Scan-Free Absorbance Spectral Imaging A(x,y, λ) of Single Live Algal Cells for Quantifying Absorbance of Cell Suspensions

Label-free, non-invasive, rapid absorbance spectral imaging A(x,y,λ) microscopy of single live cells at 1.2 μm × 1.2 μm resolution with an NA = 0.85 objective was developed and applied to unicellular green algae Chlamydomonas reinhardtii. By introducing the fiber assembly to rearrange a two-dimensional image to the one-dimensional array to fit the slit of an imaging spectrograph equipped with a CCD detector, scan-free acquisition of three-dimensional information of A(x,y,λ) was realized. The space-resolved absorbance spectra of the eyespot, an orange organelle about 1 μm, were extracted from the green-color background in a chlorophyll-rich single live cell absorbance image. Characteristic absorbance change in the cell suspension after hydrogen photoproduction in C. reinhardtii was investigated to find a single 715-nm absorption peak was locally distributed within single cells. The formula to calculate the absorbance of cell suspensions from that of single cells was presented to obtain a quantitative, parameter-free agreement with the experiment. It is quantitatively shown that the average number of chlorophylls per cell is significantly underestimated when it is evaluated from the absorbance of the cell suspensions due to the package effect.     

Multiple molecular interactions implicate the connectin/titin N2A region as a modulating scaffold for p94/calpain 3 activity in skeletal muscle

p94/calpain 3 is a skeletal muscle-specific Ca(2+)-regulated cysteine protease (calpain), and genetic loss of p94 protease activity causes muscular dystrophy (calpainopathy). In addition, a small in-frame deletion in the N2A region of connectin/titin that impairs p94-connectin interaction causes a severe muscular dystrophy (mdm) in mice. Since p94 via its interaction with the N2A and M-line regions of connectin becomes part of the connectin filament system that serves as a molecular scaffold for the myofibril, it has been proposed that structural and functional integrity of the p94-connectin complex is essential for health and maintenance of myocytes. In this study, we have surveyed the interactions made by p94 and connectin N2A inside COS7 cells. This revealed that p94 binds to connectin at multiple sites, including newly identified loci in the N2A and PEVK regions of connectin. Functionally, p94-N2A interactions suppress p94 autolysis and protected connectin from proteolysis. The connectin N2A region also contains a binding site for the muscle ankyrin repeat proteins (MARPs), a protein family involved in the cellular stress responses. MARP2/Ankrd2 competed with p94 for binding to connectin and was also proteolyzed by p94. Intriguingly, a connectin N2A fragment with the mdm deletion possessed enhanced resistance to proteases, including p94, and its interaction with MARPs was weakened. Our data support a model in which MARP2-p94 signaling converges within the N2A connectin segment and the mdm deletion disrupts their coordination. These results also implicate the dynamic nature of connectin molecule as a regulatory scaffold of p94 functions.     

Complex II from phototrophic purple bacterium Rhodoferax fermentans displays rhodoquinol-fumarate reductase activity.

It has long been accepted that bacterial quinol-fumarate reductase (QFR) generally uses a low-redox-potential naphthoquinone, menaquinone (MK), as the electron donor, whereas mitochondrial QFR from facultative and anaerobic eukaryotes uses a low-redox-potential benzoquinone, rhodoquinone (RQ), as the substrate. In the present study, we purified novel complex II from the RQ-containing phototrophic purple bacterium, Rhodoferax fermentans that exhibited high rhodoquinol-fumarate reductase activity in addition to succinate-ubiquinone reductase activity. SDS/PAGE indicated that the purified R. fermentans complex II comprises four subunits of 64.0, 28.6, 18.7 and 17.5 kDa and contains 1.3 nmol heme per mg protein. Phylogenetic analysis and comparison of the deduced amino acid sequences of R. fermentans complex II with pro/eukaryotic complex II indicate that the structure and the evolutional origins of R. fermentans complex II are closer to bacterial SQR than to mitochondrial rhodoquinol-fumarate reductase. The results strongly indicate that R. fermentans complex II and mitochondrial QFR might have evolved independently, although they both utilize RQ for fumarate reduction.     

