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PubMed1993

Delayed hematopoietic development in osteopetrotic (op/op) mice.

Changes in structure, cellularity, hematopoietic progenitor cell and macrophage content, and osteoclast activity were investigated in the hematopoietic organs of the colony-stimulating factor 1(CSF-1)-less osteopetrotic (op/op) mouse. The data indicated that op/op mice undergo an age-related hematopoietic recovery and resolution of osteopetrosis, suggesting that the hematopoietic system has the capacity to use alternative mechanisms to compensate for the absence of an important multifunctional growth factor, CSF-1. In young animals, op/op femurs were heavily infiltrated with bone, and marrow cellularity was significantly reduced. After 6 wk of age, there was an increase in the marrow space available for hematopoiesis. The femoral cavity of op/op mice progressively enlarged, and by 22 wk of age its appearance and marrow cellularity was comparable to that of controls. The percentage of op/op mononuclear phagocytes, defined by F4/80 antigen expression, progressively increased to normal levels by 35 wk of age. There was no difference in the incidence of both primitive and mononuclear phagocyte-committed, CSF-1-responsive progenitor cells in op/op marrow, but their femoral content was significantly reduced in young mice. During the period of reduced hematopoiesis in the marrow of young op/op mice, splenic hematopoietic activity was elevated. This mutant mouse represents a system for the study of the CSF-1-independent regulatory mechanisms involved in hematopoietic regulation.

Animals↗
PubMedDate not supplied

Laser printing of pluripotent embryonal carcinoma cells.

A technique by which to print patterns and multilayers of scaffolding and living cells could be used in tissue engineering to fabricate tissue constructs with cells, materials, and chemical diversity at the micron scale. We describe here studies using a laser forward transfer technology to print single-layer patterns of pluripotent murine embryonal carcinoma cells. This report focuses on verifying cell viability and functionality as well as the ability to differentiate cells after laser transfer. We find that when cells are printed onto model tissue scaffolding such as a layer of hydrogel, greater than 95% of the cells survive the transfer process and remain viable. In addition, alkaline comet assays were performed on transferred cells, showing minimal single-strand DNA damage from potential ultraviolet-cell interaction. We also find that laser-transferred cells express microtubular associated protein 2 after retinoic acid stimulus and myosin heavy chain protein after dimethyl sulfoxide stimulus, indicating successful neural and muscular pathway differentiation. These studies provide a foundation so that laser printing may next be used to build heterogeneous multilayer cellular structures, enabling cell growth and differentiation in heterogeneous three-dimensional environments to be uniquely studied.

Animals↗
PubMed1996

Biotransformation in review: applications in ocular disease and drug design.

The purpose of this paper is to review the biochemical processes of systemic biotransformation and describe their relevance to ocular disease and drug metabolism. The diverse nature of the biochemical pathways, commonly found in enzyme metabolism, is discussed. The occurrence of these processes in the eye has significance in that the products of metabolism may accumulate locally and exert deterimental effects, presumably by altering the cellular structure and/or function of crucial visual elements. The manipulation of these metabolic pathways within the eye has ramifications in the development of novel drug design for both ocular disease treatment and, perhaps more importantly, disease prevention.

Animals↗
PubMed2005

Biological effects of helium-neon laser irradiation on normal and wounded human skin fibroblasts.

OBJECTIVE: This study aimed to investigate a number of structural, cellular, and molecular responses to heliumneon (632.8 nm) laser irradiation following a single dose of 0.5, 2.5, 5, or 10 J/cm2 on normal and wounded human skin fibroblasts. BACKGROUND DATA: Low-level laser therapy (LLLT) is a form of phototherapy, involving the application of low-power monochromatic and coherent light to injuries and lesions to stimulate healing. 1 This therapy has been successfully used for pain attenuation and to induce wound healing in nonhealing defects. 2 METHODS: Changes in normal and wounded fibroblast cell morphology were evaluated by light microscopy. Cellular parameters evaluated cell proliferation, cell viability, and cytotoxicity while molecular parameters assessed the extent of DNA damage. RESULTS: The results clearly demonstrate that LLLT has an effect on normal and wounded(3) human skin fibroblasts. The parameters showed that doses of 0.5, 2.5, 5, and 10 J/cm2 were sufficient to produce measurable changes in fibroblast cells. CONCLUSION: A dose of 10 J/cm2 appeared to produce a significant amount of cellular and molecular damage, which could be an important consideration for other therapies, such as photodynamic therapy.

