Denervation-induced skeletal muscle atrophy is associated with increased mitochondrial ROS production

Reactive oxygen species (ROS), especially mitochondrial ROS, are postulated to play a significant role in muscle atrophy. We report a dramatic increase in mitochondrial ROS generation in three conditions associated with muscle atrophy: in aging, in mice lacking CuZn-SOD (Sod1(-/-)), and in the neurodegenerative disease, amyotrophic lateral sclerosis (ALS). ROS generation in muscle mitochondria is nearly threefold higher in 28- to 32-mo-old than in 10-mo-old mice and is associated with a 30% loss in gastrocnemius mass. In Sod1(-/-) mice, muscle mitochondrial ROS production is increased >100% in 20-mo compared with 5-mo-old mice along with a >50% loss in muscle mass. ALS G93A mutant mice show a 75% loss of muscle mass during disease progression and up to 12-fold higher muscle mitochondrial ROS generation. In a second ALS mutant model, H46RH48Q mice, ROS production is approximately fourfold higher than in control mice and is associated with a less dramatic loss (30%) in muscle mass. Thus ROS production is strongly correlated with the extent of muscle atrophy in these models. Because each of the models of muscle atrophy studied are associated to some degree with a loss of innervation, we were interested in determining whether denervation plays a role in ROS generation in muscle mitochondria isolated from hindlimb muscle following surgical sciatic nerve transection. Seven days post-denervation, muscle mitochondrial ROS production increased nearly 30-fold. We conclude that enhanced generation of mitochondrial ROS may be a common factor in the mechanism underlying denervation-induced atrophy.     

HIV infection is associated with increased NTPDase activity that correlates with CD39-positive lymphocytes

Infection with the human immunodeficiency virus (HIV) results in alterations in immune cells such as an increase or decrease of cytokine secretion and immunodeficiency. HIV causes a state of chronic cellular activation that can induce apoptosis in lymphocyte T-helpers, making the patient susceptive to opportunistic infections. The biochemical mechanisms involved in this immune response to HIV have been researched. Here, we have shown for the first time that ATP and ADP hydrolysis are essential for the immune response to HIV. Our results clearly indicate an increase of NTPDase-1 (EC 3.6.1.5) activity in lymphocytes of HIV-positive patients, confirmed by an enhanced CD39 expression on its surface. These results suggest that NTPDase-1 may be important to keep an adequate balance between the generation and consumption of ATP and to preserve cellular integrity and immune response to the HIV infection.     

The presence of the cag pathogenicity island is associated with increased superoxide anion radical scavenging activity by Helicobacter pylori

Reactive oxygen species (ROS) generated by Helicobacter pylori infection have been suggested to be important factors in induction of gastric malignancies. Utilizing electron spin resonance spectrometry, H. pylori-dependent radical formation and hydroxyl- and superoxide-anion radical scavenging activity was investigated. In contrast to previous reports, we found that H. pylori does not produce ROS, but displays superoxide scavenging activity. This scavenging activity was increased in cag-positive H. pylori strains when compared to strains lacking an intact cag pathogenicity island, and was dependent on enzyme activity. We hypothesize that the increased scavenging activity of cag-positive H. pylori strains is an adaptation to the increased inflammatory response associated with the cag-positive genotype of H. pylori.     

Beclin 1 deficiency is associated with increased hypoxia-induced angiogenesis

Beclin 1, a tumor suppressor protein, acts as an initiator of autophagy in mammals. Heterozygous disruption of Beclin 1 accelerates tumor growth, but the underlying mechanisms remain unclear. We examined the role of Beclin 1 in tumor proliferation and angiogenesis, using a primary mouse melanoma tumor model. Beclin 1 (Becn1^+/-) hemizygous mice displayed an aggressive tumor growth phenotype with increased angiogenesis under hypoxia, associated with enhanced levels of circulating erythropoietin but not vascular endothelial growth factor, relative to wild-type mice. Using in vivo and ex vivo assays, we demonstrated increased angiogenic activity in Becn1^+/- mice relative to wild-type mice. Endothelial cells from Becn1^+/- mice displayed increased proliferation, migration and tube formation in response to hypoxia relative to wild-type cells. Moreover, Becn1^+/- cells subjected to hypoxia displayed increased hypoxia-inducible factor-2α (HIF-2α) expression relative to HIF-1α. Genetic interference of HIF-2α but not HIF-1α, dramatically reduced hypoxia-inducible proliferation, migration and tube formation in Becn1^+/- endothelial cells. We demonstrated that mice deficient in the autophagic protein Beclin 1 display a pro-angiogenic phenotype associated with the upregulation of HIF-2α and increased erythropoietin production. These results suggest a relationship between Beclin 1 and the regulation of angiogenesis, with implications in tumor growth and development.     

