heterochronic genes and hormones interact to control stem/progenitor cells proliferation, cell cycle exit, quiescence and differentiation and determine the proper timing of developmental transitions. Finally, we discuss the relevance of these studies to understand post-embryonic development, puberty and regeneration in humans., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The development of the central nervous system is known to result from two sequential events. First, an inductive event of the mesoderm on the overlying ectoderm that generates a neural plate that, after rolling into a neural tube, acts as the main source of neural progenitors. Second, the axial regionalization of the neural plate that will result in the specification of neurons with different anteroposterior identities. Although this description of the process applies with ease to amphibians and fish, it is more difficult to confirm in amniote embryos. Here, a specialized population of cells emerges at the end of gastrulation that, under the influence of Wnt and FGF signalling, expands and generates the spinal cord and the paraxial mesoderm. This population is known as the long-term neuromesodermal precursor (NMp). Here, we show that controlled increases of Wnt/β-catenin and FGF signalling during adherent culture differentiation of mouse embryonic stem cells (mESCs) generates a population with many of the properties of the NMp. A single-cell analysis of gene expression within this population reveals signatures that are characteristic of stem cell populations. Furthermore, when this activation is triggered in three-dimensional aggregates of mESCs, the population self-organizes macroscopically and undergoes growth and axial elongation that mimics some of the features of the embryonic spinal cord and paraxial mesoderm. We use both adherent and three-dimensional cultures of mESCs to probe the establishment and maintenance of NMps and their differentiation., (© 2014. Published by The Company of Biologists Ltd.)

Pluripotency in embryonic stem cells is maintained through the activity of a small set of transcription factors centred around Oct4 and Nanog, which control the expression of 'self-renewal' and 'differentiation' genes. Here, we combine single-cell quantitative immunofluorescence microscopy and gene expression analysis, together with theoretical modelling, to investigate how the activity of those factors is regulated. We uncover a key role for post-translational regulation in the maintenance of pluripotency, which complements the well-established transcriptional regulatory layer. Specifically, we find that the activity of a network of protein complexes involving Nanog, Oct4, Tcf3, and β-catenin suffices to account for the behavior of ES cells under different conditions. Our results suggest that the function of the network is to buffer the transcriptional activity of Oct4, which appears to be the main determinant to exit pluripotency. The protein network explains the mechanisms underlying the gain and loss of function in different mutants, and brings us closer to a full understanding of the molecular basis of pluripotency.

The maintenance of pluripotency in mouse embryonic stem cells (mESCs) relies on the activity of a transcriptional network that is fuelled by the activity of three transcription factors (Nanog, Oct4 and Sox2) and balanced by the repressive activity of Tcf3. Extracellular signals modulate the activity of the network and regulate the differentiation capacity of the cells. Wnt/β-catenin signaling has emerged as a significant potentiator of pluripotency: increases in the levels of β-catenin regulate the activity of Oct4 and Nanog, and enhance pluripotency. A recent report shows that β-catenin achieves some of these effects by modulating the activity of Tcf3, and that this effect does not require its transcriptional activation domain. Here, we show that during self-renewal there is negligible transcriptional activity of β-catenin and that this is due to its tight association with membranes, where we find it in a complex with Oct4 and E-cadherin. Differentiation triggers a burst of Wnt/β-catenin transcriptional activity that coincides with the disassembly of the complex. Our results establish that β-catenin, but not its transcriptional activity, is central to pluripotency acting through a β-catenin/Oct4 complex.

Here, we report and characterize deep sequencing data and bioinformatics analysis of small RNAs from X. tropicalis gastrula. A total of 17,553,124 reads with perfect match to the genome derived from 2,616,053 unique sequences were identified. Seventy-seven percent of theses sequences were not found in previous reports from X. tropicalis oocytes and somatic tissues. Bioinformatics analyses indicate that a large fraction of the small RNAs are PIWI-interacting RNAs. Up to 23.9% of small RNAs mapped to transposable elements and 27% to genic regions. Most of the abundant transposon-derived small RNAs are found in oocyte and gastrula libraries, suggesting that transposons also need to be silenced during early embryonic development. Importantly, novel clusters of piRNAs whose expression is activated after zygotic transcription begins were identified in the genome of X. tropicalis. Additionally, miRNAs were also identified and many of them are not present in oocytes, suggesting that miRNA expression is stage-specific. To the best of our knowledge, this is the first high throughput data release and bioinformatics characterization of small RNAs during Xenopus early embryonic development., (Copyright © 2012 Wiley Periodicals, Inc.)

