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151.
A species ofMycosphaerella with aPseudocercospora anamorph was collected on overwintered fallen leaves of Japanese beech,Fagus crenata. Based on comparison of morphology withMycosphaerella species on Fagaceae, the fungus was newly described asMycosphaerella buna. ThePseudocercospora anamorph derived from a single ascospore of the fungus was morphologically identical to an endophytic anamorph isolated from
asymptomatic living leaves of Japanese beech.
Contribution No. 150, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba 相似文献
152.
The glucose isomerase from Streptomyces olivaceoviridis E-86 was purified by chromatographic procedures, showing one single protein band in the SDS-PAGE. The enzyme had high acid stability, and there was no loss in enzyme activity at pH 5.0 after incubation at 60 degrees C for 30 hr. The enzyme had sufficients activity at 60 degrees C, pH 5.5, (which is the reaction condition for a single-step process with a glucoamylase from A. niger), and at 58 degrees C, pH 6.0, (condition with a glucoamylase from R. niveus). By using this acid-stable glucose isomerase, a single-step process to produce high-fructose corn sweetener (HFCS) from liquefied starch was formed without any reductant or other reagents for enzyme stabilization. The HFCS produced was about fifty percent fructose and less than 1.5% unknown oligosaccharides. 相似文献
153.
M Kaneko 《Mutation research》1984,131(3-4):157-161
The rate of removal of DNA adducts of several benzo[a]pyrene metabolites from nuclear DNA was compared by introducing a microsome-activating system in human fibroblast cells. Confluent human fibroblasts were exposed to benzo[a]pyrene in the presence of a microsomal activating system and DNA adducts were formed in the nuclear DNA. The adducts present in DNA were determined after 1 h of incubation and 48 h later. There was no difference in the rate of removal between 7S- and 7R -N2-[10-(7 beta, 8 alpha-trihydroxy-7,8,9,10- tetrahydrobenzo[a]pyrene)yl]deoxyguanosine, 7R -N2-[10(7beta, 8 alpha, 9 beta-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene)yl]deoxyguanosine and the covalent adduct of 9-hydroxybenzo[a]pyrene-4,5-epoxide to guanosine. This finding does not agree with the idea that metabolites forming 'persistent DNA adducts' are always responsible for the carcinogenicity of their parent compound. 相似文献
154.
Single-cell pulsed-field gel electrophoresis (SCPFGE) with dual electrode pairs was developed to detect the early stage of DNA fragmentation in human sperm. The motile sperm were purified by the commonly used density-gradient centrifugation technique and subsequent swim-up. The sperm were embedded in a thin film of agarose containing bovine trypsin (20 μg/mL) and were then lysed. Prior to SCPFGE, proteolysis of DNA-binding components, such as protamine and the nuclear matrix was essential to separate the long chain fibers from the fibrous and granular fragments derived from a single nucleus. The overall electrophoretic profiles elucidated the course of DNA fragmentation. A few large fibrous fragments were observed at the beginning of the process, however, as the fragmentation advanced, the long chain fibers decreased and shortened, and, conversely, the granular fragments increased until finally almost all the DNA was shredded. Although the ejaculate contained sperm with heterogeneous stages, the purified motile sperm exhibited several dozens of uniformly elongated fibers arising from the tangled DNA at the origin, whereas a part of these fibers gave rise to fibrous fragments beyond the tip of the elongated fibers, and their numbers and sizes varied among the sperm. Conventional intra-cytoplasmic sperm injection (ICSI) usually depends on intra-operative light microscopic observation to select a sperm for injection. The present results revealed that sperm motility could not give full assurance of DNA integrity. SCPFGE is likely to serve an important role in the preoperative differential diagnosis to determine the competence of the sperm population provided for injection. 相似文献
155.
Osamu Kaneko Lucy Gong Jingli Zhang Johanna K. Hansen Raffit Hassan Byungkook Lee Mitchell Ho 《The Journal of biological chemistry》2009,284(6):3739-3749
Ovarian cancer and malignant mesothelioma frequently express both
mesothelin and CA125 (also known as MUC16) at high levels on the cell surface.
