Analysis of telomeric single-strand overhang length in human endometrial cancers

The 3' single-strand telomeric overhang (3'-OH) is a key component of telomere structure. Although telomere length has been well analyzed in a variety of human cancers, no information is available on the 3'-OH length in cancers. In the present study, we examined the 3'-OH length in normal and malignant endometria using telomere-oligonucleotide ligation assay. Although 3'-OH lengths varied among patients, 3'-OH length observed in endometrial cancers was significantly shorter than that found in samples derived from normal endometria (P < 0.001: Student's t-test), suggesting that erosion of 3'-OH length induces impaired telomeric integrity and genomic instability, leading to carcinogenesis. Interestingly, we found that the most aggressive subtypes of endometrial cancers harbored significantly longer 3'-OH length than those with non-aggressive subtypes (P < 0.001: Sheffe's test), suggesting that cancer cells with long 3'-OH length have growth advantage due to their stabilized telomere ends. In contrast, we failed to observe an association between overall telomere length and any clinicopathological characteristics of endometrial cancers. These findings suggest that erosion of 3'-OH length, rather than overall telomere length, play roles in endometrial carcinogenesis. Furthermore, long 3'-OH may serve as a molecular marker for aggressive phenotype of tumors.     

Experimental simulation of an estrous cycle — gonadotropin surges in estradiol‐infused ovariectomized rats

Abstract

Dynamic relations between the circulating estrogen and the hypophyseal gonadotropin secretion in the estrous cycle were investigated by replacing the ovaries by an infusion pump in freely moving rats. Female rats were ovariectomized in the morning at certain stages of the 4‐day estrous cycle, and simultaneously infused with estradiol (E₂) at a constant rate of 0.35 ng/min up to 120 h through a cannula chronically inserted into the jugular vein. They were killed at 6 h‐intervals. Rats ovariectomized at the second day of diestrus and at estrus showed a sharp rise in LH 36 h and 84 h, respectively, after the initiation of E₂ infusion, when the proestrous surge would occur in normal rats. During the other periods, blood levels of LH were very low, exhibiting a small daily rise in the evening. Similarly ovariectomized rats infused with vehicle only showed a gradual rise of gonadotropin secretion, never reaching the surge level. Rats ovariectomized at proestrus and infused with E₂ showed a LH surge 12 h later as expected. However, surge‐like LH secretions followed every evening thereafter. Thus, the constant supply of E₂ alone could simulate at least one 4‐day cyclic LH surge in ovariectomized rats. E₂ infusion caused a daily peak of FSH synchronized with the LH rises, but could not suppress the post‐operative hypersecretion. It is discussed that if the suppressing effect of progesterone endogenously secreted from the ovaries is cleared, a circadian pattern of the LH/FSH surge may appear under the signal from the cerebral clock mechanism and the effect of circulating estrogen. The failure to suppress the FSH hypersecretion by E₂ might indicate the involvement of inhibin in the regulatory mechanism. Time‐course changes in uterine and vaginal weights are also dealt with and discussed in relation to the constant E₂ exposure.     

Fatty acid synthesis by spinach chloroplasts I. Property of fatty acid synthesis from acetate

Intact chloroplasts (about 70% Class I chloroplasts) isolatedfrom spinach leaves incorporated 150 nmoles of [1-¹⁴C] acetateinto fatty acids per mg chlorophyll in 1 hr at pH 8.3, 25°Cand 25,000 lux. On electron and phase-contrast microscopiescombined with hypotonic treatment of chloroplasts, this syntheticactivity was shown to be proportional to the percentage of ClassI chloroplasts in the preparation. Light was necessary for thesynthesis, the activity in the complete reaction mixture inthe dark being only 2% of that in the light. The synthetic activityincreased with increasing intensities of light to reach saturationat 6,000 lux. CoA and ATP were most effective as cofactors,HCO₃^–, HPO₄^2–, Mg^2$ and Mn^2$ were less effective.ATP could be replaced by ADP in the presence of Pi, suggestingpossible supply of ATP by photophosphorylation. Omission ofthe NADPH-generation system and NADH did not affect the synthesis,indicating sufficient provision of endogenous NADPH and NADHin intact chloroplasts under light. Addition of DTE did notcause recovery of the synthetic activity of intact chloroplastsin the dark. ¹ Present address: Radioisotope Centre, University of Tokyo,Yayoi, Bunkyo, Tokyo 113, Japan. (Received August 26, 1974; )     

