A fine-structural analysis of the fusion of myogenic cells.

The fusion of myogenic cells has been examined on the fine-structural level in muscle cell cultures of embryonic Japanese Coturnix quail. Cells, selected by light microscopy, were serially sectioned normal to their long axis. In this plane, oblique sections of cell membranes are rare and plasmalemmal profiles are more easily traced between adjacent cells. In seven cases, pairs of cells, apparently fixed in the process of fusion, are joined by a single cytoplasmic bridge. Since obliquely sectioned membranes often suggest cytoplasmic confluence, tilting stage analysis was employed to resolve cell membranes in suspect cases. In contrast to such artifacts of superposition, however, the observed intercommunicating pores are contained within a pair of culs-de-sac formed by the fused membranes of both cells. These blind pouches can be traced back between the cells to the external space. The confluent regions are clearly demarcated and they are not simply areas between vesicular profiles. The results of this analysis suggest that (a) at no time is there any loss of integrity of the cellular envelope, and (b) fusion is most probably initiated at single sites between pairs of cells, the pore enlarging, leaving first vestiges and eventually no trace of the original intervening membranes.

Developmental fate of skeletal muscle satellite cells.

Radioisotopically labeled satellite cells from clonal cultures were implanted into normal muscle of the original donor. Implanted cells invariably retained their myogenic potential by participating in the regeneration of damaged myofibers or in the development of existing fibers.

Association between depression and concurrent Type 2 diabetes outcomes varies by diabetes regimen.

AIMS: Although depression has weak associations with several Type 2 diabetes mellitus (DM) outcomes, it is possible that these associations are concentrated within certain patient subgroups that are more vulnerable to their effects. This study tested the hypothesis that depression is related to glycaemic control and diabetes-related quality of life (DQOL) in patients who are prescribed injected insulin, but not those on oral glucose-lowering agents alone. METHODS: Participants (103 on insulin, 155 on oral glucose-lowering agents alone) with Type 2 DM were recruited from a large US healthcare system and underwent assessment of glycaemic control (glycated haemoglobin; HbA(1c)), medication adherence and diabetes self-care behaviours, DQOL and depression (none, mild, moderate/severe). RESULTS: There was a significant regimen x depression interaction on HbA(1c) (P = 0.002), such that depression was associated with HbA(1c) in patients using insulin (beta = 0.35, P < 0.001) but not in patients using oral agents alone (beta = -0.08, P = NS). There was a similar interaction when quality of life was analysed as an outcome (P = 0.002). Neither effect was mediated by regimen adherence. CONCLUSIONS: The generally weak association between depression and glycaemic control is concentrated among patients who are prescribed insulin. Similarly, the association between depression and illness quality of life is strongest in patients prescribed insulin. Because this is not attributable to depression-related adherence problems, psychophysiological mechanisms unique to this group ought to be carefully investigated. Clinicians might be especially vigilant for depression in Type 2 DM patients who use insulin and consider its potential impact upon their illness course.

Salmonella Knowledge, Attitudes and Practices: A Survey of Backyard Poultry Owners Residing in Seattle, Washington and the Surrounding Metropolitan Area.

Raising poultry flocks in urban backyard settings is becoming increasingly popular across the United States, but carries a risk of zoonotic infection. In the United States from 1990 to 2014, 53 outbreaks of human salmonellosis linked to live poultry have been documented resulting in 2611 known illnesses, 387 known hospitalizations and five known deaths (Centers for Disease Control and Prevention 2015a, http://www.cdc.gov/healthypets/resources/dont-play-chicken-with-your-health-poster-24x36_508.pdf). A cross-sectional descriptive study was developed to better understand knowledge, attitudes and practices of urban backyard poultry owners regarding Salmonella risk and prevention. The study included a survey of bird health, animal husbandry and hygiene practices, and knowledge, attitudes and practices relating to Salmonella risk. Participants were videotaped while caring for their birds, and the recordings were transcribed using notational analysis to determine whether reported practices differed from observed practices. The results indicated that while a large proportion of participants knew that exposure to Salmonella is an inherent risk associated with raising poultry and harvesting eggs, their reported and observed practices would not consistently reduce risk of transmission of Salmonella and other zoonotic diseases. Approximately one in four participants reported performing practices that increase risk of inoculation, such as snuggling and kissing birds or eating/drinking near them. None of the participants were observed kissing their birds on video; however, snuggling (holding birds to clothes) or touching their face during routine care was observed in approximately two-thirds of the video recordings. The video data provided a unique opportunity to compare reported practices with actions recorded during site visits. While the differences were not statistically significant, findings from our study suggest that flock owners may not accurately report the frequency with which risky practices are performed during routine animal care. Education and outreach targeting backyard flock owners should aim to improve husbandry and hygiene practices and reduce risk of zoonotic diseases associated with raising poultry in the backyard setting.

