Proceedings of the National Academy of Sciences, 1991
A cDNA encoding a functional bradykinin receptor was isolated from a rat uterus library by a clon... more A cDNA encoding a functional bradykinin receptor was isolated from a rat uterus library by a clonal selection strategy using Xenopus laevis oocytes to assay for expression of bradykinin responses. The predicted protein is homologous to the seven transmembrane G protein-coupled superfamily of receptors. Bradykinin and its analogs stimulate a Cl- current oocytes expressing the receptor with the rank order of potency: bradykinin approximately Lys-bradykinin greater than [Tyr8]-bradykinin much greater than [Phe6]bradykinin. This is the rank order of potency observed for these compounds in competitive binding assays on soluble receptor from rat uterus. Des-Arg9-bradykinin (10 microM) elicits no response when applied to oocytes expressing the receptor; thus, the cDNA encodes a B2 type bradykinin receptor. [Thi5,8,DPhe7]bradykinin, where Thi is beta-(2-thienyl)-alanine, is a very weak partial agonist and inhibits the bradykinin-mediated ion flux, suggesting the cDNA encodes a smooth muscle...
Crisaborole topical ointment, 2% (formerly known as AN2728) is a benzoxaborole, nonsteroidal, top... more Crisaborole topical ointment, 2% (formerly known as AN2728) is a benzoxaborole, nonsteroidal, topical, anti-inflammatory phosphodiesterase 4 (PDE4) inhibitor investigational compound that recently completed phase 3 studies for the treatment of mild to moderate atopic dermatitis (AD). The unique configuration of boron within the crisaborole molecule enables selective targeting and inhibition of PDE4, an enzyme that converts the intracellular second messenger 3'5'-cyclic adenosine monophosphate (cAMP) into the active metabolite adenosine monophosphate (AMP). By inhibiting PDE4 and thus increasing levels of cAMP, crisaborole controls inflammation. The use of boron chemistry enabled synthesis of a low-molecular-weight compound (251 daltons), thereby facilitating effective penetration of crisaborole through human skin. In vitro experiments showed that crisaborole inhibits cytokine production from peripheral blood mononuclear cells in a pattern similar to other PDE4 inhibitors and...
The novel boron-containing compound, AN2728, is being investigated in phase 3 trials for atopic d... more The novel boron-containing compound, AN2728, is being investigated in phase 3 trials for atopic dermatitis treatment. It exerts its anti-inflammatory effect by inhibiting phosphodiesterase-4 (PDE4), which catalyzes the breakdown of cAMP to AMP. AN2728 competes with cAMP to inhibit the PDE4B1-catalytic domain with Ki of 173±26 nM; thus AN2728 interacts at the enzyme-active site. The X-ray structure of PDE4B-catalytic domain with AN2728 and its structural relative AN2898 reveals that the boron atom interacts with the bimetal center and occupies a position in the catalytic site similar to that of the cAMP phosphate. AN2728 has good affinity across PDE4 gene products A-D. Its selective affinity for PDE4 is 4-10-fold greater than its affinity for PDE1, 2, 3A, 6, or 7B. It is inactive on PDE3B, 5, 7A1, and 8-11. AN2728 activity increases intracellular cAMP and activates PKA, followed by phosphorylation and negative regulation of various cytokine transcription factors. AN2728 inhibits prod...
Structure-activity relationships of 6-(benzoylamino)benzoxaborole analogs were investigated for t... more Structure-activity relationships of 6-(benzoylamino)benzoxaborole analogs were investigated for the inhibition of TNF-α, IL-1β, and IL-6 from lipopolysaccharide stimulated peripheral blood mononuclear cells. Compound 1q showed potent activity against all three cytokines with IC50 values between 0.19 and 0.50μM, inhibited LPS-induced TNF-α and IL-6 elevation in mice and improved collagen-induced arthritis in mice. Compound 1q (AN4161) is considered to be a promising lead for novel anti-inflammatory agent with an excellent pharmacokinetic profile.
