Transforming growth factor-beta3 promotes mesenchymal cell proliferation and angiogenesis mediated by the enhancement of cyclin D1, Flk-1, and CD31 gene expression during CL/Fr mouse lip fusion

BACKGROUND: Cleft lip with or without cleft palate is the most common congenital anomaly in the craniofacial region. Knowledge of the molecular mechanisms behind normal lip fusion can contribute to better intervention and improved functional clinical outcome. Transforming growth factor-beta3 (TGF-beta3) has been implicated in lip morphogenesis. Therefore, we hypothesized that TGF-beta3 functions during lip fusion through regulation of angiogenesis and mesenchymal cell cycle progression during early developmental stages. METHODS: To test this hypothesis we used the CL/Fraser mouse model, which has a high incidence of cleft lip. Lips isolated from embryonic day (ED) 11.5 mouse embryos were allowed to develop in serum-free organ cultures in the presence or absence of TGF-beta3. The lips that developed in these cultures fused in 2 days. RESULTS: During normal development, we detected positive immunoreactions for TGF-beta3 at the site of fusion. We also detected mesenchymal cells that were immunopositive for Flk-1 and CD31, which are markers for endothelial cell precursors. Exogenous TGF-beta3 accelerated lip fusion in culture. This enhancement was associated with an increase in the number of capillary blood vessels in the lips cultured in the presence of TGF-beta3, in comparison with controls. In tandem, TGF-beta3 increased the level of expression of both Flk-1 and CD31. Our data suggest that an elevated level of TGF-beta3 may promote angiogenesis in developing lips that is mediated by increased Flk-1 and CD31 expression. We also detected increased cyclin D1 expression (a marker for cell proliferation) in the presence of TGF-beta3, which suggests that TGF-beta3 promoted cell proliferation. CONCLUSIONS: TGF-beta3 promoted cell proliferation and angiogenesis in lip mesenchymal tissues. These events led to enhanced lip fusion in the presence of TGF-beta3.     

Phospholipase Cdelta4 associates with glutamate receptor interacting protein 1 in testis

We reported previously that phospholipase C (PLC) delta4 is required for calcium mobilization in the zona pellucida-induced acrosome reaction in sperm. Here we focused on the function of the C2 domain of PLCdelta4 and report that glutamate receptor-interacting protein1 (GRIP1) was identified as a binding protein of the PLCdelta4-C2 domain on yeast two-hybrid screening. Physiological interaction of GRIP1 with PLCdelta4 in mouse testis was confirmed by immunoprecipitation with anti-PLCdelta4 antibodies and the association seemed to correlate with the maturation stage of sperm. We also determined that a PDZ-binding motif at the C-terminus of the PLCdelta4-C2 domain is responsible for GRIP1 binding, whereas the sixth or seventh PDZ domain of GRIP1 is essential and sufficient for association with the PLCdelta4-C2 domain. These results indicate that PLCdelta4 binds via its C2 domain to the PDZ6 or PDZ7 domain of GRIP1, and that this association may play a role in spermatogenesis.     

A novel phospholipase C, PLC(eta)2, is a neuron-specific isozyme

Twelve phospholipase C (PLC) isozymes have been cloned so far, and they are divided into six classes, beta-, gamma-, delta-, epsilon-, zeta-, and eta-type, on the basis of structure and activation mechanisms. Here we report the identification of a novel PLC isozyme, PLC(eta)2. PLC(eta)2 is composed of conserved domains including pleckstrin homology, EF-hand, X and Y catalytic, and C2 domains and the isozyme-specific C-terminal region. PLC(eta)2 consists of 1164 amino acids with a molecular mass of 125 kDa. The PLC activity of PLC(eta)2 was more sensitive to calcium concentration than the PLC activity of the PLCdelta-type enzyme, which is thought to be the most calcium-sensitive PLC. Immunofluorescence analysis showed that PLC(eta)2 was localized predominantly to the plasma membrane at resting state via the pleckstrin homology domain. This observation was supported by Western blot analysis of cytosol and membrane fractions. In addition, expression of PLC(eta)2 was detected after birth and showed a restricted distribution in the brain; it was particularly abundant in the hippocampus, cerebral cortex, and olfactory bulb. The pattern was similar to that of the neuronal marker microtubule-associated protein 2 by Western blot. Furthermore, in situ hybridization showed positive signals for PLC(eta)2 in pyramidal cells of the hippocampus. Finally, we found that PLC(eta)2 was expressed abundantly in neuron-containing primary culture but not in astrocyte-enriched culture. These results indicate that PLC(eta)2 is a neuron-specific isozyme that may be important for the formation and/or maintenance of the neuronal network in the postnatal brain.     

