Immunolocalization of centriole-associated striated rootlets in human submandibular gland cells with and without solitary cilia

Using an antibody specific to striated rootlets, we investigated the immuolocalization of striated rootlets in cells constituting human submandibular glands. Striated rootlets were positively stained in all cell types constituting acini, intercalated ducts, striated ducts, and interlobular ducts, but their shapes were different. The mean lengths of striated rootlets were 1.46 +/- 0.49, 3.15 +/- 1.35 and 3.99 +/- 1.02 microm in acinar secretory cells, myoepithelial cells, and columnar cells of the striated duct, respectively. The rootlets were the longest in columnar cells of the striated duct, in which paired centrioles were located in the apical cytoplasm away from nuclei. These findings suggest that striated rootlets play important roles in the positioning of centrioles in the cell. 2-8% of striated rootlets in myoepithelial cells were associated with solitary cilia, but they were not associated with solitary cilia in acinar cells and columnar cells of the striated duct. These observations suggest that striated rootlets may be associated with centrioles under normal physiological conditions, without formation of solitary cilia.     

DmGEN, a novel RAD2 family endo-exonuclease from Drosophila melanogaster

A novel endo-exonuclease, DmGEN (Drosophila Melanogaster XPG-like endonuclease), was identified in D.melanogaster. DmGEN is composed of five exons and four introns, and the open reading frame encodes a predicted product of 726 amino acid residues with a molecular weight of 82.5 kDa and a pI of 5.36. The gene locus on Drosophila polytene chromosomes was detected at 64C9 on the left arm of chromosome 3 as a single site. The encoded protein showed a relatively high degree of sequence homology with the RAD2 nucleases, especially XPG. Although the XPG-N- and XPG-I-domains are highly conserved in sequence, locations of the domains are similar to those of FEN-1 and EXO-1, and the molecular weight of the protein is close to that of EXO-1. In vitro, DmGEN showed endonuclease and 3'-5' exonuclease activities with both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), but the endonuclease action with dsDNA was quite specific: 5'-3' exonuclease activity was found to occur with nicked DNA, while dsDNA was endonucleolytically cut at 3-4 bp from the 5' end. Homologs are widely found in mammals and higher plants. The data suggest that DmGEN belongs to a new class of RAD2 nuclease.     

Immunolocalization of vascular endothelial growth factor in rat condylar cartilage during postnatal development

It is well known that angiogenesis is essential for the replacement of cartilage by bone during skeletal growth and regeneration. To address angiogenesis of endochondral ossification in the condyle, we examined the appearance of vascular endothelial growth factor (VEGF) and its receptor Flt-1 in condylar cartilage of the growing rat. The early expression of VEGF at various sites during condylar cartilage development indicates that VEGF plays a role in the regulation of angiogenesis at each site of bone formation. From the findings of Flt-1 immunoreactivity, the VEGF produced by the chondrocytes of the hypertrophic zone should contribute to the promotion of endothelial cell proliferation and to stimulate migration and activation of osteoclasts in condylar cartilage, resulting in the invasion of these cells into the mineralized zone.Junko Aoyama and Eiji Tanaka contributed equally to this work     

Size-dependent male–male competition for a spawning substrate between <Emphasis Type="Italic">Pseudorasbora parva</Emphasis> and <Emphasis Type="Italic">Pseudorasbora pumila</Emphasis>

Pseudorasbora parva, a species native to western Japan, has been accidentally introduced into eastern Japan, where P. pumila is indigenous. We investigated inter- and intraspecific, male–male competition between P. parva and P. pumila for acquisition of spawning substrates in an experimental setting. Within each species, males of larger standard length and heavier body weight were more successful in acquiring a substrate. Males of the two species competed, but the outcome was determined primarily by body weight. This interspecific, size-dependent, male–male competition might be an important factor in the species replacement of P. pumila by P. parva.     

Changes in oligosaccharide expression on plasma membrane of the mouse oocyte during fertilisation and early cleavage

It has been reported that various structural and functional changes occur on the surface of the plasma membrane of the ovum and embryo during fertilisation and cleavage in preparation for implantation. Glycoproteins are thought to be one of the factors in cell attachment. Thus, we investigated the changes in glycoprotein expression on the cell surface membrane of the mouse embryo by using lectins. Among seven types of lectin (ConA, WGA, UEA-I, MPA, LCA, DBA and PNA), the fluorescent intensities of ConA and WGA markedly increased from unfertilised ova to blastocysts. By quantitative analysis using immuno-scanning electron microscopy, the numbers of ConA-gold particles were small until 4-cell cleavage, but increased significantly at the blastocyst stage. In contrast, an increased number of WGA-gold particles was detected even at the 4-cell stage, and this increase continued to the blastocyst stage. From the above observations, we conclude that the numbers of sugar chains bound to both ConA andWGA increases with blastocyst formation and earlier expression is observed with WGA. The present study dearly shows that glycoproteins on the cell membrane surface of the mouse embryo quantitatively increase at the time of implantation, and the possibility has been indicated that glycoproteins are involved in intercellular recognition and adhesion between the embryo and endometrial epithelium.     

