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Biomedical subjects

J Crosby

Publications and source records attributed to J Crosby.

At least 19 recordsLinked to original sources
PubMed2001

beta-carboline binding to imidazoline receptors.

A series of beta-carbolines were prepared and their affinities for imidazoline (I(1) and I(2)) sites evaluated. Selected compounds were also examined at alpha(2)-adrenoceptors. Some of the beta-carbolines were found to bind with high affinity to I(2)-sites and this affinity was dependent on both the planarity of the molecule and the presence of the aryl ring substituents. Good I(1)-affinity was observed with two of the compounds but none of the tested compounds bound to alpha(2)-adrenoceptors. The hallucinogenic properties of beta-carbolines have been linked to activity at 5-HT receptors, in particular 5-HT(2), however, it is apparent from this study that many of these compounds display substantially higher affinity for the imidazoline sites. This finding, and those showing modulation of some behavioural effects of morphine by I(2)-ligands, suggests that imidazoline sites may be interesting new targets in drug abuse research.

Animals↗
PubMed2001

What do master surgeons think of surgical competence and revalidation?

BACKGROUND: There has been on-going debate and public interest in surgical competence in recent years. METHODS: A Delphi reiterative opinion survey was conducted among master surgeons on selection of surgical trainees, methods of assessment of progress of surgical trainees, and revalidation of established consultant surgeons. RESULTS: Selection-the current methods of trainee selection were considered inadequate and in need of revision. The important attributes recognized by group are cognitive factors, innate dexterity, and personality. Important aspects of personality include decision-making ability, insight, team spirit, and emotional stability. Assessment during training-the majority view was that this should be based on clinical judgement/skills, operative skills, and cognitive ability. Assessment of technical ability should be based on standardized checklists. Research within training programs was encouraged but academic achievement does not reflect surgical competence. There was a majority verdict for an exit clinical examination. Revalidation-the group agreed on the need for competence checks during the professional career of surgeons. These should cover knowledge, clinical, operative, and humanistic skills; but expressed concern on the feasibility of a revalidation system that can reliably assess the range of skills needed for surgical competence. There was a majority vote against an internal appraisal system. External assessment by nationally appointed 'assessors' was considered preferable. CONCLUSIONS: Both selection and assessment of surgical trainees require changes and standardization. Although revalidation is necessary, concern was expressed on the reliability and validity of existing and proposed systems.

Clinical Competence↗
PubMed2001

Blockade of CD49d inhibits allergic airway pathologies independent of effects on leukocyte recruitment.

Lymphocyte and/or eosinophil recruitment is dependent on the sequential interactions between adhesion molecules expressed on activated endothelial cells and both leukocyte subtypes. Endothelial P- and E-selectins mediate tethering and rolling of leukocytes through interactions with P-selectin glycoprotein ligand-1 (PSGL-1), and diapedesis subsequently occurs by engagement of endothelial vascular cell adhesion molecule-1 and CD49d (alpha(4)-integrins). The anti-inflammatory potential of interfering with these adhesive interactions was assessed with an ovalbumin challenge mouse model of asthma. Administration of a soluble form of PSGL-1 reduced eosinophils (80%) and lymphocytes (50%) in bronchoalveolar lavage fluid without affecting epithelial changes or airway hyperreactivity (AHR). In contrast, although administration of anti-CD49d monoclonal antibodies (PS/2) resulted in similar reductions in eosinophils (75%) and lymphocytes (50%), PS/2 reduced and abolished mucous cell metaplasia and AHR, respectively. Administration of both PSGL-1 and PS/2 had the additive effect of eliminating eosinophils from the airways (96% decrease), with few or no additional reductions (relative to PS/2 administration alone) in lymphocyte recruitment, mucous cell metaplasia, or AHR. These data show that eosinophils and lymphocytes differentially utilize adhesive interactions during recruitment and that the inhibition of AHR is independent of this recruitment.

