Terminal proteomics: N- and C-terminal analyses for high-fidelity identification of proteins using MS

In proteomics, MS plays an essential role in identifying and quantifying proteins. To characterize mature target proteins from living cells, candidate proteins are often analyzed with PMF and MS/MS ion search methods in combination with computational search routines based on bioinformatics. In contrast to shotgun proteomics, which is widely used to identify proteins, proteomics based on the analysis of N- and C-terminal amino acid sequences (terminal proteomics) should render higher fidelity results because of the high information content of terminal sequence and potentially high throughput of the method not requiring very high sequence coverage to be achieved by extensive sequencing. In line with this expectation, we review recent advances in methods for N- and C-terminal amino acid sequencing of proteins. This review focuses mainly on the methods of N- and C-terminal analyses based on MALDI-TOF MS for its easy accessibility, with several complementary approaches using LC/MS/MS. We also describe problems associated with MS and possible remedies, including chemical and enzymatic procedures to enhance the fidelity of these methods.     

Effects on genome constitution and novel cell wall formation caused by the addition of 5RS rye chromosome to common wheat

The cytological instability of common wheat-rye addition lines was investigated in the present study. The chromosome numbers of almost all addition lines were considerably stable, but those of CS 5R were very variable. The rye chromosome added in this line was found to be much shorter than expected. Fluorescent in situ hybridization with 5S rDNA and the centromere-specific probes clearly revealed that the short rye chromosome contains only a short arm of chromosome 5R (5RS). In this line, chromosome numbers of both 5RS and common wheat were changeable. The chromosome numbers ranged from 2n = 36 to 2n = 44 in the cells carrying two 5RS, and ranged from 2n = 31 to 2n = 44 in one 5RS cells. In addition to the chromosome instability, the multicells wrapped in a sac-like structure were frequently observed in the root meristematic tissues of CS 5RS after the enzyme treatment for chromosome preparation. Genomic in situ hybridization with rye DNA as a probe showed that all cells in sacs investigated were at the interphase stage and contained one or two 5RS chromosomes. An electron microscopic analysis revealed that the cells of CS 5RS, particularly in sacs, have abnormal (irregular and curved) cell walls. These results indicate that 5RS has (a) specific factor(s) influencing the cell wall development as well as the genome stability.     

Molecular mimicry in pauci-immune focal necrotizing glomerulonephritis

Pauci-immune focal necrotizing glomerulonephritis (FNGN) is a severe inflammatory disease associated with autoantibodies to neutrophil cytoplasmic antigens (ANCA). Here we characterize autoantibodies to lysosomal membrane protein-2 (LAMP-2) and show that they are a new ANCA subtype present in almost all individuals with FNGN. Consequently, its prevalence is nearly twice that of the classical ANCAs that recognize myeloperoxidase or proteinase-3. Furthermore, antibodies to LAMP-2 cause pauci-immune FNGN when injected into rats, and a monoclonal antibody to human LAMP-2 (H4B4) induces apoptosis of human microvascular endothelium in vitro. The autoantibodies in individuals with pauci-immune FNGN commonly recognize a human LAMP-2 epitope (designated P(41-49)) with 100% homology to the bacterial adhesin FimH, with which they cross-react. Rats immunized with FimH develop pauci-immune FNGN and also develop antibodies to rat and human LAMP-2. Finally, we show that infections with fimbriated pathogens are common before the onset of FNGN. Thus, FimH-triggered autoimmunity to LAMP-2 provides a previously undescribed clinically relevant molecular mechanism for the development of pauci-immune FNGN.     

In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy.

Intravital microscopy coupled with chronic animal window models has provided stunning insight into tumor pathophysiology, including gene expression, angiogenesis, cell adhesion and migration, vascular, interstitial and lymphatic transport, metabolic microenvironment and drug delivery. However, the findings to date have been limited to the tumor surface (< 150 microm). Here, we show that the multiphoton laser-scanning microscope can provide high three-dimensional resolution of gene expression and function in deeper regions of tumors. These insights could be critical to the development of novel therapeutics that target not only the tumor surface, but also internal regions.     

