Molecular Anatomy Research Articles

Divergent evolution in the cytoplasmic domains of PRLR and GHR genes in Artiodactyla

BackgroundProlactin receptor (PRLR) and growth hormone receptor (GHR) belong to the large superfamily of class 1 cytokine receptors. Both of them have been identified as candidate genes affecting key quantitative traits, like growth and reproduction in livestock. We have previously studied the molecular anatomy of the cytoplasmic domain of GHR in different cattle breeds and artiodactyl species. In this study we have analysed the corresponding cytoplasmic signalling region of PRLR.ResultsWe sequenced PRLR gene exon 10, coding for the major part of the cytoplasmic domain, from cattle, American bison, European bison, yak, sheep, pig and wild boar individuals. We found different patterns of variation in the two receptors within and between ruminants and pigs. Pigs and bison species have no variation within GHR exon 10, but show high haplotype diversity for the PRLR exon 10. In cattle, PRLR shows lower diversity than GHR. The Bovinae PRLR haplotype network fits better the known phylogenetic relationships between the species than that of the GHR, where differences within cattle breeds are larger than between the different species in the subfamily. By comparison with the wild boar haplotypes, a high number of subsequent nonsynonymous substitutions seem to have accumulated in the pig PRLR exon 10 after domestication.ConclusionBoth genes affect a multitude of traits that have been targets of selection after domestication. The genes seem to have responded differently to different selection pressures imposed by human artificial selection. The results suggest possible effects of selective sweeps in GHR before domestication in the pig lineage or species divergence in the Bison lineage. The PRLR results may be explained by strong directional selection in pigs or functional switching.

Mechanical strain activates a program of genes functionally involved in paracrine signaling of angiogenesis

Studies were performed to examine the extent to which mechanical stimuli mediate control of angiogenesis in bladder cells both in vitro and in vivo. Differential gene expression between control nonstretched and cyclically stretched bladder smooth muscle cells was assessed using oligonucleotide microarrays and pathway analysis by the web tool Fast Assignment and Transference of Information (FatiGO). Data showed that a substantial proportion (33 of 86) of mechanically responsive genes were angiogenesis-related and include cytokines, growth-related factors, adhesion proteins, and matricellular, signal transduction, extracellular matrix (ECM), and inflammatory molecules. Integrative knowledge of protein-protein interactions revealed that 12 mechano-sensitive gene-encoded proteins have interacting partner(s) in the vascular system confirming their potential role in paracrine regulation of angiogenesis. Angiogenic genes include matricellular proteins such as Cyr61/CCN1, CTGF/CCN2 and tenascin C, components of the VEGF and IGF systems, ECM proteins such as type I collagen and proteoglycans, and matrix metalloproteinases. In an in vivo model of bladder overdistension, 5 of 11 mechano-responsive angiogenic genes, independently tested by real-time PCR, were upregulated as a result of pressure overload including Cyr61/CCN1, CTGF/CCN2, MCP-1, VEGF-A, MMP-1, and midkine. Meanwhile, the molecular anatomy of angiogenic gene promoters reveals the presence of GA box-binding for the myc-associated zinc finger protein, MAZ, often found adjacent to binding sites for mechano-responsive transcription factors (e.g., NF-kappaB), suggesting that the coordinated activity of these factors may induce selective angiogenic gene transcription. These data suggest that mechanical control of angiogenic genes is an integral part of the adaptive and plasticity responses to mechanical overload.

Identification of a Specific Motif of the DSS1 Protein Required for Proteasome Interaction and p53 Protein Degradation

