Biomedical subjects

C J Long

Publications and source records attributed to C J Long.

At least 19 recordsLinked to original sources

PubMed2005

Disruption of the cathepsin K gene reduces atherosclerosis progression and induces plaque fibrosis but accelerates macrophage foam cell formation.

BACKGROUND: Cathepsin K (catK), a lysosomal cysteine protease, was identified in a gene-profiling experiment that compared human early plaques, advanced stable plaques, and advanced atherosclerotic plaques containing a thrombus, where it was highly upregulated in advanced stable plaques. METHODS AND RESULTS: To assess the function of catK in atherosclerosis, catK(-/-)/apolipoprotein (apo) E(-/-) mice were generated. At 26 weeks of age, plaque area in the catK(-/-)/apoE(-/-) mice was reduced (41.8%) owing to a decrease in the number of advanced lesions as well as a decrease in individual advanced plaque area. This suggests an important role for catK in atherosclerosis progression. Advanced plaques of catK(-/-)/apoE(-/-) mice showed an increase in collagen content. Medial elastin fibers were less prone to rupture than those of apoE(-/-) mice. Although the relative macrophage content did not differ, individual macrophage size increased. In vitro studies of bone marrow derived-macrophages confirmed this observation. Scavenger receptor-mediated uptake (particularly by CD36) of modified LDL increased in the absence of catK, resulting in an increased macrophage size because of increased cellular storage of cholesterol esters, thereby enlarging the lysosomes. CONCLUSIONS: A deficiency of catK reduces plaque progression and induces plaque fibrosis but aggravates macrophage foam cell formation in atherosclerosis.

Animals↗

PubMed2005

Nonstationary noise estimation in functional MRI.

An important issue in functional MRI analysis is accurate characterisation of the noise processes present in the data. Whilst conventional fMRI noise representations often assume stationarity (or time-invariance) in the noise generating sources, such approaches may serve to suppress important dynamic information about brain function. As an alternative to these fixed temporal assumptions, we present in this paper two time-varying procedures for examining nonstationary noise structure in fMRI data. In the first procedure, we approximate nonstationary behaviour by means of a collection of simple but numerous time-varying parametric models. This is accomplished through the derivation of a locally parametric AutoRegressive (AR) plus drift model which tracks temporal covariance by allowing the model parameters to evolve over time. Before exploring time variation in these parameters, window-widths (bandwidths) that are well suited to the latent time-varying noise structure must be determined. To do this, we employ a bandwidth selection mechanism based on Stein's Unbiased Risk Estimator (SURE) criterion. In the second procedure, we describe the fMRI noise using a nonparametric method based on Functional Data Analysis (FDA). This process generates well-conditioned nonstationary covariance estimates that reflect temporal continuity in the underlying data structure whilst penalizing effective model dimension. We demonstrate both methods on simulated data and investigate the presence of nonstationary noise in resting fMRI data using the whitening capabilities of the locally parametric procedure. We evaluate the comparative behaviour of the stationary and nonstationary AR-based methods on data acquired at 1.5, 3 and 7 T magnetic field strengths and show that incorporation of time variation in the AR parameters leads to an overall decrease in the level of residual structure in the data. The FDA noise modelling technique is formulated within an activation mapping procedure and compared to the SPM (Statistical Parametric Mapping) toolbox on a cognitive face recognition task. Both the SPM and FDA methods show good sensitivity on this task, but we find that inclusion of the nonstationary FDA noise model seems to improve detection power in important task-related medial temporal regions.

Algorithms↗

PubMed2001

Size-dependent control of the binding of biotinylated proteins to streptavidin using a polymer shield.

Many medical and biotechnological processes rely on controlling and manipulating the molecular-recognition capabilities of proteins. This can be achieved using small molecules capable of competing for protein binding or by changing environmental parameters that affect protein structure and hence binding. An alternative is provided by stimuli-responsive polymers that change reversibly from a water-soluble expanded coil to a water-insoluble collapsed globule upon small changes in temperature, pH or light intensity: when attached to proteins in the vicinity of their binding sites, they reversibly block and release small ligands. Here we show how this approach can be extended to achieve size-selective binding of large, macromolecular ligands. We use the thermally responsive polymer poly(N,N-diethylacrylamide) (PDEAAm), and attach it to the protein streptavidin approximately 20 A from the binding site for biotinylated proteins. Below the lower critical solution temperature of PDEAAm, the polymer is in its extended state and acts as a 'shield' to block the binding of large biotinylated proteins; above this temperature, it collapses and exposes the binding site, thereby allowing binding. We find that the degree of shielding depends on both the size of the biotinylated protein and the size of PDEAAm, suggesting that 'smart' polymer shields could be tailored to achieve a wide range of size-dependent ligand discrimination for use in affinity separations, biosensors and diagnostics technologies.

