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Biomedical subjects

K Grant

Publications and source records attributed to K Grant.

At least 19 recordsLinked to original sources
PubMed2007

Mechanisms of endothelin 1-stimulated proliferation in colorectal cancer cell lines.

BACKGROUND: The peptide endothelin (ET) 1 promotes proliferation in a number of epithelial cancers. The aim of this study was to identify the mechanism of ET-1-stimulated proliferation in colorectal cancer cells in vitro. METHODS: The effects of ET-1 on colorectal cancer cell lines HT29, LIM1215 and SW620 were studied. Cells were cultured with ET-1 plus antagonists/inhibitors to ET(A) or ET(B) receptors, G protein subtypes, phosphoinositide 3-kinase (PI3K) or protein kinase C (PKC). DNA replication and apoptosis were investigated by 5-bromo-2'-deoxyuridine incorporation and Annexin V staining. Transactivation of the epidermal growth factor (EGF) receptor was investigated by blockade of the receptor in the presence of ET-1, measurement of levels of phosphorylated EGF receptor in the presence of ET-1, and comparing the effects of ET-1 and EGF on cell proliferation. RESULTS: ET-1 significantly stimulated growth of all cell lines via ET(A) receptors. ET-1 stimulated DNA replication, not apoptosis. ET-1-stimulated growth was inhibited by antagonism of pertussis toxin-sensitive G proteins, PI3K and PKC. Inhibition of the EGF receptor reduced the effect of ET-1. ET-1 increased levels of phosphorylated EGF receptor via the ET(A) receptor. CONCLUSION: ET-1 increased DNA replication in colorectal cancer cells via the ET(A) receptor. This mitogenic action was mediated via pertussis toxin-sensitive G proteins, PI3K, PKC and transactivation of the EGF receptor.

Analysis of Variance↗
PubMed2006

Quantitative analysis of residual protein contamination on reprocessed surgical instruments.

'Ready-for-use' instruments from surgical instrument trays were examined after routine cleaning and sterilization in a blinded study. These reprocessed instruments originated from five National Health Service hospital trust sterile service departments in England and Wales. Determination of residual protein and peptide contamination was carried out by acid stripping of the instrument surfaces, hydrolysis of the constituent amino acids and quantitative total amino acid analysis. One hundred and twenty instruments were analysed, and the median levels of residual protein contamination per instrument for the individual trays were 267, 260, 163, 456 and 756 microg. Scanning electron microscopy and energy dispersive X-ray spectroscopic analyses of the instruments showed that tissue deposits were localized on surfaces, but there was no significant correlation between overall protein soiling and instrument complexity. The highest levels of residual contamination were found on instruments used for tonsillectomy and adenoid surgery.

Amino Acids↗
PubMed2006

A fluorescence polarization assay for inhibitors of Hsp90.

Hsp90 encodes a ubiquitous molecular chaperone protein conserved among species which acts on multiple substrates, many of which are important cell-signaling proteins. Inhibition of Hsp90 function has been promoted as a mechanism to degrade client proteins involved in tumorigenesis and disease progression. Several assays to monitor inhibition of Hsp90 function currently exist but are limited in their use for a drug discovery campaign. Using data from the crystal structure of an initial hit compound, we have developed a fluorescence polarization assay to monitor binding of compounds to the ATP-binding site of Hsp90. This assay is very robust (Z' > 0.9) and can detect affinity of compounds with IC50s to 40 nM. We have used this assay in conjunction with cocrystal structures of small molecules to drive a structure-based design program aimed at the discovery and optimization of a novel class of potent Hsp90 inhibitors.

Adenosine Triphosphatases↗
PubMed2006

Characterization of maleuric acid derivatives on transgenic human monoclonal antibody due to post-secretional modifications in goat milk.