CXCR5-dependent seeding of follicular niches by B and Th cells augments antiviral B cell responses

The chemokine receptor CXCR5 and its ligand CXCL13 define the structure of B cell follicles within secondary lymphoid organs. Here, we examined the impact of CXCR5 on antiviral B cell responses in vivo. CXCR5-/- mice showed a normal production of IgM and IgG acutely after infection with vesicular stomatitis virus (VSV) and developed VSV-specific germinal centers. However, impaired Ig class switch and Ab production were observed under conditions of limited availability of Ag (i.e., after immunization with nonreplicating viral particles or soluble Ag). Adoptive transfer of CXCR5-deficient, VSV-specific B and Th cells demonstrated that CXCR5 expression on both B and Th cells is required for an efficient Ig class switch. These experiments revealed that CXCR5 is critical for the coordinated interaction of antiviral T and B cells through its impact on initial B cell expansion and the recruitment of Ag-specific B and Th cells to germinal centers.     

Coordination behavior of phosphino-phosphaferrocenes: monodentate versus bidentate coordination to divalent palladium

Two novel phosphino-phosphaferrocenes [η⁵-C₅H₄(CH₂)_nPPh₂]Fe(η⁵-PC₄H₂-2,5-Cy₂) (PP1: n=1; PP2: n=2) have been designed and prepared in order to clarify weak chelate effect in the previously reported (η⁵-C₅H₄CH₂PPh₂)Fe[η⁵-PC₄H₂-2,5-((-)-menthyl)₂] (1). ³¹P NMR studies of reactions of PP1 with PdCl₂(cod) (6) revealed that PP1 showed stronger tendency to coordinate to the Pd^II center in bidentate fashion compared to 1. On the other hand, chelate effect in PP2 was negligibly weak and a reaction of PP2 with 6 in a PP2/6 = 2/1 molar ratio gave a complex PdCl₂(PP2)₂ (10) cleanly in which PP2 coordinated to the palladium center at the PPh₂ moiety as a monodentate ligand. X-ray crystal structure studies of chelate complexes PdCl₂(PP1) (7) and PdCl₂(PP2) (9) showed that 9 had deviations from an idealized geometry in the square planar complex which could be attributed to a larger chelate ring of PP2, while PP1 in 7 constructed nearly ideal geometry for the square planar complex.From comparison of the coordination behavior between 1, PP1, and PP2, it is concluded that steric bulk of (-)-menthyl groups in 1 is the main factor of the weak chelate coordination of 1.     

A novel endonucleolytic mechanism to generate the CCA 3' termini of tRNA molecules in Thermotoga maritima

The tRNA 3'-terminal CCA sequence is essential for aminoacylation of the tRNAs and for translation on the ribosome. The tRNAs are transcribed as larger precursor molecules containing 5' and 3' extra sequences. In the tRNAs that do not have the encoded CCA, the 3' extra sequence after the discriminator nucleotide is usually cleaved off by the tRNA 3' processing endoribonuclease (3' tRNase, or RNase Z), and the 3'-terminal CCA residues are added thereto. Here we analyzed Thermotoga maritima 3' tRNase for enzymatic properties using various pre-tRNAs from T. maritima, in which all 46 tRNA genes encode CCA with only one exception. We found that the enzyme has the unprecedented activity that cleaves CCA-containing pre-tRNAs precisely after the CCA sequence, not after the discriminator. The assays for pre-tRNA variants suggest that the CA residues at nucleotides 75 and 76 are required for the enzyme to cleave pre-tRNAs after A at nucleotide 76 and that the cleavage occurs after nucleotide 75 if the sequence is not CA. Intriguingly, the pre-tRNA(Met) that is the only T. maritima pre-tRNA without the encoded CCA was cleaved after the discriminator. The kinetics data imply the existence of a CCA binding domain in T. maritima 3' tRNase. We also identified two amino acid residues critical for the cleavage site selection and several residues essential for the catalysis. Analysis of cleavage sites by 3' tRNases from another eubacteria Escherichia coli and two archaea Thermoplasma acidophilum and Pyrobaculum aerophilum corroborates the importance of the two amino acid residues for the cleavage site selection.     

Peptides binding to a Gb3 mimic selected from a phage library

Peptides binding to a Gb3 mimic were selected from 12-mer peptide library. The self-assembled monolayer (SAM) of a Gb3 mimic was formed on the gold surface, and biopanning was carried out with the phage display peptide library. After three rounds of biopanning, four individual sequences were obtained from 10 phage clones, and the selected peptides having the specific 7-mer sequence (FHENWPS) showed affinities to the Gb3 mimic as strong as to RCA120. Molecular dynamics calculations suggested that the peptides bound to the Gb3 mimic by hydrophobic interaction and hydrogen bonding formation, and the cooperative interactions played an important role in the recognition. The Stx-1 binding was inhibited by the peptides.     