Several clinical trials targeting cutaneous neurofibromas (cNF) have been conducted; however, non... more Several clinical trials targeting cutaneous neurofibromas (cNF) have been conducted; however, none has resulted in meaningful changes to care. The Clinical Trial Design and Development subgroup's goals were to (1) define key considerations in the design of clinical trials for cNF, (2) summarize existing data in relation to these considerations, and (3) provide consensus recommendations about key elements of trial design to accelerate the clinical development of therapies for cNF. The subgroup, with experts from genetics, dermatology, neurology, oncology, and basic science, spanning academia, government research, and regulatory programs, and industry, reviewed published and unpublished data on clinical trials for cNF and other diseases in the skin. Discussions of these data resulted in formulation of a list of priority issues to address in order to develop efficient and effective clinical trials for cNF. The subgroup identified 2 natural history studies of cNF, 4 priority outcome...
The only therapies currently available for cutaneous neurofibromas (cNF) are procedural. The goal... more The only therapies currently available for cutaneous neurofibromas (cNF) are procedural. The goals of the Therapies Development Working Group were to (1) summarize currently available treatment options for cNF, (2) define key considerations for drug discovery and development generally, and specifically for cNF, and (3) outline recommendations for the successful development of medical therapies for cNF. The subgroup reviewed published and unpublished data on procedural, drug/device, and medical treatment approaches utilized for cNFs via literature search. The team defined disease- and patient-specific factors to consider for therapies development in a series of consensus meetings. The team identified 5 approaches entailing procedural and drug/device methods currently under study. There have been 4 clinical studies exploring various interventional therapies, from which outcomes were highly variable. The team identified 4 key factors to prioritize during the development of products for...
Reliance on just one drug to treat the prevalent tropical disease, schistosomiasis, spurs the sea... more Reliance on just one drug to treat the prevalent tropical disease, schistosomiasis, spurs the search for new drugs and drug targets. Inhibitors of human cyclic nucleotide phosphodiesterases (huPDEs), including PDE4, are under development as novel drugs to treat a range of chronic indications including asthma, chronic obstructive pulmonary disease and Alzheimer's disease. One class of huPDE4 inhibitors that has yielded marketed drugs is the benzoxaboroles (Anacor Pharmaceuticals). A phenotypic screen involving Schistosoma mansoni and 1,085 benzoxaboroles identified a subset of huPDE4 inhibitors that induced parasite hypermotility and degeneration. To uncover the putative schistosome PDE4 target, we characterized four PDE4 sequences (SmPDE4A-D) in the parasite's genome and transcriptome, and cloned and recombinantly expressed the catalytic domain of SmPDE4A. Among a set of benzoxaboroles and catechol inhibitors that differentially inhibit huPDE4, a relationship between the inh...
The Journal of pharmacology and experimental therapeutics, Sep 27, 2016
Psoriasis and atopic dermatitis are skin diseases affecting millions of patients. Here, we charac... more Psoriasis and atopic dermatitis are skin diseases affecting millions of patients. Here, we characterize benzoxaborole PDE4 inhibitors, a new topical class that has demonstrated therapeutic benefit for psoriasis and atopic dermatitis in phase 2 or phase 3 studies. Crisaborole (AN2728), compd2, compd3, and compd4 are potent PDE4 inhibitors with similar affinity for PDE4 isoforms and equivalent inhibition on the catalytic domain and the full-length enzyme. These benzoxaboroles are less active on other PDE isozymes. Compd4 binds to the catalytic domain of PDE4B2 with the oxaborole group chelating the catalytic bi-metal and overlapping with the phosphate in cAMP during substrate hydrolysis, and the interaction extends into the adenine pocket. In cell culture, benzoxaborole PDE4 inhibitors suppress the release of TNF-α, IL-23, IL-17, IFN-γ, IL-4, IL-5, IL-13 and IL-22, and these cytokines contribute to the pathologic changes in skin structure and barrier functions as well as immune dysreg...