Solution structure of ribosomal protein L16 from Thermus thermophilus HB8

Ribosomal protein L16 is an essential component of the bacterial ribosome. It organizes the architecture of aminoacyl tRNA binding site in the ribosome 50S subunit. The three-dimensional structure of L16 from Thermus thermophilus HB8 was determined by NMR. In solution, L16 forms an alpha+beta sandwich structure combined with two additional beta sheets located at the loop regions connecting the two layers. The terminal regions and a central loop region did not show any specific secondary structure. The structured part of L16 could be superimposed well on the C(alpha) model of L16 determined in the crystal structure of the ribosome 50S subunit. By overlaying the L16 solution structure onto the coordinates of the ribosome crystal structure, we constructed the combined model that represents the ribosome-bound state of L16 in the detailed structure. The model showed that L16 possesses residues in contact with helices 38, 39, 42, 43 and 89 of 23S rRNA and helix 4 of 5S rRNA. This suggests its broad effect on the ribosome architecture. Comparison of L16 with the L10e protein, which is the archaeal counterpart, showed that they share a common fold, but differ in some regions of functional importance, especially in the N-terminal region. All known mutation sites in L16 that confer resistance to avilamycin and evernimicin were positioned so that their side-chains were exposed to solvent in the internal cavity of the ribosome. This suggests the direct participation of L16 as a part of the binding site for antibiotics.     

An N-Glycosylation Site on the��-Propeller Domain of the Integrin ��5 Subunit Plays Key Roles in Both Its Function and Site-specific Modification by��1,4-N-Acetylglucosaminyltransferase III

