Biomedical subjects

C Schroeder

Publications and source records attributed to C Schroeder.

At least 19 recordsLinked to original sources

PubMed1999

Regions of endonuclease EcoRII involved in DNA target recognition identified by membrane-bound peptide repertoires.

Target sequence-specific DNA binding regions of the restriction endonuclease EcoRII were identified by screening a membrane-bound EcoRII-derived peptide scan with an EcoRII recognition site (CCWGG) oligonucleotide duplex. Dodecapeptides overlapping by nine amino acids and representing the complete protein were prepared by spot synthesis. Two separate DNA binding regions, amino acids 88-102 and amino acids 256-273, which share the consensus motif KXRXXK, emerged. Screening 570 single substitution analogues obtained by exchanging every residue of both binding sites for all other amino acids demonstrated that replacing basic residues in the consensus motifs significantly reduced DNA binding. EcoRII mutant enzymes generated by substituting alanine or glutamic acid for the consensus lysine residues in DNA binding site I expressed attenuated DNA binding, whereas corresponding substitutions in DNA binding site II caused impaired cleavage, but enzyme secondary structure was unaffected. Furthermore, Glu96, which is part of a potential catalytic motif and also locates to DNA binding site I, was demonstrated to be critical for DNA cleavage and binding. Homology studies of DNA binding site II revealed strong local homology to SsoII (recognition sequence, CCNGG) and patterns of sequence conservation, suggesting the existence of functionally related DNA binding sites in diverse restriction endonucleases with recognition sequences containing terminal C:G or G:C pairs.

Amino Acid Sequence↗

PubMed1999

Overexpression and functional characterization of kinin receptors reveal subtype-specific phosphorylation.

G protein-coupled receptors such as the receptors for bradykinin are present in low copy numbers in most natural cells. To overcome the problems associated with the analysis of these receptors at the protein level, we used highly efficient expression systems such as the baculovirus/insect cell system. However, the structural and functional statuses of recombinant receptors have often remained elusive. We have expressed the two types of human kinin receptors, B1 and B2, in Sf9 cells. Both receptors are found on the surface of infected cells where they display the same pharmacological profiles as their cognate receptors of native cells. The functional analysis of kinin receptors coupled to the intracellular signaling pathways of Sf9 cells revealed differential patterns of ligand-induced phosphorylation for the two kinin receptors. The B1 receptor failed to undergo ligand-induced phosphorylation. However the B2 receptor showed selective phosphorylation of a minor 38 kDa band and lack of phosphorylation of a dominant 33 kDa band, indicating that only a fraction of the receptor protein is functionally linked to the kinase pathway. A striking discrepancy between the number of binding sites and the amount of receptor protein per cell (molar ratio of 1:20 to 1:1000) indicated that a significant portion of kinin receptors is associated with the intracellular compartments of Sf9 cells. Pulse-chase and immunoprecipitation experiments demonstrated that the heterogeneity of recombinant receptors is not due to proteolytic processing but likely reflects incomplete or lacking N-glycosylation. We conclude that the baculovirus/Sf9 system is suitable for the recombinant expression and functional analysis of kinin receptors though limitations of the system have to be considered.

Animals↗

PubMed1999

Clinical experience with minimally invasive coronary artery and mitral valve surgery with the advantage of cardiopulmonary bypass and cardioplegic arrest using the Port Access technique.

To minimize surgical trauma, the use of Port Access cardiac surgery was initiated in patients (pts) with coronary artery disease (CAD) (42 pts) or mitral valve disease (MVD) (24 pts) in March 1996 at our institution. Altogether 42 pts (36 men, 6 women; age 31-75 years, median 59.0 years) with isolated lesions of the left anterior descending (LAD) artery underwent Port Access coronary artery surgery (PACAS). A small (5-9 cm) incision was done parasternally on top on the fourth rib. The left internal mammary artery (LIMA) was dissected through the minithoracotomy or by using an additional thoracoscopic approach. A total of 24 pts (12 men, 12 women; age 30-75 years, median 62 years) underwent Port Access mitral valve surgery (PAMVS). In these pts the procedure was performed through a small right thoracotomy (6-8 cm). In all cases, endovascular cardiopulmonary bypass (CPB) was instituted through femoral cannulation, and an additional endoaortic balloon catheter was introduced into the ascending aorta for aortic occlusion. In pts with PACAS the survival was 98% (41/42) and in the PAMVS group 100%. All pts but one survived the PACAS and are well today. There were no deaths in the PAMVS group. The hospital stay was reduced by 1 day on average after PACAS and 3 days after PAMVS. Thus in well selected patients Port Access cardiac surgery represents a safe and feasible minimally invasive surgical approach that avoids the potential complications of a sternotomy while offering the advantages and safety of CPB and cardioplegic arrest. This minimally invasive approach offers a shortened hospital stay and earlier rehabilitation.

