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PubMed2006

CART is a novel islet regulatory peptide.

CART peptides have emerged as important islet regulators. CART is expressed both in islet endocrine cells and in parasympathetic and sensory nerves innervating the islets. In adult rats the intra-islet expression of CART is limited to the somatostatin producing delta-cells, while in adult mice CART is mainly expressed in nerve fibers. During development islet CART is upregulated; in rats in almost all types of islet endocrine cells, including the insulin-producing beta-cells, and in mice mainly in the alpha-cells. This pattern of expression peaks around birth. CART is also expressed in human pancreatic nerves and in islet tumours where the expression level of CART may be related to the degree of differentiation of the tumour. Interestingly, in several rat models of type 2 diabetes CART expression is robustly upregulated in the beta-cells, and is prominent during the phase of beta cell proliferation and hypertrophy. While CART inhibits glucose stimulated insulin secretion from rat islets it augments insulin secretion amplified by cAMP. Mice lacking CART, on the other hand, have islet dysfunction, and humans with a missense mutation in the cart gene are prone to develop type 2 diabetes. These data favor a role of CART in normal islet function and in the pathophysiology of type 2 diabetes.

Animals↗

PubMed1999

The deletion of transforming growth factor-beta-induced myofibroblasts depends on growth conditions and actin organization.

Myofibroblasts are important but transient mediators of normal wound contraction and are characterized phenotypically by their high levels of alpha-smooth-muscle actin (SMA). During wound maturation, these cells disappear. We have examined the mechanisms that lead to myofibroblast deletion in a fibroblast culture model. Transforming growth factor-beta (TGF-beta) was used to increase SMA content in gingival fibroblasts (three- to sixfold). After replating TGF-beta-induced cells at low density with serum, there was a fivefold decrease in SMA protein content, SMA protein synthesis, and SMA mRNA as cells proliferated. These reductions were due to reduced SMA mRNA stability. For TGF-beta-induced cells plated at high density without serum (ie, quiescent conditions), protein content was reduced by only 20% over 12 days. TGF-beta protected SMA-positive cells against apoptosis in serum-free cultures. Those cells that were protected against apoptosis exhibited well-developed stress fibers enriched in SMA. We conclude that, in quiescent myofibroblasts, SMA protein turnover is slow, and cells are long-lived. In proliferative conditions SMA protein and mRNA turn over quickly, and the myofibroblast phenotype dissipates. The reduced apoptosis of myofibroblasts in quiescent conditions is due in part to the organization of SMA into stress fibers.

Actins↗

PubMed1994

Lattice spacing changes accompanying isometric tension development in intact single muscle fibers.

The myosin lattice spacing of single intact muscle fibers of the frog, Rana temporaria, was studied in Ringer's solution (standard osmolarity 230 mOsm) and hyper- and hypotonic salines (1.4 and 0.8 times standard osmolarity respectively) in the relaxed state, during "fixed end" tetani, and during shortening, using synchrotron radiation. At standard tonicity, a tetanus was associated with an initial brief lattice expansion (and a small amount of sarcomere shortening), followed by a slow compression (unaccompanied by sarcomere length changes). In hypertonic saline (myosin lattice compressed by 8.1%), these spacing changes were suppressed, in hypotonic saline (lattice spacing increased by 7.5%), they were enhanced. During unloaded shortening of activated fibers, a rapid lattice expansion occurred at all tonicities, but became larger as tonicity was reduced. This expansion was caused in part by the change in length of the preparation, but also by a recoil of a stressed radial compliance associated with axial force. The lattice spacing during unloaded shortening was equal to or occasionally greater than predicted for a relaxed fiber at that sarcomere length, indicating that the lattice compression associated with activation is rapidly reversed upon loss of axial force. Lattice recompression occurred upon termination of shortening under standard and hypotonic conditions, but was almost absent under hypertonic conditions. These observations indicate that axial cross-bridge tension is associated with a compressive radial force in intact muscle fibers at full overlap; however, this radial force exhibits a much greater sensitivity to lattice spacing than does the axial force.

Animals↗

PubMed2001

Experimental allergic myositis in SJL/J mouse. Reappraisal of immune reaction based on changes after single immunization.

