Biomedical subjects

R C Rose

Publications and source records attributed to R C Rose.

At least 55 records · Page 3Linked to original sources

PubMed1993

Two E2 binding sites (E2BS) alone or one E2BS plus an A/T-rich region are minimal requirements for the replication of the human papillomavirus type 11 origin.

Human papillomaviruses (HPVs) cannot be propagated in vitro, but the DNA can be replicated transiently in an assay in the presence of two trans-acting viral proteins, E1 and E2. Using this assay, we have defined the minimal cis-acting elements of the origin of replication of HPV type 11. Most HPV genomes are conserved at the origin of replication, and the core contains three E2 binding sites (E2BS) surrounding an A/T-rich spacer region. The present results show that the minimal requirement for replication is either two E2BS alone or the A/T-rich region plus one E2BS; in the latter case the relative position of the E2BS is important. In all the studies, the presence of both E1 and E2 proteins was essential for replication, yet only the E2BS was required at the origin. We have shown that E1, E2, and the origin of replication containing an E2BS from a complex in vitro, and our data are consistent with a model in which E2 acts to target E1 to the HPV type 11 replication origin.

Base Sequence↗

PubMed1993

Expression of human papillomavirus type 11 L1 protein in insect cells: in vivo and in vitro assembly of viruslike particles.

The L1 coat protein of human papillomavirus type 11 (HPV-11) was expressed in Sf-9 insect cells with the recombinant baculovirus vector Ac11L1. Viruslike particles (VLPs) were identified by electron microscopy in the nucleus and cytoplasm of Sf-9 cells infected with Ac11L1. The L1 protein was purified from Ac11L1-infected insect cells. The purified protein spontaneously assembled in vitro into various aggregates, including particles appearing similar to empty virions. Reaction of VLP-containing insect cell extracts with antisera directed against either denatured or nondenatured capsid epitopes in Western blot (immunoblot) and immuno-dot blot assays suggested that conformational epitopes present in native HPV-11 infectious virions were also present on the baculovirus-produced HPV-11 VLPs. Immuno-dot blot assays using human sera obtained from individuals with biopsy-proven condyloma acuminatum correlated closely with results previously obtained in HPV-11 whole virus particle-based enzyme-linked immunosorbent assays. These morphologic and immunologic similarities to native HPV-11 virions suggest that recombinant VLPs produced in the baculovirus system may be useful in seroepidemiology and pathogenesis studies of genital HPV infection and that they may also be potential candidates for vaccine development.

Animals↗

PubMed1993

Ascorbic acid regeneration by bovine iris-ciliary body.

Ocular tissues appear to require high levels of ascorbic acid and the elucidation of the mechanisms by which those tissues maintain the vitamin in its reduced state remains an important objective. The regeneration of ascorbate from its oxidative by-product, dehydroascorbic acid (DHAA), was studied in bovine iris-ciliary body. Iris-ciliary body was removed by scalpel, weighed, minced, and homogenized in 20 mM MOPS, 62 mM sucrose, and 0.1 mM EDTA at pH 7.0. The homogenate was centrifuged and precipitated with ammonium sulfate such that maximal DHAA reducing activity was enhanced in a 50-75% ammonium sulfate fraction. This fraction was employed for subsequent characterization of DHAA reduction by iris-ciliary body. Results indicate that the iris-ciliary body enzymatically reduces DHAA to ascorbate at a rate significantly greater than can be accounted for by a nonenzymatic glutathione-dependent mechanism. In addition, saturation kinetics are observed, and the enzymatic activity is dependent on protein concentration, DHAA concentration, and reduced glutathione (GSH) concentration. The activity is sensitive to pH, to high temperature, and to digestion by trypsin and is greatest in the presence of both GSH and NADPH. The reducing activity is therefore attributed to one or more proteins that are distinct from the known ascorbate regenerating enzyme, GSH-dependent DHAA reductase (EC 1.8.5.1).

Animals↗

PubMed1993

Lacrimal gland uptake and metabolism of ascorbic acid.

