N-terminal isotope tagging with propionic anhydride: proteomic analysis of myogenic differentiation of C2C12 cells

N-Terminal isotope tagging (NIT) is an important proteomic tool for quantifying proteins in complex mixtures. Here we describe a modified version of the isotope-coded propionylation procedure of Zhang et al. [Zhang et al., Rapid Commun. Mass Spectom. 16 (2002) 2325], which uses 'light' D0 and 'heavy' D10-propionic anhydride. The method has been extensively modified to improve both the kinetics and overall yield of propionylation. Using albumin as a model protein, the overall variation in quantification yields, calculated using several tryptic peptides, was within +/-10% (S.D. +/-0.2) error. The efficacy of the method is demonstrated by the quantitative differences obtained for vimentin in cell lysates of C2C12 myoblasts upon their myogensis to myotubules.     

Netropsin interactions in the minor groove of d(GGCCAATTGG) studied by a combination of resolution enhancement and ab initio calculations

The structure of the complex between the minor groove binder netropsin and d(GGCCAATTGG) was determined via single-crystal X-ray techniques. The structure was refined to completion using refmac5.1.24, resulting in a residual R-factor of 20.0% (including 68 water molecules). Using crystal engineering and cryocooling techniques, the resolution could be enhanced to 1.75 A, resulting in an unambiguous determination of the drug conformation and orientation. As previously noticed, bifurcated hydrogen bonds are formed between the amide nitrogen atoms of the drug and the N3 and O2 atoms of A and T base pairs, respectively, clearly cataloging the structure to class I. As the bulky NH2 group on guanine was believed to prevent binding of the drug in the minor groove, the detailed nature of several of the amidinium and guanidinium end contacts were further investigated by ab initio quantum chemical methods.     

ATP-phosphopeptide conjugates as inhibitors of Src tyrosine kinases

A number of Src SH2 domain inhibitors enhance the kinase catalytic activity by switching the closed inactive to the open active conformation. ATP-phosphopeptide conjugates were designed and synthesized as Src tyrosine kinase inhibitors based on a tetrapeptide sequence pTyr-Glu-Glu-Ile (pYEEI) and ATP to block the SH2 domain signaling and substrate phosphorylation by ATP, respectively. In general, ATP-phosphopeptide conjugates with optimal linkers such as compounds 5 and 7 (K(i) = 1.7-2.6 microM) showed higher binding affinities to the ATP-binding site relative to the other ATP-phosphopeptide conjugates having short or long linkers, 1-4 and 6, (K(i) = 10.1-16.1 microM) and ATP (K(m) = 74 microM). These ATP-phosphopeptide conjugates may serve as novel templates for designing protein tyrosine kinase inhibitors to block SH2 mediated protein-protein interactions and to counter the activation of enzyme that resulted from the SH2 inhibition.     

Construction of a vector generating both siRNA and a fluorescent reporter: a siRNA study in cultured neurons

RNA interference is an important tool for gene silencing. However, its application to primary cultured cells has been limited by low transfection efficiencies. In this work we developed a vector which encodes both siRNA and red fluorescent protein. Using this vector we could markedly suppress green fluorescent protein (GFP) and bim an endogenous gene. Primary cultured cortical neurons transfected with siRNA against doublecortin showed that doublecortin expression was significantly inhibited in nearly all the transfected neurons. This vector identifies the transfected cells and should be useful for loss-of-gene function studies in neurons.     

Acid and base secretion in the Calu-3 model of human serous cells

Submucosal glands are the primary source of airway mucus, a critical component of lung innate defenses. Airway glands are defective in cystic fibrosis (CF), showing a complete absence of secretion to vasoactive intestinal peptide or forskolin, which increase intracellular cAMP concentration. This defect is attributed to gland serous cells, which express the cystic fibrosis transmembrane conductance regulator. Calu-3 cells, which mimic many features of serous cells, secrete Cl(-) and HCO(3)(-), with HCO(3)(-) secretion predominating for forskolin stimulation and Cl(-) secretion predominating for stimuli that open basolateral K(+) channels to hyperpolarize the cells. We used pH stat and ion substitution experiments to clarify the mechanisms and consequences of these two modes of secretion. We confirm that Calu-3 cells secrete primarily HCO(3)(-) in response to forskolin. Unexpectedly, HCO(3)(-) secretion continued in response to K(+) channel openers, with Cl(-) secretion being added to it. Secretion of HCO(3)(-) from hyperpolarized cells occurs via the conversion of CO(2) to HCO(3)(-) and is reduced by approximately 50% with acetazolamide. A gap between the base equivalent current and short-circuit current was observed in all experiments and was traced to secretion of H(+) via a ouabain-sensitive, K(+)-dependent process (possibly H(+)-K(+)-ATPase), which partially neutralized the secreted HCO(3)(-). The conjoint secretion of HCO(3)(-) and H(+) may help explain the puzzling finding that mucus secreted from normal and CF glands has the same acidic pH as does mucus from glands stimulated with forskolin or ACh. It may also help explain how human airway glands produce mucus that is hypotonic.     

Occurrence of scuticociliatosis in olive flounder Paralichthys olivaceus by Phiasterides dicentrarchi (Ciliophora: scuticociliatida)

In the course of identifying scuticociliates recently obtained from systemically infected olive flounder Paralichthys olivaceus in Korea, we found a scuticociliate species whose small subunit ribosomal RNA (SS rRNA) gene was not amplified by species-specific primers previously designed for Uronema marinum and Pseudocohnilembus persalinus. By studying morphological characteristics of wet-mounted and stained specimens, we identified the species as Philasterides dicentrarchi, which has been reported to cause systemic infection in the European sea bass Dicentrarchus labrax and turbot Scophthalmus maximus. In this study, we compared morphological characteristics of our specimens with previously reported Philasterides species, including P. dicentrarchi, and sequenced the SS rRNA gene in order to design P. dicentrarchi specific primers. This is the first report on scuticociliatosis caused by P. dicentrarchi from marine fish in Asia.     

Changes in the expression of calbindin D-28k in the gerbil hippocampus following seizure

Previous studies have reported that calbindin D-28k (CB), a calcium-binding protein, containing neurons in the hippocampus play an important role in hippocampal excitability in epilepsy, because CB modulates the free calcium ion during seizure. Hence, in the present study, we investigated changes of CB expression in the hippocampus and its association in the Mongolian gerbil to identify roles of CB in epileptogenesis. CB immunoreactivity in the hippocampus was significantly lower in the pre-seizure group of seizure sensitive (SS) gerbils as compared with those seen in the seizure resistant (SR) gerbils. The distribution of CB immunoreactivity in the hippocampus showed significant difference after seizure on-set in SS gerbils. CB immunoreactivity in the hippocampal CA1, CA2 areas, and subiculum was lowest at 3h after seizure on-set; thereafter, the immunoreactivity became to increase to 12h after seizure on-set. Mossy fibers, Schaffer collaterals and dentate granule cells showed the highest CB immunoreactivity at 3h after seizure on-set; thereafter, the immunoreactivity became to decrease. In the case of the intrinsic and output connections of the hippocampus, a rapid decrease of CB serves an inhibitory function, which regulates the seizure activity and output signals from the hippocampus.