Cell Proliferation↗
PubMed2002

Literature-based evaluation of the potential risks associated with impregnation of medical devices and implants with triclosan.

BACKGROUND: This report is a review of the published literature for studies of triclosan that address mechanism of action, efficacy on skin and in the oral cavity, and the potential for development of resistance. METHODS: Triclosan citations from the past three decades were searched using Medline and other search engines. The techniques used in these studies included in vitro antimicrobial sensitivity, molecular genetics, and enzyme and membrane biochemistry. Oral cavity efficacy and resistance studies were conducted in human volunteers in trials lasting up to 7 months. Efficacy on skin was reported in clinical trials lasting up to 12 months. RESULTS: The minimal inhibitory concentration of triclosan against Staphylococcus aureus and Escherichia coli is reported to be 0.1 and 5.0 microg/mL, respectively. Triclosan acts by blocking enoyl acyl carrier protein reductase, an enzyme essential for fatty acid biosynthesis. Its biocidal activity involves a plethora of nonspecific perturbations of cellular structural elements, including the cell membrane. In the oral cavity, triclosan use was associated with significant reductions in recoverable flora; there was no evidence of resistance or emergence of opportunistic pathogens. On skin, in a neonatal intensive care unit, triclosan use was associated with a significant reduction in methicillin-resistant S. aureus (MRSA) infections, a diminished need for antibiotics, and a decreased incidence of nosocomial infections. CONCLUSION: There is currently no evidence that long-term application of triclosan products to the skin or oral cavity selects for triclosan-resistant populations. Given the short-term nature of suture use, it is highly unlikely that such use would do other than reduce the risks of postoperative infection.

Anti-Infective Agents, Local↗
PubMed2002

A phosphorylated cytoplasmic autoantigen, GW182, associates with a unique population of human mRNAs within novel cytoplasmic speckles.

A novel human cellular structure has been identified that contains a unique autoimmune antigen and multiple messenger RNAs. This complex was discovered using an autoimmune serum from a patient with motor and sensory neuropathy and contains a protein of 182 kDa. The gene and cDNA encoding the protein indicated an open reading frame with glycine-tryptophan (GW) repeats and a single RNA recognition motif. Both the patient's serum and a rabbit serum raised against the recombinant GW protein costained discrete cytoplasmic speckles designated as GW bodies (GWBs) that do not overlap with the Golgi complex, endosomes, lysosomes, or peroxisomes. The mRNAs associated with GW182 represent a clustered set of transcripts that are presumed to reside within the GW complexes. We propose that the GW ribonucleoprotein complex is involved in the posttranscriptional regulation of gene expression by sequestering a specific subset of gene transcripts involved in cell growth and homeostasis.

Amino Acid Motifs↗
PubMed1998

M phase phosphoprotein 10 is a human U3 small nucleolar ribonucleoprotein component.

We have previously developed a novel technique for isolation of cDNAs encoding M phase phosphoproteins (MPPs). In the work described herein, we further characterize MPP10, one of 10 novel proteins that we identified, with regard to its potential nucleolar function. We show that by cell fractionation, almost all MPP10 was found in isolated nucleoli. By immunofluorescence, MPP10 colocalized with nucleolar fibrillarin and other known nucleolar proteins in interphase cells but was not detected in the coiled bodies stained for either fibrillarin or p80 coilin, a protein found only in the coiled body. When nucleoli were separated into fibrillar and granular domains by treatment with actinomycin D, almost all the MPP10 was found in the fibrillar caps, which contain proteins involved in rRNA processing. In early to middle M phase of the cell cycle, MPP10 colocalized with fibrillarin to chromosome surfaces. At telophase, MPP10 was found in cellular structures that resembled nucleolus-derived bodies and prenucleolar bodies. Some of these bodies lacked fibrillarin, a previously described component of nucleolus-derived bodies and prenucleolar bodies, however, and the bulk of MPP10 arrived at the nucleolus later than fibrillarin. To further examine the properties of MPP10, we immunoprecipitated it from cell sonicates. The resulting precipitates contained U3 small nucleolar RNA (snoRNA) but no significant amounts of other box C/D snoRNAs. This association of MPP10 with U3 snoRNA was stable to 400 mM salt and suggested that MPP10 is a component of the human U3 small nucleolar ribonucleoprotein.

