The GroEL protein of Porphyromonas gingivalis accelerates tumor growth by enhancing endothelial progenitor cell function and neovascularization.

Porphyromonas gingivalis is a bacterial species that causes destruction of periodontal tissues. Additionally, previous evidence indicates that GroEL from P. gingivalis may possess biological activities involved in systemic inflammation, especially inflammation involved in the progression of periodontal diseases. The literature has established a relationship between periodontal disease and cancer. However, it is unclear whether P. gingivalis GroEL enhances tumor growth. Here, we investigated the effects of P. gingivalis GroEL on neovasculogenesis in C26 carcinoma cell-carrying BALB/c mice and chick eggs in vivo as well as its effect on human endothelial progenitor cells (EPC) in vitro. We found that GroEL treatment accelerated tumor growth (tumor volume and weight) and increased the mortality rate in C26 cell-carrying BALB/c mice. GroEL promoted neovasculogenesis in chicken embryonic allantois and increased the circulating EPC level in BALB/c mice. Furthermore, GroEL effectively stimulated EPC migration and tube formation and increased E-selectin expression, which is mediated by eNOS production and p38 mitogen-activated protein kinase activation. Additionally, GroEL may enhance resistance against paclitaxel-induced cell cytotoxicity and senescence in EPC. In conclusion, P. gingivalis GroEL may act as a potent virulence factor, contributing to the neovasculogenesis of tumor cells and resulting in accelerated tumor growth.

The essential role of transient receptor potential vanilloid 1 in simvastatin-induced activation of endothelial nitric oxide synthase and angiogenesis.

AIMS: We investigated the role of transient receptor potential vanilloid receptor type 1 (TRPV1) in simvastatin-mediated activation of endothelial nitric oxide synthase (eNOS) and angiogenesis. METHODS: Fluo-8 NW assay was for Ca(2+) detection; Griess's assay was for NO bioavailability; Western blotting and immunoprecipitation were for protein phosphorylation and interaction; tube formation and Matrigel plug assay were for angiogenesis. RESULTS: In endothelial cells (ECs), treatment with simvastatin time-dependently increased intracellular level of Ca(2+). Pharmacological inhibition or genetic disruption of TRPV1 abrogated simvastatin-mediated elevation of intracellular Ca(2+) in ECs or TRPV1-transfected HEK293 cells. Loss of TRPV1 function abolished simvastatin-induced NO production and phosphorylation of eNOS and calmodulin protein kinase II (CaMKII) in ECs and in aortas of mice. Inhibition of TRPV1 activation prevented the simvastatin-elicited increase in the formation of TRPV1-Akt-CaMKII-AMPK-eNOS complex. In mice, Matrigel plug assay showed that simvastatin-evoked angiogenesis was abolished by TRPV1 antagonist and genetic ablation of TRPV1. Additionally, our results demonstrated that TRP ankyrin 1 (TRPA1) is the downstream effector in the simvastatin-activated TRPV1-Ca(2+) signalling and in the consequent NO production and angiogenesis as evidence by that re-expression of TRPA1 further augmented simvastatin-elicited Ca(2+) influx in TRPV1-expressed HEK293 cells and ablation of TRPA1 function profoundly inhibited the simvastatin-induced increase in the phosphorylation of eNOS and CaMKII, formation of TRPV1-Akt-CaMKII-AMPK-eNOS complex, NO bioavailability, tube formation and angiogenesis in ECs or mice. CONCLUSION: Simvastatin-induced Ca(2+) influx may through the activation of TRPV1-TRPA1 signalling, which leads to phosphorylation of CaMKII, increases in the formation of TRPV1-CaMKII-AMPK-eNOS complex, eNOS activation, NO production and, ultimately, angiogenesis in ECs.

Activation of soluble guanylyl cyclase prevents foam cell formation and atherosclerosis.