Human beta-tryptase: detection and characterization of the active monomer and prevention of tetramer reconstitution by protease inhibitors

beta-Tryptase is a trypsin-like serine protease stored in mast cell secretory granules primarily as an enzymatically active tetramer. The current study aims to determine whether monomeric beta-tryptase also can exhibit enzyme activity, as suggested previously. At neutral pH beta-tryptase tetramers in the absence of heparin or dextran sulfate spontaneously convert to inactive monomers. Addition of a polyanion to these monomers at neutral pH fails to convert them back to a tetramer or to an enzymatically active state. In contrast, at acidic pH addition of a polyanion resurrects enzyme activity. Whether this activity is associated with tetramers or monomers depends on the concentration of beta-tryptase. Under the experimental conditions employed at pH 6 in the presence of heparin, the monomer concentration at which 50% conversion to tetramers occurs is 193 ng/mL. Activity against tripeptide substrates by monomers is detected at pH 6 but not at pH 7.4, whereas tetramer activity is greater at pH 7.4 than pH 6.0. Active monomers are inhibited by soybean trypsin inhibitor, bovine pancreatic trypsin inhibitor, antithrombin III, and alpha2-macroglobulin, whereas active tetramers are resistant to these inhibitors. Active monomers form complexes with these inhibitors and cleave both antithrombin III and alpha2-macroglobulin. These inhibitors also prevent reconstitution of monomers to tetramers, indicating that inactive monomers become active monomers before becoming active tetramers. The ability of tryptase monomers to become active at acidic pH raises the possibilities of expanded substrate specificities as well as inhibitor susceptibilities where the low-pH environments associated with inflammation or poor vascularity are encountered in vivo.     

Phospholipase C delta-type consists of three isozymes: bovine PLCdelta2 is a homologue of human/mouse PLCdelta4

To date, 12 phospholipase C (PLC) isozymes have been identified in mammals, and they are divided into five classes, beta-, gamma-, delta-, epsilon-, and zeta-type. PLCdelta-type is reported to be composed of four isozymes, PLCdelta1-delta4. Here we report that a screening for mouse PLCdelta2 from a BAC library with primers that amplify a specific region of bovine PLCdelta2 resulted in isolation of one clone containing the mouse PLCdelta4 gene. Furthermore, a database search revealed that there is only one gene corresponding to PLCdelta2 and PLCdelta4 in the mouse and human genomes, indicating that bovine PLCdelta2 is a homologue of human and mouse PLCdelta4. However, PLCdelta2 Western blot analysis with a widely used commercial anti-PLCdelta2 antibody showed an expression pattern distinct from that of PLCdelta4 in wild-type mice. In addition, an 80-kDa band, which was recognized by antibody against PLCdelta2, was smaller than an 85-kDa band detected by anti-PLCdelta4 antibody, and the 80-kDa band was detectable in lysates of brain, testis, and spleen from PLCdelta4-deficient mice. We also found that immunoprecipitates from brain lysates with this PLCdelta2 antibody contained no PLC activity. From these data, we conclude that bovine PLCdelta2 is a homologue of human and mouse PLCdelta4, and that three isozymes (delta1, delta3, and delta4) exist in the PLCdelta family.     

Negative regulation of the protein kinase C activator-induced ICAM-1 expression in the human bronchial epithelial cell line NCI-H292 by p44/42 mitogen-activated protein kinase

The role of p44/42 mitogen-activated protein kinase (MAPK) in the expression of intercellular adhesion molecule-1 (ICAM-1) in NCI-H292 cells, a human bronchial epithelial cell line, was analyzed. Treatment with the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) (16.2 nM) or interferon-gamma (IFN-gamma) (100 U/ml) induced phosphorylation of p44/42 MAPK. The MEK inhibitor U0126 (0.1 to 10 microM) enhanced the TPA-induced ICAM-1 expression but not the IFN-gamma-induced one. U0126 also enhanced the ICAM-1 expression induced by two other PKC activators teleocidin (22.5 nM) and aplysiatoxin (14.9 nM). Furthermore, PD98059 (0.5 to 50 microM), another MEK inhibitor, enhanced the TPA-induced ICAM-1 expression as well. The inhibitor of p38 MAPK SB203580 did not affect the TPA-induced ICAM-1 expression. BAY11-7082, an inhibitor of nuclear factor kappaB (NF-kappaB) activation, and MG132, a 26S proteasome inhibitor, reduced the TPA-induced ICAM-1 expression but not the IFN-gamma-induced one. TPA partially decreased the level of IkappaB-alpha and the reduction was further augmented by U0126 in a concentration-dependent manner. These findings suggested that, in NCI-H292 cells, p44/42 MAPK suppresses PKC activator-induced NF-kappaB activation, thus negatively regulating the PKC activator-induced ICAM-1 expression but not the IFN-gamma-induced one.