Involvement of mu- and m-calpains and protein kinase C isoforms in L8 myoblast differentiation

The objectives were to investigate the roles of different calpains and protein kinase C (PKC) isoforms in muscle differentiation. Concentrations of mu- and m-calpain increased significantly whereas PKCalpha and delta declined significantly during L8 myoblast differentiation. Both mu-calpain and m-calpain antisense oligonucleotides inhibited myotube formation and creatine kinase activity during L8 myoblast differentiation. These results implied that both mu- and m-calpain were involved in L8 myoblast differentiation. To investigate the involvement of calpain in regulation of PKC concentrations, mu-calpain antisense oligonucleotides were added to L8 myoblasts. PKCalpha remained unchanged and PKCdelta declined. By adding m-calpain antisense oligonucleotides instead, PKCalpha level remained unchanged and PKCdelta concentrations increased significantly during differentiation. These results suggest that PKCalpha, but not PKCdelta, is the substrate for mu-calpain and PKCalpha and delta are the substrates for the m-calpain. In addition, more phosphorylated myogenin was found in day 2 antisense oligonucleotides treated L8 cells. It is concluded that the decline of PKCalpha mediated by m- and mu-calpain is essential for L8 myoblast differentiation. The decline of PKC during myoblast differentiation may cause hypo-phosphorylation of myogenin, which in turn activates muscle-specific genes during myogenesis.     

Crystal structure of a SEA variant in complex with MHC class II reveals the ability of SEA to crosslink MHC molecules

Although the biological properties of staphylococcal enterotoxin A (SEA) have been well characterized, structural insights into the interaction between SEA and major histocompatibilty complex (MHC) class II have only been obtained by modeling. Here, the crystal structure of the D227A variant of SEA in complex with human MHC class II has been determined by X-ray crystallography. SEA(D227A) exclusively binds with its N-terminal domain to the alpha chain of HLA-DR1. The ability of one SEA molecule to crosslink two MHC molecules was modeled. It shows that this SEA molecule cannot interact with the T cell receptor (TCR) while a second SEA molecule interacts with MHC. Because of its relatively low toxicity, the D227A variant of SEA is used in tumor therapy.     

Construction of a reporter plasmid for screening in vivo promoter activity in Francisella tularensis

Francisella tularensis is a facultative intracellular bacterium that survives and multiplies inside macrophages. Here we constructed a new promoter probe plasmid denoted pKK214 by introduction of a promoter-less chloramphenicol acetyltransferase (cat) gene into the shuttle vector pKK202. A promoter library was created in F. tularensis strain LVS by cloning random chromosomal DNA fragments into pKK214. Approximately 15% of the recombinant bacteria showed chloramphenicol resistance in vitro. The promoter library was also used to infect macrophages in the presence of chloramphenicol and after two cycles of infection the library contained essentially only chloramphenicol resistance clones which shows that pKK214 can be used to monitor F. tularensis genes that are expressed during infection.     

Subharmonic microbubble emissions for noninvasively tracking right ventricular pressures

Right heart catheterization is often required to monitor intra-cardiac pressures in a number of disease states. Ultrasound contrast agents can produce pressure modulated subharmonic emissions that may be used to estimate right ventricular (RV) pressures. A technique based on subharmonic acoustic emissions from ultrasound contrast agents to track RV pressures noninvasively has been developed and its clinical potential evaluated. The subharmonic signals were obtained from the aorta, RV, and right atrium (RA) of five anesthetized closed-chest mongrel dogs using a SonixRP ultrasound scanner and PA4-2 phased array. Simultaneous pressure measurements were obtained using a 5-French solid state micromanometer tipped catheter. Initially, aortic subharmonic signals and systemic blood pressures were used to obtain a calibration factor in units of millimeters of mercury per decibel. This factor was combined with RA pressures (that can be obtained noninvasively) and the acoustic data from the RV to obtain RV pressure values. The individual calibration factors ranged from -2.0 to -4.0 mmHg/dB. The subharmonic signals tracked transient changes in the RV pressures within an error of 0.6 mmHg. Relative to the catheter pressures, the mean errors in estimating RV peak systolic and minimum diastolic pressures, and RV relaxation [isovolumic negative derivative of change in pressure over time (-dP/dt)] by use of the subharmonic signals, were -2.3 mmHg, -0.8 mmHg, and 2.9 mmHg/s, respectively. Overall, acoustic estimates of RV peak systolic and minimum diastolic pressures and RV relaxation were within 3.4 mmHg, 1.8 mmHg, and 5.9 mmHg/s, respectively, of the measured pressures. This pilot study demonstrates that subharmonic emissions from ultrasound contrast agents have the potential to noninvasively track in vivo RV pressures with errors below 3.5 mmHg.     