SJL/J mice have been subjected to immunization with wide varieties of antigens to produce models of autoimmune disorders including experimental myositis. They also have a defect in dysferlin gene and spontaneously develop muscle fiber degeneration, a condition akin to limb-girdle type muscular dystrophy and Miyoshi myopathy. To know whether muscle inflammation of SJL mice after immunization with muscle fractions really represents immune-mediated myositis or no more than an epiphenomenon of muscle degeneration due to dysferlin defect, we studied immunological parameters after immunization with rabbit myosin B fraction. Initial infiltration of macrophages and CD4+ lymphocytes on day 11 was followed by increase in number of CD8+ cells. Such increase was not observed in the nontreated and adjuvant controls. Some infiltrating cells were interferon gamma (IFN-gamma) positive. Furthermore, increased expression of the signal transducers and activator of transcription 1 (STAT-1) and interferon regulatory factor 1 (IRF-1) mRNA was shown in the first 2 weeks. These results indicate Th1 system activity in the muscle, rather than simple dysferlin deficiency, particularly 1-3 weeks after immunization. Thus it is concluded that an immune-mediated myositis is taking place at this stage. This model can be helpful in understanding pathomechanisms involved in the early stage of human myositides. It has also important implications concerning immune reactions associated with transplantation or gene therapy for muscular dystrophies.

Animals↗

PubMed2000

Ex-FABP: a fatty acid binding lipocalin developmentally regulated in chicken endochondral bone formation and myogenesis.

Extracellular fatty acid binding protein (Ex-FABP) is a 21 kDa lipocalin specifically binding fatty acids, expressed during chicken embryo development in hypertrophic cartilage, in muscle fibers and in blood granulocytes. In chondrocyte and myoblast cultures Ex-FABP expression is increased by inflammatory agents and repressed by anti-inflammatory agents. In adult cartilage Ex-FABP is expressed only in pathological conditions such as in dyschondroplastic and osteoarthritic chickens. The possible mammalian counterpart is the Neu-related lipocalin (NRL), a lipocalin overexpressed in rat mammary cancer; NRL is homologous to the human neutrophil gelatinase associated lipocalin (NGAL) expressed in granulocytes and in epithelial cells in inflammation and malignancy and to the Sip24 (super-inducible protein 24), an acute phase lipocalin expressed in mouse after turpentine injection. Immunolocalization and in situ hybridization showed that NRL/NGAL is expressed in hypertrophic cartilage, in forming skeletal muscle fibers and in developing heart. In adult cartilage NRL/NGAL was expressed in articular cartilage from osteoarthritic patients and in chondrosarcoma. Moreover, NRL was induced in chondrocyte and myoblast cultures by an inflammatory agent. We propose that these lipocalins (Ex-FABP, NRL/NGAL, Sip24) represent stress proteins physiologically expressed in tissues where active remodeling is taking place during development and also present in tissues characterized by an acute phase response due to pathological conditions.

Acute-Phase Reaction↗

PubMed1999

Rac-GTPase, osteoclast cytoskeleton and bone resorption.

The members of the Rho-GTPase subfamily, Rac1 and Rac2, are intimately involved in the organization of the cytoskeleton, and the p21-activated kinases or PAKs are targets of these proteins. Rac1 and Rac2 are also essential components of NADPH oxidase, the enzyme responsible for generating free radicals. The cytoskeleton modulates the adhesion of osteoclasts to bone and its subsequent resorption. These cells contain NADPH diaphorase activity, and free radicals influence bone resorption. The influence of Rac1, Rac2 and PAK1 on the cytoskeleton, resorbing activity and NADPH diaphorase activity of disaggregated rat osteoclasts was investigated by permeabilisation with saponin and introducing specific anti-Rac1, anti-Rac2 or anti-PAK1 antibodies. Rhodamine-phalloidin stain was used to identify actin in osteoclasts cultured on plastic slides, and the bone-slice method was used to measure resorption. Saponin permeabilisation did not affect the cytoskeletal organization or bone resorption. Anti-Rac antibodies caused dose- and time-dependent cytoskeletal changes. The osteoclasts rounded up and developed retraction fibers; actin rings were disrupted and large actin dots were seen at the periphery of the cells. Osteoclast resorptive activity was depressed after incubation with the antibodies. The total area resorbed by treated cells and the mean pit area were smaller than those of controls. Anti-PAK1 antibody caused similar changes. None of the antibodies altered the NADPH diaphorase activity. Thus, Rac-GTPases are present in rat osteoclasts and are involved in the organization of the actin cytoskeleton and in resorptive activity. These effects may be mediated by PAK1 kinase, but do not influence osteoclast NADPH diaphorase activity.

Actins↗

PubMed1989

The ultraviolet-irradiated hairless mouse: a model for photoaging.