Methicillin-resistant Staphylococcus aureus infection is associated with increased mortality

Methicillin resistance is a widespread and major source of treatment complication in Staphylococcus aureus infections. Whether infections with methicillin-resistant S. aureus are associated with a worse clinical outcome, such as higher mortality, has remained controversial. Analyzing data from a large, global multicenter study, Hanberger et al. demonstrate that methicillin-resistant S. aureus infections are associated with approximately 50% higher mortality in the intensive care unit and significantly more frequent among critically ill patients than infections with methicillin-susceptible S. aureus. These findings call for the implementation or continuation of active methicillin-resistant S. aureus surveillance measures.     

Beclin 1 deficiency is associated with increased hypoxia-induced angiogenesis

Beclin 1, a tumor suppressor protein, acts as an initiator of autophagy in mammals. Heterozygous disruption of Beclin 1 accelerates tumor growth, but the underlying mechanisms remain unclear. We examined the role of Beclin 1 in tumor proliferation and angiogenesis, using a primary mouse melanoma tumor model. Beclin 1 (Becn1 (+/-) ) hemizygous mice displayed an aggressive tumor growth phenotype with increased angiogenesis under hypoxia, associated with enhanced levels of circulating erythropoietin but not vascular endothelial growth factor, relative to wild-type mice. Using in vivo and ex vivo assays, we demonstrated increased angiogenic activity in Becn1 (+/-) mice relative to wild-type mice. Endothelial cells from Becn1 (+/-) mice displayed increased proliferation, migration and tube formation in response to hypoxia relative to wild-type cells. Moreover, Becn1 (+/-) cells subjected to hypoxia displayed increased hypoxia-inducible factor-2α (HIF-2α) expression relative to HIF-1α. Genetic interference of HIF-2α but not HIF-1α, dramatically reduced hypoxia-inducible proliferation, migration and tube formation in Becn1 (+/-) endothelial cells. We demonstrated that mice deficient in the autophagic protein Beclin 1 display a pro-angiogenic phenotype associated with the upregulation of HIF-2α and increased erythropoietin production. These results suggest a relationship between Beclin 1 and the regulation of angiogenesis, with implications in tumor growth and development.     

Mammary hypertrophy is not associated with increased estrogen receptors

Estrogen promotes secondary female sex characteristics, including breast enlargement. Since excessive breast hypertrophy is unrelated to elevated serum estrogen levels, it has been postulated that the enlarged breast is a hypersensitive "target organ." At the cellular level, estrogen crosses the cell membrane, is bound to a cytoplasmic estrogen receptor (ER), and induces the formation of specific anabolic proteins. In breast cancer, this estrogen receptor is regarded as a measure of the sensitivity of the cell to estrogen. To determine if mammary hypertrophy is related to an increase in the number of estrogen receptors, we assayed breast tissue, not fat, from 25 consecutive breast reductions. The median age of patients was 26 years (17 to 77 years), with 752 gm per breast removed on average. Twenty-four percent of the patients were taking estrogens, primarily birth control bills. Cellular estrogen-receptor status was measured by a standardized cytosol extraction radioactive estradiol technique. Estrogen receptors were undetectable (less than 3 fmol/mg cytosol protein) in all patients. We conclude that estrogen receptors alone, and hence estrogen, are not a determinant in mammary hypertrophy. If the enlarged breast is a "target organ," it is by another mechanism.     

Skeletal muscle atrophy is associated with an increased expression of myostatin and impaired satellite cell function in the portacaval anastamosis rat

