Targeting of nebulin fragments to the cardiac sarcomere

Nebulin, a vertebrate skeletal muscle actin binding protein, plays an important role in thin filament architecture. Recently, a number of reports have indicated evidence for nebulin expression in vertebrate hearts. To investigate the ability of nebulin to interact with cardiac myofilaments, we have expressed nebulin cDNA fragments tagged with green fluorescent protein (GFP) in chicken cardiomyocytes and PtK2 cells. Nebulin fragments from both the superrepeats and single repeats were expressed minus and plus the nebulin linker. Nebulin fragment incorporation was monitored by fluorescent microscopy and compared with the distribution of actin, alpha-actinin and titin. Expression of nebulin N-terminal superrepeats displayed a punctate cytoplasmic distribution in PtK2 cells and cardiomyocytes. Addition of the nebulin linker to the superrepeats resulted in association of the punctate staining with the myofibrils. Nebulin C-terminal superrepeats plus and minus the linker localized with stress fibers of PtK2 cells and associated with the cardiac myofilaments at the level of the Z-line. Expression of the single repeats plus and minus the nebulin linker region resulted in both a Z-line distribution and an A-band distribution. These data suggest that N-terminal superrepeat nebulin modules are incapable of supporting interactions with the cardiac myofilaments; whereas the C-terminal nebulin modules can. The expression of the N-terminal or C-terminal superrepeats did not alter the distribution of actin, alpha-actinin or titin in either atrial or ventricular cultures.     

Presence of embryonic myosin in normal postural muscles of the adult rat

Indirect immunofluorescence was used to localize embryonic myosin heavy chains in soleus, adductor longus, tibialis anterior, plantaris, and extensor digitorum longus muscles of 6-month-old rats. A monoclonal antibody (2B6), specifically recognizing rat embryonic myosin, was applied to unfixed, transverse, frozen sections. The number of embryonic myosin-positive (EMP) extrafusal fibers was expressed as a percentage of the total number of fibers. EMP extrafusal fibers were only seen in the soleus and adductor longus muscles, both postural muscles. Approximately 1% of the soleus muscle fibers appeared positively stained for embryonic myosin. The majority of such fibers had a small diameter (<500 ), appeared intensely fluorescent, and typically contained central nuclei. Re-expression of embryonic myosin due to spontaneous fiber denervation is not a likely factor in this study, since alpha-bungarotoxin and N-CAM localization were restricted to the motor end-plate region of EMP fibers. Since embryonic myosin was shown to disappear in all normal-sized myofibers by 2 to 3 months of age, the results suggest that the EMP extrafusal fibers seen in postural muscles of 6 to 12-month-old animals are regenerating myofibers. We speculate that a small number of muscle fibers may be regenerating in normal, adult postural muscles, in response to fiber damage possibly caused by excessive recruitment or overloading.     

Synthesis of platelet-derived growth factor by cells of splenic red pulp in normal rats

A population of cells in the spleens of normal rats was found to contain platelet-derived growth factor (PDGF) B chain mRNA. These cells were found predominantly in the red pulp and nuclear morphology of some was consistent with that of macrophages. Similar cells were also shown by immunocytochemical staining to contain PDGF-AB/BB. These PDGF-positive cells were also found almost exclusively in the red pulp. It has been suggested by others that PDGF plays an important role in the function of the lymphohemopoietic microenvironment.     