To evaluate the importance of 1- and 24-hydroxylation of 25-hydroxyvitamin D3 on skeletal mineral... more To evaluate the importance of 1- and 24-hydroxylation of 25-hydroxyvitamin D3 on skeletal mineralization, male and female rats from vitamin D-deficient mothers were administered from weaning either 100 pmol/day of 25-hydroxyvitamin D3, 50 pmol/day of 1,25-dihydroxyvitamin D3, or 100 pmol/day of 24,24-difluoro-25-hydroxyvitamin D3 as their sole source of vitamin D. A separate group of rats did not receive any vitamin D. 1,25-Dihydroxyvitamin D3 was given by constant infusion at a dose that normalized plasma calcium concentrations and produced normal body weight gains. Skeletal mineralization was studied by determining femur organic and ash weights. Femurs were obtained from male rats 6 wk after weaning, from female rats at conception, at the end of lactation, and 6 wk after lactation, and from weanling pups born to the female rats. No striking differences in femur organic and ash weights were found between 25-hydroxyvitamin D3 groups and either the 1,25-dihydroxyvitamin D3 group or t...
Current opinion in investigational drugs (London, England : 2000), 2010
Brain and spinal cord injuries result in cognitive and/or sensorimotor impairments that can signi... more Brain and spinal cord injuries result in cognitive and/or sensorimotor impairments that can significantly diminish the quality of life for the patient and their carers, and result in healthcare system costs totaling in the billions. The current gold-standard of acute care for spinal cord injury is to administer high doses of glucocorticoids within 8 h of injury; administration after 8 h may be without effect or detrimental to the outcome of the patient. Therefore, improved pharmacological approaches for limiting the extent of tissue damage and neurological dysfunction in the acute injury setting are urgently needed. Early intervention in CNS injury by antagonizing or controlling the post-injury inflammatory process with pharmaceutical agents is a major focus of current clinical and preclinical investigations. In this editorial overview, recent clinical trials and preclinical studies of brain and spinal cord injuries are discussed, including studies focusing on the use of broad-spect...
Multicellular organisms must have means of preserving their genomic integrity or face catastrophi... more Multicellular organisms must have means of preserving their genomic integrity or face catastrophic consequences such as uncontrolled cell proliferation or massive cell death. One response is a modification of nuclear proteins by the addition and removal of polymers of ADP-ribose that modulate the properties of DNA-binding proteins involved in DNA repair and metabolism. These ADP-ribose units are added by poly(ADP-ribose) polymerase (PARP) and removed by poly(ADP-ribose) glycohydrolase. Although budding yeast Saccharomyces cerevisiae does not possess proteins with significant sequence similarity to the human PARP family of proteins, we identified novel small molecule inhibitors against two family members, PARP1 and PARP2, using a cell-based assay in yeast. The assay was based on the reversal of growth inhibition caused by the heterologous expression of either PARP1 or PARP2. Validation of the assay was achieved by showing that the growth inhibition was relieved by a mutation in a sin...
Brazilian journal of medical and biological research = Revista brasileira de pesquisas médicas e biológicas / Sociedade Brasileira de Biofísica ... [et al.], 1994
Peptides corresponding to sequences derived from predicted extra- and intracellular loops of the ... more Peptides corresponding to sequences derived from predicted extra- and intracellular loops of the rat bradykinin receptor were analyzed for interspecies homology as well as for matches within the present dataset of protein sequences to provide a theoretical basis for the specific recognition of the native cognate protein by antibodies raised against these antigens. Application of polyclonal antibodies raised against the selected peptides allowed the immunocytochemical localization of the native receptor protein in cells of rat and human origin. The detection of the molecule was achieved by different immunohisto- and immunocytochemical methods in combination with light, fluorescence, confocal optical laser and electron microscopy. These results were compared to localization studies by autoradiography. Distribution and subcellular localization were determined in human neutrophils, human epithelial carcinoma cells (A431) and in rat kidney tissue.