Proliferation and differentiation of satellite cells are critical in the regeneration of atrophied muscle following immobilization and aging. We hypothesized that impaired satellite cell function is responsible for the atrophy of skeletal muscle also seen in cirrhosis. Myostatin and insulin-like growth factor 1 (IGF1) have been identified to be positive and negative regulators, respectively, of satellite cell function. Using a rat model of cirrhosis [portacaval anastamosis (PCA)] and sham-operated controls, we examined the expression of myostatin, its receptor activinR2b, and its downstream messenger cyclin-dependent kinase inhibitor p21 (CDKI p21) as well as IGF1 and its receptor in the gastrocnemius muscle. Expression of PCNA, a marker of proliferation, and myogenic regulatory factors (myoD, myf5, and myogenin), markers of differentiation of satellite cells, were also measured. Real- time PCR for mRNA and Western blot assay for protein quantification were performed. PCA rats had lower body weight and gastrocnemius weight compared with sham animals (P < 0.05). PCNA and myogenic regulatory factors were lower in PCA rats (P < 0.05). Myostatin, activinR2b, and CDKI p21 were higher in the PCA animals (P < 0.05). The expression of IGF1 and its receptor was lower in liver and skeletal muscle of PCA animals (P < 0.05). These data suggest that skeletal muscle atrophy seen in the portacaval shunted rats is a consequence of impaired satellite cell proliferation and differentiation mediated, in part, by higher myostatin and lower IGF1 expression.     

Spinal muscular atrophy associated with progressive myoclonic epilepsy is caused by mutations in ASAH1

Spinal muscular atrophy (SMA) is a clinically and genetically heterogeneous disease characterized by the degeneration of lower motor neurons. The most frequent form is linked to mutations in SMN1. Childhood SMA associated with progressive myoclonic epilepsy (SMA-PME) has been reported as a rare autosomal-recessive condition unlinked to mutations in SMN1. Through linkage analysis, homozygosity mapping, and exome sequencing in three unrelated SMA-PME-affected families, we identified a homozygous missense mutation (c.125C>T [p.Thr42Met]) in exon 2 of ASAH1 in the affected children of two families and the same mutation associated with a deletion of the whole gene in the third family. Expression studies of the c.125C>T mutant cDNA in Farber fibroblasts showed that acid-ceramidase activity was only 32% of that generated by normal cDNA. This reduced activity was able to normalize the ceramide level in Farber cells, raising the question of the pathogenic mechanism underlying the CNS involvement in deficient cells. Morpholino knockdown of the ASAH1 ortholog in zebrafish led to a marked loss of motor-neuron axonal branching, a loss that is associated with increased apoptosis in the spinal cord. Our results reveal a wide phenotypic spectrum associated with ASAH1 mutations. An acid-ceramidase activity below 10% results in Farber disease, an early-onset disease starting with subcutaneous lipogranulomata, joint pain, and hoarseness of the voice, whereas a higher residual activity might be responsible for SMA-PME, a later-onset phenotype restricted to the CNS and starting with lower-motor-neuron disease.     

Radiation-induced lung adenocarcinoma is associated with increased frequency of genes inactivated by promoter hypermethylation

Epigenetic inactivation of genes by promoter hypermethylation, a major mechanism in the initiation and progression of tobacco-induced cancer, has also been associated with lung cancer induced through environmental and occupational exposures. Our previous study of gene methylation in workers from the MAYAK nuclear enterprise identified a significantly higher prevalence for methylation of the p16 gene (CDKN2A) in adenocarcinomas from workers compared to tumors from non-worker controls. The purpose of this investigation was to determine whether genes in addition to p16 are "targeted" for silencing and whether overall gene methylation was more common in radiation-induced adenocarcinoma. A significant increase in the prevalence of methylation of GATA5 was seen in tumors from workers compared to tumors from controls. The prevalence for methylation of PAX5 beta and H-cadherin did not differ in tumors from workers and controls. Evaluating the frequency for methylation of a five-gene panel revealed that 93% of adenocarcinomas from workers compared to 66% of tumors from controls were methylated for at least one gene. Moreover, a twofold increase was seen in the number of tumors methylated for three or more genes for tumors from workers compared to controls. Increased frequency for inactivation of genes by promoter hypermethylation and targeting of tumor suppressor genes such as GATA5 may be factors that contribute to the increased risk for lung cancer associated with radiation exposure.     

