Biomedical subjects

K Nomoto

Publications and source records attributed to K Nomoto.

At least 73 records · Page 4Linked to original sources

PubMed2000

Ineffective control of murine cytomegalovirus by IE1-specific cytotoxic T lymphocytes during protracted infection in the lung.

Interstitial pneumonia caused by cytomegalovirus (CMV) is a fatal disease in immunocompromised patients. In order to examine the defense mechanism against the virus in the lung, we employed an intratracheal infection model in susceptible mice. In mice infected intratracheally with murine CMV, a protracted infection was observed where infectious virus was detected up to 21 days of infection. During this prolonged infection, massive accumulation in the lung of CD8+ T cells with activated phenotypes occurred and these CD8+ T cells showed direct ex vivo cytolytic activity against target cells pulsed with the nonamer peptide derived from IE1 protein of the virus, which has been shown to be the dominant epitope recognized by most of virus-specific CTL. Moreover, adoptive transfer of in vitro induced IE1 peptide-specific CTL line showed no anti-virus effect in the lung, although they were effective in the spleen. Hence, there is reason to assume the IE1-specific CTL induced in vivo or in vitro plays limited roles during the prolonged infection in the lung.

Animals↗

PubMed2000

Protective effect of a traditional Japanese medicine, Bu-zhong-yi-qi-tang (Japanese name: Hochu-ekki-to), on the restraint stress-induced susceptibility against Listeria monocytogenes.

In this study, the effect of traditional Japanese (Chinese) medicine, Bu-zhong-yi-qi-tang (Japanese name: Hochu-ekki-to, HOT), on the restraint stress treatment (RST)-induced susceptibility against Listeria monocytogenes (L. monocytogenes) was examined. When RST was performed every day for 10 h from the day of infection, the bacterial numbers were increased at 3 and 5 days after the infection. Oral pretreatment with HOT for 7 days prevented such increases. Pretreatment with HOT prevented the suppression of antigen-specific IFN-gamma production by RST. HOT also prevented suppression of macrophage accumulation, including MHC-class II positives, in the peritoneal cavity and their bactericidal activity by RST. HOT suppressed the serum corticosterone level elevated by RST in infected mice. Taken together, the suppression of corticosterone using HOT participates in the prevention of suppressions of the bactericidal activity of macrophages, migration of macrophages and antigen-specific IFN-gamma production of Th1 cells by RST. Our findings suggest that HOT is a useful drug for patients suffering from stress disease to reduce the susceptibility to bacterial infection.

Animals↗

PubMed2000

Inability of cyclophosphamide-induced tolerance to permit engraftment of pluripotent stem cells contained in a moderate number of syngeneic bone marrow cells.

We have previously described cyclophosphamide (CP)-induced tolerance that comprises an intravenous (i.v.) injection of 1 x 10(8) allogeneic spleen cells (SC) followed, 2 days later, by an intraperitoneal (i.p.) administration of 200 mg/kg of CP. By using this method, we were able to readily induce both long-lasting mixed chimerism and skin allograft tolerance in most of H-2 matched combinations, but not in H-2 mismatched combinations. In the present study, we have investigated whether the treatment with SC followed by 200 mg/kg CP can permit engraftment of syngeneic pluripotent hematopoietic stem cells (HSC), and whether CP itself can permit the HSC engraftment. Recipient B6 (H-2b; Ly-5.2) mice received 1 x 10(7), 5 x 10(7) or 1 x 10(8) SC from syngeneic B6.Ly-5.1 (H-2b; Ly-5.1) mice and administered 2 day later 200 mg/kg CP. Using anti-Ly-5.1 and Ly-5.2 mAbs and a flow cytometry, the origins of lymphoid and myeloid cells injecting the recipients were observed over time. Chimerism was at least initially detectable in all groups treated with SC alone (without 200 mg/kg CP), but became undetectable by 32 weeks after the treatment with SC alone. In recipient B6 mice treated with B6.Ly-5.1 SC and 200 mg/kg CP, on the other hand, lymphoid-chimerism was detectable at 32 weeks in a transferred cell dose-dependent manner, but granulocyte-chimerism indicating pluripotent HSC engraftment disappeared earlier than 32 weeks after the treatment. In order to further evaluate the effect of CP itself on HSC engraftment, recipient B6 mice were given various doses of CP (50-400 mg/kg) and were injected with T cell-depleted 1 x 10(7) BMC from B6.Ly-5.1 mice. Multilineage mixed chimerism over 32 weeks was induced in only one of 11 B6 mice treated with 400 mg/kg CP followed by B6.Ly-5.1 BMC, although lymphoid chimerism was induced temporarily in recipient B6 mice treated with 200 mg/kg CP followed by B6.Ly-5.1 BMC and persistently in most of recipient B6 mice treated with 400 mg/kg CP followed by B6.Ly-5.1 BMC.