Recently we reported that N-glycans on the β-propeller domain of the integrin α5 subunit (S-3,4,5) are essential for α5β1 heterodimerization, expression, and cell adhesion. Herein to further investigate which N-glycosylation site is the most important for the biological function and regulation, we characterized the S-3,4,5 mutants in detail. We found that site-4 is a key site that can be specifically modified by N-acetylglucosaminyltransferase III (GnT-III). The introduction of bisecting GlcNAc into the S-3,4,5 mutant catalyzed by GnT-III decreased cell adhesion and migration on fibronectin, whereas overexpression of N-acetylglucosaminyltransferase V (GnT-V) promoted cell migration. The phenomenon is similar to previous observations that the functions of the wild-type α5 subunit were positively and negatively regulated by GnT-V and GnT-III, respectively, suggesting that the α5 subunit could be duplicated by the S-3,4,5 mutant. Interestingly GnT-III specifically modified the S-4,5 mutant but not the S-3,5 mutant. This result was confirmed by erythroagglutinating phytohemagglutinin lectin blot analysis. The reduction in cell adhesion was consistently observed in the S-4,5 mutant but not in the S-3,5 mutant cells. Furthermore mutation of site-4 alone resulted in a substantial decrease in erythroagglutinating phytohemagglutinin lectin staining and suppression of cell spread induced by GnT-III compared with that of either the site-3 single mutant or wild-type α5. These results, taken together, strongly suggest that N-glycosylation of site-4 on the α5 subunit is the most important site for its biological functions. To our knowledge, this is the first demonstration that site-specific modification of N-glycans by a glycosyltransferase results in functional regulation.Glycosylation is a crucial post-translational modification of most secreted and cell surface proteins (1). Glycosylation is involved in a variety of physiological and pathological events, including cell growth, migration, differentiation, and tumor invasion. It is well known that glycans play important roles in cell-cell communication, intracellular signal transduction, protein folding, and stability (2, 3).Integrins comprise a family of receptors that are important for cell adhesion. The major function of integrins is to connect cells to the extracellular matrix, activate intracellular signaling pathways, and regulate cytoskeletal formation (4). Integrin α5β1 is well known as a fibronectin (FN)³ receptor. The interaction between integrin α5 and FN is essential for cell migration, cell survival, and development (5–8). In addition, integrins are N-glycan carrier proteins. For example, α5β1 integrin contains 14 and 12 putative N-glycosylation sites on the α5 and β1 subunits, respectively. Several studies suggest that N-glycosylation is essential for functional integrin α5β1. When human fibroblasts were cultured in the presence of 1-deoxymannojirimycin, which prevents N-linked oligosaccharide processing, immature α5β1 integrin appeared on the cell surface, and FN-dependent adhesion was greatly reduced (9). Treatment of purified integrin α5β1 with N-glycosidase F, which cleaves between the innermost N-acetylglucosamine (GlcNAc) and asparagine N-glycan residues of N-linked glycoproteins, prevented the inherent association between subunits and blocked α5β1 binding to FN (10).A growing body of evidence indicates that the presence of the appropriate oligosaccharide can modulate integrin activation. N-Acetylglucosaminyltransferase III (GnT-III) catalyzes the addition of GlcNAc to mannose that is β1,4-linked to an underlying N-acetylglucosamine, producing what is known as a “bisecting” GlcNAc linkage as shown in Fig. 1B. GnT-III is generally regarded as a key glycosyltransferase in N-glycan biosynthetic pathways and contributes to inhibition of metastasis. The introduction of a bisecting GlcNAc catalyzed by GnT-III suppresses additional processing and elongation of N-glycans. These reactions, which are catalyzed in vitro by other glycosyltransferases, such as N-acetylglucosaminyltransferase V (GnT-V), which catalyzes the formation of β1,6 GlcNAc branching structures (Fig. 1B) and plays important roles in tumor metastasis, do not proceed because the enzymes cannot utilize the bisected N-glycans as a substrate. Introduction of the bisecting GlcNAc to integrin α5 by overexpression of GnT-III resulted in decreased in ligand binding and down-regulation of cell adhesion and migration (11–13). Contrary to the functions of GnT-III, overexpression of GnT-V promoted integrin α5β1-mediated cell migration on FN (14). These observations clearly demonstrate that the alteration of N-glycan structure affected the biological functions of integrin α5β1. Similarly characterization of the carbohydrate moieties in integrin α3β1 from non-metastatic and metastatic human melanoma cell lines showed that expression of β1,6 GlcNAc branched structures was higher in metastatic cells compared with non-metastatic cells, confirming the notion that the β1,6 GlcNAc branched structure confers invasive and metastatic properties to cancer cells. In fact, Partridge et al. (15) reported that GnT-V-modified N-glycans containing poly-N-acetyllactosamine, the preferred ligand for galectin-3, on surface receptors oppose their constitutive endocytosis, promoting intracellular signaling and consequently cell migration and tumor metastasis.Open in a separate windowFIGURE 1.Potential N-glycosylation sites on the α5 subunit and its modification by GnT-III and GnT-V. A, schematic diagram of potential N-glycosylation sites on the α5 subunit. Putative N-glycosylation sites are indicated by triangles, and point mutations are indicated by crosses (N84Q, N182Q, N297Q, N307Q, N316Q, N524Q, N530Q, N593Q, N609Q, N675Q, N712Q, N724Q, N773Q, and N868Q). B, illustration of the reaction catalyzed by GnT-III and GnT-V. Square, GlcNAc; circle, mannose. TM, transmembrane domain.In addition, sialylation on the non-reducing terminus of N-glycans of α5β1 integrin plays an important role in cell adhesion. Colon adenocarcinomas express elevated levels of α2,6 sialylation and increased activity of ST6GalI sialyltransferase. Elevated ST6GalI positively correlated with metastasis and poor survival. Therefore, ST6GalI-mediated hypersialylation likely plays a role in colorectal tumor invasion (16, 17). In fact, oncogenic ras up-regulated ST6GalI and, in turn, increased sialylation of β1 integrin adhesion receptors in colon epithelial cells (18). However, this is not always the case. The expression of hyposialylated integrin α5β1 was induced by phorbol esterstimulated differentiation in myeloid cells in which the expression of the ST6GalI was down-regulated by the treatment, increasing FN binding (19). A similar phenomenon was also observed in hematopoietic or other epithelial cells. In these cells, the increased sialylation of the β1 integrin subunit was correlated with reduced adhesiveness and metastatic potential (20–22). In contrast, the enzymatic removal of α2,8-linked oligosialic acids from the α5 integrin subunit inhibited cell adhesion to FN (23). Collectively these findings suggest that the interaction of integrin α5β1 with FN is dependent on its N-glycosylation and the processing status of N-glycans.Because integrin α5β1 contains multipotential N-glycosylation sites, it is important to determine the sites that are crucial for its biological function and regulation. Recently we found that N-glycans on the β-propeller domain (sites 3, 4, and 5) of the integrin α5 subunit are essential for α5β1 heterodimerization, cell surface expression, and biological function (24). In this study, to further investigate the underlying molecular mechanism of GnT-III-regulated biological functions, we characterized the N-glycans on the α5 subunit in detail using genetic and biochemical approaches and found that site-4 is a key site that can be specifically modified by GnT-III.     

