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Recent advances in quantitative proteomic technology have enabled the large-scale validation of biomarkers. We here performed a quantitative proteomic analysis of membrane fractions from colorectal cancer tissue to discover biomarker candidates, and then extensively validated the candidate proteins identified. A total of 5566 proteins were identified in six tissue samples, each of which was obtained from polyps and cancer with and without metastasis. GO cellular component analysis predicted that 3087 of these proteins were membrane proteins, whereas TMHMM algorithm predicted that 1567 proteins had a transmembrane domain. Differences were observed in the expression of 159 membrane proteins and 55 extracellular proteins between polyps and cancer without metastasis, while the expression of 32 membrane proteins and 17 extracellular proteins differed between cancer with and without metastasis. A total of 105 of these biomarker candidates were quantitated using selected (or multiple) reaction monitoring (SRM/MRM) with stable synthetic isotope-labeled peptides as an internal control. The results obtained revealed differences in the expression of 69 of these proteins, and this was subsequently verified in an independent set of patient samples (polyps (n= 10), cancer without metastasis (n= 10), cancer with metastasis (n= 10)). Significant differences were observed in the expression of 44 of these proteins, including ITGA5, GPRC5A, PDGFRB, and TFRC, which have already been shown to be overexpressed in colorectal cancer, as well as proteins with unknown function, such as C8orf55. The expression of C8orf55 was also shown to be high not only in colorectal cancer, but also in several cancer tissues using a multicancer tissue microarray, which included 1150 cores from 14 cancer tissues. This is the largest verification study of biomarker candidate membrane proteins to date; our methods for biomarker discovery and subsequent validation using SRM/MRM will contribute to the identification of useful biomarker candidates for various cancers. Data are available via ProteomeXchange with identifier PXD000851.

Distribution of norbin protein in rats was characterized by immunohistochemical study. It was distributed not only in whole brain but also in peripheral nervous system. The protein was localized in the somata, except for nuclei, and dendrites of neurons. Subcellular fractionation revealed that norbin is a cytosolic protein. Prominent distribution was observed in dendrites of dendritic outgrowth in Purkinje cells. Norbin should play a role in somatodendritic functions of neurons.

In some pedigrees of familial Alzheimer's disease (FAD), three mutations of β amyloid precursor protein (APP) have been found at the Val717residue (to Ile, Phe, or Gly) and these mutations increase the secretion of Aβ42(43). To study the specificity of the effects of these mutations on APP processing, we transiently expressed APP genes with mutations of Val717to Lys, Ser, Glu, or Cys in COS cells. The three familial AD-linked mutations increased the levels or ratios of Aβ42(43), whereas the secretion of Aβ40 was decreased. Other mutations irrelevant to FAD except Val717to Lys had little effect on the ratio of Aβ42(43). Substitution to Lys decreased the secretion of Aβ42(43); substitution to Glu or Gly decreased the amount of intracellular C-terminal fragment produced by α-secretase, whereas it was increased by mutations to Phe, Cys, or Lys. However, the levels of secretion of soluble APP were constant, but a substitution to Glu reduced it. These results suggest a specific role of the Val717residue in APP processing and, especially, in γ-cleavage.

The gene encoding of the 65 kDa isoform of the γ-aminobutyric acid (GABA)-synthesizing enzyme, glutamic acid decarboxylase (GAD), GAD65, was targeted in mice by homologous recombination. Viable GAD65 −/− mice were obtained with the expected mendelian frequency and displayed no gross morphological defects. Despite the complete loss of GAD65 mRNA and protein in a homozygous mutant, there was no difference in GABA content in the brains of GAD65 +/+, +/−, and −/− mice. As for the other 67 kDa isoform (GAD67), the levels of mRNA and protein were largely unchanged by the GAD65 mutation. General behavior, including locomotor activity and performance in the Morris water maze task, appeared normal, but seizures were more easily induced by picrotoxin and pentylenetetrazol: the latencies to seizures induced by picrotoxin were shorter and the dose of pentylenetetrazol required for induction of seizures was lower.

Tetraethylammonium (TEA) induces long-term potentiation (LTP)-like synaptic enhancement in rat hippocampal slices. To find the genes related to this phenomenon, subtraction screening was performed between the mRNA of TEA-treated slices and that of untreated whole brain. One of the clones induced by the TEA treatment, named as norbin, was expressed only in neural tissues. The predicted protein sequence of norbin consisted of 729 amino acids, and no homologies in the sequence were found with known genes or proteins. Overexpression of norbin in cultured Neuro 2a cells by cDNA transfection induced neurite-outgrowth. Since in the course of neural plasticity the formation of new synapses should occur, the neurite-outgrowth-related protein, norbin, might play an important role in neural plasticity.

In the mammalian nervous system, several basic helix-loop-helix (bHLH) proteins have been identified that may play essential roles in neurogenesis or neural development. This paper describes a novel bHLH protein in rat brain, which has a bHLH domain highly homologous with that of MATH-2 and NeuroD. On the other hand, the amino-acid sequence of the other regions had little homology with those of the known proteins. This protein consists of 381 amino acids and was detected only in neural tissue, indicating that it has an important role specific to neuronal activities.

A partial 5′-region cDNA (11.5 kb) of chicken breast muscle connectin/titin encoding 3752 amino acids was sequenced. The predicted amino acid sequence contains 31 immunoglobulin C2 motifs and 10 interdomains. The sequence suggests a skeletal muscle type of connectin isoforms. Immunoelectron microscopic studies using antisera raised against several products of the cDNA fragments expressed inE. colirevealed that a region of some 800 amino acids from the N terminus of connectin is involved in its binding to the Z line in a sarcomere.