PubMed Health⌕ Search

Biomedical subjects

J D Sutherland

Publications and source records attributed to J D Sutherland.

At least 19 recordsLinked to original sources
PubMed1997

Killing two birds with one stone: a chemically plausible scheme for linked nucleic acid replication and coded peptide synthesis.

To understand how life began, we must explain the origins of nucleic acid replication and genetically coded peptide synthesis. Neither of these is easy to explain individually; here, we propose a chemically plausible scheme for the evolution of a process that simultaneously produced both polymers. Later, two separate machineries could have evolved from the linked process.

DNA Replication↗
PubMed1996

The transcriptional factor CF2 is a mediator of EGF-R-activated dorsoventral patterning in Drosophila oogenesis.

Establishment of dorsoventral polarity during Drosophila oogenesis requires localized intercellular communication between the follicular cells and the oocyte. This is initiated by the transmission of a "dorsal signal" from the oocyte to the anterior dorsal follicle cells by the EGF receptor (EGF-R) pathway and is followed by transmission of a second signal from the ventral follicle cells back to the embryo. We show that the zinc finger transcription factor CF2 participates in these processes. CF2 is suppressed by EGF-R signaling in the anterior dorsal follicle cells. Altered expression patterns of CF2 result in specific dorsoventral patterning defects in egg chambers and in embryos, as demonstrated phenotypically and with molecular markers. CF2 appears to act as a repressor of dorsal follicle cell fates and specifically as a repressor of the rhomboid gene transcription.

Animals↗
PubMed1996

Technetium-99m-MAG3 renal studies in spinal cord injury patients: normal range, reproducibility, and change as a function of duration and level of injury.

UNLABELLED: The normal range, reproducibility, and change as a function of duration and level of injury for Tc-99m-MAG3 renal studies were quantitated in spinal cord injury (SCI) patients. METHODS: Five SCI patients without evidence of renal disease in each of four groups: less than 2 months, 2-12 months, 1-2 years, and greater than 2 years from time of injury, were each studied twice. There were at least two patients with paraplegia and two with tetraplegia in each group. Renal clearance (camera based method), percent function in each kidney, time of peak renal parenchymal activity, and half time of parenchymal activity following the peak were evaluated. The peak and half times were determined with regions of interest (ROIs) over the entire kidney and over just the cortex. All results were compared to normal ranges previously established in normal subjects of the same age range using the same methodology. RESULTS: Renal clearance in the less than 2 month SCI patients was not significantly different from normal subjects in either paraplegic or in tetraplegic individuals. However, clearance in tetraplegics was increased by 28.5% at 2-12 month, increased by 50.6% at 1-2 years, and decreased by 25.9% at greater than 2 years compared to normal subjects (all P < 0.02). Clearance in those with paraplegia showed a similar, but less marked, trend (P = NS). The time of peak parenchymal activity when measured with cortical ROIs did not vary among patient groups or level of injury, but was increased compared to normal subjects (P < 0.05). The percent function in each kidney and half time following the peak were symmetrical, did not differ among patient groups or with level of injury, and did not differ from normal subjects. The parenchymal peak time was significantly shorter with cortical rather than renal ROIs in all patient groups (P < 0.05). In serial studies in the same patient the percent standard deviation in total renal clearance was less than between single studies in different patients, but the decrease was significant for only the right kidney (P < 0.03), and the decrease was not as great as in normal subjects. In addition, the percent standard deviation for percent function in each kidney was significantly less than the percent standard deviations in single studies (P < 0.02). There were no significant differences between intra- and interpatient studies for any other parameter. CONCLUSION: We conclude that: (1) renal clearance measured with Tc-99m-MAG3 in tetraplegic patients increases significantly during the first 2 years following injury and decreases significantly thereafter; there is a similar, but much less marked, trend in paraplegics, (2) parenchymal peak times with cortical ROIs occur later for SCI patients than for normal subjects, and (3) there is more intrapatient variation in total renal clearance and percent renal clearance on a side in SCI patients than in normal subjects suggesting that it may be harder to study SCI patients reproducibly. These findings should be taken into account when performing and interpreting Tc-99m-MAG3 renal studies in SCI patients.

Adult↗
PubMed1995

Amino-acid substitutions in the cleavage site of acyl-coenzyme A:isopenicillin N acyltransferase from Penicillium chrysogenum: effect on proenzyme cleavage and activity.

