Judith Lippke - Academia.edu (original) (raw)

Papers by Judith Lippke

Research paper thumbnail of The effect of cadmium toxicity on macromolecular synthesis in the Chinese hamster ovary cell /

Thesis (M.S.)--University of Rhode Island. Includes bibliographical references (leaves 50-53).

Research paper thumbnail of Erythropoietic Cell Cultures From Chick Embryos

The Journal of Cell Biology, 1972

Erythropoietic cell cultures from very early chick blastoderms survive for several days They show... more Erythropoietic cell cultures from very early chick blastoderms survive for several days They show four to seven doublings of the erythroid cells and the appropriate morphological changes from proerythroblasts to mature erythrocytes Cell cycle times are the same as zn ovo for the first day of culture, but slow down thereafter The hemoglobins of both the primitive and the definitive red cell series are produced. 5-Bromodeoxyuridine added to the cultures inhibits differentiation and hemoglobin synthesis, though not cell division, but quite soon the cells cease being sensitive The effect of the drug can be reversed by the addition of thymidine.

Research paper thumbnail of Differentiation in the early chick embryo: Effects of bromodeoxyuridine on erythropoiesis

Developmental Biology, 1974

Organ cultures of chick embryos at the definitive streak stage will normally show the first appea... more Organ cultures of chick embryos at the definitive streak stage will normally show the first appearance of hemoglobin in erythroid cells after 20 hr of culture. Addition of 5-bromodeoxyuridine (BrdU) will prevent hemoglobin formation when added at the beginning of culture, but the ...

Research paper thumbnail of Dna Sequences Encoding CMH-1, a Cysteine Protease Involved in Apoptosis

Research paper thumbnail of Altered cytokine export and apoptosis in mice deficient in interleukin-1 beta converting enzyme

Science (New York, N.Y.), Jan 31, 1995

The interleukin-1 beta (IL-1 beta) converting enzyme (ICE) processes the inactive IL-1 beta precu... more The interleukin-1 beta (IL-1 beta) converting enzyme (ICE) processes the inactive IL-1 beta precursor to the proinflammatory cytokine. Adherent monocytes from mice harboring a disrupted ICE gene (ICE-/-) did not export IL-1 beta or interleukin-1 alpha (IL-1 alpha) after stimulation with lipopolysaccharide. Export of tumor necrosis factor-alpha and interleukin-6 (IL-6) from these cells was also diminished. Thymocytes from ICE-/- mice were sensitive to apoptosis induced by dexamethasone or ionizing radiation, but were resistant to apoptosis induced by Fas antibody. Despite this defect in apoptosis, ICE-/- mice proceed normally through development.

Research paper thumbnail of Processing and Activation of CMH-1 by Granzyme B

Journal of Biological Chemistry, 1996

Granzyme B plays an essential role in cytotoxic T lymphocyte (CTL)-mediated cell killing. Recent ... more Granzyme B plays an essential role in cytotoxic T lymphocyte (CTL)-mediated cell killing. Recent studies suggest that granzyme B may exert its effect by cleaving and activating CPP32, a member of the interleukin-1 beta-converting enzyme/Ced-3 family of cysteine proteases. We have examined the processing and activation of CMH-1, a close homologue of CPP32, by granzyme B in vitro. We have found that granzyme B specifically cleaves CMH-1 at Asp198-Ser199 between the p20 and p12 and activates the cysteine protease. Cleavage between p20 and the prosequence of CMH-1 at Asp23-Ala24 is autocatalytic and is not required for CMH-1 activity in vitro. The cleavage and activation of CMH-1 by granzyme B in vitro sugge st that, in addition to CPP32, CMH-1 may also play a role in CTL-mediated cell killing.

Research paper thumbnail of Identification and Characterization of CPP32/Mch2 Homolog 1, a Novel Cysteine Protease Similar to CPP32

Journal of Biological Chemistry, 1996

We have identified and characterized a novel cysteine protease named CMH-1 that is a new member o... more We have identified and characterized a novel cysteine protease named CMH-1 that is a new member of the interleukin 1 beta converting enzyme (ICE) family of proteases with substrate specificity for Asp-X. CMH-1 has the highest similarity to CPP32 (52% amino acid identity) and MCH2 (31% identical). CMH-1 shares conserved amino acid residues that form the core structure of ICE as well as those residues involved in catalysis and in the P1 aspartate binding. Overexpression of CMH-1 in COS cells resulted in the processing of CMH-1 and the induction of apoptosis of transfected cells. Coexpression of CMH-1 with poly(ADP-ribose) polymerase (PARP) also resulted in a specific cleavage of PARP. Purified recombinant CMH-1 cleaved PARP but not interleukin 1 beta precursor in vitro.

