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Biomedical subjects

R E Stephens

Publications and source records attributed to R E Stephens.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1995

Comparison of constitutive cytokine release in high and low histologic grade AIDS-related Kaposi's sarcoma cell strains and in sera from HIV+/KS+ and HIV+/KS- patients.

Kaposi's sarcoma (KS) is both an AIDS-defining disease and the most common HIV-associated malignancy. A cytokine-mediated pathogenesis for AIDS-KS is implicated because AIDS-KS-derived cell strains both respond to and express a variety of cytokines. We have reported the establishment of several (n = 18) AIDS-KS cell strains and determined that reduced exogenous growth factors are necessary to sustain proliferation in isolates from high histologic grade KS lesions. This current investigation explored the possibility that there are histologic grade-associated differences in either the qualitative and/or quantitative constitutive release of AIDS-KS growth stimulatory cytokines. Our findings showed that the incorporation of HTLV-II cytokine-rich conditioned media induced both qualitative and significant quantitative cytokine release, suggesting that exogenous growth promoters stimulate constitutive cytokine release. ELISA of our AIDS-KS cell strains demonstrated constitutive release of IL-6 (seven of seven), FGF-2 (five of seven), GM-CSF (three of seven), and IL-1 beta (one of seven). None of our AIDS-KS cell strains constitutively released detectable levels of Onco-M, IL-4, PDGF, TNF-alpha, or TNF-beta. In addition, we report that the method of cytokine result quantitation significantly affects reported cytokine levels. We determined that there was no significant histologic grade-dependent difference in the constitutive release of soluble cytokines by in vitro grown cultures of AIDS-KS cells. The presence of HIV influenced the sera cytokine profiles by elevating IL-6 and decreasing PDGF concentrations of HIV+ individuals relative to HIV- healthy controls. However, the presence of KS was not associated with unique serum cytokine profiles, because no differences were noted in comparisons of HIV+/KS+ versus HIV+/KS- individuals. Our findings suggest that the local environment is key in modulating AIDS-KS cytokine expression and that KS growth-promoting factors function at the local or paracrine, not the systemic, level. In conclusion, our previous results demonstrated a histologic grade-associated difference in the in vitro growth capacity of AIDS-KS cells; with high histologic grade isolates displaying a marked growth advantage during culture in minimally supplemented media. Findings from this current study reveal that although the potential for a constitutive growth loop exists in the high-grade isolates, it is not reflected in the free levels of soluble cytokines secreted into the culture medium.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Transcriptional control of tektin A mRNA correlates with cilia development and length determination during sea urchin embryogenesis.

Previous studies have shown that tektin A, one of three integral filamentous protein components of outer doublet microtubules, is synthesized in sea urchins in an amount correlating to the length of embryonic cilia initially assembled or experimentally regenerated. To investigate further the molecular mechanism for the regulation of tektin synthesis, tektin cDNA clones were used to assess mRNA levels during ciliogenesis, zinc-induced animalization, deciliation-induced regeneration and theophylline-induced elongation. Possibly involved in centriole replication, low, near-constant levels of mRNA for all three tektins are present in the unfertilized egg and during cleavage stages. Preceded by new synthesis of tektin B and C mRNAs, tektin A mRNA is up-regulated during ciliogenesis, but only tektin A mRNA levels correlate directly with ciliary length in animalized embryos; the others augment larger, non-limiting pools of tektins B and C. Tektin mRNAs decrease to steady-state levels after ciliogenesis, but are up-regulated again when the embryos are deciliated, correlating with the length of cilia to be deployed. In a species where a 3-fold ciliary length increase can be induced by theophylline treatment of zinc-arrested embryos, the mRNAs accumulate to proportionately higher levels during arrest but are not translated until induction, whereupon they decrease inversely with ciliary elongation. This suggests transcriptional control with respect to mRNA amounts but post-transcriptional control with respect to the expression of this phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Correlation between AIDS-related Kaposi sarcoma histological grade and in vitro behavior: reduced exogenous growth factor requirements for isolates from high grade lesions.

Kaposi sarcoma, the most common AIDS-associated malignancy, affects 10-30% of all AIDS patients. To date, research into the biological characteristics of AIDS-related Kaposi sarcoma (AIDS-KS) derived cell lines has been based on cultures established from skin explants or pleural effusions/peritoneal fluids. We have established several AIDS-KS lines from biopsy confirmed oral mucosal and epidermal AIDS-KS lesions and have found a correlation between AIDS-KS lesional grade and in vitro cellular growth characteristics. In comparison to epidermal AIDS-KS lesions, mucosal AIDS-KS lesions frequently possessed both a more advanced histologic grade and demonstrated a greater capacity to proliferate in minimal medium. We report the ability of AIDS-KS isolates from high grade lesions to sustain proliferation (greater than 60 population doubling levels) in medium not supplemented with endothelial cell growth supplement and/or cytokine rich conditioned medium. These findings indicate that AIDS-KS cells isolated from high grade lesions have reduced requirements for exogenously provided growth supplements, and suggest that increased autologous cytokine production accompanies AIDS-KS lesional progression.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Rapid induction of a hyperciliated phenotype in zinc-arrested sea urchin embryos by theophylline.