The hairless mouse is proving to be a relevant model for the systematic study of photoaging. As in humans, with chronic ultraviolet radiation, these mice develop elastic fiber hyperplasia, followed by elastosis and ultrastructural degradation. Collagen is damaged and its metabolism is altered, while the normally low levels of proteoglycans and glycosaminoglycans are greatly increased. With this model we have described the effects on dermal connective tissue of UVB (290 to 320 nm), UVA (320 to 400 nm), and the combination of the two. We have also assessed the protective effects of sunscreens. We found that a significant amount of photodamage was repaired when ultraviolet radiation was stopped. Subepidermally in a former region of elastosis, a band of new normal dermis was laid down. Enhancement of the repair was achieved with topical all-trans-retinoic acid in a time- and dose-dependent manner. Retinoic acid was also found to induce angiogenesis in unirradiated mice.

Aging↗

PubMed1997

Strategies for the depyrogenation of contaminated immunoglobulin G solutions by histidine-immobilized hollow fiber membrane.

The depyrogenation of different IgG solutions using the histidine-linked hollow fiber membrane developed in our laboratory is presented here. Three strategies for endotoxin (ET) removal were investigated according to the immobilized histidine's ability to bind different immunoglobulins: (1) ET removal from 1 mg/ml non histidine-binding mouse monoclonal IgG1 (MabCD4) solution was achieved in the presence of acetate buffer (pH 5.0) without any protein loss. (2) For contaminated human IgG, combined adsorption of ET and IgG in the presence of MOPS of Tris buffer was tested, followed by differential elution using increasing salt concentrations. This attempt was not successful since ET were quantitatively found in the IgG elution fraction. (3) Alternatively, it was proposed to adsorb selectively ET in the presence of acetate buffer (pH 5.0) under non binding conditions for human IgG. Human IgG could then be purified if necessary with the same membrane in the presence of MOPS buffer (pH 6.5). With a 1 m2 histidine-PEVA module under these operating conditions, it is estimated that the depyrogenation of 3 l of 1 mg/ml IgG (human or murine) solution containing 80 EU/ml of ET should be possible.

Adsorption↗

PubMed2001

Early anchoring collagen fibers at the bone-tendon interface are conducted by woven bone formation: light microscope and scanning electron microscope observation using a canine model.

To clarify the early process of recovery at the bone-tendon interface, we used light microscopy and SEM to examine the process of anchoring of collagen fibers to bone in a canine model. At two weeks, tendon, scar tissue, woven bone and lamellar bone were present at the insertion site. SEM revealed anchoring of collagen fibril bundles of the scar to the woven bone. By 4 weeks, the number of anchoring fibers had increased and a parallel arrangement of fibers was observed. SEM demonstrated deep penetration of fibers into the woven bone layer. In addition, the fibers were observed to project into and intermingle with the scar tissue. By 6 weeks, the anchoring fibers had developed fully and were distributed densely over the interface. SEM also revealed that the collagen fibril bundles in the scar tissue had connected with the collagen fibrils of the woven bone by way of the anchoring bundles. The woven bone was identifiable throughout the early stages of recovery as the interface between soft tissue and hard tissue. Throughout all experimental periods, no staining was observed at the interface of the tendon and bone by Saffranin-O. The formation of woven bone was important during early recovery of the tendon-bone interface prior to the completion of fibrocartilage-mediated insertion.

Animals↗

PubMed2003

Functional differentiation of multiple climbing fiber inputs during synapse elimination in the developing cerebellum.

We studied how physiological properties of cerebellar climbing fiber (CF) to Purkinje cell (PC) synapses change during developmental transition from multiple to mono CF innervation onto each PC. From P3 to P6, differences in the strengths of multiple CFs became larger. Around P10, each PC was either monoinnervated by one strong CF (CF-mono) or multiply innervated by one strong CF (CF-multi-S) plus a few weaker CFs (CF-multi-W). We show that simultaneous release of multiple vesicles per site occurs normally from CF-multi-S, CF-mono, and mature CFs, but less frequently from CF-multi-W and neonatal CFs. We also present evidence suggesting that weaker CFs with lower probability of multivesicular release would be withdrawn preferentially. The results suggest that differentiation into strong and weak CFs with high and low probabilities of multivesicular release precedes developmental CF synapse elimination.

Afferent Pathways↗

PubMed1998

The origin and fate of pioneer myotomal cells in the avian embryo.

