Temporal and Spatial Expression of the {alpha}-Amylase Gene during Seed Germination in Rice and Barley

We have analyzed the in situ hybridization of     

Anaerobic Coryneforms Isolated from Human Bone Marrow and Skin

Eighteen isolates of anaerobic coryneforms from human bone marrow and skin and four type strains of Propionibacterium were studied chemically, biochemically and antigenically. All of the isolates were identified as Propionibacterium acnes; of the 18 isolates, 16 belonged to serotype I and two to serotype II. By means of gas liquid chromatography and mass spectral analysis, a large amount of iso-type fatty acids, such as iso-pentadecanoic and iso-heptadecanoic acids were detected in whole cells of isolates and type strains. Antitumor and adjuvant effects of the isolates and type strains were found to differ considerably among the strains. One of the isolates, P. acnes C-7, which showed potent biological activities was fractionated by hot phenol-water extraction. The resulting insoluble middle layer was found the most effective in tumor protection, adjuvant action in immune response and phagocytic activity in mice.     

A proteomic profile of synoviocyte lesions microdissected from formalin-fixed paraffin-embedded synovial tissues of rheumatoid arthritis

Background

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation of the synovial joints. Early intervention followed by early diagnosis can result in disease remission; however, both early stage diagnosis and provision of effective treatment have been impeded by the heterogeneity of RA, which details of pathological mechanism are unclear. Regardless of numerous investigations of RA by means of genomic and proteomic approaches, proteins interplaying in RA synovial tissues that contain various types of synoviocytes, are not yet sufficiently understood. Hence we have conducted an HPLC/mass spectrometry-based exploratory proteomic analysis focusing on synoviocyte lesions laser-microdissected (LMD) from formalin-fixed paraffin-embedded (FFPE) synovial tissues (RA, n = 15; OA, n = 5), where those of Osteoarthritis (OA) were used as the control.

Results

A total of 508 proteins were identified from the RA and OA groups. With the semi-quantitative comparisons, the spectral index (SpI), log2 protein ratio (R_SC) based on spectral counting, and statistical G-test, 98 proteins were found to be significant (pair-wise p < 0.05) to the RA synovial tissues. These include stromelysin-1 (MMP3), proteins S100-A8 and S100-A9, plastin-2, galectin-3, calreticulin, cathepsin Z, HLA-A, HLA-DRB1, ferritin, neutrophil defensin 1, CD14, MMP9 etc.

Conclusions

Our results confirmed the involvement of known RA biomarkers such as stromelysin-1 (MMP3) and proteins S100-A8 and S100-A9, and also that of leukocyte antigens such as HLA-DRB1. Network analyses of protein–protein interaction for those proteins significant to RA revealed a dominant participation of ribosome pathway (p = 5.91 × 10⁻⁴⁵), and, interestingly, the associations of the p53 signaling (p = 2.34 × 10⁻⁵). An involvement of proteins including CD14, S100-A8/S100-A9 seems to suggest an activation of the NF-kB/MAPK signaling pathway. Our strategy of laser-microdissected FFPE-tissue proteomic analysis in Rheumatoid Arthritis thus demonstrated its technical feasibility in profiling proteins expressed in synovial tissues, which may play important roles in the RA pathogenesis.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9091-8) contains supplementary material, which is available to authorized users.     

Nano-LC-MS/MS for Quantification of Lyso-Gb3 and Its Analogues Reveals a Useful Biomarker for Fabry Disease