Animals↗

PubMed2000

Identification of multiple urechistachykinin peptides, gene expression, pharmacological activity, and detection using mass spectrometric analyses.

Urechistachykinin I and II (Uru-TK I and II) are invertebrate tachykinin-related peptides (TRPs), which have been isolated from echiuroid worms. The cDNA sequence encoding the Uru-TK I and II revealed that the precursor also encoded five TRP-like peptides. Here, we report the characterization of these Uru-TK-like peptides named as Uru-TK III-VII. Northern and Southern blot analyses demonstrated that Uru-TK mRNA is localized in nerve tissue. In addition, the presence of the Uru-TK-like peptides as matured forms in the nerve tissue was detected by mass spectrometric analysis, and identified these peptides were shown to exhibit a contractile activity on cockroach hindgut that was as potent as that of Uru-TK II. Furthermore, synthetic Uru-TK-like peptide analogs which contained Met-NH2 instead of Arg-NH2 at their C-termini were shown to possess a potential to bind to a mammalian tachykinin receptor, indicating that Uru-TK-like peptides are likely to correspond to vertebrate tachykinins, except for the difference at the C-terminal residue. These findings show that Uru-TK-like peptides are essentially equivalent to Uru-TK I and II, leading to the proposal that Uru-TK-like peptides play an essential role as invertebrate tachykinin neuropeptides.

Animals↗

PubMed2000

Characterization of CD4- CD8- CD3+ T-cell receptor-alphabeta+ T cells in murine cytomegalovirus infection.

In this study, we have investigated that after the intraperitoneal infection with murine cytomegalovirus (MCMV), the CD3+ CD4- CD8-(double negative; DN) T-cell receptor (TCR)alphabeta+ T cells increased in peritoneal cavity, liver and spleen in both resistant C57BL/6 and susceptible BALB/c mice. The total cellular population of these cells showed peak levels around day 5 after infection in all the three investigated organs and the following phenotypical and functional characteristics emerged. The peritoneal DN TCRalphabeta+ T cells expressed highly skewed TCRVbeta8 on day 5 after infection compared with the uninfected mice, but those in spleen and liver showed moderate and low skewed TCRVbeta8, respectively. The percentages of NK1.1+ DN TCRalphabeta+ T cells gradually decreased as did modulation of some of their activation markers consistent with an activated cell phenotype. The peritoneal DN TCRalphabeta+ T cells on day 5 after infection expressed the genes of interferon-gamma (IFN-gamma), tumour necrosis factor-alpha, Eta-1 (early T-cell activation-1) and MCP-1 (monocyte chemoattractant protein 1) but lacked expression of interleukin-4 (IL-4). After in vitro stimulation with phorbol 12-myristate 13-acetate and calcium ionophore in the presence of Brefeldin A, higher frequencies of intracellular IFN-gamma+ DN TCRalphabeta+ T cells were detected in all three investigated organs of infected mice compared with those of uninfected mice. Stimulation of peritoneal DN TCRalphabeta+ T cells with plate-bound anti-TCRbeta monoclonal antibodies showed proliferation and also produced IFN-gamma but not IL-4. These results suggest that DN TCRalphabeta+ T cells were activated and may have an antiviral effect through producing IFN-gamma and some macrophage-activating factors during an early phase of MCMV infection.

Animals↗

PubMed2000

Vgamma1+ gammadelta T cells play protective roles at an early phase of murine cytomegalovirus infection through production of interferon-gamma.

