Validation of HB-EGF and amphiregulin as targets for human cancer therapy

Aberrant expression levels of epidermal growth factor receptor (EGFR) and its cognate ligands have been recognized as one of the causes of cancer progression. To investigate the validity of EGFR ligands as targets for cancer therapy, we examined the expression of EGFR ligands and in vitro anti-tumor effects of small interference RNA (siRNA) for EGFR ligands in various cancer cells. HB-EGF expression was dominantly elevated in ovarian, gastric, and breast cancer, melanoma and glioblastoma cells, whereas amphiregulin was primarily expressed in pancreatic, colon, and prostate cancer, renal cell carcinoma and cholangiocarcinoma cells. Transfection of siRNAs for HB-EGF or amphiregulin into these cells significantly increased the numbers of apoptotic cells with attenuation of EGFR and ERK activation. In lung cancer cells, any EGFR ligand was not recognized as a validated target for cancer therapy. These results suggest that HB-EGF and amphiregulin are promising targets for cancer therapy.     

Evaluation of O,O-Dialkyl O-Cyanophenyl Phosphates and Phosphorothioates as Insecticides

Various O,O-dialkyl O-cyanophenyl phosphates and phosphorothioates were prepared and their biological activities were examined. Among them, O,O-dimethyl O- (4-chloro-2-cyanophenyl) phosphorothioate was found to have selective and high toxicity to houseflies. O,O-Dimethyl O- (4-cyanophenyl) phosphorothioate, O,O-diethyl O- (4-cyanophenyl) phosphorothioate and O,O-diethyl O- (2-chloro-4-cyanophenyl) phosphorothioate showed high insecticidal activty to American cockroaches, though the former two were not so effective to houseflies. The dimethyl esters of these series exhibited markedly lowered mammalian toxicity. Among the O-ethyl O-cyanophenyl phenylphosphonothioates, O-ethyl O- (2-chloro-4-cyanophenyl) phenylphosphonothioate was highly effective to mites, while less effective to insects.     

Shematrin: a family of glycine-rich structural proteins in the shell of the pearl oyster Pinctada fucata

Random sequencing of molecules from a cDNA library constructed from mantle mRNA of the pearl oyster Pinctada fucata was used to obtain information on organic matrix proteins in the shell. In the determined sequences, we identified 7 distinct cDNAs encoding similar glycine-rich domains. Complete sequence analysis of these cDNAs showed that the predicted sequences of the proteins, which we named shematrins, possessed similar domains comprising repeat sequences of two or more glycines, followed by a hydrophobic amino acid. In addition, in shematrin-1, -2 and -3, a repeat domain designated as XGnX (where X is a hydrophobic amino acid) was conserved. It is of further note that all the shematrin proteins have RKKKY, RRKKY or RRRKY as their C-terminal sequence. According to northern blot analysis, all shematrins are exclusively expressed in the mantle, and particularly in the edge region of the mantle; furthermore, peptide fragments similar to shematrin-1 and -2 were detected in the prismatic layer of shells by MALDI-TOF/TOF MS analysis. These findings suggest that many of shematrins are synthesized in the mantle edge and secreted into the prismatic layer of the shell, where the protein family is thought to provide a framework for calcification.     

Cell signaling pathways mediating epidermal growth factor stimulation of Na:K:2Cl cotransport activity in rabbit corneal epithelial cells