Ascorbic acid is thought to contribute to protection against the potentially damaging effects of radiation, oxygen toxicity, and abrasion in the eye. The anterior surface of the cornea is particularly subject to insult from each threat. We considered the possibility that the lacrimal gland of pigs has transport and/or metabolic capability to sequester the reduced or oxidized form of ascorbic acid and prepare it for secretion in the tears. Slices of fresh lacrimal gland were incubated in a physiologic buffer and exposed to < or = 12 microM 14C-labeled ascorbic acid or dehydro-l-ascorbic acid over 40-min incubation periods. Dehydro-l-ascorbic acid was taken up to a greater extent than the reduced compound. 14C-Label recovered from the tissue was at least 75% in the form of ascorbic acid after incubation with either substrate. Uptake of both the reduced and oxidized substrates proceeded to a tissue to medium ratio in excess of unity; the former was prevented by the presence of nonlabeled ascorbate in the bathing medium, but was unaffected by the removal of Na+ from the bath. The uptake of both substrates was less after inhibition of cellular metabolic energy. The lacrimal gland in this diurnal animal species has transport and metabolic capabilities that could serve in secretion of ascorbic acid into tears. This might help to protect the corneal epithelium against various forms of damage.

Animals↗

PubMed1993

Biology of free radical scavengers: an evaluation of ascorbate.

Reactive free radical species (R.) are associated with several forms of tissue damage and disease, and also with the process of aging. Protection is thought to be available in the form of endogenous compounds that react with and thereby "scavenge" the R.. Because many R. are reactive forms of oxygen, an effective scavenger is often referred to as an antioxidant. To be an effective antioxidant physiologically, a substance must have certain chemical and biological properties: it must be present in adequate amounts in the body; it must react with a variety of R.; it must be suitable for compartmentation; it must be readily available; it might be suitable for regeneration; it must be conserved by the kidneys; and it must have tolerable toxicity. Several water-soluble candidates are mentioned, with most having no more than one or two of the attributes listed. Ascorbic acid is discussed in detail, and an analysis is made of whether it has the properties mentioned.

Animals↗

PubMed1992

Short term effects of oxidized ascorbic acid on bovine corneal endothelium and human placenta.

Studies on the toxic effects of dehydro-L-ascorbic acid (DHAA) have been extended to include evaluations over time periods up to 3 hr. and to test for specific effects on a membrane transport protein, a membrane-bound enzyme and a soluble intracellular enzyme. In studies on cultured corneal endothelial cells, DHAA concentrations of 1, 2, and 5 mM over 3 hr. had an inhibitory effect on subsequent uptake of DHAA present at a tracer level. Surviving fragments of human placenta and alkaline phosphatase activity of the placental brush-border membrane were susceptible to the effect of DHAA at a high concentration (10 mM). Because intracellular metabolism of DHAA was not affected, and an increase in membrane permeability was not detected, it is concluded that a specific membrane transport protein might be the site of DHAA-induced damage. These studies support the concept that the oxidized form of ascorbic acid (vitamin C) has potential toxic effects on biological systems and suggests that proteins that mediate transport and metabolism may be sites where DHAA causes damage.

Alkaline Phosphatase↗

PubMed1992

Antibody response to human papillomavirus (HPV) type 11 in children with juvenile-onset recurrent respiratory papillomatosis (RRP).

We previously established, using an ELISA, the presence of specific antibodies directed at human papillomavirus (HPV) type 11 virions in the sera of patients with condylomata acuminata, mostly a disease of young adults that, like recurrent respiratory papillomatosis (RRP), is caused by two closely related HPVs, types 6 and 11. The present study was done to investigate if children with RRP can make viral-specific antibodies to an infection that is acquired at birth. Using the same ELISA, we studied the sera of 32 children with biopsy-documented juvenile-onset RRP and compared them to the sera of 31 control children. The median (and interquartile range) of the OD values in the controls and the cases was 0.078 (0.003, 0.101) and 0.230 (0.063, 0.725), respectively, a statistically significant difference (P = 0.001). Among the cases, there was no difference in seroreactivity between children with HPV-11-induced RRP and those with HPV-6-induced RRP (P = 0.31). Since HPV-11 viral particles do bind to the ELISA plate and remain intact and accessible to antibodies, we conclude that children with RRP, like adults with condylomata acuminata, develop antibodies directed at HPV-11 virions.