Animals↗
PubMed2001

Intrinsic AHR in IL-5 transgenic mice is dependent on CD4(+) cells and CD49d-mediated signaling.

Overexpression of interleukin (IL)-5 by the airway epithelium in mice using the rat CC10 promoter (NJ.1726 line) leads to several histopathologies characteristic of human asthma, including airway hyperreactivity (AHR). We investigated the contribution of B and T cells, as well as CD4 expression, to the development of AHR in IL-5 transgenic mice. NJ.1726 mice on a T cell or CD4 knockout background, but not on a B cell knockout background, lost intrinsic AHR. These effects occurred without decreases in IL-5 or eosinophils. We further investigated the contribution of alpha(4)-integrin signaling to the development of AHR in IL-5 transgenic mice through the administration of anti-CD49d (alpha(4)-integrin) antibody (PS/2). Administration of PS/2 resulted in immediate (16-h) inhibition of AHR. The inhibition of AHR was not associated with a decrease in airway eosinophils. These studies demonstrate that, despite the presence of increased levels of IL-5 and eosinophils in the lungs of NJ.1726 mice, CD4(+) cells and alpha(4)-integrin signaling are necessary for the intrinsic AHR that develops in IL-5 transgenic mice.

Animals↗
PubMed2001

Solid phase synthesis of 4-hydroxycinnamic acid and its derivatives for potential use in combinatorial chemistry: a novel route for the synthesis of 4-hydroxycinnamoyl coenzyme A and NMDA receptor antagonists.

Synthesis of 4-hydroxycinnamic acid 6 and its N-hydroxysuccinimide ester 8 has been carried out in high yield on solid support. Further development allowed the synthesis of 4-hydroxycinnamoyl CoA 1 in excellent overall yield. The utility of solid phase as a method for the synthesis of 4-hydroxycinnamic acid derivatives was demonstrated by the synthesis of a number of compounds including the NMDA receptor antagonists, N-(phenylalkyl)cinnamides 9 and 10.

Acyl Coenzyme A↗
PubMed2000

Isolation of RP-HPLC pure clonidine-displacing substance from NG108-15 cells.

A crude extract of clonidine-displacing substance (CDS) has previously been extracted from the NG108-15 cell line. This study aimed to purify CDS extracted from this cell line further, by the technique of reverse phase-HPLC (RP-HPLC), and subsequently determine whether this refined CDS bears any similarity to CDS's extracted from other tissues. Crude CDS was extracted from NG108-cells and fractionated by RP-HPLC eluting with a linear gradient of methanol (5-65%; 1 ml min(-1) flow rate) over 50 min., and collected at 1 min. intervals. The pharmacological activities of the CDS fractions were determined by their abilities to displace bound [3H]clonidine to alpha(2)-adrenoceptors in rat brain membranes. RP-HPLC analysis of CDS revealed a pharmacologically active fraction distinct from agmatine, eluting at 24 min, corresponding to an absorbance peak observed at this time. Collectively, these results confirmed that CDS was present in the NG108-15 cell line. However, the RP-HPLC analysis showed the pharmacological activity to elute at a more hydrophobic gradient than previously observed with CDS's extracted from bovine tissues. These results support the notion of the existence of several CDS's.

Animals↗
PubMed2000

Task-based learning: the answer to integration and problem-based learning in the clinical years.