An inverse correlation between expression of a preprocathepsin B-related protein with cysteine-rich sequences and steroid 11beta -hydroxylase in adrenocortical cells

A cDNA encoding a secretory protein hitherto unknown was cloned from mouse adrenocortical cells by subtractive hybridization between the cells without and with expressing steroid 11beta-hydroxylase (Cyp11b-1), a marker for the functional differentiation of cells in the zonae fasciculata reticularis (zFR). The deduced protein consisting of 466 amino acids contained a secretory signal, epidermal growth factor-like repeats, and a proteolytically inactive cathepsin B-related sequence. The amino acid sequence was 89% identical with that of human tubulointerstitial nephritis antigen-related protein. Among the mouse organs examined, adrenal glands prominently expressed its mRNA. The mRNA and its encoded protein were detected in the outer adrenocortical zones that do not express Cyp11b-1, i.e. the zona glomerulosa and the undifferentiated cell zone, while being undetectable in zFR that express Cyp11b-1. The new protein was designated as adrenocortical zonation factor 1 (AZ-1). Clonal lines with different levels of AZ-1 expression were established from Y-1 adrenocortical cells that originally express Cyp11b-1 but little AZ-1. Analyses of the clonal lines revealed that Cyp11b-1 is detected in the clonal lines maintaining little AZ-1 expression and becomes undetectable in those expressing AZ-1. On the other hand, irrespective of the AZ-1 expression, all clones expressed cholesterol side-chain cleavage enzyme, which occurs throughout the cortical zones. These results demonstrated that adrenocortical cells expressing AZ-1 do not express Cyp11b-1, whereas those with little AZ-1 express this zFR marker in vitro and in vivo, implying a putative role of AZ-1 in determining the zonal differentiation of adrenocortical cells.     

Crystal structure of serine dehydratase from rat liver

SDH (L-serine dehydratase, EC 4.3.1.17) catalyzes the pyridoxal 5'-phosphate (PLP)-dependent dehydration of L-serine to yield pyruvate and ammonia. Liver SDH plays an important role in gluconeogenesis. Formation of pyruvate by SDH is a two-step reaction in which the hydroxyl group of serine is cleaved to produce aminoacrylate, and then the aminoacrylate is deaminated by nonenzymatic hydrolysis to produce pyruvate. The crystal structure of rat liver apo-SDH was determined by single isomorphous replacement at 2.8 A resolution. The holo-SDH crystallized with O-methylserine (OMS) was also determined at 2.6 A resolution by molecular replacement. SDH is composed of two domains, and each domain has a typical alphabeta-open structure. The active site is located in the cleft between the two domains. The holo-SDH contained PLP-OMS aldimine in the active site, indicating that OMS can form the Schiff base linkage with PLP, but the subsequent dehydration did not occur. Apo-SDH forms a dimer by inserting the small domain into the catalytic cleft of the partner subunit so that the active site is closed. Holo-SDH also forms a dimer by making contacts at the back of the clefts so that the dimerization does not close the catalytic cleft. The phosphate group of PLP is surrounded by a characteristic G-rich sequence ((168)GGGGL(172)) and forms hydrogen bonds with the amide groups of those amino acid residues, suggesting that the phosphate group can be protonated. N(1) of PLP participates in a hydrogen bond with Cys303, and similar hydrogen bonds with N(1) participating are seen in other beta-elimination enzymes. These hydrogen bonding schemes indicate that N(1) is not protonated, and thus, the pyridine ring cannot take a quinone-like structure. These characteristics of the bound PLP suggest that SDH catalysis is not facilitated by forming the resonance-stabilized structure of the PLP-Ser aldimine as seen in aminotransferases. A possible catalytic mechanism involves the phosphate group, surrounded by the characteristic sequence, acting as a general acid to donate a proton to the leaving hydroxyl group of serine.     

