Effects of N(G)-monomethyl-L-arginine on Ca(2+) current and nitric-oxide synthase in rat ventricular myocytes

The effects of N(G)-monomethyl-L-arginine (L-NMMA), a nitric-oxide synthase (NOS) inhibitor, on the L-type Ca(2+) current (ICa) and NO effects on NOS were determined in rat ventricular myocytes. L-NMMA (10 and 100 microM) had no significant effect on basal ICa, but in a cAMP-stimulated condition due to forskolin (1 microM) or milrinone (10 microM), a cGMP-inhibited cAMP-phosphodiesterase (PDE), L-NMMA (10 and 100 microM) concentration dependently augmented ICa. The enhancing effects of L-NMMA (10 and 100 microM) on ICa were not seen in the presence of either a nonselective inhibitor of PDE, 3-isobutyl-1-methylxanthine (20 microM), resulting in a stimulated ICa condition or a cGMP-dependent protein kinase activator, 8-bromo-cGMP (200 microM). 8-Bromo-cGMP (200 microM) inhibited 100 microM L-NMMA-induced ICa increase in the simultaneous application of forskolin (1 microM). Acetylcholine (ACh; 1 and 3 microM) inhibited 1 microM forskolin-stimulated ICa in a concentration-dependent manner, but this inhibitory action of ACh was significantly attenuated by the additional application of L-NMMA (100 microM). In the continuing presence of both L-NMMA (100 microM) and forskolin (1 microM), ACh (6 microM) had no inhibitory effect on ICa. In another series of experiments with isolated ventricular myocytes, we obtained both the positive staining of NADPH-diaphorase activity and the expression of the endothelial isoform of NOS. These data suggest that the effect of L-NMMA on ICa in a cAMP-stimulated condition with or without cholinergic inhibition is due to inhibition (acute effects) of a cGMP-stimulated cAMP-PDE via inhibition of the endothelial isoform of NOS.     

Endoscopic therapy for Barrett's esophagus

There are many foreign reports about the endoscopic ablation therapy for Barrett's esophagus. Endoscopic ablation therapy include thermal therapy (electrocoagulation, laser etc.), photodynamic therapy or endoscopic resection and so on. Ablation of Barrett's esophagus by these therapy in combination with adequate acid suppression lead to mucosal replacement by squamous epithelium. But the true value of these endoscopic therapy has not been fully investigated. Further studies are required.     

Dramatic improvement in Down syndrome-associated cognitive impairment with donepezil

OBJECTIVE: To report 2 cases of patients with Down syndrome and severe cognitive impairment who gained dramatic improvements in quality of life (QOL) upon donepezil treatment. CASE SUMMARIES: Case 1. A 38-year-old woman with Down syndrome, diagnosed with secondary progressive dementia when her mental state had deteriorated rapidly after graduation from junior high school, started donepezil treatment. The loading dose was 3 mg/day and was increased to 5 mg/day for maintenance. One month after the dose was increased, adverse effects such as soft stool and urinary incontinence appeared. These adverse effects disappeared when the dose was decreased again to 3 mg/day. Her QOL improved dramatically with this minimal dose. She recovered verbal and written communication skills that she had lost for the past 21 years. Case 2. A 22-year-old man with Down syndrome, who had been diagnosed as having severe mental retardation, was put on donepezil therapy. Both loading and maintenance doses were 3 mg/day. His QOL had also dramatically improved, with some recovery in verbal communication. Transient agitation/violence and transient muscle weakness appeared during the first few months of treatment. DISCUSSION: Patients with Down syndrome may be more sensitive to donepezil therapy than others and may benefit from this medicine, although they may also have adverse effects more frequently. CONCLUSIONS: Donepezil may be a useful medicine for some patients with Down syndrome with severe cognitive impairment or mental retardation if the adverse effects are manageable.     

Clinical outline of Barrett's esophagus

Barrett's esophagus is one of the important gastrointestinal disease in Europe and the United States. It was recognized as not only complication of reflux esophagitis, but also pre-malignant lesion of adenocarcinoma. Recently, realization of Barrett's esophagus is attentioned in Japan, because increasing of reflux esophagitis for westernization of eating habit, living habit and aging of the population. In this section, we present general consideration and clinical research of Barrett's esophagus. We expect Barrett's esophagus will gradually increase near the future and need to research abundantly about controversial point of Barrett's esophagus. We also think it is important to accumulate intensively the clinical data of Barrett's esophagus patients.     

Expression of Recombination Activating Genes in Germinal Center B Cells: Involvement of Interleukin 7 (IL-7) and the IL-7 Receptor

Mouse germinal center (GC) B cells have been shown to undergo secondary V(D)J (V, variable; D, diversity; J, joining) recombination (receptor editing) mediated by the reexpressed products of recombination activating gene (RAG)-1 and RAG-2. We show here that interleukin (IL)-7 as well as IL-4 was effective in inducing functional RAG products in mouse IgD⁺ B cells activated via CD40 in vitro. Blocking of the IL-7 receptor (IL-7R) by injecting an anti– IL-7R monoclonal antibody resulted in a marked suppression of the reexpression of RAG-2 and subsequent V(D)J recombination in the draining lymph node of immunized mice, whereas RAG-2 expression was not impaired in immunized IL-4–deficient mice. Further, these peripheral B cells activated in vitro or in vivo were found to express IL-7R. These findings indicate a novel role for IL-7 and IL-7R in inducing receptor editing in GC B cells.     

