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Biomedical subjects

R E Stephens

Publications and source records attributed to R E Stephens.

At least 19 recordsLinked to original sources
PubMed1999

Anatomy of the internal branch of the superior laryngeal nerve.

The purpose of this study was to determine the length and distribution of the branches of the internal branch of the superior laryngeal nerve (ibSLN) and describe the initial afferent pathway for the laryngeal cough reflex (LCR). On 25 sides of 19 cadaver specimens, the ibSLN and its branches were dissected from the greater cornu of the hyoid to the mucosa of the larynx and laryngopharynx. The location of these terminal fibers were confirmed by direct observation and fiberoptic laryngoscopy. In 21 specimens, the ibSLN coursed 6.95+/-3.71 mm before piercing the thyrohyoid membrane and splitting into superior, middle, and inferior rami. Four specimens split proximal to the thyrohyoid membrane. The superior ramus distributed to the mucosa of the piriform recess. In this study the large, middle ramus was a new finding and distributed branches to the mucosa of the vestibule of the larynx, specifically the quadrangular membrane. The length of the ibSLN from the greater cornu to the end of the middle ramus at quadrangular membrane was 28.52+/-4.61 mm. The termination of these fibers were confirmed by observation and direct laryngoscopy. The middle ramus probably conveyed the afferent component of the laryngeal cough reflex. The inferior ramus did not distribute to the vestibular mucosa.

Afferent Pathways↗
PubMed1999

Assessing the laryngeal cough reflex and the risk of developing pneumonia after stroke.

OBJECTIVE: To determine the effectiveness of a new reflex cough test, using nebulized tartaric acid, in the evaluation of the laryngeal cough reflex and the development of aspiration pneumonia. STUDY DESIGN: In this two-phase study, the cough test assessed the cough reflex in 161 stroke subjects. Phase 1 was a double-blinded prospective study of 40 subjects scheduled to have both modified barium swallow and the reflex cough test. Phase 1 subjects with an abnormal cough test showed an increased pneumonia incidence, and therefore, phase 2 was not blinded. In phase 2, 121 subjects were evaluated using the cough test; 38 received a modified barium swallow. Test results were compared using the Fisher exact test. RESULTS: A total of 131 subjects from both phases had a normal reflex cough test; none developed pneumonia (p < .01). Thirty subjects from both phases had abnormal reflex cough test results; 5 developed pneumonia. Modified barium swallow findings did not reliably indicate the risk for developing pneumonia. Specificity of a normal reflex cough test was 100%. CONCLUSION: The reflex cough test reliably evaluated the laryngeal cough reflex and the associated risk of developing aspiration pneumonia in stroke patients. Testing the laryngeal cough reflex may significantly reduce morbidity, mortality, and costs in stroke patients.

Aged↗
PubMed1998

Electrophoretic resolution of tubulin and tektin subunits by differential interaction with long-chain alkyl sulfates.

Tubulin dimers, formed from globular alpha and beta subunits, and the tektins, three equimolar alpha-helical proteins that form filaments, mutually associate to form the junctional regions of doublet and triplet microtubules. When evaluated by SDS-PAGE, the apparent molecular weights of these proteins can deviate substantially from their sequence molecular weights in a manner sensitive to both the source of SDS and the species of origin. The electrophoretic mobility of sperm tail flagellar tubulins and tektins from an echinoderm and a mollusc were studied systematically using detergent-free stacking and resolving gels with a running buffer containing pure sodium dodecyl sulfate augmented with fixed amounts of C10, C14, C16, or C18 alkyl sulfates. Although having no systematic effect on molecular weight standards, the presence of alkyl sulfates of increasing chain length progressively exaggerated the separation of tubulin subunits, similarly facilitated the separation of two normally comigrating tektins, yet minimally influenced the relative migration of adequately separated tektins. This phenomenon is most likely due to preferential binding of longer chain alkyl sulfates by specific hydrophobic regions of these otherwise similar proteins. The use of binary mixtures of pure alkyl sulfates, required in the running buffer alone, may prove useful for reproducibly separating other proteins that characteristically bind SDS anomalously.

Dimerization↗
PubMed1998

Tektins as structural determinants in basal bodies.