Skeletal muscle satellite cells: changes in proliferation potential as a function of age.

Skeletal muscle satellite cells were isolated from soleus and extensor digitorum longus muscles of Sprague-Dawley rats at selected ages between 6 days and 30 months and grown in cell culture. Cells at each donor age were monitored individually to determine the number of progeny they were capable of producing. Under identical in vitro conditions, the average number of progeny produced was inversely proportional to donor age. Hence, the proliferation potential of satellite cells decreases with age.

Effects of endotoxin on neutrophil-mediated ischemia/reperfusion injury in the rat heart in vivo.

We have previously shown that a nonlethal dose of lipopolysaccharide (LPS) decreases L-selectin expression of neutrophils (PMNs), thereby preventing PMN-mediated reperfusion injury in the isolated heart. In the present study we determined whether or not that dose of LPS would protect hearts during in vivo ischemia and reperfusion by preventing PMN-induced reperfusion injury. Rats receiving saline vehicle showed marked myocardial injury (necrotic area/area at risk = 82%+/-2%) and significant depression in left ventricular function as assessed in the isolated isovolumic heart preparation at constant flow rates of 5, 10, 15, and 20 ml/min. The administration of LPS (100 microg/kg body wt) 7 hr prior to ischemia resulted in a reduction in myocardial damage (necrotic area/area at risk = 42%+/-3%) and preservation of function. Myocardial function was similar to that of sham ischemic saline- and LPS-treated rats. Moreover, PMN infiltration as determined by histology was quantitatively more severe in hearts of saline-treated rats than in hearts of LPS-treated rats. Isolated hearts from vehicle- and LPS-treated animals undergoing sham ischemia in vivo recovered to the same extent after in vitro ischemia/reperfusion, suggesting that LPS did not induce protection by altering intrinsic properties of the heart. Our results indicate that LPS-induced protection of the heart from in vivo PMN-mediated ischemia/reperfusion injury may be due to decreased L-selectin expression of PMNs in LPS-treated animals.

An ultrastructural study of peripheral neurons and associated non-neural structures in the bivalve mollusc, Spisula solidissima.

The ultrastructural morphology of peripheral neurons and associated structures in the bivalve mollusc. Spisula solidissima have been studied in an effort to describe the synaptic topography and to provide anatomical correlates of previous physiological observations. The somata of the peripheral neurons are located within the perineurium at branch points of the siphonal nerves. There are many fiber-fiber synaptic contacts which are characterized by isolated sites of contact with membrane specialization and unilateral accumulation of synaptic vesicles. There are also synaptic contacts involving the somata, both axo-somatic and somato-axonic, the two being distinguishable on the basis of the polarity of vesicle accumulation. All of the observed synaptic profiles were similar in appearance regardless of the neuron loci involved. Much of the non-synaptic soma surface is covered with processes of glial cells. Likewise, in many cases, individual fibers and groups of fibers are encases with glial processes. Unique clusters of membrane bound, pigment containing glial like cells occur throughout the nervous system of Spisula. The heterogenous appearance of the inclusions and the presence of lysosome-like bodies in the cytoplasm of these cells suggest a possible phagocytic role.

Estimation of central venous pressure by ultrasound of the internal jugular vein.

This article describes a simple, new technique using ultrasound (US) to estimate central venous pressure (CVP). The sonographic patterns of the internal jugular vein (IJV) with a low, normal, and elevated CVP are also described. Although bedside visual inspection of the height of the jugular veins as an estimate of CVP has been an integral part of the physical examination, its major limitation has been that the jugular veins are not always observable. In obese patients, a layer of fat often obscures the jugular pulsations. US has proven to be a powerful tool to noninvasively visualize neck veins in the emergency department. Bedside US of the IJV, performed by emergency physicians, provides immediate, important information that cannot be obtained without invasive catheters.

The effect of Marcaine on muscle and non-muscle cells in vitro.

