Isolation and1H-NMR spectroscopic identification of the glucose-containing lipid-linked precursor oligosaccharide ofN-linked carbohydrate chains

The lipid-linked precursor ofN-type glycoprotein oligosaccharides was isolated from porcine thyroid microsomes after in cubation with UDP[³H] Glucose. The carbohydrate was released from dolichol pyrophosphate by mild acid hydrolysis, purified by gel filtration and characterized by 500-MHz¹H-NMR spectroscopy in combination with enzymatic degradation. The parent oligosaccharide was found to be Glc₃Man₉Glc-NAc₂. The three glucose residues are present in the linear sequence Glcα1-2Glα1-3 Glc, the latter being α(1-3)-linked to one of the mannose residues. In order to establish the branch location of the triglucosyl unit, the parent compound was digested with jack-bean α-mannosidase. The oligosaccharide product was purified by gel filtration, and identified by¹H-NMR as Glc₃Man₅GlcNAc₂ lacking the mannose residues A, D₂, B and D₃. Therefore, the structure of the precursor oligosaccharide is as follows: $$\begin{gathered} c b a D_1 C 4 \hfill \\ Glc\alpha 1 - 2Glc\alpha 1 - 3Glc\alpha 1 - 3Man\alpha 1 - 2Man\alpha 1 - 2Man\alpha 1 \hfill \\ 3 \swarrow 3 2 1 \hfill \\ Man\alpha 1 - 2Man\alpha 1 Man\beta 1 - 4GlcNAc\beta 1 - 4GlcNAc \hfill \\ D_{2 } A 3 6 \hfill \\ Man\alpha 1 \hfill \\ 6 \hfill \\ Man\alpha 1 - 2Man\alpha 1 \nwarrow 4 \hfill \\ D_3 B \hfill \\ \end{gathered} $$      

Myosin regulatory light chain modulates the Ca2+ dependence of the kinetics of tension development in skeletal muscle fibers.

To determine the role of myosin regulatory light chain (RLC) in modulating contraction in skeletal muscle, we examined the rate of tension development in bundles of skinned skeletal muscle fibers as a function of the level of Ca(2+) activation after UV flash-induced release of Ca(2+) from the photosensitive Ca(2+) chelator DM-nitrophen. In control fiber bundles, the rate of tension development was highly dependent on the concentration of activator Ca(2+) after the flash. There was a greater than twofold increase in the rate of tension development when the post-flash [Ca(2+)] was increased from the lowest level tested (which produced a steady tension that was 42% of maximum tension) to the highest level (producing 97% of maximum tension). However, when 40-70% of endogenous myosin RLC was extracted from the fiber bundles, tension developed at the maximum rate, regardless of the post-flash concentration of Ca(2+). Thus, the Ca(2+) dependence of the rate of tension development was eliminated by partial extraction of myosin RLC, an effect that was partially reversed by recombination of RLC back into the fiber bundles. The elimination of the Ca(2+) dependence of the kinetics of tension development was specific to the extraction of RLC rather than an artifact of the co-extraction of both RLC and Troponin C, because the rate of tension development was still Ca(2+) dependent, even when nearly 50% of endogenous Troponin C was extracted from fiber bundles fully replete with RLC. Thus, myosin RLC appears to be a key component in modulating Ca(2+) sensitive cross-bridge transitions that limit the rate of force development after photorelease of Ca(2+) in skeletal muscle fibers.     

Effects of endurance exercise on isomyosin patterns in fast- and slow-twitch skeletal muscles

Although endurance training has been shown to profoundly affect the oxidative capacity of skeletal muscle, little information is available concerning the impact of endurance training on skeletal muscle isomyosin expression across a variety of muscle fiber types. Therefore, a 10-wk running program (1 h/day, 5 days/wk, 20% grade, 1 mile/h) was conducted to ascertain the effects of endurance training on isomyosin expression in the soleus, vastus intermedius (VI), plantaris (PLAN), red and white medial gastrocnemius (RMG and WMG), and red and white vastus lateralis muscles (RVL and WVL). Evidences of training were noted by the presence of a resting and a submaximal exercise bradycardia, as well as an enhancement in peak O2 consumption in the trained rodents relative to the nontrained controls. No evidence for skeletal muscle hypertrophy was observed subsequent to training when muscle weight was normalized to body weight. Shifts in the isomyosin profile of the trained VI, RMG, RVL, and PLAN were seen relative to the nontrained controls. Specifically, training affected the slow myosin (SM) composition of the VI by decreasing the relative content of the SM2 isoform by 14% while increasing that of the SM1 isoform (P less than 0.05). In addition, training elicited various degrees of a fast to slower myosin transformation in the RMG, RVL, and PLAN. All three muscles showed a significant reduction in the fast myosin 2 isoform (P less than 0.05), with significant increases in intermediate myosin in the RVL and PLAN along with elevations in SM2 in the RMG and PLAN (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Microarray expression analysis of effects of exercise training: increase in atrial MLC-1 in rat ventricles