Improvement of Automatic In-Gel Digestion by in Situ Alkylation of Proteins

We have recently improved the automation of an in-gel digestion system, DigestPro 96, using in situ alkylation of proteins with acrylamide, conducted during one-dimensional (1D) SDS-PAGE. The improved method included the processes of destaining, dehydration, trypsin digestion, and extraction but excluded the reduction and alkylation steps following staining of proteins with CBB. The extracted peptide mixtures were directly loaded onto a micro C18 LC column of the mass spectrometer. The resultant spectra were processed with “Mascot” search engine to estimate the sequence coverage of the bovine serum albumin (BSA). The original method, designed for Laemmli 1D SDS gel applications, consisted of reduction and post-alkylation with iodoacetamide, which produced carboxyamidemethyl (CAM; –S–CH₂CONH₂) derivatives. The original method also included a desalting step essential for mass spectrometry, especially matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We compared the original and improved methods using BSA (3 pmol loaded to the gel, one third of digested peptide mixture injected into LC-MS). The original method yielded both CAM and propionicamide (PAM; –S–CH₂CH₂CONH₂) derivatives. The source of PAM derivatives is the unpolymerized acrylamide formed during electrophoresis. The sequence coverage of CAM derivatives of BSA by the original method was 10% with desalting and 19% without desalting. The sequence coverage of PAM derivative by the improved method was 32%. Our results clearly show the advantage of our improved automated in-gel digestion method for in situ PAM alkylated protein with respect to peptide recovery, compared with the original method with CAM post-alkylation.     

Hypoxia followed by reoxygenation induces secretion of cyclophilin A from cultured rat cardiac myocytes

We previously reported that hypoxia followed by reoxygenation (hypoxia/reoxygenation) rapidly activated intracellular signaling such as mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated protein kinase (ERK) 1/2, p38MAPK, and stress-activated protein kinases (SAPKs). To investigate the humoral factors which mediate cardiac response to hypoxia/reoxygenation, we analyzed the conditioned media from cardiac myocytes subjected to hypoxia/reoxygenation by two-dimensional electrophoresis and mass spectrometry. We identified cyclophilin A (CyPA) as one of the proteins secreted from cardiac myocytes in response to hypoxia/reoxygenation. Hypoxia/reoxygenation induced the expression of CyPA and its cell surface receptor CD147 on cardiac myocytes in vitro. This was also confirmed by ischemia/reperfusion in vivo. Recombinant human (rh) CyPA activated ERK1/2, p38MAPK, SAPKs, and Akt in cultured cardiac myocytes. Furthermore, CyPA significantly increased Bcl-2 in cardiac myocytes. These data strongly suggested that CyPA is released from cardiac myocytes in response to hypoxia/reoxygenation and may protect cardiac myocytes from oxidative stress-induced apoptosis.     

Proteome analysis of a rat liver nuclear insoluble protein fraction and localization of a novel protein, ISP36, to compartments in the interchromatin space

A rat liver nuclear insoluble protein fraction was analyzed to investigate candidate proteins participating in nuclear architecture formation. Proteins were subjected to two-dimensional separation by reversed-phase HPLC in 60% formic acid and SDS/PAGE. The method produced good resolution of insoluble proteins. One hundred and thirty-eight proteins were separated, and 28 of these were identified. The identified proteins included one novel protein, seven known nuclear proteins and 12 known nuclear matrix proteins. The novel 36 kDa protein was further investigated for its subnuclear localization. The human ortholog of the protein was expressed in Escherichia coli and antibodies were raised against the recombinant protein. Exclusive localization of the protein to the nuclear insoluble protein fraction was confirmed by cell fractionation followed by immunoblotting. Immunostaining of mouse C3H cells suggested that the 36 kDa protein was a constituent of an insoluble macromolecular complex spread throughout the interchromatin space of the nucleus. The protein was designated 'interchromatin space protein of 36 kDa', ISP36.     