Antibodies, Viral↗

PubMed1992

Antibody-mediated neutralization of human papillomavirus type 11 (HPV-11) infection in the nude mouse: detection of HPV-11 mRNAs.

The nu/nu mouse xenograft is the only experimental system permitting the growth of human papillomaviruses (HPV). Previous studies demonstrating inhibition of HPV-11 infection by antibodies against HPV-11 virions have used indirect markers of infection, such as graft size and histopathologic features. The presence of HPV-11 mRNAs was used as a direct marker of infection: Infectious HPV-11 was incubated with rabbit serum raised against purified HPV-11 virions or with the corresponding preimmune serum (controls) before use in the mouse xenograft model, and HPV-11 mRNAs were detected by a method using reverse transcription and amplification by polymerase chain reaction. Graft size, histopathologic features, and the presence of capsid antigen were also assessed. Six weeks after infection, 1 of 23 grafts in the test group contained HPV-11 mRNAs compared with 19 of 20 controls (P less than .001). Therefore, antibody-mediated inhibition of infection by HPV-11 leads to blockade of genomic expression and is thus consistent with active prevention of viral penetration, that is, neutralization.

Animals↗

PubMed1992

Tissue-mediated regeneration of ascorbic acid: is the process enzymatic?

Ascorbic acid's function in animals is attributed in part to the ease with which it reduces potentially damaging components, such as reactive free radicals. After more than six decades of speculation and laboratory efforts, the mechanisms by which ascorbic acid is maintained in the useful, reduced state remain uncertain. Previous attempts to isolate the enzymes that reduce the partially and the fully oxidized metabolites of vitamin C are reviewed. Some speculation on why dehydroascorbate reductase (EC 1.8.5.1) has not been purified from animal tissues is presented.

Animals↗

PubMed1991

Detection of human papillomavirus types 6 and 11 E4 gene products in condylomata acuminatum.

Polyclonal antiserum to an Escherichia coli-produced beta-galactosidase/E4 fusion protein of human papillomavirus type 6b (antiserum 256), and affinity purified HPV 11 anti-E4 antibodies were tested for reactivity in Western blots with bacterially expressed trpE/E4 fusion proteins of HPV types 6b, 11, 16, and 18. To further characterize the affinity purified anti-E4 antibodies, a dot-immunobinding assay was performed using overlapping synthetic HPV 11 E1E4 peptides as antigens. Protein extracts of condylomata acuminatum from 18 patients containing HPV type 6 or 11 DNA sequences were tested in Western blots using antiserum 256 or affinity purified HPV 11 anti-E4 antibodies. In the Western blots of the trpE proteins, antiserum 256 identified the HPV types 6b and 11 fusion proteins; the affinity purified HPV 11 anti-E4 antibodies identified only the HPV 11 fusion protein. In the dot-immunobinding assay, three HPV 11 peptides were recognized, each containing a shared 8 amino acid sequence that differs significantly from the corresponding sequences of HPV types 6b, 16, or 18. In the Western blots of protein extracts from 18 condylomata acuminatum samples shown to contain HPV types 6 or 11 DNA, putative E4 gene products were identified in six samples by antiserum 256. The affinity purified HPV 11 anti-E4 antibodies identified putative E4 gene products in one of these same six lesions, which was shown to contain HPV 11 sequences by the Southern blot method. All six samples containing E4 gene products were from women. Three of these women were pregnant, one had serum antibodies to the human immunodeficiency virus, and one was a renal transplant recipient receiving glucocorticoids.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1991

Ocular ascorbate transport and metabolism.