Attenuation of exercise-induced heat shock protein 72 expression blunts improvements in whole-body insulin resistance in rats with type 2 diabetes

Heat shock proteins (HSPs) play an important role in insulin resistance and improve the cellular stress response via HSP induction by exercise to treat type 2 diabetes. In this study, the effects of exercise-induced HSP72 expression levels on whole-body insulin resistance in type 2 diabetic rats were investigated. Male 25-week-old Otsuka Long-Evans Tokushima Fatty rats were divided into three groups: sedentary (Sed), trained in a thermal-neutral environment (NTr: 25 °C), and trained in a cold environment (CTr: 4 °C). Exercise training was conducted 5 days/week for 10 weeks. Rectal temperature was measured following each bout of exercise. An intraperitoneal glucose tolerance test (IPGTT) was performed after the training sessions. The serum, gastrocnemius muscle, and liver were sampled 48 h after the final exercise session. HSP72 and heat shock cognate protein 73 expression levels were analyzed by Western blot, and serum total cholesterol, triglyceride (TG), and free fatty acid (FFA) levels were measured. NTr animals exhibited significantly higher body temperatures following exercise, whereas, CTr animals did not. Exercise training increased HSP72 levels in the gastrocnemius muscle and liver, whereas, HSP72 expression was significantly lower in the CTr group than that in the NTr group (p < 0.05). Glucose tolerance improved equally in both trained animals; however, insulin levels during the IPGTT were higher in CTr animals than those in NTr animals (p < 0.05). In addition, the TG and FFA levels decreased significantly only in NTr animals compared with those in Sed animals. These results suggest that attenuation of exercise-induced HSP72 expression partially blunts improvement in whole-body insulin resistance and lipid metabolism in type 2 diabetic rats.     

Effects of Melanogenesis‐Inducing Nitric Oxide and Histamine on the Production of Eumelanin and Pheomelanin in Cultured Human Melanocytes

Melanin pigments produced in human melanocytes are classified into two categories; black coloured eumelanin and reddish‐yellow pheomelanin. Stimulation of melanocytes with α‐melanocyte‐stimulating hormone (α‐MSH), one of several melanogenic factors, has been reported to enhance eumelanogenesis to a greater degree than pheomelanogenesis, which contributes to hyperpigmentation in skin. Nitric oxide (NO) and histamine are also melanogenesis‐stimulating factors that are released from cells surrounding melanocytes following ultraviolet (UV) irradiation. In this study, the effects of NO and histamine on the ratio of eumelanin and pheomelanin were examined in human melanocytes, and then compared with that of α‐MSH. The amounts of eumelanin and pheomelanin were quantified using high‐performance liquid chromatography analysis after oxidation and hydrolysis of melanin. Melanogenesis was induced by the addition of α‐MSH, NO, or histamine to melanocytes. The amount of eumelanin production significantly increased with independent stimulation by these melanogenic factors, especially histamine, while that of pheomelanin significantly increased with α‐MSH and NO, but only slightly with histamine. As a result, the ratio of eumelanin and pheomelanin increased significantly with the addition of NO or histamine. These results suggest that NO and histamine, as in the case of α‐MSH, may contribute to UV‐induced hyperpigmentation by enhancing eumelanogenesis.     

Alternate muscle activity observed between knee extensor synergists during low-level sustained contractions.

To determine quantitatively the features of alternate muscle activity between knee extensor synergists during low-level prolonged contraction, a surface electromyogram (EMG) was recorded from the rectus femoris (RF), vastus lateralis (VL), and vastus medialis (VM) in 11 subjects during isometric knee extension exercise at 2.5% of maximal voluntary contraction (MVC) for 60 min (experiment 1). Furthermore, to examine the relation between alternate muscle activity and contraction levels, six of the subjects also performed sustained knee extension at 5.0, 7.5, and 10.0% of MVC (experiment 2). Alternate muscle activity among the three muscles was assessed by quantitative analysis on the basis of the rate of integrated EMG sequences. In experiment 1, the number of alternations was significantly higher between RF and either VL or VM than between VL and VM. Moreover, the frequency of alternate muscle activity increased with time. In experiment 2, alternating muscle activity was found during contractions at 2.5 and 5.0% of MVC, although not at 7.5 and 10.0% of MVC, and the number of alternations was higher at 2.5 than at 5.0% of MVC. Thus the findings of the present study demonstrated that alternate muscle activity in the quadriceps muscle 1) appears only between biarticular RF muscle and monoarticular vasti muscles (VL and VM), and its frequency of alternations progressively increases with time, and 2) emerges under sustained contraction with force production levels < or =5.0% of MVC.