Brazilian journal of medical and biological research = Revista brasileira de pesquisas médicas e biológicas / Sociedade Brasileira de Biofísica ... [et al.], 1994
Brazilian journal of medical and biological research = Revista brasileira de pesquisas médicas e biológicas / Sociedade Brasileira de Biofísica ... [et al.], 1994
1. To identify and isolate cDNAs encoding rat and human bradykinin-B2 receptor subtypes we isolat... more 1. To identify and isolate cDNAs encoding rat and human bradykinin-B2 receptor subtypes we isolated a human bradykinin receptor cDNA homologous to a rat B2 receptor cDNA. 2. The cDNA was expressed in the bradykinin receptor negative cell line, CHO; membranes prepared from these cells bound bradykinin and had specificity similar to that of the known rat B2 receptor. In addition, the expressed receptor has a low affinity for des-Arg9-bradykinin. Thus, the cDNA encodes a human B2-bradykinin receptor. 3. Comparison of the human and rat cDNAs suggested that the human and rat genes are composed of three exons. Cloning, sequencing and characterization of parts of the human and rat B2-bradykinin receptor genes demonstrated the postulated three-exon structure. This structure includes two 5' exons upstream of the most favorable translation initiation methionine in exon-3. 4. The two 5' exons each contain methionines, which if independently spliced to the third exon, would yield an ope...
This study examines whether 1,25-dihydroxyvitamin D3 or 24,24-difluoro-25-hydroxyvitamin D3, an a... more This study examines whether 1,25-dihydroxyvitamin D3 or 24,24-difluoro-25-hydroxyvitamin D3, an analogue of 25-hydroxyvitamin D3 blocked from undergoing 24-hydroxylation, can maintain normal growth and reproduction in the female rat. Vitamin D-deficient weanling rats were maintained from weaning through mating, pregnancy, and lactation with either 1,25-dihydroxyvitamin D3 (given by continuous subcutaneous infusion), 24,24-difluoro-25-hydroxyvitamin D3, 25-hydroxyvitamin D3, or vehicle. Body weight, plasma calcium levels, estrous cycling time, ability to give birth to live pups, litter weight, number of pups per litter, dam plasma calcium level during lactation, and pup growth to 9 wk of age were recorded. No striking differences were observed between the 25-hydroxyvitamin D3 groups and either the 1,25-dihydroxyvitamin D3 group or the 24,24-difluoro-25-hydroxyvitamin D3 group. However, significant differences in most parameters were observed between the vitamin D-deficient and metabo...
Proceedings of the National Academy of Sciences, 1991
A cDNA encoding a functional bradykinin receptor was isolated from a rat uterus library by a clon... more A cDNA encoding a functional bradykinin receptor was isolated from a rat uterus library by a clonal selection strategy using Xenopus laevis oocytes to assay for expression of bradykinin responses. The predicted protein is homologous to the seven transmembrane G protein-coupled superfamily of receptors. Bradykinin and its analogs stimulate a Cl- current oocytes expressing the receptor with the rank order of potency: bradykinin approximately Lys-bradykinin greater than [Tyr8]-bradykinin much greater than [Phe6]bradykinin. This is the rank order of potency observed for these compounds in competitive binding assays on soluble receptor from rat uterus. Des-Arg9-bradykinin (10 microM) elicits no response when applied to oocytes expressing the receptor; thus, the cDNA encodes a B2 type bradykinin receptor. [Thi5,8,DPhe7]bradykinin, where Thi is beta-(2-thienyl)-alanine, is a very weak partial agonist and inhibits the bradykinin-mediated ion flux, suggesting the cDNA encodes a smooth muscle...
Crisaborole topical ointment, 2% (formerly known as AN2728) is a benzoxaborole, nonsteroidal, top... more Crisaborole topical ointment, 2% (formerly known as AN2728) is a benzoxaborole, nonsteroidal, topical, anti-inflammatory phosphodiesterase 4 (PDE4) inhibitor investigational compound that recently completed phase 3 studies for the treatment of mild to moderate atopic dermatitis (AD). The unique configuration of boron within the crisaborole molecule enables selective targeting and inhibition of PDE4, an enzyme that converts the intracellular second messenger 3'5'-cyclic adenosine monophosphate (cAMP) into the active metabolite adenosine monophosphate (AMP). By inhibiting PDE4 and thus increasing levels of cAMP, crisaborole controls inflammation. The use of boron chemistry enabled synthesis of a low-molecular-weight compound (251 daltons), thereby facilitating effective penetration of crisaborole through human skin. In vitro experiments showed that crisaborole inhibits cytokine production from peripheral blood mononuclear cells in a pattern similar to other PDE4 inhibitors and...
