Traveling Theta Waves along the Entire Septotemporal Axis of the Hippocampus

A topographical relationship exists between the hippocampus-entorhinal cortex and the neocortex. However, it is not known how these anatomical connections are utilized during information exchange and behavior. We recorded theta oscillations along the entire extent of the septotemporal axis of the hippocampal CA1 pyramidal layer. While the frequency of theta oscillation remained same along the entire long axis, the amplitude and coherence between recording sites decreased from dorsal to ventral hippocampus (VH). Theta phase shifted monotonically with distance along the longitudinal axis, reaching ～180° between the septal and temporal poles. The majority of concurrently recorded units were phase-locked to the local field theta at all dorsoventral segments. The power of VH theta had only a weak correlation with locomotion velocity, and its amplitude varied largely independently from theta in the dorsal part. Thus, theta oscillations can temporally combine or segregate neocortical representations along the septotemporal axis of the hippocampus.     

Contribution of FSH and triiodothyronine to the development of circadian clocks during granulosa cell maturation

The involvement of FSH and triiodothyronine (T(3)) in circadian clocks was investigated using immature granulosa cells of ovaries during the progress of cell maturation. Granulosa cells were prepared from preantral follicles of mouse Period2 (Per2)-dLuc reporter gene transgenic rats injected subcutaneously with the synthetic nonsteroidal estrogen diethylstilbestrol. Analysis of the cellular clock of the immature granulosa cells was performed partly using a serum-free culture system. Several bioluminescence oscillations of Per2-dLuc promoter activity were generated in the presence of FSH + fetal bovine serum, but not in the presence of either FSH or serum. As revealed by bioluminescence recording and analysis of clock gene expression, the granulosa cells lack the functional cellular clock at the immature stage, although Lhr was greatly expressed during the period of cell maturation. The granulosa cells gained a strong circadian rhythm of bioluminescence during stimulation with FSH, whereas LH reset the cellular clock of matured granulosa cells. During strong circadian rhythms of clock genes, the Star gene showed significant expression in matured granulosa cells. In contrast, T(3) showed an inhibitory effect on the development of the functional cellular clock during the period of cell maturation. These results indicate that FSH provides a cue for the development of the functional cellular clock of the immature granulosa cells, and T(3) blocks the development of the cellular clock.     

Role for furin in tumor necrosis factor alpha-induced activation of the matrix metalloproteinase/sphingolipid mitogenic pathway

Neutral sphingomyelinase (nSMase), the initial enzyme of the sphingolipid signaling pathway, is thought to play a key role in cellular responses to tumor necrosis factor alpha (TNF-alpha), such as inflammation, proliferation, and apoptosis. The mechanism of TNF-alpha-induced nSMase activation is only partly understood. Using biochemical, molecular, and pharmacological approaches, we found that nSMase activation triggered by TNF-alpha is required for TNF-alpha-induced proliferation and in turn requires a proteolytic cascade involving furin, membrane type 1 matrix metalloproteinase (MT1-MMP), and MMP2, and leading finally to extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and DNA synthesis, in smooth muscle cells (SMC) and fibroblasts. Pharmacological and molecular inhibitors of MMPs (batimastat), furin (alpha1-PDX inhibitor-transfected SMC), MT1-MMP (SMC overexpressing a catalytically inactive MT1-MMP), MMP2 (fibroblasts from MMP2(-/-) mice), and small interfering RNA (siRNA) strategies (siRNAs targeting furin, MT1-MMP, MMP2, and nSMase) resulted in near-complete inhibition of the activation of nSMase, sphingosine kinase-1, and ERK1/2 and of subsequent DNA synthesis. Exogenous MT1-MMP activated nSMase and SMC proliferation in normal but not in MMP2(-/-) fibroblasts, whereas exogenous MMP2 was active on both normal and MMP2(-/-) fibroblasts. Altogether these findings highlight a pivotal role for furin, MT1-MMP, and MMP2 in TNF-alpha-induced sphingolipid signaling, and they identify this system as a possible target to inhibit SMC proliferation in vascular diseases.     

CD4 mimics targeting the mechanism of HIV entry

A structure–activity relationship study was conducted of several CD4 mimicking small molecules which block the interaction between HIV-1 gp120 and CD4. These CD4 mimics induce a conformational change in gp120, exposing its co-receptor-binding site. This induces a highly synergistic interaction in the use in combination with a co-receptor CXCR4 antagonist and reveals a pronounced effect on the dynamic supramolecular mechanism of HIV-1 entry.     