The interaction between mesothelin and CA125 may facilitate the implantation
and peritoneal spread of tumors by cell adhesion, whereas the detailed nature
of this interaction is still unknown. Here, we used truncated mutagenesis and
alanine replacement techniques to identify a binding site on mesothelin for
CA125. We examined the molecular interaction by Western blot overlay assays
and further quantitatively analyzed by enzyme-linked immunosorbent assay. We
also evaluated the binding on cancer cells by flow cytometry. We identified
the region (296–359) consisting of 64 amino acids at the N-terminal of
cell surface mesothelin as the minimum fragment for complete binding activity
to CA125. We found that substitution of tyrosine 318 with an alanine abolished
CA125 binding. Replacement of tryptophan 321 and glutamic acid 324 with
alanine could partially decrease binding to CA125, whereas mutation of
histidine 354 had no effect. These results indicate that a
conformation-sensitive structure of the region (296–359) is required and
sufficient for the binding of mesothelin to CA125. In addition, we have shown
that a single chain monoclonal antibody (SS1) recognizes this CA125-binding
domain and blocks the mesothelin-CA125 interaction on cancer cells. The
identified CA125-binding domain significantly inhibits cancer cell adhesion
and merits evaluation as a new therapeutic agent for preventing or treating
peritoneal malignant tumors.Ovarian cancer largely is confined to the peritoneal cavity for much of its
natural history (1). Peritoneal
mesothelioma is a highly invasive tumor originating from the mesothelial
linings of the peritoneum (2).
The development of effective drug regimens against ovarian cancer and
mesothelioma has proven extremely difficult.Mesothelin was first identified in 1992 by the monoclonal antibody
(mAb)2 K1 that was
generated by the immunization of mice with human ovarian carcinoma (OVCAR-3)
cells (3). The mesothelin gene
encodes a 71-kDa precursor protein that is processed to a 40-kDa protein
termed mesothelin, which is a glycosylphosphatidylinositol (GPI)-anchored
glycoprotein present on the cell surface
(4). Mesothelin is a
differentiation antigen that is present on a restricted set of normal adult
tissues such as the mesothelium. In contrast, it is overexpressed in a variety
of cancers including mesothelioma, ovarian cancer, and pancreatic cancer
(5). In addition, mesothelin is
also expressed on the surface of non-small cell lung cancer cells
(6,
7), especially most lung
adenocarcinomas (8).We and others have shown that mesothelin is shed from tumor cells
(9,
10), and antibodies specific
for mesothelin are elevated in the sera of patients with mesothelioma and
ovarian cancer (11). Shed
serum mesothelin has been approved by the United States Food and Drug
Administration (FDA) as a new diagnostic biomarker in mesothelioma. In a Phase
I clinical study of an intrapleural interferon-β gene transfer using an
adenoviral vector in patients with mesotheliomas, we found that antitumor
immune responses targeting mesothelin were elicited in several patients
(12). A recent study indicated
that anti-mesothelin antibodies and circulating mesothelin relate to the
clinical state in ovarian cancer patients
(13). Pastan and colleagues
(14) developed an immunotoxin
(SS1P) with a Fv for mesothelin. Two Phase I clinical trials were completed at
the National Cancer Institute (National Institutes of Health, Bethesda, MD)
and there was sufficient antitumor activity of SS1P to justify a Phase II
trial. A chimeric antibody containing the mouse SS1 Fv for mesothelin was also
developed and is currently examined in a Phase I clinical trial for ovarian
cancer, mesothelioma, pancreatic cancer, and non-small cell lung cancer
(15).Mucins are heavily glycosylated proteins found in the mucus layer or at the
cell surface of many epitheliums
(16). There are two
structurally distinct families of mucins, secreted and membrane-bound forms.