Research paper thumbnail of New methods for detection of low levels of DNA damage in human populations

Environmental Health Perspectives, 1983

The use of a postlabeling method to characterize and to detect infrequent base modifications in D... more The use of a postlabeling method to characterize and to detect infrequent base modifications in DNA is outlined. This method has the advantage that low levels of DNA modifications, approximately 1 modified base per 105 nucleotides, can be detected. Moreover, a broad spectrum of modification can be identified by using this methodology. The basis for the method involves transfer of a radioactive phosphate from the y position of ATP to the 5'-hydroxyl terminus of 3'-phosphoryl nucleotides that are derived from modified DNA by appropriate nuclease digestion.

Research paper thumbnail of Methods for Amplifying Hepatitis C Virus Nucleic Acids

Research paper thumbnail of III, Phenotypic heterogeneity in cultured human head and neck squamous cell carcinoma lines with low-Level methotrexate resistance

Cancer Research

Low-level methotrexate (MTX) resistance (less than 20-fold) was induced by gradual selection pres... more Low-level methotrexate (MTX) resistance (less than 20-fold) was induced by gradual selection pressure in four human head and neck squamous cell carcinoma lines established in culture from biopsies of patients not previously treated with MTX. Each parental and resistant line was characterized with respect to MTX uptake and polyglutamylation, dihydrofolate reductase (DHFR) content, and growth rate. Relative DHFR gene copy numbers and amounts of DHFR-related cytoplasmic messenger RNA were analyzed by plasmid complementary DNA hybridization in a dot blot assay and were correlated with the amount of gene product. The resistant lines were not cloned in order to simulate in vitro the conditions which might exist in an in vivo setting, where multiple resistant subpopulations of cells may be present in a tumor. The study was restricted to cells with low-level resistance since these are likely to be the clinically most relevant type. Of the four resistant lines characterized, one showed a sev...

Research paper thumbnail of Isolation ofIntact High-Molecular-Weight

A method for obtaining high-molecular-weight chromosomal DNA from Bacteroides intermedius and Bac... more A method for obtaining high-molecular-weight chromosomal DNA from Bacteroides intermedius and Bacteroides gingivalis is described. This technique is a modification of the guanidine isothiocyanate isolation procedure for RNA and should be useful for isolating intact DNA from organisms with high endogenous nuclease activity.

Research paper thumbnail of French CK. Isolation of intact high-molecular weight DNA by using guanidine isothiocyanate

Applied and Environmental Microbiology

A method for obtaining high-molecular-weight chromosomal DNA from Bacteroides intermedius and Bac... more A method for obtaining high-molecular-weight chromosomal DNA from Bacteroides intermedius and Bacteroides gingivalis is described. This technique is a modification of the guanidine isothiocyanate isolation procedure for RNA and should be useful for isolating intact DNA from organisms with high endogenous nuclease activity.

Research paper thumbnail of Expression and characterization of human FKBP52, an immunophilin that associates with the 90-kDa heat shock protein and is a component of steroid receptor complexes

Proceedings of the National Academy of Sciences of the United States of America, Jan 15, 1992

Using an FK506 affinity column to identify mammalian immunosuppressant-binding proteins, we ident... more Using an FK506 affinity column to identify mammalian immunosuppressant-binding proteins, we identified an immunophilin with an apparent M(r) approximately 55,000, which we have named FKBP52. We used chemically determined peptide sequence and a computerized algorithm to search GenPept, the translated GenBank data base, and identified two cDNAs likely to encode the murine FKBP52 homolog. We amplified a murine cDNA fragment, used it to select a human FKBP52 (hFKBP52) cDNA clone, and then used the clone to deduce the hFKBP52 sequence (calculated M(r) 51,810) and to express hFKBP52 in Escherichia coli. Recombinant hFKBP52 has peptidyl-prolyl cis-trans isomerase activity that is inhibited by FK506 and rapamycin and an FKBP12-like consensus sequence that probably defines the immunosuppressant-binding site. FKBP52 is apparently common to several vertebrate species and associates with the 90-kDa heat shock protein (hsp90) in untransformed mammalian steroid receptor complexes. The putative im...

