Angiogenic endothelium-specific nestin expression is enhanced by the first intron of the nestin gene

Nestin is a member of intermediate filaments abundantly expressed in neural stem cells and glioblastomas. The nestin gene has four exons and three introns, and neural cell-specific expression is regulated by the second intron. We previously reported that nestin was invariably detected in the tumor endothelium in gliomas even though tumor cells were negative for nestin. In the present study, we further confirmed nestin immunostaining in tumor endothelium of a variety of common cancers, including lung, stomach, colon, and cervical carcinomas. We examined an endothelium-specific regulator using human umbilical vein endothelial cells (HUVECs) and human glioblastoma-derived U251 cells. In a luciferase reporter assay, the first intron plus 5' upstream promoter (5'UP) gave the highest activity, followed by 5'UP, and the second intron plus 5'UP. However, the assay values were much lower by HUVEC extracts than by U251 cell extracts. Although green fluorescent protein expression was positive over all U251 cells under either the first intron, second intron, or ubiquitously active CAG promoter, the fluorescence in HUVECs was limited to a few cells even under the first intron. This difference came from the growth feature of HUVECs which exhibit growth arrest by contact inhibition. We found that the nestin expression was specific to proliferative endothelium, by using proliferation markers in hemangioblastomas and in situ hybridization. Using an endothelial tube formation assay, tyrosine kinase domain-deleted VEGF receptor KDR effectively abolished the tube formation under the first intron. We suggest that the nestin expression in tumor endothelium is enhanced by the first intron.     

Preparation of ceramic microspheres for in situ radiotherapy of deep-seated cancer

Radiotherapy is one of the most effective treatments for cancers. However, external irradiation provides only small doses to deep-seated cancers, and often causes damage to healthy tissues. It has been reported that 20-30 microm diameter 17Y(2)O(3)-19Al(2)O(3)-64SiO(2) (mol%) glass microspheres are useful for the in situ irradiation of cancers. Yttrium-89 (89Y) in this glass can be neutron bombarded to form the beta-emitter 90Y (half-life=64.1h). When injected in the vicinity of the cancer, such activated glass microspheres can provide a large localized dose of beta-radiation. The Y(2)O(3) content of the glass in the microspheres is limited to only 17 mol%. Chemically durable microspheres with a higher Y(2)O(3) content need to be developed. Phosphorus-31 (31P) with 100% natural abundance can also be activated by neutron bombardment to form the beta-emitter 32P (half-life=14.3d). Chemically durable microspheres containing a high phosphorus content are expected to be more effective for cancer treatment. We prepared pure Y(2)O(3) and YPO(4) microspheres using a high-frequency induction thermal plasma melting technique, and investigated the resulting structure and chemical durability. We successfully prepared smooth, highly spherical polycrystalline Y(2)O(3) and YPO(4) microspheres with diameters in the range 20-30 microm. Both the Y(2)O(3) and YPO(4) microspheres showed high chemical durability in saline solutions buffered at pH=6 and 7. These microspheres are expected to be more effective than the conventional glass microspheres for the in situ radiotherapy of cancer.     

Caenorhabditis elegans RBX1 is essential for meiosis, mitotic chromosomal condensation and segregation, and cytokinesis

BACKGROUND: The RING-H2 finger protein RBX1 (ROC1/HRT1) is a common subunit of SKP1-CDC53/CUL1-F-box (SCF), other cullins and von Hippel-Lindau (VHL) tumour suppressor E3 ubiquitin ligase complexes. RBX1 protein sequences are highly conserved in various species, including yeasts, Drosophila melanogaster, mice and humans. In Saccharomyces cerevisiae, RBX1 is essential for the G1/S transition. RESULTS: Caenorhabditis elegans RBX1 is strongly expressed in early embryos and in the gonad, including meiotic cells. Depletion of RBX1 by RNA-mediated interference (RNAi) caused pronounced defects in the first meiotic division. Several irregular phenotypes were identified in embryos that escaped from meiotic arrest: defects in mitotic chromosomal condensation and segregation, abnormal chromosome bridges, giant nuclei, abnormal cortical protrusion, multinucleate cells and defects in germ cell proliferation. Moreover, histone H3 phosphorylation at Ser10 and Ser28 was significantly reduced in these embryos. The histone H3 phosphorylation defect of embryos was rescued by the additional depletion of protein phosphatase 1 (GLC7alpha/beta) by RNAi. CONCLUSION: These results indicate that the RBX1 protein participates in diverse functions relevant to chromosome metabolism and cell cycle control.     