CA125 (also known as MUC16) was first identified in 1981 by OC125, a mAb that
had been developed from mice immunized with human ovarian cancer cells
(17). The first cDNA clones
were reported in 2001 (18,
19). CA125 is a very large
membrane-bound cell surface mucin, with an average molecular mass between 2.5
and 5 million daltons. It is also heavily glycosylated with both
O-linked and N-linked oligosaccharides
(20). The peptide backbone of
CA125 is composed of the N-terminal region, extensive Ser/Thr/Pro-rich tandem
repeats (TR) with 156 amino acids each with both N- and
O-glycosylations, a SEA domain with high levels of
O-glycosylation and a C-terminal region with a short cytoplasmic tail
(19). The SEA domain was first
identified as a module commonly found in sea urchin sperm protein,
enterokinase and agrin (21,
22). The significance of the
SEA domain in CA125 is not clear.CA125 was originally used as a biomarker in ovarian cancer due to its high
expression in ovarian carcinomas and that it is shed into the serum
(23). A majority (88%) of
mesotheliomas are also CA125 positive on the cell membrane
(24). It was shown that 25% of
peritoneal mesotheliomas have high CA125 expression
(25). The intensity of CA125
membranous expression is indistinguishable between ovarian carcinomas and
peritoneal mesotheliomas. Gene expression analysis using the SAGE tag data
base has shown that mesothelioma has the second highest co-expression of CA125
and mesothelin after ovarian cancer
(26). Rump and colleagues
(26) have shown that
mesothelin binds to CA125 and that this interaction may mediate cell adhesion.
Scholler et al. (27)
recently showed that CA125/mesothelin-dependent cell attachment could be
blocked with anti-CA125 antibodies. Because mesothelin is present on
peritoneal mesothelium, there may be an important role for the
mesothelin-CA125 interaction in the tumorigenesis of ovarian cancer and
mesothelioma in the peritoneal cavity. The mesothelin binding site on CA125
may lie within the 156-amino acid TR units, indicating multimeric binding of
mesothelin to CA125. It has been found that the extraordinarily abundant
N-glycans on CA125, presumably in the TR region, are required for
binding to both glycosylated and non-glycosylated mesothelin
(28).Here, we identified the binding site of CA125 on mesothelin by use of
truncated mutagenesis and alanine replacement approaches. We measured binding
qualitatively by Western blot overlay assays and quantitatively by
enzyme-linked immunosorbent assay (ELISA). We also evaluated the interaction
of CA125 and mesothelin on cancer cells by flow cytometry. Furthermore, we
have shown that a single chain mAb (SS1) recognized the CA125-binding domain
and blocked the mesothelin-CA125 interaction on cancer cells. The identified
CA125-binding domain-Fc fusion protein also significantly inhibited cancer
cell adhesion. Our results suggest that conformation-sensitive structures of
the region (296–359) are required and sufficient for specific binding of
mesothelin to CA125. The domain proteins or the antibodies that block the
mesothelin-CA125 interaction merit evaluation as new therapeutic agents in
treating peritoneal malignant tumors. 相似文献
156.
157.
Chloroplasts consist of six morphologically distinct compartments. Each compartment has a specific set of polypeptides that perform distinct biochemical functions. We report here the identification of a membrane-associated protein with a novel localization. This protein was synthesized as a 37 kDa precursor and was processed to a mature protein of 30 kDa after being imported into isolated pea chloroplasts. Fractionation of chloroplasts showed that the 30 kDa mature protein was associated with both of the envelope membranes as well as with thylakoid membranes. Immunocyto-chemical localization of the 30 kDa protein revealed that the protein occurred in clusters in the vicinity of both the envelope and the thylakoid. Possible functions of this 30 kDa protein, inferred from its novel localization pattern, are discussed.Abbreviations CAB
light-harvesting chlorophyll a/b-binding protein of photosystem II
- prCAB
precursor protein to CAB
- SS
small subunit of ribulose-1,5-bisphosphate carboxylase
- prSS
precursor protein to SS
- RCF
relative centrifugation force 相似文献
158.