Abstract Deleted in Split hand/Split foot 1 ( DSS1) was previously identified as a novel 12- O-tetradecanoylphorbol-13-acetate (TPA)-inducible gene with possible involvement in early event of mouse skin carcinogenesis. The mechanisms by which human DSS1 (HsDSS1) exerts its biological effects via regulation of the ubiquitin-proteasome system (UPS) are currently unknown. Here, we demonstrated that HsDSS1 regulates the human proteasome by associating with it in the cytosol and nucleus via the RPN3/S3 subunit of the 19S regulatory particle (RP). Molecular anatomy of HsDSS1 revealed an RPN 3/S3- interacting motif (R3IM), located at amino acid residues 15 to 21 of the NH 2 terminus. Importantly, negative charges of the R3IM motif were demonstrated to be required for proteasome interaction and binding to poly-ubiquitinated substrates. Indeed, the R3IM motif of HsDSS1 protein alone was sufficient to replace the ability of intact HsDSS1 protein to pull down proteasome complexes and protein substrates with high-molecular mass ubiquitin conjugates. Interestingly, this interaction is highly conserved throughout evolution from humans to nematodes. Functional study, lowering the levels of the endogenous HsDSS1 using siRNA, indicates that the R3IM/proteasome complex binds and targets p53 for ubiquitin-mediated degradation via gankyrin-MDM2/HDM2 pathway. Most significantly, this work indicates that the R3IM motif of HsDSS1, in conjunction with the complexes of 19S RP and 20S core particle (CP), regulates proteasome interaction through RPN3/S3 molecule, and utilizes a specific subset of poly-ubiquitinated p53 as a substrate.

Functional characterization of individual genomic condensin-binding sites in Saccharomyces cerevisiae using minichromosome mitotic segregation stability assay

Proper chromatin compaction in mitosis (condensation) is required for equal chromosome distribution and precise genetic information inheritance. Protein complex named condensin is responsible for the mitotic condensation, it also individualizes chromosomes, and ensures chromatin separation between sister chromatids in mitosis as well as proper mitotic spindle tension. Mitotic condensin function depends on recognition of the specific binding sites on the chromosome. Mechanism of condensin binding on the individual sites of the mitotic chromosomes, as well as molecular anatomy of these sites remains to be unclear. Even less known is how condensin binding on the individual sites helps separating chromosomes in anaphase. In current paper using minichromosome test, we analyze seven individual condensin binding sites in Saccharomyces cerevisiae found in previous all-genome CHIP on CHIP screening in our lab. This approach allowed us to find out what was the individual contribution of condensin binding sites in securing mitotic stability of the minichromosomes.

Crystallographically Proven Nanometer‐Sized Gold Thiolate Cluster Au102(SR)44: Its Unexpected Molecular Anatomy and Resulting Stereochemical and Bonding Consequences

A stereochemical analysis of the recent crystal-structure determination by Kornberg and co-workers of the first known thiolate-ligated gold nanocluster (see image) revealed two major surprises, which are discussed in this Highlight article.

Molecular anatomy of the cytoplasmic domain of bovine growth hormone receptor, a quantitative trait locus

Quantitative trait loci (QTL) studies have indicated growth hormone receptor (GHR) as a candidate gene affecting cattle milk yield and composition. In order to characterize genetic variation at GHR in cattle, we studied European and East African breeds with different histories of selection, and Bos grunniens, Ovis aries, Sus scrofa, Bison bison and Rangifer tarandus as references. We sequenced most of the cytoplasmic domain (900 bp of exon 10), 89 bp of exon 8, including the putative causative mutation for the QTL effect, and 390 bp of intron 8 for comparison. In the cytoplasmic domain, seven synonymous and seven non-synonymous single nucleotide polymorphisms (SNP) were identified in cattle. Three non-synonymous SNPs were found in sheep and one synonymous SNP in yak, while other studied species were monomorphic. Three major haplotypes were observed, one unique to African breeds, one unique to European breeds and one shared. Bison and yak haplotypes are derivatives of the European haplotype lineage. Most of the exon 10 non-synonymous cattle SNPs appear at phylogenetically highly conserved sites. The polymorphisms in exon 10 cluster around a ruminant-specific tyrosine residue, suggesting that this site may act as an additional signalling domain of GHR in ruminants. Alternative explanations for the persistent polymorphism include balancing selection, hitch-hiking, pleiotropic or sexually antagonistic fitness effects or relaxed functional constraints.