Cytokines decrease sGC in pulmonary artery smooth muscle cells via NO-dependent and NO-independent mechanisms

Exposure of rat pulmonary artery smooth muscle cells (rPASMC) to cytokines leads to nitric oxide (NO) production by NO synthase 2 (NOS2). NO stimulates cGMP synthesis by soluble guanylate cyclase (sGC), a heterodimer composed of alpha(1)- and beta(1)-subunits. Prolonged exposure of rPASMC to NO decreases sGC subunit mRNA and protein levels. The objective of this study was to determine whether levels of NO produced endogenously by NOS2 are sufficient to decrease sGC expression in rPASMC. Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) increased NOS2 mRNA levels and decreased sGC subunit mRNA levels. Exposure of rPASMC to IL-1beta and TNF-alpha for 24 h decreased sGC subunit protein levels and NO-stimulated sGC enzyme activity. L-N(6)-(1-iminoethyl)lysine (NOS2 inhibitor) or 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (sGC inhibitor) partially prevented the cytokine-mediated decrease in sGC subunit mRNA levels. However, cytokines also decreased sGC subunit mRNA levels in PASMC derived from NOS2-deficient mice. These results demonstrate that levels of NO and cGMP produced in cytokine-exposed PASMC are sufficient to decrease sGC subunit mRNA levels. In addition, cytokines can decrease sGC subunit mRNA levels via NO-independent mechanisms.     

Cytokine expression in rat molar gingival periodontal tissues after topical application of lipopolysaccharide

It is well known that proinflammatory cytokines produced by host cells play an important role in periodontal tissue destruction. However, the localization of the cytokines in in vivo periodontal tissues during development of periodontal disease has not been determined. Immunohistochemical expression of proinflammatory cytokines including IL-1!, IL-1#, and TNF-! was examined at 1 and 3 h, and 1, 2, 3, and 7 days after topical application of lipopolysaccharide (LPS; 5 mg/ml in physiological saline) from E. coli into the rat molar gingival sulcus. In the normal periodontal tissues, a small number of cytokine-positive epithelial cells were seen in the junctional epithelium (JE), oral sulcular and oral gingival epithelium, in addition to macrophages infiltrating in the subjunctional epithelial area and osteoblasts lining the alveolar bone surface. Epithelial remnants of Malassez existing throughout periodontal ligament were intensely positive for IL-1# but negative for the other two cytokines. At 3 h after the LPS treatment, almost all cells in the JE were strongly positive for the cytokines examined. In addition, several cytokine-positive cells, including neutrophils, macrophages, and fibroblasts, were seen in the subjunctional epithelial connective tissue. At day 2, expression of the cytokines in the JE gradually decreased, while cytokine-positive cells in the connective tissue increased in number. Positive staining of the cytokines was seen in osteoclasts and preosteoclasts which appeared along the alveolar bone margin in this period. The number of cytokine-positive cells decreased by day 7. These findings indicate that, in addition to macrophages, neutrophils, and fibroblasts, the JE cells are a potent source of TNF-!, IL-1!, and IL-1# reacting to LPS application, and suggest that JE cells may play an important role in the first line of defense against LPS challenge, and the proinflammatory cytokines transiently produced by various host cells may be involved in the initiation of inflammation and subsequent periodontal tissue destruction.     

Involvement of vertebrate polkappa in Rad18-independent postreplication repair of UV damage

DNA damage, which is left unrepaired by excision repair pathways, often blocks replication, leading to lesions such as breaks and gaps on the sister chromatids. These lesions may be processed by either homologous recombination (HR) repair or translesion DNA synthesis (TLS). Vertebrate Polkappa belongs to the DNA polymerase Y family, as do most TLS polymerases. However, the role for Polkappa in vertebrate cells is unclear because of the lack of reverse genetic studies. Here, we generated cells deficient in Polkappa (polkappa cells) from the chicken B lymphocyte line DT40. Although purified Polkappa is unable to bypass ultraviolet (UV) damage, polkappa cells exhibited increased UV sensitivity, and the phenotype was suppressed by expression of human and chicken Polkappa, suggesting that Polkappa is involved in TLS of UV photoproduct. Defects in both Polkappa and Rad18, which regulates TLS in yeast, in DT40 showed an additive effect on UV sensitivity. Interestingly, the level of sister chromatid exchange, which reflects HR-mediated repair, was elevated in normally cycling polkappa cells. This implies functional redundancy between HR and Polkappa in maintaining chromosomal DNA. In conclusion, vertebrate Polkappa is involved in Rad18-independent TLS of UV damage and plays a role in maintaining genomic stability.