Cytomegalovirus (CMV) causes severe opportunistic infection in immunocompromised hosts. The importance of conventional alphabeta T cells in protection against CMV infection has been well documented. However, the role of the second T-cell population (which express the gammadelta T-cell receptor) in CMV infection is not known. In the present study, we analysed the function and protective role of gammadelta T cells in a murine cytomegalovirus (MCMV) infection model. After intraperitoneal infection with MCMV, the number of gammadelta T cells increased in the liver and peritoneal cavity from day 3, and reached a peak on day 5. The gammadelta T cells showed an activated T-cell phenotype and predominantly expressed Vgamma1, which is known to be expressed by heat-shock protein 65 (hsp 65)-specific gammadelta T cells. Analysis of cytokine expression demonstrated that the MCMV-induced gammadelta T cells expressed interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) but not interleukin-4 (IL-4), implying their participation in the cell-mediated immune response against MCMV. Depletion of gammadelta T cells by anti-T-cell receptor (TCR) gammadelta monoclonal antibody (mAb) treatment resulted in significant increase of virus titre and decrease of IFN-gamma in the liver on day 3 after MCMV infection, which further supports the importance of gammadelta T cells in early protection against infection. Finally, the MCMV-induced gammadelta T cells produced IFN-gamma in vitro in response to hsp 65. Our results suggest that gammadelta T cells participate in early protection against MCMV infection through recognition of hsp 65 and production of IFN-gamma.

Animals↗

PubMed2000

Unusual cytotoxic activities of thymus-independent, self-antigen-specific CD8(+) T cells.

We compared the cytotoxic activities of thymus-dependent and thymus-independent CD8(+) T cells. Thymus-dependent CD8(+) T cells, which are foreign antigen specific, acquired cytotoxic activity to tumor cells with a basal dose of the antigen peptides and to hybridoma cells expressing anti-TCR mAb only after differentiation into effector cytotoxic T lymphocytes (CTL). In contrast, thymus-independent CD8(+) T cells, which have been shown to be self-antigen specific, never showed cytotoxic activity to the target cells with a basal dose of the self-antigen peptide, while they could lyse hybridoma cells expressing anti-TCR mAb even without prior antigenic stimulation. Furthermore, the ex vivo cytotoxic activity of thymus-independent CD8(+) T cells was also observed against the target cells with high doses of the antigen peptides, which were not lysed by freshly isolated thymus-dependent CD8(+) T cells. Thus it is revealed that thymus-independent, self-antigen-specific CD8(+) T cells already acquire mature CTL functions in situ but have an increased threshold of TCR-mediated signaling for activation. These differences in cytotoxic activities between thymus-dependent and thymus-independent CD8(+) T cells suggest distinct roles of the two subsets of CD8(+) T cells in vivo.

Animals↗

PubMed2000

Heterogeneous response patterns of alveolar macrophages from patients with lung cancer by stimulation with interferon-gamma.

BACKGROUND: Macrophages are considered to play an important role in the host defense against malignant tumors. In this study, cytotoxic activity of alveolar macrophages (AM) derived from 32 patients with lung cancer was investigated. METHODS: AM were aseptically obtained by lavage from resected lung and subsequently tested for cytolytic activity against QG56, a lung squamous cell line, following treatment with recombinant interferon-gamma (IFN-gamma). RESULTS: In seven patients (21.9%), AM showed no cytotoxicity even though AM were incubated with IFN-gamma. In 20 (62.5%), AM showed substantial cytotoxicity in response to IFN-gamma in a dose-dependent manner. In the other five (15.6%), relatively strong cytotoxicity was observed even without preincubation with IFN-gamma. Such a heterogeneous profile of the cytotoxicity of AM might be a reflection of various activated states of AM since the potential of cytotoxicity and that of IL-1 secretion were almost parallel. Both IFN-gamma dependent and -independent cytotoxicity were partially blocked either by anti-tumor necrosis factor-alpha (TNF-alpha) antibody or by the inhibitor of nitric oxide synthesis. However, those activities were completely abrogated by both treatments. Since the supernatant of AM culture exhibited TNF-alpha-mediated but not NO-mediated cytolysis, TNF-alpha could mediate a bystander killing whereas NO acts in close contact with tumor cells. CONCLUSION: The AM have anti-tumor cytotoxicity in lung cancer although the cytolytic potential is heterogeneous and that the tumor lysis by AM is mediated by both TNF-alpha and NO production.

Adenocarcinoma↗

PubMed2000

beta-estradiol suppresses T cell-mediated delayed-type hypersensitivity through suppression of antigen-presenting cell function and Th1 induction.