GUDMAP

In late 2004, an International Consortium of research groups were charged with the task of producing a high-quality molecular anatomy of the developing mammalian urogenital tract (UGT). Given the importance of these organ systems for human health and reproduction, the need for a systematic molecular and cellular description of their developmental programs was deemed a high priority. The information obtained through this initiative is anticipated to enable the highest level of basic and clinical research grounded on a 21st-century view of the developing anatomy. There are three components to the Genitourinary Developmental Molecular Anatomy Project GUDMAP; all of these are intended to provide resources that support research on the kidney and UGT. The first provides ontology of the cell types during UGT development and the molecular hallmarks of those cells as discerned by a variety of procedures, including in situ hybridization, transcriptional profiling, and immunostaining. The second generates novel mouse strains. In these strains, cell types of particular interest within an organ are labeled through the introduction of a specific marker into the context of a gene that exhibits appropriate cell type or structure-specific expression. In addition, the targeting construct enables genetic manipulation within the cell of interest in many of the strains. Finally, the information is annotated, collated, and promptly released at regular intervals, before publication, through a database that is accessed through a Web portal. Presented here is a brief overview of the Genitourinary Developmental Molecular Anatomy Project effort.

The evolution of nervous system centralization

It is yet unknown when and in what form the central nervous system in Bilateria first came into place and how it further evolved in the different bilaterian phyla. To find out, a series of recent molecular studies have compared neurodevelopment in slow-evolving deuterostome and protostome invertebrates, such as the enteropneust hemichordate Saccoglossus and the polychaete annelid Platynereis. These studies focus on the spatially different activation and, when accessible, function of genes that set up the molecular anatomy of the neuroectoderm and specify neuron types that emerge from distinct molecular coordinates. Complex similarities are detected, which reveal aspects of neurodevelopment that most likely occurred already in a similar manner in the last common ancestor of the bilaterians, Urbilateria. This way, different aspects of the molecular architecture of the urbilaterian nervous system are reconstructed and yield insight into the degree of centralization that was in place in the bilaterian ancestors.

The counter-creationism handbook

Preface Acknowledgments How to Use This Book List of Creationist Claims Introduction 1. Philosophy and Theology Ethics Epistemology Foundation of Knowledge Theory of Science Scientific Method Theology 2. Biology Abiogenesis Genetics Molecular Biology Physiology and Anatomy Behavior and Cognition Botany Ecology and Population Biology Developmental Biology Systematics Evolution 3. Paleontology Physical Anthropology Transitional Fossils Fossil Record Methodology 4. Geology Geochronology Geological Column Sedimentation Evaporation Glaciation Mountain Building Erosion Geophysics and Plate Tectonics 5. Astronomy and Cosmology Earth Moon Planets and Solar System Sun and Stars Cosmology 6. Physics and Mathematics Second Law of Thermodynamics and Information Theory First Law of Thermodynamics Radiometric Decay 7. Miscellaneous Antievolution History Linguistics Folklore 8. Biblical Creationism Biblical Creationism Generally Biblical Accuracy Young-Earth Creationism Age of the Universe Death and the Fall Created Kinds Flood Miscellaneous Young-Earth Creationism Day-Age Creationism Geocentrism 9. Intelligent Design ID as Science Detecting Design First Cause Anthropic Principle Meta-arguments 10. Other Creationism Vedic Creationism Native North American Creationism Islamic Creationism Bibliography Index

Are renal proximal tubular epithelial cells constantly prepared for an emergency? Focus on “The proliferation capacity of the renal proximal tubule involves the bulk of differentiated epithelial cells”

a human kidney contains approximately one million functional units (the nephrons) that consist of a filter (the glomerulus) and a processing portion (the proximal, intermediate, and distal tubule). The glomeruli produce ∼180 liters of primary filtrate every day of which only 1 to 2 liters are

Automated recognition of cell phenotypes in histology images based on membrane- and nuclei-targeting biomarkers

