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Biomedical subjects

David J Adams

Publications and source records attributed to David J Adams.

72 records · Page 4Linked to original sources
PubMed2003

Synthesis, structure elucidation, in vitro biological activity, toxicity, and Caco-2 cell permeability of lipophilic analogues of alpha-conotoxin MII.

The alpha-conotoxin MII is a two disulfide bridge containing, 16 amino acid long peptide toxin isolated from the marine snail Conus magus. This toxin has been found to be a highly selective and potent inhibitor of neuronal nicotinic acetylcholine receptors (nAChRs) of the subtype alpha3beta2. To improve the bioavailability of this peptide, two lipidic analogues of MII have been synthesized, the first by coupling 2-amino-d,l-dodecanoic acid (Laa) to the N terminus (LaaMII) and the second by replacing Asn5 in the MII sequence with this lipoamino acid (5LaaMII). Both lipidic linear peptides were then oxidized under standard conditions. (1)H NMR shift analysis of these peptides and comparison with the native MII peptide showed that the tertiary structure of the N-conjugated analogue, LaaMII, was consistent with that of the native conotoxin, whereas the 5LaaMII analogue formed the correct disulfide bridges but failed to adopt the native helical tertiary structure. The N terminus conjugate was also found to inhibit nAChRs of the subtype alpha3beta2 with equal potency to the parent peptide, whereas the 5LaaMII analogue showed no inhibitory activity. The active LaaMII analogue was found to exhibit significantly improved permeability across Caco-2 cell monolayers compared to the native MII, and both peptides showed negligible toxicity.

Amino Acid Sequence↗
PubMed2003

The structure of the zinc finger domain from human splicing factor ZNF265 fold.

Identification of the protein domains that are responsible for RNA recognition has lagged behind the characterization of protein-DNA interactions. However, it is now becoming clear that a range of structural motifs bind to RNA and their structures and molecular mechanisms of action are beginning to be elucidated. In this report, we have expressed and purified one of the two putative RNA-binding domains from ZNF265, a protein that has been shown to bind to the spliceosomal components U1-70K and U2AF35 and to direct alternative splicing. We show that this domain, which contains four highly conserved cysteine residues, forms a stable, monomeric structure upon the addition of 1 molar eq of Zn(II). Determination of the solution structure of this domain reveals a conformation comprising two stacked beta-hairpins oriented at approximately 80 degrees to each other and sandwiching the zinc ion; the fold resembles the zinc ribbon class of zinc-binding domains, although with one less beta-strand than most members of the class. Analysis of the structure reveals a striking resemblance to known RNA-binding motifs in terms of the distribution of key surface residues responsible for making RNA contacts, despite a complete lack of structural homology. Furthermore, we have used an RNA gel shift assay to demonstrate that a single crossed finger domain from ZNF265 is capable of binding to an RNA message. Taken together, these results define a new RNA-binding motif and should provide insight into the functions of the >100 uncharacterized proteins in the sequence data bases that contain this domain.

Amino Acid Sequence↗
PubMed2003

Callosal neglect.

BACKGROUND: According to the interhemispheric inhibition model of neglect, the uninjured hemisphere inhibits (via the corpus callosum) the injured hemisphere but the injured hemisphere can no longer inhibit the opposite hemisphere, which becomes hyperactive and produces an ipsilesional attentional bias. Alternatively, according to the compensation hypothesis, the uninjured hemisphere helps compensate for the damaged hemisphere, which is impaired in directing attention to contralateral stimuli. If the inhibition model of neglect is correct, callosal disconnection should reduce neglect. If the compensation model is correct, however, it may increase or induce neglect. PATIENT: A 32-year-old woman, at age 14 years, developed a right frontal astrocytoma and was treated with surgery and radiation but had a cardiopulmonary arrest secondary to aspiration. Subsequent imaging studies revealed damage to the frontal, parietal, and occipital regions of the right hemisphere and damage to the temporal region of the left hemisphere. After discharge, she was able to return to school and drive a car, without any evidence of neglect. About 10 years later, she developed complex partial and atonic seizures that were multifocal and medically intractable. She underwent a complete section of her corpus callosum at age 31 years. RESULTS: One year after the callosal section, she demonstrated (1) diminished spontaneous saccades to the left, hypometric leftward saccades, and left gaze impersistence; (2) left arm hemispatial limb akinesia; (3) unilateral spatial neglect; and (4) motor and cognitive impersistence. CONCLUSION: In patients with right hemisphere injury, callosal section may induce or enhance motor-intentional deficits and hemispatial neglect.

Adult↗
PubMed2003

Disruption of the gene encoding the ChiB1 chitinase of Aspergillus fumigatus and characterization of a recombinant gene product.