AIMS: Soluble guanylyl cyclase (sGC) is a key modulator in the regulation of vascular tone. However, its role and involving mechanism in cholesterol metabolism of macrophages and atherosclerosis remain unclear. METHODS: Oil red O staining, Dil-oxidized low-density lipoprotein (oxLDL)-binding assay and cholesterol efflux assay were performed in biology of foam cells. Levels of cytokines or intracellular lipid were evaluated by ELISA or colorimetric kits. Expression of gene or protein was determined by quantitative real-time PCR or Western blotting. Histopathology was examined by haematoxylin and eosin staining. RESULTS: Soluble guanylyl cyclase was expressed in macrophages of mouse atherosclerotic lesions. Treatment with 1H-[1, 2, 4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, sGC inhibitor) exacerbated oxLDL-induced cholesterol accumulation in macrophages. In contrast, 3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1, sGC activator) attenuated the oxLDL-induced cholesterol accumulation because of increased cholesterol efflux. Additionally, YC-1 dose dependently increased the protein expression of ATP-binding cassette transporter A1 (ABCA1) but did not alter that of scavenger receptor class A (SR-A), CD36, SR-BI or ABCG1. Moreover, YC-1-upregulated ABCA1 level depended on liver X receptor α (LXRα). Inhibition of the LXRα-ABCA1 pathway by LXRα small interfering RNA (siRNA), ABCA1 neutralizing antibody or ABCA1 siRNA abolished the effect of YC-1 on cholesterol accumulation and cholesterol efflux. In vivo, YC-1 retarded the development of atherosclerosis, accompanied by reduced serum levels of cholesterol and pro-inflammatory cytokines, in apolipoprotein E-deficient mice. CONCLUSION: Activation of sGC by YC-1 leads to LXRα-dependent upregulation of ABCA1 in macrophages and may confer protection against atherosclerosis.

Activation of transient receptor potential vanilloid 1 decreases endothelial nitric oxide synthase phosphorylation at Thr497 by protein phosphatase 2B-dependent dephosphorylation of protein kinase C.

AIMS: We investigated the effects and underlying molecular mechanism of transient receptor potential vanilloid 1 (TRPV1), a calcium (Ca(2+) )-permeable non-selective cation channel, on phosphorylation of endothelial nitric oxide synthase (eNOS) at threonine 497 (Thr497) in bovine aortic endothelial cells (BAECs) and in mice. METHODS: Western blotting and immunoprecipitation were used for the evaluation of protein phosphorylation; protein phosphatase 2B (PP2B) activity was assessed by convention kit; Griess assay was for NO production; tube formation and Matrigel plug assay were used for angiogenesis. RESULTS: In BAECs, treatment with the TRPV1 ligand evodiamine decreased the phosphorylation of eNOS at Thr497, protein kinase Cα (PKCα) at Serine 657 (Ser657) and PKCß2 at Ser660. Evodiamine increased protein phosphatase 2B (PP2B) activity and promoted the formation of a PP2B-PKC complex. Inhibition of TRPV1 activation by the pharmacological antagonists, removal of extracellular Ca(2+) or pharmacological inhibition of PI3K/Akt/calmodulin-dependent protein kinase II/AMP-activated protein kinase signalling pathway abolished the evodiamine-induced alterations in phosphorylation of eNOS at Thr497, PKCα at Ser657, PKCß2 at Ser660 and PP2B activity, as well as the formation of a PP2B-PKC complex. Inhibition of PP2B activation partially reduced the evodiamine-induced NO bioavailability and tube formation in endothelial cells (ECs) and angiogenesis in mice. Moreover, evodiamine decreased the phosphorylation of eNOS at Thr497, PKCα at Ser657 and PKCß2 at Ser660 in apolipoprotein E (ApoE)-deficient mouse aortas but not TRPV1-deficient or ApoE/TRPV1 double-knockout mice. CONCLUSION: TRPV1 activation in ECs may elicit a Ca(2+) -dependent effect on PP2B-PKC signalling, which leads to dephosphorylation of eNOS at Thr497 in ECs and in mice.

Essential role of transient receptor potential vanilloid type 1 in evodiamine-mediated protection against atherosclerosis.