The novel boron-containing compound, AN2728, is being investigated in phase 3 trials for atopic d... more The novel boron-containing compound, AN2728, is being investigated in phase 3 trials for atopic dermatitis treatment. It exerts its anti-inflammatory effect by inhibiting phosphodiesterase-4 (PDE4), which catalyzes the breakdown of cAMP to AMP. AN2728 competes with cAMP to inhibit the PDE4B1-catalytic domain with Ki of 173±26 nM; thus AN2728 interacts at the enzyme-active site. The X-ray structure of PDE4B-catalytic domain with AN2728 and its structural relative AN2898 reveals that the boron atom interacts with the bimetal center and occupies a position in the catalytic site similar to that of the cAMP phosphate. AN2728 has good affinity across PDE4 gene products A-D. Its selective affinity for PDE4 is 4-10-fold greater than its affinity for PDE1, 2, 3A, 6, or 7B. It is inactive on PDE3B, 5, 7A1, and 8-11. AN2728 activity increases intracellular cAMP and activates PKA, followed by phosphorylation and negative regulation of various cytokine transcription factors. AN2728 inhibits prod...
Structure-activity relationships of 6-(benzoylamino)benzoxaborole analogs were investigated for t... more Structure-activity relationships of 6-(benzoylamino)benzoxaborole analogs were investigated for the inhibition of TNF-α, IL-1β, and IL-6 from lipopolysaccharide stimulated peripheral blood mononuclear cells. Compound 1q showed potent activity against all three cytokines with IC50 values between 0.19 and 0.50μM, inhibited LPS-induced TNF-α and IL-6 elevation in mice and improved collagen-induced arthritis in mice. Compound 1q (AN4161) is considered to be a promising lead for novel anti-inflammatory agent with an excellent pharmacokinetic profile.
Several clinical trials targeting cutaneous neurofibromas (cNF) have been conducted; however, non... more Several clinical trials targeting cutaneous neurofibromas (cNF) have been conducted; however, none has resulted in meaningful changes to care. The Clinical Trial Design and Development subgroup's goals were to (1) define key considerations in the design of clinical trials for cNF, (2) summarize existing data in relation to these considerations, and (3) provide consensus recommendations about key elements of trial design to accelerate the clinical development of therapies for cNF. The subgroup, with experts from genetics, dermatology, neurology, oncology, and basic science, spanning academia, government research, and regulatory programs, and industry, reviewed published and unpublished data on clinical trials for cNF and other diseases in the skin. Discussions of these data resulted in formulation of a list of priority issues to address in order to develop efficient and effective clinical trials for cNF. The subgroup identified 2 natural history studies of cNF, 4 priority outcome...
The only therapies currently available for cutaneous neurofibromas (cNF) are procedural. The goal... more The only therapies currently available for cutaneous neurofibromas (cNF) are procedural. The goals of the Therapies Development Working Group were to (1) summarize currently available treatment options for cNF, (2) define key considerations for drug discovery and development generally, and specifically for cNF, and (3) outline recommendations for the successful development of medical therapies for cNF. The subgroup reviewed published and unpublished data on procedural, drug/device, and medical treatment approaches utilized for cNFs via literature search. The team defined disease- and patient-specific factors to consider for therapies development in a series of consensus meetings. The team identified 5 approaches entailing procedural and drug/device methods currently under study. There have been 4 clinical studies exploring various interventional therapies, from which outcomes were highly variable. The team identified 4 key factors to prioritize during the development of products for...