A fully human antibody to tumor necrosis factor-alpha was expressed in the mammary glands of transgenic goats. The goat expressed antibody (gAb) is heterogeneous and has several isoforms due to typical cellular post-translational modifications. In addition, one post-secretional modification on gAb was discovered by high-resolution cation exchange chromatography (CIEX). The presence of these variants in the final product was shown to be dependent upon the initial milk storage and traditional purification methodologies used. These observations allow for the development of new sample recovery and purification processes to eliminate these variants. Various enzymatic treatments were used to characterize different gAb heavy chain C-terminal lysine and sialic acid variants. In addition, an unknown derivative with the additional mass of 140 Da was found in transgenic gAb using mass spectrometry (MS). The modification sites were identified as the N-termini of gAb light chains and heavy chains using Q-TOF MS. Characterization of transgenic gAb isoforms was facilitated by utilizing different enzymes, CIEX and MS techniques. A maleuric acid modification on the N-terminal portion of gAb was shown to be consistent with the available data characterizing this new derivative of transgenic gAb isoforms in goat milk.

Animals↗
PubMed2003

Endothelin-1: a multifunctional molecule in cancer.

Endothelin-1 is a small vasoconstrictor peptide that was first identified in 1988. Here we review the evidence implicating ET-1 in tumorigenesis. In particular, we concentrate on the role of ET-1 in mitogenesis, apoptosis, angiogenesis, tumour invasion and metastasis, and discuss the potential for endothelin-system modulation as an adjuvant therapeutic strategy.

Apoptosis↗
PubMed2001

Characterization of noncovalent complexes of recombinant human monoclonal antibody and antigen using cation exchange, size exclusion chromatography, and BIAcore.

The binding of fully human monoclonal antibodies (MAbs) D2E7 and 2SD4 to their antigen, human tumor necrosis factor-alpha (TNFalpha), was investigated by BIAcore, cation exchange (CIEX), and size exclusion liquid chromatography (SEC) using ultraviolet and laser light scattering detectors. D2E7 has a higher affinity for TNFalpha than 2SD4 and the two antibodies (Abs) differ by 12 amino acids in the antigen (Ag) binding regions. A BIAcore biosensor instrument was used to determine the association, k(on) and dissociation, k(off), rate constants for the binding of TNFalpha to D2E7 and 2SD4. The HPLC methods were used to resolve and to study D2E7, 2SD4, and TNFalpha molecules and the noncovalent complexes of D2E7 and 2SD4 with TNFalpha. The CIEX method demonstrated that all D2E7 charged-variants bound TNFalpha equally well. There was no preferential binding for any one of D2E7 charged-variants to TNFalpha. D2E7 and 2SD4 Abs were resolved by the CIEX method. When a mixture of D2E7 and 2SD4 was mixed with excess TNFalpha, D2E7. TNFalpha complexes were formed before any 2SD4. TNFalpha complexes. Thus, the CIEX method was able to rank the affinities of the MAbs. D2E7 and TNFalpha formed complexes of 600-5000 kDa. The molecular weights of various D2E7. TNFalpha complexes were determined by a SEC method with light scattering (LS) and refractive index (RI) detectors. Upon overnight incubation, a 598-kDa complex emerged as the most stable and the only D2E7. TNFalpha complex. The molar ratio of D2E7 to TNFalpha in this complex was approximately 1:1. Based on molecular weights and the molar ratio, an immune complex, consisting of alternating three D2E7 and three TNFalpha molecules, is proposed as the most stable complex.

Antibodies, Monoclonal↗
PubMed2001

Myelinated dendrites in the mormyrid electrosensory lobe.