Here, we report and characterize deep sequencing data and bioinformatics analysis of small RNAs from Xenopus tropicalis gastrula. A total of 17,553,124 reads with perfect match to the genome derived from 2,616,053 unique sequences were identified. Seventy-seven percent of theses sequences were not found in previous reports from X. tropicalis oocytes and somatic tissues. Bioinformatics analyses indicate that a large fraction of the small RNAs are PIWI-interacting RNAs. Up to 23.9% of small RNAs mapped to transposable elements and 27% to genic regions. Most of abundant transposable derived small RNAs are found in oocyte and gastrula libraries, suggesting that transposon needs to be silenced also during early development. Additionally, miRNAs were identified and many of them are not present in oocytes, suggesting that miRNA expression is stage specific. To the best of our knowledge, this is the first high throughput data release and bioinformatics characterization of small RNAs during Xenopus development., (Copyright © 2012 Wiley Periodicals, Inc.)

Next generation sequencing technologies may now be applied to the study of transcriptomics. RNA-Seq or RNA sequencing employs high-throughput sequencing of complementary DNA fragments delivering a transcriptional profile. In this chapter, we aim to provide a starting point for Xenopus researchers planning on starting an RNA-Seq transcriptomics study. We begin by providing a section on template isolation and library preparation. The next section comprises the main bioinformatics procedures that need to be performed for raw data processing, normalization, and differential gene expression. Finally, we have included a section on studying deep sequencing results in Xenopus, which offers general guidance as to what can be done in this model.

Transposable elements (retrotransposons and DNA transposons) comprise a large proportion of animal genomes, for example 20% in D. melanogaster, 36% in X. tropicalis and 45% in humans. After invading a new genome, the transposable element increases its copy number and subsequently accumulates mutations. These may eventually result in inactive copies. Until recent days transposons have been considered "junk" DNA and no clear function have been assigned for this important amount of information on genomes.

Transposable elements comprise a large proportion of animal genomes. Transposons can have detrimental effects on genome stability but also offer positive roles for genome evolution and gene expression regulation. Proper balance of the positive and deleterious effects of transposons is crucial for cell homeostasis and requires a mechanism that tightly regulates their expression. Herein we describe the expression of DNA transposons of the Tc1/mariner superfamily during Xenopus development. Sense and antisense transcripts containing complete Tc1-2_Xt were detected in Xenopus embryos. Both transcripts were found in zygotic stages and were mainly localized in Spemann's organizer and neural tissues. In addition, the Tc1-like elements Eagle, Froggy, Jumpy, Maya, Xeminos and TXr were also expressed in zygotic stages but not oocytes in X. tropicalis. Interestingly, although Tc1-2_Xt transcripts were not detected in Xenopus laevis embryos, transcripts from other two Tc1-like elements (TXr and TXz) presented a similar temporal and spatial pattern during X. laevis development. Deep sequencing analysis of Xenopus tropicalis gastrulae showed that PIWI-interacting RNAs (piRNAs) are specifically derived from several Tc1-like elements. The localized expression of Tc1-like elements in neural tissues suggests that they could play a role during the development of the Xenopus nervous system.

Proliferation and cell fate determination in the developing embryo are extrinsically regulated by multiple interactions among diverse secreted factors, such as Sonic Hedgehog (SHh), which act in a concentration-dependent manner. The fact that SHh is secreted as a lipid-modified protein suggests the existence of a mechanism to regulate its movement across embryonic fields. We have previously shown that heparan sulfate proteoglycans (HSPGs) are required for SHh binding and signalling. However, it was not determined which specific HSPG was responsible for these functions. Here we evaluated the contribution of perlecan on SHh localization and activity. To understand the mechanism of action of perlecan at the cellular level, we studied the role of perlecan-SHh interaction in SHh activity using both cell culture and biochemical assays. Our findings show that perlecan is a crucial anchor and modulator of SHh activity acting as an extracellular positive regulator of SHh.