AIM: We investigated whether transient receptor potential vanilloid type 1 (TRPV1) was involved in the therapeutic effect of evodiamine, a main bioactive component in the fruit of Evodiae rutaecarpa, on the development of atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice and ApoE(-/-)TRPV1(-/-) mice. METHODS: Histopathology was examined by haematoxylin and eosin staining, levels of cytokines and mediators were evaluated by ELISA kits, and protein expression was determined by Western blotting. RESULTS: Chronic administration with evodiamine (10 mg kg(-1) body weight) reduced the size of atherosclerotic lesions and alleviated the hyperlipidaemia and systemic inflammation, as well as hepatic macrovesicular steatosis, in ApoE(-/-) mice. Treating ApoE(-/-) mice with evodiamine enhanced hepatic cholesterol clearance, as revealed by upregulation of hepatic low-density lipoprotein receptor and ATP-binding cassette (ABC) transporters ABCG5, ABCG8 and cholesterol 7α-hydrolase. Genetic deletion of TRPV1 in ApoE(-/-) mice promoted the progression of atherosclerosis; elevated the serum levels of cholesterol, cytokines and chemokines; and exacerbated hepatic macrovesicular steatosis. Moreover, genetic deletion of TRPV1 abrogated the evodiamine-evoked atheroprotection but not anti-obesity effect in ApoE(-/-) mice. CONCLUSION: Evodiamine may confer novel TRPV1-dependent atheroprotection and TRPV1-independent anti-obesity action.

Prior exercise training alleviates the lung inflammation induced by subsequent exposure to environmental cigarette smoke.

AIM: Environmental cigarette smoke (CS) contains many compounds that are harmful to the respiratory system and lead to chronic lung inflammation and other lung diseases. Exercise training is known to confer protection against diseases with chronic inflammation by reducing inflammatory response in human or experimental animals. In this study, we investigated the preventive effect of exercise training against lung inflammation induced by environmental CS. METHODS AND RESULTS: In this study, two groups of mice received air exposure with (the exercise group) or without (the control group) exercise training for 8 weeks and another two groups received air exposure for the first 4 weeks and CS exposure for the following 4 weeks with (the exercise+CS group) or without (the CS group) exercise training for 8 weeks. As compared with lung tissues of control and exercise groups, those of the CS group showed significantly increased bronchoalveolar-capillary permeability, inflammatory cell infiltration, epithelial thickening, expression of proliferating cell nuclear antigen, mucin 2, cytokines, chemokines, adhesion molecules and activation of NF-κB. These CS-induced pathophysiologic consequences were largely prevented in the exercise + CS group. CONCLUSION: Collectively, prior exercise training may protect against lung inflammation induced by environmental CS in mice by attenuating the activation of NF-κB and the production of inflammatory mediators.

An efficient transfection method for mouse embryonic stem cells.

Embryonic stem (ES) cells are considered to have potentials for tissue regeneration and treatment of diverse human diseases. ES cells are capable of indefinite renewal and proliferation, which can be induced to differentiate into tissues of all three germ lines. Despite these exciting potential, it remains unclear as to how the renewal and differentiation programs are operated and regulated at the genetic level. Genetic manipulation such as delivery of exogenous gene expression or knockdown with small interfering RNA (siRNA) is commonly used in most of cancer or transformed cells but relatively rare in ES cells. In this study, we compare the transfection efficacies of several liposome-based transfection methods by introduction of a plasmid encoding enhanced green fluorescent protein (EGFP) into mouse ES (mES) cells. Our results show that transfection by Effectene achieves the efficiency of >98% in CCE and >80% in D3 cells. The optimal ratio of DNA:Effectene for EGFP transfection is between 1:4 and 1:8. Transient-expressed EGFP or endogenous protein kinase A (PKA) were significantly knocked down by Effectene transfection of specific siRNA. High EGFP level expression and accumulation in mES cells induces minor cytotoxicity but can be reduced by introducing siRNA of EGFP. Further, this transfection method did not significantly affect mES properties of proliferation or differentiation. Our results provide an optimal protocol to achieve an efficient transfection for mES cells.

Adenovirus-mediated overexpression of catalase attenuates oxLDL-induced apoptosis in human aortic endothelial cells via AP-1 and C-Jun N-terminal kinase/extracellular signal-regulated kinase mitogen-activated protein kinase pathways.

In a variety of vascular disorders, endothelial cells (ECs) are exposed to high levels of reactive oxygen species (ROS) generated intercellularly. Recently, several anti-oxidants, including catalase, have been suggested to be cytoprotective against the development of atherosclerosis. The object of this study was to investigate whether adenovirus-mediated gene transfer of catalase in ECs can attenuate ROS production and cell apoptosis under oxidized low density lipoprotein (oxLDL) stimulation. Adenovirus-mediated gene transfer of human catalase gene (Ad-Cat) resulted in a high level of catalase overexpression in human arterial EC (HAEC), which manifested a time-dependent increase in cell viability under the exposure of oxLDL and decreased oxLDL-induced apoptosis. Phosphorylation studies of ERK1/2, JNK, and p38, three subgroups of mitogen activator protein kinase demonstrated that catalase overexpression suppressed JNK phosphorylation and increased ERK1/2 phosphorylation. NF-kappaB and AP-1 were induced after the exposure of HAECs to oxLDL. While catalase overexpression was found to inactivate AP-1, it had no effect on NF-kappaB activity. These results provide the evidence that overexpression of catalase in ECs attenuates ROS production and cell apoptosis under oxLDL stimulation. The protective effect is mediated through the downregulation of JNK and the upregulation of ERK1/2 phosphorylation as well as AP-1 inactivation. This observation supports the feasibility of catalase gene transfer to human endothelium to protect against oxidant injury.