Metabolic changes in early childhood using LCModel with corrected water scaling method

Purpose:

To examine metabolic changes of the brain in early infancy measured by the LCModel with the water scaling method (LCModel‐WS), and to determine whether the unsuppressed water signal (UWS) on the MR console and the area of the unsuppressed water peak (AUW) in the LCModel can be used to correct metabolite concentrations.

Materials and Methods:

MR spectroscopy was performed on a 1.5 Tesla MR scanner. To determine whether UWS and AUW increases linearly with PD and exp(‐TE/T2), these values were measured using three phantoms with different PD and T2 values. UWS and AUW were also measured (PRESS, TR = 5000 ms, TE = 30 ms, VOI = 4.5 mL) in 57 pediatric controls (aged 2 weeks to 15 years).

Results:

Phantom studies revealed UWS and AUW increases linearly with PD and exp(‐TE/T2). UWS and AUW were high in controls younger than 2 years of age, but gradually decreased to become almost constant after 4 years (UWS = 504 × 10³, AUW = 2.05 × 10⁷). AUW was linearly proportional to UWS in controls. These indicated that metabolite concentrations should be multiplied by the ratio of UWS/504 × 10³ or AUW/2.05 × 10⁷. Age dependent metabolite concentrations corrected by the ratio were obtained.

Conclusion:

Both UWS and AUW can be used to correct metabolite concentrations; these corrections can significantly improve quantification of metabolites' concentration in early childhood. J. Magn. Reson. Imaging 2012;35:174‐180. © 2011 Wiley Periodicals, Inc.     

Molecular cloning and nucleotide sequence of complementary DNAs encoding human short chain acyl-coenzyme A dehydrogenase and the study of the molecular basis of human short chain acyl-coenzyme A dehydrogenase deficiency.

Complementary DNAs encoding the precursor of human placental short chain acyl-coenzyme A (CoA) dehydrogenase (SCAD) (EC 1.3.99.2) were cloned and sequenced. The cDNA inserts in these clones were 1,852 bases in length combined, and encoded the entire 412-amino acid precursor SCAD (mol wt 44,303). This sequence included the 24-amino acid leader peptide moiety (mol wt 2,576) and 388 amino acids corresponding to the mature protein (mol wt 41,727). The comparison of SCAD and medium chain acyl-CoA dehydrogenase sequences revealed a high degree of homology, suggesting that these enzymes evolved from a common ancestral gene and belong to a gene family. We also studied mutant human SCAD in cultured skin fibroblasts from three patients with hereditary SCAD deficiency. Labeling fibroblast cultures with [35S]-methionine followed by immunoprecipitation with anti-SCAD antibody revealed that a normal size variant SCAD protein was synthesized. In all of the three SCAD-deficient cell lines, the size of variant SCAD mRNA as determined by Northern blotting using one of the normal SCAD cDNA as a probe was also normal, and no difference was observed on Southern blots in the restriction patterns of mutant genomic DNA using EcoRI, TaqI, HincII, and BamHI. These results suggest that the defects in SCAD in these cell lines are caused by a point mutation.     

Changing Blue Fluorescent Protein to Green Fluorescent Protein Using Chemical RNA Editing as a Novel Strategy in Genetic Restoration

Using the transition from cytosine of BFP (blue fluorescent protein) gene to uridine of GFP (green fluorescent protein) gene at position 199 as a model, we successfully controlled photochemical RNA editing to effect site‐directed deamination of cytidine (C) to uridine (U). Oligodeoxynucleotides (ODNs) containing 5′‐carboxyvinyl‐2′‐deoxyuridine (^CVU) were used for reversible photoligation, and single‐stranded 100‐nt BFP DNA and in vitro‐transcribed full‐length BFP mRNA were the targets. Photo‐cross‐linking with the responsive ODNs was performed using UV (366 nm) irradiation, which was followed by heat treatment, and the cross‐linked nucleotide was cleaved through photosplitting (UV, 312 nm). The products were analyzed using restriction fragment length polymorphism (RFLP) and fluorescence measurements. Western blotting and fluorescence‐analysis results revealed that in vitro‐translated proteins were synthesized from mRNAs after site‐directed RNA editing. We detected substantial amounts of the target‐base‐substituted fragment using RFLP and observed highly reproducible spectra of the transition‐GFP signal using fluorescence spectroscopy, which indicated protein stability. ODNc restored approximately 10% of the C‐to‐U transition. Thus, we successfully used non‐enzymatic site‐directed deamination for genetic restoration in vitro. In the near future, in vivo studies that include cultured cells and model animals will be conducted to treat genetic disorders.