An ultrastructural and immunohistochemical study of extracellular matrix in meningiomas

Extracellular matrix of meningiomas was studied by light and electron microscopy with the aid of immunohistochemical techniques. Special attention was paid to the distribution of type I, III, IV, V collagens and laminin with a comparison between meningothelial and fibroblastic types. Connective tissue fibers and basement membrane were not found among the tumor cells in the meningothelial type, but were found in the fibroblastic type. The immunolocalizations were consistently demonstrated extracellularly, but were not within the cytoplasm. Type I, III and V collagens were usually demonstrated in the fibrous septum in the meningothelial type, while they were localized among the tumor cells in the fibroblastic type. Furthermore, type IV collagen and laminin were demonstrated within the vascular walls or around the syncytium in the meningothelial type, while they were localized among the tumor cells in the fibroblastic type. In both types the expression of type IV collagen and laminin was closely related to the distribution of basement membrane. Although meningothelial and fibroblastic meningiomas showed quite different distribution of extracellular matrices, the profile of collagen types expressed by these two basic types was essentially the same. The cellular derivation of meningiomas was discussed with particular attention to the structure of human arachnoid villi and meninges.     

Relationship between sleep duration and cause-specific mortality in diabetic men and women based on self-reports

Sleep and Biological Rhythms - Sleep duration could affect glucose tolerance and mortality. However, the impact that sleep duration has on prognosis of people with diabetes is unclear. A cohort of...     

Localization of MCM2-7, Cdc45, and GINS to the site of DNA unwinding during eukaryotic DNA replication

Little is known about the architecture and biochemical composition of the eukaryotic DNA replication fork. To study this problem, we used biotin-streptavidin-modified plasmids to induce sequence-specific replication fork pausing in Xenopus egg extracts. Chromatin immunoprecipitation was employed to identify factors associated with the paused fork. This approach identifies DNA pol alpha, DNA pol delta, DNA pol varepsilon, MCM2-7, Cdc45, GINS, and Mcm10 as components of the vertebrate replisome. In the presence of the DNA polymerase inhibitor aphidicolin, which causes uncoupling of a highly processive DNA helicase from the stalled replisome, only Cdc45, GINS, and MCM2-7 are enriched at the pause site. The data suggest the existence of a large molecular machine, the "unwindosome," which separates DNA strands at the replication fork and contains Cdc45, GINS, and the MCM2-7 holocomplex.     

Complete nucleotide sequence of the prophage VT2-Sakai carrying the verotoxin 2 genes of the enterohemorrhagic Escherichia coli O157:H7 derived from the Sakai outbreak

The enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain RIMD 0509952, derived from an outbreak in Sakai city, Japan, in 1996, produces two kinds of verotoxins, VT1 and VT2, encoded by the stx1 and stx2 genes. In the EHEC strains, as well as in other VT-producing E. coli strains, the toxins are encoded by lysogenic bacteriophages. The EHEC O157:H7 strain RIMD 0509952 did not produce plaque-forming phage particles upon inducing treatments. We have determined the complete nucleotide sequence of a prophage, VT2-Sakai, carrying the stx2A and stx2B genes on the chromosome, and presumed the putative functions of the encoded proteins and the cis-acting DNA elements based on sequence homology data. To our surprise, the sequences in the regions of VT2-Sakai corresponding to the early gene regulators and replication proteins, and the DNA sequences recognized by the regulators share very limited homology to those of the VT2-encoding 933W phage carried by the EHEC O157:H7 strain EDL933 reported by Plunkett et al. (J. Bacteriol., p1767-1778, 181, 1999), although the sequences corresponding to the structural components are almost identical. These data suggest that these two phages were derived from a common ancestral phage and that either or both of them underwent multiple genetic rearrangements. An IS629 insertion was found downstream of the stx2B gene and upstream of the lysis gene S, and this might be responsible for the absence of plaque-forming activity in the lysate obtained after inducing treatments.     

Small aggregates of platelets can be detected sensitively by a flow cytometer equipped with an imaging device: mechanisms of epinephrine-induced aggregation and antiplatelet effects of beraprost

BACKGROUND: Although cross-talks between platelets and other blood cells are important in vivo, laboratory platelet aggregation tests have been performed mainly with the use of platelet-rich plasma (PRP) as samples. Methods that enable an efficient and sensitive detection of platelet aggregates in whole blood are being developed. METHODS: A flow cytometer equipped with an imaging device, the flow imaging cytometer 2 (FIC2), was used to detect platelet aggregates in whole blood. RESULTS: The FIC2 provides a resolution that is high enough to differentiate platelet aggregates from single platelets or other blood cells. Epinephrine elicited platelet aggregate formation in hirudin plus argatroban-treated whole blood, but not in PRP. The reconstitution study revealed that a small amount of adenosine diphosphate (ADP) from erythrocytes may play an important role in epinephrine-induced platelet aggregation (in whole blood), through mediation of P2Y1 receptors. When the inhibitory effect of beraprost, an antiplatelet agent, on platelet aggregation was assessed, analysis of whole blood samples with FIC2 proved to be the most sensitive among the methods available. CONCLUSIONS: FIC2 is a promising device for detection of platelet aggregates in whole blood, with wide basic and clinical applications.     