Site-directed mutagenesis of the penDE gene and expression in Escherichia coli has produced recombinant acylcoenzyme A:isopenicillin N acyltransferase (re-AT) containing amino-acid substitutions in the proenzyme cleavage site (decreases) region (Asp-Gly102 decreases Cys103-Thr-Thr). The effect of these substitutions on proenzyme cleavage and AT activity has been investigated. The re-AT with substitutions at Cys103 (Cys103-->Ser, Cys103-->Ala and Cys103-->Trp) were uncleaved and inactive. Substitutions at Asp101 and Gly102 (Asp101-->Gly, Gly102-->Ala, Gly102-->Val, Gly102-->Met, Gly102-->Val and Asp101Gly102-->GlyPhe) did not prevent proenzyme cleavage or abolish AT activity. Thr105-->Ser and Thr105-->Ala substitutions did not prevent proenzyme cleavage or AT activity; however, AT containing Thr105-->Val resulted in a significant inhibition of proenzyme cleavage.

Acyltransferases↗
PubMed1995

Drosophila hormone receptor 38: a second partner for Drosophila USP suggests an unexpected role for nuclear receptors of the nerve growth factor-induced protein B type.

In Drosophila the response to the hormone ecdysone is mediated in part by Ultraspiracle (USP) and ecdysone receptor (EcR), which are members of the nuclear receptor superfamily. Heterodimers of these proteins bind to ecdysone response elements (EcREs) and ecdysone to modulate transcription. Herein we describe Drosophila hormone receptor 38 (DHR38) and Bombyx hormone receptor 38 (BHR38), two insect homologues of rat nerve growth factor-induced protein B (NGFI-B). Although members of the NGFI-B family are thought to function exclusively as monomers, we show that DHR38 and BHR38 in fact interact strongly with USP and that this interaction is evolutionarily conserved. DHR38 can compete in vitro against EcR for dimerization with USP and consequently disrupt EcR-USP binding to an EcRE. Moreover, transfection experiments in Schneider cells show that DHR38 can affect ecdysone-dependent transcription. This suggests that DHR38 plays a role in the ecdysone response and that more generally NGFI-B type receptors may be able to function as heterodimers with retinoid X receptor type receptors in regulating transcription.

Amino Acid Sequence↗
PubMed1995

A heuristic approach to the analysis of enzymic catalysis: reaction of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-alpha-aminobutyrate and delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-allylglycine catalyzed by isopenicillin N synthase isozymes.

Isopenicillin N synthase (IPNS) catalyzes the oxidative cyclization of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine to isopenicillin N. It is proposed that the multiple products produced from certain substrate analogues result from pathway branching after formation of a ferryl oxene intermediate. We have been interested in ascertaining the reasons for multiple product formation. One possibility is that the products are predisposed toward formation once the beta-lactam ring and the ferryl oxene are produced. Alternately, the products may be persuaded into being by the enzyme restricting conformations such that otherwise less favorable chemistry can take place. With the existing description of the IPNS catalytic cycle, this fundamental question has not been answerable. We describe here the application of a heuristic method to resolve this key issue. It was reasoned that by comparing the ratios of products formed by a set of perturbed IPNS variants it might be possible to generate qualitative information about the relative magnitude of certain activation parameters. If certain product ratios are affected but others are not, then it should be possible to say which steps in the reaction are dictated merely by chemical fundamentals and which steps are directly effected by the enzyme. In this paper we report the high-level expression, purification, and characterization of four IPNS isozymes. Comparison of the product ratios obtained on incubation of unnatural substrate analogues with four IPNS isozymes corresponding to perturbed active site variants shows substantial variation in some cases and little in others. Interpretation of the results obtained with delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-alpha-aminobutyrate (ACAB) allows conclusions to be drawn regarding the role of the enzyme in restricting available conformations of the natural substrate to disfavor certain otherwise chemically favorable pathways and hence products. The results obtained with delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-allylglycine, while rather more complex, substantiate the conclusions drawn from the ACAB data. A major conclusion is that, in the oxidation of ACV, IPNS is a negative catalyst of cepham formation but a positive catalyst of penam formation.

Acremonium↗
PubMed1994

Acyl-coenzyme A: isopenicillin N acyltransferase from Penicillium chrysogenum: effect of amino acid substitutions at Ser227, Ser230 and Ser309 on proenzyme cleavage and activity.