The gene encoding a major, inducible 45 kDa chitinase of Aspergillus fumigatus was cloned and analysis of the deduced amino acid sequence identified a chitinase of the fungal/bacterial class which was designated ChiB1. Recombinant ChiB1, expressed in Pichia pastoris, was shown to function by a retaining mechanism of action. That is, the beta-conformation of the chitin substrate linkage was preserved in the product in a manner typical of family 18 chitinases. Cleavage patterns with the N-acetylglucosamine (GlcNAc) oligosaccharide substrates GlcNAc(4), GlcNAc(5) and GlcNAc(6) indicated that the predominant reaction involved hydrolysis of GlcNAc(2) from the non-reducing end of each substrate. Products of transglycosylation were also identified in each incubation. Following disruption of chiB1 by gene replacement, growth and morphology of disruptants and of the wild-type strain were essentially identical. However, during the autolytic phase of batch cultures the level of chitinase activity in culture filtrate from a disruptant was much lower than the activity from the wild-type. The search for chitinases with morphogenetic roles in filamentous fungi should perhaps focus on chitinases of the fungal/plant class although such an investigation will be complicated by the identification of at least 11 putative active site domains for family 18 chitinases in the A. fumigatus TIGR database (http://www.tigr.org/).

Aspergillus fumigatus↗
PubMed2003

Phase I evaluation of prolonged-infusion gemcitabine with fludarabine for relapsed or refractory acute myelogenous leukemia.

PURPOSE: The purpose of this study was to determine the maximum tolerated duration of infusion of gemcitabine at 10 mg/m(2)/min in combination with fludarabine at 25 mg/m(2) daily for 5 days in the treatment of relapsed or refractory acute myelogenous leukemia. EXPERIMENTAL DESIGN: Eighteen patients with relapsed or refractory acute myelogenous leukemia were enrolled. The median age was 54.5 years (range, 21-80 years). Patients received a 30-min infusion of fludarabine at 25 mg/m(2) daily for 5 days. i.v. gemcitabine was given as a single infusion at 10 mg/m(2)/min with the duration adjusted following a modified continuous reassessment method. RESULTS: After 18 patients, the maximum recommended duration of infusion of gemcitabine in combination with fludarabine was selected as a 15-h infusion given at 10 mg/m(2)/min (9,000 mg/m(2)). Severe stomatitis or esophagitis was the most common nonhematological dose-limiting toxicity. Myelosuppression was universal. Febrile neutropenia was common, and 3 of 18 (17%) patients developed bacteremia. Occasional nausea, vomiting, or diarrhea was also reported. There were three complete responses and two partial responses for an overall response rate of 28%. CONCLUSIONS: Prolonged-infusion gemcitabine at a fixed dose rate of 10 mg/m(2)/min for 15 h with 25 mg/m(2)/day fludarabine for 5 days is a tolerable induction regimen for relapsed or refractory leukemia. Stomatitis, esophagitis, febrile neutropenia, and myelosuppression should be anticipated; however, this regimen may be beneficial in patients with relapsed or refractory leukemia.

Adult↗
PubMed2002

Tools for targeted manipulation of the mouse genome.

In the postgenomic era the mouse will be central to the challenge of ascribing a function to the 40,000 or so genes that constitute our genome. In this review, we summarize some of the classic and modern approaches that have fueled the recent dramatic explosion in mouse genetics. Together with the sequencing of the mouse genome, these tools will have a profound effect on our ability to generate new and more accurate mouse models and thus provide a powerful insight into the function of human genes during the processes of both normal development and disease.

Animals↗
PubMed2002

Omega-conotoxin CVID inhibits a pharmacologically distinct voltage-sensitive calcium channel associated with transmitter release from preganglionic nerve terminals.

Neurotransmitter release from preganglionic parasympathetic neurons is resistant to inhibition by selective antagonists of L-, N-, P/Q-, R-, and T-type calcium channels. In this study, the effects of different omega-conotoxins from genus Conus were investigated on current flow-through cloned voltage-sensitive calcium channels expressed in Xenopus oocytes and nerve-evoked transmitter release from the intact preganglionic cholinergic nerves innervating the rat submandibular ganglia. Our results indicate that omega-conotoxin CVID from Conus catus inhibits a pharmacologically distinct voltage-sensitive calcium channel involved in neurotransmitter release, whereas omega-conotoxin MVIIA had no effect. omega-Conotoxin CVID and MVIIA inhibited depolarization-activated Ba(2+) currents recorded from oocytes expressing N-type but not L- or R-type calcium channels. High affinity inhibition of the CVID-sensitive calcium channel was enhanced when position 10 of the omega-conotoxin was occupied by the smaller residue lysine as found in CVID instead of an arginine as found in MVIIA. Given that relatively small differences in the sequence of the N-type calcium channel alpha(1B) subunit can influence omega-conotoxin access (Feng, Z. P., Hamid, J., Doering, C., Bosey, G. M., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 15728-15735), it is likely that the calcium channel in preganglionic nerve terminals targeted by CVID is a N-type (Ca(v)2.2) calcium channel variant.