The ontogeny of the myotome was investigated using [3H]thymidine or Brdu treatment in conjunction with 1,1', di-octadecyl-3, 3, 3', 3',-tetramethylindo-carbocyanine perchlorate (DiI) labeling and expression of specific markers. We have identified a subset of early post-mitotic cells that is present in the dorsomedial aspect of epithelial somites and is homogeneously distributed along their entire rostrocaudal extent. The post-mitotic quality of this cell subset enabled us to trace their fate in time-course experiments. Following initial somite dissociation, this epithelial post-mitotic layer bends underneath the medial portion of the nascent dermomyotome. Then, these cells progressively lose epithelial arrangement and migrate in a rostral direction where they accumulate temporarily. Subsequently, these early post-mitotic precursors extend processes that reach both rostral and caudal edges of each segment. Medial somite-derived myofibers also fill the entire mediolateral extent of the segment and reach the dorsomedial lip of the dermomyotome, thus forming the primary myotome. During this process, their large nuclei localize to a narrow stripe in the middle of the nascent myotome. Consistent with the proliferation studies, DiI labeling of the medial epithelial somite cells gave rise to a primary myotomal structure, and continuous pulsing of the DiI-injected embryos with radioactive thymidine revealed that these fibers indeed developed from post-mitotic progenitors. As these early post-mitotic cells that arise prior to somite dissociation are the first wave of progenitors that constitutes the myotome, we have termed them avian muscle pioneers. We propose that the primary myotome formed by the muscle pioneers constitutes a longitudinal scaffold that serves as a substrate for the addition of subsequent waves of myotomal cells.

Animals↗

PubMed1997

Anatomical variants of brain structure: confused spatial relationship of the fornix to the corpus callosum and anterior commissure.

How the developing nerve fibers are guided to and able to find their target is currently a matter of research. As examples of the false guidance of axons, anatomical variants of the spatial relationship of the columna fornicis to the corpus callosum and the anterior commissure are demonstrated. In a 60 year-old female patient, some of the fibers of the genu corporis callosi were found to be entrapped by a fornix fiber bundle. The brain of a 20 year-old man showed that the unilateral anterior commissure ran posterior to the columna fornicis. These changes were clinically insignificant.

Adult↗

PubMed1998

The structure and function of periodontal ligament cells in acellular cementum in rat molars.

To elucidate the structure and function of periodontal ligament cells at the periodontal ligament-cementum interface in advanced acellular cementogenesis, the cervical regions of molars in rats aged 6 weeks were observed by light and electron microscopy. The light and transmission electron microscopy showed the periodontal ligament cells to be elongated between dense, well-developed principal fibers. The transmission and scanning electron microscopy showed that these cells extended wing-like projections from the lateral surface, forming cylindrical compartments surrounding the principal fibers. In addition, finger-like projections extended toward the cementum from the cementum-facing ends. The main results suggest the following: at the periodontal ligament-cementum interface, the periodontal ligament cells maintain the architecture of the principal fibers by means of extracellular compartments. The arrangement of finger-like projections results in the formation of acellular cementum containing only Sharpey's fibers as a fibrous component.

Animals↗

PubMed2002

Synthesis and biological evaluation of cyclopenta[c]thiophene related compounds as new antitumor agents.

A series of 22 cyclopenta[c]thiophene related compounds was obtained by the pharmacomodulation of 6-amino-5,6-dihydro-4H-cyclopenta[c]thiophen-4-ones 1a-g. All compounds were evaluated for potential anticancer activity in the NCI's in vitro human disease-oriented tumor cell line screening panel that consisted of 60 human tumor cell lines arranged in nine subpanels, representing diverse histologies. Among these tested compounds, seven were found to be cytotoxic, especially against leukemia cell lines, allowing us to point out some structure-activity relationships. These derivatives were further evaluated for potential in vivo anticancer activity in the hollow fiber assay developed at the NCI, which selected two compounds, 1f and 3a for standard xenograft testing.

Antineoplastic Agents↗

PubMed1995

Development of a submicrometer optical fiber oxygen sensor.

A submicrometer optical fiber oxygen sensor has been fabricated, based on the fluorescence quenching of tris-(1,10-phenanthroline)ruthenium(II) chloride in the presence of oxygen or dissolved oxygen. The Ru compound has been incorporated into acrylamide polymer that is attached covalently to a silanized optical fiber tip surface by photoinitiated polymerization. Leaching of the sensing reagent from the polymer host matrix has been minimized by the optimization of the ratio between the acrylamide monomer and the cross-linker, N,N-methylenebisacrylamide. The sensor is fully reversible and highly reproducible. A standard deviation of approximately 2% for 10 consecutive fluorescence measurements has been observed for several oxygen concentrations. The sample volume required for measurements is 100 fL. An absolute detection limit of 1 x 10(-17) mol is achieved. This is an improvement by a factor of 10(6) as compared to other existing optical fiber oxygen sensors.