Hemocyanin, the oxygen transporter metallo-glycoprotein from mollusks, shows strong relationship between its notable structural features and intrinsic immunomodulatory effects. Here we investigated the individual contribution of CCHA and CCHB subunits from Concholepas hemocyanin (CCH) to in vivo humoral immune response and their pre-clinical evaluation as immunotherapeutic agent in a mice bladder cancer model, in relation to their biochemical properties. To this end, subunits were purified and well characterized. Homogeneous subunits were obtained by anionic exchange chromatography, and its purity assessed by electrophoretic and immunochemical methods. While each CCH subunit contains eight functional units showing partial cross reaction, the vibrational spectral analysis showed several spectral differences, suggesting structural differences between them. In addition, we demonstrated differences in the carbohydrate content: CCHA had a 3.6% w/w sugar with both N- and O-linked moieties. In turn, CCHB had a 2.5% w/w sugar with N-linked, while O-linked moieties were nearly absent. Considering these differences, it was not possible to predict a priori whether the immunogenic and immunotherapeutic properties of subunits might be similar. Surprisingly, both subunits by itself induced a humoral response, and showed an antitumor effect in the bladder carcinoma cell line MBT-2. However, when immunologic parameters were analyzed, CCHA showed better efficiency than CCHB. No allergic reactions or any toxic effects were observed in mice treated with CCHA, sustaining its potential therapeutic use. Our study supports that CCHA subunit accounts for the most important features involved in the immunogenicity of CCH, such as better hydrophilicity and higher content of carbohydrates.

Background: Recent evidence from global studies of gene expression indicates that transcriptomes are more complex than expected. Xenopus has been typically used as a model organism to study early embryonic development, particularly dorso-ventral patterning. In order to identify novel transcripts involved in dorso-ventral patterning, we compared dorsal and ventral transcriptomes of Xenopus tropicalis at the gastrula stage using serial analysis of gene expression (SAGE)., Results: Of the experimental tags, 54.5% were confidently mapped to transcripts and 125 showed a significant difference in their frequency of occurrence between dorsal and ventral libraries. We selected 20 differentially expressed tags and assigned them to specific transcripts using bioinformatics and reverse SAGE. Five mapped to transcripts with known dorso-ventral expression and the frequency of appearance for these tags in each library is in agreement with the expression described by other methods. The other 15 tags mapped to transcripts with no previously described asymmetric expression along the dorso-ventral axis. The differential expression of ten of these novel transcripts was validated by in situ hybridization and/or RT-PCR. We can estimate that this SAGE experiment provides a list of at least 86 novel transcripts with differential expression along the dorso-ventral axis. Interestingly, the expression of some novel transcripts was independent of beta-catenin., Conclusions: Our SAGE analysis provides a list of novel transcripts with differential expression in the dorso-ventral axis and a large number of orphan tags that can be used to identify novel transcripts and to improve the current annotation of the X. tropicalis genome.

Purpose: We determined the antitumor properties of a newly available hemocyanin obtained from the Chilean gastropod Concholepas concholepas (Biosonda Corp., Santiago, Chile) in a syngeneic heterotopic mouse bladder carcinoma model. Since keyhole limpet hemocyanin (Pierce, Rockford, Illinois) is used increasingly in biomedicine as a carrier for vaccines and an immunotherapeutic agent for bladder transitional cell carcinoma, there is a growing interest in finding new substances that share its potent immunomodulatory properties. Considering that keyhole limpet hemocyanin and Concholepas concholepas hemocyanin differ significantly, it was not possible to predict a priori the antitumor properties of Concholepas concholepas hemocyanin., Materials and Methods: C3H/He mice were primed with Concholepas concholepas hemocyanin before subcutaneous implantation of mouse bladder tumor-2 cells. Treatment consisted of a subcutaneous dose of Concholepas concholepas hemocyanin (1 mg or 100 mug) at different intervals after implantation. Keyhole limpet hemocyanin and phosphate buffered saline served as positive and negative controls, respectively. In addition, experiments were designed to determine which elements of the immune response were involved in its adjuvant immunostimulatory effect., Results: Mice treated with Concholepas concholepas hemocyanin showed a significant antitumor effect, as demonstrated by decreased tumor growth and incidence, prolonged survival and lack of toxic effects. These effects were similar to those achieved with keyhole limpet hemocyanin. We found that each hemocyanin increased natural killer cell activity but the effect of Concholepas concholepas hemocyanin was stronger. Analysis of serum from treated mice showed an increased interferon-gamma and low interleukin-4, which correlated with antibody isotypes, confirming that hemocyanins induce a T helper type 1 cytokine profile., Conclusions: To our knowledge our results are the first demonstration of the antitumor effect of a hemocyanin other than keyhole limpet hemocyanin. They suggest that this is an ancient conserved immunogenic mechanism shared by those hemocyanins that is able to enhance T helper type 1 immunity and lead to antitumor activity. Therefore, Concholepas concholepas hemocyanin may be an alternative candidate for providing safe and effective immunotherapy for human superficial bladder cancer.