This is the third paper in a series on the morphology, immunohistochemistry, and synaptology of the mormyrid electrosensory lateral line lobe (ELL). The ELL is a highly laminated, cerebellum-like structure in the rhombencephalon that subserves an active electric sense: Objects in the nearby environment are detected on the basis of changes in the reafferent electrosensory signals that are generated by the animal's own electric organ discharge. This paper concentrates on the intermediate (cell and fiber) layer of the medial zone of the ELL and pays particular attention to the large multipolar neurons of this layer (LMI cells). LMI cells are gamma-aminobutyric acid (GABA)ergic and have one axon and three to seven proximal dendrites that all become myelinated after their last proximal branching point. The axon projects to the contralateral homotopic region and has ipsilateral collaterals. Both ipsilaterally and contralaterally, it terminates in the deep and superficial granular layers. The myelinated dendrites end in the deep granular layer, where they most likely do not make postsynaptic specializations, but do make presynaptic specializations, similar to those of the LMI axons. Because it is not possible to distinguish between axonal and dendritic LMI terminals in the granular layer, the authors refer to both as LMI terminals. These are densely filled with small, flattened vesicles and form large appositions with ELL granular cell somata and dendrites with symmetric synaptic membrane specializations. LMI cells do not receive direct electrosensory input on their somata, but electrophysiological recordings suggest that they nevertheless respond strongly to electrosensory signals (Bell [1990] J. Neurophysiol. 63:303-318). Consequently, the authors speculate that the myelinated dendrites of LMI cells are excited ephaptically (i.e., by electric field effects) by granular cells, which, in turn, are excited via mixed synapses by mormyromast primary afferents. The authors suggest that this ephaptic activation of the GABAergic presynaptic terminals of the myelinated dendrites may trigger immediate synaptic release of GABA and, thus, may provide a very fast local feedback inhibition of the excited granular cells in the center of the electrosensory receptive field. Subsequent propagation of the dendritic excitation down the myelinated dendrites to the somata and axon hillocks of LMI cells probably generates somatic action potentials, resulting in the spread of inhibition through axonal terminals to a wide region around the receptive field center and in the contralateral ELL. Similar presynaptic myelinated dendrites that subserve feedback inhibition, until now, have not been described elsewhere in the brain of vertebrates.

Animals↗
PubMed2000

sem-4 promotes vulval cell-fate determination in Caenorhabditis elegans through regulation of lin-39 Hox.

Vulval cell-fate determination in Caenorhabditis elegans requires the action of numerous gene products, including components of the Ras/Raf/MAPK signaling cascade and the hox gene lin-39. sem-4 encodes a zinc finger protein with previously characterized roles in fate specification of sex myoblasts, coelomocytes, and multiple neuronal lineages in C. elegans (M. Basson and R. Horvitz, 1996, Genes Dev. 10, 1953-1965). By characterizing three new alleles of sem-4 that we identified in a screen for vulval-defective mutants, we determined that loss of sem-4 activity results in abnormal specification of the secondary vulval cell lineages. We analyzed sem-4 interactions with other genes involved in vulval differentiation and determined that sem-4 does not function directly in the Ras-mediated signal transduction pathway but acts in close association with and upstream of lin-39 to promote vulval cell fate. We demonstrate that sem-4 regulates lin-39 expression and propose that sem-4 is a regulator of lin-39 in the vulval cell-fate determination pathway that may act to link lin-39 to incoming signals.

Alleles↗
PubMed2000

An outbreak of type C botulism in 12 horses and a mule.

A USDA Early Response Team investigated deaths of several horses and a mule in northern Arizona at the request of local animal health officials. Thirteen animals (12 horses and 1 mule) housed at 5 facilities in a 7.4 square mile area died between August 1998 and January 1999. Clinical signs consisted of muscular weakness that rapidly progressed to lateral recumbency. Ten animals had paresis of the tongue, throat, or lips. Affected animals appeared alert and were interested in eating and drinking, even while recumbent. All 13 animals were euthanatized. Clostridium botulinum type C was isolated from feces or intestinal contents from 3 affected horses. Preformed toxin was detected in samples of soil and bird droppings collected from a nearby horse burial site. It was hypothesized that the outbreak was a result of birds, presumably ravens, feeding at the burial site and at horse facilities in the area that transferred toxin to the affected animals.

Animals↗
PubMed2000

The midbrain precommand nucleus of the mormyrid electromotor network.