Human disc degeneration is associated with increased MMP 7 expression

During intervertebral disc (IVD) degeneration, normal matrix synthesis decreases and degradation of disc matrix increases. A number of proteases that are increased during disc degeneration are thought to be involved in its pathogenesis. Matrix metalloproteinase 7 (MMP 7) (Matrilysin, PUMP-1) is known to cleave the major matrix molecules found within the IVD, i.e., the proteoglycan aggrecan and collagen type II. To date, however, it is not known how its expression changes with degeneration or its exact location. We investigated the localization of MMP 7 in human, histologically graded, nondegenerate, degenerated and prolapsed discs to ascertain whether MMP 7 is up-regulated during disc degeneration. Samples of human IVD tissue were fixed in neutral buffered formalin, embedded in paraffin, and sections stained with hematoxylin and eosin to score the degree of morphological degeneration. Immunohistochemistry was performed to localize MMP 7 in 41 human IVDs with varying degrees of degeneration. We found that the chondrocyte-like cells of the nucleus pulposus and inner annulus fibrosus were MMP 7 immunopositive; little immunopositivity was observed in the outer annulus. Nondegenerate discs showed few immunopositive cells. A significant increase in the proportion of MMP 7 immunopositive cells was seen in the nucleus pulposus of discs classified as showing intermediate levels of degeneration and a further increase was seen in discs with severe degeneration. Prolapsed discs showed more MMP 7 immunopositive cells compared to nondegenerated discs, but fewer than those seen in cases of severe degeneration.     

Human disc degeneration is associated with increased MMP 7 expression

During intervertebral disc (IVD) degeneration, normal matrix synthesis decreases and degradation of disc matrix increases. A number of proteases that are increased during disc degeneration are thought to be involved in its pathogenesis. Matrix metalloproteinase 7 (MMP 7) (Matrilysin, PUMP-1) is known to cleave the major matrix molecules found within the IVD, i.e., the proteoglycan aggrecan and collagen type II. To date, however, it is not known how its expression changes with degeneration or its exact location. We investigated the localization of MMP 7 in human, histologically graded, nondegenerate, degenerated and prolapsed discs to ascertain whether MMP 7 is up-regulated during disc degeneration. Samples of human IVD tissue were fixed in neutral buffered formalin, embedded in paraffin, and sections stained with hematoxylin and eosin to score the degree of morphological degeneration. Immunohistochemistry was performed to localize MMP 7 in 41 human IVDs with varying degrees of degeneration. We found that the chondrocyte-like cells of the nucleus pulposus and inner annulus fibrosus were MMP 7 immunopositive; little immunopositivity was observed in the outer annulus. Nondegenerate discs showed few immunopositive cells. A significant increase in the proportion of MMP 7 immunopositive cells was seen in the nucleus pulposus of discs classified as showing intermediate levels of degeneration and a further increase was seen in discs with severe degeneration. Prolapsed discs showed more MMP 7 immunopositive cells compared to nondegenerated discs, but fewer than those seen in cases of severe degeneration.     

Lymphocyte DNA damage is associated with increased aortic intima-media thickness

OBJECTIVES: To investigate whether there is an association between lymphocyte DNA damage and aortic intima-media thickness (IMT). METHODS: We studied 70 patients (mean age: 41.6+/-10 years) who underwent transesophageal echocardiography for various indications. Four different grades were determined according to intima-media thickness (IMT) of thoracic aorta measured by transesophageal echocardiography. DNA damage was assessed by alkaline single cell electrophoresis (comet) assay in peripheral lymphocytes. Plasma level of total antioxidant status (TAS) was determined by using automated measurement method. High sensitive C-reactive protein and other biochemical markers were studied in all subjects. RESULTS: The major increase in lymphocyte DNA damage was observed in patients with grade 4 IMT when compared with other groups (p<0.001, for all). Lymphocyte DNA damage of patients with grade 1 IMT was also lower than patients with grade 2 IMT (p=0.013) and patients with grade 3 IMT (p<0.001). Lymphocyte DNA damage of patients with grade 2 IMT was found at low level compared with patients with grade 3 IMT (p=0.012) as well. In multiple linear regression analysis, IMT was independently correlated with lymphocyte DNA damage (beta=0.515, p<0.001), TAS level (beta=-420, p<0.001), total cholesterol (beta=0.407, p<0.001) and LDL cholesterol level (beta=287, p=0.020). CONCLUSION: Lymphocyte DNA damage may be an independent predictor for the grade of thoracic IMT, and may play a role in pathogenesis of thoracic atherosclerosis besides TAS and cholesterol levels.     