Hedgehog (Hh) proteins are morphogens involved in short- and long-range effects during early embryonic development. Genetic analysis in fly and vertebrate embryos showed that heparan sulfate proteoglycans (HSPGs) are required for Hh transport and signaling. To further understand how HSPGs regulate Sonic hedgehog (Shh), we performed experiments using cell culture and biochemical assays. When the synthesis of HSPGs was reduced, a decrease in Shh activity was observed. Contrary to that, addition of a peptide that competes the binding of Shh to HSPGs resulted in augmentation of Shh activity. From these results, we concluded that HSPGs exert positive and negative effects in Shh activity. This dual effect correlates with the finding that Shh interacts preferentially with two HSPGs. The current model for the role of HSPGs in Shh diffusion is discussed in view of our findings., (Copyright 2005 Wiley-Liss, Inc.)

We describe here the structure of the hemocyanin from the Chilean gastropod Concholepas concholepas (CCH), emphasizing some attributes that make it interesting among molluscan hemocyanins. CCH exhibits a predominant didecameric structure as revealed by electron microscopy and a size of 8 MDa by gel filtration, and, in contrast with other mollusc hemocyanins, its stabilization does not require additional Ca(2+) and/or Mg(2+) in the medium. Polyacrylamide gel electrophoresis studies, analyses by a MonoQ FPLC column, and Western blots with specific monoclonal antibodies showed that CCH is made by two subunits noncovalently linked, named CCH-A and CCH-B, with molecular masses of 405 and 350 kDa, respectively. Interestingly, one of the subunits undergoes changes within the macromolecule; we demonstrated that CCH-A has an autocleavage site that under reducing conditions is cleaved to yield two polypeptides, CCH-A1 (300 kDa) and CCH-A2 (108 kDa), whereas CCH-B remains unchanged. The CCH-A nick occurs at 4 degrees C, increases at 37 degrees C, and is not inhibited by the addition of protease inhibitors and/or divalent cations. Since the CCH structure is a heterodimer, we investigated whether subunits would be either intermingled, forming heterodecamers, or assembled as two homogeneous decamers. Light scattering and electron microscope studies of the in vitro reassociation of purified CCH subunits demonstrated that the sole addition of Mg(2+) is needed for its reassembly into the native decameric molecule; no homodecamer reorganization was found with either CCH-A or CCH-B subunits alone. Our evidence showed that C. concholepas hemocyanin is an unusual example of heterodecameric organization.

We studied the reactivity of mouse monoclonal antibodies (MAbs) against the hemocyanin from the Chilean marine gastropod Concholepas concholepas (CCH). This protein has been successfully used as a carrier to produce antibodies to haptens and peptides. All MAbs (13) belonging to IgG subclass exhibit dissociation constants (K(d)) from 1 x 10(-7) M to 1 x 10(-9) M. MAbs were characterized by enzyme-linked immunosorbant assay (ELISA) using CCH treated with different procedures, including dissociation into CCH-A and CCH-B subunits, Western blot, enzymatic digestion, chemical deglycosylation, and thermal denaturation. MAbs were classified into three categories, according to subunit specificity by ELISA. The epitope distribution shows that CCH subunits display common epitopes (group I, 5 MAbs, 1H5, 2A8, 3A5, 3B3, and 3E3), as well as specific epitopes for CCH-A subunits (group II, 3 MAbs, 1B8, 4D8, and 8E5) and for CCH-B subunits (group III, 5 MAbs, 1A4, 1E4, 2H10, 3B7, and 7B4). The results can be summarized as follows: (1). six antibodies react with thermal denatured CCH, suggesting that they recognize linear epitopes, whereas seven recognize conformational epitopes; (2). oxidation of carbohydrate moieties does not affect the binding of the MAbs; (3). enzymatic digestion of CCH decreases the reactivity of all antibodies irrespective of the protease used (elastase or trypsin); (4). bringing together the above data, in addition to epitopic complementarity analysis, we identified 12 different epitopes on the CCH molecule recognized by these MAbs. The anti-CCH MAbs presented here can be useful tools to understand the subunit organization of the CCH and its complex structure, which can explain its immunogenic and immunostimulating properties in mammals.