The functional role of the midbrain precommand nucleus (PCN) of the electromotor system was explored in the weakly electric mormyrid fish Gnathonemus petersii, using extracellular recording of field potentials, single unit activity, and microstimulation in vivo. Electromotor-related field potentials in PCN are linked in a one-to-one manner and with a fixed time relationship to the electric organ discharge (EOD) command cycle, but occur later than EOD command activity in the medulla. It is suggested that PCN electromotor-related field potentials arise from two sources: (1) antidromically, by backpropagation across electrotonic synapses between PCN axons and command nucleus neurons, and (2) as corollary discharge-driven feedback arriving from the command nucleus indirectly, via multisynaptic pathways. PCN neurons can be activated by electrosensory input, but this does not necessarily activate the whole motor command chain. Microstimulation of PCN modulates the endogenous pattern of electromotor command in a way that can mimic the structure of certain stereotyped behavioral patterns. PCN activity is regulated, and to a certain extent synchronized, by corollary discharge feedback inhibition. However, PCN does not generally function as a synchronized pacemaker driving the electromotor command chain. We propose that PCN neurons integrate information of various origins and individually relay this to the command nucleus in the medulla. Some may also have intrinsic, although normally nonsynchronized, pacemaker properties. This descending activity, integrated in the electromotor command nucleus, will play an important modulatory role in the central pattern generator decision process.

Action Potentials↗
PubMed2000

Reversible associative depression and nonassociative potentiation at a parallel fiber synapse.

The electrosensory lobe (ELL) of mormyrid electric fish is one of several cerebellum-like sensory structures in fish that remove predictable features of the sensory inflow. This adaptive process obeys anti-Hebbian rules and appears to be mediated by associative depression at the synapses between parallel fibers and Purkinje-like cells of ELL. We show here that there is also a nonassociative potentiation at this synapse that depends only on the repeated occurrence of the EPSP. The depression can be reversed by the potentiation and vice versa. Finally, we show that the associative depression requires NMDA receptor activation, changes in postsynaptic calcium, and the occurrence of a postsynaptic dendritic spike within a few milliseconds following EPSP onset.

2-Amino-5-phosphonovalerate↗
PubMed2000

Intracellular targets of cyclin-dependent kinase inhibitors: identification by affinity chromatography using immobilised inhibitors.

BACKGROUND: Chemical inhibitors of cyclin-dependent kinases (CDKs) have great therapeutic potential against various proliferative and neurodegenerative disorders. Olomoucine, a 2,6,9-trisubstituted purine, has been optimized for activity against CDK1/cyclin B by combinatorial and medicinal chemistry efforts to yield the purvalanol inhibitors. Although many studies support the action of purvalanols against CDKs, the actual intracellular targets of 2,6, 9-trisubstituted purines remain unverified. RESULTS: To address this issue, purvalanol B (95. ) and an N6-methylated, CDK-inactive derivative (95M. ) were immobilized on an agarose matrix. Extracts from a diverse collection of cell types and organisms were screened for proteins binding purvalanol B. In addition to validating CDKs as intracellular targets, a variety of unexpected protein kinases were recovered from the 95. matrix. Casein kinase 1 (CK1) was identified as a principal 95. matrix binding protein in Plasmodium falciparum, Leishmania mexicana, Toxoplasma gondii and Trypanosoma cruzi. Purvalanol compounds also inhibit the proliferation of these parasites, suggesting that CK1 is a valuable target for further screening with 2,6,9-trisubstituted purine libraries. CONCLUSIONS: That a simple batchwise affinity chromatography approach using two purine derivatives facilitated isolation of a small set of highly purified kinases suggests that this could be a general method for identifying intracellular targets relevant to a particular class of ligands. This method allows a close correlation to be established between the pattern of proteins bound to a small family of related compounds and the pattern of cellular responses to these compounds.

Amino Acid Sequence↗
PubMed2000

Photosensitized light-induced damage of IRBP (interphotoreceptor retinoid-binding protein): effects on binding properties.