Amino Acid Sequence↗
PubMed2003

Dynamin participates in focal extracellular matrix degradation by invasive cells.

The degradation of extracellular matrix (ECM) by matrix metalloproteases is crucial in physiological and pathological cell invasion alike. Degradation occurs at specific sites where invasive cells make contact with the ECM via specialized plasma membrane protrusions termed invadopodia. Herein, we show that the dynamin 2 (Dyn2), a GTPase implicated in the control of actin-driven cytoskeletal remodeling events and membrane transport, is necessary for focalized matrix degradation at invadopodia. Dynamin was inhibited by using two approaches: 1) expression of dominant negative GTPase-impaired or proline-rich domain-deleted Dyn2 mutants; and 2) inhibition of the dynamin regulator calcineurin by cyclosporin A. In both cases, the number and extension of ECM degradation foci were drastically reduced. To understand the site and mechanism of dynamin action, the cellular structures devoted to ECM degradation were analyzed by correlative confocal light-electron microscopy. Invadopodia were found to be organized into a previously undescribed ECM-degradation structure consisting of a large invagination of the ventral plasma membrane surface in close spatial relationship with the Golgi complex. Dyn2 seemed to be concentrated at invadopodia.

Cell Line, Tumor↗
PubMed2003

Differential requirement for the nonhelical tailpiece and the C terminus of the myosin rod in Caenorhabditis elegans muscle.

Myosin heavy chain (MHC) is a large, multidomain protein important for both cellular structure and contraction. To examine the functional role of two C-terminal domains, the end of the coiled-coil rod and the nonhelical tailpiece, we have generated constructs in which residues within these domains are removed or mutated, and examined their behavior in Caenorhabditis elegans striated muscle. Genetic tests demonstrate that MHC lacking only tailpiece residues is competent to support the timely onset of embryonic contractions, and therefore viability, in animals lacking full-length MHC. Antibody staining experiments show that this truncated molecule localizes as wild type in early stages of development, but may be defective in processes important for thick filament organization later in embryogenesis. Ultrastructural analysis reveals thick filaments of normal morphology in disorganized arrangement, as well as occasional abnormal assemblages. In contrast, molecules in which the four terminal residues of the coiled coil are absent or mutated fail to rescue animals lacking endogenous MHC. Loss of these four residues is associated with delayed protein localization and delayed contractile function during early embryogenesis. Our results suggest that these two MHC domains, the rod and the tailpiece, are required for distinct steps during muscle development.

Amino Acid Sequence↗
PubMed2005

Plasticity of B cell receptor internalization upon conditional depletion of clathrin.

B cell antigen receptor (BCR) association with lipid rafts, the actin cytoskeleton, and clathrin-coated pits influences B cell signaling and antigen presentation. Although all three cellular structures have been separately implicated in BCR internalization, the relationship between them has not been clearly defined. In this study, internalization pathways were characterized by specifically blocking each potential mechanism of internalization. BCR uptake was reduced by approximately 70% in B cells conditionally deficient in clathrin heavy chain expression. Actin or raft antagonists were both able to block the residual, clathrin-independent BCR internalization. These agents also affected clathrin-dependent internalization, indicating that clathrin-coated pits, in concert with mechanisms dependent on rafts and actin, mediate the majority of BCR internalization. Clustering G(M1) gangliosides enhanced clathrin-independent BCR internalization, and this required actin. Thus, although rafts or actin independently did not mediate BCR internalization, they apparently cooperate to promote some internalization even in the absence of clathrin. Simultaneous inhibition of all BCR uptake pathways resulted in sustained tyrosine phosphorylation and activation of the extracellular signal-regulated kinase (ERK), strongly suggesting that downstream BCR signaling can occur without receptor translocation to endosomes and that internalization leads to signal attenuation.

