Quantitative assessment of changes in cell growth, size and morphology during telomere-initiated cellular senescence in Saccharomyces cerevisiae.

Telomerase-deficient cells of the budding yeast S. cerevisiae experience progressive telomere shortening and undergo senescence in a manner similar to that seen in cultured human fibroblasts. The cells exhibit a DNA damage checkpoint-like stress response, undergo changes in size and morphology, and eventually stop dividing. In this study, a new assay is described that allowed quantitation of senescence in telomerase-deficient est2 cells with applied statistics. Use of the new technique revealed that senescence was strongly accelerated in est2 mutants that had homologous recombination genes RAD51, RAD52 or RAD54 co-inactivated, but was only modestly affected when RAD55, RAD57 or RAD59 were knocked out. Additionally, a new approach for calculating population doublings indicated that loss of growth capacity occurred after approximately 64 generations in est2 cells but only 42 generations in est2 rad52 cells. Phase contrast microscopy experiments demonstrated that senescing est2 cells became enlarged in a time-dependent manner, ultimately exhibiting a 60% increase in cell size. Progressive alterations in physical properties were also observed, including striking changes in light scattering characteristics and cellular sedimentation rates. The results described herein will facilitate future studies of genetic and environmental factors that affect telomere shortening-associated cell senescence rates using the yeast model system.

TFE3 is a bHLH-ZIP-type transcription factor that regulates the mammalian Golgi stress response.

The Golgi stress response is a mechanism by which, under conditions of insufficient Golgi function (Golgi stress), the transcription of Golgi-related genes is upregulated through an enhancer, the Golgi apparatus stress response element (GASE), in order to maintain homeostasis in the Golgi. The molecular mechanisms associated with GASE remain to be clarified. Here, we identified TFE3 as a GASE-binding transcription factor. TFE3 was phosphorylated and retained in the cytoplasm in normal growth conditions, whereas it was dephosphorylated, translocated to the nucleus and activated Golgi-related genes through GASE under conditions of Golgi stress, e.g. in response to inhibition of oligosaccharide processing in the Golgi apparatus. From these observations, we concluded that the TFE3-GASE pathway is one of the regulatory pathways of the mammalian Golgi stress response, which regulates the expression of glycosylation-related proteins in response to insufficiency of glycosylation in the Golgi apparatus.

Modification of gel architecture and TBE/TAE buffer composition to minimize heating during agarose gel electrophoresis.

Agarose gel electrophoresis of DNA and RNA is routinely performed using buffers containing either Tris, acetate, and EDTA (TAE) or Tris, borate, and EDTA (TBE). Gels are run at a low, constant voltage (∼10 V/cm) to minimize current and asymmetric heating effects, which can induce band artifacts and poor resolution. In this study, alterations of gel structure and conductive media composition were analyzed to identify factors causing higher electrical currents during horizontal slab gel electrophoresis. Current was reduced when thinner gels and smaller chamber buffer volumes were used, but was not influenced by agarose concentration or the presence of ethidium bromide. Current was strongly dependent on the amount and type of EDTA used and on the concentrations of the major acid-base components of each buffer. Interestingly, resolution and the mobilities of circular versus linear plasmid DNAs were also affected by the chemical form and amount of EDTA. With appropriate modifications to gel structure and buffer constituents, electrophoresis could be performed at high voltages (20-25 V/cm), reducing run times by up to 3-fold. The most striking improvements were observed with small DNAs and RNAs (10-100 bp): high voltages and short run times produced sharper bands and higher resolution.

Factors affecting chemical-based purification of DNA from Saccharomyces cerevisiae.

Extraction of high molecular weight chromosomal DNA from yeast cells is a procedure that is performed frequently for experiments involving polymerase chain reaction (PCR), Southern blotting and other DNA analysis techniques. We have investigated several parameters affecting DNA yield and quality, using a simple chemical-based purification procedure that was modelled on alkaline lysis methods developed for bacterial cells. The three major steps of the procedure, cell lysis, protein removal and DNA precipitation, were optimized by testing the impacts of several chemicals, including sodium dodecyl sulphate (SDS), sodium hydroxide, Tris buffer, sodium acetate and potassium acetate. Other parameters, such as the effect of elevated temperatures on cell lysis, were also investigated. A rapid, optimized protocol was derived for the purification of DNA from small cell cultures that can be readily digested with restriction enzymes and used as a template for PCR. Average yield was calculated to be approximately 1.7 µg DNA/10(8) cells, which is similar to the theoretical maximum amount obtainable from haploid yeast cells.