PURPOSE: To determine if IRBP (interphotoreceptor retinoid-binding protein) is damaged following irradiation by visible light in the presence of bound all-trans retinal. METHODS: Following irradiation of the IRBP-all-trans retinal complex, the retinal was removed and damage to IRBP measured as loss of titratable thiol groups, loss of tryptophan fluorescence, and changes in retinol-binding-induced fluorescence. RESULTS: IRBP irradiated by itself showed only minimal loss of tryptophan fluorescence; this loss was substantially increased by irradiation in the presence of all-trans retinal. Thiol groups and retinol-binding activity were also shown to be reduced. The damage to IRBP seemed to involve photosensitization by the all-trans retinal, which was in turn protected from bleaching by the IRBP. The binding affinity was shown to be reduced ten-fold following irradiation. CONCLUSION: In the eye, IRBP can stabilise vitamin A and debatably may be responsible for transport of different forms of vitamin A between the photoreceptor cells and pigment epithelium. If this is the case, it would play a key role in rhodopsin regeneration after bleaching. IRBP also appears to be necessary to sustain photoreceptor cells. Light was shown to cause photosensitized damage to IRBP, and thus might impair the regeneration process and photoreceptor viability.

Animals↗
PubMed2000

Infection of polarized primary epithelial cells from rat uterus with Chlamydia trachomatis: cell-cell interaction and cytokine secretion.

PROBLEM: The objective of this study was to examine the susceptibility of rat uterine epithelial cells (UEC) to infection with Chlamydia trachomatis and to study the epithelial-stromal interactions following infection. METHOD OF STUDY: UEC were isolated from adult rats and grown in culture. Polarized, confluent monolayers of UEC were infected with 10(6) IFU/well C. trachomatis (MoPn). In order to confirm infection, MoPn was labeled with a fluorescent tracking dye, PKH-26, and then used in epithelial cell infections. Transepithelial resistances were measured prior to and following infection to test the effect of Chlamydia on the integrity of the epithelial monolayers. In other experiments, polarized epithelial cultures were infected in the presence and absence of stromal cells. Media was collected from the apical and basolateral compartments of the cultures before and after infection and analyzed for cytokines IL-1alpha and TNF-alpha. RESULTS: Epithelial cell cultures infected with PKH-26 labeled MoPn were examined 4-5 days later. Bacterial inclusions were detected inside epithelial cells indicating infection had occurred. Co-localization of PKH-26 labeled bacteria with FITC-labelled anti-Chlamydia antibody on the epithelial cells confirmed infection. No changes were found in resistance across the monolayers of epithelial cells in the presence or absence of infection. ELISA results indicate that UEC secrete IL-1alpha constitutively in citro. Stromal cells secrete very little IL-1alpha. When stromal cells were co-incubated with epithelial cells there was a decrease in the amount of IL-1alpha secreted by epithelial cells 48 hr post-infection. On the other hand, maximum TNF-alpha was found in stromal cells. both with and without infection. Epithelial cells, in these studies made very little TNF-alpha. CONCLUSIONS: These results show that primary rat epithelial cells can be infected with Chlamydia in vitro. Epithelial and stromal cells from uteri of adult rats make IL-1alpha and TNF-alpha in vitro both prior to and following infection with Chlamydia. This system can be used to analyze the role played by epithelial-stromal interactions in providing protection on this mucosal surface.

Animals↗
PubMed2000

Rapid activation of GABAergic interneurons and possible calcium independent GABA release in the mormyrid electrosensory lobe.