BackgroundThree-dimensional in vitro culture of cancer cells are used to predict the effects of prospective anti-cancer drugs in vivo. In this study, we present an automated image analysis protocol for detailed morphological protein marker profiling of tumoroid cross section images.MethodsHistologic cross sections of breast tumoroids developed in co-culture suspensions of breast cancer cell lines, stained for E-cadherin and progesterone receptor, were digitized and pixels in these images were classified into five categories using k-means clustering. Automated segmentation was used to identify image regions composed of cells expressing a given biomarker. Synthesized images were created to check the accuracy of the image processing system.ResultsAccuracy of automated segmentation was over 95% in identifying regions of interest in synthesized images. Image analysis of adjacent histology slides stained, respectively, for Ecad and PR, accurately predicted regions of different cell phenotypes. Image analysis of tumoroid cross sections from different tumoroids obtained under the same co-culture conditions indicated the variation of cellular composition from one tumoroid to another. Variations in the compositions of cross sections obtained from the same tumoroid were established by parallel analysis of Ecad and PR-stained cross section images.ConclusionProposed image analysis methods offer standardized high throughput profiling of molecular anatomy of tumoroids based on both membrane and nuclei markers that is suitable to rapid large scale investigations of anti-cancer compounds for drug development.

The molecular anatomy of spontaneous germline mutations in human testes.

The frequency of the most common sporadic Apert syndrome mutation (C755G) in the human fibroblast growth factor receptor 2 gene (FGFR2) is 100–1,000 times higher than expected from average nucleotide substitution rates based on evolutionary studies and the incidence of human genetic diseases. To determine if this increased frequency was due to the nucleotide site having the properties of a mutation hot spot, or some other explanation, we developed a new experimental approach. We examined the spatial distribution of the frequency of the C755G mutation in the germline by dividing four testes from two normal individuals each into several hundred pieces, and, using a highly sensitive PCR assay, we measured the mutation frequency of each piece. We discovered that each testis was characterized by rare foci with mutation frequencies 103 to >104 times higher than the rest of the testis regions. Using a model based on what is known about human germline development forced us to reject (p < 10−6) the idea that the C755G mutation arises more frequently because this nucleotide simply has a higher than average mutation rate (hot spot model). This is true regardless of whether mutation is dependent or independent of cell division. An alternate model was examined where positive selection acts on adult self-renewing Ap spermatogonial cells (SrAp) carrying this mutation such that, instead of only replacing themselves, they occasionally produce two SrAp cells. This model could not be rejected given our observed data. Unlike the disease site, similar analysis of C-to-G mutations at a control nucleotide site in one testis pair failed to find any foci with high mutation frequencies. The rejection of the hot spot model and lack of rejection of a selection model for the C755G mutation, along with other data, provides strong support for the proposal that positive selection in the testis can act to increase the frequency of premeiotic germ cells carrying a mutation deleterious to an offspring, thereby unfavorably altering the mutational load in humans. Studying the anatomical distribution of germline mutations can provide new insights into genetic disease and evolutionary change.

Forward to the Special Issue on the Prefrontal Cortex and Working Memory: In Memory of Patricia S. Goldman-Rakic

Forward to the Special Issue on the Prefrontal Cortex and Working Memory: In Memory of Patricia S. Goldman-Rakic

Single Molecular Anatomy of Host–Guest Chemistry Based on Atomic Force Microscopy Study of Cyclodextrin–Ferrocene Molecular Interaction

The formation process of a typical host–guest compound, β-cyclodextrin–ferrocene system, in various solutions was analyzed on the basis of the results of unbinding-force measurement using atomic force microscopy (AFM). The ratio of the formation numbers determined by AFM is consistent with the complexation constant obtained from the macroscopic analysis. We also analyzed the unbinding force on the basis of the mechanism of surface tension, and solvophobic factors, the intrinsic van der Waals interaction and the effective length of surface tension for ferrocene, were evaluated at the single-molecule level.