Light germination ofAnthoceros miyabeanus spores

The effects of light on the spore germination of a hornwort species,Anthoceros miyabeanus Steph., were investigated. Spores of this species were photoblastic, but their sensitivities to light quality were different. Under either continuous white, red or diffused daylight, more than 80% of the spores germinated, but under blue light none or a few of them germinated. Under continuous far-red light or in total darkness, the spores did not germinate at all.Anthoceros spores required red light irradiation for a very long duration, i.e., over 12–24 hr of red light for saturated germination. However, the spore germination showed clear photo-reversibility by repeated irradiation of red and far-red light. The germination pattern clearly varied with the light quality. There were two fundamental patterns; (1) cell mass type in white or blue light: spores divide before germination, and the sporelings divide frequently and form 1–2 rhizoids soon after germination, and (2) germ tube type in red light: spores germinate without cell division, and the single-cell sporelings elongate without cell division and rhizoid formation.     

A novel type of family 19 chitinase from Aeromonas sp. No.10S-24. Cloning, sequence, expression, and the enzymatic properties.

A family 19 chitinase gene from Aeromonas sp. No.10S-24 was cloned, sequenced, and expressed in Escherichia coli. From the deduced amino acid sequence, the enzyme was found to possess two repeated N-terminal chitin-binding domains, which are separated by two proline-threonine rich linkers. The calculated molecular mass was 70 391 Da. The catalytic domain is homologous to those of plant family 19 chitinases by about 47%. The enzyme produced alpha-anomer by hydrolyzing beta-1,4-glycosidic linkage of the substrate, indicating that the enzyme catalyzes the hydrolysis through an inverting mechanism. When N-acetylglucosamine hexasaccharide [(GlcNAc)6] was hydrolyzed by the chitinase, the second glycosidic linkage from the nonreducing end was predominantly split producing (GlcNAc)2 and (GlcNAc)4. The evidence from this work suggested that the subsite structure of the enzyme was (-2)(-1)(+1)(+2)(+3)(+4), whereas most of plant family 19 chitinases have a subsite structure (-3)(-2)(-1)(+1)(+2)(+3). Thus, the Aeromonas enzyme was found to be a novel type of family 19 chitinase in its structural and functional properties.     

In vitro morphogenetic response and distribution of endogenous plant hormones in hypocotyl segments of snapdragon (Antirrhinum majus L.)

Snapdragon seedlings, 20 mm in length, were cut into 5 segments from the cotyledon to the root, which were cultured in vitro on hormone-free MS medium. Adventitious shoot formation was highest in the basal hypocotyl segments with stimulation by the addition of BA. Endogenous cytokinins were higher in the basal hypocotyl segments than in the two upper hypocotyl segments, whereas auxin content was higher in the two upper than in the basal hypocotyl segments. Ratios of cytokinins to auxin were also the highest in the basal hypocotyl segment. A general principle in in vitro culture that a high concentration of cytokinin and a low concentration of auxin promotes the induction of shoot morphogenesis was confirmed from measurements of endogenous growth regulator concentrations.