Undersulfation of heparan sulfate restricts differentiation potential of mouse embryonic stem cells

Heparan sulfate proteoglycans, present on cell surfaces and in the extracellular matrix, interact with growth factors and morphogens to influence growth and differentiation of cells. The sulfation pattern of the heparan sulfate chains formed during biosynthesis in the Golgi compartment will determine the interaction potential of the proteoglycan. The glucosaminyl N-deacetylase/N-sulfotransferase (NDST) enzymes have a key role during biosynthesis, greatly influencing total sulfation of the heparan sulfate chains. The differentiation potential of mouse embryonic stem cells lacking both NDST1 and NDST2 was studied using in vitro differentiation protocols, expression of differentiation markers, and assessment of the ability of the cells to respond to growth factors. The results show that NDST1 and NDST2 are dispensable for mesodermal differentiation into osteoblasts but necessary for induction of adipocytes and neural cells. Gene expression analysis suggested a differentiation block at the primitive ectoderm stage. Also, GATA4, a primitive endoderm marker, was expressed by these cells. The addition of FGF4 or FGF2 together with heparin rescued the differentiation potential to neural progenitors and further to mature neurons and glia. Our results suggest that the embryonic stem cells lacking both NDST1 and NDST2, expressing a very low sulfated heparan sulfate, can take the initial step toward differentiation into all three germ layers. Except for their potential for mesodermal differentiation into osteoblasts, the cells are then arrested in a primitive ectoderm and/or endoderm stage.     

The BUME method: a novel automated chloroform-free 96-well total lipid extraction method for blood plasma

Lipid extraction from biological samples is a critical and often tedious preanalytical step in lipid research. Primarily on the basis of automation criteria, we have developed the BUME method, a novel chloroform-free total lipid extraction method for blood plasma compatible with standard 96-well robots. In only 60 min, 96 samples can be automatically extracted with lipid profiles of commonly analyzed lipid classes almost identically and with absolute recoveries similar or better to what is obtained using the chloroform-based reference method. Lipid recoveries were linear from 10-100 μl plasma for all investigated lipids using the developed extraction protocol. The BUME protocol includes an initial one-phase extraction of plasma into 300 μl butanol:methanol (BUME) mixture (3:1) followed by two-phase extraction into 300 μl heptane:ethyl acetate (3:1) using 300 μl 1% acetic acid as buffer. The lipids investigated included the most abundant plasma lipid classes (e.g., cholesterol ester, free cholesterol, triacylglycerol, phosphatidylcholine, and sphingomyelin) as well as less abundant but biologically important lipid classes, including ceramide, diacylglycerol, and lyso-phospholipids. This novel method has been successfully implemented in our laboratory and is now used daily. We conclude that the fully automated, high-throughput BUME method can replace chloroform-based methods, saving both human and environmental resources.     

The effect of prolyl oligopeptidase inhibition on extracellular acetylcholine and dopamine levels in the rat striatum