A high-resolution anatomical ontology of the developing murine genitourinary tract

Cataloguing gene expression during development of the genitourinary tract will increase our understanding not only of this process but also of congenital defects and disease affecting this organ system. We have developed a high-resolution ontology with which to describe the subcompartments of the developing murine genitourinary tract. This ontology incorporates what can be defined histologically and begins to encompass other structures and cell types already identified at the molecular level. The ontology is being used to annotate in situ hybridisation data generated as part of the Genitourinary Development Molecular Anatomy Project (GUDMAP), a publicly available data resource on gene and protein expression during genitourinary development. The GUDMAP ontology encompasses Theiler stage (TS) 17–27 of development as well as the sexually mature adult. It has been written as a partonomic, text-based, hierarchical ontology that, for the embryological stages, has been developed as a high-resolution expansion of the existing Edinburgh Mouse Atlas Project (EMAP) ontology. It also includes group terms for well-characterised structural and/or functional units comprising several sub-structures, such as the nephron and juxtaglomerular complex. Each term has been assigned a unique identification number. Synonyms have been used to improve the success of query searching and maintain wherever possible existing EMAP terms relating to this organ system. We describe here the principles and structure of the ontology and provide representative diagrammatic, histological, and whole mount and section RNA in situ hybridisation images to clarify the terms used within the ontology. Visual examples of how terms appear in different specimen types are also provided.

Molecular Anatomy of the Alkaliphilic Xylanase from Bacillus halodurans C-125

Two regions in xylanase A from Bacillus halodurans C-125 (XynA), an alkaliphilic xylanase, were identified to be responsible for its activity at basic pH by comparing the dissociation constants of the XynA proton donor Glu residue (pK(e2) and pK(es2)) with those of xylanase B from Clostridium stercorarium F9 (XynB) and their mutants constructed by substituting either Ser137/Asn127 of XynA/XynB or the 4th loop, designed based on the structural difference close to the proton donor. The substitution of XynB at Asn127 into Ser increased pK(e2) by 0.37. The effect is explained that the positive charge of His126 likely affects the proton donor via Asn127 and a water molecule in XynB, resulting in a decrease in pK(e2), whereas such interactions were not observed with Ser. The substitution of XynB at the 4th loop into XynA (XynB Loop4A) increased the pK(e2) and pK(es2) values by 0.29 and 0.62, respectively. The effect of the 4th loop in XynA is likely due to a hydrogen bond between Asp199 in the loop and Tyr239, which interacts with both the proton donors Glu195 and Arg204, with flexibility of the loop. Both the mutations independently affected the increases in pK(e2).

Bi-Directional Notch Signaling Via Signal-Sending and Signal-Receiving Microdomains of the Partnered Immunological Synapses during Th:DC Interaction.

The Notch pathway regulates adaptive immune responses, yet the temporal development of a specific molecular anatomy underlying the directionality of Notch signaling, central to cell-fate decisions, remains unknown. Using the development of the functional immune synapse (IS) of the human physiological T-helper lymphocyte (Th): Dendritic cell (DC) interaction as our model, we followed the temporal accumulation of Notch signaling components, unprocessed and processed, in the developing ThIS and the apposed DCIS of these cells by 2D and 3D immunofluorescense microscopy. Downstream Notch targets in both cell types were followed, as well. We demonstrate that Th-Notch1 receptor and DC-Notch ligands (Delta-like1, Jagged-1) cluster in their apposed central-supramolecular-activation-clusters (cSMAC), whereas DC-Notch1 receptor and Th-Notch ligands cluster in their apposed peripheral-SMAC in an anti-parallel arrangement to that seen in the cSMAC. The resultant accumulation in both cell types of processed nuclear Notch receptor, its ligands, as well as HES-1 and phosphorylated-STAT3, supports antiparallel, reciprocal Notch signal propagation in the DC-to-Th direction via the cSMAC and Th-to-DC direction via the pSMAC. The imposed asymmetric recruitment of the components of Notch pathway, therefore, provides a novel bi-directional route by which the partnered ThIS and DCIS regulate Notch-mediated immune responses. Our data indicate that terminally differentiated immune cells communicate bidirectionally using unidirectional Notch signaling platforms that are spatially segregated into reciprocally signaling microdomains. Significantly, our observations of bidirectional Notch signaling indicate that the heterologous Th:DC interaction is cooperative, requiring reciprocal information transfer across both cell types to mount an appropriate immune response.

