Serial section method for cytofluorometric determinations of the DNA content of component cells of the human cochlea

We describe a method for measuring the DNA content of the component cells of the organ of Corti using serial sections of human cochleae obtained at autopsy. Cochleae were fixed in Carnoy's solution and embedded in Acrytron E, a water-miscible methacrylate resin. A procedure was developed to reduce the background fluorescence in methacrylate-embedded sections; the resin was pretreated with ion-exchange resin (Amberlite IRA-410). Experiments showed that pretreatment reduce the background fluorescence practically to zero. Seventy 3 microns-thick serial sections were prepared on fluorescence free glass slides and stained with azocarmin G and acriflavine-Feulgen. After postirradiation using blue excitation light, the amount of Feulgen-DNA present in the target nucleus in each section was determined using a microfluorometer. The amount of DNA in the entire nucleus was determined by adding together the DNA content of the segments of the nucleus. The characteristic appearance of the organ of Corti made it easy to detect these cells; under green excitation light the cells of this organ exhibited red cytoplasmic azocarmin-G fluorescence. Due to the relatively wide internuclear spaces, cytofluorometry fo individual nuclei could be performed without interference from the neighboring cells. Our technique using serial sections allowed us to measure the DNA content of individual cells and obtain histological information about particular cells and their neighboring cells. Several polyploid cells were found among the Hensen's cells in the cochlea, while all other component cells of the organ of Corti were diploid.     

Effect of adoptive T-cell immunotherapy on immunological parameters and prognosis in patients with advanced pancreatic cancer

Background aimsImmunotherapy is effective for many types of cancer, but its benefits in advanced pancreatic cancer, which has a poor prognosis, are not well established. In this study, the authors examined the effects of adoptive T-cell immunotherapy (ATI) on immune cell profiles and prognosis in patients with unresectable advanced pancreatic cancer.MethodsSeventy-seven patients with unresectable advanced pancreatic cancer were treated with six cycles of αβ T cells alone or in combination with chemotherapy or chemoradiation. Immune cell profiles in peripheral blood samples obtained before and after treatment were comprehensively evaluated by flow cytometry. Furthermore, associations between changes in immune cell frequencies and prognosis were determined.ResultsATI prolonged survival to 18.7 months compared with previous estimates of 6.2–11.1 months for patients treated with chemotherapy alone. ATI decreased CD3+CD4+CD8? T cell frequency in peripheral blood and increased CD3+CD4?CD8+ T cell frequency. An increase in CD3+ T cells and CD3+TCRγδ? T cells in peripheral blood after treatment was associated with a good prognosis.ConclusionsATI altered the immune profile in peripheral blood, including CD3+CD4?CD8+ T cells, and improved prognosis in pancreatic cancer.     

Purification and characterization of an alpha-amylase of Pichia burtonii isolated from the traditional starter "murcha" in Nepal

Among more than 20 yeast strains isolated from the traditional starter "murcha" in Nepal, we characterized a yeast that might be involved in saccharification. This strain, identified as Pichia burtonii, produced an extracellular amylolytic enzyme when cultured in the presence of starch in the medium. Since no amylase secreted by P. burtonii has yet been reported, we purified the enzyme and determined its N-terminal amino acid sequence. Together with the results of a hydrolyzing activity assay toward various substrates, it was found to be an alpha-amylase. The purified enzyme, named Pichia burtonii alpha-amylase (PBA), was a glycoprotein with an apparent molecular mass of 51 kDa. Enzyme activity was optimal at pH 5.0 at 40 degrees C. The enzyme retained 80% of its original activity after incubation under the optimal pH condition at 50 degrees C for 30 min. The activity was inhibited by metal ions such as Cd(2+), Cu(2+), Hg(2+), Al(3+), and Zn(2+).     

Alteration of gene expression in the colon of colorectal cancer model rat by dietary sodium gluconate

Butyrate induces apoptosis of various cancer cell lines in a p53-independent manner and inhibits the proliferation of cancer cells. In a previous report, we reported a significant reduction in tumor incidence in rat colon as a result of dietary sodium gluconate (GNA). The stimulation of apoptosis through enhanced butyrate production in the large intestine was involved in the antitumorigenic effect of GNA. In the present study, a cDNA microarray analysis was performed to investigate the particular mechanism involved in the antitumorigenic effect of GNA. Some up-regulated genes suggested by microarray analysis were further evaluated using real-time PCR. A microarray revealed that GNA regulates the expression of retinoic acid receptor (RAR) and retinoid X receptor (RXR), and several genes known as the target of retinoids in cancer cells. In other words, the antitumorigenic effect of GNA may involve the regulation of the retinoid signaling pathway by butyrate in a retinoid-independent manner.     

Crystallization and preliminary crystallographic analysis of the N-terminal domain of PriA from Escherichia coli

PriA, a DEXH-type DNA helicase, binds specifically to the 3' end of DNA through its N-terminal domain, and is a candidate sensor protein that recognizes arrested DNA replication forks in bacteria. We crystallized an N-terminal fragment of PriA in the absence and the presence of oligonucleotides to elucidate the structural basis for the specific recognition of the 3' terminus of DNA.     

Differential regulation of osteoclastogenesis by Notch2/Delta-like 1 and Notch1/Jagged1 axes

Introduction

Osteoclastogenesis plays an important role in the bone erosion of rheumatoid arthritis (RA). Recently, Notch receptors have been implicated in the development of osteoclasts. However, the responsible Notch ligands have not been identified yet. This study was undertaken to determine the role of individual Notch receptors and ligands in osteoclastogenesis.