Reliance on just one drug to treat the prevalent tropical disease, schistosomiasis, spurs the sea... more Reliance on just one drug to treat the prevalent tropical disease, schistosomiasis, spurs the search for new drugs and drug targets. Inhibitors of human cyclic nucleotide phosphodiesterases (huPDEs), including PDE4, are under development as novel drugs to treat a range of chronic indications including asthma, chronic obstructive pulmonary disease and Alzheimer's disease. One class of huPDE4 inhibitors that has yielded marketed drugs is the benzoxaboroles (Anacor Pharmaceuticals). A phenotypic screen involving Schistosoma mansoni and 1,085 benzoxaboroles identified a subset of huPDE4 inhibitors that induced parasite hypermotility and degeneration. To uncover the putative schistosome PDE4 target, we characterized four PDE4 sequences (SmPDE4A-D) in the parasite's genome and transcriptome, and cloned and recombinantly expressed the catalytic domain of SmPDE4A. Among a set of benzoxaboroles and catechol inhibitors that differentially inhibit huPDE4, a relationship between the inh...
The Journal of pharmacology and experimental therapeutics, Sep 27, 2016
Psoriasis and atopic dermatitis are skin diseases affecting millions of patients. Here, we charac... more Psoriasis and atopic dermatitis are skin diseases affecting millions of patients. Here, we characterize benzoxaborole PDE4 inhibitors, a new topical class that has demonstrated therapeutic benefit for psoriasis and atopic dermatitis in phase 2 or phase 3 studies. Crisaborole (AN2728), compd2, compd3, and compd4 are potent PDE4 inhibitors with similar affinity for PDE4 isoforms and equivalent inhibition on the catalytic domain and the full-length enzyme. These benzoxaboroles are less active on other PDE isozymes. Compd4 binds to the catalytic domain of PDE4B2 with the oxaborole group chelating the catalytic bi-metal and overlapping with the phosphate in cAMP during substrate hydrolysis, and the interaction extends into the adenine pocket. In cell culture, benzoxaborole PDE4 inhibitors suppress the release of TNF-α, IL-23, IL-17, IFN-γ, IL-4, IL-5, IL-13 and IL-22, and these cytokines contribute to the pathologic changes in skin structure and barrier functions as well as immune dysreg...
To evaluate the importance of 1- and 24-hydroxylation of 25-hydroxyvitamin D3 on skeletal mineral... more To evaluate the importance of 1- and 24-hydroxylation of 25-hydroxyvitamin D3 on skeletal mineralization, male and female rats from vitamin D-deficient mothers were administered from weaning either 100 pmol/day of 25-hydroxyvitamin D3, 50 pmol/day of 1,25-dihydroxyvitamin D3, or 100 pmol/day of 24,24-difluoro-25-hydroxyvitamin D3 as their sole source of vitamin D. A separate group of rats did not receive any vitamin D. 1,25-Dihydroxyvitamin D3 was given by constant infusion at a dose that normalized plasma calcium concentrations and produced normal body weight gains. Skeletal mineralization was studied by determining femur organic and ash weights. Femurs were obtained from male rats 6 wk after weaning, from female rats at conception, at the end of lactation, and 6 wk after lactation, and from weanling pups born to the female rats. No striking differences in femur organic and ash weights were found between 25-hydroxyvitamin D3 groups and either the 1,25-dihydroxyvitamin D3 group or t...
Current opinion in investigational drugs (London, England : 2000), 2010
Brain and spinal cord injuries result in cognitive and/or sensorimotor impairments that can signi... more Brain and spinal cord injuries result in cognitive and/or sensorimotor impairments that can significantly diminish the quality of life for the patient and their carers, and result in healthcare system costs totaling in the billions. The current gold-standard of acute care for spinal cord injury is to administer high doses of glucocorticoids within 8 h of injury; administration after 8 h may be without effect or detrimental to the outcome of the patient. Therefore, improved pharmacological approaches for limiting the extent of tissue damage and neurological dysfunction in the acute injury setting are urgently needed. Early intervention in CNS injury by antagonizing or controlling the post-injury inflammatory process with pharmaceutical agents is a major focus of current clinical and preclinical investigations. In this editorial overview, recent clinical trials and preclinical studies of brain and spinal cord injuries are discussed, including studies focusing on the use of broad-spect...
