Serum metabolomics as a novel diagnostic approach for pancreatic cancer

Pancreatic cancer is one of the leading causes of cancer-related death, and there is currently little hope of a cure because there are no effective biomarkers for its early detection. Therefore, the search for novel biomarkers that would allow the early detection of pancreatic cancer is ongoing. In this study, the differences between the metabolomes of pancreatic cancer patients with Stage III, Stage IVa, or Stage IVb disease (n = 20) and healthy volunteers (n = 9) were evaluated by metabolomics, which is the endpoint of the Omics cascade and therefore the last step in the cascade before the phenotype. In our experimental conditions using gas chromatography mass spectrometry (GC/MS), a total of 60 metabolites were detected in serum, and the levels of 18 of the 60 metabolites were significantly changed in pancreatic cancer patients compared with those in healthy volunteers. Then, Principal Component Analysis (PCA), which is a basic form of Multiple Classification Analysis, was performed, and the PCA scores plots based on the 60 metabolites highlighted the metabolomic differences between the pancreatic cancer patients and healthy volunteers. The differences between different stages of pancreatic cancer were also assessed by Partial Least Squares Discriminant Analysis (PLS-DA), which is one of Multiple Classification Analysis, and we found that it was possible to discriminate among the Stage III, Stage IVa, and Stage IVb groups. In addition, values of the 9 metabolites in 1 Stage I pancreatic cancer patient were similar to those obtained from the Stage III, Stage IVa, and Stage IVb pancreatic cancer patients. Our findings will aid the discovery of novel biomarkers that allow the early detection of pancreatic cancer by metabolomic approaches.     

Energy and Electron Transfer Systems of Chlamydomonas reinhardi. I. Photosynthetic and Respiratory Cytochrome Systems of the Pale Green Mutant

The role of cytochromes in photosynthetic electron transfer system has been studied using the pale green mutant of Chlamydomonas reinhardi (ATCC 18302). The existence of cytochromes b₅₆₃ and f is confirmed, while no significant amount of ascorbate-reducible cytochrome b₅₅₉ is detected in this mutant. The presence of cytochrome c and a small amount of a-type cytochrome is determined in these cells.     

Distribution,abundance, and food habits of the snailfishLiparis tanakai in the Suo Sea,Seto Inland Sea

In order to examine the distribution, abundance, and food habits of the snailfish,Liparis tanakai, in the Suo Sea, western Seto Inland Sea of Japan, experimental fishing surveys were made using trawl nets on the night (1930–0340 h) of 26 May and 2 and 3 June, 1986. The study area was subdivided into 18 areas, each sampled by one trawl tow of 35 min duration. A total of 190 fish was collected during the surveys, and most fish (78.9%) were obtained from muddy substrata ranging in depth from 30 to 40 m at the middle area of the Suo Sea. The total length of the fish in the samples ranged from 72 to 273 mm. The overall length-frequency distribution formed a unimodal curve, the mode occurring at the class of 100–130 mm in total length. Of 151 fish examined, 146 (96.7%) had stomachs full of food, including a variety of small crustaceans and fish. Shrimps, especiallyCrangon affinis, ranked highest in importance in the diet ofL. tanakai. These biological data are compared with those of conspecifics from Sendai Bay, northern Honshu of Japan.     

Patch clamp studies on V-type ATPase of vacuolar membrane of haploid Saccharomyces cerevisiae. Preparation and utilization of a giant cell containing a giant vacuole