Yosuke Demizu Yu‐u Yabuki Mitsunobu Doi Yukiko Sato Masakazu Tanaka Masaaki Kurihara 《Journal of peptide science》2012,18(7):466-475
A pair of l ‐leucine (l ‐Leu) and d ‐leucine (d ‐Leu) was incorporated into α‐aminoisobutyric acid (Aib) peptide segments. The dominant conformations of four hexapeptides, Boc‐l ‐Leu‐Aib‐Aib‐Aib‐Aib‐l ‐Leu‐OMe (1a), Boc‐d ‐Leu‐Aib‐Aib‐Aib‐Aib‐l ‐Leu‐OMe (1b), Boc‐Aib‐Aib‐l ‐Leu‐l ‐Leu‐Aib‐Aib‐OMe (2a), and Boc‐Aib‐Aib‐d ‐Leu‐l ‐Leu‐Aib‐Aib‐OMe (2b), were investigated by IR, 1H NMR, CD spectra, and X‐ray crystallographic analysis. All peptides 1a,b and 2a,b formed 310‐helical structures in solution. X‐ray crystallographic analysis revealed that right‐handed (P) 310‐helices were present in 1a and 1b and a mixture of right‐handed (P) and left‐handed (M) 310‐helices was present in 2b in their crystalline states. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
159.
Okada-Katsuhata Y Yamashita A Kutsuzawa K Izumi N Hirahara F Ohno S 《Nucleic acids research》2012,40(3):1251-1266
Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that detects and degrades mRNAs containing premature termination codons (PTCs). SMG-1-mediated Upf1 phosphorylation takes place in the decay inducing complex (DECID), which contains a ribosome, release factors, Upf1, SMG-1, an exon junction complex (EJC) and a PTC-mRNA. However, the significance and the consequence of Upf1 phosphorylation remain to be clarified. Here, we demonstrate that SMG-6 binds to a newly identified phosphorylation site in Upf1 at N-terminal threonine 28, whereas the SMG-5:SMG-7 complex binds to phosphorylated serine 1096 of Upf1. In addition, the binding of the SMG-5:SMG-7 complex to Upf1 resulted in the dissociation of the ribosome and release factors from the DECID complex. Importantly, the simultaneous binding of both the SMG-5:SMG-7 complex and SMG-6 to phospho-Upf1 are required for both NMD and Upf1 dissociation from mRNA. Thus, the SMG-1-mediated phosphorylation of Upf1 creates a binding platforms for the SMG-5:SMG-7 complex and for SMG-6, and triggers sequential remodeling of the mRNA surveillance complex for NMD induction and recycling of the ribosome, release factors and NMD factors. 相似文献
160.
Identification of Tau and MAP2 as novel substrates of Rho-kinase and myosin phosphatase 总被引:2,自引:0,他引:2
Amano M Kaneko T Maeda A Nakayama M Ito M Yamauchi T Goto H Fukata Y Oshiro N Shinohara A Iwamatsu A Kaibuchi K 《Journal of neurochemistry》2003,87(3):780-790
Rho-kinase and myosin phosphatase are implicated in the phosphorylation-state of myosin light chain downstream of Rho, which is thought to induce smooth muscle contraction and stress fibre formation in non-muscle cells. Here, we found that microtubule-associated proteins, Tau and MAP2, interacted with the myosin-binding subunit (MBS) of myosin phosphatase, and were the possible substrates of both Rho-kinase and myosin phosphatase. We determined the phosphorylation sites of Tau (Thr245, Thr377, Ser409) and MAP2 (Ser1796) by Rho-kinase. We also found that Rho-kinase phosphorylated Tau at Ser262 to some extent. Phosphorylation by Rho-kinase decreased the activity of Tau to promote microtubule assembly in vitro. Substitutions of Ala for Ser/Thr at the phosphorylation sites of Tau (Tau-AAA) did not affect the activity to promote microtubule assembly, while substitutions of Asp for Ser/Thr (Tau-DDD), which are expected to mimic the phosphorylation-state of Tau, slightly reduced the activity. When Tau, or mutated forms of Tau, were expressed in PC12 cells, followed by treatment with cytochalasin D, they promoted extension of the cell process in a cytochalasin-dependent manner. However, Tau-DDD showed the weaker activity in this capacity than wild-type Tau or Tau-AAA. These results suggest that the phosphorylation-state of these residues of Tau affects its activity both in vitro and in vivo. Thus, it is likely that the Rho-kinase/MBS pathway regulates not only the actin-myosin system but also microtubule dynamics. 相似文献