Cleavage of Disulfide Bonds of Wheat Glutenin by Mercuric Chloride

The dissociation of wheat glutenin into subunits was observed by treatment with a small amount of mercuric chloride under moderate conditions, suggesting that the cleavage of inter-polypeptide chain disulfide bonds in the glutenin might occur. The dissociation into the subunits was examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The electrophoretic patterns of the glutenin treated with mercuric chloride were essentially similar to those of the glutenin treated with 2-mercaptoethanol. Silver nitrate also had the same effects as mercuric chloride, and p-chloromercuribenzoate and N-ethylmaleimide showed no effect on the dissociation of the glutenin. Complete dissociation was achieved when the glutenin solution containing 0.5% SDS and 0.01 m phosphate buffer (pH 7.0) was incubated with 10^?3 m mercuric chloride (about four moles per mole of disulfide groups) at 30°C for 20 hr. Partial dissociation was also observed after 30 min incubation. Increasing temperature and SDS concentration promoted the rate of the dissociation of the glutenin by mercuric chloride.     

Molecular Weight Determination of Component Polypeptides of Glutenin after Fractionation by Gel Filtration

Reduced and cyanoethylated glutenin was fractionated into three fractions (F I, F II and F III) by gel filtration on Sephadex G–100 in 0.1 m acetic acid. The molecular weight determination was made with these three fractions by sedimentation equilibrium in 6.5 m guanidine hydrochloride containing 0.01 m acetic acid. The molecular weight obtained was 44,000 for F II, and 32,000 for F III. F I showed a distribution of molecular weight due to the aggregation. The average molecular weight of F I was 52,000, being 27,000 at the meniscus and 98,000 at the bottom. The estimation of molecular weight by SDS–PAGE* gave overestimated values for glutenin polypeptides, as was already reported for gliadin.     

Personalized medicine and proteomics: lessons from non-small cell lung cancer

Personalized medicine allows the selection of treatments best suited to an individual patient and disease phenotype. To implement personalized medicine, effective tests predictive of response to treatment or susceptibility to adverse events are needed, and to develop a personalized medicine test, both high quality samples and reliable data are required. We review key features of state-of-the-art proteomic profiling and introduce further analytic developments to build a proteomic toolkit for use in personalized medicine approaches. The combination of novel analytical approaches in proteomic data generation, alignment and comparison permit translation of identified biomarkers into practical assays. We further propose an expanded statistical analysis to understand the sources of variability between individuals in terms of both protein expression and clinical variables and utilize this understanding in a predictive test.     

alpha-Synuclein affects the MAPK pathway and accelerates cell death

Insoluble alpha-synuclein accumulates in Parkinson's disease, diffuse Lewy body disease, and multiple system atrophy. However, the relationship between its accumulation and pathogenesis is still unclear. Recently, we reported that overexpression of alpha-synuclein affects Elk-1 phosphorylation in cultured cells, which is mainly performed by mitogen-activated protein kinases (MAPKs). We further examined the relationship between MAPK signaling and the effects of alpha-synuclein expression on ecdysone-inducible neuro2a cell lines and found that cells expressing alpha-synuclein had less phosphorylated MAPKs. Moreover, they showed significant cell death when the concentration of serum in the culture medium was reduced. Under normal serum conditions, the addition of the MAPK inhibitor U0126 also caused cell death in alpha-synuclein-expressing cells. Transfection of constitutively active MEK-1 resulted in MAPK phosphorylation in alpha-synuclein-expressing cells and improved cell viability even under reduced serum conditions. Thus, we conclude that alpha-synuclein regulates the MAPK pathway by reducing the amount of available active MAPK. Our findings suggest a mechanism for pathogenesis and thus offer therapeutic insight into synucleinopathies.     

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole induces apoptosis and necrosis with activation of different caspases in rat splenocytes

A dietary carcinogen, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) at 20 microM activates caspase-3-like proteases as an apoptotic marker in rat splenocytes. The present study demonstrated 100 microM Trp-P-1 induced necrosis with activation of caspase-3-like proteases. The activation in necrosis and apoptosis resulted from the activation of caspase-9 and caspase-8, respectively. Thus, Trp-P-1 induces apoptosis and necrosis with the activation of different caspases.     

Non-offspring nursing by a nulliparous pregnant female just before first parturition in free-ranging Japanese macaques

A nulliparous pregnant Japanese macaque, Macaca fuscata, suckled her 1-year-old sister 44 days before her own parturition. Other Japanese macaque yearlings did not hold the nipples of this nulliparous female in their mouths. Although this non-offspring nursing before first parturition did not improve the rate of milk secretion, the nulliparous female may have improved her indirect fitness and her suckling behavior.     

Dendritic cell immunoactivating receptor,a novel C-type lectin immunoreceptor,acts as an activating receptor through association with Fc receptor gamma chain

An increasing number of C-type lectin receptors are being discovered on dendritic cells, but their signaling abilities and underlying mechanisms require further definition. Among these, dendritic cell immunoreceptor (DCIR) induces negative signals through an inhibitory immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic tail. Here we identify a novel C-type lectin receptor, dendritic cell immunoactivating receptor (DCAR), whose extracellular lectin domain is highly homologous to that of DCIR. DCAR is expressed similarly in tissues to DCIR, but its short cytoplasmic portion lacks signaling motifs like ITIM. However, a positively charged arginine residue is present in the transmembrane region of the DCAR, which may explain its association with Fc receptor gamma chain and its stable expression on the cell surface. Furthermore, cross-linking of DCAR in the presence of gamma chain activates calcium mobilization and tyrosine phosphorylation of cellular proteins. These signals are mediated by the immunoreceptor tyrosine-based activating motif (ITAM) of the gamma chain. Thus, DCAR is closely related to DCIR, but it introduces activating signals into antigen-presenting cells through its physical and functional association with ITAM-bearing gamma chain. The identification of this activating immunoreceptor provides an example of signaling via a dendritic cell-expressed C-type lectin receptor.     

Histone H3 is aberrantly phosphorylated in glutamine-repeat diseases

Double-labeling immunohistochemical studies staining with anti-ubiquitin and anti-phosphoserine antibodies and application of an enzymatic dephosphorylation technique reveal neuronal inclusions and affected nuclei to be aberrantly phosphorylated in brain tissues with patients with glutamine-repeat diseases. Regional distribution of the phosphorylated nuclei in neurons correlates with the pathology. To identify the target nuclear protein, transient expression of Huntington's disease exon 1 gene containing an expanded glutamine repeat was generated in a cell culture and nuclear inclusions were isolated with a fluorescence-activated cell sorting system. Immunoblotting studies of the aggregated nuclear proteins using anti-phosphoserine antibody demonstrate the protein of the aberrant phosphorylation as histone H3. The immunoblots of control and diseased brain tissues demonstrate that the phosphorylation of histone H3 is commonly increased in the diseased brains. Aberrant phosphorylation of histone H3 is surmised to be a shared pathological process in glutamine-repeat diseases.