INTRODUCTION: Integrated teaching and problem-based learning (PBL) are powerful educational strategies. Difficulties arise, however, in their application in the later years of the undergraduate medical curriculum, particularly in clinical attachments. Two solutions have been proposed - the use of integrated clinical teaching teams and time allocated during the week for PBL separate from the clinical work. Both approaches have significant disadvantages. Task-based learning (TBL) is a preferred strategy. In TBL, a range of tasks undertaken by a doctor are identified, e.g. management of a patient with abdominal pain, and these are used as the focus for learning. Students have responsibility for integrating their learning round the tasks as they move through a range of clinical attachments in different disciplines. They are assisted in this process by study guides. METHOD: The implementation of TBL is described in one medical school. One hundred and thirteen tasks, arranged in 16 groups, serve to integrate the student learning as they rotate through 10 clinical attachments. RESULTS: This trans-disciplinary approach to integration, which incorporates the principles of PBL offers advantages to both teachers and students. It recognizes that clinical attachments in individual disciplines can offer rich learning opportunities and that such attachments can play a role in an integrated, as well as in a traditional, curriculum. In TBL, the contributions of the clinical attachments to the curriculum learning outcomes must be clearly defined and tasks selected which will serve as a focus for the integration of the students' learning over the range of attachments.

Clinical Clerkship↗
PubMed1999

A chain initiation factor common to both modular and aromatic polyketide synthases.

Antibiotic-producing polyketide synthases (PKSs) are enzymes responsible for the biosynthesis in Streptomyces and related filamentous bacteria of a remarkably broad range of bioactive metabolites, including antitumour aromatic compounds such as mithramycin and macrolide antibiotics such as erythromycin. The molecular basis for the selection of the starter unit on aromatic PKSs is unknown. Here we show that a component of aromatic PKS, previously named 'chain-length factor', is a factor required for polyketide chain initiation and that this factor has decarboxylase activity towards malonyl-ACP (acyl carrier protein). We have re-examined the mechanism of initiation on modular PKSs and have identified as a specific initiation factor a domain of previously unknown function named KSQ, which operates like chain-length factor. Both KSQ and chain-length factor are similar to the ketosynthase domains that catalyse polyketide chain extension in modular multifunctional PKSs and in aromatic PKSs, respectively, except that the ketosynthase domain active-site cysteine residue is replaced by a highly conserved glutamine in KSQ and in chain-length factor. The glutamine residue is important both for decarboxylase activity and for polyketide synthesis.

Acyl Carrier Protein↗
PubMed1999

Extraction of active clonidine-displacing substance from bovine lung and comparison with clonidine-displacing substance extracted from other tissues.

Crude methanolic clonidine-displacing substance (CDS) extracted from bovine lung competed for radioligand binding from alpha2-adrenoceptors and I2-sites present in rat brain membranes, and from I1-sites present in rat brain and kidney membranes. There was no difference in the competition of [3H]clonidine binding to alpha2-adrenoceptors present in either rat or rabbit brain membranes by the crude CDS extract and therefore either tissue could be used to estimate the number of units of CDS present in extracts. Further purification by reverse phase high performance liquid chromatography (RP-HPLC), with UV detection, of extracts obtained from bovine lung, brain and rat brain exhibited similar three-peak profiles, previously reported. Corresponding fractions competed for radioligand binding to alpha2-adrenoceptors present in rat brain membranes, eluting between 19 and 23 min, which corresponded with the middle peak of the three-peaks. Therefore, we propose the CDS-like material eluting from all these tissues to be similar. Interestingly, CDS extracted from bovine adrenal glands under the same conditions showed a similar three-peak profile, but did not repeat the displacement of binding just at 19-23 min, but at every time point after 4 min. This suggests this tissue could represent a source of CDS in this species.

Adrenal Glands↗
PubMed1999

Plasmodium vivax merozoite surface protein-3 contains coiled-coil motifs in an alanine-rich central domain.