Zinc ions, present since fertilization, will arrest embryos of the sea urchin Tripneustes gratilla at the hatched blastula stage, but such embryos lack long cilia, the usual characteristic of animalized embryos. Eventually these embryos will express a minimal long cilia phenotype, with a mean population < 1.4 times that of control blastulae, but only after contemporaneous control embryos gastrulate. Theophylline will rapidly but minimally animalize embryos when added after hatching, but its inductive ability decreases as the embryos gastrulate. Theophylline-animalized embryos produce cilia whose mean population length is > 1.5 times that of control blastulae. At any point from the time of hatching up until control complete gastrulation, theophylline added to zinc-arrested embryos will induce a hyperciliated phenotype with a mean population length nearly 3 times that of control blastulae. The elongation of cilia is immediate and significantly exceeds the rate of normal ciliary regeneration. Initially requiring the presence of theophylline, the hyperciliated phenotype becomes stable as control embryos begin gastrulation and, when deciliated, the induced embryos will regenerate hyperlong cilia in the absence of theophylline. The time dependence for both induction and phenotype stability would suggest that certain timing mechanisms are still operative in zinc-arrested embryos. This inducible system should facilitate studies of length control during ciliary elongation and regeneration.

Animals↗
PubMed1994

Cultured AIDS-related Kaposi's sarcoma cells retain a proliferative bioenergetic profile but demonstrate reduced cytoprotective capabilities.

Features of AIDS-related Kaposi's sarcoma (AIDS-KS), such as the multifocal presentation at mucosal and epidermal sites subjected to trauma, suggest that AIDS-KS is initially a reactive hyperplasia that subsequently progresses to a neoplasia. It is recognized that there is an association between sustained inflammatory states and the subsequent development of neoplasia (e.g., ulcerative colitis/colonic adenocarcinoma). Furthermore, patients who develop AIDS-KS experience both a constant immune stimulation due to sustained high levels of virus-induced cytokines and, because of a sparing effect on their phagocytic cells, retention of the phagocytic inflammatory response. A component of phagocytic activation is the initiation of the oxidative burst, resulting in the generation of reactive oxygen species (ROS), which can be mutagenic to host cells if released beyond the phagolysosome and not inactivated. Our results demonstrate that cultured AIDS-KS cells possess decreased cytoprotective capabilities. Relative to either dermal fibroblasts, or human microvascular endothelial cells (HMECs), AIDS-KS cells contained significantly lower levels of glutathione, a tripeptide integral in both cytoprotection and maintenance of cellular thiol status. While HMECs increased catalase activity during culture in the cytokine-rich KS milieu (control medium supplemented with conditioned medium from MOT, an HTLV II-infected cell line), AIDS-KS cells demonstrated reduced catalase function under these conditions. Furthermore, HMEC cultures showed an inherent biochemical responsiveness, by increasing catalase activity following exposure to exogenous H2O2. In contrast, the catalase activity of AIDS-KS cells decreased following H2O2 challenge. Our results show that an inherent deficiency in cellular cytoprotection is present in AIDS-KS cells and suggest that oxidant stress may function in the development and progression of AIDS-KS.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Metabolism of substance P and neurokinin A by human vascular endothelium and smooth muscle.

Analysis of SP and NKA metabolism by human vascular endothelium, relative to that in human plasma, identified integrative, multiple pathways for the processing of circulating SP (but not NKA) by angiotensin-converting enzyme (ACE; EC 3.4.15.1), dipeptidyl(amino)peptidase IV (DAP IV; EC 3.4.14.5), and aminopeptidase M (AmM; EC 3.4.11.2). In contrast, SP and NKA, which may diffuse into or be neurally released within the vessel wall, were both metabolized by smooth muscle neutral endopeptidase-24.11 (NEP-24.11; EC 3.4.24.11). Collectively, these studies indicate peptide-specific and site-specific differential processing of SP and NKA by human plasma and vasculature.

Cells, Cultured↗
PubMed1994

Ohioensins and pallidisetins: novel cytotoxic agents from the moss Polytrichum pallidisetum.

Bioassay-directed fractionation of an EtOH extract of the moss Polytrichum pallidisetum (Polytrichaceae) led to the isolation of three novel benzonaphthoxanthenones, 1-O-methylohioensin B [6], 1-O-methyldihydroohioensin B [7] and 1,14-di-O-methyldihydroohioensin B [8], and two novel cinnamoyl bibenzyls, pallidisetin A [9] and pallidisetin B [10]. Their structures and relative stereochemistry were established by spectral analyses and chemical correlation. Compounds 6-10 exhibited cytotoxic activity against the human tumor cell lines RPMI-7951 melanoma and U-251 glioblastoma multiforme. These two types of compounds could hypothetically be derived from cinnamic acid and bibenzyls through different biogenetic pathways.

Animals↗