Search PubMed⌕ Search

Biomedical subjects

K D Holmes

Publications and source records attributed to K D Holmes.

12 recordsLinked to original sources
PubMed2005

The NR1-4 C-terminus interferes with N-methyl-D-aspartate receptor-mediated excitotoxicity: evidence against a typical T/SXV-PDZ interaction.

The N-methyl-D-aspartate receptor (NMDAR) plays a key role in the neural plasticity that underlies learning and memory in vivo. The plasticity exhibited by NMDARs may also contribute to disease pathogenesis, as a number of disorders are caused or exacerbated by exaggerated NMDAR activity. The NMDAR is composed of two obligatory types of subunits, NR1 and NR2. These transmembrane proteins include large intracellular C-termini that have yet to be fully characterized. We have developed a three-color fluorescence system in order to visualize NMDAR expression in living cells. Using excitotoxicity as a proxy for exaggerated NMDAR activity, we analyzed the effect of over-expressing NR1-4 and NR2A C-terminal domains on exaggerated NMDAR function. We demonstrate that a determinant within the C-terminal domain of NR1-4 (C02') is important for NMDAR excitotoxicity, whereas no novel determinants were identified in the NR2A C-terminus. Through the use of heterologous cells, and by examining the interaction between the prototypical NMDAR-binding partner postsynaptic density-95 (PSD-95), we show that this effect is unlikely to be mediated through a classical interaction with PSD-95.

Animals↗
PubMed2002

The C-terminal C1 cassette of the N -methyl-D-aspartate receptor 1 subunit contains a bi-partite nuclear localization sequence.

The N -methyl-D-aspartate receptor (NMDAR) is a multimeric transmembrane protein composed of at least two subunits. One subunit, NR1, is derived from a single gene and can be subdivided into three regions: the N-terminal extracellular domain, the transmembrane regions, and the C-terminal intracellular domain. The N-terminal domain is responsible for Mg2+ metal ion binding and channel activity, while the transmembrane domains are important for ion channel formation. The intracellular C-terminal domain is involved in regulating receptor activity and subcellular localization. Our recent experiments indicated that the intracellular C-terminal domain, when expressed independently, localizes almost exclusively in the nucleus. An examination of the amino acid sequence reveals the presence of a putative nuclear localization sequence (NLS) in the C1 cassette of the NR1 intracellular C-terminus. Using an expression vector designed to test whether a putative NLS sequence is a valid, functional NLS, we have demonstrated that a bi-partite NLS does in fact exist within the NR1-1 C-terminus. Computer algorithms identified a putative helix-loop-helix motif that spanned the C0C1 cassettes of the C-terminus. These data suggest that the NR1 subunit may represent another member of a family of transmembrane proteins that undergo intramembrane proteolysis, releasing a cytosolic peptide that is actively translocated to the nucleus leading to alterations in gene regulation.

Amino Acid Sequence↗
PubMed2001

Distribution of an NMDA receptor:GFP fusion protein in sensory neurons is altered by a C-terminal construct.

The NMDA receptor plays an important role in mediating sensory input to the spinal cord. Domains within the C-terminus of the NMDA receptor bind to cytoskeletal proteins and facilitate membrane targeting and synaptic clustering, and may participate in regulation of receptor function. One strategy to manipulate NMDA receptor function is to express C-terminal constructs in neurons to disrupt synaptic clustering via competition for binding motifs in cytoskeletal proteins and postsynaptic densities. Biolistic particle-mediated gene transfer was used to deliver plasmid DNA into organotypic cultures of dorsal root ganglia (DRG). Fusion proteins consisting of recombinant (r)NMDA receptor subunit 1-1 (rNR1-1) deletion constructs and enhanced green fluorescent protein (GFP) were expressed in sensory neurons and demonstrated unique distribution patterns within the cell. Expression of the full length rNR1-1:GFP construct was cytosolic and localized to membranous patches similar to endogenous NR1-1 protein expression in sensory neurons. Expression of a construct containing only the C-terminus, GFP:C0C1C2, demonstrated nuclear and membranous localization. When the GFP:C0C1C2 construct was co-expressed with rNR1-1 in sensory neurons, membranous localization of rNR1-1 was disrupted. In contrast, co-expression of a C-terminal cassette lacking the C1 exon cassette, GFP:C0C2, with rNR1-1 did not alter the membranous distribution of rNR1-1. This observation verifies the utility of a gene transfer strategy to diminish membranous NR1-1 content by expressing a construct containing the C1 exon cassette.

Active Transport, Cell Nucleus↗
PubMed2000

Biolistic-mediated interleukin 4 gene transfer prevents the onset of type 1 diabetes.

We tested the efficacy of biolistic-mediated gene transfer as a noninvasive therapy for type 1 diabetes (T1D) in nonobese diabetic (NOD) mice by expression of murine interleukin 4 (mIL-4) cDNA. Epidermal delivery of 2 microg of DNA yielded transient detection of serum mIL-4, using a conventional cDNA expression vector. A vector stabilized by incorporation of the Epstein-Barr virus (EBV) EBNA1/oriP episomal maintenance replicon produced higher levels of serum mIL-4 that persisted for 12 days after inoculation. Although biolistic inoculation of either vector reduced insulitis and prevented diabetes, the protracted mIL-4 expression afforded by the EBV vector resulted in Th2-type responses in the periphery and pancreas and more significant protection from the onset of diabetes. Our studies demonstrate the efficacy of biolistic gene delivery of stabilized cytokine expression as a viable therapeutic approach to prevent the onset of T1D.