Age estimation of museum wool textiles from Ovis aries using deamidation rates utilizing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Cultural heritage contains a large number of precious proteinaceous specimens, such as wool and silk textiles, leather objects, paper, paint, coatings, binders (and associated adhesives), etc. To minimize the degradation of and to preserve these artifacts, it is desirable to understand the fundamental factors that cause their degradation, to identify the deterioration markers that determine their degradation stage and their age, and to use technologies that can provide this information rapidly while consuming a minimal amount of sample. There are several forces that cause protein degradation, including amino acid racemization, protein deamidation, and protein truncation. The purpose of this paper is to study protein deamidation using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for high-throughput dating of museums wool specimens. For proof of concept, several well-dated sheep's wool textiles from museum collections were analyzed. For wool samples aged from the present to ~400 years ago, the deamidation of two asparagine-containing peptides obtained from the tryptic digest of sheep wool were found to behave linearly in time, indicating that they could act as a potential biomarker of aging for wool samples.

[Reappearance of t(12;17)-positive primary myelofibrosis following Ph+ CML cell reduction by imatinib].

A 67-years old woman was referred to our hospital in October 1992 with thrombocytopenia and splenomegaly. A bone marrow biopsy revealed decreased cellularity, with moderately increased reticulin fibrosis and discrete dysmorphic megakaryocytes but no signs of dysplasia in the erythroid or the myeloid lineages. The karyotype of the bone marrow cells was t(12;17) (q24;q11). She was diagnosed as having agnogenic myeloid metaplasia. The patient received only blood transfusions until November 1998 when leukocytosis with immature cells started to appear. The bone marrow aspiration analysis showed increased cellularity and chromosomal analysis demonstrated the presence of t(9;22) (q34;q11) without any t(12;17) (q24;q11) abnormality. Because IFN therapy and oral administration of hydroxyurea did not show any cytological effect, administration of imatinib mesylate was started from December 2001. The Ph-positive cells as demonstrated by the FISH method had decreased to 7% by April 2003. But the t(12;17)(q24;q11) positive clones, which were observed on the first admission, again appeared in the peripheral blood, whereas Ph clones were detected in only one out of 24 cells examined. During the course of treatment with imatinib mesylate for chronic myelogenous leukemia which developed from agnogenic myeloid metaplasia accompanied with t(12;17)(q24;q11) translocation, the co-existence of two clones derived from, possibly, stem cells was identified.

[Gastric relapse of stage I ocular adnexal mucosa-associated lymphoid tissue lymphoma].

A 57-year-old male with right ocular adnexal mucosa-associated lymphoid tissue lymphoma (MALT lymphoma) was successfully treated with local radiation therapy. The gastroendoscopic examination revealed a slight inflammatory change of the gastric mucosa, and the urease test was positive. Eradication therapy against Helicobacter pylori was successfully done, however, the patient developed gastric MALT lymphoma two years after the initial treatment. Southern blot analysis of the immunoglobulin heavy chain gene rearrangement revealed that the lymphoma cells from the ocular adnexal and gastric MALT lymphomas were identical, indicating that the gastric MALT lymphoma was not the primary but the metastatic region from the ocular adnexal MALT lymphoma. Further, immunohistochemical staining demonstrated the expression of integrin alpha4beta 7 on ocular adnexal MALT lymphoma cells, which is essential for the adhesion of lymphocytes to gastrointestinal mucosa. These results indicate that ocular adnexal MALT lymphoma cells can metastasize to the stomach, depending on the adhesional function of integrin alpha4 beta7.

[Factor XI deficiency caused by a mutation of Gly400Val].

The patient described herein is a 69-year-old Japanese woman with a history of excessive bleeding after left heminephrectomy for a malignant renal tumor at 31 years of age. Her parents, who do not have abnormal bleeding, are first cousins. Her factor XI activity was less than 1% of normal with an prolonged activated partial thromboplastin time (APTT) of 74.3 seconds. Analysis of the patient's factor XI genes revealed homozygosity for a valine substituting for the wild-type glycine at amino acid 400 (Gly400Val). The patient has two children, neither of whom has abnormal bleeding and whose factor XI activities are 62% and 57% of normal, with APTT levels within normal limits in both cases. We herein report on a Japanese family with factor XI deficiency caused by Gly400Val mutation.

[Superior mesenteric and portal vein thrombosis in a polycythemia vera patient with JAK2 mutation].

