Biomedical subjects

William Welch

Publications and source records attributed to William Welch.

18 recordsLinked to original sources

PubMed2006

The interaction of an impermeant cation with the sheep cardiac RyR channel alters ryanoid association.

In previous studies, we have demonstrated that the interaction of ryanoids with the sarcoplasmic reticulum Ca(2+)-release channel [ryanodine receptor (RyR)] incorporated into planar lipid bilayers reduced the effectiveness of tetraethylammonium (TEA(+)) as a blocker of K(+) translocation (J Gen Physiol 117: 385-393, 2001). In the current study, we investigated both the effect of TEA(+) on [(3)H]ryanodine binding and the actions of this impermeant cation on the interaction of the reversible ryanoid 21-amino-9alpha-hydroxyryanodine with individual, voltage-clamped RyR channels. A dose-dependent inhibition of [(3)H]ryanodine binding was observed in the presence of TEA(+), suggesting that the cation and alkaloid compete for access to a common site of interaction. Single channel studies gave further insights into the mechanism of the competition between the two classes of ligands. TEA(+) decreases the association rate of 21-amino-9alpha-hydroxyryanodine with its receptor, whereas the dissociation rate of the ryanoid from the channel was unaffected. Our results demonstrate that TEA(+) inhibits both K(+) translocation through RyR, and ryanoid interaction at the high affinity ryanodine site on the channel. These actions involve binding of TEA(+) to different, but weakly interacting, sites in the RyR channel.

Animals↗

PubMed2006

Convergence of heat shock protein 90 with ubiquitin in filamentous alpha-synuclein inclusions of alpha-synucleinopathies.

Heat shock proteins (Hsps) facilitate refolding of denatured polypeptides, but there is limited understanding about their roles in neurodegenerative diseases characterized by misfolded proteins. Because Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy are alpha-synucleinopathies characterized by filamentous alpha-synuclein (alpha-syn) inclusions, we assessed which Hsps might be implicated in these disorders by examining human brain samples, transgenic mouse models, and cell culture systems. Light and electron microscopic multiple-label immunohistochemistry showed Hsp90 was the predominant Hsp examined that co-localized with alpha-syn in Lewy bodies, Lewy neurites, and glial cell inclusions and that Hsp90 co-localized with alpha-syn filaments of Lewy bodies in PD. Hsp90 levels were most predominantly increased in PD brains, which correlated with increased levels of insoluble alpha-syn. These alterations in Hsp90 were recapitulated in a transgenic mouse model of PD-like alpha-syn pathologies. Cell culture studies also revealed that alpha-syn co-immunoprecipitated preferentially with Hsp90 and Hsc70 relative to other Hsps, and exposure of cells to proteasome inhibitors resulted in increased levels of Hsp90. These data implicate predominantly Hsp90 in the formation of alpha-syn inclusions in PD and related alpha-synucleinopathies.

Adult↗

PubMed2005

The Gln4863Ala mutation within a putative, pore-lining trans-membrane helix of the cardiac ryanodine receptor channel alters both the kinetics of ryanoid interaction and the subsequent fractional conductance.

The specific, high-affinity interaction of the plant toxin ryanodine with its molecular target the ryanodine receptor channel (RyR) has been instrumental in RyR research. Alanine scanning of putative pore regions of mouse RyR2 has highlighted the amino acid Gln4863, predicted to lie within trans-membrane helix TM10, as an important determinant of ryanodine binding. We have investigated the effects of several ryanodine derivatives, guanidinopropionylryanodine, 21-p-nitrobenzoylamino-9alpha-hydroxyryanodine, 8beta-amino-9alpha-hydroxyryanodine, and 21-amino-9alpha-hydroxyryanodine, with the mouse Q4863A RyR2 mutant at the single-channel level. Our results demonstrate that the rate of dissociation of all ryanoids investigated is increased by the mutation. The modification of channel function after ryanoid binding is qualitatively similar for wild-type and mutant, but in several cases, single-channel conductances were increased with Q4863A. These novel findings have been interpreted within the framework of existing comparative molecular field analysis studies on ryanoids. We suggest that replacement of a glutamine by an alanine residue at position 4863 causes RyR2 to simultaneously alter interactions with both ends of the ryanoid molecule.