Adult↗

PubMed1998

Cooperative binding properties of restriction endonuclease EcoRII with DNA recognition sites.

EcoRII is a member of the expanding group of type IIe restriction endonucleases that share the distinguishing feature of requiring cooperativity between two recognition sites in their substrate DNA. To determine the stoichiometry of the active DNA-enzyme complex and the mode of cooperative interaction, we have investigated the dependence of EcoRII cleavage on the concentration of EcoRII dimers. Maximal restriction was observed at dimer/site ratios of 0.25 and 0. 5. The molecular weight of the DNA-enzyme complex eluted from a gel filtration column also corresponds to a dimeric enzyme structure bound to two substrate sites. We conclude that one EcoRII dimer is sufficient to interact cooperatively with two DNA recognition sites. A Lac repressor "barrier" bound between two normally reactive EcoRII sites did not inhibit restriction endonuclease activity, indicating that cooperativity between EcoRII sites is achieved by bending or looping of the intervening DNA stretch. Comparative cleavage of linear substrates with differently spaced interacting sites revealed an inverse correlation between cleavage rate and site distance. At the optimal distance of one helical turn, EcoRII cleavage is independent of the orientation of the recognition sequence in the DNA double strand.

Bacterial Proteins↗

PubMed1998

Subtype-specific endothelin-A and endothelin-B receptor desensitization correlates with differential receptor phosphorylation.

In the rat cardiovascular system endothelin-1 (ET-1) elicits prolonged physiologic responses mediated by the ETA receptor, whereas the effects mediated by the ETB receptor are transient. The molecular mechanisms for the subtype-specific responses are not yet clear. However, post-translational modifications such as phosphorylation and palmitoylation may play an important role. In Sf9 cells overexpressing the human ETA and ETB receptors, both subtypes are palmitoylated. However, only the ETB but not the ETA receptor is phosphorylated in a ligand-dependent manner. Because phosphorylation is believed to play an important role in ligand-dependent receptor inactivation, we analyzed whether the differential phosphorylation of the ETA and ETB receptors reflects a differential mechanism of receptor inactivation. Using a modified inositol phosphate accumulation assay, we analyzed CHO cells that expressed the ETA or ETB receptor. The ETB receptor was deactivated almost completely within 5 min after agonist stimulation, whereas stimulation of the ETA receptor resulted in sustained activation, i.e., > 90% of the initial activity was maintained after 5 min of ligand stimulation and > 30% after 20 min. A strong correlation was observed between the time course of ETA receptor inactivation and ETA receptor internalization. The endogenous ETA receptor in Rat-1 cells produced a prolonged stimulation of phospholipase C similar to that seen in CHO cells. Therefore, the sustained signaling activity of the ETA receptor is not a property only of recombinant cell lines. Together, our data suggest rapid ETB receptor inactivation due to phosphorylation and delayed ETA receptor inactivation by internalization. These mechanisms adequately reflect the differential response patterns of the ET receptors under physiologic conditions.

Animals↗

PubMed1998

So this is what it's like: struggling to survive in pediatric intensive care.

This is the story of my daughter's first 3 months of life, a time when we both struggled to survive while she was all but subsumed by scientific technology. Part I describes some common and dangerous failures of relationship which occur in busy hospitals. Part II describes the feelings of hope that arose in me not from any improvement in my child's condition, but from the ability of a few people to step outside of technology and science and risk what the others could not--authentic and caring engagement.

Anecdotes as Topic↗

PubMed1997

Subtype-specific desensitization of human endothelin ETA and ETB receptors reflects differential receptor phosphorylation.