Geographical distribution and disease associations of the CD45 exon 6 138G variant

CD45 is crucial for normal lymphocyte signalling, and altered CD45 expression has major effects on immune function. Both mice and humans lacking CD45 expression are severely immunodeficient, and single-nucleotide polymorphisms in the CD45 gene that cause altered splicing have been associated with autoimmune and infectious diseases. Recently, we identified an exon 6 A138G polymorphism resulting in an increased proportion of activated CD45RO T cells and altered immune function. Here we report a significantly reduced frequency of the 138G allele in hepatitis C Japanese patients and a possibly reduced frequency in type I diabetes. The allele is widely distributed in the Far East and India, indicating that it may have a significant effect on disease burden in a large part of the human population.     

Genetic variability in the extracellular matrix protein as a determinant of risk for developing HTLV-I-associated neurological disease

Aggrecan, which is a well-known proteoglycan in joint cartilage, also exists in the spinal cord and plays an important role in maintaining water content in the extracellular matrix structure. In this study, we first examined the variable number of tandem repeat (VNTR) polymorphism of the aggrecan gene in 227 HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients, in 217 HTLV-I-infected healthy carriers (HCs), and in 85 normal controls. The VNTR allele 28 (1,630 bp) was more frequently observed in HAM/TSP patients than in HCs (χ ²=12.02, p=0.0005, odds ratio 1.79, 95% C.I. 1.29–2.50) and in controls (χ ²=13.43, p=0.0002, odds ratio 2.54, 95% C.I. 1.52–4.25), although this allele was not related to disease progression or to HTLV-I provirus load. We also found that the aggrecan concentration in cerebrospinal fluid (CSF) from rapidly progressive HAM/TSP patients was significantly higher than in slowly progressive patients (corrected p=0.0145) but not in infected non-inflammatory neurological other disease controls (OND) (corrected p=0.078). We then analyzed this aggrecan VNTR polymorphism in the different set of patients with HAM/TSP (n=58) and healthy carriers (n=70). This analysis, again, revealed that allele 28 was detected more frequently in HAM/TSP group than in HCs (χ ²=11.03, p=0.0009, odd ratio 3.04, 95% C.I. 1.55–5.97). The reproducibility of our study was regarded as a second- or third-class association by comparing combined p values and the Better Associations for Disease and GEnes (BADGE) system. Our results suggest that aggrecan polymorphism can be a novel genetic risk factor for developing HAM/TSP.Financial support: Grant in Aid for Research on Brain Science of the Ministry of Health, Labor and Welfare, Japan.     

A conserved lysine residue in the crenarchaea-specific loop is important for the crenarchaeal splicing endonuclease activity

In Archaea, splicing endonuclease (EndA) recognizes and cleaves precursor RNAs to remove introns. Currently, EndAs are classified into three families according to their subunit structures: homotetramer, homodimer, and heterotetramer. The crenarchaeal heterotetrameric EndAs can be further classified into two subfamilies based on the size of the structural subunit. Subfamily A possesses a structural subunit similar in size to the catalytic subunit, whereas subfamily B possesses a structural subunit significantly smaller than the catalytic subunit. Previously, we solved the crystal structure of an EndA from Pyrobaculum aerophilum. The endonuclease was classified into subfamily B, and the structure revealed that the enzyme lacks an N-terminal subdomain in the structural subunit. However, no structural information is available for crenarchaeal heterotetrameric EndAs that are predicted to belong to subfamily A. Here, we report the crystal structure of the EndA from Aeropyrum pernix, which is predicted to belong to subfamily A. The enzyme possesses the N-terminal subdomain in the structural subunit, revealing that the two subfamilies of heterotetrameric EndAs are structurally distinct. EndA from A. pernix also possesses an extra loop region that is characteristic of crenarchaeal EndAs. Our mutational study revealed that the conserved lysine residue in the loop is important for endonuclease activity. Furthermore, the sequence characteristics of the loops and the positions towards the substrate RNA according to a docking model prompted us to propose that crenarchaea-specific loops and an extra amino acid sequence at the catalytic loop of nanoarchaeal EndA are derived by independent convergent evolution and function for recognizing noncanonical bulge-helix-bulge motif RNAs as substrates.