A 47-year-old woman was admitted to our hospital in December 1994 with polycythemia. The patient's red blood cell volume was 33 ml/kg and bone marrow cytology was able to rule out other myeloproliferative diseases such as chronic myelogenous leukemia, essential thrombocytosis and myelofibrosis. The patient was diagnosed as having polycythemia vera. She had undergone only phlebotomy until 1999 when the thrombocytosis appeared, subsequent to which she was treated with oral hydroxyurea. However, in March 2006, she developed upper abdominal pain and was admitted to our hospital on March 14th, 2006. Computed tomography scan revealed thromboses in the portal and superior mesenteric veins. Anticoagulation therapy delivered intravenously via the superior mesenteric vein dramatically improved her symptoms and liver function. She is currently on anticoagulation therapy in our outpatient clinic.

Crystallization and preliminary X-ray crystallographic studies of pig heart carbonyl reductase.

Pig heart carbonyl reductase (PHCR), which belongs to the short-chain dehydrogenase/reductase (SDR) family, has been crystallized by the hanging-drop vapour-diffusion method. Two crystal forms (I and II) have been obtained in the presence of NADPH. Form I crystals belong to the tetragonal space group P4(2), with unit-cell parameters a = b = 109.61, c = 94.31 A, and diffract to 1.5 A resolution. Form II crystals belong to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 120.10, c = 147.00 A, and diffract to 2.2 A resolution. Both crystal forms are suitable for X-ray structure analysis at high resolution.

[Anaphylactic reaction to oxaliplatin--a case of colon cancer].

Oxaliplatin (L-OHP) is a new third-generation platinum which is efficacious in treating advanced unresectable recurrent colorectal cancer as a first-line regimen. The marketing authorization was given in Japan in March, 2005. Its increased use has resulted in rare serious adverse effects, including anaphylactic shock. We experienced a case that developed anaphylactic shock by L-OHP. We report a 69-year-old man who was treated for recurrent colorectal cancer who underwent systemic chemotherapy with FOLFOX 4. After eight cycles he developed severe L-OHP associated neuropathy and lung metastases was a progressive tendency. The FOLFOX 4 regimen was discontinued and another modality, FOLFIRI regimen, was used. After eight cycles of FOLFIRI regimen, lung and liver metastases showed progressive disease for response assessment by RECIST criteria. Although a patient was stopped L-OHP for neurotoxicity, neuropathy was disappeared after 4 months interval. Therefore, we reintroduced L-OHP, FOLFOX 4 regimen. Anaphylactic shock occurred in the second cycle of reintroduction of the FOLFOX 4 regimen (total 10 cycles), 30 minutes after infusion of L-OHP. L-OHP infusion was immediately withdrawn and he was treated with intravenous hydroxyzine hydrochloride and methylprednisolone. The anaphylaxis symptoms resolved in 30 minutes. Chemotherapy based on L-OHP for unresectable recurrence colorectal cancer causes anaphylactic shock as a rare severe complication. The prediction factor is not proved. We should take steps for early detection of anaphylactic reaction and perform the appropriate treatment.

Interleukin-2 gene transfer potentiates the alpha-galactosylceramide-stimulated antitumor effect by the induction of TRAIL in NKT and NK cells in mouse models of subcutaneous and metastatic carcinoma.

Alpha-Galactosylceramide (alpha-GalCer) is a potent CD1d ligand that activates natural killer like T-cells (NKT), leading to the production of helper T (Th) 1 and Th2 cytokines that mediate various immunemodulatory and antitumor effects. Here, we determined whether the administration of adenovirus-vector-encoding mouse interleukin-2 (AdmIL-2) can augment the antitumor effect of alpha-GalCer on subcutaneous and metastatic tumors in mice. Mice were intraperitoneally injected with alpha-GalCer on days 7, 10 and 13 after tumor inoculation, with or without intratumoral injection of AdmIL-2 on day 7. alpha-GalCer treatment increased the serum levels of interferon-gamma, while intratumoral injection of AdmIL-2 elevated serum IL-2 levels. A combination of alpha-GalCer and AdmIL-2 (alpha-GalCer/AdmIL-2) inhibited the in vivo tumor growth and improved the survival of tumor-bearing mice, as compared to the use of a single agent. Experiments on spontaneous metastasis models revealed that alpha-GalCer/AdmIL-2 reduced lung metastasis and prolonged survival, as compared to control groups. In addition, the splenic and liver mononuclear cells from mice treated with alpha-GalCer/AdmIL-2 showed enhanced cytolytic activity against NK-sensitive YAC-1 and NK-resistant 3LL tumors. Moreover, alpha-GalCer/AdmIL-2 treatment expanded the absolute numbers of lung and liver NK, NKT and T-cells as well as the TNF-related apoptosis-inducing ligand (TRAIL) expression of these cells. This study shows the efficacy of alpha-GalCer/AdmIL-2 immunomodulatory therapy, and provides a cellular mechanism on how it exerts the antitumor effects.