Research paper thumbnail of Nonstructural protein 5A (NS5A) and human replication protein A increase the processivity of hepatitis C virus NS5B polymerase activity in vitro

Journal of virology, 2015

The precise role(s) and topological organization of different factors in the hepatitis C virus (H... more The precise role(s) and topological organization of different factors in the hepatitis C virus (HCV) RNA replication complex are not well understood. In order to elucidate the role of viral and host proteins in HCV replication, we have developed a novel in vitro replication system that utilizes a rolling-circle RNA template. Under close-to-physiological salt conditions, HCV NS5BΔ21, an RNA-dependent RNA polymerase, has poor affinity for the RNA template. Human replication protein A (RPA) and HCV NS5A recruit NS5BΔ21 to the template. Subsequently, NS3 is recruited to the replication complex by NS5BΔ21, resulting in RNA synthesis stimulation by helicase. Both RPA and NS5A(S25-C447), but not NS5A(S25-K215), enabled the NS5BΔ21-NS3 helicase complex to be stably associated with the template and synthesize RNA product in a highly processive manner in vitro. This new in vitro HCV replication system is a useful tool that may facilitate the study of other replication factors and aid in the d...

Research paper thumbnail of Nucleotide-binding domains of cystic fibrosis transmembrane conductance regulator, an ABC transporter, catalyze adenylate kinase activity but not ATP hydrolysis

The Journal of biological chemistry, Jan 17, 2006

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-bin... more The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter family. CFTR consists of two transmembrane domains, two nucleotide-binding domains (NBD1 and NBD2), and a regulatory domain. Previous biochemical reports suggest NBD1 is a site of stable nucleotide interaction with low ATPase activity, whereas NBD2 is the site of active ATP hydrolysis. It has also been reported that NBD2 additionally possessed adenylate kinase (AK) activity. Knowledge about the intrinsic biochemical activities of the NBDs is essential to understanding the Cl(-) ion gating mechanism. We find that purified mouse NBD1, human NBD1, and human NBD2 function as adenylate kinases but not as ATPases. AK activity is strictly dependent on the addition of the adenosine monophosphate (AMP) substrate. No liberation of [(33)P]phosphate is observed from the gamma-(33)P-labeled ATP substrate in the presence or absence of AMP. AK activity is intrinsic to bo...

Research paper thumbnail of New Approaches to DNA Damage and Repair: The Ultraviolet Light Example

Molecular and Cellular Mechanisms of Mutagenesis, 1982

DNA fragments of defined sequence are used as probes to study DNA damage and repair. The case of ... more DNA fragments of defined sequence are used as probes to study DNA damage and repair. The case of ultraviolet light is presented and includes the following: (a) Description of the distribution of cyclobutane pyrimidine dimers within defined DNA sequences. Considerations of the effect of neighboring base composition, dose rate, and double- or single-stranded property of the DNA are discussed. (b) Dissection of the anatomy of the incision event and subsequent repair steps. A three-step incision model for repair of cyclobutane dimers by the Micrococcus luteus repair enzymes will be presented. The steps are (1) recognition of the lesion and N-glycosylase scission, (2) cleavage of the phosphodiester bond 3' to the newly created apyrimidinic site, and (3) scission of the apyrimidinic sugar on the 5' side. (c) Use of human alphoid sequences as indicators of DNA damage in intact human cells. (d) Biological significance of a novel ultraviolet light-induced photoproduct. This photoproduct occurs at pyrimidine-cytosine sequences and may have a significant biological role.

Research paper thumbnail of Crystal Structure of the Ectodomain Complex of the CGRP Receptor, a Class-B GPCR, Reveals the Site of Drug Antagonism

Structure, 2010

Dysregulation of the calcitonin gene-related peptide (CGRP), a potent vasodilator, is directly im... more Dysregulation of the calcitonin gene-related peptide (CGRP), a potent vasodilator, is directly implicated in the pathogenesis of migraine. CGRP binds to and signals through the CGRP receptor (CGRP-R), a heterodimer containing the calcitonin receptor-like receptor (CLR), a class B GPCR, and RAMP1, a receptor activity-modifying protein. We have solved the crystal structure of the CLR/RAMP1 N-terminal ectodomain heterodimer, revealing how RAMPs bind to and potentially modulate the activities of the CLR GPCR subfamily. We also report the structures of CLR/RAMP1 in complex with the clinical receptor antagonists olcegepant (BIBN4096BS) and telcagepant (MK0974). Both drugs act by blocking access to the peptide-binding cleft at the interface of CLR and RAMP1. These structures illustrate, for the first time, how small molecules bind to and modulate the activity of a class B GPCR, and highlight the challenges of designing potent receptor antagonists for the treatment of migraine and other class B GPCR-related diseases.