Prolyl oligopeptidase (PREP, EC 3.4.21.26) inhibitors have potential as cognition enhancers, but the mechanism of action behind the cognitive effects remains unclear. Since acetylcholine (ACh) and dopamine (DA) are known to be associated with the regulation of cognitive processes, we investigated the effects of two PREP inhibitors on the extracellular levels of ACh and DA in the rat striatum using in vivo microdialysis. KYP-2047 and JTP-4819 were administered either as a single systemic dose (50 μmol/kg～17 mg/kg i.p.) or directly into the striatum by retrodialysis via the microdialysis probe (12.5, 37.5 or 125 μM at 1.5 μl/min for 60 min). PREP inhibitors had no significant effect on striatal DA levels after systemic administration. JTP-4819 significantly decreased ACh levels both after systemic (by ～25%) and intrastriatal (by ～30-50%) administration. KYP-2047 decreased ACh levels only after intrastriatal administration by retrodialysis (by ～40-50%) when higher drug levels were reached, indicating that higher brain drug levels are needed to modulate ACh levels than to inhibit PREP. This result does not support the earlier hypothesis that the positive cognitive effects of PREP inhibitors in rodents would be mediated through the cholinergic system. In vitro specificity studies did not reveal any obvious off-targets that could explain the observed effect of KYP-2047 and JTP-4819 on ACh levels, instead confirming the concept that these compounds have a high selectivity towards PREP.     

The use of PIB-PET as a dual pathological and functional biomarker in AD

Amyloid imaging with positron emission tomography (PET) is presently used in Alzheimer's disease (AD) research. In this study we investigated the possibility to use early frames (ePIB) of the PIB scans as a rough index of CBF by comparing normalised early PIB values with cerebral glucose metabolism (rCMRglc). PIB-PET and FDG-PET were performed in 37 AD patients, 21 subjects with mild cognitive impairment (MCI) and 6 healthy controls (HC). The patients were divided based on their PIB retention (amyloid load) as either PIB positive (PIB+) or PIB negative (PIB−). Data of the unidirectional influx K₁ from a subset of the subjects including 7 AD patients and 3 HC was used for correlative analysis. Data was analysed using regions of interest (ROI) analysis. A strong, positive correlation was observed across brain regions between K₁ and ePIB (r = 0.70; p ≤ 0.001). The ePIB values were significantly lower in the posterior cingulate (p ≤ 0.001) and the parietal cortices (p = 0.002) in PIB+ subjects compared to PIB−, although the group difference were stronger for rCMRglc in cortical areas (p ≤ 0.001). Strong positive correlations between ePIB and rCMRglc were observed in all cortical regions analysed, especially in the posterior cingulate and parietal cortices (p ≤ 0.001). A single dynamic PIB-PET scan may provide information about pathological and functional changes (amyloidosis and impaired blood flow). This might be important for diagnosis of AD, enrichment of patients in clinical trials and evaluation of treatment effects. This article is part of a Special Issue entitled: Imaging Brain Aging and Neurodegenerative disease.     

Carnosine treatment largely prevents alterations of renal carnosine metabolism in diabetic mice

Recently, we identified an allelic variant of human carnosinase 1 (CN1) that results in increased enzyme activity and is associated with susceptibility for diabetic nephropathy in humans. Investigations in diabetic (db/db) mice showed that carnosine ameliorates glucose metabolism effectively. We now investigated the renal carnosinase metabolism in db/db mice. Kidney CN1 activity increased with age and was significantly higher in diabetic mice compared to controls. Increased CN1 activity did not affect renal carnosine levels, but anserine concentrations were tenfold lower in db/db mice compared to controls (0.24±0.2 vs. 2.28±0.3 nmol/mg protein in controls; p<0.001). Homocarnosine concentrations in kidney tissue were low in both control and db/db mice (below 0.1 nmol/mg protein, p=n.s.). Carnosine treatment for 4 weeks substantially decreased renal CN1 activity in diabetic mice (0.32±0.3 in non-treated db/db vs. 0.05±0.05 μmol/mg/h in treated db/db mice; p<0.01) close to normal activities. Renal anserine concentrations increased significantly (0.24±0.2 in non-treated db/db vs. 5.7±1.2 μmol/mg/h in treated db/db mice; p<0.01), while carnosine concentrations remained unaltered (53±6.4 in non-treated vs. 61±15 nmol/mg protein in treated db/db mice; p=n.s.). Further, carnosine treatment halved proteinuria and reduced vascular permeability to one-fifth in db/db mice. In renal tissue of diabetic mice carnosinase activity is significantly increased and anserine concentrations are significantly reduced compared to controls. Carnosine treatment largely prevents the alterations of renal carnosine metabolism.