Successful treatment of chronic myeloproliferative disease-unclassifiable (CMPD-U) with no chromosomal abnormalities by imatinib mesylate.

We report a chronic myeloproliferative disease-unclassifiable (CMPD-U) patient who achieved hematological remission following imatinib mesylate (imatinib). Chromosomal and molecular analyses demonstrated no genetic abnormalities of c-abl, bcr-abl, c-kit or platelet-derived growth factor receptor (PDGFR) genes from hematopoietic cells. Although there has been one report of CMPD-U patient with chromosomal abnormalities of the PDGFR gene having complete hematologic responses upon treatment with imatinib, there have not been similar reports of patients without chromosomal abnormalities. This is the first case report of a CMPD-U patient with no chromosomal abnormalities who completely responded to treatment with imatinib.

Successful treatment of autoimmune pancreatitis complicated with autoimmune thrombocytopenia and interstitial pneumonia by prednisolone.

We report the second patient diagnosed with autoimmune pancreatitis complicated with autoimmune thrombocytopenia and interstitial pneumonia. The patient was treated with prednisolone and responded favorably. We demonstrated that anti-platelet (PLT) antibody of the patient was IgG4 and that it may react with HLA, not specific antigen, on both pancreas and PLT.

Optical oxygen-sensing properties of porphyrin derivatives anchored on ordered porous aluminium oxide plates.

An optical oxygen-sensing activity of anchored porphyrin derivatives on ordered porous aluminium oxide plates was studied in relevance to development of new oxygen-sensing systems. Porphyrin derivatives, 5,10,15,20-tetrakis(4-carboxylundecane-1-oxy)porphyrin, 5-[4-(11-carboxylundecane-1-oxy)-10,15,20-triphenyl]porphyrin, 5-(4-carboxylphenyl)-10,15,20-triphenylporphyrin, and their platinum complexes, 5,10,15,20-tetrakis(4-carboxylundecane-1-oxy)porphyrinatoplatinum(II), 5-[4-(11-carboxylundecane-1-oxy)-10,15,20-triphenyl]porphyrinatoplatinum(II), 5-(4-carboxylphenyl)-10,15,20-triphenylporphyrinatoplatinum(II), were synthesized and anchored by an equilibrium adsorption method on aluminium oxide plates, which were prepared by an anodic oxidation. The excitation spectra of the porphyrin-anchored layers showed a broadened and blue-shifted Soret band compared with the corresponding porphyrins in DMSO. The luminescence intensity decreased with increasing oxygen concentrations. The oxygen-sensing ability estimated from I(0)/I(100) (I(0) and I(100) denote the luminescence intensity in 0 and 100% oxygen) was 9.08, 6.78, 8.71, 81.9, 35.5, and 39.1, which are greater than those of corresponding porphyrin derivatives in DMSO under the measured conditions, and indicates the remarkable enhancement effect of platinum(II). Non-linear Stern-Volmer plots were well fitted by the two component system to give the oxygen-sensitive constant (K(SV1)/%(-1)), the oxygen-insensitive constant (K(SV2)/%(-1)), and the former contribution (f(1)): 0.232, 3.32 x 10(-2), and 0.642; 0.141, 2.05 x 10(-2), and 0.687; 0.143, 1.05 x 10(-2), and 0.882; 17.3, 7.04 x 10(-3), and 0.980; 10.2, 1.43 x 10(-2), and 0.935; 16.3, 8.35 x 10(-3), and 0.954. The response time for the change of the atmospheric gas from argon to oxygen was 9.4 s, 12.5 s, 9.6 s, 5.0 s, 8.9 s, and 4.6 s, indicating the shortening effect of platinum. The reverse effect of platinum was observed in the change from oxygen to argon: 15.5 s, 17.0 s, 20.8 s, 667.4 s, 590.1 s, and 580.4 s, indicating the specific interaction of oxygen to the platinum(II) center.