We characterized the signaling and ion transport pathways that mediate epidermal growth factor receptor physiological control in SV40-immortalized rabbit corneal epithelial cells (tRCEC). Our evaluation employed single-cell fluorescence imaging to measure the intracellular [Na⁺]_i in these cells loaded with the Na⁺ sensitive dye, SBFI. EGF (1 to 5 ng/ml) transiently increased [Na⁺]_i from 10 mm to as much as 35 mm after 25 min, which was followed by a decline towards its control value. These increases waned at higher EGF concentrations up to 50 ng/ml. Both inhibition of EGF receptor-linked tyrosine kinase activity (50 μm RG-13022) and cPLA₂ activity (10 μm AACOCF₃) obviated EGF-induced increases in [Na⁺]_i. In contrast, PGE₂ (10 μg/ml) and cAMP (2 mm) increased [Na⁺]_i by 25 mm. Inhibition of NKCC activity through exposure to either Cl-free Ringers or 300 μm furosemide in NaCl Ringers eliminated EGF-induced increases in [Na⁺]_i. Similarly, EGF failed to increase [Na⁺]_i following inhibition of: 1) PKA activity (10 μm H-89); 2) Erk1/2 (15 μm PD98059) or 3) p38 (15 μm SB203580) activity. Stimulation protein kinase C activity (0.1 μm PMA) transiently increased [Na⁺]_i followed by a decline towards its baseline value. EGF-induced increases in [Na⁺]_i were unaltered by inhibition of K⁺ conductance (100 μm 4-AP). Taken together, EGF stimulates Erk1/2; p38 and cPLA₂ activity. Their stimulation increases PGE₂ and cAMP levels resulting in PKA and NKCC activation. Received: 18 December 2000/Revised: 24 May 2001     

Polypeptides coded for by the region pre-S and gene S of hepatitis B virus DNA with the receptor for polymerized human serum albumin: expression on hepatitis B particles produced in the HBeAg or anti-HBe phase of hepatitis B virus infection

There are four polypeptides coded for by the region Pre-S and gene S on DNA of hepatitis B virus that carry the receptor for polymerized human serum albumin (poly-HSA), i.e., P31 and P39, as well as their glycosylated counterparts P35 and P43. With the use of monoclonal antibodies directed to Pre-S(1) sequence and Pre-S(2) sequence (bearing the receptor for poly-HSA), the content of these polypeptides, as well as their expression on the surface, was determined for hepatitis B particles of various categories. P39 and P43, carrying both Pre-S(1) and Pre-S(2) sequences, were contained abundantly in Dane and tubular particles, and to a much lesser extent in small spherical particles, all of which were purified from plasma containing hepatitis B e antigen (HBeAg). P31 and P35, carrying Pre-S(2) but not Pre-S(1) sequence, were contained comparably in these three categories of hepatitis B particles. In remarkable contrast, small spherical particles derived from plasma containing antibody to HBeAg were very low in the content of any Pre-S polypeptides. P31 and P39 showed higher activities for poly-HSA receptor than their glycosylated versions. When Dane particles were digested with trypsin, the poly-HSA receptor was deprived in parallel with the loss of antigenicity for Pre-S(2) sequence. The antigenicity for Pre-S(1) sequence was much less affected, and that for the product of gene S was virtually unchanged by the digestion.     

Vascular endothelial growth factor system in the cow oviduct: a possible involvement in the regulation of oviductal motility and embryo transport