Methods

Mouse bone marrow-derived macrophages or human peripheral blood monocytes were used as osteoclast precursors and cultured with receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) to induce osteoclasts. Osteoclasts were detected by tartrate-resistant acid phosphatase (TRAP) staining. K/BxN serum-induced arthritic mice and ovariectomized mice were treated with anti-mouse Delta-like 1 (Dll1) blocking monoclonal antibody (mAb).

Results

Blockade of a Notch ligand Dll1 with mAb inhibited osteoclastogenesis and, conversely, immobilized Dll1-Fc fusion protein enhanced it in both mice and humans. In contrast, blockade of a Notch ligand Jagged1 enhanced osteoclastogenesis and immobilized Jagged1-Fc suppressed it. Enhancement of osteoclastogenesis by agonistic anti-Notch2 mAb suggested that Dll1 promoted osteoclastogenesis via Notch2, while suppression by agonistic anti-Notch1 mAb suggested that Jagged1 suppressed osteoclastogenesis via Notch1. Inhibition of Notch signaling by a gamma-secretase inhibitor suppressed osteoclastogenesis, implying that Notch2/Dll1-mediated enhancement was dominant. Actually, blockade of Dll1 ameliorated arthritis induced by K/BxN serum transfer, reduced the number of osteoclasts in the affected joints and suppressed ovariectomy-induced bone loss.

Conclusions

The differential regulation of osteoclastogenesis by Notch2/Dll1 and Notch1/Jagged1 axes may be a novel target for amelioration of bone erosion in RA patients.     

Evaluation of an oral function promotion programme for the independent elderly in Japan

Objectives: The purpose of this study was to provide an oral function promotion programme for the independent elderly and evaluate the changes in oral health status and oral function. Background: Few studies have scientifically analysed and evaluated the effectiveness of oral function promotion programmes provided for the independent elderly. Materials and methods: The subjects were independent elderly females (mean age: 74.6 ± 6.3) recruited from senior citizens’ centres in Tokyo. The intervention group (n = 79) received a 3‐month oral function promotion programme, which included facial muscle and tongue exercises and salivary gland massages. The control group (n = 62) did not receive this programme. Results: In the intervention group, the tongue coating scores decreased and the organoleptic score of oral malodour fell. The amount of food debris in the oral cavity decreased and the tongue dryness improved. Furthermore, the salivary flow rate increased. The length of time for maintaining the tongue in the forward position increased from 11.2 s to 18.7 s, and the number of times for moving the tip of the tongue in a clockwise circular motion, counter‐clockwise circular motion and side‐to‐side motion within 30 s, increased from 14.5 to 20.6, 14.5 to 20.2, and 17.2 to 23.3 respectively. The number of times for movement of the lips significantly improved from 23.0 to 28.8 and the pronunciation of words was observed to be clearer. Conclusion: An oral function promotion programme was effective in improving the oral health status and oral function of an independent elderly population.     

Archetype JC virus efficiently propagates in kidney-derived cells stably expressing HIV-1 Tat

Pathogenic JCV with rearranged regulatory regions (PML-type) causes PML, a demyelinating disease, in the brains of immunocompromised patients. On the other hand, archetype JCV persistently infecting the kidney is thought to be converted to PML-type virus during JCV replication in the infected host under immunosuppressed conditions. In addition, Tat protein, encoded by HIV-1, markedly enhances the expression of a reporter gene under control of the JCV late promoter.
In order to examine the influence of Tat on JCV propagation, we used kidney-derived COS-7 cells, which only permit archetype JCV, and established COS-tat cells, which express HIV-1 Tat stably. We found that the extent of archetype JCV propagation in COS-tat cells is significantly greater than in COS-7 cells. On the other hand, COS-7 cells express SV40 T antigen, which is a strong stimulator of archetype JCV replication. The expression of SV40 T antigen was enhanced by HIV-1 Tat slightly according to real-time RT-PCR, this was not closely related to JCV replication in COS-tat cells. The efficiency of JCV propagation depended on the extent of expression of functional Tat. To our knowledge, this is the first report of increased production of archetype JCV in a culture system using cell lines stably expressing HIV-1 Tat. We propose here that COS-tat cells are a useful tool for studying the role of Tat in archetype JCV replication in the development of PML.     

Direct Optical Microscopic Observation of the Microtubule Polymerization Intermediate Sheet Structure in the Presence of Gas7

The process of microtubule elongation is thought to consist of two stages—formation of a tubulin sheet structure and its closure into a tube. However, real-time observation of this process has been difficult. Here, by utilizing phospho-tau binding protein Gas7 (growth-arrest-specific protein 7), we visualized the polymer transformation process by dark-field microscopy. Upon elongation, thin and flexible structures, often similar to a curved hook, appeared at the end of microtubules. Electron microscopic observations supported the idea that these flexible structures are tubulin sheets. They maintained their length until they gradually became thick and rigid beginning in the central portion, resulting in straight microtubules. In the absence of Gas7, the sheet-like structure was rarely observed; moreover, when observed, it was fragile and engaged in typical dynamic instability. With Gas7, no catastrophe was observed. These results suggest that Gas7 enhances microtubule polymerization by stabilizing sheet intermediates and is a useful tool for analyzing microtubule transformation.