BACKGROUND: Although an immunomodulatory role for estrogens has long been demonstrated by experimental and clinical observations, the mechanism by which estrogens exert their effect on T cells has not been clearly defined. METHODS: In this study we analyzed the effects of beta-estradiol (E2), at its contraceptive dose, on the delayed-type hypersensitivity (DTH) to purified protein derivatives (PPD) and associated immune response in female mice. RESULTS: E2 treatment decreased PPD-specific DTH response, which coincided with a decrease in the leukocytes numbers in the draining lymph nodes (DLN) and spleen compared with control mice. E2 treatment also suppressed the in vitro PPD-specific proliferative response of DLN and spleen cells from PPD-primed mice. The analysis of production and gene expression of cytokines by DLN cells demonstrated that E2 treatment suppressed IL-2 and IFN-gamma production in response to PPD in vitro. In contrast, IL-4 and IL-10 gene expression by DLN cells of E2-treated mice, taken 24 h after in vivo restimulation of mice with PPD, was enhanced. Furthermore, we found that spleen APC from E2-treated mice failed to induce optimum proliferation of the PPD-primed T cells in response to PPD in vitro. The impaired APC function by E2 was not due to induction of suppressor cell activity because addition of the normal spleen APC to APC from E2-treated mice restored the proliferative response of the PPD-primed T cells in response to PPD. CONCLUSION: Our results suggest that the E2-mediated inhibition of DTH reaction is due to a combination of the down regulation of APC function and deviation of the immune response from Th1-type to Th2-type.

Animals↗

PubMed2000

Mediation of the immunomodulatory effect of beta-estradiol on inflammatory responses by inhibition of recruitment and activation of inflammatory cells and their gene expression of TNF-alpha and IFN-gamma.

BACKGROUND: Estrogen has long been reported to show immunomodulatory effects on immune responses, yet, its specific anti-inflammatory mechanism is not clear. METHODS: In this study, we analyzed the effects of beta-estradiol (E2), at its contraceptive dose, on both T cell-independent and T cell-dependent inflammations, and the associated immune mechanism, in female mice. The T cell-independent inflammation was locally induced either with an intradermal injection of olive oil in the footpad, or by an intraperitoneal injection of proteose peptone (PP). The T cell-dependent inflammation was induced by an intraperitoneal injection of the purified protein derivatives (PPD). RESULTS: While E2 inhibited olive oil-induced inflammation as monitored by the decrease in footpad swelling, it did not affect the gene expression of monocyte chemoattractant protein-1 and IL-6 by cells at the inflammatory locus. E2 also inhibited PP-induced inflammation as monitored by the decrease in the number of inflammatory peritoneal exudate cells (PEC) coinciding with a marked decrease in the number of macrophages and granulocytes (Gr. 1+). While E2 did not affect the gene expression of monocyte chemoattractant protein-1 and IL-6 by PP-elicited PEC, it decreased both gene expression and production of TNF-alpha. E2 also decreased the number of cells expressing the lymphocyte function-activated protein-1 in PP-elicited PEC, but not for CD62L. In purified protein derivative-induced T cell-dependent inflammation, E2 decreased the total cellularity of PEC and the relative numbers of CD3+ and CD4+ T cells, and the number of cells expressing the lymphocyte activation markers CD40, CD44, CD69 and IL-2Ralpha in PEC. Furthermore, while E2 did not affect the gene expression of the early T lymphocyte activation protein-1 by PEC, it decreased the gene expression of INF-gamma. CONCLUSION: Collectively, the results suggest that E2-mediated inhibition of inflammatory responses may be due to a combination of suppression of homing and activation of inflammatory cells and their production of TNF-alpha and IFN-gamma.

Adjuvants, Immunologic↗

PubMed2000

Changes in the leukocyte distribution and surface expression of adhesion molecules accompanied with hypothalamically induced restlessness in the cat.

One type of emotional behavior called restlessness occurs when the anteromedial hypothalamus is stimulated in cats. We examined the changes in the distribution and surface expression of adhesion molecules in leukocytes accompanied with restlessness. Mature female cats were used for this study. The cats were stimulated with 60 Hz sine wave train pulses (20-90 microA, 10 s in duration, at 5-min intervals) for 60 min. Samples of blood were collected from 30 min before stimulation up to several hours after the final stimulation. The number of granulocytes increased just after stimulation, while at the same time the expression of L-selectin decreased. On the other hand, the number of CD4+ and CD8+ T lymphocytes decreased at 1-2 h after the end of the stimulation, while the expression of L-selectin increased. In addition, the expression of LFA-1 and VLA-4 did not change. These data suggest that hypothalamically elicited restlessness is thus accompanied by a leukocyte distribution change, which might be mediated by changes in the expression of L-selectin on leukocytes. Plasma cortisol increased during stimulation in restlessness. However, during in vitro culture experiments, cortisol did not alter the expression of leukocyte L-selectin which thus indicated that cortisol does not directly affect the surface expression of L-selectin. These results thus suggest that hypothalamically induced restlessness is a useful stress model for psychoneuroimmunological studies.