Characterization of obestatin- and ghrelin-producing cells in the gastrointestinal tract and pancreas of rats: an immunohistochemical and electron-microscopic study

Both ghrelin and obestatin are derived from preproghrelin by post-translational processing. We have morphologically characterized the cells that produce obestatin and ghrelin in new-born and adult Sprague-Dawley rats that were freely fed, fasted, or subjected to gastric bypass surgery or reserpine treatment. Tissue samples collected from the gastrointestinal tract and pancreas were examined by double-immunofluorescence staining, immunoelectron microscopy, and conventional electron microscopy. Obestatin was present in the stomach, duodenum, jejunum, colon, and pancreas. In the stomach, differences were noted in the development of obestatin- and preproghrelin-immunreactive (IR) cells on the one hand and ghrelin-IR cells on the other, particularly 2 weeks after birth. Preproghrelin- and obestatin-IR cells were more numerous than ghrelin-IR cells in the stomach, suggesting the lack of ghrelin in some A-like cells. Most obestatin-producing cells in the stomach were distributed in the basal part of the oxyntic mucosa; these cells co-localized with chromogranin A (pancreastatin) and vesicle monoamine transporters type 1 and 2, but not with serotonin or histidine decarboxylase. Immunoelectron microscopy revealed the obestatin- and ghrelin-producing cells to be A-like cells, characterized by numerous highly electron-dense granules containing ghrelin and obestatin. Some granules exhibited an even electron density with thin electron-lucent halos, suggestive of monoamines. Feeding status, gastric bypass surgery, and reserpine treatment had no obvious effect on the A-like cells. In the pancreas, obestatin was present in the peripheral part of the islets, with a distribution distinct from that of glucagon-producing A cells, insulin-producing beta cells, and cells producing pancreatic polypeptide Y. Thus, obestatin and ghrelin co-localize with an anticipated monoamine in A-like cells in the stomach, and obestatin is found in pancreatic islets. This study was supported by a grant from the Cancer Foundation of St. Olav’s Hospital, Trondheim, Norway.     

Identification of neutrophils in the nonsensory epithelium of the vomeronasal organ in virus-antibody-free rats

Cells infiltrating the nonsensory epithelium of the vomeronasal organ of virus-antibody-free rats exhibited surface immunoreactivity for ₂-microglobulin and immunoglobulin (Ig) E. They were further characterized by using immunohistochemical techniques with antibodies to cell-specific markers or histochemical techniques for immunocytes with surface receptors for IgE. Localization of intracellular granules immunoreactive for lactoferrin and CD18, a leukocyte adhesion molecule, unequivocally identified these cells as neutrophils. The low number of IgA-and IgG-immunoreactive B lymphocytes, T lymphocytes, and accessory immunocytes in the vomeronasal organ as well as the rest of the nasal cavity confirmed the absence of infection. We hypothesize that the operation of the vomeronasal pump induces repeated episodes of transient focal ischemia followed by reperfusion, which results in release of neutrophil chemoattractants and modulation of adhesion factors that regulate the extravasation and migration of neutrophils into the nonsensory epithelium. The distribution of immunoreactivity for interleukin 8 suggests that it is not the primary neutrophil chemoattractant in this system while that of CD18 suggests its active involvement in neutrophil extravasation. In addition to their role in immune surveillance, neutrophils may stimulate ion/water secretion into the vomeronasal lumen, affecting the perireceptor processes regulating stimulus access and clearance from the sensory epithelium.     

Suramin-induced mucopolysaccharidosis in rat incisor

Two weeks after a single injection of suramin, the secretory and post-secretory ameloblasts of the rat incisor were filled with large lysosome-like vacuoles. At the light-microscope level, these vacuoles were positively stained with Alcian blue when MgCl₂ was used at a critical electrolyte concentration varying between 0.1 and 0.3 M, whereas no staining appeared when MgCl₂ varied between 0.7 and 0.9 M. Hyaluronidase digestion markedly reduced but did not totally abolish the staining, indicating that glycosaminoglycans were accumulated inside these vacuoles. Examination of these cells with the electron microscope revealed a polymorphic population of large vesicles, filled to various degrees with cetylpyridinium chloride (CPC)-positive and malachite green aldehyde (MGA)-positive material. The same pattern was observed in secretory odontoblasts but to a lesser extent. In the extracellular matrix, suramin-induced alterations appeared as large defects occurring during enamel formation. In predentin and dentin, the number and/or size of electron-dense aggregates resulting from CPC and MGA fixation, were enhanced in the suramin-injected rats. These aggregates were largely reduced or suppressed respectively by hyaluronidase digestion and chloroform/methanol treatment of the sections. The accumulation of glycosaminoglycans and phospholipids reported here inside ameloblasts and odontoblasts and in predentin and dentin supports the occurrence of suramin-induced mucopolysaccharidosis and lipidosis in this experimental animal model.     