Actins↗
PubMed2006

The regulation of microtubule dynamics in Saccharomyces cerevisiae by three interacting plus-end tracking proteins.

Microtubule plus-end tracking proteins (+TIPs) are a diverse group of molecules that regulate microtubule dynamics and interactions of microtubules with other cellular structures. Many +TIPs have affinity for each other but the functional significance of these associations is unclear. Here we investigate the physical and functional interactions among three +TIPs in S. cerevisiae, Stu2, Bik1, and Bim1. Two-hybrid, coimmunoprecipitation, and in vitro binding assays demonstrate that they associate in all pairwise combinations, although the interaction between Stu2 and Bim1 may be indirect. Three-hybrid assays indicate that these proteins compete for binding to each other. Thus, Stu2, Bik1, and Bim1 interact physically but do not appear to be arranged in a single unique complex. We examined the functional interactions among pairs of proteins by comparing cytoplasmic and spindle microtubule dynamics in cells lacking either one or both proteins. On cytoplasmic microtubules, Stu2 and Bim1 act cooperatively to regulate dynamics in G1 but not in preanaphase, whereas Bik1 acts independently from Stu2 and Bim1. On kinetochore microtubules, Bik1 and Bim1 are redundant for regulating dynamics, whereas Stu2 acts independently from Bik1 and Bim1. These results indicate that interactions among +TIPS can play important roles in the regulation of microtubule dynamics.

Cell Cycle Proteins↗
PubMed2006

Actin bodies in yeast quiescent cells: an immediately available actin reserve?

Most eukaryotic cells spend most of their life in a quiescent state, poised to respond to specific signals to proliferate. In Saccharomyces cerevisiae, entry into and exit from quiescence are dependent only on the availability of nutrients in the environment. The transition from quiescence to proliferation requires not only drastic metabolic changes but also a complete remodeling of various cellular structures. Here, we describe an actin cytoskeleton organization specific of the yeast quiescent state. When cells cease to divide, actin is reorganized into structures that we named "actin bodies." We show that actin bodies contain F-actin and several actin-binding proteins such as fimbrin and capping protein. Furthermore, by contrast to actin patches or cables, actin bodies are mostly immobile, and we could not detect any actin filament turnover. Finally, we show that upon cells refeeding, actin bodies rapidly disappear and actin cables and patches can be assembled in the absence of de novo protein synthesis. This led us to propose that actin bodies are a reserve of actin that can be immediately mobilized for actin cables and patches formation upon reentry into a proliferation cycle.

Actin Cytoskeleton↗
PubMed2025

Knowledge-enhanced protein subcellular localization prediction from 3D fluorescence microscope images.

MOTIVATION: Pinpointing the subcellular location of proteins is essential for studying protein function and related diseases. Advances in spatial proteomics have shown that automatic recognition of protein subcellular localization from images could highly facilitate protein translocation analysis and biomarker discovery, but existing machine-learning works have been mostly limited to processing 2D images. By contrast, 3D images have higher spatial resolution and allow researchers to observe cellular structures in their natural context, but currently, there are only a few studies of 3D image processing for protein distribution analysis due to the lack of data and complexity of modeling. RESULTS: We developed a knowledge-enhanced protein subcellular localization model, KE3DLoc, which could recognize distribution patterns in 3D fluorescence microscope images using deep learning methods. The model designs an image feature extraction module that incorporates information from 3D and 2D projected cells and implements asymmetric loss and confidence weights to address data imbalance and weak cell annotation issues. Besides, considering that the biological knowledge in the Gene Ontology (GO) database can provide valuable support for protein location understanding, the KE3DLoc model incorporates a novel knowledge enhancement module that optimizes the protein representation by related knowledge graphs derived from the GO. Since the image module and the knowledge module calculate features from different levels, KE3DLoc designs protein ID aggregation to enhance the consistency of protein features across different cells. Experimental results on three public datasets have demonstrated that the KE3DLoc significantly outperforms existing methods and provides valuable insights for spatial proteomics research. AVAILABILITY AND IMPLEMENTATION: All datasets and codes used in this study are available at GitHub: https://github.com/PRBioimages/KE3DLoc.