Research paper thumbnail of actinomycetemcomitans, Bacteroides intermedius, and Bacteroides gingivalis A Cross-reactivity Study of Whole Genomic DNA Probes for Haemophilus

Research paper thumbnail of Development of a sensitive RT-PCR method for amplifying and sequencing near full-length HCV genotype 1 RNA from patient samples

Virology Journal, 2013

Background: Direct-acting antiviral (DAAs) agents for hepatitis C virus (HCV) span a variety of t... more Background: Direct-acting antiviral (DAAs) agents for hepatitis C virus (HCV) span a variety of targets, including proteins encoded by the NS3/4A, NS4B, NS5A, and NS5B genes. Treatment with DAAs has been shown to select variants with sequence changes in the HCV genome encoding amino acids that may confer resistance to the treatment. In order to assess these effects in patients, a Reverse Transcription Polymerase Chain Reaction (RT-PCR) method was developed to sequence these regions of HCV from patient plasma. Methods: A method was developed to amplify and sequence genotype 1 HCV RNA from patient plasma. Optimization of HCV RNA isolation, cDNA synthesis, and nested PCR steps were performed. The optimization of HCV RNA isolation, design of RT-PCR primers, optimization of RT-PCR amplification conditions and reagents, and the evaluation of the RT-PCR method performance is described.

Research paper thumbnail of Development of methotrexate resistance in a human squamous cell carcinoma of the head and neck in culture

Proceedings of the National Academy of Sciences, 1984

Four methotrexate (MTX)-resistant sublines of a human squamous cell carcinoma (SCC15) were estab-

Research paper thumbnail of The effect of cadmium toxicity on macromolecular synthesis in the Chinese hamster ovary cell /

Thesis (M.S.)--University of Rhode Island. Includes bibliographical references (leaves 50-53).

Research paper thumbnail of Erythropoietic Cell Cultures From Chick Embryos

The Journal of Cell Biology, 1972

Erythropoietic cell cultures from very early chick blastoderms survive for several days They show... more Erythropoietic cell cultures from very early chick blastoderms survive for several days They show four to seven doublings of the erythroid cells and the appropriate morphological changes from proerythroblasts to mature erythrocytes Cell cycle times are the same as zn ovo for the first day of culture, but slow down thereafter The hemoglobins of both the primitive and the definitive red cell series are produced. 5-Bromodeoxyuridine added to the cultures inhibits differentiation and hemoglobin synthesis, though not cell division, but quite soon the cells cease being sensitive The effect of the drug can be reversed by the addition of thymidine.

Research paper thumbnail of Differentiation in the early chick embryo: Effects of bromodeoxyuridine on erythropoiesis

Developmental Biology, 1974

Organ cultures of chick embryos at the definitive streak stage will normally show the first appea... more Organ cultures of chick embryos at the definitive streak stage will normally show the first appearance of hemoglobin in erythroid cells after 20 hr of culture. Addition of 5-bromodeoxyuridine (BrdU) will prevent hemoglobin formation when added at the beginning of culture, but the ...

Research paper thumbnail of Dna Sequences Encoding CMH-1, a Cysteine Protease Involved in Apoptosis

Research paper thumbnail of Altered cytokine export and apoptosis in mice deficient in interleukin-1 beta converting enzyme

Science (New York, N.Y.), Jan 31, 1995

The interleukin-1 beta (IL-1 beta) converting enzyme (ICE) processes the inactive IL-1 beta precu... more The interleukin-1 beta (IL-1 beta) converting enzyme (ICE) processes the inactive IL-1 beta precursor to the proinflammatory cytokine. Adherent monocytes from mice harboring a disrupted ICE gene (ICE-/-) did not export IL-1 beta or interleukin-1 alpha (IL-1 alpha) after stimulation with lipopolysaccharide. Export of tumor necrosis factor-alpha and interleukin-6 (IL-6) from these cells was also diminished. Thymocytes from ICE-/- mice were sensitive to apoptosis induced by dexamethasone or ionizing radiation, but were resistant to apoptosis induced by Fas antibody. Despite this defect in apoptosis, ICE-/- mice proceed normally through development.