Fiber Optic Technology↗

PubMed2006

A novel strategy to construct a flat-lying DNA monolayer on a mica surface.

Flat-lying, densely packed DNA monolayers in which DNA chains are well organized have been successfully constructed on a mica surface by dropping a droplet of a DNA solution on a freshly cleaved mica surface and subsequently transferring the mica to ultrapure water for developing. The formation kinetics of such monolayers was studied by tapping mode atomic force microscopy (TMAFM) technique. A series of TMAFM images of DNA films obtained at various developing times show that before the sample was immersed into water for developing the DNA chains always seriously aggregated by contacting, crossing, or overlapping and formed large-scale networks on the mica surface. During developing, the fibers of DNA networks gradually dispersed into many smaller fibers up to single DNA chains. At the same time, the fibers or DNA chains also experienced rearrangement to decrease electrostatic repulsion and interfacial Gibbs free energy. Finally, a flat-lying, densely packed DNA monolayer was formed. A formation mechanism of the DNA monolayers was proposed that consists of aggregation, dispersion, and rearrangement. The effects of both DNA and Mg2+ concentration in the formation solution on DNA monolayer formation were also investigated in detail.

Aluminum Silicates↗

PubMed1997

Identification of a delta-TIP cDNA clone and determination of related A and D genome subfamilies in Gossypium species.

Tonoplast intrinsic proteins (TIPs) have been implicated in the process of cell elongation, such as occurs in the developing cotton fiber. We have isolated a cDNA clone (997 bp in length) from a cotton (Gossypium hirsutum L.) library which putatively encodes a protein of 248 residues (Mr 25079) with 85% identity to Arabidopsis delta-TIP. The derived amino acid sequence included two conserved sequences associated with major intrinsic proteins (SGxHxNPA at residues 78 to 85, NPA residues at 197 to 199) and a cysteine residue at 116 which is reported to bind mercury in Arabidopsis delta-TIP. The polymerase chain reaction was used to generate partial genomic clones of the cotton delta-TIP. In comparison to other genomic TIP sequences, the number (two) and position of the introns were conserved in cotton. Comparing the TIP sequences from cotton revealed two subfamilies, which were consistently distinguished by a Tsp45I restriction site polymorphism. This polymorphism was used to demonstrate that TIP subfamilies were specific to either the A or D genomes of Gossypium. When delta-TIP DNA fragments were amplified from cDNA of fiber 14 days after anthesis, the A and D were found, indicating the presence of delta-TIP transcripts in these elongating cells.

Amino Acid Sequence↗

PubMed2002

Developmental and tissue-specific expression of CaMV 35S promoter in cotton as revealed by GFP.

The CaMV 35S promoter is the most commonly used promoter for driving transgene expression in plants. Though it is presumed to be a constitutive promoter, some reports suggest that it is not expressed in all cell types. In addition, the information available on its expression profile in all possible cell and tissue types and during early stages of development is incomplete. We present here a detailed expression profile of this promoter investigated using the green fluorescent protein (GFP) gene as a reporter system in cotton during embryo development, and in all the vegetative and floral cell and tissue types. GFP expression was not detected during the early stages of embryogenesis. The first perceptible GFP expression was observed in a small area at the junction of hypocotyl and cotyledons in embryos at around 13 days after anthesis. The GFP fluorescence progressively became stronger and expanded throughout the cotyledon and hypocotyl as embryo development advanced. After germination, varying levels of promoter activity were observed in all cell and tissue types in the hypocotyl, cotyledon, stem, leaf, petiole, and root. The promoter was also expressed in all floral parts. Although cotton pollen exhibited a low level of greenish autofluorescence, it was possible to discern GFP-dependent fluorescence in some of the pollen from all the T0 plants examined. Developing cotton fibers also exhibited GFP fluorescence suggesting that the 35S promoter was active in these specialized epidermal cells. Thus, we show that the expression of the 35S promoter was developmentally regulated during embryogenesis and that beyond a certain stage during embryogenesis, the promoter was expressed in most cell and tissue types in cotton albeit at different levels.

Caulimovirus↗