Alanine↗

PubMed2005

Voltage-sensitive equilibrium between two states within a ryanoid-modified conductance state of the ryanodine receptor channel.

We have investigated the influence of transmembrane holding potential on the kinetics of interaction of a cationic ryanoid, 8beta-amino-9alpha-hydroxyryanodine, with individual ryanodine receptor (RyR) channels and on the functional consequences of this interaction. In agreement with previous studies involving cationic, neutral, and anionic ryanoids, both rates of association and dissociation of the ligand are sensitive to transmembrane potential. A voltage-sensitive equilibrium between high- and low-affinity forms of the receptor underlies alterations in rates of association and dissociation of the ryanoid. The interaction of 8beta-amino-9alpha-hydroxyryanodine with RyR influences the rate of cation translocation through the channel. With this ryanoid bound, the channel fluctuates between two clearly resolved subconductance states (alpha and beta). We interpret this observation as indicating that with 8beta-amino-9alpha-hydroxyryanodine bound, the pore of the RyR channel exists in two essentially isoenergetic conformations with differing ion-handling properties. The equilibrium between the alpha- and beta-states of the RyR-8beta-amino-9alpha-hydroxyryanodine complex is sensitive to transmembrane potential. However, the mechanisms determining this equilibrium differ from those responsible for the voltage-sensitive equilibrium between high- and low-affinity forms of the receptor.

Animals↗

PubMed2005

Ovarian cancer is a heterogeneous disease.

The purpose of this study was to systematically evaluate the molecular profiles of different histologic types of epithelial ovarian cancer before the disease has metastasized beyond the ovary. Stage 1 epithelial ovarian cancers were chosen for analysis of early genetic events associated with different cell types. Allelotyping of 47 cases was performed using 224 polymorphic markers. Analysis with Fisher's exact test found markers specific for grade 3 tumors and clear cell histology. Hierarchal clustering analysis using dChip software revealed that the pattern of allele loss in eight regions on four chromosomes led to grouping of grade 3 tumors, endometrioid (grades 1 and 2) tumors, and clear cell tumors. We conclude that ovarian cancer is a heterogeneous disease in which histologic phenotypes correlate with distinct genetic patterns.

Adenocarcinoma, Clear Cell↗

PubMed2005

The carotid-vertebral space: an 'extended' lateral window to the ventromedial cranial base and lower craniocervical junction.

We describe a unique method of accessing the ventromedial skull base and lower craniocervical junction. Our method employs a trajectory between that of the more anterior transoral or retropharyngeal approaches and the various posterior or posterolateral skull base approaches. This "extended" lateral approach allows surgeons to resect very large tumors of the skull base through a single incision. The operative field is more extensive than that achieved with other approaches; it extends from the cerebellar hemisphere to the extradural ventral upper cervical spine, and it provides access to tissue outside the spinal canal, such as the ventral strap muscles. We describe our use of this approach during a single-stage resection of a large hemangiopericytoma in a 37-year-old man.

Acyclovir↗

PubMed2004

Emissions and demonstration of an emission control technology for small two-stroke utility engines.

Small utility engines represent an important contribution to the emissions inventory and have been subjected to increasingly stringent regulations in recent years. For this project, a Tanaka two-stroke engine was tested in its original condition and with a modified fuel/oil injection system. The modified fuel/oil injection system applied to the Tanaka two-stroke engine resulted in significant emissions reductions of approximately 52% for carbon monoxide (CO), 70% for total hydrocarbons (THC), 70% for particulate matter (PM), and 67% for the regulated THC + nitrogen oxides metric. This technology met the California Air Resources Board's 2000 model-year regulations for all pollutants, with the exception of slightly higher PM emissions. Two additional two-stroke engines were tested under a new condition and after at least 100 hr of use to examine the effects of deterioration on in-use, two-stroke engines. For one engine, CO and PM emissions more than tripled after 162 hr of operation in the field, with smaller increases also observed for THC (20%). For the second engine, significant repairs were required throughout the 100 operating hours, which counteracted the effects of the emissions deterioration and resulted in lower CO and THC emissions.