NO modulates monocyte chemotactic protein-1 expression in endothelial cells under cyclic strain.

Endothelial cells (ECs) under hemodynamic forces increase intracellular reactive oxygen species (ROS) that modulate gene expression. We previously showed that NO attenuated the shear flow-induced gene level. The present study explored the role of endothelial NO in cyclic strain-treated ECs. Treatment of ECs with S-nitroso-N-acetylpenicillamine (SNAP), an NO donor, reduced cyclic strain-induced monocyte chemotactic protein (MCP)-1 expression. Conversely, exposure of ECs to an NO synthase inhibitor augmented MCP-1 mRNA levels. NO attenuated the binding of activator protein-1 to the 12-O-tetradecanoylphobol-13-acetate-responsive element (TRE) in the MCP-1 promoter region. ECs overexpressed with endothelial NO synthase (eNOS) inhibited cyclic strain-induced MCP-1 expression and MCP-1 promoter (-540 bp) activity. Consistently, ECs treated with SNAP or infected with adenovirus carrying eNOS reduced strain-induced superoxide levels. These strain-induced superoxide and MCP-1 expressions were greatly blunted by treating ECs with an NADPH oxidase inhibitor, diphenyleneiodonium chloride or apocynine, but not with a xanthine oxidase inhibitor. ECs infected with adenovirus carrying the dominant-negative mutant of Rac (RacN17), a component of NADPH oxidase, reduced the strain-induced superoxide and MCP-1 expression. In contrast, ECs transfected with a constitutively active Rac (RacV12) increased MCP-1 and 4x TRE promoter activities. However, ECs cotransfected with eNOS and RacV12 reduced those promoter activities. Consistently, the increases of superoxide levels and MCP-1 expression by overexpression of RacV12 were abolished after infecting ECs with eNOS. Our results show that NO from eNOS-inhibiting redox-sensitive MCP-1 expression is mediated via Rac-dependent NADPH oxidase by reducing ROS. This study provides a molecular basis to support the notion that endothelial NO acts as an antioxidant by negatively regulating redox-sensitive gene expression in ECs constantly under hemodynamic influence.

Adenovirus-mediated heme oxygenase-1 gene transfer inhibits the development of atherosclerosis in apolipoprotein E-deficient mice.

BACKGROUND: Increasing evidence supports the role of heme oxygenase-1 (HO-1) in cytoprotective response and iron homeostasis. The object of this study was to investigate whether adenovirus-mediated gene transfer of HO-1 in arteries reduces iron overload and inhibits lesion formation in apolipoprotein E (apoE)-deficient mice. METHODS AND RESULTS: Infection of rat aortic smooth muscle cells with adenovirus carrying the human HO-1 gene (Adv-HO-1) resulted in a high-level expression of HO-1 protein, which effectively reduced the hemin-induced iron overload in these cells. Adenovirus-mediated gene transfer in arteries in vivo was achieved by direct injection of Adv-HO-1 into the left ventricles of anesthetized animals. Transgene was expressed in the endothelium and aortic lesion of apoE-deficient mice after they had received recombinant adenovirus for 1 week and gradually decayed during the next 5 weeks. When young apoE-deficient mice (14 weeks old) received Adv-HO-1 (2.5 x 10(9) pfu) for 6 weeks, lesions that developed in the aortic root or aortic arch were significantly smaller than those in control littermates receiving empty viral vector. Furthermore, the iron deposition as well as tissue iron content was much less in aortic tissue of Adv-HO-1-treated mice. The inhibitory effect of HO-1 gene transfer on the progression of advanced lesions was also observed in older apoE-deficient mice (20 weeks old) receiving Adv-HO-1 intraventricularly. CONCLUSIONS: Overexpression of HO-1 in vascular cells facilitates iron metabolism and attenuates development of atherosclerosis in apoE-deficient mice.