Acrylamides↗

PubMed2000

Founder's Award, Society for Biomaterials. Sixth World Biomaterials Congress 2000, Kamuela, HI,May 15-20, 2000. Really smart bioconjugates of smart polymers and receptor proteins.

Over the past 18 years we have been deeply involved with the synthesis and applications of stimuli-responsive polymer systems, especially polymer-biomolecule conjugates. This article summarizes our work with one of these conjugate systems, specifically polymer-protein conjugates. We include conjugates prepared by random polymer conjugation to lysine amino groups, and also those prepared by site-specific conjugation of the polymer to specific amino acid sites that are genetically engineered into the known amino acid sequence of the protein. We describe the preparation and properties of thermally sensitive random conjugates to enzymes and several affinity recognition proteins. We have also prepared site-specific conjugates to streptavidin with temperature-sensitive polymers, pH-sensitive polymers, and light-sensitive polymers. The preparation of these conjugates and their many fascinating applications are reviewed in this article.

Acrylamides↗

PubMed2000

Fibroblast growth factor-2 promotes keratan sulfate proteoglycan expression by keratocytes in vitro.

Keratocytes of the corneal stroma produce a specialized extracellular matrix responsible for corneal transparency. Corneal keratan sulfate proteoglycans (KSPG) are unique products of keratocytes that are down-regulated in corneal wounds and in vitro. This study used cultures of primary bovine keratocytes to define factors affecting KSPG expression in vitro. KSPG metabolically labeled with [(35)S]sulfate decreased during the initial 2-4 days of culture in quiescent cultures with low serum concentrations (0.1%). Addition of fetal bovine serum, fibroblast growth factor-2 (FGF-2), transforming growth factor beta, or platelet derived growth factor all stimulated cell division, but only FGF-2 stimulated KSPG secretion. Combined with serum, FGF-2 also prevented serum-induced KSPG down-regulation. KSPG secretion was lost during serial subculture with or without FGF-2. Expression of KSPG core proteins (lumican, mimecan, and keratocan) was stimulated by FGF-2, and steady state mRNA pools for these proteins, particularly keratocan, were significantly increased by FGF-2 treatment. KSPG expression therefore is supported by exogenous FGF-2 and eliminated by subculture of the cells in presence of serum. FGF-2 stimulates KSPG core protein expression primarily through an increase in mRNA pools.

Animals↗

PubMed1999

Protein electrostatic surface distribution can determine whether calcium oxalate crystal growth is promoted or inhibited.

Acidic proteins found in mineralized tissues act as nature's crystal engineers, where they play a key role in promoting or inhibiting the growth of minerals such as hydroxyapatite and calcium oxalate. Despite their importance in such fundamental physiological processes as bone and tooth formation, however, there is remarkably little known of the protein structure-function relationships that govern crystal recognition. We have taken a model system approach to elucidate some of the relationships between protein surface chemistry and secondary crystal growth of biological minerals. We show here that the distribution of electrostatic surface charge on our model protein, Protein G, determined whether the secondary growth of calcium oxalate, the principal mineral phase of kidney stones, was promoted or inhibited when the proteins were preadsorbed at low and equivalent surface coverages of <10%. The native Protein G, which contains 10 surface carboxylates, increased the rate of calcium oxalate growth from aqueous solution under constant composition conditions up to 97%, whereas a site-directed mutant with six of the surface charges removed inhibited the growth rate by 60%. The adsorption isotherms of both proteins were determined and suggested that the differences in electrostatic surface properties also lead to differences in protein orientation on the crystal surface. These results demonstrate that differences in electrostatic surface potential of proteins can directly determine whether secondary calcium oxalate growth is promoted or inhibited, and a model is proposed that suggests the distribution of carboxylate residues determines the interrelated binding orientation and exposed surface chemistry of the adsorbed Protein G.

Amino Acid Sequence↗

PubMed1999

Ag+ alters cell growth, neurite extension, cardiomyocyte beating, and fertilized egg constriction.