Splenic atrophy in experimental stroke is accompanied by increased regulatory T cells and circulating macrophages

Induction of stroke not only produces local ischemia and brain damage, but also has profound effects on peripheral immune responses. In the current study, we evaluated effects on spleen and blood cells 4 days after stroke induction. Surprisingly, there was a less inflammatory cytokine profile in the middle cerebral artery occlusion-affected right brain hemisphere at 96 h compared with earlier time points. Moreover, our results demonstrate that stroke leads to splenic atrophy characterized by a reduction in organ size, a drastic loss of splenocyte numbers, and induction of annexin V+ and TUNEL+ cells within the spleen that are in the late stages of apoptosis. The consequence of this process was to reduce T cell proliferation responses and secretion of inflammatory cytokines, resulting in a state of profound immunosuppression. These changes produced a drastic reduction in B cell numbers in spleen and blood, and a novel increase in CD4+FoxP3+ regulatory T cells. Moreover, we detected a striking increase in the percentage of nonapoptotic CD11b+ VLA-4-negative macrophages/monocytes in blood. Immunosuppression in response to brain injury may account for the reduction of inflammatory factors in the stroke-affected brain, but also potentially could curtail protective immune responses in the periphery. These findings provide new evidence to support the contention that damage to the brain caused by cerebral ischemia provides a powerful negative signal to the peripheral immune system that ultimately induces a drastic state of immunosuppression caused by cell death as well as an increased presence of CD4+FoxP3+ regulatory T cells.     

Increased urinary protein excretion in the "normal" range is associated with increased renin-angiotensin system activity

Increased levels of albuminuria and proteinuria, both linked to augmented renin-angiotensin system (RAS) activity, are associated with adverse kidney and cardiovascular events. However, the relationship between variations in urinary albumin excretion (UAE) and total protein excretion (UTPE) in the normal range and RAS activity is unclear. We examined the association between UAE and UTPE and the hemodynamic response to angiotensin II (ANG II) challenge, a well-accepted indirect measure of RAS activity, in healthy individuals with normal UAE and UTPE. Forty subjects (15 men, 25 women; age 38 ± 2 yr; UAE, 3.32 ± 0.55 mg/day; UTPE, 56.8 ± 3.6 mg/day) were studied in high-salt balance. Blood pressure (BP), arterial stiffness determined by applanation tonometry, and circulating RAS components were measured at baseline and in response to graded ANG II infusion. The primary outcome was the BP response to ANG II challenge at 30 and 60 min. UAE was associated with a blunted diastolic BP response to ANG II infusion (30 min, P = 0.005; 60 min, P = 0.17), a relationship which remained even after adjustment (30 min, P < 0.001; 60 min, P = 0.035). Similar results were observed with UTPE (30 min, P = 0.031; 60 min, P = 0.001), even after multivariate analysis (30 min, P = 0.008; 60 min, P = 0.001). Neither UAE nor UTPE was associated with systolic BP, circulating RAS components, or arterial stiffness responses to ANG II challenge. Among healthy individuals with UAE and UTPE in the normal range, increased levels of these measures were independently associated with a blunted diastolic BP response to ANG II, indicating increased vascular RAS activity, which is known to be deleterious to both renal and cardiac function.     

Hyperalgesia during naloxone-precipitated withdrawal from morphine is associated with increased on-cell activity in the rostral ventromedial medulla

Hyperresponsiveness to noxious stimulation (hyperalgesia) is observed with naloxone-precipitated morphine withdrawal in several experimental models, and may be due to changes in central nervous system neurons. Previous studies have demonstrated that certain neurons in the rostral ventromedial medulla (on-cells) discharge just prior to nocifensive withdrawal reflexes and are inhibited by morphine. Because the tail flick latency (TFL) is shorter when on-cells are active, it has been proposed that on-cells facilitate nocifensive reflexes. The present study examined the hypothesis that the hyperalgesia observed following naloxone-precipitated withdrawal from morphine is caused by increased on-cell discharge. Rats were maintained in a lightly anesthetized state with chloral hydrate. Administration of saline (1.25 cc, i.v.) or morphine sulfate (1.25 mg/kg, i.v.) was followed by naloxone (1.0 mg/kg, i.v.). On- and off-cell activity was continuously recorded and was correlated with TFL and paw withdrawal threshold (PWT). As previously reported, morphine increased off-cell activity, blocked on-cell activity, and suppressed the tail flick and paw withdrawal reflexes. When naloxone was given after morphine, TFL and PWT were reduced to values significantly below baseline (hyperalgesia). Both spontaneous and reflex-related on-cell activity increased to levels greater than the premorphine baseline. Spontaneous off-cell activity decreased abruptly to near zero when morphine was followed by naloxone. Linear regression analysis during the hyperresponsive state revealed a significant correlation between increased on-cell activity and reduced TFL, but not between decreased off-cell activity and TFL. These findings are consistent with the hypothesis that on-cells facilitate spinal nocifensive reflexes, and that the naloxone-precipitated hyperalgesia is at least in part accounted for by increased on-cell activity. A neural model of opiate dependence, tolerance, and withdrawal is proposed.