Animals↗

PubMed2000

Assessment of aortic atherosclerosis and carotid atherosclerosis in coronary artery disease.

The present study investigated the relationship between aortic atherosclerosis and carotid atherosclerosis, and studied the effects of coronary risk factors for these arteries. The subjects consisted of 78 patients with coronary artery disease (CAD) and 69 patients without CAD. All subjects underwent enhanced computed tomography and B-mode ultrasonography within a short time period to determine the extent of aorta and carotid atherosclerosis. Significant correlations between maximal aortic wall thickness (MAWT) and aortic wall volume (AWV) with carotid intima-media thickness (IMT) were demonstrated. MAWT, AWV and IMT were significantly higher in patients with CAD compared with controls (p=0.009, p=0.024, p=0.001, respectively). Furthermore, there were significant differences in MAWT, AWV and IMT among groups classified by the number of coronary artery stenoses, and no significant differences among groups classified by risk factors, but it was shown that MAWT, AWV and IMT increased gradually as the risk factors increased in number. MAWT, AWV and IMT had positive correlations with age, systolic blood pressure and triglyceride, and a negative correlation with high density lipoprotein-cholesterol. This study demonstrated that both aortic atherosclerosis and carotid atherosclerosis are closely correlated with coronary atherosclerosis, and that the atherosclerosis indices are independently associated with age and hyperlipidemia.

Aortic Diseases↗

PubMed2000

Antitumor activity of interleukin-12 against murine bladder cancer.

PURPOSE: We investigated the antitumor activity of interleukin-12 (IL-12) against MBT-2, a murine bladder carcinoma, to clarify whether or not IL-12 is effective against urothelial tumors. MATERIALS AND METHODS: MBT-2, a murine carcinogen-induced, poorly differentiated transitional cell carcinoma of C3H/He origin, was used. Three or 10 days after the subcutaneous administration of MBT-2 cells, C3H/He mice were injected intraperitoneally with IL-12 five times per wk. for 2 wk. Tumor growth was measured twice weekly. Spleen cells from the C3H/He mice that had rejected MBT-2 after the IL-12 treatment were examined for MBT-2-specific cytolytic T lymphocytes (CTL) activity and cytokine production. RESULTS: Tumor growth and acceptance was obviously suppressed when C3H/He mice were treated with IL-12 from 3 days after the tumor inoculation. In the spleen cells from the C3H/He mice that had rejected MBT-2, MBT-2-specific CTL activity and secretion of IL-2 and interferon (IFN)-gamma were clearly detected. However, the established MBT-2 tumor cells were not rejected when C3H/He mice were given IL-12 from 10 days after the tumor inoculation, although the tumor growth was transiently suppressed during the IL-12 treatment. CONCLUSION: These data demonstrate that IL-12 is considerably effective against murine bladder cancer and suggest the clinical application of IL-12 against human bladder cancer.

Adjuvants, Immunologic↗

PubMed2000

[Utility of Ringer's acetate solution as an intraoperative fluid during cardiovascular surgery with cardiopulmonary bypass].

This study aimed to clarify the difference in the effects of Ringer's acetate (AR) and Ringer's lactate (LR) administration during cardiovascular surgery with cardiopulmonary bypass. We evaluated their effects on intra and postoperative metabolism, liver functions, blood gas and hemodynamic states. Twenty patients were divided into two groups; AR group (n = 10) and LR group (n = 10). Intraoperative serum D-lactate levels in LR group were significantly higher than those in AR group from the beginning of the operation to awakening. Serum acetate levels showed no increase in both groups. The arterial ketone body ratio (AKBR) in AR group was higher than that in LR group, but the difference was not significant. Serum glutamic pyruvic transaminase (GPT) and alkaline phosphatase (ALP) levels in LR group were significantly higher than those in AR group from the induction of the anesthesia. It has been reported that acetate has a greater vasodialatory effect than lactate. However, our findings indicate no significant difference in hemodynamics between the two groups. These results suggest that AR may be more useful than LR during cardiovascular surgery with cardiopulmonary bypass.

Adult↗

PubMed2000

[Living donor liver transplantation in Kyushu University].