BACKGROUND: The Russian Space Agency uses electrochemically generated silver ions (Ag+) to purify drinking water for their space station, Mir, and their portion of the International Space Station. U.S. EPA guidelines allow 10.6 micromol x L(-1) Ag+ in human drinking water for up to 10 d. Studies correlate Ag+ exposure with tissue dysfunction in humans, rats, and mice, and with altered ion transport, skeletal muscle contraction, and embryonic cell constriction in other animal cells. Ag+ effects on cell shape change-related functions have not been assessed. METHODS: Immortalized embryonic human intestinal epithelial cells, freshly explanted embryonic avian nerve cells and cardiomyocytes, and marine fertilized eggs were grown in vitro in medium containing AgNO3. RESULTS: Intestinal cells detach from the substratum and viable cell number decreases by 5-6 d at 5 micromol x L(-1) AgNO3, and faster at higher concentrations. Microtubules appear unaltered in adherent cells. Detached cells are nonviable. Neurite outgrowth and glial cell migration from dorsal root ganglia are inhibited by 3 d at 15 micromol x L(-1) AgNO3 or greater. Contractions stop temporarily in most cardiomyocytes by 5 min at 5 micromol x L(-1) AgNO3 or more, but some cardiomyocytes beat 3 times faster than normal at 7.5-20 micromol x L(-1) AgNO3. Picomolar Ag+ increases marine egg polar lobe constriction within an hour, even in the absence of microtubules. CONCLUSION: Ag+ alters animal cell growth and shape changes by a MT-independent mechanism. This is the first report of Ag+ effects on vertebrate neurite outgrowth, glial cell migration, or cardiomyocyte beat rate.

Animals↗

PubMed1998

Thromboxane modulating agents. 4. Design and synthesis of 3-(2-[[(4-chlorophenyl)sulfonyl]-amino]ethyl)benzenepropanoic acid derivatives as potent thromboxane receptor antagonists.

The design of a series of thromboxane receptor antagonists based on 3-(2-[[(4-chlorophenyl)sulfonyl]amino]ethyl)benzenepropanoic acid (1) is described. Addition of an arylmethyl group at the 5-position of 1 gave exceptionally potent agents in vitro and in vivo, with 13a (UK-147,535) giving complete blockade of the TxA2 receptor for greater than 12 hours in dogs, following an oral dose of 0.1 mg/kg.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1998

Ecological validity of neuropsychological tests: focus on the California Verbal Learning Test and the Wisconsin Card Sorting Test.

To date, few studies have assessed the ecological validity of current measures of memory and executive functioning, particularly the California Verbal Learning Test (CVLT) and the Wisconsin Card Sorting Test (WCST). A battery of tests, including the CVLT and the WCST, was administered to 28 severely head-injured adults who were 1 year or more postinjury. Hierarchical linear regression was performed to assess the relationship between these cognitive tests and work functioning while statistically controlling for physical disability, emotional distress, and memory compensation strategies utilized by the participants. Results suggest that the CVLT was best at predicting performance on the job; however, the CVLT and the WCST were about equally predictive of the type of position held by a participant. Hence, this study demonstrates the ecological validity of measures of memory and executive functioning for predicting work-related skills.

Journal Article↗

PubMed1997

Thromboxane modulating agents. 3. 1H-imidazol-1-ylalkyl- and 3-pyridinylalkyl-substituted 3-[2-[(arylsulfonyl)amino]ethyl]benzenepropanoic acid derivatives as dual thromboxane synthase inhibitor/thromboxane receptor antagonists.

The design of a series of dual thromboxane synthase inhibitor/thromboxane receptor antagonists based on a 3-[2-[(arylsulfonyl)amino]ethyl]benzenepropanoic acid thromboxane receptor antagonist template is described. Introduction of a 5-(1H-imidazol-1-ylmethyl), a 5-(3-pyridinyl-methyl), or a 5-(3-pyridinyloxy) substituent leads to dual agents with thromboxane synthase inhibitory activity comparable with that of dazmegrel (7). In addition, 3-pyridinylalkyl substituents also make a significant contribution to thromboxane receptor binding. Oral administration of compound 74 (5 mg/kg) to conscious dogs produces long-lasting thromboxane synthase inhibition and thromboxane receptor blockade as measured by inhibition of U46619-induced platelet aggregation ex vivo.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1997

Effective treatment of minor head injury and understanding its neurological consequences.

Minor head injury (MHI) is typically associated with coma and posttraumatic amnesia of short duration. Resulting impairment has been found in neurological and cognitive functioning, although these deficits tend to be brief in duration. A review of the literature reveals that although considerable research has been done on MHI, there remain a few critical issues that require resolution. First, one of the more noticeable problems relates to the fact that MHI does not have well-defined lower limits, which can lead to individuals being categorized as having an MHI despite never enduring trauma to the brain. The second problem relates to the failure of researchers to differentiate between cognitive deficits and postconcussion symptoms (PCS) when assessing outcome. Because PCS can occur in the absence of head injury and are often present beyond the period of cognitive recover from MHI, the two clearly result from different factors. Finally, many preinjury factors (age, personality) and postinjury factors (stress, neurological complications) interact with cognitive functioning and significantly affect recovery, which confounds the study of outcome. These problems are reviewed and discussed.