A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has provided a myriad of applications for biological systems. Over the last several years, mutagenesis studies have improved folding properties of GFP (refs 1,2). However, slow maturation is still a big obstacle to the use of GFP variants for visualization. These problems are exacerbated when GFP variants are expressed at 37 degrees C and/or targeted to certain organelles. Thus, obtaining GFP variants that mature more efficiently is crucial for the development of expanded research applications. Among Aequorea GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive, and uniquely quenched by chloride ion (Cl-). For YFP to be fully and stably fluorescent, mutations that decrease the sensitivity to both pH and Cl- are desired. Here we describe the development of an improved version of YFP named "Venus". Venus contains a novel mutation, F46L, which at 37 degrees C greatly accelerates oxidation of the chromophore, the rate-limiting step of maturation. As a result of other mutations, F64L/M153T/V163A/S175G, Venus folds well and is relatively tolerant of exposure to acidosis and Cl-. We succeeded in efficiently targeting a neuropeptide Y-Venus fusion protein to the dense-core granules of PC12 cells. Its secretion was readily monitored by measuring release of fluorescence into the medium. The use of Venus as an acceptor allowed early detection of reliable signals of fluorescence resonance energy transfer (FRET) for Ca2+ measurements in brain slices. With the improved speed and efficiency of maturation and the increased resistance to environment, Venus will enable fluorescent labelings that were not possible before.     

Phototactic responses of four marine dinoflagellates with different types of eyespot and chloroplast

Great structural variety is seen in the eyespot of dinoflagellates, a structure involved in phototaxis. Although there are several works on the phototactic responses in some species of dinoflagellates, none of the dinoflagellates used in these studies possessed an eyespot and, therefore, we have no knowledge of the relationship between eyespot type and phototactic response. In this study, we determined wavelength dependency curves for phototaxis in four marine dinoflagellates that possess a different type of either eyespot or chloroplast. These include: (i) a dinoflagellate possessing a peridinin-containing ohioroplast with an eyespot (Scrippsiella hexapraecingula Horiguchi et Chihara); (ii) a dinoflagellate containing a diatom endosymbiont and with the type B eyespot sensu Dodge (1984; (Peridinium foli-aceum (Stein) Biecheler); (iii) a dinoflagellate with peri-dinin-containing chloroplasts, but lacking an eyespot (Atexandrium hiranoi Kita et Fukuyo); and (iv) a dinoflagellate with fucoxanthin, 19′-hexanoyloxyfucoxanthin and 19′-butanoyloxyfucoxanthin, but lacking an eyespot (Gymnodinium mikimotoi Miyabe et Kominami ex Oda), Regardless of the eyespot or the chloroplast type, all four dinoflagellates showed similar wavelength dependency curves for phototaxis, with sensitivity between 380 and 520 nm, the highest peak at approximately 440 or 460 nm and smaller peaks or shoulders at 400–420 nm and 480–500 nm. Substantial peaks have also been noted in the ultraviolet range (260–280 nm). The ultrastructural study of the eye-spot of Scrippsiella hexapraecingula revealed that the eyespot consists of two layers of lipid globules and probably acts as a quarter-wave stack antenna.     

VP-16-induced nucleotide pool changes and poly(ADP-ribose) synthesis: the role of VP-16 in interphase death

Exposure of human promyelocytic leukemia cell line (HL-60) to VP-16 resulted in accumulation of DNA strand breaks. Concomitantly, intracellular NAD levels fell at 1 h, followed by declines in ATP at 2 h and in GTP, CTP, and UTP at 3 h. Furthermore, marked morphological changes, such as loss of microvilli or bleb formation, appeared at 4 h and cell death by 8-10 h. The addition of an inhibitor of poly(ADP-ribose) polymerase, 3-aminobenzamide (5 mM), theophylline (2 mM), or thymidine (1 mM), prevented these sequential reductions of nucleotide pools and cell death. In fact, the activation of poly(ADP-ribose) synthesis was detectable within a few hours after treatment with VP-16, although it was smaller than that induced by N-methyl-N'-nitro-N-nitrosoguanidine. These results may suggest the possible role of activation of poly(ADP-ribosyl)ation in VP-16-induced nucleotide pool changes and subsequent interphase death.