Animals↗
PubMed2000

A multi-mutant herpes simplex virus vector has minimal cytotoxic effects on the distribution of filamentous actin, alpha-actinin 2 and a glutamate receptor in differentiated PC12 cells.

To develop effective gene therapy techniques that target populations of neurons in the spinal cord, suitable vectors must be developed that will undergo efficient, retrograde transport from an appropriate peripheral site and will not be cytotoxic. Our previous work (LeVatte et al, 1998a) has demonstrated that a replication defective herpes simplex virus vector 14Hdelta3vhsZ, that has been substantially detoxified, is retrogradely transported from peripheral sites and can infect large numbers of the targeted spinal neurons. We plan to develop targeted gene therapy approaches designed to modulate the excitatory glutamatergic methyl-D-aspartate (NMDA) receptor in spinal cord neurons as a means of ameliorating a form of episodic high blood pressure that occurs after spinal cord injury. In this report, we demonstrate that, in differentiated PC12 cells, a neuronal-like cell line, the virus vector does not appear to alter aspects of the cytoskeletal architecture important to the proper distribution of the NMDA receptor. In turn, the distribution of endogenous NMDA receptor 1 subunit protein (NMDAR1) or a transfected NMDAR1-green fluorescent fusion protein was also found to be unaltered after vector infection. However, whereas endogenous NMDAR1 distribution was maintained, vector infection did tend to reduce the level of its expression. This drop in endogenous NMDAR1 expression coincided with the expression of the HSV immediate early genes ICP0 and ICP27 over the first 24-48 h. These results indicate that the 14Hdelta3vhsZ herpes simplex virus vector is suitable to use in future strategies to alter the level of gene expression in targeted populations of spinal cord neurons.

Actin Cytoskeleton↗
PubMed1991

Beta-blocker therapy: identification and management of side effects.

The purpose of this study was to develop and test a new beta-Blocker Visual Analog Scale designed to identify and quantify the impact that the side effects of beta-blocker therapy have on people's lives, and the self-management practices people use to mediate their influence. Instruments included the 20-item beta-Blocker Visual Analog Scale and the Profile of Mood States. Subjects had hypertension; 51 men were involved in a larger study involving antihypertensive medications and exercise, and 19 men and women were receiving beta-blocker therapy as first-line drug of choice. Estimates of internal consistency reliability, content validity, and concurrent and discriminant validity were moderately strong. The most problematic side effects were related to lack of sleep, vivid or active dreams, lack of energy and pep, diminished interest in sexual activity, and changes in vision. Among self-management practices used to mediate side effects were planning rest and activity periods, thinking carefully before reacting, and seeking out others for support.

Adaptation, Psychological↗
PubMed1990

Tolerance for rectosigmoid distention in irritable bowel syndrome.

Two hypotheses were tested: (a) lowered tolerance for balloon distention of the rectosigmoid in patients with irritable bowel syndrome is caused by a psychological tendency to exaggerate the painfulness of any aversive stimulus, and (b) contractions elicited by balloon distention are responsible for pain reports. Tolerance for stepwise distention of a balloon in the rectosigmoid was compared with tolerance for holding one hand in ice water in 16 irritable bowel patients, 10 patients with functional bowel disorder who did not satisfy restrictive criteria for irritable bowel, 25 lactose malabsorbers, and 18 asymptomatic controls. Contractile activity was measured 5 cm above and 5 cm below the distending balloon. Psychometric tests were used to assess neuroticism, anxiety, and depression, and a standardized psychiatric interview was administered. Patients with irritable bowel syndrome had significantly lower tolerance for balloon distention but not ice water, and balloon tolerance was not correlated with neuroticism or other psychological traits measured. Rectosigmoid and rectal motility were also not related to tolerance for balloon distention. Both hypotheses were rejected. A peripheral mechanism such as altered receptor sensitivity may be the cause of distention pain in irritable bowel syndrome.

Colon, Sigmoid↗
PubMed1989

Three-point alignment of zygoma fractures with miniplate fixation.

Over the years, depression of the malar eminence has been one of the most persistent complications of zygoma fractures, regardless of the fixation technique used. The use of a sublabial incision for direct visualization and alignment of the fracture lines at the infraorbital rim and lateral maxillary buttress is described. When coupled with single miniplate fixation of the zygoma across the frontozygomatic suture, the sublabial approach results in improved reduction of the fractures with rigid stabilization. Our experience with this approach, as well as with two-point wire fixation and with miniplate fixation without sublabial exposure, is reviewed.

Adolescent↗
PubMed1988

Tracheobronchial foreign bodies.

A retrospective review of 88 cases of foreign body aspiration was undertaken. The patients ranged in age from 5 months to 73 years; the peak incidences of foreign body aspiration occurred in children less than 3 years of age and in adults older than 50 years. Sixty-one of the 88 patients were male. Physical examination was abnormal in 61% of patients. The most common radiographic abnormality was inspiratory-expiratory abnormality, seen in 27% of patients. Rigid endoscopy under general anesthesia was the preferred method for removal of the aspirated material. Multiple foreign bodies were found in 5% of the patients. Tracheobronchial foreign bodies should, therefore, be strongly suspected in susceptible patient populations who present with a suggestive history, even when no physical or radiographic evidence can be seen. Patients should be carefully examined for multiple foreign bodies at the time of rigid endoscopic removal.

Adolescent↗
PubMedDate not supplied

Pseudomembranous colitis.

Explore the source record for details and available documents.

Enterocolitis, Pseudomembranous↗