Endothelins regulate blood pressure in mammals through G protein-coupled receptors. Two receptor subtypes, ETA and ETB, exist which differ by their agonist profiles. Here we show subtype-specific differences in the inactivation of these endothelin receptors. Using a modified inositol phosphate accumulation assay, we found that stimulation of ETA by endothelin-1 results in sustained activation of the subtype, retaining >30% of its initial activity even 20 min after agonist administration, whereas the ETB rapidly deactivated after agonist stimulation, losing >80% of its initial activity within 5 min after endothelin application. The discrepancy in receptor inactivation is reflected by subtype-specific differences in receptor phosphorylation. Whereas ETA failed to undergo ligand-induced phosphorylation, the ETB was rapidly phosphorylated in response to agonist stimulation. By contrast, the kinetics of ligand-induced internalization were essentially identical for the receptor subtypes, suggesting endothelin receptor internalization being independent of ligand-induced receptor phosphorylation. Interestingly, a strong correlation was observed between the time course of ETA inactivation and ETA internalization. Therefore, our data suggest a subtype-specific inactivation of human endothelin receptors: fast receptor phosphorylation in the case of ETB and slow receptor internalization in the case of ETA. Subtype-specific modulation of endothelin receptors may account for the short-term hypotensive effects of endothelins via rapidly down-regulating ETB receptors and the long-lasting hypertensive effects due to sustained ETA activation.

Animals↗

PubMed1997

Cloning and functional characterization of the ornithokinin receptor. Recognition of the major kinin receptor antagonist, HOE140, as a full agonist.

Kinins are proinflammatory peptides that dilate vessels, increase vascular permeability, contract smooth muscles, and provoke pain. The known mammalian kinin receptors are classified as two subtypes, i.e. the B1 receptor triggered by [des-Arg9]bradykinin and inhibited by [des-Arg9,Leu8]bradykinin, and the B2 receptor stimulated by bradykinin and antagonized by HOE140. Here we report the cloning of a non-mammalian kinin receptor gene amplified from genomic chicken DNA. The protein predicted from the open reading frame shows 31 and 49% sequence identity to the human B1 and B2 receptors, respectively, suggesting that it represents a G protein-coupled receptor of the kinin receptor family. The recombinantly expressed chicken receptor had IC50 values of 4.7 nM for the authentic ligand, ornithokinin ([Thr6,Leu8]bradykinin), 3.8 nM for HOE140, and >/=10 microM for bradykinin, [des-Arg9]bradykinin, and [des-Arg9,Leu8]bradykinin. Ornithokinin and HOE140 at nanomolar concentrations stimulated intracellular inositol phosphate accumulation and induced a significant transient rise in intracelluar free Ca2+, whereas bradykinin was ineffective even at 100 nM. Hence the principal B2 receptor antagonist HOE140 is a potent agonist of the chicken kinin receptor. This unique pharmacological profile classifies the ornithokinin receptor as a novel subtype among kinin receptors and will facilitate further molecular studies on ligand binding and receptor activation.

Adrenergic beta-Antagonists↗

PubMed1997

Focus luminescence assay: macroscopically visualized foci of human cytomegalovirus and varicella zoster virus infection.

The plaques or foci of certain viruses due to their small size have to be counted microscopically, e.g., human cytomegalovirus (HCMV). The focus luminescence assay (FLA) described below generates macroscopic images as a result of the magnification due to scattered emitted light, and provides a hard copy using autoradiography or video imaging. Foci are detected according to an immunohistochemical protocol with horseradish peroxidase or alkaline phosphatase antibody conjugates which convert substrate into a luminescent product. Detection of varicella zoster virus (VZV) foci developed with a specific substrate-enhancer combination was so sensitive that 20-times lower primary antibody concentrations were effective than those required for conventional immunohistochemical staining. This method for HCMV and VZV may allow quantitative infectivity and focus reduction assays for viruses which produce little or no CPE.

Acyclovir↗

PubMed1997

Relationship between human genotype and phenotype of N-acetyltransferase (NAT2) as estimated by discriminant analysis and multiple linear regression: 1. Genotype and N-acetylation in vivo.

Twenty-six healthy Caucasian subjects were evaluated for polymorphic N-acetyltransferase (NAT2) metabolic activity in vivo by sulfamethazine phenotyping and for their respective NAT2 genotype. Application of discriminant analysis allowed the separation of the rapid and slow acetylators solely on the base of their respective mutation pattern with identical results as achieved by the classical method of discrimination according to the phenotyping results. Multiple linear regression analysis was used to obtain a quantitative relationship between allelic pattern and the phenotypic outcome. It is shown that the computation methods produce relationships enabling the influence of particular mutations and/or allelic configurations on the metabolic activity in vivo to be estimated. This may be important in cases of discordant or overlapping phenotype and genotype results as well as in investigating the NAT2 polymorphism as a risk factor for cancer and other disease in epidemiological studies.

Acetylation↗

PubMed1997

Different modes of inhibition by adamantane amine derivatives and natural polyamines of the functionally reconstituted influenza virus M2 proton channel protein.