Amino Acid Sequence↗
PubMed2002

Isolation, structure, and activity of GID, a novel alpha 4/7-conotoxin with an extended N-terminal sequence.

Using assay-directed fractionation of Conus geographus crude venom, we isolated alpha-conotoxin GID, which acts selectively at neuronal nicotinic acetylcholine receptors (nAChRs). Unlike other neuronally selective alpha-conotoxins, alpha-GID has a four amino acid N-terminal tail, gamma-carboxyglutamate (Gla), and hydroxyproline (O) residues, and lacks an amidated C terminus. GID inhibits alpha 7 and alpha 3 beta 2 nAChRs with IC(50) values of 5 and 3 nm, respectively and is at least 1000-fold less potent at the alpha 1 beta 1 gamma delta, alpha 3 beta 4, and alpha 4 beta 4 combinations. GID also potently inhibits the alpha 4 beta 2 subtype (IC(50) of 150 nm). Deletion of the N-terminal sequence (GID Delta 1-4) significantly decreased activity at the alpha 4 beta 2 nAChR but hardly affected potency at alpha 3 beta 2 and alpha 7 nAChRs, despite enhancing the off-rates at these receptors. In contrast, Arg(12) contributed to alpha 4 beta 2 and alpha 7 activity but not to alpha 3 beta 2 activity. The three-dimensional structure of GID is well defined over residues 4-19 with a similar motif to other alpha-conotoxins. However, despite its influence on activity, the tail appears to be disordered in solution. Comparison of GID with other alpha 4/7-conotoxins which possess an NN(P/O) motif in loop II, revealed a correlation between increasing length of the aliphatic side-chain in position 10 (equivalent to 13 in GID) and greater alpha 7 versus alpha 3 beta 2 selectivity.

Amino Acid Sequence↗
PubMed2002

A new level of conotoxin diversity, a non-native disulfide bond connectivity in alpha-conotoxin AuIB reduces structural definition but increases biological activity.

alpha-Conotoxin AuIB and a disulfide bond variant of AuIB have been synthesized to determine the role of disulfide bond connectivity on structure and activity. Both of these peptides contain the 15 amino acid sequence GCCSYPPCFATNPDC, with the globular (native) isomer having the disulfide connectivity Cys(2-8 and 3-15) and the ribbon isomer having the disulfide connectivity Cys(2-15 and 3-8). The solution structures of the peptides were determined by NMR spectroscopy, and their ability to block the nicotinic acetylcholine receptors on dissociated neurons of the rat parasympathetic ganglia was examined. The ribbon disulfide isomer, although having a less well defined structure, is surprisingly found to have approximately 10 times greater potency than the native peptide. To our knowledge this is the first demonstration of a non-native disulfide bond isomer of a conotoxin exhibiting greater biological activity than the native isomer.

Amino Acid Sequence↗
PubMed2002

Phase I evaluation of prolonged-infusion gemcitabine with irinotecan for relapsed or refractory leukemia or lymphoma.

PURPOSE: To estimate the maximum-tolerated duration of infusion of gemcitabine at 10 mg/m(2)/min in combination with irinotecan at 40 mg/m(2) daily for 3 days in the treatment of relapsed or refractory acute leukemia or lymphoma. PATIENTS AND METHODS: Patients with leukemia or lymphoma were escalated in separate strata. Stratum I consisted of 11 patients, median age of 47 years (range, 18 to 68 years), with relapsed or refractory leukemia. Stratum II contained nine patients, median age of 48 years (range, 39 to 68 years), who had refractory non-Hodgkin's lymphoma. Patients received irinotecan at 40 mg/m(2) daily for 3 days, beginning just before the first dose of gemcitabine. Gemcitabine was given at 10 mg/m(2)/min, with the total duration adjusted following a modified continuous reassessment model. RESULTS: Severe myelosuppression and stomatitis/esophagitis were the most serious hematologic and nonhematologic toxicities. Several patients developed febrile neutropenia, nausea, or vomiting. In both strata, the maximum recommended duration of infusion of gemcitabine was 12 hours delivered at 10 mg/m(2)/min (7,200 mg/m(2)). The overall response rate for one cycle of this therapy in this phase I trial for patients with leukemia was 18% (95% confidence interval, 8% to 45%), and for those with lymphoma, 33% (95% confidence interval, 17% to 66%). CONCLUSION: A prolonged infusion of gemcitabine at 10 mg/m(2)/min for 12 hours with 3 days of irinotecan at 40 mg/m(2)/d is a tolerable induction regimen for patients with acute leukemia or lymphoma. Stomatitis/esophagitis should be anticipated; however, this regimen may induce responses in patients with difficult-to-treat hematologic malignancies.