The effect of the local anaesthetic Marcaine on muscle and non-muscle cell types was examined using an in vitro assay. Each of the cell types examined (myotubes, myoblasts, fibroblasts and liver parenchyma) expressed morphological alterations when incubated in Marcaine-medium. Myotubes were the most sensitive of the cells studied and exhibited several pronounced membrane structural changes after short incubation periods in Marcaine-medium. The toxic effects of Marcaine were irreversible and the myotubes continued to degenerate despite being placed in fresh medium. Myoblasts and non-muscle cells, however, demonstrated a rapid recovery when removed from the Marcaine-medium. Since Marcaine is thought to complete with Ca++ for specific sites on cell membranes, it is proposed that the differential effects which were observed are dependent upon the level of calcium related activities being carried out by the cells.

Microvessel endothelial cell transdifferentiation: phenotypic characterization.

Human dermal microvessel endothelial cells (MEC) have two basic functions: maintenance of tissue homeostasis and facilitation of inflammatory responses. The former requires that the endothelium expresses traits of an epithelium, while inflammatory reactions are associated with intimal disruption. Acute inflammation transiently alters endothelium, whereas chronic inflammation may result in vessel reorganization and MEC mesenchymalization. Foreskin MEC in vitro undergo a similar epithelial-mesenchymal modulation. In the presence of cAMP, cultivated dermal MEC exhibit the structural and functional characteristics of an epithelium. MEC grown in cAMP-deficient medium initially have a "transitional" configuration and are subsequently transformed into mesenchymal cells. If cAMP is replaced by histamine, MEC maintain a stable intermediate transitional configuration. Transitional MEC refed cAMP-supplemented medium revert to an epithelial phenotype, whereas parallel cultures fed cAMP-deficient medium are transformed into mesenchymal cells. Phenotypic modulation can be induced without cell division and thus provides a unique example of direct transdifferentiation. Our data furthermore suggest that this transdifferentiation results in the acquisition of properties usually attributed to cells of the reticuloendothelial system.

Ethanolamine and choline transport in cultured bovine aortic endothelial cells.

The transport of the polar head groups, ethanolamine and choline, was examined in cultured bovine aortic endothelial cells. Both ethanolamine and choline are taken up by high- and low-affinity systems. The K'm and V'max for the Na+-dependent, high-affinity ethanolamine and choline transport system are 3.0 and 3.0 microM and 5.4 and 7.3 pmol/mg protein/min, respectively. Ethanolamine and choline competitively influence one another's transport as the presence of 50 microM ethanolamine increases the K'm but not the V'max of choline uptake. Likewise, 50 microM choline increases the K'm but not the V'max of ethanolamine transport. The concentration of ethanolamine that inhibits maximal velocity of 5 microM choline by 50% is 9.7 microM, while 12 microM choline inhibits 5 microM ethanolamine maximal velocity by 50%. Uptake of both head groups is only partially Na+-dependent and is inhibited similarly by 2-methylethanolamine and 2,2-dimethylethanolamine at all concentrations examined. Hemicholinium-3, a classic inhibitor of high-affinity, Na+-dependent choline transport, reduces both ethanolamine and choline accumulation in a concentration-dependent fashion, but has a greater effect on choline transport at higher concentrations. The major portion of these data is consistent with our hypothesis that the uptake of physiological concentrations of ethanolamine and choline may occur through the same transport system. However, the results of the effect of hemicholinium-3 and the extent of Na+-dependency of choline and ethanolamine uptake could be interpreted as meaning that separate transport systems for choline and ethanolamine exist which cross react or that a single transport system exists which has separate active sites for the two compounds.

Histamine-modulated transdifferentiation of dermal microvascular endothelial cells.

Homeostatic and inflammatory functions of skin microvessels are tightly regulated by vasoactive amines. Following stimulation with histamine, dermal microvascular endothelial cells (MEC) undergo a rapid change in phenotype (transdifferentiation) and subsequently exhibit an enhanced rate of growth. To elucidate mechanisms regulating MEC transdifferentiation, this study investigated the functional relationships among vimentin, Ca2+, and protein kinase C (PKC) in histamine-modulated dermal MEC in vitro. Distribution of vimentin and PKC in foreskin-derived MEC cultivated in a modified Iscove's medium was assessed with immunocytochemistry. Calcium ion kinetics in histamine-treated MEC were analyzed using the Ca2+ probe Fluo-3 in conjunction with interactive laser cytometry. Histamine, acting through H-1 receptors, produces a rapid (less than 100 ms) and differential elevation of free calcium in each of three cytological compartments defined by the vimentin cytoskeleton in epithelial MEC. A distinctive compartmentalized and nonuniform distribution of PKC precisely coincides with that observed for free-Ca2+ released in response to histamine. The studies reveal that histamine modulation of the MEC phenotype is associated with a rapid patterned reorganization of the vimentin skeleton. It is hypothesized that histamine induces vimentin post-translational modifications by activating a spatially localized interaction among cytoplasmic free Ca2+, PKC, and the vimentin matrix. The results further suggest that vimentin, in addition to its structural role, may participate in signal transduction and gene regulation processes in effecting MEC transdifferentiation.