Air Pollutants↗

PubMed2004

A model of the putative pore region of the cardiac ryanodine receptor channel.

Using the bacterial K+ channel KcsA as a template, we constructed models of the pore region of the cardiac ryanodine receptor channel (RyR2) monomer and tetramer. Physicochemical characteristics of the RyR2 model monomer were compared with the template, including homology, predicted secondary structure, surface area, hydrophobicity, and electrostatic potential. Values were comparable with those of KcsA. Monomers of the RyR2 model were minimized and assembled into a tetramer that was, in turn, minimized. The assembled tetramer adopts a structure equivalent to that of KcsA with a central pore. Characteristics of the RyR2 model tetramer were compared with the KcsA template, including average empirical energy, strain energy, solvation free energy, solvent accessibility, and hydrophobic, polar, acid, and base moments. Again, values for the model and template were comparable. The pores of KcsA and RyR2 have a common motif with a hydrophobic channel that becomes polar at both entrances. Quantitative comparisons indicate that the assembled structure provides a plausible model for the pore of RyR2. Movement of Ca2+, K+, and tetraethylammonium (TEA+) through the model RyR2 pore were simulated with explicit solvation. These simulations suggest that the model RyR2 pore is permeable to Ca2+ and K+ with rates of translocation greater for K+. In contrast, simulations indicate that tetraethylammonium blocks movement of metal cations.

Bacterial Proteins↗

PubMed2003

Residue Gln4863 within a predicted transmembrane sequence of the Ca2+ release channel (ryanodine receptor) is critical for ryanodine interaction.

Despite the pivotal role of ryanodine in ryanodine receptor (RyR) research, the molecular basis of ryanodine-RyR interaction remains largely undefined. We investigated the role of the proposed transmembrane helix TM10 in ryanodine interaction and channel function. Each amino acid residue within the TM10 sequence, 4844IIFDITFFFFVIVILLAIIQGLII4867, of the mouse RyR2 was mutated to either alanine or glycine. Mutants were expressed in human embryonic kidney 293 cells, and their properties were assessed. Mutations D4847A, F4850A, F4851A, L4858A, L4859A, and I4866A severely curtailed the release of intracellular Ca2+ in human embryonic kidney 293 cells in response to extracellular caffeine and diminished [3H]ryanodine binding to cell lysates. Mutations F4846A, T4849A, I4855A, V4856A, and Q4863A eliminated or markedly reduced [3H]ryanodine binding, but cells expressing these mutants responded to extracellular caffeine by releasing stored Ca2+. Interestingly these two groups of mutants, each with similar properties, are largely located on opposite sides of the predicted TM10 helix. Single channel analyses revealed that mutation Q4863A dramatically altered the kinetics and apparent affinity of ryanodine interaction with single RyR2 channels and abolished the effect of ryanodol, an analogue of ryanodine, whereas the single channel conductance of the Q4863A mutant and its responses to caffeine, ATP, and Mg2+ were comparable to those of the wild type channels. Furthermore the effect of ryanodine on single Q4863A mutant channels was influenced by the transmembrane holding potential. Together these results suggest that the TM10 sequence and in particular the Q4863 residue constitute an important determinant of ryanodine interaction.

Amino Acid Sequence↗

PubMed2003

An anionic ryanoid, 10-O-succinoylryanodol, provides insights into the mechanisms governing the interaction of ryanoids and the subsequent altered function of ryanodine-receptor channels.

