A low copy number central sequence with strict symmetry and unusual chromatin structure in fission yeast centromere.

Fission yeast centromeres vary in size but are organized in a similar fashion. Each consists of two distinct domains, namely, the approximately 15-kilobase (kb) central region (cnt+imr), containing chromosome-specific low copy number sequences, and 20- to 100-kb outer surrounding sequences (otr) with highly repetitive motifs common to all centromeres. The central region consists of an inner asymmetric sequence flanked by inverted repeats that exhibit strict identity with each other. Nucleotide changes in the left repeat are always accompanied with the same changes in the right. The chromatin structure of the central region is unusual. A nucleosomal nuclease digestion pattern formed on unstable plasmids but not on stable chromosome. DNase I hypersensitive sites correlate with the location of tRNA genes in the central region. Autonomously replicating sequences are also present in the central region. The behavior of truncated minichromosomes suggested that the central region is essential, but not sufficient, to confer transmission stability. A portion of the outer repetitive region is also required. A larger outer region is necessary to ensure correct meiotic behavior. Fluorescence in situ hybridization identified individual cens. In the interphase, they cluster near the nuclear periphery. The central sequence (cnt+imr) may play a role in positioning individual chromosomes within the nucleus, whereas the outer regions (otr) may interact with each other to form the higher-order complex structure.     

Potentiation by BL191 of differentiation of neuroblastoma cells induced by dibutyryl cAMP and prostaglandin E1

BL191, a newly developed phosphodiesterase inhibitor, markedly potentiated a differentiation of neuroblastoma cell clones (Neuro2a, NS-20Y, and N1E115) induced by dibutyryl cyclic adensoine 3′:5′-monophosphate(dibutyryl cAMP) and prostaglandin E₁ (PGE₁). BL191 (1 mM) inhibited DNA synthesis more strongly when used together with PGE₁ (0.5 μg/ml) and dibutyryl cAMP (0.5 mM) than papaverine (1.6 μg/ml) alone did. The inhibition rates of DNA synthesis were 72.5% for N1E-115, 75.3% for Neuro2a, and 82.5% for NS-20Y. After the treatment with BL191. PGE₁, and dibutyryl cAMP for 48 h all of three cell lines became enlarged and flattened, and extended long processes. The specific activities of choline acetyl transferase (EC 2.3.1.9) of NS-20Y and dopamine β-hydroxylase (EC 1.14.17.1) of N1E-115 increased about 3-fold as compared to the controls. The tumorigenicities of Neuro2a and N1E-115 cells were decreased, but not of NS-20Y. These data suggest the heterogenous responsiveness in neuroblastoma cells to drug treatment.     

Mycobiont diversity and first evidence of mixotrophy associated with Psathyrellaceae fungi in the chlorophyllous orchid Cremastra variabilis

Mixotrophy (MX, also called partial mycoheterotrophy) in plants is characterized by isotopic abundances that differ from those of autotrophs. Previous studies have evaluated mycoheterotrophy in MX plants associated with fungi of similar ecological characteristics, but little is known about the differences in the relative abundances of ¹³C and ¹⁵N in an orchid species that associates with several different mycobionts species. Since the chlorophyllous orchid Cremastra variabilis Nakai associates with various fungi with different ecologies, we hypothesized that it may change its relative abundances of ¹³C and ¹⁵N depending on the associated mycobionts. We investigated mycobiont diversity in the chlorophyllous orchid C. variabilis together with the relative abundance of ¹³C and ¹⁵N and morphological underground differentiation (presence or absence of a mycorhizome with fungal colonization). Rhizoctonias (Tulasnellaceae, Ceratobasidiaceae, Sebacinales) were detected as the main mycobionts. High differences in δ¹³C values (– 34.7? to?– 27.4 ‰) among individuals were found, in which the individuals associated with specific Psathyrellaceae showed significantly high relative abundance of ¹³C. In addition, Psathyrellaceae fungi were always detected on individuals with mycorhizomes. In the present study, MX orchid association with non-rhizoctonia saprobic fungi was confirmed, and the influence of mycobionts on morphological development and on relative abundance of ¹³C and ¹⁵N was discovered. Cremastra variabilis may increase opportunities to gain nutrients from diverse partners, in a bet-hedging plasticity that allows colonization of various environmental conditions.

     

Effects of Dietary Methionine and Cystine on Endogenous Hypercholesterolemia in Hypothyroid Rats

The effects on cholesterolemia of dietary additions (1.2%) of methionine and cystine to a 20% casein diet were studied in both euthyroid and thiouracil-induced hypothyroid rats. The hypothyroid rats lapsed into endogenous hypercholesterolemia, which was due to an increase in the very-low-density lipoprotein plus low-density lipoprotein-cholesterol [(VLDL+LDL)-Ch] concentration with no change in the high-density lipoprotein-cholesterol (HDL-Ch) concentration. These lipoprotein changes in hypothyroid rats resulted in a marked (5-fold) increase in the atherogenic index [AI, (VLDL-LDL)-Ch/HDL-Ch] when compared to that of elutyroid rats. Methionine reduced the hypercholesterolemia in the hypothyroid state by suppressing the elevation in (VLDL + LDL)-Ch with no significant reduction in HDL-Ch, resulting in a notable fall of AI, while methionine showed no significant effect on cholesterolemia and AI in the euthyroid state. Cystine induced hypercholesterolemia due to a significant elevation of HDL-Ch in the euthyroid state, but the amino acid showed no significant effect on cholesterolemia and hence AI in the hypothyroid state. These results suggest that methionine overcomes changes in the parameters involved in Ch biodynamics that cause hypercholesterolemia in the hypothyroid state, whereas cystine counterbalances the parameter changes and results in diminution of its hypercholesterolemic effect in the hypothyroid state.     