Innervation of the anococcygeus muscle of the rat

Summary The innervation of the anococcygeus muscle of the rat was investigated with regard to the histochemical features of nerve fibers within the muscle and to the location of the postganglionic autonomic neurons which are the source of these fibers. Acetylcholinesterase-positive fibers and catecholaminergic fibers are abundant in the anococcygeus as well as the related retractor penis muscle. Neuronal somata, either between muscle bundles of the anococcygeus or in the connective tissue sheath, are also acetylcholinesterase-positive. Nerve fibers and a minority of the ganglion cells in the anococcygeus and retractor penis muscles are immunoreactive for vasoactive intestinal polypeptide. Injection of the retrogradely transported dye Fluorogold into the anococcygeus muscle filled neurons in the abdominopelvic sympathetic chain, pelvic plexus and a small number of neurons in the inferior mesenteric ganglion. In the pelvic plexus, some neurons were located in the major pelvic ganglion but most were found along the main penile nerve and its branches to the anococcygeus muscle. Immunocytochemistry of these identified neurons indicates that about one half of them are positive for vasoactive intestinal polypeptice. These results raise the possibility that both acetylcholine and vasoactive intestinal polypeptide are important neurotransmitters in autonomic nerves to the anococcygeus muscle.     

Calmodulin in rat enterocyte: an immunogold electron-microscope study

Immunogold labeling of ultrathin sections of the epithelium of rat small intestine has been used to obtain insights into the ultrastructural localization and possible function of calmodulin in the enterocyte. Calmodulin is found mainly overlying the periphery of the microvillous core, in agreement with the location of the 110-kDa calmodulin complex. Extremely small amounts of calmodulin can be detected along the interdigitating basolateral membrane. This immunogold electron-microscope study suggests that calmodulin plays an important role in regulating the mechanochemical activity of myosin I but not in processes associated with the basolateral membrane of rat enterocyte.     

Adrenocortical atrophy of hypophysectomized rats can be reduced by corticotropin-releasing hormone (CRH)

Summary The effects of high dose injections of corticotropin-releasing hormone (CRH) on the adrenal cortex of hypophysectomized rats were studied at the light-and electron-microscopical levels. Adrenocortical atrophy induced by hypophysectomy could be reduced by daily i.p. injection of 10 g (3 nmol) CRH given for 3 days starting at day 5 after the operation. The cortex broadened, mostly because of hypertrophy of the zona fasciculata. Blood vessels were enlarged. Although the adrenocortical cells of hypophysectomized rats showed features of a functionally suppressed state, such as tubular mitochondria, the cells of CRH-treated animals showed characteristics of stimulated cells. The inner membrane of the mitochondria formed the typical densely packed vesicles of adrenocortical cells that are active in steroidogenesis. Lipid droplets were found to be reduced, and the cells developed filopodia at their surface. These morphological observations indicate that CRH influences the adrenal cortex via extrapituitary mechanisms.     