The primary afferent fibers from the electroreceptors of mormyrid electric fish terminate centrally in the granular layer of the electrosensory lobe (ELL). This study examines the excitatory and inhibitory processes that take place in this layer using an in vitro slice preparation and field potentials evoked by stimulation of primary afferent fibers in the deep fiber layer of ELL. The postsynaptic response to stimulation of the afferent fibers was still present after blocking chemical transmission in three different ways: by adding glutamate receptor antagonists to the medium, by substituting a nominally calcium-free medium for normal medium, and by blocking calcium channels with cadmium. Blockade of chemical transmission was demonstrated by disappearance of control responses to parallel fiber stimulation. The continued presence of a postsynaptic response in the absence of chemical excitation is consistent with previous anatomic and physiological evidence for electrical synapses between afferent fibers and granular cells in ELL. Granular cell activation by primary afferent fibers was followed by a powerful, short-latency inhibition mediated by GABA and GABA(A) receptors, as indicated by a large increase in the postsynaptic response to afferent fiber stimulation following application of the GABA(A) receptor antagonist, bicuculline. Bicuculline caused a marked increase of the postsynaptic response even after chemical synaptic excitation had been blocked by glutamate receptor antagonists, by a calcium-free medium, or by cadmium. Thus activation of the inhibitory interneurons responsible for GABA release did not require chemical excitation. Nonchemical excitation of the inhibitory interneurons could be mediated either by electrical synapses between afferent fibers and inhibitory interneurons, or by nonsynaptic activation of the large GABAergic terminals that are known to be present on granular cells. The marked increase of the postsynaptic response caused by bicuculline in a calcium-free medium or in the presence of cadmium suggests that the release of GABA by inhibitory terminals was not entirely dependent on calcium influx. This effect of bicuculline on the postsynaptic response in a calcium-free medium or in the presence of cadmium was markedly reduced by prior addition of the GABA transporter antagonist, nipecotic acid. Thus calcium-independent release of GABA may occur in ELL and may be partly dependent on reversal of a GABA transporter. Rapid and powerful inhibition at the first stage in the processing of electrosensory information could serve to enhance the small differences in latency among afferent fibers that appear to encode small differences in stimulus intensity.

Animals↗
PubMed2000

Audit of emergency medical admissions during the winter 1997/1998.

OBJECTIVES: To test the hypothesis that a significant cause of rising medical admissions over the Christmas and New Year period is the failure of social and medical systems which support patients at home for most of the year; and to understand the reasons for admission during the winter months of an at risk population and to identify possible avoidable admissions. DESIGN: Structured interviews were conducted with patients within 48 hours of admission to collect information on home circumstances and contact with social, medical and primary care in the year prior to admission. In cases where the patient was unable to provide this information, case notes and nursing notes were reviewed. Discharge diagnosis was recorded at the end of the study period. SUBJECTS: Five hundred and thirty nine patients defined as an "at risk group" who were admitted to hospital as emergency medical admissions between 1st December 1997 and 31st January 1998. RESULTS: Subjects were predominantly female (58%). The major diagnostic groups were cardiac (27%) and respiratory (22%). Respondents indicated no change in primary care in the period preceding their admission, with the exception of day centre support. Sixty seven percent of patients fully interviewed received help from carers, friends or relatives with daily tasks and only seven (3%) reported receiving less input prior to admission. Forty five percent of these patients had seen a GP within the month prior to their admission. CONCLUSIONS: The study results showed no breakdown in either social or family support. In the group interviewed, the proportion who had experienced any decrease in social support during the period prior to admission was very small.

Aged↗
PubMed1999

Characterization of recombinant human monoclonal tissue necrosis factor-alpha antibody using cation-exchange HPLC and capillary isoelectric focusing.

Cation-exchange liquid chromatography (CIEX) and capillary isoelectric focusing (cIEF) methods have been developed for the routine analysis of a recombinant, human anti-tumor necrosis factor monoclonal antibody D2E7. Both of these methods can separate heavy-chain C-terminal variants of this antibody. Various enzymatic digestion methods have also been developed for the identification of the antibody C-termini lysine (Lys) variants. A comparison of conventional CIEX-HPLC and cIEF methods has been made for the analysis of antibodies. cIEF can also be used to determine the isoelectric points (pI) of antibody variants based on the use of internal pI standards. Different C-termini Lys variants have been separated and collected from the CIEX column and subsequently analyzed by cIEF and mass spectrometry.

Antibodies, Monoclonal↗