Colocalization and interaction of cyclooxygenase-2 with caveolin-1 in human fibroblasts.

Results from our previous study suggest that cyclooxygenase-2 (COX-2) induced by phorbol 12-myristate 13-acetate (PMA) may be localized to caveolae-like structures (Liou, J.-Y., Shyue, S.-K., Tsai, M.-J., Chung, C.-L., Chu, K.-Y., and Wu, K. K. (2000) J. Biol. Chem. 275, 15314-15320). In this study, we determined subcellular localization of COX-2 and caveolin-1 by confocal microscopy. COX-2 in human foreskin fibroblasts stimulated by PMA (100 nm) or interleukin-1beta (1 ng/ml) for 6 h was localized to plasma membrane in addition to endoplasmic reticulum and nuclear envelope. Caveolin-1 was localized to plasma membrane, and image overlay showed colocalization of COX-2 with caveolin-1. This was confirmed by the presence of COX-2 and caveolin-1 in the detergent-insoluble membrane fraction of cells stimulated by PMA. Immunoprecipitation showed complex formation of COX-2 with caveolin-1 in a time-dependent manner. A larger quantity of COX-2 was complexed with caveolin-1 in PMA-treated than in interleukin-1beta-treated cells. Purified COX-2 complexed with glutathione S-transferase-fused caveolin-1, which was not inhibited by the scaffolding domain peptide. Caveolin-1-bound COX-2 was catalytically active, and its activity was not inhibited by the scaffolding domain peptide. These results suggest that COX-2 induced by PMA and interleukin-1beta is colocalized with caveolin-1 in the segregated caveolae compartment. Because caveolae are rich in signaling molecules, this COX-2 compartment may play an important role in diverse pathophysiological processes.

Selective augmentation of prostacyclin production by combined prostacyclin synthase and cyclooxygenase-1 gene transfer.

BACKGROUND: We tested the hypothesis that combined cyclooxygenase-1 (COX-1) and prostacyclin synthase (PGIS) gene transfer selectively augments prostacyclin production without a concurrent overproduction of other prostanoids. METHODS AND RESULTS: ECV304 cells were transfected with bicistronic pCOX-1/PGIS versus pCOX-1 or pPGIS, and prostanoids were analyzed. Contrary to the high prostaglandin E2 synthesis in pCOX-1 transfected cells, selective prostacyclin formation was noted with bicistronic plasmid transfection. Next, we determined the optimal ratio of Ad-COX-1 to Ad-PGIS by transfecting human umbilical vein endothelial cells with various titers of these 2 adenoviral constructs and determined the level of protein expression and prostanoid synthesis. Our results show that optimal ratios of adenoviral titers to achieve a large prostacyclin augmentation without overproduction of prostaglandin E2 or F2alpha were 50 to 100 plaque forming units (pfu) of Ad-COX-1 to 50 pfu of Ad-PGIS per cell. A higher Ad-PGIS to Ad-COX-1 ratio caused a paradoxical decline in prostacyclin synthesis. CONCLUSIONS: Prostacyclin synthesis can be selectively augmented by cotransfecting endothelial cells with an optimal ratio of COX-1 to PGIS. Combined COX-1 and PGIS gene transfer has the potential for therapeutic augmentation of prostacyclin.

Colocalization of prostacyclin synthase with prostaglandin H synthase-1 (PGHS-1) but not phorbol ester-induced PGHS-2 in cultured endothelial cells.

The subcellular colocalization of prostacyclin synthase (PGIS) with prostaglandin H synthase (PGHS) has not been delineated. To test the hypothesis that its colocalization with PGHS is crucial for prostacyclin synthesis, we determined subcellular locations of PGIS, PGHS-1, and PGHS-2 in bovine aortic endothelial cells by immunofluorescent confocal microscopy. PGIS and PGHS-1 were colocalized to nuclear envelope (NE) and endoplasmic reticulum (ER) in resting and adenovirus-infected bovine aortic endothelial cells. PGIS and PGHS-2 were also colocalized to ER in serum-treated or adenovirus-cyclooxygenase-2-infected cells. By contrast, PGIS was not colocalized with PGHS-2 in cells induced with phorbol 12-myristate 13-acetate where PGHS-2 was visualized primarily in vesicle-like structures. The lack of colocalization was accompanied by failed prostacyclin production. Resting ECV304 cells did not produce prostacyclin and had no detectable PGHS-1 and PGIS proteins. Confocal analysis showed abnormal colocalization of PGIS and PGHS-1 to a filamentous structure. Interestingly, the abundant PGIS and PGHS-1 expressed in adenovirus-infected ECV304 cells were colocalized to NE and ER, which synthesized a large quantity of prostacyclin. These findings underscore the importance of colocalization of PGHS and PGIS to ER and NE in prostacyclin synthesis.