Prion domain interaction responsible for species discrimination in yeast [PSI+] transmission

BACKGROUND: The yeast [PSI+] factor is transmitted by a prion mechanism involving self-propagating Sup35 aggregates. As with mammalian prions, a species barrier prevents prion transmission between yeast species. The N-terminal of Sup35 of Saccharomyces cerevisiae, necessary for [PSI+], contains two species-signature elements-a Gln/Asn-rich region (residues 1-41; designated NQ) that is followed by oligopeptide repeats (designated NR). RESULTS: In this study, we show that S. cerevisiae[PSI+] is transmissible through plasmid shuffling and cytoplasmic transfer to heterotypic Sup35s whose NQ is replaced with the S. cerevisiae NQ. In addition to homology, the N-terminal location is essential for NQ mediated susceptibility to [PSI+] transmission amongst heterotypic Sup35s. In vitro, a swap of NQ of S. cerevisiae Sup35 led to cross seeding of amyloid formation. CONCLUSIONS: These findings suggest that NQ discriminates self from non-self, and is sufficient to initiate [PSI+] transmission irrespective of whether NR is heterotypic. NR as well as NQ alone coalesces into existing [PSI+] aggregates, showing their independent potentials to interact with the identical sequence in the [PSI+] conformer. The role of NQ and NR in [PSI+] prion formation is discussed.     

Exacerbation of rheumatoid arthritis following Helicobacter pylori eradication: disruption of established oral tolerance against heat shock protein?

A 62-year-old Japanese woman with RA received an eradication therapy against Helicobacter pylori in November 1999. Eight weeks later, successful eradication was confirmed by negative results for rapid urease test, pathologic findings, and a fall in anti-H. pylori IgG antibody titer. During the course, parameters for RA activity were exacerbated: C-reactive protein 1.1-4.2 mg/dL, rheumatoid arthritis precipitation antigen 2560-5120 dils., erythrocyte sedimentation rate 52-123 mm/h, and complements CH50 50 to over 60 U/mL. Lansbury index increased from 70% to 105%. Two more weeks later, the patient noticed right shoulder pain. She also complained of bilateral gonalgia two months later, and physical examination revealed increased fluid in the knee joints. Prednisolone was required to control the disease activity. The results of this case suggested that RA patients might experience a deleterious effect on the disease activity following H. pylori eradication possibly through disruption of the established oral tolerance against stress protein such as mycobacterial heat shock protein 65.     

Regulation of the type I IFN induction: a current view

The type I IFN-alpha/beta gene family was identified about a quarter of a century ago as a prototype of many cytokine gene families, which led to the subsequent burst of studies on molecular mechanisms underlying cytokine gene expression and signaling. Although originally discovered for their activity to confer an antiviral state on cells, more evidence has recently been emerging regarding IFN-alpha/beta actions on cell growth, differentiation and many immunoregulatory activities, which are of even greater fundamental biological significance. Indeed, much attention has recently been focused on the induction and function of the IFN-alpha/beta system regulated by Toll-like receptors (TLRs), which are critical for linking the innate and adaptive immunities. The understanding of the regulatory mechanisms of IFN-alpha/beta gene induction by TLRs and viruses is an emerging theme, for which much new insight has been gained over the past few years.     