Leukemia inhibitory factor relaxes arteries through endothelium-dependent mechanism

Leukemia inhibitory factor (LIF) is a cytokine, which inhibits angiogenesis and decreases endothelial cell proliferation and migration, suggesting that LIF may modulate vascular tone. In this study, we examined the effects of LIF on the tone of rat arteries. The isometric tension of ring preparations from rat superior mesenteric arteries was continuously measured. LIF relaxed the mesenteric arteries in a dose-dependent manner, when the arterial rings were precontracted with phenylephrine. The relaxation was totally inhibited by mechanical removal of endothelium. N(G)-nitro-L-arginine methyl ester did not affect the relaxation by LIF. Ca(2+)-dependent K channel (KCa) blockers, apamin with charybdotoxin, inhibited the relaxation by LIF. Catalase, an enzyme which scavenges hydrogen peroxide, also inhibited the relaxation by LIF. Endothelium-derived hyperpolarizing factor relaxes smooth muscle cells and the effect is blocked by KCa and catalase. Our results suggest that LIF regulates vascular tone through the effect of this factor.     

Chromosomal enrichment and activation of the aurora B pathway are coupled to spatially regulate spindle assembly

Chromatin-induced spindle assembly depends on regulation of microtubule-depolymerizing proteins by the chromosomal passenger complex (CPC), consisting of Incenp, Survivin, Dasra (Borealin), and the kinase Aurora B, but the mechanism and significance of the spatial regulation of Aurora B activity remain unclear. Here, we show that the Aurora B pathway is suppressed in the cytoplasm of Xenopus egg extract by phosphatases, but that it becomes activated by chromatin via a Ran-independent mechanism. While spindle microtubule assembly normally requires Dasra-dependent chromatin binding of the CPC, this function of Dasra can be bypassed by clustering Aurora B-Incenp by using anti-Incenp antibodies, which stimulate autoactivation among bound complexes. However, such chromatin-independent Aurora B pathway activation promotes centrosomal microtubule assembly and produces aberrant achromosomal spindle-like structures. We propose that chromosomal enrichment of the CPC results in local kinase autoactivation, a mechanism that contributes to the spatial regulation of spindle assembly and possibly to other mitotic processes.     

Feeding habits of juvenile slime flounder <Emphasis Type="Italic">Microstomus achne</Emphasis> in the coastal area of southern Hokkaido

A total of 45 juvenile [30.0–57.4 mm total length (TL)] slime flounder Microstomus achne were collected in the coastal area of southern Hokkaido from April to July in 2001 and April to June in 2002. Their diets were analyzed. Slime flounder juveniles of 30.0–39.9 mm TL fed predominantly on small crustaceans (gammarid amphipods, harpacticoids and cumaceans) and those of 40.0–57.4 mm TL on gammarid amphipods, cumaceans and polychaetes. The major prey items changed with growth from small crustaceans (e.g., harpacticoids) to polychaetes, although gammarid amphipods were the major prey items throughout the juvenile period (30.0–57.4 mm TL).     

Two birds with one stone--dealing with nuclear transport and spindle assembly

Spindle assembly and nuclear transport both utilize the same simple device: Ran-GTP-sensitive interaction of importin beta and its cargo proteins. In this issue of Cell, report that one of these cargos required for spindle assembly turns out to be Rae1, previously known as an mRNA export protein. This study reveals the importance of RNAs in spindle structure.     

Xenopus Shugoshin 2 regulates the spindle assembly pathway mediated by the chromosomal passenger complex

Shugoshins (Sgo) are conserved proteins that act as protectors of centromeric cohesion and as sensors of tension for the machinery that eliminates improper kinetochore-microtubule attachments. Most vertebrates contain two Sgo proteins, but their specific functions are not always clear. Xenopus laevis Sgo1, XSgo1, protects centromeric cohesin from the prophase dissociation pathway. Here, we report the identification of XSgo2 and show that it does not regulate cohesion. Instead, we find that it participates in bipolar spindle assembly. Both Sgo proteins interact physically with the Chromosomal Passenger Complex (CPC) containing Aurora B, a key regulator of mitosis, but the functional consequences of such interaction are distinct. XSgo1 is required for proper localization of the CPC while XSgo2 positively contributes to its activation and the subsequent phosphorylation of at least one key substrate for bipolar spindle assembly, the microtubule depolymerizing kinesin MCAK (Mitotic Centromere-Associated Kinesin). Thus, the two Xenopus Sgo proteins have non-overlapping functions in chromosome segregation. Our results further suggest that this functional specificity could rely on the association of XSgo1 and XSgo2 with different regulatory subunits of the PP2A complex.     

Structural basis for recognition of H3T3ph and Smac/DIABLO N-terminal peptides by human Survivin

Survivin is an inhibitor of apoptosis family protein implicated in apoptosis and mitosis. In apoptosis, it has been shown to recognize the Smac/DIABLO protein. It is also a component of the chromosomal passenger complex, a key player during mitosis. Recently, Survivin was identified in vitro and in vivo as the direct binding partner for phosphorylated Thr3 on histone H3 (H3T3ph). We have undertaken structural and binding studies to investigate the molecular basis underlying recognition of H3T3ph and Smac/DIABLO N-terminal peptides by Survivin. Our crystallographic studies establish recognition of N-terminal Ala in both complexes and identify intermolecular hydrogen-bonding interactions in the Survivin phosphate-binding pocket that contribute to H3T3ph mark recognition. In addition, our calorimetric data establish that Survivin binds tighter to the H3T3ph-containing peptide relative to the N-terminal Smac/DIABLO peptide, and this preference can be reversed through structure-guided mutations that increase the hydrophobicity of the phosphate-binding pocket.