Tektins, present as three equimolar 47-55 kDa protein components, form highly insoluble protofilaments that are integral to the junctional region of outer doublet microtubules in cilia and flagella. To identify and quantify tektins in other compound microtubules such as centrioles or basal bodies, a rabbit antiserum was raised against tektin filaments isolated from Spisula solidissima (surf clam) sperm flagellar outer doublets and affinity-purified with nitrocellulose blot strips of tektins resolved by SDS- or SDS-urea-PAGE. These antibodies recognized analogous tektins in axonemes of organisms ranging from ctenophores to higher vertebrates. Quantitative immunoblotting established that outer doublet tektins occur in a 1:17 weight ratio to tubulin. Cilia and basal apparatuses were prepared from scallop gill epithelial cells; cilia and deciliated cells were prepared from rabbit trachea. Tektins were detected by immunoblotting in basal body-enriched preparations while tektins were localized to individual basal bodies by immunofluorescence. Supported by greater fluorescence in basal bodies than in adjacent axonemes in tracheal cells, analysis of basal apparatuses demonstrated both a proportionately greater ratio of tektin to tubulin (approximately 1:13) and two distinct solubility classes of tektins, consistent with tektins comprising the B-C junction of triplets in addition to the A-B junction as in doublets.

Animals↗
PubMed1998

X-ray induced DNA double-strand breaks in human sperm.

A methodology for quantifying DNA double-strand breaks in human sperm is described. Sperm from three healthy human donors on three separate days each were irradiated with 12.5, 25, 50 and 100 cGy X-rays. Linear dose-response effects were observed in migrated DNA from sperm nuclei when electrophoresed under neutral conditions. RNase and proteinase K treatments for longer duration were necessary, to decondense the chromatin and presumably to release the broken DNA for migration in the electrophoretic field in a dose-dependent manner. An increase in DNA migration was observed with as low as 12.5 cGy, but damage was observed in all samples at 25 cGy. No evidence of repair of these X-ray-induced DNA double-strand breaks was observed during a 2 h period.

Adult↗
PubMed1997

Microgel electrophoresis: sensitivity, mechanisms, and DNA electrostretching.

Based on the treatment of microgels to remove proteins, we speculate that proteins may be bound to DNA in the microgels even after electrophoresis. We speculate that some DNA single-strand breaks may be a reflection of these protein-DNA complexes. We suggest methods to limit such artifacts, and present data demonstrating a lymphocyte DNA double-strand break sensitivity of 12.5 rads and day-to-day reproducibility of microgel electrophoresis using these principles. Extending these principles, we describe DNA behavior during alkaline and neutral microgel electrophoresis based on observations of the stained DNA and its migration patterns. During microgel electrophoresis, individual DNA molecules behave as if anchored at one end while the other end is free to migrate in response to the electric field. We capitalize on this behavior by developing a neutral microgel method to stretch chromosomes.

DNA Damage↗
PubMed1997

Synthesis and turnover of embryonic sea urchin ciliary proteins during selective inhibition of tubulin synthesis and assembly.

When ciliogenesis first occurs in sea urchin embryos, the major building block proteins, tubulin and dynein, exist in substantial pools, but most 9 + 2 architectural proteins must be synthesized de novo. Pulse-chase labeling with [3H]leucine demonstrates that these proteins are coordinately up-regulated in response to deciliation so that regeneration ensues and the tubulin and dynein pools are replenished. Protein labeling and incorporation into already-assembled cilia is high, indicating constitutive ciliary gene expression and steady-state turnover. To determine whether either the synthesis of tubulin or the size of its available pool is coupled to the synthesis or turnover of the other 9 + 2 proteins in some feedback manner, fully-ciliated mid- or late-gastrula stage Strongylocentrotus droebachiensis embryos were pulse labeled in the presence of colchicine or taxol at concentrations that block ciliary growth. As a consequence of tubulin autoregulation mediated by increased free tubulin, no labeling of ciliary tubulin occurred in colchicine-treated embryos. However, most other proteins were labeled and incorporated into steady-state cilia at near-control levels in the presence of colchicine or taxol. With taxol, tubulin was labeled as well. An axoneme-associated 78 kDa cognate of the molecular chaperone HSP70 correlated with length during regeneration; neither colchicine nor taxol influenced the association of this protein in steady-state cilia. These data indicate that 1) ciliary protein synthesis and turnover is independent of tubulin synthesis or tubulin pool size; 2) steady-state incorporation of labeled proteins cannot be due to formation or elongation of cilia; 3) substantial tubulin exchange takes place in fully-motile cilia; and 4) chaperone presence and association in steady-state cilia is independent of background ciliogenesis, tubulin synthesis, and tubulin assembly state.