Symposium on ‘The Anatomy of Tissue Engineering’

Symposium on ‘The Anatomy of Tissue Engineering’

A Beautiful Science

I am often struck by the incredible beauty of the images published in each month's issue of the Journal of Histochemistry and Cytochemistry. Similarly, my office is decorated with framed photomicrographs of multilabeled brain sections, brightly fluorescent neural stem cells, and quantum dot stained embryonic mouse brain. Devoid of their scientific content, these images remain aesthetically pleasing (Figure 1). I suspect that you too, as a reader of the Journal of Histochemistry and Cytochemistry, were drawn to the field of histology and molecular anatomy in part because of the allure of images revealed through the microscope. Like artists, we spend countless hours in the planning and production of our “creations” before exhibiting them to the public in the form of published images. Despite this similarity to artists, I take offense when scientific colleagues sometimes refer to our discipline as an “art” or to its practitioners as “artists.” Inherent in the description of histochemists as artists is the concept that the creative end-product is in some way unique and/or irreproducible. In contrast, science by its very nature must be reproducible. Figure 1 Multi-label detection of glial fibrillary acidic protein (GFAP; aqua), vimentin (red), and cell nuclei (blue) in neural precursor cell culture (A); neuronal nuclear antigen (green), GFAP (yellow), and microtubule-associated protein two (red) in mouse ... Imagine in your mind a Pablo Picasso, Vincent Van Gogh, or Jackson Pollock painting. Each of these artists has an identity and style that is unique and cannot be reproduced in a meaningful fashion by following a “paint by numbers” template. In contrast, the images created by a Paul Nakane, Ron Van Noorden, or Julia Polak must be capable of exact replication by someone adequately trained in the discipline following a careful set of detailed instructions. Otherwise, the Journal of Histochemistry and Cytochemistry could not be considered a scientific journal and those of us in the field would lose the right to call ourselves scientists. Why then are we so often called artists by other scientists who would never refer to themselves in this fashion? I feel there are two main factors perpetuating this misperception, “them” and “us.” The nonhistochemist can readily appreciate the beauty in the images we produce but might perhaps be unwilling or reluctant to invest the time and effort to consider the many variables that must be controlled if reproducible and high-quality molecular detection is to be achieved. Brilliant investigators capable of manipulating genomes and creating unique organisms through rigorous science often put little thought into the scientific protocols and methods necessary for adequately investigating the cell biological, morphological, and pathological consequences of their genetic manipulations. Putting tissues in containers of formalin and setting them on a shelf for later “analysis” is simply not good scientific practice. Labeling what we do as “art” absolves those not willing to approach molecular morphology as a scientific discipline, because after all, “it's art, not science.” Unfortunately, “we” are also part of the problem. In my opinion, histochemists have alternately presented our scientific discipline in two distinct fashions: either as so complex and variable as to be beyond mere scientific mortals and thus best left to us highly trained “artists”; or as so simple as to be unworthy of consideration by our more talented and often better-funded molecular biology colleagues and thus best left to us “technicians.” In truth, histology is a science with a long history of rigorous investigation of the parameters and variables that affect the quality and reproducibility of our data. The data we generate from our activities, often in the form of beautiful images, must withstand careful scientific scrutiny and prove useful for testing scientifically relevant hypotheses. Histochemistry must be unequivocally recognized as a scientific discipline whose practitioners are trained in the art of beautiful science.

Single Molecular Anatomy of Solvophobic Effects in Host-Guest Interactions Based on Surface Tension Using Atomic Force Microscopy

The effectiveness of the mechanism of surface tension to analyze the host-guest interaction was demonstrated by measuring the unbinding force of the beta-Cyclodextrin (CyD)--adamantane molecules in a liquid environment at the single-molecule level. The contribution of the solvophobic effect was quantitatively distinguished from the intrinsic van der Waals force, and the effective length that produces the detected force through employing the notion of surface tension was determined to be , which is consistent with the perimeter of the adamantane molecule, . Specific strong interaction of water molecules was also confirmed on the nanoscale.