Plasmodium merozoites are covered with a palisade layer of proteins that are arranged as organized bundles or appear as protruding spikes by electron microscopy. Here we present a third Plasmodium vivax merozoite surface protein, PvMSP-3, which is associated with but not anchored in the merozoite membrane. Serum from a P. vivax immune squirrel monkey was used to screen a lambdagt11 P. vivax genomic DNA (gDNA) library. Plaque-selected antibodies from clone no. 6.1, and rabbit antisera against its encoded protein, produced a pattern in immunofluorescence assays (IFAs) that is consistent with a localization at the surface of mature schizonts and free merozoites. Specific antisera also agglutinated merozoites and recognized a protein of 150 000 Da by SDS-PAGE. The complete msp-3 gene and flanking sequences were cloned from a P. vivax lambda Dash II gDNA library and also partly characterized by RACE (rapid amplification of cDNA ends). The immediate upstream sequence contains non-coding repeats and a putative protein encoding open reading frame (ORF), which are also present on the msp-3 5'RACE gene product. Pvmsp-3 encodes a protein with a calculated mass of 89 573 Da, which has a potential signal peptide and a major central alanine-rich domain (31%) that exhibits largely alpha-helical secondary structure and is flanked by charged regions. The protein does not have a putative transmembrane domain or a consensus sequence for a glycosylphosphatidylinositol (GPI) anchor modification. However, the alanine-rich domain has heptad repeats that are predicted to form coiled-coil tertiary structures, which mediate protein-protein interactions. PvMSP-3 is structurally related to P. falciparum MSP-3 and the 140000 Da MSP of P. knowlesi. Characterization of PvMSP-3, thus, also begins to define a new interspecies family of evolutionarily related Plasmodium merozoite proteins.

Alanine↗
PubMed1999

Isomerization of urocanic acid after ultraviolet radiation is influenced by skin pigmentation.

Exposure to ultraviolet (UV) radiation may induce erythema, DNA damage and suppression of immune responses. Melanin pigmentation offers protection against the first two of these effects, but immunosuppression seems to occur irrespective of the subject's pigmentation. Cis-urocanic acid (cis-UCA), produced by isomerization of trans-UCA in the stratum corneum on UV exposure, initiates some of the immunomodulatory effects of UV radiation. In the present study the relationship between skin pigmentation and UCA isomerization has been examined in 28 healthy individuals of skin types I-IV. Pigmentation is measured in five areas of not recently exposed back skin before irradiation with 0, 0.45, 0.9, 1.8 and 3.6 standard erythema dose (SED) of filtered broadband UV-B (1 SED = 10 mJ cm-2 at 298 nm). The concentration of UCA isomers is measured immediately after the irradiation. With 3.6 SED, the relative production of cis-UCA is close to the maximum obtainable, irrespective of skin type. A significant negative correlation is found between pigmentation and relative production of cis-UCA at 0.45 and 1.8 SED, and between pigmentation and absolute production of cis-UCA at 0.45 SED. At doses of 0.45 and 0.9 SED the relative and absolute production of cis-UCA are higher in the group with skin types I and II when compared with the group with skin types III and IV. The higher isomerization in the lightly pigmented subjects than in the more pigmented ones may indicate that people with fair skin are at a relatively higher risk of immunosuppression when exposed to low doses of UV radiation.

Adolescent↗
PubMed1999

The role of urocanic acid in UVB-induced suppression of immunity to Trichinella spiralis infection in the rat.

The naturally occurring trans-isomer of urocanic acid (trans-UCA), found in the stratum corneum, absorbs ultraviolet light (UV) and isomerizes to the cis-form. Cis-UCA has been shown to impair some cellular immune responses, and has been proposed as an initiator of the suppression that follows UV irradiation. UVB exposure leads to an increase in cis-UCA in the skin of rats from about 10% to 40% of the total UCA. Previously it has been demonstrated that UVB lowers immune responses to Trichinella spiralis after oral infection of rats with the parasitic worm. In the present study we investigated the role of cis-UCA in the control of this parasitic infection. Rats were infected orally with T. spiralis and injected with different doses of cis- or trans-UCA subcutaneously. Mitogenic responses and the mixed lymphocyte reaction were not affected by either isomer. In contrast, the number of T. spiralis larvae in muscle tissue of infected rats was increased significantly in the cis-UCA-treated animals compared with the trans-UCA-treated animals. In addition, delayed-type hypersensitivity (DTH) to T. antigen in infected rats was significantly impaired by cis-UCA but not by trans-UCA. If rats were injected with a monoclonal antibody with specificity for cis-UCA 2 hr prior to UVB exposure, the UVB-induced suppression in DTH to T. spiralis and the increase in larvae counts were significantly inhibited compared with rats that were similarly injected with a control antibody. Thus cis-UCA can inhibit the specific resistance to parasitic infections and acts as an important mediator of UVB-induced suppression of immunity to T. spiralis in the rat.