Microscopy, Fluorescence↗
PubMed2001

Consciousness.

Consciousness is topical, for reasons including its renewed respectability among psychologists, rapid progress in the neuroscience of perception, memory and action, advances in artificial intelligence and dissatisfaction with the dualistic separation of mind and body. Consciousness is an ambiguous term. It can refer to (i) the waking state; (ii) experience; and (iii) the possession of any mental state. Self-consciousness is equally ambiguous, with senses including (i) proneness to embarrassment in social settings; (ii) the ability to detect our own sensations and recall our recent actions; (iii) self-recognition; (iv) the awareness of awareness; and (v) self-knowledge in the broadest sense. The understanding of states of consciousness has been transformed by the delineation of their electrical correlates, of structures in brainstem and diencephalon which regulate the sleep-wake cycle, and of these structures' cellular physiology and regional pharmacology. Clinical studies have defined pathologies of wakefulness: coma, the persistent vegetative state, the 'locked-in' syndrome, akinetic mutism and brain death. Interest in the neural basis of perceptual awareness has focused on vision. Increasingly detailed neuronal correlates of real and illusory visual experience are being defined. Experiments exploiting circumstances in which visual experience changes while external stimulation is held constant are tightening the experimental link between consciousness and its neural correlates. Work on unconscious neural processes provides a complementary approach. 'Unperceived' stimuli have detectable effects on neural events and subsequent action in a range of circumstances: blindsight provides the classical example. Other areas of cognitive neuroscience also promise experimental insights into consciousness, in particular the distinctions between implicit and explicit memory and deliberate and automatic action. Overarching scientific theories of consciousness include neurobiological accounts which specify anatomical or physiological mechanisms for awareness, theories focusing on the role played by conscious processes in information processing and theories envisaging the functions of consciousness in a social context. Whether scientific observation and theory will yield a complete account of consciousness remains a live issue. Physicalism, functionalism, property dualism and dual aspect theories attempt to do justice to three central, but controversial, intuitions about experience: that it is a robust phenomenon which calls for explanation, that it is intimately related to the activity of the brain and that it has an important influence on behaviour.

Animals↗
PubMedDate not supplied

Changes and changing concepts in the biology of group A streptococci and in the epidemiology of streptococcal infections.

Concepts of the cellular structure of group A streptococci have been modified by the recognition of surface fimbriae and by problems with the older view of a layered arrangement of cell wall components. Evidence of genetic drift of serologic types and of some increase in the prevalence of erythromycin-resistant strains has appeared. A curiois bimodal age distribution for streptococcal pharyngitis and an increase in the prevalence of non-group A strains has been detected. Despite repeated challenges, the value of throat cultures for confirming the possibility of streptococcal pharyngitis remains firmly established; throat cultures are superior to saliva cultures, which more frequently detect non-group A streptococci. Difficulties in detecting subclinical steptococcal infections and in defining streptococcal infection in children in endemic situations (particularly in separating true streptococcal infection from carriage of streptococci in individuals whose illness is due to some other agent) continue to present problems to clinicians and epidemiologists. The value of school culture surveys and of treatment of asymptomatic family contacts of cases remains uncertain. A 20% bacteriologic failure rate has been reported after intramuscular treatment with benzathine penicillin, but no in vitro evidence of resistance to penicillin has appeared. No adequate explanations are available for the decline in the problem of rheumatic fever in industrialized countries, for its increased recognition in tropical countries, for the failure of rheumatic fever to occur after streptococcal pyoderma, or for the emergence of group B Streptococcus as a predominant bacterial pathogen in newborn infants.

Adolescent↗
PubMed1994

Genetic analysis of Rough sheath1 developmental mutants of maize.