1. The concept is reviewed that the eye is subject to photo-oxidative damage through chemical free radical species that interact with sensitive tissue components. 2. The role of ascorbic acid may be to protect the eye by scavenging free radicals. 3. Ascorbic acid is present at a high concentration in various ocular compartments of diurnal animals, regardless of whether the animal synthesizes the compound or extracts it from the diet. 4. Ascorbic acid accumulates in the eye by active transport through the iris-ciliary body into aqueous humor, and subsequent transport into the lens and cornea. 5. Conservation of ascorbic acid occurs by reduction of dehydro-L-ascorbic acid and the ascorbate free radical through processes that appear to be enzymatic.

Animals↗

PubMed1991

Use of human papillomavirus type 11 virions in an ELISA to detect specific antibodies in humans with condylomata acuminata.

Human papillomavirus types 6 and 11 (HPV-6 and HPV-11) are the major aetiological agents of condylomata acuminata. Serological studies of this disease have been difficult to perform and interpret because native, type-specific antigens have not been available. In particular, since these viruses have not been propagated in vitro and sufficient quantities of virions are not present in lesions, virus particles have been difficult to obtain. In the present study, we used HPV-11 particles, obtained from human tumours produced in athymic mice, as antigen in an ELISA to compare antibody responses between 46 patients with biopsyproven condylomata acuminata and 44 controls. The median [interquartile range] of the absorbance values for the condylomata acuminata and the control groups were respectively 0.324 [0.183, 1.029] and 0.118 [0.047, 0.286] (P = 0.0001). Thirty-three per cent of the absorbance values in the condylomata acuminata group were higher than any of those of the control group. Sera from patients whose biopsies contained the papillomavirus common antigen were more reactive than sera from patients whose biopsies did not contain it (P = 0.0014). This study demonstrates the presence of specific antibodies directed at native HPV-11 viral particles in the sera of patients with condylomata acuminata, and describes a test which can be used in future serological studies of this common sexually transmitted disease.

Adult↗

PubMed1991

Ascorbic acid uptake and metabolism by corneal endothelium.

Ascorbic acid is concentrated in various ocular compartments where it is thought to protect diurnal animal species against damaging effects of ultraviolet radiation. The authors evaluated the possibility that corneal endothelial cells have specific transport and/or metabolic properties that deliver ascorbic acid to the stroma. Bovine corneal endothelial cells were grown to confluence in multiple-well plates. Individual groups of cells (approximately 10(4)) were then incubated at various times at 34 degrees C in a physiologic buffer that contained a 10 microM level of 14C-labeled ascorbic acid or the oxidized product, dehydro-L-ascorbic acid. Endothelial cells take up dehydro-L-ascorbic acid at least seven times as rapidly as they take up ascorbic acid. After 30 sec of incubation with 14C-dehydro-L-ascorbic acid, most of the label accumulated in the cell is in the reduced form. Uptake is inhibited by cyanide and iodoacetamide but is unaffected by ouabain. Exposure of cultured cells to various intermediates in the energy metabolism pathways reduced uptake of ascorbic acid but had a minor effect on uptake of the oxidized molecule. These results suggest that the cornea has transport and metabolic capacity to extract dehydro-L-ascorbic acid from aqueous humor and reduce it, thus providing a source of ascorbic acid for corneal protection. This also would maintain "total" ascorbic acid of aqueous humor in the reduced state.

2,3-Diketogulonic Acid↗

PubMed1990

Ascorbic acid metabolism in protection against free radicals: a radiation model.

The role of ascorbic acid in scavenging free radicals was evaluated in a model of mammalian colonic epithelium homogenized in physiologic buffer and exposed to ionizing radiation. Ascorbic acid interacts with hydroxyl free radicals, resulting in production of the ascorbate free radical (AFR). Colonic mucosa contains a soluble factor that is heat sensitive, PCA precipitable and is contained within 1,000 MW dialysis tubing; it uses GSH and cysteine to reduce AFR. The factor from rat colon is fractionated between 55 and 70% saturation with solid (NH4)2SO4; a 3-4 fold increase in enzyme activity was achieved. We suggest that the factor is a cytosolic enzyme appropriately referred to as soluble AFR-reductase. This information provides insight into the mechanism by which ascorbic acid protects against damage by hydroxyl free radicals.