The influenza virus M2 protein, target of the antiviral drugs amantadine and rimantadine, forms a proton channel which functions during virus uncoating and maturation by modifying the pH in virions as well as in trans-Golgi vesicles. We studied the influence of different ionic gradients on the inhibition of the proton translocation activity of isolated, baculovirus-expressed M2 protein reconstituted into liposomes. Two distinct patterns of inhibition were observed. A group of amphiphilic amines including amantadine, cyclooctylamine and rimantadine inhibited M2 effectively in the presence of physiological Na+ concentrations. The 10-fold greater activity of rimantadine over amantadine and the 100-fold stronger effect of cyclooctylamine compared to cyclopentylamine matched the relative activities in influenza virus-infected cells. A completely different inhibitory pattern emerged for the polyamines spermine, spermidine and putrescine. Polyamines have recently been identified as the 'intrinsic' rectifiers of a class of potassium channels and shown to interact with acidic amino acid residues lining and flanking the channel pore. In the presence of a physiological Na+/K+ gradient their minimal inhibitory concentrations for influenza virus M2 protein were 100, 400 and 500 microM, polyamine levels reported to exist in oocytes. In conditions depleted for Na+, polyamines inhibited M2 at concentrations two to three orders of magnitude lower. The data suggest that influenza virus M2 protein possesses a binding site for polyamines, distinct from the amantadine binding site, which is normally masked by Na+ and which could be targeted by selective antiviral inhibitors.

Adamantane↗

PubMed1997

The early experience of a voluntary small group insurance program utilizing managed care plans.

HealthCare Group of Arizona (HCGA), a state-sponsored, voluntary health insurance purchasing program offering prepaid health plans to small businesses, became operational in 1988. This article summarizes the results from a wide-ranging evaluation of that program and discusses their implications. In general, enrollees were satisfied with their experience in their plans. HCGA did not appear to attract an adverse mix of health risks, and service utilization rates were consistent with HMO industry averages. However, these findings varied across health plans and the marketing approaches they adopted. Enrollment growth in HCGA has been steady, but premium subsidies may be necessary if HCGA is to substantially increase its enrollment of low-wage, uninsured workers.

Adolescent↗

PubMed1996

Palmitoylation of endothelin receptor A. Differential modulation of signal transduction activity by post-translational modification.

Post-translational modifications such as phosphorylation and palmitoylation play important roles for the function and regulation of receptors coupled to heterotrimeric guanyl nucleotide-binding proteins. Here we demonstrate that the human endothelin receptor A (ETA) incorporates [3H]palmitate. Mutation of a cluster of five cysteine residues present in the cytoplasmic tail of ETA into serine or alanine residues completely prevented palmitoylation of the receptor. The ligand binding affinity of the non-palmitoylated ETA mutants was essentially unchanged as compared to the palmitoylated wild type ETA suggesting that the replacement of the cysteine residues did not alter the overall structure of the receptor. Furthermore, the ligand-induced stimulation of adenylyl cyclase by the mutant ETA was unaffected by the mutation. In contrast, the mutated non-palmitoylated receptors but not the wild type receptor failed to stimulate phosphatidylinositol hydrolysis by phospholipase C activation upon challenge by endothelin-1. Furthermore, the mutant receptors failed to stimulate the ligand-induced transient increase in the cytoplasmic calcium seen with the wild type ETA. Endothelin-1 induced mitogenic stimuli via the wild type receptors but not through the mutated receptors suggesting an important role for phospholipase C in this signal transduction pathway. The differential regulation of distinct signal transduction pathways by post-translational modification suggests that palmitoylation of the ETA provides a novel mechanism of modulating ETA receptor activity.

Amino Acid Sequence↗

PubMed1996

Income, health status, and insurance coverage of small group employees in a voluntary purchasing arrangement.

The ongoing health care reform discussion has highlighted the problems of insuring small group employees. Several state and private initiatives have attempted to address some of these problems through the formation of voluntary small group purchasing arrangements. This article uses data from one such initiative, Health Care Group of Arizona (HCGA), to describe the income, health status, and prior insurance of small group employees who enrolled in prepaid health plans through HCGA. It also compares employee enrollees to nonenrollees along these dimensions. The findings suggest that HCGA enrollees had relatively low incomes and that about three-quarters were without health insurance prior to enrollment. Higher income employee enrollees were more likely to report health conditions at enrollment even after controlling for other factors including age. Enrollees were less likely than nonenrollees to have prior health insurance but were more likely to be drawn from lower income groups and to report recent health conditions.