Analysis of Variance↗
PubMed1999

Sunscreen protection against cis-urocanic acid production in human skin.

Commercial sunscreens may offer some protection from immunosuppression induced by ultraviolet (UV) radiation, but agreement concerning the degree of protection is lacking. Cis-urocanic acid, formed by the photoisomerization of transurocanic acid is considered an important mediator of the cutaneous immunomodulation resulting from exposure to UV radiation. We investigated the effect of sunscreens on the isomerization of urocanic acid in 17 human subjects. Two sunscreens containing chemical filters, sun protection factor (SPF) 4 and SPF 10, and a SPF 10 sunscreen with a physical filter were applied at a thickness of 2 mg/cm2. The effect of a thin layer (0.5 mg/cm2) of the chemical SPF 10 sunscreen was also evaluated, as the amount of sunscreen applied in practice may be considerably less than recommended. All areas were irradiated with a single UV dose of 3.6 SED (standard erythema doses). In irradiated unprotected skin the median net production of cis-urocanic acid was 52% (relative amount). In the sites treated with the chemical sunscreens, the production of cis-urocanic acid was 7.4% (SPF 4) and 3.5% (SPF 10), and isomerization was thus reduced more efficiently at a higher SPF (p<0.01). The physical sunscreen reduced the formation of cis-UCA to 15%, and was significantly less effective than both the chemical SPF 10 sunscreen (p<0.01) and the SPF 4 sunscreen (p<0.01). The production of cis-urocanic acid in the area treated with the thin layer of the chemical SPF 10 sunscreen was 22%. The protection against the production of cis-urocanic acid was therefore reduced significantly (p<0.01) when the sunscreen was applied in an amount lower than recommended.

Administration, Cutaneous↗
PubMed1998

Acylation of Streptomyces type II polyketide synthase acyl carrier proteins.

Acyl derivatives of type II PKS ACPs are required for in vitro studies of polyketide biosynthesis. The presence of an exposed cysteine residue prevented specific chemical acylation of the phosphopantetheine thiol of the actinorhodin PKS holo ACP. Acylation studies were further complicated by intramolecular disulphide formation between cysteine 17 and the phosphopantetheine. The presence of this intramolecular disulphide was confirmed by tryptic digestion of the ACP followed by ESMS analysis of the fragments. An act Cys17Ser ACP was engineered by site-directed mutagenesis. S-Acyl adducts of act C17S, oxytetracycline and griseusin holo ACPs were rapidly formed by reaction with hexanoyl, 5-ketohexanoyl and protected acetoacetyl imidazolides. Comparisons with type 11 FAS ACPs were made.

Acyl Carrier Protein↗
PubMed1998

Catalytic self-acylation of type II polyketide synthase acyl carrier proteins.