Modulation of inducible nitric oxide synthase induction by prostaglandin E2 in macrophages: distinct susceptibility in murine J774 and RAW 264.7 macrophages.

Prostaglandin E2 (PGE2) is the major cyclooxygenase metabolite in macrophages with complex proinflammatory and immunoregulatory properties. In the present study, we have compared the modulatory role of PGE2/cAMP-dependent signaling on induced nitric oxide (NO) production in two murine macrophages, J774 and RAW 264.7. With no effect on NO release by itself, PGE2 co-addition with lipopolysaccharide (LPS) resulted in a concentration-dependent enhancement in NO release and inducible NO synthase induction in J774, but not in RAW 264.7, macrophages. The potentiation effect of PGE2 in J774 cells was still seen when applied within 9 h after LPS treatment. Whereas RAW 264.7 macrophages release PGE2 with greater extent than J774 macrophages in response to LPS, indomethacin and NS-398, upon abolishing LPS-induced PGE2 release, caused a more obvious inhibition of NO release from J774 than RAW 264.7 cells. Thus, we suggest a higher positive modulatory role of PGE2--either endogenous or exogenous--on NO formation in J774 cells. Supporting these findings, exogenous PGE2 triggers cAMP formation in J774 cells with higher potency and efficacy. Of interest, dBcAMP also elicits higher sensitivity in potentiating NO release in J774 cells. We conclude that the opposite effect of PGE2/cAMP signaling on macrophage NO induction depends on its signaling efficacy and might be associated with the difference in endogenous PGE2 levels.

Origins and antiquity of X-linked triallelic color vision systems in New World monkeys.

It is known that the squirrel monkey, marmoset, and other related New World (NW) monkeys possess three high-frequency alleles at the single X-linked photopigment locus, and that the spectral sensitivity peaks of these alleles are within those delimited by the human red and green pigment genes. The three alleles in the squirrel monkey and marmoset have been sequenced previously. In this study, the three alleles were found and sequenced in the saki monkey, capuchin, and tamarin. Although the capuchin and tamarin belong to the same family as the squirrel monkey and marmoset, the saki monkey belongs to a different family and is one of the species that is most divergent from the squirrel monkey and marmoset, suggesting the presence of the triallelic system in many NW monkeys. The nucleotide sequences of these alleles from the five species studied indicate that gene conversion occurs frequently and has partially or completely homogenized intronic and exonic regions of the alleles in each species, making it appear that a triallelic system arose independently in each of the five species studied. Nevertheless, a detailed analysis suggests that the triallelic system arose only once in the NW monkey lineage, from a middle wavelength (green) opsin gene, and that the amino acid differences at functionally critical sites among alleles have been maintained by natural selection in NW monkeys for >20 million years. Moreover, the two X-linked opsin genes of howler monkeys (a NW monkey genus) were evidently derived from the incorporation of a middle (green) and a long wavelength (red) allele into one chromosome; these two genes together with the (autosomal) blue opsin gene would immediately enable even a male monkey to have trichromatic vision.

Molecular genetics of spectral tuning in New World monkey color vision.

Although most New World monkeys have only one X-linked photopigment locus, many species have three polymorphic alleles at the locus. The three alleles in the squirrel monkey and capuchin have spectral peaks near 562, 550, and 535 nm, respectively, and the three alleles in the marmoset and tamarin have spectral peaks near 562, 556, and 543 nm, respectively. To determine the amino acids responsible for the spectral sensitivity differences among these pigment variants, we sequenced all exons of the three alleles in each of these four species. From the deduced amino acid sequences and the spectral peak information and from previous studies of the spectral tuning of X-linked pigments in humans and New World monkeys, we estimated that the Ala --> Ser, Ile --> Phe, Gly --> Ser, Phe --> Tyr, and Ala --> Tyr substitutions at residue positions 180, 229, 233, 277, and 285, respectively, cause spectral shifts of about 5, -2, -1, 8, and 15 nm. On the other hand, the substitutions His --> Tyr, Met --> Val or Leu, and Ala --> Tyr at positions 116, 275, and 276, respectively, have no discernible spectral tuning effect, though residues 275 and 276 are inside the transmembrane domains. Many substitutions between Val and Ile or between Val and Ala have occurred in the transmembrane domains among the New World monkey pigment variants but apparently have no effect on spectral tuning. Our study suggests that, in addition to amino acid changes involving a hydroxyl group, large changes in residue size can also cause a spectral shift in a visual pigment.