Using a high level Escherichia coli expression system for the Penicillium chrysogenum penDE gene, we have produced acyl-coenzyme A: isopenicillin N acyltransferase (AT) enzymes containing amino acid substitutions at three conserved Ser residues. Chosen for study based on amino acid sequence homologies to other proteins, Ser227, Ser230 and Ser309 were changed to Cys or Ala to assess amino acid side chain involvement in proenzyme cleavage and AT enzyme mechanism. Substitutions at Ser230 had no effect on proenzyme cleavage, acyl-coenzyme A: IPN acyltransferase (IAT) or acyl-coenzyme A:6-aminopenicillanic acid acyltransferase (AAT) activities. While Ser227-->Cys had no effect, Ser227-->Ala produced uncleaved proenzyme lacking both AAT and IAT activities, suggesting that the presence of a nucleophilic side chain at this residue is required for proenzyme cleavage and AT activity. Substitution of Ser309-->Cys did not appreciably prevent proenzyme cleavage, IAT or AAT activity. Recombinant AT (recAT) proenzyme containing Ser309-->Ala was cleaved; however, IAT and AAT activities were not observed. This separation of proenzyme cleavage from IAT and AAT activities has not been previously observed, and suggests that Ser309 is involved in substrate acylation.

Acyltransferases↗
PubMed1993

The requirement for subunit interaction in the production of Penicillium chrysogenum acyl-coenzyme A:isopenicillin N acyltransferase in Escherichia coli.

Subunit interaction in the formation of active acyl-coenzyme A:isopenicillin N acyltransferase (AT) has been investigated. Various AT derivatives were produced from altered Penicillium chrysogenum penDE genes placed in Escherichia coli expression systems. The regions of penDE encoding the alpha (11 kDa) and beta (29 kDa) AT subunits were separated at the DNA level by linker insertion at the region encoding Gly102/Cys103. Synthesis of AT from the resulting two-cistron mRNA resulted in active alpha,beta-heterodimeric recombinant AT (reAT), containing subunits of 11 and 29 kDa (similar to wild-type AT). Complete separation of the alpha and beta subunits was performed by placing the region of penDE encoding each subunit on different plasmids. Production of either subunit in the absence of the other did not form active reAT. However, cotransformation of E. coli with two plasmids, each encoding a different AT subunit, produced reAT having acyl-coenzyme A:6-aminopenicillanic acid (acyl-CoA:6-APA) AT activity. Mutation of penDE replacing Thr105 with Asn resulted in inactive and uncleaved reAT. Coexpression of this mutant penDE with a penDE derivative encoding the beta subunit in E. coli produced acyl-CoA:6-APA AT activity. These results suggest that the formation of reAT involves cooperative folding events between the subunits. In vitro transcription/translation was used to determine the origin of the AT hydrolase activity that cleaves the 40-kDa precursor polypeptide. The appearance of a 29-kDa protein (and presumably the corresponding 11-kDa protein, although not observable) from the 40-kDa in vitro translated protein provides further evidence that AT hydrolysis is an autocatalytic event.

Acyl Coenzyme A↗
PubMed1993

On the production of alpha, beta-heterodimeric acyl-coenzyme A: isopenicillin N-acyltransferase of Penicillium chrysogenum. Studies using a recombinant source.

A high level E. coli expression system has been constructed for the Penicillium chrysogenum penDE gene, which encodes the acyl-coenzyme A: isopenicillin N-acyltransferase (AT) enzyme. Induction of overexpression of recombinant AT (recAT) by increasing the growth temperature of the host adversely affected solubility and activity of the AT enzyme. Addition of isopropylthio-beta-D-galactopyranoside (IPTG) at decreased growth temperatures (less than 32 degrees C) resulted in the overproduction of soluble, active recAT. When purified to homogeneity, recAT was an alpha, beta-heterodimer, comprised of 11 kDa (alpha) and 29 kDa (beta) subunits, derived from a 40 kDa precursor polypeptide by a posttranslational cleavage. The recAT enzyme contained both the acyl-coenzyme A: isopenicillin N-acyltransferase and the acyl-coenzyme A: 6-aminopenicillanic acid acyltransferase activities. The processing event that generated the two subunits of recAT from the 40 kDa precursor polypeptide occurred between Gly102/Cys103. This expression system produced a large amount of soluble, active recAT that is identical to native AT, making it a suitable source of AT enzyme for further characterization.

Acyltransferases↗
PubMed1993

Detection of jeopardized myocardium with Tl-201 myocardial perfusion imaging. Comparison of early and late reinjection protocols.

