Customized prefabricated neovascularized free flaps

Neovascularization of tissues into which a vascular pedicle has been implanted can result in the creation of a flap, or free flap, which is supported by those vessels as a neopedicle. This phenomenon allows the construction of customized prefabricated free flaps from tissue without restriction to naturally occurring vascular territories.     

Overexpression of endothelial nitric oxide synthase attenuates cardiac hypertrophy induced by chronic isoproterenol infusion.

Endogenous nitric oxide (NO) inhibits the contractile response to beta-adrenergic stimulation, but its effect on cardiac hypertrophy mediated by beta-adrenoceptors remains unclear. The present study was designed to determine whether overproduction of endothelial NO synthase (eNOS) could inhibit cardiac hypertrophy induced by chronic isoproterenol (ISO) infusion (30mg/kg per day) using eNOS overexpressing (eNOS-Tg) mice and wild-type (WT) mice. In a separate group, WT mice were treated with ISO and hydralazine to decrease blood pressure to the same levels in eNOS-Tg mice. The eNOS expression, NOS activity, and cGMP levels in the heart were remarkably higher in eNOS-Tg mice than in WT mice. ISO increased both heart weight and the heart/body weight ratio, which were significantly attenuated in eNOS-Tg mice compared with WT or hydralazine-treated WT mice. Histological examination revealed that the extent of fibrosis was not significantly different among the 3 groups, and that the increase in myocyte size was more than 10% lower in eNOS-Tg than in the other groups. In addition, up-regulated expression of atrial natriuretic peptide mRNA associated with cardiac hypertrophy was significantly inhibited in eNOS-Tg mice during ISO infusion. These results indicate that endogenous NO might act as a negative modulator for the hypertrophic response to beta-adrenergic stimulation.     

Metformin increases degradation of phospholamban via autophagy in cardiomyocytes

Phospholamban (PLN) is an effective inhibitor of the sarco(endo)plasmic reticulum Ca²⁺ ATPase (SERCA). Here, we examined PLN stability and degradation in primary cultured mouse neonatal cardiomyocytes (CMNCs) and mouse hearts using immunoblotting, molecular imaging, and [³⁵S]methionine pulse-chase experiments, together with lysosome (chloroquine and bafilomycin A1) and autophagic (3-methyladenine and Atg5 siRNA) antagonists. Inhibiting lysosomal and autophagic activities promoted endogenous PLN accumulation, whereas accelerating autophagy with metformin enhanced PLN degradation in CMNCs. This reduction in PLN levels was functionally correlated with an increased rate of SERCA2a activity, accounting for an inotropic effect of metformin. Metabolic labeling reaffirmed that metformin promoted wild-type and R9C PLN degradation. Immunofluorescence showed that PLN and the autophagy marker, microtubule light chain 3, became increasingly colocalized in response to chloroquine and bafilomycin treatments. Mechanistically, pentameric PLN was polyubiquitinylated at the K3 residue and this modification was required for p62-mediated selective autophagy trafficking. Consistently, attenuated autophagic flux in HECT domain and ankyrin repeat-containing E3 ubiquitin protein ligase 1-null mouse hearts was associated with increased PLN levels determined by immunoblots and immunofluorescence. Our study identifies a biological mechanism that traffics PLN to the lysosomes for degradation in mouse hearts.Phospholamban (PLN) is a 52-amino acid peptide located in the sarcoplasmic reticulum (SR) membrane in cardiac, slow-twitch skeletal, and smooth muscle, where it exists as a monomer or pentamer. Whereas monomeric PLN physically interacts with sarco(endo)plasmic reticulum Ca²⁺ ATPase type 2a (SERCA2a) to antagonize its function, pentameric PLN complexes are thought to be a reservoir of inactive PLN (1–3). The physical interaction between SERCA2a and PLN reduces the apparent affinity of SERCA2a for Ca²⁺, thereby making SERCA2a less active in transporting Ca²⁺ from the cytoplasm to the lumen of the SR at the same concentration of cytoplasmic Ca²⁺. The physical interaction between the two proteins is regulated by phosphorylation of PLN at Ser16 by protein kinase A or at Thr17 by Ca²⁺/calmodulin-dependent protein kinase II (2). Phosphorylation of PLN reduces its affinity for SERCA2a, thereby increasing SERCA2a activity (2). Evidence from transgenic mice also supports the inhibitory function of PLN. Although targeted PLN deletion enhances baseline cardiac performance, cardiac-specific overexpression of superinhibitory forms of PLN leads to decreases in the affinity of SERCA2a for Ca²⁺ (2). These observations underscore the primary role of PLN as a regulator of SERCA2a activity and, therefore, as a crucial regulator of cardiac contractility. PLN inhibition of SERCA2a can be reversed by either external (i.e., activation of β-adrenergic receptors) or internal (i.e., increased intracellular Ca²⁺ concentration) stimuli.Previous studies identified three PLN mutations in families of patients with hereditary dilated cardiomyopathy. These mutations, the substitution of Cys for Arg9 (R9C) (4), Arg14 deletion (RΔ14) (5), and the substitution of TGA for TAA in the Leu39 codon, creating a stop codon (L39stop) (6), also lead to dilated cardiomyopathy in transgenic mice. At the cellular level, ectopically expressed RΔ14 and L39stop PLN mutants localize at the plasma membrane in HEK-293T cells, cultured mouse neonatal cardiomyocytes, and cardiac fibroblasts, whereas wild-type and the R9C mutant reside within the endoplasmic reticulum (ER)/SR (6, 7). These data, together with a recent study by Sharma et al. (8), suggest a highly ordered trafficking of PLN, ultimately ensuring correct localization, and thus function, within the SR. However, PLN trafficking and degradation mechanisms in mammalian cardiomyocytes have not been clearly established.Protein degradation and clearance of damaged organelles are critical for cellular physiology, and failure in proper clearance has been shown to have pathological repercussions (9). Autophagy is a major mechanism that mediates protein and organelle degradation in response to external and internal signals. External stimulation through pharmacological agonists, such as metformin and rapamycin, promotes autophagy via AMP-activated protein kinase (AMPK) and mammalian target of rapamycin signal pathways, whereas amino acid starvation and an increased intracellular AMP/ATP ratio serve as internal signals to promote autophagy via the Ca²⁺/Calmodulin-dependent kinase kinase-β (10). Steps in the autophagy pathway involve nucleation of targeted macromolecules on the ER membrane, trafficking of autophagosomes to lysosomes and, finally, fusion of the autophagosome-lysosome, resulting in targeted protein degradation (11). In the heart, autophagy plays a crucial role in response to insults, in part by relieving ER stress (12) and removing damaged mitochondria (13). Loss of autophagy could result in irreversible apoptosis and reduced cardiac functioning (14).To characterize PLN degradation, we conducted a series of assays in cultured mouse neonatal cardiomyocytes (CMNCs) and the hearts of HECT domain and ankyrin repeat-containing E3 ubiquitin protein ligase 1 (Hace1)-null mice. Our results show that PLN degradation required both polyubiquitinylation and p62-mediated selective autophagy in CMNCs. Loss of HACE1 was associated with increased PLN levels, supporting the notion that selective autophagy modulates PLN degradation in vivo. Metformin promoted wild-type and R9C PLN degradation through autophagic pathways, resulting in metformin-induced inotropic enhancement.     