Maize Rough sheath1 (Rs1) mutants are dominant and cause a proliferation of sheath-like tissue at the base of the blade and throughout the ligular region. They also cause ligule displacement, a chaotic pattern of vasculature and abnormal cellular structure of vascular bundles. The affected region of Rs1-O leaves displays genetic and morphological attributes of both sheath and auricle, suggesting an overlap of these genetic programs. The rs1 locus maps approximately 26 map units distal to opaque2 (o2) on chromosome 7S, defining a new distal-most locus on the genetic map. Three mutant alleles, Rs1-O, Rs1-1025 and Rs1-Z, all display similar phenotypes. The mutations are completely dominant and the Rs1-O phenotype is not affected by dosage of the chromosome arm carrying the rs1+ allele, indicating that these alleles are neomorphic. Analysis of genetic mosaics showed that the Rs1-O phenotype is non-cell-autonomous, suggesting that intercellular signals convey the phenotype. Rs1 mutant phenotypes are affected by modifiers present in particular genetic backgrounds. An enhancer of Rs1-O was identified; segregation data imply a single recessive gene, ers1. Rs1 mutants were also found to enhance the expression of unlinked rs2 and Rs4 mutants, suggesting that these mutations affect similar developmental processes. We discuss the phenotypic and genetic similarities between Rs1 and Knotted 1 (Kn1) mutants that led to the identification of rs1 as a kn1-like homeobox gene (unpublished data).

Crosses, Genetic↗
PubMed2001

Ethylnitrosourea-induced mutation in mice leads to the expression of a novel protein in the eye and to dominant cataracts.

A novel ENU-induced mutation in the mouse leading to a nuclear and zonular opacity of the eye lens (Aey1) was mapped to chromosome 1 between the markers D1Mit303 and D1Mit332. On the basis of the chromosomal position, the gamma-crystallin encoding gene cluster (Cryg) and the betaA2-crystallin encoding gene Cryba2 were tested as candidate genes. An A --> T mutation destroys the start codon of the Cryge gene in the mutants; this mutation was confirmed by the absence of a restriction site for NcoI in the corresponding genomic fragment of homozygous mutants. The next in-frame start codon is 129 bp downstream; this predicted truncated gammaE-crystallin consists of 131 amino acids, resulting in a molecular mass of 14 kD. However, another open reading frame was observed just 19 bp downstream of the regular Cryge start codon, resulting in a protein of 119 amino acids and a calculated molecular weight of 13 kD. Western blot analysis using polyclonal antibodies against gamma-crystallins or the novel Aey1-specific protein demonstrated the specific expression of the Aey1 protein in the cataractous lenses only; the truncated form of the gammaE-crystallin could not be detected. Therefore, it is concluded that the novel protein destroys the sensitive cellular structure of the eye lens.

Amino Acid Sequence↗
PubMed2002

Ethylnitrosourea-induced base pair substitution affects splicing of the mouse gammaE-crystallin encoding gene leading to the expression of a hybrid protein and to a cataract.

A novel ENU-induced mutation in the mouse leading to a nuclear and cortical opacity of the eye lens (ENU418) was mapped to proximal chromosome 1 by a genome-wide mapping approach. It suggests that the cluster of gamma-crystallin encoding genes (Cryg) and the betaA2-crystallin encoding gene Cryba2 are excellent candidate genes. An A --> G exchange in the middle of intron 1 of the Cryge gene was found as the only alteration cosegregating with the cataractous phenotype. The mutation was confirmed by the presence of a novel restriction site for ApaI in the corresponding genomic DNA fragment. The mutation represses splicing of intron 1; the additional 92 bp in the corresponding cDNA leads to a frameshift and the expression of a novel hybrid protein containing 3 amino acids of the gammaE-crystallin at the N terminus, but 153 novel amino acids. The Cryge(ENU418) protein has a calculated molecular mass of approximately 15.6 kD and an alkaline isoelectric point (pH 10.1) and is predicted to have two hydrophobic domains. Western blot analysis using a polyclonal antibody against the hydrophilic C-terminal part of the Cryge(ENU418)-specific protein demonstrated its stable expression in the cataractous lenses; it was not found in the wild types. Histological analysis of the cataractous lenses indicated that the expression of the new protein disrupts the cellular structure of the eye lens.

Alkylating Agents↗