Previous studies have shown that endurance exercise training increases myocardial contractility. We have previously described training-induced alterations in myocardial contractile function at the cellular level, including an increase in the Ca(2+) sensitivity of tension. To determine the molecular mechanism(s) of these changes, oligonucleotide microarrays were used to analyze the gene expression profile in ventricles from endurance-trained rats. We used an 11-wk treadmill training protocol that we have previously shown results in increased contractility in cardiac myocytes. After the training, the hearts were removed and RNA was isolated from the ventricles of nine trained and nine control rats. With the use of an Affymetrix Rat Genome U34A Array, we detected altered expression of 27 genes. Several genes previously found to have increased expression in hypertrophied myocardium, such as atrial natriuretic factor and skeletal alpha-actin, were decreased with training in this study. From the standpoint of altered contractile performance, the most significant finding was an increase in the expression of atrial myosin light chain 1 (aMLC-1) in the trained ventricular tissue. We confirmed microarray results for aMLC-1 using RT-PCR and also confirmed a training-induced increase in aMLC-1 protein using two-dimensional gel electrophoresis. aMLC-1 content has been previously shown to be increased in human cardiac hypertrophy and has been associated with increased Ca(2+) sensitivity of tension and increased power output. These results suggest that increased expression of aMLC-1 in response to training may be responsible, at least in part, for previously observed training-induced enhancement of contractile function.     

Altered expression of skeletal muscle myosin isoforms in cancer cachexia

Cachexia is commonly seen in cancer and ischaracterized by severe muscle wasting, but little is known about theeffect of cancer cachexia on expression of contractile protein isoforms such as myosin. Other causes of muscle atrophy shift expression ofmyosin isoforms toward increased fast (type II) isoform expression. Weinjected mice with murine C-26 adenocarcinoma cells, a tumor cell linethat has been shown to cause muscle wasting. Mice were killed 21 daysafter tumor injection, and hindlimb muscles were removed. Myosin heavychain (MHC) and myosin light chain (MLC) content was determined inmuscle homogenates by SDS-PAGE. Body weight was significantly lower intumor-bearing (T) mice. There was a significant decrease in muscle massin all three muscles tested compared with control, with the largestdecrease occurring in the soleus. Although no type IIb MHC was detectedin the soleus samples from control mice, type IIb comprised 19% of thetotal MHC in T soleus. Type I MHC was significantly decreased in T vs. control soleus muscle. MHC isoform content was not significantly different from control in plantaris and gastrocnemius muscles. Thesedata are the first to show a change in myosin isoform expression accompanying muscle atrophy during cancer cachexia.

     

Increased expression of costimulatory markers CD134 and CD80 on interleukin-17 producing T cells in patients with systemic lupus erythematosus

Introduction  

There is growing evidence that interleukin 17 (IL-17) producing T cells are involved in the pathogenesis of systemic lupus erythematosus (SLE). Previous studies showed that increased percentages of T-cell subsets expressing the costimulatory molecules CD80 and CD134 are associated with disease activity and renal involvement in SLE. The aim of this study was to investigate the distribution and phenotypical characteristics of IL-17 producing T-cells in SLE, in particular in patients with lupus nephritis, with emphasis on the expression of CD80 and CD134.     

Baseline predictors of response and discontinuation of tumor necrosis factor-alpha blocking therapy in ankylosing spondylitis: a prospective longitudinal observational cohort study

Introduction  

Identifying ankylosing spondylitis (AS) patients who are likely to benefit from tumor necrosis factor-alpha (TNF-α) blocking therapy is important, especially in view of the costs and potential side effects of these agents. Recently, the AS Disease Activity Score (ASDAS) has been developed to assess both subjective and objective aspects of AS disease activity. However, data about the predictive value of the ASDAS with respect to clinical response to TNF-α blocking therapy are lacking. The aim of the present study was to identify baseline predictors of response and discontinuation of TNF-α blocking therapy in AS patients in daily clinical practice.     

Advanced glycation endproducts are increased in rheumatoid arthritis patients with controlled disease

Introduction  

Advanced glycation end products (AGEs) are produced and can accumulate during chronic inflammation, as might be present in patients with rheumatoid arthritis (RA). AGEs are involved in the development of cardiovascular disease. The aim of this study is to evaluate whether AGEs are increased in patients with long-standing RA and whether AGE accumulation is related to disease activity, disease severity and measures of (premature) atherosclerosis, such as endothelial activation, endothelial dysfunction and intima media thickness (IMT).