Biomarkers useful for diagnosis and evaluation of treatment for patients with Fabry disease are urgently needed. Recently, plasma globotriaosylsphingosine (lyso-Gb3) and lyso-Gb3-related analogues have attracted attention as promising biomarkers of Fabry disease. However, the plasma concentrations of lyso-Gb3 and its analogues are extremely low or below the detection limits in some Fabry patients as well as in healthy subjects. In this paper, we introduce the novel application of a nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) system to the measurement of lyso-Gb3 and its analogues in plasma. Nano-LC-MS/MS requires smaller amounts of samples and is more sensitive than conventional techniques. Using this method, we measured the plasma concentrations of lyso-Gb3 and its analogues in 40 healthy subjects, 5 functional variants (males with E66Q), and various Fabry patients (9 classic Fabry males/9 mutations; 7 later-onset Fabry males/5 mutations; and 10 Fabry females/9 mutations). The results revealed that the mean lyso-Gb3 and lyso-Gb3(-2) concentrations in all the Fabry patient subgroups were statistically higher, especially in the classic Fabry males, than those in the functional variants and healthy subjects. The plasma concentrations of lyso-Gb3 and its analogues in healthy subjects, functional variants, and some Fabry patients with specific mutations (R112H and M296I) that cannot be established by conventional techniques were successfully determined by means of nano-LC-MS/MS. The lyso-Gb3 and lyso-Gb3(-2) concentrations in male patients with these mutations were lower than those in most Fabry patients having other mutations, but higher than those in the functional variants and healthy subjects. This new method is expected to be useful for sensitive determination of the plasma concentrations of lyso-Gb3 and its analogues. This study also revealed that not only lyso-Gb3 but also lyso-Gb3(-2) in plasma is a useful biomarker for the diagnosis of Fabry disease.     

Brown Adipose Tissue in Cetacean Blubber

Brown adipose tissue (BAT) plays an important role in thermoregulation in species living in cold environments, given heat can be generated from its chemical energy reserves. Here we investigate the existence of BAT in blubber in four species of delphinoid cetacean, the Pacific white-sided and bottlenose dolphins, Lagenorhynchus obliquidens and Tursiops truncates, and Dall’s and harbour porpoises, Phocoenoides dalli and Phocoena phocoena. Histology revealed adipocytes with small unilocular fat droplets and a large eosinophilic cytoplasm intermingled with connective tissue in the innermost layers of blubber. Chemistry revealed a brown adipocyte-specific mitochondrial protein, uncoupling protein 1 (UCP1), within these same adipocytes, but not those distributed elsewhere throughout the blubber. Western blot analysis of extracts from the inner blubber layer confirmed that the immunohistochemical positive reaction was specific to UCP1 and that this adipose tissue was BAT. To better understand the distribution of BAT throughout the entire cetacean body, cadavers were subjected to computed tomography (CT) scanning. Resulting imagery, coupled with histological corroboration of fine tissue structure, revealed adipocytes intermingled with connective tissue in the lowest layer of blubber were distributed within a thin, highly dense layer that extended the length of the body, with the exception of the rostrum, fin and fluke regions. As such, we describe BAT effectively enveloping the cetacean body. Our results suggest that delphinoid blubber could serve a role additional to those frequently attributed to it: simple insulation blanket, energy storage, hydrodynamic streamlining or contributor to positive buoyancy. We believe delphinoid BAT might also function like an electric blanket, enabling animals to frequent waters cooler than blubber as an insulator alone might otherwise allow an animal to withstand, or allow animals to maintain body temperature in cool waters during sustained periods of physical inactivity.     

Entropic Enhancement of Protein-DNA Affinity by Oxygen-to-Sulfur Substitution in DNA Phosphate

Dithioation of DNA phosphate is known to enhance binding affinities, at least for some proteins. We mechanistically characterized this phenomenon for the Antennapedia homeodomain-DNA complex by integrated use of fluorescence, isothermal titration calorimetry, NMR spectroscopy, and x-ray crystallography. By fluorescence and isothermal titration calorimetry, we found that this affinity enhancement is entropy driven. By NMR, we investigated the ionic hydrogen bonds and internal motions of lysine side-chain NH₃⁺ groups involved in ion pairs with DNA. By x-ray crystallography, we compared the structures of the complexes with and without dithioation of the phosphate. Our NMR and x-ray data show that the lysine side chain in contact with the DNA phosphate becomes more dynamic upon dithioation. Our thermodynamic, structural, and dynamic investigations collectively suggest that the affinity enhancement by the oxygen-to-sulfur substitution in DNA phosphate is largely due to an entropic gain arising from mobilization of the intermolecular ion pair at the protein-DNA interface.