Research paper thumbnail of Processing and Activation of CMH-1 by Granzyme B

Journal of Biological Chemistry, 1996

Granzyme B plays an essential role in cytotoxic T lymphocyte (CTL)-mediated cell killing. Recent ... more Granzyme B plays an essential role in cytotoxic T lymphocyte (CTL)-mediated cell killing. Recent studies suggest that granzyme B may exert its effect by cleaving and activating CPP32, a member of the interleukin-1 beta-converting enzyme/Ced-3 family of cysteine proteases. We have examined the processing and activation of CMH-1, a close homologue of CPP32, by granzyme B in vitro. We have found that granzyme B specifically cleaves CMH-1 at Asp198-Ser199 between the p20 and p12 and activates the cysteine protease. Cleavage between p20 and the prosequence of CMH-1 at Asp23-Ala24 is autocatalytic and is not required for CMH-1 activity in vitro. The cleavage and activation of CMH-1 by granzyme B in vitro sugge st that, in addition to CPP32, CMH-1 may also play a role in CTL-mediated cell killing.

Research paper thumbnail of Identification and Characterization of CPP32/Mch2 Homolog 1, a Novel Cysteine Protease Similar to CPP32

Journal of Biological Chemistry, 1996

We have identified and characterized a novel cysteine protease named CMH-1 that is a new member o... more We have identified and characterized a novel cysteine protease named CMH-1 that is a new member of the interleukin 1 beta converting enzyme (ICE) family of proteases with substrate specificity for Asp-X. CMH-1 has the highest similarity to CPP32 (52% amino acid identity) and MCH2 (31% identical). CMH-1 shares conserved amino acid residues that form the core structure of ICE as well as those residues involved in catalysis and in the P1 aspartate binding. Overexpression of CMH-1 in COS cells resulted in the processing of CMH-1 and the induction of apoptosis of transfected cells. Coexpression of CMH-1 with poly(ADP-ribose) polymerase (PARP) also resulted in a specific cleavage of PARP. Purified recombinant CMH-1 cleaved PARP but not interleukin 1 beta precursor in vitro.

Research paper thumbnail of New methods for detection of low levels of DNA damage in human populations

Environmental Health Perspectives, 1983

The use of a postlabeling method to characterize and to detect infrequent base modifications in D... more The use of a postlabeling method to characterize and to detect infrequent base modifications in DNA is outlined. This method has the advantage that low levels of DNA modifications, approximately 1 modified base per 105 nucleotides, can be detected. Moreover, a broad spectrum of modification can be identified by using this methodology. The basis for the method involves transfer of a radioactive phosphate from the y position of ATP to the 5'-hydroxyl terminus of 3'-phosphoryl nucleotides that are derived from modified DNA by appropriate nuclease digestion.

Research paper thumbnail of Methods for Amplifying Hepatitis C Virus Nucleic Acids

Research paper thumbnail of III, Phenotypic heterogeneity in cultured human head and neck squamous cell carcinoma lines with low-Level methotrexate resistance

Cancer Research

Low-level methotrexate (MTX) resistance (less than 20-fold) was induced by gradual selection pres... more Low-level methotrexate (MTX) resistance (less than 20-fold) was induced by gradual selection pressure in four human head and neck squamous cell carcinoma lines established in culture from biopsies of patients not previously treated with MTX. Each parental and resistant line was characterized with respect to MTX uptake and polyglutamylation, dihydrofolate reductase (DHFR) content, and growth rate. Relative DHFR gene copy numbers and amounts of DHFR-related cytoplasmic messenger RNA were analyzed by plasmid complementary DNA hybridization in a dot blot assay and were correlated with the amount of gene product. The resistant lines were not cloned in order to simulate in vitro the conditions which might exist in an in vivo setting, where multiple resistant subpopulations of cells may be present in a tumor. The study was restricted to cells with low-level resistance since these are likely to be the clinically most relevant type. Of the four resistant lines characterized, one showed a sev...