Journal Article↗

PubMed1996

Minor head injury: attempts at clarifying the confusion.

Traumatic brain injury (TBI) refers to a broad range of neurological, cognitive and emotional factors that result from the application of a mechanical force to the head. Mechanical force can be applied on a continuum from none to very severe, and the extent of brain injury is related to the severity of this force. A review of the literature reveals that, while considerable research has been done on minor head injury, there remain several major sources of confusion. First, one of the most noticeable problems relates to the fact that the mild head injury has lower limits which are vaguely defined. This leads to individuals being categorized as having sustained a mild TBI despite minimal or no neurological damage being present. A second source of confusion in the literature is related to the failure to differentiate between cognitive consequences of TBI and post-concussion symptoms (PCS). Since PCS can occur in the absence of head injury, and are often present beyond the period of cognitive recovery from mild TBI, the two clearly result from different factors. Researchers have often failed to separate these two factors when studying recovery of function, and this has led to varying findings on outcome. Finally, many pre-injury factors (age, education, emotional adjustment) and post-injury factors (pain, family support, stress) interact with cognitive functioning and significantly affect recovery from TBI. These problems are reviewed and discussed.

Brain Concussion↗

PubMed1996

Relationship between reaction time and neuropsychological test performance.

Reaction time (RT) has been used to discriminate individuals with brain damage from controls, but little is known about the relationship between RT and neuropsychological (NP) tests. This relationship was examined 3 ways. First, the mean, median, and SD scores of simple and choice RT for 213 referrals were used as potential predictors of NP impairment in discriminant analyses; cross-validation produced a significant hit rate. Second, the hit rate (NP impairment vs. no) using RT was compared to hit rates using fluency, IQ, and memory measures; the RT rate did not significantly exceed the others. Third, the abilities of RT and of a NP impairment score to classify 28 subjects into brain injury and control groups were compared; the hit rates were not significantly different. Results are interpreted to indicate moderate agreement between RT and NP tests regarding impairment classifications and to suggest roles for RT in NP assessment.

Journal Article↗

PubMed1996

Visual reaction time and its relationship to neuropsychological test performance.

The purpose of this study was to investigate the relationship between simple and choice reaction time (RT) measures and neuropsychological test performance (as assessed by the Impairment Index of the Halstead-Reitan Neuropsychological Test Battery). Both median RT scores and intraindividual variability of RT scores were evaluated in three groups: impaired TBI, nonimpaired TBI, and normal controls. In all three groups, there was a statistically significant correlation between both reaction time measures and level of cognitive functioning. In addition, SRT, speed of information processing (as measured by CRT), and intraindividual variability of RT scores continued to be impaired in many brain-damaged individuals who, according to the Impairment Index, had fully recovered from their cognitive deficits. Median RT scores were better able to discriminate between impaired TBI patients and normal controls; but intraindividual variability of RT scores was better able to discriminate between nonimpaired TBI patients and normal controls. It was concluded that, by adding the two short RT tests to the neuropsychological test battery, more accurate predictions can be made about a patient's level of cognitive functioning and the nature of treatment needed for continued recovery.

Journal Article↗

PubMed1996

Autonomous parvovirus transduction of a gene under control of tissue-specific or inducible promoters.

Several classes of viruses are in use, or are being developed, as gene therapy vectors. Viruses with small genomes containing few essential genes have the advantage of requiring only simple complementation systems to allow packaging of foreign DNA, substituted for the entire viral coding sequences. Retroviruses and the dependent parvovirus AAV (adeno-associated virus) have been used in this way, and both possess an efficient integration mechanism which should allow long-term expression of transduced genes. In some situations, however, long-term persistence may be undesirable and there is a need for small, non-integrating viral vectors. Autonomous parvoviruses, such as LuIII, have potential as such vectors for short-term expression of therapeutic genes. We previously described recombinants of LuIII that transduced reporter genes, expressed using the viral constitutive promoter, P4. We have now generated several recombinants containing regulated promoters. A virus including a liver-specific enhancer directed 10- to 20-fold preferential expression of the luciferase reporter in transduced human hepatoma (HepG2) versus HeLa cells. In additional LuIII recombinants, the luciferase reporter was linked with chimeric promoters containing binding sequences for either the yeast GAL4 protein or the bacterial tetracycline repressor. Luciferase expression was strongly activated when these viruses were used to infect cells containing a cognate trans-activator (GAL4 or tTA, a tetracycline repressor fusion with VP16 of herpes simplex), introduced by transfection. The response to tTA could be abolished, or reduced in a graded manner, by exposure of the infected cells to tetracycline. Further results suggested that an increase in basal expression, apparently mediated by the viral left terminal inverted repeat, could be minimized by interposing polyadenylation signals between this sequence and the promoter. These results confirm that appropriate transcriptional regulation can be achieved for genes transduced by an autonomous parvovirus vector. Such vectors therefore show promise for the delivery of therapeutic genes in situations requiring cell-specific, short-term expression, eg in targeting suicide genes for ablation of cancer cells.