Effects of endotoxin on neutrophil-mediated I/R injury in isolated perfused rat hearts.

With the use of a syngeneic model, we demonstrate that rat polymorphonuclear neutrophils (PMNs) exacerbate ischemia-reperfusion injury in the isolated rat heart. However, PMNs (19 x 10(6) cells) from lipopolysaccharide (LPS)-treated rats (LPS-PMNs; 100 mg/kg administered 7 h before exsanguination) induce less reperfusion injury in the isolated heart. Average recovery of left ventricular developed pressure after 20 min of ischemia and 60 min of reperfusion was 51 +/- 4% in hearts receiving PMNs from saline-treated control rats (saline-PMNs) versus 78 +/- 2% in hearts receiving LPS-PMNs. Ischemic hearts reperfused with LPS-PMNs recovered to the same extent as did hearts reperfused with Krebs buffer only. LPS-PMNs and saline-PMNs showed no difference in basal or phorbol ester-induced superoxide production. Whereas twice the number of LPS-PMNs was positive for nitroblue tetrazolium, the percent positive for L-selectin, a receptor integral in PMN-adhesion to endothelium, was 50% less in LPS-PMNs than in controls. After reperfusion, three-fourths of the saline-PMNs remained within the hearts, whereas only one-fourth of LPS-PMNs were trapped. These data suggest that PMNs from LPS-treated rats do not exacerbate ischemia-reperfusion injury as do control PMNs, possibly, due to impaired PMN adhesion to endothelium as a result of decreased L-selectin receptors.

Ethanolamine metabolism in cultured bovine aortic endothelial cells.

The role of extracellular ethanolamine in phospholipid synthesis was examined in cultured bovine aortic endothelial cells. Serine and ethanolamine were both readily accumulated by these cells and incorporated into phospholipid. Exposing cells to extracellular ethanolamine for 4-6 weeks had no effect on cell growth, yet increased the phosphatidylethanolamine content of these cells by 31% as compared to control cells. The intracellular content of ethanolamine was measured by high performance liquid chromatography, and results showed that the ethanolamine-treated cells contained a significantly greater amount of free ethanolamine compared to control cells (0.62 +/- 0.07 nmol/mg of protein versus 0.27 +/- 0.05 nmol/mg of protein, respectively). Ethanolamine-treated cells also had decreased accumulation and incorporation into lipid of [3H]ethanolamine throughout a 48-h incubation and increased K'm and V'max parameters of ethanolamine transport as compared to control cells. Studies were also done to examine the effect of ethanolamine on the generation of free ethanolamine from phosphatidylserine. In pulse-chase experiments with [3H]serine, a physiological concentration of ethanolamine (25 microM) decreased the amount of 3H-labeled phosphatidylethanolamine produced from 3H-labeled phosphatidylserine by 12 h as compared to the amount of 3H-labeled phosphatidyl-ethanolamine produced in the absence of ethanolamine in the chase incubation. Furthermore, ethanolamine-treated cells accumulated 20% less labeled ethanolamine in the aqueous pool from [3H]serine after 24 h of incubation than did control cells. These results can be explained by isotope dilution with the ethanolamine pool that accumulates in these cells with time when exposed to media supplemented with a physiological concentration of ethanolamine and by an effect of ethanolamine on ethanolamine generation from phosphatidylserine. The results show that an extracellular source of ethanolamine significantly influences the phospholipid metabolism of cultured bovine aortic endothelial cells.

Modulation of the insulin-like growth factor system by chronic alcohol feeding.