A method for obtaining giant protoplasts of Escherichia coli (the spheroplast incubation (SI) method: Kuroda et al. (Kuroda, T., Okuda, N., Saitoh, N., Hiyama, T., Terasaki, Y., Anazawa, H., Hirata, A., Mogi, T., Kusaka, I., Tsuchiya, T., and Yabe, I. (1998) J. Biol. Chem. 273, 16897-16904) was adapted to haploid cells of Saccharomyces cerevisiae. The yeast cell grew to become as large as 20 micrometer in diameter and to contain an oversized vacuole inside. A patch clamp technique in the whole cell/vacuole recording mode was applied for the vacuole isolated by osmotic shock. At zero membrane potential, ATP induced a strong current (as high as 100 pA; specific activity, 0.1 pA/micrometer(2)) toward the inside of the vacuole. Bafilomycin A(1,) a specific inhibitor of the V-type ATPase, strongly inhibited the activity (K(i) = 10 nM). Complete inhibition at higher concentrations indicated that any other ATP-driven transport systems were not expressed under the present incubation conditions. This current was not observed in the vacuoles prepared from a mutant that disrupted a catalytic subunit of the V-type ATPase (RH105(Deltavma1::TRP)). The K(m) value for the ATP dose response of the current was 159 microM and the H(+)/ATP ratio estimated from the reversible potential of the V-I curve was 3.5 +/- 0.3. These values agreed well with those previously estimated by measuring the V-type ATPase activity biochemically. This method can potentially be applied to any type of ion channel, ion pump, and ion transporter in S. cerevisiae, and can also be used to investigate gene functions in various organisms by using yeast cells as hosts for homologous and heterogeneous expression systems.     

The ALG-2-interacting protein Alix associates with CHMP4b,a human homologue of yeast Snf7 that is involved in multivesicular body sorting

Alix (ALG-2-interacting protein X) is a 95-kDa protein that interacts with an EF-hand type Ca(2+)-binding protein, ALG-2 (apoptosis-linked gene 2), through its C-terminal proline-rich region. In this study, we searched for proteins that interact with human AlixDeltaC (a truncated form not containing the C-terminal region) by using a yeast two-hybrid screen, and we identified two similar human proteins, CHMP4a and CHMP4b (chromatin-modifying protein; charged multivesicular body protein), as novel binding partners of Alix. The interaction of Alix with CHMP4b was confirmed by a glutathione S-transferase pull-down assay and by co-immunoprecipitation experiments. Fluorescence microscopic analysis revealed that CHMP4b transiently expressed in HeLa cells mainly exhibited a punctate distribution in the perinuclear area and co-localized with co-expressed Alix. The distribution of CHMP4b partly overlapped the distributions of early and late endosomal marker proteins, EEA1 (early endosome antigen 1) and Lamp-1 (lysosomal membrane protein-1), respectively. Transient overexpression of CHMP4b induced the accumulation of ubiquitinated proteins as punctate patterns that were partly overlapped with the distribution of CHMP4b and inhibited the disappearance of endocytosed epidermal growth factor. In contrast, stably expressed CHMP4b in HEK293 cells was observed diffusely in the cytoplasm. Transient overexpression of AlixDeltaC in stably CHMP4b-expressing cells, however, induced formation of vesicle-like structures in which CHMP4b and AlixDeltaC were co-localized. SKD1(E235Q), a dominant negative form of the AAA type ATPase SKD1 that plays critical roles in the endocytic pathway, was co-immunoprecipitated with CHMP4b. Furthermore, CHMP4b co-localized with SKD1(E235Q) as punctate patterns in the perinuclear area, and Alix was induced to exhibit dot-like distributions overlapped with SKD1(E235Q) in HeLa cells. These results suggest that CHMP4b and Alix participate in formation of multivesicular bodies by cooperating with SKD1.     

Dominant negative mutant of a lipoprotein-specific molecular chaperone,LolA, tightly associates with LolCDE

Periplasmic molecular chaperone LolA and the inner membrane ATP binding cassette transporter LolCDE are essential for ATP-dependent release of outer membrane-directed lipoproteins from the inner membrane of Escherichia coli. A LolA(F47E) mutant carrying a Phe to Glu mutation at position 47 was defective in the release of lipoproteins from spheroplasts and proteoliposomes reconstituted with LolCDE. When incubated with proteoliposomes containing LolCDE, LolA remained in the supernatant whereas LolA(F47E) bound to proteoliposomes. This tight association of LolA(F47E) with LolCDE caused a dominant negative phenotype in vivo, suggesting that the LolA-LolCDE interaction is critical for lipoprotein release.     

Equine follicle-stimulating hormone: molecular cloning of beta subunit and biological role of the asparagine-linked oligosaccharide at asparagine(56) of alpha subunit.