Animals↗
PubMed1996

Selective incorporation of architectural proteins into terminally differentiated molluscan gill cilia.

Incubation of excised gills from the bay scallop Aequipecten irradians with 3H-leucine demonstrates that many ciliary structural proteins can attain a degree of labeling approaching that previously reported for sea urchin or surf clam embryos undergoing ciliary turnover or regeneration. This labeling is not a consequence of any predominant incorporation into new cilia at the meristematic growth tips of the gill since tissue regions of varying maturity incorporate label into the same proteins at similar levels, with the most mature region having the highest incorporation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorographic analysis of isolated cilia, separated into detergent-soluble membrane/matrix and detergent-insoluble 9+2 axoneme fractions, reveals that 1) tubulin in the membrane/matrix fraction is labeled whereas tubulin in the axoneme is not; 2) no labeled dynein heavy chains are seen in either fraction; 3) the most heavily labeled axonemal components do not appear to any significant extent in the membrane/matrix fraction; and 4) after thermal depolymerization of the microtubules, nearly all labeled proteins reside in the in-soluble ninefold ciliary remnant, the most prominent being tektin A, an integral component of outer doublet microtubules. Further fractionation of the remnant with sarkosyl-urea to produce tektin filaments demonstrates two solubility classes of tekin A, only the more soluble of which is labeled. Very similar selective architectural protein labeling patterns have been reported for steady-state cilia of sea urchin embryos, and this may indicate a widespread turnover or exchange mechanism characteristic of cilia heretofore considered static.

Animals↗
PubMed1996

Substance P and neurokinin A metabolism by cultured human skeletal muscle myocytes and fibroblasts.

A recent study determined that cultured human skeletal muscle adult myoblasts, myotubes, and fibroblasts degraded angiotensins and kinins via neutral endopeptidase-24.11 (NEP-24.11: EC 3.4.24.11) and aminopeptidase N (APN: EC 3.4.11.2). Due to the possible importance of other peptides to skeletal muscle blood flow and function, the present study looked specifically at the metabolism of the neurokinins substance P (SP) and neurokinin A (NKA) by skeletal muscle peptidases. The results show that SP is degraded not only by NEP-24.11, but also sequentially by dipeptidyl(amino)peptidase IV (DAP IV: EC 3.4.14.5)/APN. NKA is unaffected by DAP IV but is metabolized by NEP-24.11 and APN. NEP-24.11 was inhibited by phosphoramidon (IC50 = 80 nM), thiorphan and ZINCOV, DAP IV by diprotin A (IC50 = 8 microM), and APN by amastatin (IC50 = 50 nM) and bestatin (IC50 = 100 microM). Skeletal muscle myocyte and fibroblast metabolism of SP and NKA may regulate local skeletal muscle vascular and extravascular functions including SP- and NKA-mediated nerve-induced vasodilation. Inhibition of both NEP-24.11 and DAP IV/APN may increase skeletal muscle blood flow and decrease peripheral vascular resistance via potentiation of local neurokinin levels.

Adult↗
PubMed1995

Thyronines and probucol inhibition of human capillary endothelial cell-induced low density lipoprotein oxidation.

Oxidized lipoproteins have been implicated as important factors in the pathogenicity of atherosclerosis. Thus, antioxidants play a significant role in inhibiting a critical step in atheroma progression. Previously, we demonstrated that thyronine analogs inhibit Cu(2+)-induced low density lipoprotein (LDL) oxidation. In the present study, we examined the effect of thyronine analogs on endothelial cell (EC)-induced LDL oxidation. LDL was incubated with or without EC in the presence or absence of various concentrations of thyronine, vitamin C, or probucol at 37 degrees in a humidified atmosphere (95% air, 5% CO2). Thyronine analogs, probucol, and vitamin C inhibited EC-induced LDL oxidation in a concentration-dependent manner. The concentration of each agent (microM) producing 50% inhibition (IC50) of EC-induced LDL oxidation for thiobarbituric acid reactive substances (TBARS) and electrophoretic mobility, respectively, was as follows: 0.294 and 0.417 for levothyroxine (L-T4); 0.200 and 0.299 for L-triiodothyronine (L-T3); 0.125 and 0.264 for dextro-thyroxine (D-T4); 0.203 and 0.304 for reversed triiodothyronine (rT3); 1.02 and 1.44 for probucol; and 13.6 and 14.9 for vitamin C. Thyroid binding globulin (TBG) inhibited EC-induced LDL oxidation; further, thyronines bound to TBG exhibited more antioxidant activity than unbound thyronines. Pretreatment of EC with any of the thyronines decreased the ability of EC to oxidize LDL. Also, our results showed that a synergistic interaction exists between vitamin C and T4 in the inhibition of EC-induced LDL oxidation. The T4 and TBG concentrations that inhibited LDL oxidation were in the physiological range. We conclude that T4, like the pharmacological agent probucol, reduces oxidative modification of LDL and thus may act as a natural inhibitor of atherogenesis.