Reinjection of a second dose of Tl-201 before redistribution imaging has significantly improved the ability of myocardial perfusion imaging to detect jeopardized myocardium. However, PET studies and the results from coronary artery angioplasty and bypass surgery indicate that Tl-201 studies still underestimate the number of ischemic areas at stress that represent viable myocardium. To determine whether an imaging protocol using an early reinjection of Tl-201 immediately after acquisition of the stress images would improve the detection of jeopardized myocardium, 138 patients were studied with an early reinjection protocol and the incidence and magnitude of reversible defects were compared by SPECT to a second group of 143 patients who underwent the standard late reinjection protocol. Two observers independently evaluated 19 standard myocardial segments by visual examination in each patient. They determined the presence or absence of reversible defects, and, when present, their magnitude. The frequency of reversible defects always was less with early reinjection compared to the standard late reinjection; the difference reached statistical significance for one of the two observers (P < 0.05). It is concluded that early reinjection of Tl-201 in myocardial perfusion imaging does not increase and may decrease the sensitivity of the study for jeopardized myocardium.

Adenosine↗
PubMed1993

The autonomous self.

Although the nature of the self has become a central issue in psychoanalysis, a comprehensive theory of its origin and development is needed. In an effort to elaborate one such theory, the author first reviews the history of psychoanalytic conceptualizations of the self. He then examines extra-clinical evidence from literature, philosophy, and the social sciences to gain additional insight into the persistent features of the self. Finally, he outlines some unique characteristics of the human self in light of new insights from modern evolutionary biology.

Biological Evolution↗
PubMed1991

Site-specific forced misincorporation mutagenesis using modified T7 DNA polymerase.

A new method for forced misincorporation site-specific mutagenesis is described. The method uses an exonuclease-deficient modified version of T7 DNA polymerase in the presence of one dNTP to force a misincorporation. Analysis by PAGE is used to monitor the efficiency of such misincorporation reactions. Brief extension of the terminally mismatched primer/template using the same enzyme in the presence of all four dNTPs is followed by chase/ligation using unmodified T7 DNA polymerase and T4 DNA ligase to give heteroduplex DNA. We have applied the method to mutagenesis of the Lac Z region of M13 and found that, using strand selection, efficiencies of mutagenesis at one site are greater than 50%. When the mutating dNTP is complementary to a neighbouring homopolymeric tract on the template, multiple mutation is observed and efficiences are lower. The method is more general than internal mismatch mutagenesis and, because of its rapidity, is more expedient than existing methods of forced misincorporation mutagenesis.

Amino Acid Sequence↗
PubMed1991

Evaluation of anionic histological dyes as co-injectable cell markers in pre-implantation mouse embryos.

The enzyme horseradish peroxidase (HRP) is a widely used microinjectable cell marker for studying cell position, lineage, and migration in many kinds of animal embryos. Marked cells are easily identified because they darken when exposed to a chromophore and an HRP substrate such as hydrogen peroxide. This assay, however, requires cytochemical fixation. Thus, when HRP-marked cells need to be identified prior to fixation, visible co-injectants such as dyes and fluorescent substances have been used with HRP. Fluorescent substances have limitations because their excitation could be harmful to the marked cells. Visible but non-fluorescent co-injectants, however, would permit visualization of HRP-marked cells without inflicting such damage. We tested the compatibility of several histological dyes and electrolytic carriers with HRP iontophoresed as a cell marker in 2-cell mouse embryos. The dyes tested were Evans Blue, Cibacron Blue F3GA, Fast Green FCF, and Patent Blue Violet; the electrolytic carriers were KCl, K2SO4, CH3CO2K, and KH2PO4. The combination found most useful was Patent Blue Violet in K2SO4. Survival of embryos incubated to the blastocyst stage following injection with HRP + Patent Blue Violet in K2SO4 at the 2-cell stage was significantly greater than that of embryos injected with any other dye. Although the proportion of embryos undergoing the 8-cell-to-morula transition was somewhat decreased by this treatment, the proportion of embryos reaching the blastocyst stage was comparable to that in the uninjected (control) group. Our results indicate that Patent Blue Violet is a useful, HRP-co-injectable dye for short-term cell marking in preimplantation mouse embryos.

Animals↗
PubMed1990

Acyl coenzyme A: 6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum and Aspergillus nidulans.

A study of the final stages of the biosynthesis of the penicillins in Penicillium chrysogenum has revealed two types of enzyme. One hydrolyses phenoxymethyl penicillin to 6-aminopenicillanic acid (6-APA). The other, also obtained from Aspergillus nidulans, transfers a phenylacetyl group from phenylacetyl CoA to 6-APA. The acyltransferase, purified to apparent homogeneity, had a molecular mass of 40 kDa. It also catalyses the conversion of isopenicillin N (IPN) to benzylpenicillin (Pen G) and hydrolyses IPN to 6-APA. In the presence of SDS it dissociates, with loss of activity, into fragments of ca 30 and 10.5 kDa, but activity is regained when these fragments recombine in the absence of SDS.

Acyl Coenzyme A↗
PubMed1990

High level expression in Escherichia coli of a fungal gene under the control of strong promoters.