Cell Line↗

PubMed1995

Memory remediation after severe closed head injury: notebook training versus supportive therapy.

This study evaluated the effectiveness of a 9-week memory notebook treatment for closed-head-injured (CHI) participants with documented memory deficits. Eight participants who had sustained a severe CHI more than 2 years earlier were allocated to receive either notebook training or supportive therapy. Memory outcome indicators, which differed in sensitivity to detect everyday memory failures (EMFs), were administered before treatment, immediately after treatment, and at a 6-month follow-up. At posttreatment, the notebook training group reported significantly fewer observed EMFs on a daily checklist measure than the supportive therapy group. Although in the same direction, this finding no longer reached significance at follow-up. No significant treatment effects were found for the laboratory-based memory measures at posttreatment or follow-up. Although the present results are to be considered preliminary because of the small sample size, they suggest that notebook training has the potential to help individuals compensate for everyday memory problems and that the methods used to measure training efficacy are important.

Activities of Daily Living↗

PubMed1995

Engineered chimeric streptavidin tetramers as novel tools for bioseparations and drug delivery.

We report the construction of chimeric streptavidin tetramers that are composed of subunits of both wild-type (WT) streptavidin and genetically-engineered streptavidin variants designed for enhanced bioseparation and drug delivery performance. Subunit mixing is accomplished by guanidine thiocyanateinduced denaturation of an equimolar mixture of WT streptavidin and the respective site-directed mutant, followed by renaturation and reassociation of mixed tetramers. In the first example, we demonstrate the mixing of WT subunits with an Asn49Cys (N49C) mutant. The WT/n49C tetramers can be used for site-specific and stoichiometric attachment of therapeutics/imaging agents or targeting proteins through the genetically-engineered thiol while retaining unhindered access to biotin-binding at the WT subunits. Second, we demonstrate that the His127Cys mutation (H127C) results in a streptavidin mutant that forms a disulfide-linked dimer under non-reducing conditions. Mixing of H127C and WT streptavidin subunits results in chimeric tetramers where both the stoichiometry (WT:H127C::1:1) and subunit architecture is controlled by the unique disulfide bridge engineered into H127C. In the third example, WT subunits were mixed with the subunits of a site-directed mutant, Trp120Ala (W120A), which displays a biotin dissociation constant that is enhanced by more than 10(4) compared to WT streptavidin. The W120 biotin-binding affinity is sufficiently high (Ka approximately equal to 10(7) M-1) to immobilize the mutant on a biotinagarose affinity chromatography column, but the engineered off-rate allows for facile elution with excess biotin at physiological pH, whereas WT streptavidin is irreversibly immobilized on the column. We demonstrate that the purified WT/W120A chimeric tetramers combine the advantages of both subunits, allowing for irreversible immobilization of biotinylated targets at the WT subunit, while retaining the reversible separation capabilities of the W120A subunits via biotin-agarose affinity chromatography.

Dimerization↗

PubMed1994

Characterization of adenosine receptors in the rat isolated aorta.

1. Adenosine and its analogues relaxed the isolated rat aorta by an endothelium-dependent mechanism with an order of potency of 5'-N-ethylcarboxamidoadenosine (NECA) > 2-(p-(2-carboxy-ethyl)phenethylamino)-5'-N-ethylcarboxamidoadenosi ne (CGS 21680) > adenosine = N6-(2-(4-amino-phenyl)ethyl)adenosine (APNEA) = N6-cyclopentyladenosine (CPA) > 5'-methylthioadenosine (MTA), although the maximal response achieved by CGS 21680 was less than that achieved by NECA. 2. Both 8-sulphophenyltheophylline (8-SPT) and MTA antagonized responses to the adenosine analogues, but there were some anomolous features of this antagonism and NECA was inhibited more powerfully than the other agonists. This suggests that as well as A2a receptors mediating relaxation, the rat aorta may relax to adenosine analogues by other mechanisms.

Adenosine↗