Research paper thumbnail of Isolation ofIntact High-Molecular-Weight

A method for obtaining high-molecular-weight chromosomal DNA from Bacteroides intermedius and Bac... more A method for obtaining high-molecular-weight chromosomal DNA from Bacteroides intermedius and Bacteroides gingivalis is described. This technique is a modification of the guanidine isothiocyanate isolation procedure for RNA and should be useful for isolating intact DNA from organisms with high endogenous nuclease activity.

Research paper thumbnail of French CK. Isolation of intact high-molecular weight DNA by using guanidine isothiocyanate

Applied and Environmental Microbiology

A method for obtaining high-molecular-weight chromosomal DNA from Bacteroides intermedius and Bac... more A method for obtaining high-molecular-weight chromosomal DNA from Bacteroides intermedius and Bacteroides gingivalis is described. This technique is a modification of the guanidine isothiocyanate isolation procedure for RNA and should be useful for isolating intact DNA from organisms with high endogenous nuclease activity.

Research paper thumbnail of Expression and characterization of human FKBP52, an immunophilin that associates with the 90-kDa heat shock protein and is a component of steroid receptor complexes

Proceedings of the National Academy of Sciences of the United States of America, Jan 15, 1992

Using an FK506 affinity column to identify mammalian immunosuppressant-binding proteins, we ident... more Using an FK506 affinity column to identify mammalian immunosuppressant-binding proteins, we identified an immunophilin with an apparent M(r) approximately 55,000, which we have named FKBP52. We used chemically determined peptide sequence and a computerized algorithm to search GenPept, the translated GenBank data base, and identified two cDNAs likely to encode the murine FKBP52 homolog. We amplified a murine cDNA fragment, used it to select a human FKBP52 (hFKBP52) cDNA clone, and then used the clone to deduce the hFKBP52 sequence (calculated M(r) 51,810) and to express hFKBP52 in Escherichia coli. Recombinant hFKBP52 has peptidyl-prolyl cis-trans isomerase activity that is inhibited by FK506 and rapamycin and an FKBP12-like consensus sequence that probably defines the immunosuppressant-binding site. FKBP52 is apparently common to several vertebrate species and associates with the 90-kDa heat shock protein (hsp90) in untransformed mammalian steroid receptor complexes. The putative im...

Research paper thumbnail of Nonstructural protein 5A (NS5A) and human replication protein A increase the processivity of hepatitis C virus NS5B polymerase activity in vitro

Journal of virology, 2015

The precise role(s) and topological organization of different factors in the hepatitis C virus (H... more The precise role(s) and topological organization of different factors in the hepatitis C virus (HCV) RNA replication complex are not well understood. In order to elucidate the role of viral and host proteins in HCV replication, we have developed a novel in vitro replication system that utilizes a rolling-circle RNA template. Under close-to-physiological salt conditions, HCV NS5BΔ21, an RNA-dependent RNA polymerase, has poor affinity for the RNA template. Human replication protein A (RPA) and HCV NS5A recruit NS5BΔ21 to the template. Subsequently, NS3 is recruited to the replication complex by NS5BΔ21, resulting in RNA synthesis stimulation by helicase. Both RPA and NS5A(S25-C447), but not NS5A(S25-K215), enabled the NS5BΔ21-NS3 helicase complex to be stably associated with the template and synthesize RNA product in a highly processive manner in vitro. This new in vitro HCV replication system is a useful tool that may facilitate the study of other replication factors and aid in the d...

Research paper thumbnail of Nucleotide-binding domains of cystic fibrosis transmembrane conductance regulator, an ABC transporter, catalyze adenylate kinase activity but not ATP hydrolysis

The Journal of biological chemistry, Jan 17, 2006

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-bin... more The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter family. CFTR consists of two transmembrane domains, two nucleotide-binding domains (NBD1 and NBD2), and a regulatory domain. Previous biochemical reports suggest NBD1 is a site of stable nucleotide interaction with low ATPase activity, whereas NBD2 is the site of active ATP hydrolysis. It has also been reported that NBD2 additionally possessed adenylate kinase (AK) activity. Knowledge about the intrinsic biochemical activities of the NBDs is essential to understanding the Cl(-) ion gating mechanism. We find that purified mouse NBD1, human NBD1, and human NBD2 function as adenylate kinases but not as ATPases. AK activity is strictly dependent on the addition of the adenosine monophosphate (AMP) substrate. No liberation of [(33)P]phosphate is observed from the gamma-(33)P-labeled ATP substrate in the presence or absence of AMP. AK activity is intrinsic to bo...