Circulating CD34+ hematopoietic progenitors in the harvesting peripheral blood stem cells; enhancement by recombinant human granulocyte colony-stimulating factor

The number of circulating progenitor cells increases during the period of hematopoietic recovery following myeloablative therapy. These progenitor cells were used for autologous transplantation in order to reconstitute hematopoiesis. As an indicator of the circulating progenitor cells, the number of granulocyte-macrophage colony forming units (CFU-GM), which is measured by means of a long-term cell culture, has been widely used. Recently, a cell surface marker, CD34, which can easily be measured by means of flowcytometry, was found to represent immature hematopoietic progenitor cells, which are very close to stem cells. Therefore, the relationship between the number of CD34 positive cells (CD34⁺ cells) and the number of CFU-GM in the peripheral blood following chemotherapy was studied in 9 patients selected to undergo autotransplantation. The number of peripheral blood CD34+ cells was found to be significantly correlated with that of CFU-GM (r = 0.81). When four out of 9 patients received recombinant human granulocyte-colony stimulating factor (rG-CSF) administration, a significant increase in the release of peripheral blood CD34⁺ cells as well as peripheral blood CFU-GM was observed (P<0.01). Thus, the measurement of CD34⁺ cells is useful for predicting the number of circulating CFU-GM.     

Protein Production in Chemically Defined Medium by Bacillus brevis No. 47

A simple chemically defined medium was devised for exoprotein production by Bacillus brevis No. 47. About 2 mg/ml of proteins was produced in the synthetic medium containing 4% glucose and 1% ammonium sulfate. An essential component of fermentation medium was Ca salt which is required by this organism for assimilating glucose.

Studies on the effects of various medium components on protein production revealed that the conditions appropriate for growth are also suitable for protein accumulation. Some compounds, especially inhibitors of cell wall synthesis and certain detergents, were found to enhance protein production.     

Carbohydrate Composition during Germination and Outgrowth of Ascospores of Saccharomyces cerevisiae

Single spores of Saccharomyces cerevisiae were examined to distinguish changes in the synthesis and degradation of intracellular and wall carbohydrates during germination and outgrowth. Intracellular carbohydrate was fractionated into trehalose and glycogen. Trehalose degradation occurred during germination and outgrowth. The intracellular glycogen was degraded during germination and then synthesized during outgrowth. Wall carbohydrate was fractionated into glycogen, glucan and mannan. The wall glycogen and the KOH-soluble glucan were degraded during germination and then synthesized prior to and during outgrowth, respectively. The major component of the KOH-insoluble glucan in the wall is β-1,3-glucan. The glucan and mannan were synthesized during outgrowth.

The study revealed that the development of a vegetative cell from a spore follows rapid decreases in the amounts of trehalose, glycogen and KOH-soluble glucan during germination, and great increases in the amounts of glycogen, β-1,3-glucan and mannan during outgrowth.     

A Hereditary Enteropathy Caused by Mutations in the SLCO2A1 Gene,Encoding a Prostaglandin Transporter

Previously, we proposed a rare autosomal recessive inherited enteropathy characterized by persistent blood and protein loss from the small intestine as chronic nonspecific multiple ulcers of the small intestine (CNSU). By whole-exome sequencing in five Japanese patients with CNSU and one unaffected individual, we found four candidate mutations in the SLCO2A1 gene, encoding a prostaglandin transporter. The pathogenicity of the mutations was supported by segregation analysis and genotyping data in controls. By Sanger sequencing of the coding regions, 11 of 12 other CNSU patients and 2 of 603 patients with a diagnosis of Crohn’s disease were found to have homozygous or compound heterozygous SLCO2A1 mutations. In total, we identified recessive SLCO2A1 mutations located at seven sites. Using RT-PCR, we demonstrated that the identified splice-site mutations altered the RNA splicing, and introduced a premature stop codon. Tracer prostaglandin E2 uptake analysis showed that the mutant SLCO2A1 protein for each mutation exhibited impaired prostaglandin transport. Immunohistochemistry and immunofluorescence analyses revealed that SLCO2A1 protein was expressed on the cellular membrane of vascular endothelial cells in the small intestinal mucosa in control subjects, but was not detected in affected individuals. These findings indicate that loss-of-function mutations in the SLCO2A1 gene encoding a prostaglandin transporter cause the hereditary enteropathy CNSU. We suggest a more appropriate nomenclature of “chronic enteropathy associated with SLCO2A1 gene” (CEAS).     

Distribution of thymocytes expressing gamma delta receptors in the murine thymus during development

We have examined the appearance of thymocytes expressing gamma delta TCR within the developing thymus by using immunohistochemical techniques and flow cytometry in conjunction with the mAb 3A10, which recognizes a determinant associated with the constant region of the delta-chain. gamma delta+ Cells were first detected at day 16 of gestation, attained maximal levels at day 17 of gestation, and declined thereafter. By using the Ulex europeus agglutinin to identify medullary epithelial cells in situ, we observed a striking colocalization of gamma delta+ thymocytes and U. europeus agglutinin-positive medullary epithelial cells during late fetal and neonatal periods of development. In the thymuses of adult mice, gamma delta+ thymocytes were scattered throughout cortical and medullary areas of the thymus and most concentrated in the subcapsular areas of the thymus. Ultrastructural immunohistochemistry confirmed the close association between medullary thymic epithelial cells and gamma delta+ thymocytes in the neonatal thymus and also showed that some TCR-gamma delta molecules were patched to areas of contact with medullary epithelial cells. In contrast to the cellular distribution of either CD3 molecules or the TCR-alpha beta, where extensive intracellular labeling of thymocytes has been observed, cytoplasmic accumulation of delta-chain was not detected.