Anticholesteremic Agents↗
PubMed1995

Ciliogenesis in sea urchin embryos--a subroutine in the program of development.

One major milestone in the development of the sea urchin embryo is the assembly of a single cilium on each blastomere just before hatching. These cilia are constructed both from pre-existing protein building blocks, such as tubulin and dynein, and from a number of 9 + 2 architectural elements that are synthesized de novo at ciliogenesis. The finite or quantal synthesis of certain key architectural proteins is coincident with ciliary elongation and proportional to ciliary length. Upon deciliation, the synthesis of architectural proteins occurs anew, a new cilium grows, and the stores of various building blocks are replenished. This routine of coordinated ciliary gene expression may be replayed experimentally many times without delaying normal development. The ability to regenerate cilia has allowed elucidation of these various protein synthetic relationships and has led to the discovery of the pathways by which membrane-associated tubulin and axoneme-associated architectural proteins are conveyed into the highly compartmentalized growing cilium. The sea urchin embryo thus provides a very convenient model system for studies of ciliary assembly and maintenance, coordinate gene expression and membrane dynamics.

Animals↗
PubMed1995

Dynein inner arm heavy chain identification in cAMP-activated flagella using class-specific polyclonal antibodies.

While studying cAMP-dependent dynein alpha-heavy chain phosphorylation, we found previously [Stephens and Prior, 1992: J. Cell Sci. 103:999-1012] that high salt extraction of sperm flagella from the mussel Mytilus edulis or the clam Spisula solidissima removed most visible dynein arms, accompanied by an amount of Mg+2-ATPase that correlated with the mass of dynein alpha- and beta-heavy chains removed. However, although almost devoid of ATPase activity, such extracted axonemes retained one third of the heavy chain mass as two sets of electrophoretically-distinct, vanadate-cleavable, non-phosphorylated proteins. To explore the nature of these dynein-like proteins, antibodies to the alpha- and beta-heavy chains were blot affinity-purified from a rabbit antiserum raised against gradient-purified Spisula 18-20S flagellar outer arm dynein. Although able to recognize common epitopes of the opposite chain type, neither the alpha- nor the beta-heavy chain antibody recognized the tightly-bound proteins in either species, proving that they are immunologically distinct. While the beta-antibody recognized its heavy chain homolog in gill cilia, the alpha-antibody did not, demonstrating immunological distinction between flagellar and ciliary dynein alpha-heavy chains. Immunization of a mouse with nitrocellulose strips containing one of the two tightly-bound Spisula flagellar proteins produced an antiserum that cross-reacted with each tightly-bound protein in both species and also recognized alpha- and beta-heavy chains. The anti-molluscan serum cross-reacted strongly with sea urchin sperm flagellar dynein B-, C-, and D-bands, considered to be inner arm components, but not with sea urchin outer arm alpha- or beta-heavy chains. These data indicate that the electrophoretically and immunologically distinct, tightly-bound proteins of molluscan flagella are inner arm dynein heavy chains.

Animals↗
PubMed1995

Cultured AIDS-related Kaposi's sarcoma (AIDS-KS) cells demonstrate impaired bioenergetic adaptation to oxidant challenge: implication for oxidant stress in AIDS-KS pathogenesis.