Animals↗

PubMed1990

Expression of the full-length products of the human papillomavirus type 6b (HPV-6b) and HPV-11 L2 open reading frames by recombinant baculovirus, and antigenic comparisons with HPV-11 whole virus particles.

The L2 open reading frames (ORFs) of human papillomavirus (HPV) types 6b and 11 were expressed as full-length non-fusion proteins in Spodoptera frugiperda (Sf-9) cells using recombinant baculovirus. Both proteins were detected on Western blots as immunoreactive bands which migrated with apparent Mrs of 76K and 78K, respectively, and contained both cross-reactive and type-specific epitopes, as determined by polyclonal antisera directed against defined subregions of the HPV-6b and HPV-11 L2 ORFs. In addition, the minor capsid protein of HPV-11 particles co-migrates with the HPV-11 L2 ORF product and is immunoreactive with HPV-11 L2-specific antisera. These observations indicate that the anomalous electrophoretic mobilities of papillomavirus L2 ORF proteins can be explained without invoking post-transcriptional processing events and that the minor capsid protein of HPV-11 is antigenically and biophysically related to the HPV-11 L2 ORF product.

Animals↗

PubMed1990

Intestinal absorption and metabolism of ascorbic acid in rainbow trout.

Transport and metabolism of the reduced and oxidized forms of vitamin C were evaluated in trout intestine in vitro. Transepithelial fluxes of [14C]ascorbic acid were determined with the substrate present at 10 microM. The mucosa-to-serosa flux was fourfold greater than the serosa-to-mucosa flux with ascorbic acid present in both bathing solutions. Cellular accumulation of the reduced molecule occurred with a final tissue-to-medium ratio of 1.64. Ascorbic acid influx across the brush-border membrane was determined under conditions that inhibit transport of other nutrients. Influx was not reduced because of the presence of the D- or L-stereoisomeric form of glucose but was highly dependent on the presence of Na in the bathing medium. Brush-border influx was saturable with a Km of 220 microM. Short-term (8 min) exposure of intestinal loops to the oxidized form of vitamin C, dehydro-L-ascorbic acid, was followed by high-performance liquid chromatography analysis of the 14C label present in the tissue. Total uptake of substrate was greater from the serosal solution than from the mucosal solution; in either situation most of the 14C label was found in the reduced state. It is concluded that transport and metabolic capabilities are present in the trout intestine to absorb oxidized and reduced forms of vitamin C and to help regulate the redox potential of circulating ascorbic acid by recycling dehydro-L-ascorbic acid.

Absorption↗

PubMed1990

Spontaneous decay of oxidized ascorbic acid (dehydro-L-ascorbic acid) evaluated by high-pressure liquid chromatography.

We applied high-pressure liquid chromatography to assess the decomposition of the oxidized form of vitamin C, dehydro-L-ascorbic acid. We selected experimental conditions that might represent a wide variety of clinical and research procedures. Decay of dehydro-L-ascorbic acid proceeded much more rapidly at high pH (7-8) than at low pH (3-5) and was more rapid at 37 or 45 degrees C than at 0 or 23 degrees C. When evaluated at pH 6.6, the percent decay was somewhat more rapid from an initial concentration of 1000 mumol/L than at 5-10 mumol/L. The analytical procedure (HPLC) provided useful information about the rate of decay under various conditions. This may facilitate future biological and clinical studies that require a distinction between the oxidized and reduced forms of vitamin C.

Ascorbic Acid↗

PubMed1990

Infective endocarditis due to the CDC group M6 bacillus.

Although staphylococcal endocarditis has occasionally been associated with a TTP-like syndrome, a similar syndrome has not been reported with endocarditis due to aerobic gram-negative rods. We report a case of subacute bacterial endocarditis with a thrombocytopenic syndrome that at first resembled TTP which was due to an unusual gram-negative rod. This case emphasizes the need for repeated examination of the bacteremic patient to detect the changing murmurs of endocarditis.

Aged↗