We have investigated the interactions of a novel anionic ryanoid, 10-O-succinoylryanodol, with individual mammalian cardiac muscle ryanodine receptor channels under voltage clamp conditions. As is the case for all ryanoids so far examined, the interaction of 10-O-succinoylryanodol with an individual RyR channel produces profound alterations in both channel gating and rates of ion translocation. In the continued presence of the ryanoid the channel fluctuates between periods of normal and modified gating, indicating a reversible interaction of the ligand with its receptor. Unlike the majority of ryanoids, we observe a range of different fractional conductance states of RyR in the presence of 10-O-succinoylryanodol. We demonstrate that 10-O-succinoylryanodol is a very flexible molecule and propose that each fractional conductance state arises from the interaction of a different conformer of the ryanoid molecule with the RyR channel. The probability of channel modification by 10-O-succinoylryanodol is dependent on the transmembrane holding potential. Comparison of the voltage dependence of channel modification by this novel anionic ryanoid with previous data obtained with cationic and neutral ryanoids reveals that the major influence of transmembrane potential on the probability of RyR channel modification by ryanoids results from an alteration in receptor affinity. These investigations also demonstrate that the charge of the ryanoid has a major influence on the rate of association of the ligand with its receptor indicating that ionic interactions are likely to be involved in this reaction.

Animals↗

PubMed2003

Substrate specificity and kinetics for VP14, a carotenoid cleavage dioxygenase in the ABA biosynthetic pathway.

The plant growth regulator, abscisic acid (ABA), is synthesized via the oxidative cleavage of an epoxy-carotenoid. Specifically, a double bond is cleaved by molecular oxygen and an aldehyde is formed at the site of cleavage in both products. The Vp14 gene from maize encodes an oxidative cleavage enzyme for ABA biosynthesis and the recombinant VP14 protein catalyzes the cleavage reaction in vitro. The enzyme has a strict requirement for a 9-cis double bond adjacent to the site of cleavage (the 11-12 bond), but shows some plasticity in other features of carotenoids that are cleaved. A kinetic analysis with the 9-cis isomer of five carotenoids displays several substrate activity relationships. One of the carotenoids was not readily cleaved, but inhibited the cleavage of another substrate in mixed assays. Of the remaining four carotenoids used in this study, three of the substrates have similar V(max) values. The V(max) for the cleavage of one carotenoid substrate was significantly higher. Molecular modeling and several three-dimensional quantitative substrate-activity relationship programs were used to analyze these results. In addition to a 9-cis double bond, the presence and orientation of the ring hydroxyl affects substrate binding or the subsequent cleavage. Additional variations that affect substrate cleavage are proposed.

Abscisic Acid↗

PubMed2003

Structural characteristics that govern binding to, and modulation through, the cardiac ryanodine receptor nucleotide binding site.

Comparative molecular field analysis (CoMFA) predicts that the large electrostatic field around the phosphate groups of ATP plays a crucial role in stabilizing the open state of the cardiac ryanodine receptor (RyR) channel. We therefore investigated the effects of adenosine-5'-(beta,gamma-methylenetriphosphate) (AMP-PCP), an ATP analog with lower negative charge in this region, on the gating of the cardiac RyR channel. In the presence of 10 microM cytosolic Ca2+, AMP-PCP exhibited approximately 50% of the efficacy of ATP and optimal doses increased open probability (Po) to only 0.441 +/- 0.156 (n = 4), thus confirming the predictive ability of our preliminary CoMFA model. We also reveal that AMP-PCP has a higher affinity than ATP for the cardiac RyR, demonstrating that the structural properties required for tight binding to RyR differ from those necessary for recruiting long open states and high Po values. CoMFA identified very strong correlations between the structures of adenine-based ligands and their affinity for RyR and different (but also highly significant) correlations between structure and the ability to activate the channel. Analysis indicates that ATP may be more effective than other adenine nucleotides because it can convert the greatest amount of binding energy into conformational changes that stabilize the open channel state.

Adenine Nucleotides↗

PubMed2003

Use of Tisseel fibrin sealant in neurosurgical procedures: incidence of cerebrospinal fluid leaks and cost-benefit analysis in a retrospective study.