Comparison of rat mesenchymal stem cells derived from bone marrow,synovium, periosteum,adipose tissue,and muscle

Mesenchymal stem cells (MSCs) are increasingly being reported as occurring in a variety of tissues. Although MSCs from human bone marrow are relatively easy to harvest, the isolation of rodent MSCs is more difficult, thereby limiting the number of experiments in vivo. To determine a suitable cell source, we isolated rat MSCs from bone marrow, synovium, periosteum, adipose, and muscle and compared their properties for yield, expansion, and multipotentiality. After two passages, the cells in each population were CD11b (−), CD45 (−), and CD90 (+). The colony number per nucleated cells derived from synovium was 100-fold higher than that for cells derived from bone marrow. With regard to expansion potential, synovium-derived cells were the highest in colony-forming efficiency, fold increase, and growth kinetics. An in vitro chondrogenesis assay demonstrated that the pellets derived from synovium were heavier, because of their greater production of cartilage matrix, than those from other tissues, indicating their superiority in chondrogenesis. Synovium-derived cells retained their chondrogenic potential after a few passages. The Oil Red-O positive colony-rate assay demonstrated higher adipogenic potential in synovium- and adipose-derived cells. Alkaline phosphatase activity was greater in periosteum- and muscle-derived cells during calcification. The yield and proliferation potential of rat MSCs from solid tissues was much better than those from bone marrow. In particular, synovium-derived cells had the greatest potential for both proliferation and chondrogenesis, indicating their usefulness for cartilage study in a rat model. This study was supported in part by grants from the Japan Latest Osteoarthritis Society and from the Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone in Tokyo Medical and Dental University (to T.M.), and by the Japan Society for the Promotion of Science (grant no. 18591657 to I.S.). Recombinant human bone morphogenetic protein-2 was kindly provided by Astellas Pharma.     

Cryo-electron microscopy of myelin treated with detergents

After detergent extraction, myelin lamellae disaggregate into diverse globular fragments that correspond to the globular structures observed in normal cryofixed myelin. The interaction of these structural units in the bilayer is probably the morphological basis that provides the stability of the myelin lamellae and their lamination.     

Phenotypic modulation of smooth muscle cells during the formation of neointimal thickenings in the rat carotid artery after balloon injury: an electron-microscopic and stereological study

The formation of neointimal thickenings in the rat carotid artery after balloon injury was studied by a combination of electron-microscopic and stereological methods. All smooth muscle cells in the normal media had a contractile phenotype, the cytoplasm being dominated by myofilaments. Seven days after endothelial denudation, the smooth muscle cells in the innermost part of the media had assumed a synthetic phenotype by loss of myofilaments and formation of a large endoplasmic reticulum and Golgi complex. These cells moved through fine openings in the internal elastic lamina and gave rise to a growing neointima by proliferation and secretion of extracellular matrix components. Fourteen days after the operation, the neointima had almost reached its final size, and mitoses were no longer noted. Nevertheless, the cells maintained a synthetic phenotype with prominent secretory organelles, although myofilaments had started to become more abundant again. They were surrounded by an extracellular matrix made up of collagen fibrils and coalescing patches of elastin. Thirty-five days after the operation, an endothelial cell layer had reformed and covered most of the luminal vessel surface. In parallel, the smooth muscle cells in the neointima had returned to a contractile phenotype with a cytoplasm dominated by myofilaments. These findings provide a morphological basis for further analysis of the cellular and molecular interactions involved in the formation of neointimal thickenings after endothelial injury, and for the search for agents interfering with this process.     

Tumor necrosis factor-α in macrophages of heart,liver, kidney,and in the pituitary gland

The relationship between myonuclear number, cellular size, succinate dehydrogenase activity, and myosin type was examined in single fiber segments (n=54; 9±3 mm long) mechanically dissected from soleus and plantaris muscles of adult rats. One end of each fiber segment was stained for DNA before quantitative photometric analysis of succinate dehydrogenase activity; the other end was double immunolabelled with fast and slow myosin heavy chain monoclonal antibodies. Mean±S.D. cytoplasmic volume/myonucleus ratio was higher in fast and slow plantaris fibers (112±69 vs. 34±21x10³ m³) than fast and slow soleus fibers (40±20 vs. 30±14x10³ m³), respectively. Slow fibers always had small volumes/myonucleus, regardless of fiber diameter, succinate dehydrogenase activity, or muscle of origin. In contrast, smaller diameter (<70 m) fast soleus and plantaris fibers with high succinate dehydrogenase activity appeared to have low volumes/myonucleus while larger diameter (>70 m) fast fibers with low succinate dehydrogenase activity always had large volume/myonucleus. Slow soleus fibers had significantly greater numbers of myonuclei/mm than did either fast soleus or fast plantaris fibers (116±51 vs. 55±22 and 44±23), respectively. These data suggest that the myonuclear domain is more limited in slow than fast fibers and in the fibers with a high, compared to a low, oxidative metabolic capability.     