Research paper thumbnail of New Approaches to DNA Damage and Repair: The Ultraviolet Light Example

Molecular and Cellular Mechanisms of Mutagenesis, 1982

DNA fragments of defined sequence are used as probes to study DNA damage and repair. The case of ... more DNA fragments of defined sequence are used as probes to study DNA damage and repair. The case of ultraviolet light is presented and includes the following: (a) Description of the distribution of cyclobutane pyrimidine dimers within defined DNA sequences. Considerations of the effect of neighboring base composition, dose rate, and double- or single-stranded property of the DNA are discussed. (b) Dissection of the anatomy of the incision event and subsequent repair steps. A three-step incision model for repair of cyclobutane dimers by the Micrococcus luteus repair enzymes will be presented. The steps are (1) recognition of the lesion and N-glycosylase scission, (2) cleavage of the phosphodiester bond 3' to the newly created apyrimidinic site, and (3) scission of the apyrimidinic sugar on the 5' side. (c) Use of human alphoid sequences as indicators of DNA damage in intact human cells. (d) Biological significance of a novel ultraviolet light-induced photoproduct. This photoproduct occurs at pyrimidine-cytosine sequences and may have a significant biological role.

Research paper thumbnail of Crystal Structure of the Ectodomain Complex of the CGRP Receptor, a Class-B GPCR, Reveals the Site of Drug Antagonism

Structure, 2010

Dysregulation of the calcitonin gene-related peptide (CGRP), a potent vasodilator, is directly im... more Dysregulation of the calcitonin gene-related peptide (CGRP), a potent vasodilator, is directly implicated in the pathogenesis of migraine. CGRP binds to and signals through the CGRP receptor (CGRP-R), a heterodimer containing the calcitonin receptor-like receptor (CLR), a class B GPCR, and RAMP1, a receptor activity-modifying protein. We have solved the crystal structure of the CLR/RAMP1 N-terminal ectodomain heterodimer, revealing how RAMPs bind to and potentially modulate the activities of the CLR GPCR subfamily. We also report the structures of CLR/RAMP1 in complex with the clinical receptor antagonists olcegepant (BIBN4096BS) and telcagepant (MK0974). Both drugs act by blocking access to the peptide-binding cleft at the interface of CLR and RAMP1. These structures illustrate, for the first time, how small molecules bind to and modulate the activity of a class B GPCR, and highlight the challenges of designing potent receptor antagonists for the treatment of migraine and other class B GPCR-related diseases.

Research paper thumbnail of actinomycetemcomitans, Bacteroides intermedius, and Bacteroides gingivalis A Cross-reactivity Study of Whole Genomic DNA Probes for Haemophilus

Research paper thumbnail of Development of a sensitive RT-PCR method for amplifying and sequencing near full-length HCV genotype 1 RNA from patient samples

Virology Journal, 2013

Background: Direct-acting antiviral (DAAs) agents for hepatitis C virus (HCV) span a variety of t... more Background: Direct-acting antiviral (DAAs) agents for hepatitis C virus (HCV) span a variety of targets, including proteins encoded by the NS3/4A, NS4B, NS5A, and NS5B genes. Treatment with DAAs has been shown to select variants with sequence changes in the HCV genome encoding amino acids that may confer resistance to the treatment. In order to assess these effects in patients, a Reverse Transcription Polymerase Chain Reaction (RT-PCR) method was developed to sequence these regions of HCV from patient plasma. Methods: A method was developed to amplify and sequence genotype 1 HCV RNA from patient plasma. Optimization of HCV RNA isolation, cDNA synthesis, and nested PCR steps were performed. The optimization of HCV RNA isolation, design of RT-PCR primers, optimization of RT-PCR amplification conditions and reagents, and the evaluation of the RT-PCR method performance is described.

Research paper thumbnail of Development of methotrexate resistance in a human squamous cell carcinoma of the head and neck in culture

Proceedings of the National Academy of Sciences, 1984

Four methotrexate (MTX)-resistant sublines of a human squamous cell carcinoma (SCC15) were estab-