Despite its recognition as the most prevalent HIV associated cancer, speculation still abounds regarding the pathogenesis of AIDS-related Kaposi's sarcoma (AIDS-KS). However, it has been established that both cytokines, e.g. IL-6, and HIV-associated products, e.g., Tat, are integral in AIDS-KS cellular proliferation. Further, both experimental and clinical evidence is accumulating to link reactive oxygen intermediates (ROI) with both cytokine induction (primarily via nuclear factor-kappa B[NF-kappa B] dependent routes) as well as the subsequent cytokine, tumor necrosis factor alpha (TNF alpha) stimulation of HIV replication. Features of AIDS-KS patients, such as retention of phagocytes, presence of sustained immunostimulation, and a frequent history of KS lesions arising at traumatized sites, make oxidant stress a viable clinical factor in AIDS-KS development. Time course nucleotide profile analyses show that AIDS-KS cells have an inherent, statistically significant, biochemical deficit, even prior to oxidant stress, due to 1) a more glycolytic bioenergetic profile, resulting in lower levels of high energy phosphates (impairing capacity for glutathione [GSH] synthesis and DNA repair); 2) lower levels of NADPH (compromising the activities of GSSG reductase and peroxidase function of catalase); and 3) reduced levels of GSH (impeding both GSH peroxidase and GSH-S-transferases). Following exposure to physiologically relevant levels of H2O2, only the human microvascular endothelial cells (a putative AIDS-KS progenitor cell) responded with bioenergetic adaptations that reflected co-ordination of energy generating and cytoprotective pathways, e.g., retention of the cellular energy charge, increased NAD+, and an accentuation of the ATP, NADPH, and total adenine nucleotide differences relative to AIDS-KS cells. Also, some of the AIDS-KS strains retained intracellular GSSG subsequent to oxidant challenge, inviting the formation of deleterious protein mixed disulfides. While the results of our study address some AIDS-KS issues, they also raise an etiological question, i.e., Does the inability to tolerate oxidant stress arise in conjunction with AIDS-KS neoplastic development, or is it pre-existing in the population at risk? Regardless, use of antioxidant therapy (low risk/ potentially high benefit) in both the "at risk" population as well as in those individuals with active disease may prove a useful preventative and/or treatment modality.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Human microvascular endothelial cell-extracellular matrix interaction in cellular growth state determination.

We introduce two methods, both of which are based on cellular-extracellular matrix interaction, which will facilitate the study of human microvascular endothelial cells. One method describes the means to obtain a G1 population baseline in human microvascular endothelial cells. Because of the contribution of the extracellular matrix in endothelial cell growth, synchronization in G1 was possible only after the incorporation of angiostatic levels of heparin and hydrocortisone into the extracellular matrix. In the second method, we demonstrate that selective perturbation of human microvascular endothelial cell-extracellular matrix interactions results in the induction of a transitional growth state, between proliferative and differentiated growth states, in human microvascular endothelial cells. In the functional, microtubule formation assays, transitional growth state endothelial cells display rates that are indermediate between those obtained from differentiated and proliferative endothelial cells. Our results demonstrate the importance of the human microvascular endothelial cell-extracellular matrix interaction in the determination of cellular growth state. Our findings also imply that responsiveness of microvascular endothelial cells to their cellular-extracellular matrix environs is highest during the differentiated growth state.

Adolescent↗
PubMed1995

Angiotensin and bradykinin metabolism by peptidases identified in cultured human skeletal muscle myocytes and fibroblasts.

Angiotensin (ANG) and kinin metabolizing enzymes, angiotensin-converting enzyme (ACE; EC 3.4.15.1), neutral endopeptidase-24.11 (NEP-24.11; EC 3.4.24.11), and aminopeptidase M (AmM; EC 3.4.11.2), have recently been identified in a purified skeletal muscle glycoprotein fraction. We have analyzed the cellular localization of these enzymes. In cultured human skeletal muscle adult myoblasts, myotubes, and fibroblasts, kinins and angiotensins were metabolized by NEP-24.11 and AmM but not by ACE. NEP-24.11 degraded ANG II, ANG III. and bradykinin (BK) and converted ANG I to the active metabolite ANG(1-7). ANG III was converted to the novel ANG IV metabolite [des-Arg1]ANG III by AmM. These data suggest that, due to their abundance in the body, skeletal muscle myocytes and fibroblasts may play a major role in modulation of the systemic and local effects of angiotensins and kinins. This role could be particularly important in individuals receiving treatment with ACE inhibitors.

Aminopeptidases↗