Equine FSH (eFSH) and eCG are members of the glycoprotein hormone family. These proteins are heterodimeric, composed of noncovalently associated alpha and beta subunits. We have previously reported that recombinant eCG has potent LH- and FSH-like activities and that the oligosaccharide at Asn(56) of the alpha subunit plays an indispensable role in expressing LH- but not FSH-like activity. In the present study, we cloned eFSH beta subunit cDNA and expressed wild-type recombinant eFSH and a partially deglycosylated mutant FSH (eFSH alpha56/beta) to investigate the biological role of the oligosaccharide at Asn(56) in FSH activity. The wild-type eFSH and eCG stimulated estradiol production in a dose-dependent manner in the primary cultures of rat granulosa cells, indicating that these equine gonadotropins have FSH activity. Partially deglycosylated eCG (eCG alpha56/beta) also stimulated estradiol production, confirming that the FSH-like activity of eCG is resistant to the removal of the N-linked oligosaccharide. Partially deglycosylated eFSH (eFSH alpha56/beta), however, did not show any FSH activity, indicating that the oligosaccharide at Asn(56) was necessary for eFSH. Thus, FSH-like activities of two gonadotropins, eCG and eFSH, are evoked through the distinct molecular mechanisms regarding the biological role of oligosaccharide at Asn(56) of the alpha subunit.     

Duplication detection in Japanese Duchenne muscular dystrophy patients and identification of carriers with partial gene deletions using pulsed-field gel electrophoresis

DNA samples from 21 unrelated Japanese patients with Duchenne muscular dystrophy (DMD) with nondeletion-type abnormality in the dystrophin gene and three samples from possible deletion carriers were analyzed using pulsed-field gel electrophoresis (PFGE). Among the 21 patients, 7 were found to carry partial duplications of the dystrophin gene spanning 50–400 kb. Of these 7 patients, 4 carried duplications corresponding to the major hot-spot regions for deletions (7.5–8.5 kb from the 5 end of cDNA), whereas two cases contained duplications in a region about 10 kb from the 5 end of cDNA, where causative mutations are reported to be rare. Only 1 case was found to contain a duplication of a region about 1 kb from the 5 end of cDNA, which is the reported duplication prone region. A combination of Southern blot analyses of conventional agarose gel electrophoresis and PFGE was confirmed to be useful, not only for detecting duplications and deletions, per se, but also for identifying carriers in the affected family.     

Action of chondroitinases. I. The mode of action of two chondroitinase-AC preparations of different origin.

The modes of action of two chondroitinases-AC [EC 4.2.2.5] from Arthrobacter aurescens and Flavobacterium heparinum were examined. By comparison of the increase of viscosity and by analyses of digests using paper chromatography and gel filtration, it was shown that the Arthrobacter enzyme (A-Chase) degrades the substrate by a stepwise attack, while the Flavobacterium enzyme (F-Chase) exhibits a more random attack, though the first attack of both enzymes is of endo-type. Further study was carried out of the initial rate and final extent of the enzymic degradation of various mucopolysaccharides. The order of the initial rates at which the native mucopolysaccharides are degraded was similar for both enzymes. For A-Chase, the initial rate and final extent of degradation of modified chondroitin sulfate C or chondroitin methyl ester, however, were low compared with those of F-Chase. These results also suggested that A-Chase degrades the substrate by stepwise attack and F-Chase by random attack.     

Activation of telomerase is induced by a natural antigen in allergen-specific memory T lymphocytes in bronchial asthma.

The function of the immune system is known to be dependent on the cellular differentiation and clonal expansion of allergen-specific lymphocytes. Telomerase, a ribonucleoprotein enzyme, is believed to be essential for the indefinite proliferation of human cells. To clarify whether telomerase is involved in the pathogenesis of immune diseases as well as of malignancies, we investigated the upregulation of telomerase activity in allergen-specific T lymphocytes. Upregulation of telomerase in allergen-sensitized lymphocytes was induced not only by artificial mitogenic stimulations but also by the natural antigen, house dust mite, which causes allergic diseases. Moreover, the upregulation of telomerase activity in memory T cells activated during allergen-specific immune responses might be associated with the enduring allergen-specific atopic propensity in asthmatics.