Adult↗
PubMed2002

Solution structure of mu-conotoxin PIIIA, a preferential inhibitor of persistent tetrodotoxin-sensitive sodium channels.

Mu-conotoxins are peptide inhibitors of voltage-sensitive sodium channels (VSSCs). Synthetic forms of mu-conotoxins PIIIA and PIIIA-(2-22) were found to inhibit tetrodotoxin (TTX)-sensitive VSSC current but had little effect on TTX-resistant VSSC current in sensory ganglion neurons. In rat brain neurons, these peptides preferentially inhibited the persistent over the transient VSSC current. Radioligand binding assays revealed that PIIIA, PIIIA-(2-22), and mu-conotoxins GIIIB discriminated among TTX-sensitive VSSCs in rat brain, that these and GIIIC discriminated among the corresponding VSSCs in human brain, and GIIIA had low affinity for neuronal VSSCs. (1)H NMR studies found that PIIIA adopts two conformations in solution due to cis/trans isomerization at hydroxyproline 8. The major trans conformation results in a three-dimensional structure that is significantly different from the previously identified conformation of mu-conotoxins GIIIA and GIIIB that selectively target TTX-sensitive muscle VSSCs. Comparison of the structures and activity of PIIIA to muscle-selective mu-conotoxins provides an insight into the structural requirements for inhibition of different TTX-sensitive sodium channels by mu-conotoxins.

Amino Acid Sequence↗
PubMed2002

Human non-pregnancy ribonuclease with anti-Kaposi's sarcoma activity.

Our demonstration of a 19kDa anti-Kaposi's sarcoma (KS) ribonuclease (RNase) in urine from a non-pregnant female may provide at least part of the explanation for the low incidence of KS in human females. N-terminal sequence analysis and isoelectric focusing of the purified RNase, coupled with the very low levels of anti-KS activity noted for recombinant forms of human eosinophil derived neurotoxin and human eosinophil cationic protein, suggest that the 19kDa enzyme is an eosinophilic protein whose potent anti-KS activity is dependent upon post-translational modifications that occur only in human cells.

Adenocarcinoma↗
PubMed2002

Phase I evaluation of prolonged-infusion gemcitabine with mitoxantrone for relapsed or refractory acute leukemia.

PURPOSE: To ascertain the maximum tolerated duration of infusion of gemcitabine at 10 mg/m(2)/min in combination with mitoxantrone at 12 mg/m(2) daily for 3 days in the treatment of acute leukemia. PATIENTS AND METHODS: Thirty-four patients were enrolled. Stratum I consisted of 26 patients, median age 50 years (range, 25 to 71 years), with relapsed or refractory leukemia. Stratum II contained eight patients, median age 62.5 years (range, 38 to 83 years), who had received fewer than three cycles of myelotoxic therapy for chronic myeloid leukemia or myelodysplasia that had evolved into leukemia. Patients received mitoxantrone at 12 mg/m(2) daily for 3 days. After the first mitoxantrone dose, gemcitabine was provided intravenously at 10 mg/m(2)/min with the duration adjusted by following a continuous reassessment model. RESULTS: Severe myelosuppression, and stomatitis or esophagitis were the most common hematologic and nonhematologic dose-limiting toxicities. Several patients developed febrile neutropenia, nausea, or vomiting. In both strata, the maximum recommended duration of infusion of gemcitabine was 12 hours (7,200 mg/m(2)). The mean steady-state concentration of gemcitabine was 24.72 micromol/L and varied over a fivefold range among patients. Overall response rates in this phase I trial for strata I and II were 42% and 63%, respectively. CONCLUSION: Prolonged-infusion gemcitabine at a fixed dose rate of 10 mg/m(2)/min for 12 hours with 12 mg/m(2)/d mitoxantrone for 3 days is a tolerable induction regimen and achieves plasma concentrations sufficient for maximal intracellular activation. Stomatitis or esophagitis should be anticipated; however, this regimen may induce significant responses in patients with difficult-to-treat leukemias.