Prostacyclin synthase active sites. Identification by molecular modeling-guided site-directed mutagenesis.

Prostacyclin synthase (PGIS), a cytochrome P450 enzyme, catalyzes the biosynthesis of a physiologically important molecule, prostacyclin. In this study we have used a molecular modeling-guided site-directed mutagenesis to predict the active sites in substrate binding pocket and heme environment of PGIS. A three-dimensional model of PGIS was constructed using P450BM-3 crystal structure as the template. Our results indicate that residues Ile67, Val76, Leu384, Pro355, Glu360, and Asp364, which were suggested to be located at one side of lining of the substrate binding pocket, are essential for catalytic activity. This region containing beta1-1, beta1-2, beta1-3, and beta1-4 strands is predicted well by the model. At the heme region, Cys441 was confirmed to be the proximal axial ligand of heme iron. The conserved Phe and Arg in P450BM-3 were substituted by Leu112 and Asp439, respectively in PGIS. Alteration of Leu112 to Phe retained the activity, indicating that Leu112 is a functional substitution for Phe. In contrast, mutant Asp439 --> Ala exhibited a slight increase in activity. This result implies a difference in the heme region between P450BM-3 and PGIS. Our results also indicate that stability of PGIS expression is not affected by heme site or active site mutations.

Adaptive evolution of color vision genes in higher primates.

The intron 4 sequences of the three polymorphic alleles at the X-linked color photo-pigment locus in the squirrel monkey and the marmoset reveal that the alleles in each species are exceptionally divergent. The data further suggest either that each triallelic system has arisen independently in these two New World monkey lineages, or that in each species at least seven deletions and insertions (14 in the two species) in intron 4 have been transferred and homogenized among the alleles by gene conversion or recombination. In either case, the alleles in each species apparently have persisted more than 5 million years and probably have been maintained by overdominant selection.

Intronic gene conversion in the evolution of human X-linked color vision genes.

Human red and green visual pigment genes are X-linked duplicate genes. To study their evolutionary history, introns 2 and 4 (1,987 and 1,552 bp, respectively) of human red and green pigment genes were sequenced. Surprisingly, we found that intron 4 sequences of these two genes are identical and that the intron 2 sequences differ by only 0.3%. The low divergences are unexpected because the duplication event producing the two genes is believed to have occurred before the separation of the human and Old World monkey (OWM) lineages. Indeed, the divergences in the two introns are significantly lower than both the synonymous divergence (3.2% +/- 1.1%) and the nonsynonymous divergence (2.0% +/- 0.5%) in the coding sequences (exons 1-6). A comparison of partial sequences of exons 4 and 5 of human and OWM red and green pigment genes supports the hypothesis that the gene duplication occurred before the human-OWM split. In conclusion, the high similarities in the two intron sequences might be due to very recent gene conversion, probably during evolution of the human lineage.

Weak male-driven molecular evolution in rodents.

In humans and rodents the male-to-female ratio of mutation rate (alpha m) has been suggested to be extremely large, so that the process of nucleotide substitution is almost completely male-driven. However, our sequence data from the last intron of the X chromosome-linked (Zfx) and Y chromosome-linked (Zfy) zinc finger protein genes suggest that alpha m is only approximately 2 in rodents with a 95% confidence interval from 1 to 3. Moreover, from published data on oogenesis and spermatogenesis we estimate the male-to-female ratio of the number of germ cell divisions per generation to be approximately 2 in rodents, confirming our estimate of alpha m and suggesting that errors in DNA replication are the primary source of mutation. As the estimated alpha m for rodents is only one-third of our previous estimate of approximately 6 for higher primates, there appear to be generation-time effects--i.e., alpha m decreases with decreasing generation time.