OBJECTIVE: To determine the clinical efficacy and cost effectiveness of using the fibrin sealant (FS) Tisseel (Baxter Healthcare Corp., Deerfield, IL) for patients undergoing anterior cranial base, infratemporal, and retromastoid surgical procedures. METHODS: A retrospective review was performed, comparing two matched populations of patients who underwent surgical procedures using anterior cranial, infratemporal, or retromastoid approaches to intracranial pathological lesions. The incidences of cerebrospinal fluid (CSF) leaks in matched groups treated with the FS Tisseel or treated without FS were compared. The costs of Tisseel use were examined in comparison with the costs of postoperative management of CSF leaks and/or tension pneumocranium with spinal drainage and occasionally surgical reexploration, when lumbar drainage failed. RESULTS: Patients who received the FS Tisseel exhibited no detectable postoperative CSF leaks or tension pneumocranium. Patients who did not receive Tisseel demonstrated 4 to 16% incidences of postoperative leaks, depending on the surgical approach used. The costs of treating those leaks far exceeded the costs of using Tisseel, even if it were used indiscriminately for all patients. CONCLUSION: This retrospective review indicates that the FS Tisseel reduces the incidence of postoperative CSF leaks and tension pneumocranium while reducing overall management costs. Further prospective study is needed to determine which patients can benefit most from FS use.

Adolescent↗

PubMed2002

Bow hunter's syndrome in the setting of contralateral vertebral artery stenosis: evaluation and treatment options.

STUDY DESIGN: A case report. OBJECTIVE: To illustrate a rare case of bow hunter's syndrome in a patient with significant contralateral vertebral artery (VA) occlusive disease. SUMMARY OF BACKGROUND DATA: Bow hunter's syndrome is an uncommon condition in which the VA is symptomatically occluded during neck rotation. This case is interesting in that the patient had what appeared to be a normal right VA and occluded left VA when the head was in the neutral position. When the head was rotated 45 degrees to the left, the patient's right VA was occluded (bow hunter's finding), and it became apparent that the left VA was not completely occluded (as it appeared in the neutral position angiogram) but rather was 90% stenosed. The complete occlusion appearance in the neutral position was an angiographic phenomenon caused by competitive flow through the open right VA. When the patient rotated his head to the left, he occluded his right VA and had insufficient blood flow through the left VA, thus creating a symptomatic ischemic state. METHODS: This case was studied using dynamic computed tomography imaging, single-photon emission computed tomography, transcranial Doppler ultrasound, brain stem auditory evoked potentials, and dynamic range-of-motion cerebral angiography. RESULTS: The patient demonstrated bow hunter's syndrome as documented on clinical examination and history. Transcranial Doppler studies, dynamic computed tomography scanning, and cerebral/cervical angiography confirmed the diagnosis and revealed an interesting angiographic pattern, which explained the patient's symptoms and findings only when angiographic flow patterns were taken into consideration. CONCLUSIONS: Bow hunter's syndrome should be suspected when a patient presents with reproducible vertebrobasilar symptoms on rotating the neck. Quantitative documentation using imaging and electroneurophysiologic tests is important when assessing this subjective process. Careful evaluation of the angiographic imagescan often help explain an odd flow pattern and provide the physician with a range of treatment options.

Blood Flow Velocity↗

PubMed2002

Influence of filgrastim (granulocyte colony-stimulating factor) on human immunodeficiency virus type 1 RNA in patients with cytomegalovirus retinitis.

Filgrastim, or granulocyte colony-stimulating factor, reverses neutropenia associated with human immunodeficiency virus type 1 (HIV-1) and cytomegalovirus (CMV) infections. During a trial of anti-CMV retinitis therapies coadministered with antiretroviral therapy, 2-4 plasma specimens of HIV-1 RNA were collected from 36 HIV-1-infected patients receiving filgrastim to prevent neutropenia and from 36 patients not receiving filgrastim. For both groups, the crude mean and mean rate of change of HIV-1 log(10) RNA levels were similar. Adjustment for covariates (CD4(+) T cell lymphocytes, virus load at enrollment, level of neutropenia and antiretroviral therapy [mainly non-highly active antiretroviral therapy], and anti-CMV therapy during follow-up) resulted in a mean log(10) HIV-1 RNA level for individuals receiving filgrastim versus those not receiving the drug of 5.11 versus 4.87 (P=.12) and respective log mean rates of change per month of -0.08 versus -0.21 (P=.08). This latter difference has borderline statistical significance, which suggests that filgrastim may reduce the decline of HIV-1 RNA loads.