We performed living donor liver transplantation (LDLT) for 40 patients at Kyushu University Hospital, Fukuoka Japan during the period from October 1996 to April 2000. The patients consisted of 32 adults and 8 children with a mean age of 35.8 years (range: 1 year and 10 months to 65 years old). The underlying liver diseases of the 40 patients included the fulminant hepatic failure (n = 14), biliary atresia (n = 7), liver cirrhosis (HCV) (n = 6), primary biliary cirrhosis (n = 5), primary sclerosing cholangitis (n = 2), familiar amyloidotic polyneuropathy (n = 2), Alagille syndrome (n = 1), glycogen storage disease (n = 1), huge hepatic hemangiomas (n = 1), and Wilson's disease (n = 1). All liver grafts were obtained from each patient's family members except for one domino transplant donor's case, comprised of 13 parents, 13 sons and daughters, 11 brothers and sisters, and 3 wives. The donors are presently all doing well. The patient survival rate is presently 92.5%.

Adolescent↗

PubMed1999

Roles of CD4+ and CD8+ T cells in discordant skin xenograft rejection.

An essential role of murine CD4+ T cells in immune reactivity and skin graft rejection in discordant xenogeneic combinations have been reported. Our study was conducted to further clarify the roles of CD4+ and CD8+ T cells in discordant skin xenograft rejection, by using CD4 and CD8 knockout [C57BL/6 Cr Slc (B6; H-2b) background] mice. When human skins were grafted on CD8 knockout mice or B6 mice, both hosts rejected human skin grafts within 12 days after grafting. By contrast, survival of human skin grafts was significantly prolonged in CD4 knockout mice (mean survival times=19.3+/-(SD) 1.6 days; median 19 days). Fully allogeneic C3H/He Slc (H-2k) skin grafts were rejected within 14 days in CD4 knockout mice, suggesting that non-CD4+ T cells in CD4 knockout mice were immunocompetent for allograft rejection. In spleens of these recipient mice, CD8+ T cells seemed to be activated 10 days after human skin grafting. Immunohistological analysis revealed the infiltration of CD8+ T cells at the site of transplanted human skin on CD4 knockout mice. To further examine the role of CD8+ T cells in CD4 knockout mice, human skin grafting was performed on day 0 followed by administration of anti-CD8 monoclonal antibody on days 0, 5, and 14. The administration of anti-CD8 monoclonal antibodies caused the significant prolongation of human skin graft survival. These results indicate the following two conclusions: (1) CD4+ T cells have an essential role in rejecting discordant human skin xenografts rapidly and (2) however, CD8+ T cells also are capable of rejecting discordant human skin xenografts.

Animals↗

PubMed1999

The importance of C-terminal residues of vertebrate and invertebrate tachykinins for their contractile activities in gut tissues.

The C-terminal residues of mammalian tachykinins and urechistachykinins (Uru-TKs), tachykinin-related peptides of echiuroid worm origin, were substituted for each other. Their contractile effects were assayed on the cockroach hindgut and the guinea pig ileum. [Met(10)] substitution of Uru-TKs caused a 1000 times lower activity on the hindgut, but a 1000 times higher activity on the ileum. In contrast, [Arg(11)]substance P (SP) was 100 times more and 400 times less potent than SP on the hindgut and ileum, respectively. A SP antagonist blocked these Uru-TK activities on the hindgut. These results demonstrated that the C-terminal Met-NH(2) is necessary for ileum contraction and the Arg-NH(2) is required for hindgut contraction, which was caused by binding to the cockroach's neurokinin-like receptor.

Amino Acid Substitution↗

PubMed1999

Phosphodiesterase 4 conformers: preparation of recombinant enzymes and assay for inhibitors.

Cyclic nucleotides are key regulators of many cellular processes. Their immediate action is terminated through the activity of phosphodiesterases, a diverse family of enzymes. This diversity has given rise to drug discovery opportunities, and assay technology is therefore of key importance. Inhibitors of the cyclic-AMP-specific phosphodiesterases (the PDE4 family) are drug candidates for a variety of inflammatory disorders. However, PDE4 inhibitors, besides their immunomodulatory effects, also cause side effects including nausea and emesis. Recently, it has been suggested that PDE4 exists in two different conformations with respect to inhibition by the prototypical compound rolipram. Inhibition of the low-affinity conformer is thought to give rise to anti-inflammatory effects, and inhibition of the high-affinity conformer to side effects. Therefore, a selective inhibitor of the low-affinity conformer may have clinical utility. Methods are described to prepare recombinant forms of PDE4B that allow screening for compounds that could preferentially inhibit the low-affinity conformer. Furthermore, conditions for an efficient, scintillation proximity, microtiter plate-based assay are described, providing a considerable advance over previous assays in terms of throughput and automatability.

3',5'-Cyclic-AMP Phosphodiesterases↗