A recent report (Patino et al., (1989) FEMS Microbiol. Lett. 58, 139-144) described the low level expression, in Escherichia coli, of the Isopenicillin N Synthase (IPNS) gene from Cephalosporium acremonium under the control of strong promoters. We report here our work on the expression of the IPNS gene. Plasmids containing the IPNS gene under the control of the trp or trc promoters directed synthesis of high levels of active IPNS in E. coli. Constitutive and inductive high level IPNS expression systems have been developed. Importantly, the expression vectors do not encode beta-lactamase so IPNS activity can be determined directly by biological assays. Analysis by nmr verified that the IPNS produced from these expression systems catalysed the conversion of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (LLD-ACV) to isopenicillin N in high yield.

Acremonium↗
PubMed1988

Lyophilized Clostridium perfringens 3 alpha- and Clostridium bifermentans 7 alpha-hydroxysteroid dehydrogenases: two new stable enzyme preparations for routine bile acid analysis.

Preparations of 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) from Clostridium perfringens were successfully lyophilized into a stable powder form. Purification of the enzyme was achieved using triazine dye affinity chromatography. C. perfringens 3 alpha-hydroxysteroid dehydrogenase was purified 24-fold using Reactive Red 120 (Procion Red) -cross-linked agarose (70% yield). Quantitative measurement of bile acids with the purified enzymes, 3 alpha-hydroxysteroid dehydrogenase and 7 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.159) from Clostridium bifermentans (strain F-6), was achieved spectrophotometrically. Standard curves with chenodeoxycholic acid (CDC) and cholic acid were linear within a concentration range of 20-100 microM. Analysis of mixtures of ursodeoxycholic acid and CDC showed the additive nature of the 3 alpha-hydroxysteroid dehydrogenase and showed also that 7 alpha-hydroxyl groups were independently quantified by the 7 alpha-hydroxysteroid dehydrogenase. Bile acids in Folch extracts of human bile samples were measured using purified preparations of Pseudomonas testosteroni 3 alpha-hydroxysteroid dehydrogenase, C. perfringens 3 alpha-hydroxysteroid dehydrogenase, Escherichia coli 7 alpha-hydroxysteroid dehydrogenase and C. bifermentans (strain F-6) 7 alpha-hydroxysteroid dehydrogenase. Statistical comparison validated the use of C. perfringens 3 alpha- and C. bifermentans 7 alpha-hydroxysteroid dehydrogenases for the quantification of bile acids in bile.

3-Hydroxysteroid Dehydrogenases↗
PubMed1987

Oxidation of primary bile acids by a 7 alpha-hydroxysteroid dehydrogenase elaborating Clostridium bifermentans soil isolate.

A gram-positive, rod-shaped anaerobe (strain F-6) was isolated from soil. This organism was identified by cellular morphology as well as fermentative and biochemical data as Clostridium bifermentans. Strain F-6 formed 7-ketolithocholic acid from chenodeoxycholic acid and 7-ketodeoxycholic acid from cholic acid in whole cell cultures, but did not transform deoxycholic acid, ursodeoxycholic acid, or ursocholic acid. This reaction is reversible. The structures of 7-ketolithocholic acid and 7-ketodeoxycholic acid were verified by mass spectroscopy and by thin-layer chromatography using Komarowsky's spray reagent. When incubated with the strain F-6 glycine and taurine conjugates of the primary bile acids were partially hydrolyzed and transformed to 7-keto products. Optimal yields of 7-ketolithocholic acid and 7-ketodeoxycholic acid were obtained after 78 h of incubation. Culture pH changed with time and was characterized by an initial drop (1.1 pH units) and a gradual increase back to the starting pH (7.3). Corroborating these observations, an inducible, NADP-dependent, 7 alpha-hydroxysteroid dehydrogenase was demonstrated in cell extracts of strain F-6. A trace of NAD-dependent 7 alpha-hydroxysteroid dehydrogenase was also found. A substantial increase in the specific activity of the NADP-dependent 7 alpha-hydroxysteroid dehydrogenase was observed when either 7-ketolithocholic acid, chenodeoxycholic acid, or deoxycholic acid was included in the growth medium. Optimal induction of the NADP-dependent 7 alpha-hydroxysteroid dehydrogenase was achieved with 0.3-0.4 mM 7-ketolithocholic acid. Production of the enzyme(s) was optimal at 6-8 h of growth and the 7 alpha-hydroxysteroid dehydrogenases had a pH optimum of approximately 11.(ABSTRACT TRUNCATED AT 250 WORDS)

Bile Acids and Salts↗