Insulin-like growth factor (IGF)-I is a potent anabolic agent that plays an important role in regulating muscle protein balance. Alterations in one or more of the various components of the IGF system may be in part responsible for the muscle wasting that accompanies chronic alcohol consumption. The purpose of the present study was to characterize changes in the growth hormone-IGF axis produced by chronic alcohol consumption in rats. After 8 weeks of alcohol feeding, the IGF-I concentration was decreased in plasma (31%) as well as in the liver and skeletal muscle (40-50%), compared with pair-fed control animals. In addition, alcohol consumption decreased IGF-I mRNA abundance in liver and muscle (approximately 50%). IGF-I content in duodenum and kidney, however, was not altered by alcohol feeding. Concomitantly, the relative concentration of IGF binding protein (IGFBP)-1 was increased in plasma, liver, and muscle of alcohol-fed rats, compared with control values. In contrast, no changes in the plasma concentrations of IGFBP-2, -3, or -4 were detected in alcohol-fed rats at this time point Previous studies have indicated that elevations in glucocorticoids or decreases in insulin or growth hormone might be responsible for the decrease in IGF-I and/or the increase in IGFBP-1 in other catabolic conditions. However, there was no difference in the plasma concentrations of these hormones between alcohol-fed and control animals in this study. These data indicate that chronic alcohol feeding in rats decreases IGF-I and increases IGFBP-1 in the circulation and in skeletal muscle and that these changes appear to be independent of changes in classical hormonal regulators of the IGF system. The observed alterations in the IGF system are consistent with a reduction in the anabolic actions of IGF-I induced by chronic alcohol consumption.

Macrophages and smooth muscle cells express lipoprotein lipase in human and rabbit atherosclerotic lesions.

Lipoprotein lipase (LPL; EC 3.1.1.34) may promote atherogenesis by producing remnant lipoproteins on the endothelial surface and by acting on lipoproteins in the artery wall. In vitro, smooth muscle cells and macrophages synthesize LPL, but in human carotid lesions only a few smooth muscle cells were reported to contain LPL protein. Endothelial cells do not synthesize LPL in vitro, but in normal arteries intense immunostaining for LPL is present on the endothelium. We used Northern blot analysis, in situ hybridization, and immunocytochemistry of human and rabbit arteries to determine cellular distribution and the site of the synthesis of LPL in atherosclerotic lesions. Northern blot analysis showed that LPL mRNA was detectable in macrophage-derived foam cells isolated from arterial lesions of "ballooned" cholesterol-fed rabbits. In situ hybridization studies of atherosclerotic lesions with an antisense riboprobe showed a strong hybridization signal for LPL mRNA in some, but not all, lesion macrophages, which were mostly located in the subendothelial and edge areas of the lesions. Also, some smooth muscle cells in lesion areas also expressed LPL mRNA. Immunocytochemistry of frozen sections of rabbit lesions with a monoclonal antibody to human milk LPL showed intense staining for LPL protein in macrophage-rich intimal lesions. The results suggest that lesion macrophages and macrophage-derived foam cells express LPL mRNA and protein. Some smooth muscle cells in the lesion areas also synthesize LPL. These data are consistent with an important role for LPL in atherogenesis.

Macrophage colony-stimulating factor mRNA and protein in atherosclerotic lesions of rabbits and humans.

In this study, the authors demonstrate the expression of mRNA and the presence of protein for macrophage colony-stimulating factor (MCSF) in atherosclerotic lesions from humans and rabbits. In situ hybridization of serial sections of human fatty streaks demonstrated expression of MCSF mRNA by cells dispersed throughout the lesions. Immunocytochemical staining with a panel of MCSF-specific antibodies showed extensive cell-associated staining of all of the cell types in the lesions. Immunocytochemical studies of atherosclerotic lesions from Watanabe heritable hyperlipidemic (WHHL) and cholesterol-fed rabbits demonstrated a similar cell-associated pattern of staining. There was no MCSF-specific staining of aortas from normal rabbits or of cultured aortic smooth muscle cells from either humans or rabbits. Macrophage-derived foam cells (MFC) were isolated from the aortas of ballooned, cholesterol-fed rabbits. A Northern blot demonstrated that RNA isolated from the MFC hybridized with a human cDNA probe for MCSF. RNA from alveolar macrophages isolated simultaneously from the same rabbits did not hybridize with the MCSF probe. Conditioned media from an 18- to 24-hour incubation of the MFC contained colony-stimulating activity as demonstrated in a mouse bone marrow culture assay. Most of this colony-stimulating activity was neutralized by preincubating the conditioned media with an MCSF-specific antibody.