Multicellular organisms must have means of preserving their genomic integrity or face catastrophi... more Multicellular organisms must have means of preserving their genomic integrity or face catastrophic consequences such as uncontrolled cell proliferation or massive cell death. One response is a modification of nuclear proteins by the addition and removal of polymers of ADP-ribose that modulate the properties of DNA-binding proteins involved in DNA repair and metabolism. These ADP-ribose units are added by poly(ADP-ribose) polymerase (PARP) and removed by poly(ADP-ribose) glycohydrolase. Although budding yeast Saccharomyces cerevisiae does not possess proteins with significant sequence similarity to the human PARP family of proteins, we identified novel small molecule inhibitors against two family members, PARP1 and PARP2, using a cell-based assay in yeast. The assay was based on the reversal of growth inhibition caused by the heterologous expression of either PARP1 or PARP2. Validation of the assay was achieved by showing that the growth inhibition was relieved by a mutation in a sin...
Brazilian journal of medical and biological research = Revista brasileira de pesquisas médicas e biológicas / Sociedade Brasileira de Biofísica ... [et al.], 1994
Peptides corresponding to sequences derived from predicted extra- and intracellular loops of the ... more Peptides corresponding to sequences derived from predicted extra- and intracellular loops of the rat bradykinin receptor were analyzed for interspecies homology as well as for matches within the present dataset of protein sequences to provide a theoretical basis for the specific recognition of the native cognate protein by antibodies raised against these antigens. Application of polyclonal antibodies raised against the selected peptides allowed the immunocytochemical localization of the native receptor protein in cells of rat and human origin. The detection of the molecule was achieved by different immunohisto- and immunocytochemical methods in combination with light, fluorescence, confocal optical laser and electron microscopy. These results were compared to localization studies by autoradiography. Distribution and subcellular localization were determined in human neutrophils, human epithelial carcinoma cells (A431) and in rat kidney tissue.
Brazilian journal of medical and biological research = Revista brasileira de pesquisas médicas e biológicas / Sociedade Brasileira de Biofísica ... [et al.], 1994
Brazilian journal of medical and biological research = Revista brasileira de pesquisas médicas e biológicas / Sociedade Brasileira de Biofísica ... [et al.], 1994
1. To identify and isolate cDNAs encoding rat and human bradykinin-B2 receptor subtypes we isolat... more 1. To identify and isolate cDNAs encoding rat and human bradykinin-B2 receptor subtypes we isolated a human bradykinin receptor cDNA homologous to a rat B2 receptor cDNA. 2. The cDNA was expressed in the bradykinin receptor negative cell line, CHO; membranes prepared from these cells bound bradykinin and had specificity similar to that of the known rat B2 receptor. In addition, the expressed receptor has a low affinity for des-Arg9-bradykinin. Thus, the cDNA encodes a human B2-bradykinin receptor. 3. Comparison of the human and rat cDNAs suggested that the human and rat genes are composed of three exons. Cloning, sequencing and characterization of parts of the human and rat B2-bradykinin receptor genes demonstrated the postulated three-exon structure. This structure includes two 5' exons upstream of the most favorable translation initiation methionine in exon-3. 4. The two 5' exons each contain methionines, which if independently spliced to the third exon, would yield an ope...
This study examines whether 1,25-dihydroxyvitamin D3 or 24,24-difluoro-25-hydroxyvitamin D3, an a... more This study examines whether 1,25-dihydroxyvitamin D3 or 24,24-difluoro-25-hydroxyvitamin D3, an analogue of 25-hydroxyvitamin D3 blocked from undergoing 24-hydroxylation, can maintain normal growth and reproduction in the female rat. Vitamin D-deficient weanling rats were maintained from weaning through mating, pregnancy, and lactation with either 1,25-dihydroxyvitamin D3 (given by continuous subcutaneous infusion), 24,24-difluoro-25-hydroxyvitamin D3, 25-hydroxyvitamin D3, or vehicle. Body weight, plasma calcium levels, estrous cycling time, ability to give birth to live pups, litter weight, number of pups per litter, dam plasma calcium level during lactation, and pup growth to 9 wk of age were recorded. No striking differences were observed between the 25-hydroxyvitamin D3 groups and either the 1,25-dihydroxyvitamin D3 group or the 24,24-difluoro-25-hydroxyvitamin D3 group. However, significant differences in most parameters were observed between the vitamin D-deficient and metabo...
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