Caspase activation throughout the first wave of spermatogenesis in the rat

Early in postnatal life, the first wave of spermatogenesis is accompanied by an initial wave of germ cell apoptosis. This may reflect an adjustment in the number of germ cells that can be adequately maintained by Sertoli cells. Two major pathways (intrinsic and extrinsic) are involved in the process of caspase activation and apoptosis in mammalian cells. The extrinsic pathway is characterized by the oligomerization of death receptors such as FAS or tumor necrosis factor, followed by the activation of caspase-8 and caspase-3. The intrinsic pathway involves the activation of procaspase-9, which in turn activates caspase-3. Extensive information is available concerning apoptotic inducers and their possible mechanisms in the adult rat. However, no data exist regarding the molecular and cellular mechanisms governing physiological cell death during puberty in the male rat. We have studied caspase activation throughout the first wave of spermatogenesis in the rat under physiological conditions, by combining the TUNEL procedure with the localization of active caspases in germ cells. We observed TUNEL-positive germ cells in rats of 5–40 days of age, the highest number being found in 25-day-old rats. TUNEL-positive and caspase-3-positive germ cells appeared as long chains of interconnected germ cells in 25-day-old rats. Caspase activation was assayed by either immunohistochemistry with antibodies against active caspase-3, -8, and -9, or by determining enzymatic activity in seminiferous tubules extracts. Both techniques showed activation of caspase-3, -8, and -9 in 25-day-old rats and low enzymatic activity at other ages. Confocal scanning laser microscopy indicated that active caspase-3, -8, and -9 co-localized with TUNEL-positive cells. Thus, caspase-3, -8, and -9 are active in apoptotic germ cells during the first wave of rat spermatogenesis. The extrinsic pathway of apoptosis may therefore play an important role in germ cell apoptosis during puberty in the rat.This work was financed by a research grant from FONDECYT (1040800) to R.D.M.     

The subsets of keratinocytes responsible for covering open wounds in neonatal rat skin

Full-thickness excisional wounds were made on the dorsal skin of 1-day-old rats to elucidate from where the cells move into the defect and what kinds of cells they are. Immunohistochemical analyses of the wound sites revealed that the following two subsets of keratinocytes were the major contributors to reepithelialization: first, the cells at the forefront of the migrating epithelium, termed leading edge cells, which expressed K14 keratin, known as basal cell-specific keratin, but not K6 or K10 keratins, so that they had probably moved from the basal cell layer; and, second, the cells tentatively termed immature spinous cells, which expressed K14 and K6 but not K10, and formed an ingrowth region following the leading edge cells. These two kinds of cells moved to the open wound area, as a multilayered cell sheet. Fluorescent phalloidin staining experiments indicated that actin filaments became concentrated in the leading edge cells within 6 h postwounding (PW), whereas weak signals of actin filaments were detected in the immature spinous cells. Taken together, the present findings support the view that wound covering in neonatal rat skin is accomplished by a movement en masse of keratinocytes from the bottom half of the surrounding epidermis.     