Acute Disease↗
PubMed2002

Basal nonselective cation permeability in rat cardiac microvascular endothelial cells.

The presence of a basal nonselective cation permeability was mainly investigated in primary cultures of rat cardiac microvascular endothelial cells (CMEC) by applying both the patch-clamp technique and Fura-2 microfluorimetry. With low EGTA in the pipette solution, the resting membrane potential of CMEC was -21.2 +/- 1.1 mV, and a Ca(2+)-activated Cl(-) conductance was present. When the intracellular Ca(2+) was buffered with high EGTA, the membrane potential decreased to 5.5 +/- 1.2 mV. In this condition, full or partial substitution of external Na(+) by NMDG(+) proportionally reduced the inward component of the basal I-V relationship. This current was dependent on extracellular monovalent cations with a permeability sequence of K(+) > Cs(+) > Na(+) > Li(+) and was inhibited by Ca(2+), La(3+), Gd(3+), and amiloride. The K(+)/Na(+) permeability ratio, determined using the Goldman-Hodgkin-Katz equation, was 2.01. The outward component of the basal I-V relationship was reduced when intracellular K(+) was replaced by NMDG(+), but was not sensitive to substitution by Cs(+). Finally, microfluorimetric experiments indicated the existence of a basal Ca(2+) entry pathway, inhibited by La(3+) and Gd(3+). The basal nonselective cation permeability in CMEC could be involved both in the control of myocardial ionic homeostasis, according to the model of the blood-heart barrier, and in the modulation of Ca(2+)-dependent processes.

Animals↗
PubMed2001

Association of a functional inducible nitric oxide synthase promoter variant with complications in type 2 diabetes.

Complications of diabetes have a genetic influence. Since increased inducible nitric oxide synthase (iNOS) gene ( NOS2A) expression can contribute to tissue damage, NOS2A is a worthy candidate for such a role. We therefore tested a 4-bp insertion/deletion (+/-) polymorphism 0.7 kb upstream of NOS2A for association with complications in type 2 diabetes patients, and also performed transient transfection experiments to examine the effect of this variant on promoter activity in kidney cells in culture. We investigated 379 Caucasian type 2 diabetes patients of British/European descent, 93 of whom had microalbuminuria, 26 overt nephropathy, 46 retinopathy, and 73 clinical neuropathy. Genotyping for the variant was carried out by PCR and automated Genescan analysis. Transient transfection studies involved the renal HEK 293 cell line and luciferase reporter gene constructs containing 1.1 kb of 5'-flanking DNA from '+' or '-' allele homozygotes. We found that the '+' allele frequency in patients without microalbuminuria was 12%, but was 23% in those with microalbuminuria ( P=0.0005), and was 26% in those with nephropathy ( P=0.0007), 22% in those with retinopathy ( P=0.037), and 23% in those with neuropathy ( P=0.045). The odds ratios for homozygote +/+ to have microalbuminuria or nephropathy were 2.4 (95% CI 1.4-4.2, P=0.0023) and 5.4 (95% CI 1.8-16, P=0.0009), respectively. Luciferase reporter gene constructs containing 1 kb of NOS2A promoter DNA for each allele were made and sequence analysis confirmed that the +/- variation was the only sequence difference present. Transient transfection of these into HEK 293 cells revealed 25 times higher reporter gene activity for the '+' allele compared with the '-' allele. Gel shift analysis with 30mer oligonucleotides corresponding to each allele showed specific binding to nuclear extracts, being greater for the '+' allele. Thus the '+' allele of the NOS2A promoter variant may confer higher iNOS expression, and could contribute to complications of type 2 diabetes, especially in the approximately 5% of patients homozygous for this variant.

Albuminuria↗
PubMed1998

Inducible chitinolytic system of Aspergillus fumigatus.

Incubation of Aspergillus fumigatus NCPF 2140 in growth medium containing 1% chitin as sole carbon source led to induction of specific extracellular chitinolytic activity of 1.5 mumol GlcNAc released min-1 (mg protein)-1. The effect was repressed by the inclusion of GlcNAc in the medium, indicating regulation by a negative feedback mechanism. Extracellular chitinase activity was inhibited by allosamidin (IC50 0.12 microM). Multiple chitinolytic enzymes were detected on zymograms of extracellular preparations; levels of individual enzymes induced were dependent upon whether cells were incubated with purified colloidal chitin or a crude preparation of crystalline chitin. A major, inducible, 45 kDa chitinase was purified using ammonium sulphate precipitation, chitin affinity chromatography and a novel procedure involving the electroelution of the enzyme from a substrate gel containing glycol chitin. The enzyme is a glycoprotein with endochitinase activity.

Acetylglucosamine↗