Adolescent↗

PubMed1995

Type III restriction endonucleases translocate DNA in a reaction driven by recognition site-specific ATP hydrolysis.

Type III restriction/modification systems recognize short non-palindromic sequences, only one strand of which can be methylated. Replication of type III-modified DNA produces completely unmethylated recognition sites which, according to classical mechanisms of restriction, should be signals for restriction. We have shown previously that suicidal restriction by the type III enzyme EcoP15I is prevented if all the unmodified sites are in the same orientation: restriction by EcoP15I requires a pair of unmethylated, inversely oriented recognition sites. We have now addressed the molecular mechanism of site orientation-specific DNA restriction. EcoP15I is demonstrated to possess an intrinsic ATPase activity, the potential driving force of DNA translocation. The ATPase activity is uniquely recognition site-specific, but EcoP15I-modified sites also support the reaction. EcoP15I DNA restriction patterns are shown to be predetermined by the enzyme-to-site ratio, in that site-saturating enzyme levels elicit cleavage exclusively between the closest pair of head-to-head oriented sites. DNA restriction is blocked by Lac repressor bound in the intervening sequence between the two EcoP15I sites. These results rule out DNA looping and strongly suggest that cleavage is triggered by the close proximity of two convergently tracking EcoP15I-DNA complexes.

Adenosine Triphosphatases↗

PubMed1995

Restriction endonucleases functionally interacting with two DNA sites.

Simultaneous interaction with two recognition sites was found to be a precondition for DNA cleavage by certain type-II and type-III restriction endonucleases. Nevertheless, the molecular mechanisms of the protein-DNA interaction are different between members of both classes of enzymes.

Binding Sites↗

PubMed1995

Hyperexpressed EcoRII renatured from inclusion bodies and native enzyme both exhibit essential cooperativity with two DNA sites.

EcoRII was the first restriction endonuclease (ENase) reported needing the cooperative interaction with at least two DNA sites for activity. We constructed an EcoRII-overproducing strain of Escherichia coli by placing the coding sequence under control of the T7 gene 10 regulatory elements. The yield of EcoRII expression could be increased to about 10% of total soluble cellular protein. Inclusion bodies are formed that mainly consist of insoluble EcoRII molecules. After solubilization by 6 M guanidine hydrochloride refolding of the enzyme was achieved by dilution into appropriate buffer. The endonuclease was purified to homogeneity from both the soluble protein fraction and the protein renatured from inclusion bodies. Their identity was proven by circular dichroism and analysis of enzyme activity with respect to the special substrate requirements of EcoRII. It is shown that EcoRII cleavage of oligodeoxyribonucleotide duplexes (oligo duplexes) with only one recognition site follows a sigmoidal concentration dependence, i.e., they cannot be cleaved below a distinct low DNA concentration where simultaneous interaction with two substrate molecules is no longer possible. We demonstrate that the restriction of oligo duplexes containing two recognition sites does not show this concentration dependence, confirming an intramolecular site cooperativity.

Base Sequence↗

PubMed1995

The significance of distance and orientation of restriction endonuclease recognition sites in viral DNA genomes.

Studies on phage T3 and T7 have shown that these viruses avoid restriction not only by the phage-coded Ocr (and S-adenosylmethionine hydrolase) protein functions or by the complete loss of specific recognition sites for certain restriction endonucleases from their genomes, but also that there are two additional modes: resistance towards EcoP15 (which recognizes a non-symmetrical sequence) is achieved by an identical orientation of all the recognition sites in the virus genome (strand bias) and in the case of EcoRII by the extreme reduction in number and thereby greater distance between recognition sites in the genome. These observations led to the discovery that certain restriction endonucleases require the simultaneous cooperation with two DNA sites for their function, as well as to the ongoing elucidation of the molecular modes of action of these enzymes. Type II and type III enzymes display fundamentally different mechanisms of protein-DNA interaction. For EcoRII we favor a model of simultaneous binding of two DNA sites to a dimeric enzyme molecule (neighbouring sites of the same, looping, DNA molecule or sites located on different DNA molecules), while the action of EcoP15 seems to conform with a tracking-collision model of two enzyme molecules bound to inversely oriented recognition sites. In addition to podoviruses T3 and T7, strand bias of recognition sequences for different type III DNA modification-restriction enzymes is also observed in the inoviruses M13, IKE and PF3.

Bacteriophage T3↗