Implantation in vitro: co-culture of rat blastocyst and epithelial cell vesicles

Implantation of blastocysts involves conversion of maternal and embryonic cell surfaces from a nonadhesive to an adhesive state in response to the internally driven developmental program or to externally generated factors. However, the intricacies of the cellular and subcellular changes that promote the attachment are not known, because these changes are difficult to determine in situ because of the nonaccessibility of the site. To overcome this, an in vitro model of implantation was developed by co-culturing rat blastocysts and uterine epithelial cells of the same gestational age (day 5 postcoitum; plug day as day 1) in drops hanging from the lid of a Petri dish. The system was used to study the changes on the surface membranes of the cells of the trophectoderm and uterine epithelium and to evaluate the antiadhesive activity of the newly designed test substances. The isolated epithelial cell vesicles were co-cultured with zona-free blastocysts in the microdrops (40–50 µl) hanging from the lid of a 60-mm Petri dish. The lid was placed over the lower dish, which was presaturated with the medium. The culture was examined 48 h later to determine the site of adhesion of epithelial cell vesicles with the trophoblasts lining the blastocyst. The cell-cell adhesion was monitored on a computerized image analyzer. To validate the adhesion of blastocysts and epithelial cell vesicles in co-culture, the expression of a cell adhesion molecule, uvomorulin, was studied using immunocytochemical technique after incubating with antiuvomorulin antibody. Intense staining was noted on the membrane surfaces at the site of attachment of the blastocyst and cell vesicles.The authors express their sincere thanks to the Ministry of Health and Family Welfare, Government of India, for their financial support     

Injections of leptin into rat ventromedial hypothalamus increase adipocyte apoptosis in peripheral fat and in bone marrow

The accumulation of fat cells (adipocytes) in bone marrow is now thought to be a factor contributing to age-related bone loss. Women with osteoporosis have higher numbers of marrow adipocytes than women with healthy bone, and bone formation rate is inversely correlated with adipocyte number in bone tissue biopsies from both men and women. Adipogenic differentiation of bone marrow stromal cells increases with age, but the factors regulating populations of mature adipocytes are not well understood. Leptin is thought to regulate adipose tissue mass via its receptors in the ventromedial hypothalamus (VMH). We have therefore tested the hypothesis that stimulation of leptin receptors in the VMH regulates adipocyte number in bone marrow. Results indicate that unilateral twice-daily injections of leptin into the rat VMH for only 4 or 5 days cause a significant reduction in the number of adipocytes in peripheral fat pads and bone marrow and indeed eliminate adipocytes almost entirely from bone marrow of the proximal tibia. Osteoblast surface is not affected with leptin treatment. Apoptosis assays performed on bone marrow samples from control and treated rats have revealed a significant increase in protein concentration of the apoptosis marker caspase-3 with leptin treatment. We conclude that stimulation of leptin receptors in the VMH significantly decreases the adipocyte population in bone marrow, primarily through apoptosis of marrow adipocytes. Elimination of marrow adipocytes via this central pathway may represent a useful strategy for the treatment and prevention of osteoporosis.     

Production of an endothelial cell migratory signal in rat endometrium during early pregnancy

Rat endometrial explants were cultured in a three-dimensional collagen/endothelial cell matrix to measure angiogenic activity, as represented by migration of vascular endothelial cells towards the explants. Minimal endothelial cell migratory activity was observed with endometrial explants taken during the four-day oestrous cycle and days 3 and 4 of pregnancy. In contrast, a surge of endothelial cell migration occurred in response to endometrial explants taken from day-5-pregnant rats. Activity was found in explants taken approximately 5 h prior to implantation, but returned to minimal levels by day 6 of pregnancy. Endothelial cell migration remained minimal in response to both implantation and intersite tissue explants taken from days 6 and 7 of pregnancy. Endometrium from ovariectomised rats produced no endothelial cell migratory activity as measured by this technique. However, near preimplantation levels of endothelial cell migratory activity could be induced in ovariectomised rat endometrium by administering progesterone for 72 hours. Oestrogen given in conjunction with progesterone had no additional effect. These results demonstrate the presence of an endometrial signal that controls endothelial cell migration, and demonstrate this activity can be induced by progesterone without the addition of oestrogen.