AIDS-Related Opportunistic Infections↗

PubMed2002

Quantitative relationships between ryanoids, receptor affinity and channel conductance.

The review examines the relationship between the structure of several ryanodine analogs and (A) binding, (B) channel conductance, and (C) ligand binding kinetics. Comparative molecular field analysis (CoMFA) and comparative molecular similarity analysis (CoMSIA) are used to quantitatively assign structural correlations. Hydrogen bond donating (but not accepting) ability was found to be highly correlated with ligand affinity. Analysis of the correlation between hydrophobicity and ligand affinity indicates that, in general, deviation from the amphipathic nature of ryanodine weakens binding. Affinities and binding kinetics obtained in vivo are comparable to those obtained in the less-than-physiological in vitro conditions. Therefore, the structure-activity relationships surveyed are relevant to the living cell. The review presents arguments favoring the propositions that (A) the pyrrole is a major factor orienting the ligand in the receptor binding site and (B) that ryanoids alter ryanodine receptor function through allosteric mechanisms.

Animals↗

PubMed2002

Excess noise in modified conductance states following the interaction of ryanoids with cardiac ryanodine receptor channels.

The interaction of ryanodine with the ryanodine receptor (RyR) produces profound changes in channel function. Open probability increases dramatically and conductance is reduced. In this report we describe differences in the properties of reduced conductance states produced by the interaction of ryanodine derivatives with RyR channels. Some reduced conductance states are considerably noisier than the normal open state of the RyR channel. Inspection and analysis of these events reveals that the excess noise arises from transitions between two conductance states. Following the interaction of certain ryanodine derivatives, RyR channels undergo transitions between two conformations with slightly different ion-handling properties.

Animals↗

PubMed2002

Use of filgrastim as adjuvant therapy in patients with AIDS-related cytomegalovirus retinitis.

BACKGROUND: Neutropenia in AIDS predisposes to bacterial infection. Granulocyte colony-stimulating factor (filgrastim) can reverse neutropenia. OBJECTIVE: To determine the effects of filgrastim on bacterial infections, hospitalization, and mortality in patients with cytomegalovirus retinitis and AIDS. METHODS: Using a person-time analysis, a retrospective cohort study of filgrastim adjuvant therapy in three multicenter clinical trials of anti-cytomegalovirus therapy during the period 1990-1997 measured filgrastim use, bacterial infections, and mortality. RESULTS: Of 719 patients, 379 patients used filgrastim for 31% of the follow-up time. There was an inverse relationship between the 389 confirmed bacterial infections, including 186 bacteremias, and absolute neutrophil counts. Before adjustment for CD4 T-cells counts and antibiotic/antiretroviral therapy, filgrastim was associated with reduced risk of catheter-related bacteremia [relative risk (RR), 0.52; P = 0.02] and repeat bacterial infection (RR, 0.41; P = < 0.01). After adjustment, the RR of catheter-related bacteremia with filgrastim use was decreased (RRadj, 0.69; P = 0.16) and the RR of repeat bacterial infection with filgrastim use was of marginal significance (RRadj, 0.57; P = 0.07), possibly due to the confounding effect of trimethoprim-sulfamethoxazole on all bacteremia (RRadj, 0.55; P = < 0.01). Unrelated to bacteremia, filgrastim use was associated with a 56% reduction in mortality (P < 0.01). CONCLUSIONS: There was a large survival benefit associated with filgrastim use in this study but the reasons for this benefit are unclear. Although a reduction in crude risk of some bacterial infections with filgrastim use was detected, after adjustment for potentially confounding factors these risks were smaller and no longer statistically significant.

AIDS-Related Opportunistic Infections↗