Vascular endothelial growth factor (VEGF) is a potent angiogenic and permeability enhancing factor, which shows the highest activity in the oviduct during the periovulatory period of the estrous cycle in cattle. It has also been shown that the contraction activity of oviduct is highest during the periovulatory period. The present study therefore focused on the possible involvement of VEGF in the regulation of biosynthesis and secretion of contraction-relaxation-related substances in the cow oviduct. Possible autonomous VEGF system in the oviduct as well as its endocrine control was also studied. Bovine oviductal epithelial cells (BOEC) in the second passage were cultured with VEGF (1 ng/ml) alone or with luteinizing hormone (LH; 10 ng/ml), estradiol 17-beta (E2; 1 ng/ml), and/or progesterone (P4; 1 ng/ml). The levels of prostaglandins (PGs), endothelin-1 (ET-1), and angiotensin II (Ang II) in the medium were measured using second antibody enzymeimmunoassay (EIA). The mRNA expressions for cycloxygenase-2 (Cox-2), prostaglandin F synthase (PGFS), prostaglandin E synthase (PGES), prepro-ET-1, endothelin converting enzyme-1 (Ece-1), angiotensin converting enzyme-1 (Ace-1), VEGF and its receptors were investigated using real-time RT-PCR. The results indicate that, (1) VEGF dose-dependently stimulated the release of prostaglandin E2 (PGE2), prostaglandin F2alpha (PGF2alpha), and ET-1, but not Ang II. VEGF and VEGF with LH, E2, and P4 upregulated mRNA expression for biosynthesis cascade of PG, ET-1 as well as their release. However, only the combination of VEGF with LH, E2, and P4 upregulated mRNA for Ace-1 and Ang II release, but not VEGF alone. (2) Treatments of LH, with E2 and/or P4 increased the mRNA expression for VEGF, Flk-1 and Flt-1, and (3) VEGF itself downregulated the expression of mRNA for VEGF, and LH, E2, and P4 enhanced this downregulatory effect. The results of the present study provide the first evidence that (1) VEGF directly stimulates the biosynthesis and release of PGE2, PGF2alpha, and ET-1 in the bovine oviduct, (2) LH stimulates the oviductal VEGF system, and (3) VEGF downregulates the oviductal VEGF system and this downregulation was further intensified in the presence of LH. The data suggest that the preovulatory LH-surge, together with increasing E2 secretion from the Graffian follicle and basal P4 levels from the regressing corpus luteum (CL), upregulates the oviductal VEGF system, inducing the maximum oviductal production of contraction-relaxation-related substances for active oviduct contraction and rapid transport of gametes to the fertilization site. However, the oviductal VEGF elevation caused by the LH-surge, appears to downregulate the oviductal VEGF system immediately after ovulation thereby may contribute to suppress oviductal contraction to secure slow transport of the embryo to the uterus at the optimal time.     

The ICOS molecule plays a crucial role in the development of mucosal tolerance

The ICOS molecule stimulates production of the immunoregulatory cytokine IL-10, suggesting an important role for ICOS in controlling IL-10-producing regulatory T cells and peripheral T cell tolerance. In this study we investigate whether ICOS is required for development of oral, nasal, and high dose i.v. tolerance. Oral administration of encephalitogenic myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide to ICOS-deficient (ICOS-/-) mice did not inhibit experimental autoimmune encephalomyelitis (EAE), T cell proliferation, or IFN-gamma production, in striking contrast to wild-type mice. Similarly, intranasal administration of MOG(35-55) before EAE induction suppressed EAE and T cell responses in wild-type, but not in ICOS-/-, mice. In contrast, ICOS-/- mice were as susceptible as wild-type mice to high dose tolerance. These results indicate that ICOS plays an essential and specific role in mucosal tolerance and that distinct costimulatory pathways differentially regulate different forms of peripheral tolerance. Surprisingly, CD4+ cells from MOG-fed wild-type and ICOS-/- mice could transfer suppression to wild-type recipients, indicating that functional regulatory CD4+ cells can develop in the absence of ICOS. However, CD4+ T cells from MOG-fed wild-type mice could not transfer suppression to ICOS-/- recipients, suggesting that ICOS may have a key role in controlling the effector functions of regulatory T cells. These results suggest that stimulating ICOS may provide an effective therapeutic approach for promoting mucosal tolerance.     

Using evolutionary rates to investigate protein functional divergence and conservation. A case study of the carbonic anhydrases

Functional constraints on proteins limit their evolutionary rates at specific sites. These constraints allow for the interpretation of conserved residues and sites with a rate change as those most likely underlying the functional similarities and differences among protein subfamilies, respectively. This study describes new likelihood-ratio tests (LRTs) that complement existing ones for the identification of both conserved and rate change sites. These identifications are validated by the recovery of residues that are known from existing biochemical and structural information to be critical for the functional similarities and differences among carbonic anhydrases (CAs). In combination with this other information, these LRTs also support a unique antioxidant defense role for the puzzling CA III. As illustrated by the CAs, these LRTs, in combination with other biological evidence, offer a powerful and cost-effective approach for testing hypotheses, making predictions, and designing experiments in protein functional studies.