Effects of various compounds on histidine decarboxylase activity: Active site mapping

The effect of about one hundred compounds on the activity of histidine decarboxylase partially purified from whole bodies of fetal rats was determined. Most of them at their 10 mM concentration had little effect on the enzyme activity; but 12 compounds inhibited the enzyme to a greater extent than 30%. Among these, except for -methylhistidine that has been known to be a strong and specific inhibitor, DOPA, homocysteine, cysteine, methionine and urocanic acid were the best inhibitors; -phenyllactic acid, phenylpyruvic acid and carnosine were less strong inhibitors; valine, oxaloacetic acid andN -methylimidazole acetic acid were weak inhibitors. Histamine had no inhibitory action. Thus, the substrate binding site of histidine decarboxylase is very rigid and specific forl-histidine.     

Persistent enhancement of transmitter release accompanying long-term potentiation in the guinea pig hippocampus

In order to examine temporal changes in enhancement of transmitter release during long-term potentiation (LTP), we examined amplitude fluctuation of excitatory postsynaptic potentials (EPSPs) for longer periods than 2 h after tetanic stimulation (up to 4 h in the longest observation). The relative magnitude of excitatory postsynaptic potentiation (EPSP) fluctuation (coefficient of variation, CV) reduced throughout the observation periods in association with an increase in EPSP amplitude after tetanic stimulation. The reciprocals of squared CVs (= mean²/variance) were almost in proportion to the magnitude of LTP, and the ratio of 1/CV² and the LTP magnitude did not change significantly for up to 4 h. These findings suggest that a prolonged enhancement of transmitter release from presynaptic terminals underlies LTP, and the relative contribution of this presynaptic enhancement does not change significantly for 2 h (maybe up to 4 h, or longer) after tetanic stimulation.     

Culture of chondrocytes in fibroin-hydrogel sponge

Fibroin-hydrogel sponge and collagen gel were used as scaffold for in vitro cartilage regeneration. Fibroin-hydrogel sponge was formed by phase separation from freezed fibroin solution. Chondrocytes were harvested from proximal humerus, distal femur and proximal tibia of 4-week-old Japanese white rabbits and inoculated in the fibroin-hydrogel sponge and collagen gel. Those constructs were cultured in DMEM supplemented with 10% FCS and 50 ml L-ascorbate at 37 degrees C. Histological observation, measurement of sulfated glycosaminoglycan and cell density were carried out at 3, 7, and 14 days after the cultivation. Well-defined cartilage tissue can be seen both in the fibroin-hydrogel sponge and in the collagen gel. The matrix was intensely stained by safranin-O and showed a metachromatic reaction in both group. However, the quantity of sulfated glycosaminoglycan and cell density of the fibroin-hydrogel sponge group were increased more rapidly than these of the collagen gel group. Thus, the chondrocytes proliferated in the fibroin sponge without losing their differentiated phenotype. It is possible that culture environment in the fibroin sponge was suitable for chondrocytes regeneration.     

Heterosynaptic expression of depolarization-induced suppression of inhibition (DSI) in rat hippocampal cultures

Depolarization-induced suppression of inhibition (DSI) is a transient suppression of the inhibitory synaptic transmission, observed in the hippocampus and the cerebellum, upon postsynaptic depolarization. Using rat hippocampal cultures, we examined whether DSI is confined to the inhibitory synapses on the depolarized neuron or, if DSI can spread to those on neighboring non-depolarized neurons. Whole-cell recordings were performed in 108 neuronal pairs with the following synaptic responses. Stimulation of one neuron evoked the inhibitory autaptic currents (IACs) recurrently in that neuron and also elicited the inhibitory postsynaptic currents (IPSCs) orthodromically in the other neuron. In 38 of 108 pairs, the postsynaptic depolarization caused transient suppression of IPSCs (homosynaptic DSI). In 11 of the 38 pairs exhibiting the homosynaptic DSI, the depolarization also induced suppression of IACs (heterosynaptic DSI). The heterosynaptic DSI, like the homosynaptic DSI, depended on depolarizing pulse duration and was blocked by a phorbol ester. These results suggest that DSI can spread to the synapses on a neighboring non-depolarized neuron in rat hippocampal cultures.