BACKGROUND: Aromatic polyketides are synthesised in streptomycetes by the successive condensation of simple carboxylic acids, catalysed by multienzyme complexes--the polyketide synthases (PKSs). Polyketide assembly intermediates are covalently linked as thioesters to the holo-acyl carrier protein (ACP) subunit of these type II PKSs. The ACP is primed for chain elongation by the transfer of malonate from malonyl CoA. Malonylation of fatty acid synthase (FAS) ACPs is catalysed by specific malonyl transferase (MT) enzymes. The type II PKS gene clusters apparently lack genes encoding such MT proteins, however. It has been proposed that the MT subunit of the FAS in streptomycetes catalyses malonylation of both FAS and PKS ACPs in vivo. RESULTS: We demonstrate that type II PKS ACPs catalyse self-malonylation upon incubation with malonyl CoA in vitro. The self-malonylation reaction of the actinorhodin C17S holo-ACP has a K(m) for malonyl CoA of 219 microM and a kcat of 0.34 min-1. Complete acylation of the PKS ACPs was observed with malonyl, methylmalonyl and acetoacetyl CoAs. No reaction was observed with acetyl and butyryl CoAs and FAS ACPs did not react with any of the substrates. Recombinant FAS MT from Streptomyces coelicolor did not accelerate the rate of malonylation. CONCLUSIONS: The catalytic self-acylation of type II PKS ACPs is an unprecedented reaction. We propose a reaction mechanism in which conserved arginines form a salt bridge with the acyl moiety and sequester it from bulk solvent. This work suggests that the beta-ketoacyl synthase, chain length factor and ACP may constitute a truly minimal PKS in vivo.

Acyl Carrier Protein↗
PubMed1998

MCAT is not required for in vitro polyketide synthesis in a minimal actinorhodin polyketide synthase from Streptomyces coelicolor.

BACKGROUND: It has been proposed that Streptomyces malonyl CoA: holo acyl carrier protein transacylases (MCATs) provide a link between fatty acid and polyketide biosynthesis. Two recent studies have provided evidence that the presence of MCAT is essential for polyketide synthesis to proceed in reconstituted minimal polyketide synthases (PKSs). In contrast to this, we previously showed that the holo acyl carrier proteins (ACPs) from type II PKSs are capable of catalytic self-malonylation in the presence of malonyl CoA, which suggests that MCAT might not be necessary for polyketide biosynthesis. RESULTS: We reconstituted a homologous actinorhodin (act) type II minimal PKS in vitro. When act holo-ACP is present in limiting concentrations, MCAT is required by the synthase complex in order for polyketide biosynthesis to proceed. When holo-ACP is present in excess, however, efficient polyketide synthesis proceeds without MCAT. The rate of polyketide production increases with holo-ACP concentration, but at low ACP concentration or equimolar AC:KS:CLF (KS, ketosynthase; CLF, chain length determining factor) concentrations this rate is significantly lower than expected, indicating that free holo-ACP is sequestered by the KS/CLF complex. CONCLUSIONS: The rate of polyketide biosynthesis is dictated by the ratio of holo-ACP to KS and CLF, as well as by the total protein concentration. There is no absolute requirement for MCAT in polyketide biosynthesis in vitro, although the role of MCAT during polyketide synthesis in vivo remains an open question. MCAT might be responsible for the rate enhancement of malonyl transfer at very low free holo-ACP concentrations or it could be required to catalyse the transfer of malonyl groups from malonyl CoA to sequestered holo-ACP.

Acyl-Carrier Protein S-Malonyltransferase↗
PubMed1998

Chemometric analysis of high performance liquid chromatography-diode array detection-electrospray mass spectrometry of 2- and 3-hydroxypyridine.

Triply coupled high performance liquid chromatography using diode array detection and positive ion electrospray mass spectrometry of 2- and 3-hydroxypyridine is presented. Considerations of the physical method for coupling the two detectors, the influence of pH on retention times, the cone voltage of the mass spectrometer and the linear concentration ranges are described. Data from both detectors are aligned and interpolated. The analyte mass spectra are reduced to 20 significant masses. Principal components plots on the raw, normalised and standardised data, derivatives to determine composition 1 regions, deconvolution and procrustes analysis to compare data from both detectors are discussed. Common trends in both mass spectral and diode array chromatograms are interpreted. This paper represents a new approach to common processing of chromatographic data from two detectors.

Antioxidants↗