No V(H) somatic hypermutation was detected in B-cells of a patient with macroglobulinemia due to splenic marginal zone lymphoma

B-cell diseases are classified on the basis of the normal differentiation stages. We report here a case of a patient with a long history of leukocytosis, splenomegaly without lymphadenopathy, and hyperviscosity symptoms. Clinically, the patient's diagnosis was leukemic Waldenstrom macroglobulinemia. Chromosomal analysis revealed translocation t(2;7)(p11;q22) along with disease progression. Death occurred from pulmonary infection at 46 months after the initial presentation. At autopsy, malignant lymphocytes were found in the marginal areas of the spleen with spreading to the bone marrow and the liver. The histologic findings were consistent with splenic marginal zone lymphoma. We examined the sequences of the immunoglobulin V(H) gene in cells from the initial peripheral blood and from the spleen at autopsy and found that the sequences were identical and had no somatic hypermutation. Macroglobulinemia can occur in various B-cell disorders, including splenic marginal zone lymphoma, even with the transformation of unmutated B-lymphocytes.     

Der Einfluß der Nebennierenexstirpation auf die Arbeitsfähigkeit des Muskels

Zusammenfassung Nach Entfernung beider Nebennieren durch Thermokauterisieren lebten Frösche durchschnittlich 36,5 Stunden.Isolierte Wadennmuskeln nebennierenloser Tiere ermüden etwas schneller und vermögen eine etwas kleinere Arbeit zu leisten als die Muskeln normaler Frösche. Der Unterschied ist aber zu klein, um einen sicheren Schluß auf eine Beeinflussung der Muskeltätigkeit durch das Adrenalin zuzulassen.     

Methanol in urine as a biological indicator of occupational exposure to methanol vapor

Summary The exposure-excretion relationship and possible health effects of exposure to methanol vapor were studied in 33 exposed workers during the second half of 2 working weeks. Urinary methanol concentrations were also determined in 91 nonexposed subjects. The geometric mean value for methanol in urine samples from the latter was < 2 mg/1 (95% upper limit of normal, < 5 mg/l) when log-normal distribution was assumed. Among the exposed workers, the methanol level in urine samples collected prior to the work shift exceeded the 95% upper limit of normal. The time-weighted average intensity of exposure to methanol vapor was measured using personal sampling devices (in which water severed as an absorbent) in 48 cases of methanol exposure (i.e., 2 of the 33 exposed workers failed to provide urine samples, whereas 17 subjects were examined twice). Methanol concentrations in urine were determined in samples collected at the end of the shift from the 48 exposed cases as well as from 30 nonexposed controls. There was a significant correlation between the exposure to methanol vapor at concentrations of up to 5,500 ppm and the levels of methanol measured in the shift-end urine samples. The calculation indicated that a mean level of 42 mg methanol/l urine (95% confidence range, 26–60 mg/kg) was excreted in the shift-end urine sample following 8 h exposure to methanol at 200 ppm (the current occupational exposure limit). Dimmed vision and nasal irritation were among the most frequent symptoms complained during work. Three cases showing clinical signs of borderline significance were identified.