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Biomedical subjects

R C Rose

Publications and source records attributed to R C Rose.

At least 19 recordsLinked to original sources
PubMed1995

Analysis of water-soluble antioxidants by high-pressure liquid chromatography.

The measurement of endogenous substances that function as biological antioxidants is of importance because the values obtained might be an index of future health. We quantified three water-soluble antioxidants by high-pressure liquid chromatography with electrochemical detection (h.p.l.c.-e.c.). Current-voltage relationships made at various settings of the D2 porous graphite electrode help to identify ascorbic acid, glutathione and uric acid. The antioxidants are determined simultaneously and without need for derivatization. The method is seen to be useful for comparison of normal rat liver with liver that had undergone oxidative stress through ischaemia. Antioxidant levels in liver, kidney, pancreas and intestinal mucosa are presented and compared with literature values. Endogenous contents of oxidized forms of ascorbic acid and glutathione become apparent following exposure of tissue samples to a strong reductant such as 2-mercapthoethanol.

Animals↗
PubMed1994

Human papillomavirus (HPV) type 11 recombinant virus-like particles induce the formation of neutralizing antibodies and detect HPV-specific antibodies in human sera.

Recombinant human papillomavirus type 11 (HPV-11) virus-like particles (VLPs) were tested for their ability to induce the formation of neutralizing antibodies, and were also tested for serodiagnostic capabilities in an ELISA in comparison with HPV-11 whole virions. VLPs, purified by CsCl density gradient centrifugation from the cell-free supernatant of Ac11L1-infected Sf9 suspension cell cultures, were used to immunize rabbits and anti-VLP antibodies were tested in the athymic mouse model of HPV-11 infection. Pretreatment of infectious HPV-11 virions with the immune serum of VLP-treated animals caused a marked reduction of graft growth (P < 10(-4)) and viral gene expression (P < 10(-4)), similar to the effects obtained using whole virion postimmune serum, and consistent with immune neutralization. To assess the serodiagnostic capabilities of VLPs, a VLP ELISA was developed and used to analyse sera that were tested previously in an HPV-11 whole virion ELISA. Specific antibodies were detected in 49% of patients' sera (P = 2 x 10(-4)), and individual VLP seroreactivities correlated with those previously obtained using whole virions as the antigen (r = 0.87; P < 10(-6)). These results indicate that recombinant VLPs can be used to elicit a neutralizing antibody response, and can substitute faithfully for native virions in the development of HPV-serodiagnostic immunoassays.

Animals↗
PubMed1994

Serological differentiation of human papillomavirus types 11, 16 and 18 using recombinant virus-like particles.

The L1 major capsid protein-coding sequences of human papillomavirus (HPV) types 11, 16 and 18 were expressed in the baculovirus system. Virus-like particles (VLPs) were purified from recombinant-infected Spodoptera frugiperda Sf9 cells and cell-free culture supernatants. Rabbits immunized with purified VLPs developed antibodies that reacted only with the specific VLP type used as the immunogen. In addition, rabbit antibodies raised against infectious HPV-11 virions only reacted with HPV-11 L1 VLPs and not with VLPs derived from either HPV-16 or HPV-18. These results suggest that HPV-11, HPV-16 and HPV-18 virions are antigenically distinct from one another. This observation should be considered in future studies of immune responses to HPV.

Animals↗
PubMed1994

Evaluation of temperature sensitivity of human papillomavirus type 11 by using the human xenograft severe combined immunodeficiency mouse model.

The temperature sensitivity of human papillomavirus type 11 was evaluated by using a human xenograft severe combined immunodeficiency mouse model. Incubation of the virus for 1 h at a temperature higher than 56 degrees C but lower than 72 degrees C was sufficient to inhibit the virally induced growth of infected human tissue. However, 100 degrees C was necessary to completely inactivate HPV type 11 genome expression.

Animals↗
PubMed1993

Cerebral metabolism of oxidized ascorbate.

The brain has a high level of ascorbic acid which is thought to act as a reducing agent, e.g. in protecting tissues against oxidative stress. The mechanism by which ascorbate is maintained in the useful, reduced state in the CNS is evaluated herein. Cerebrum from rat or calf was minced and homogenized in buffer. The endogenous levels of ascorbic acid, dehydro-L-ascorbic acid (DHAA) and reduced glutathione (GSH) were determined by HPLC with coulometric electrochemical detection. We also quantitated tissue capacity to regenerate ascorbic acid from DHAA, which is a product of electron transfer reactions of ascorbic acid. The homogenate was fractionated by centrifugation in steps up to 110,000 x g and dialyzed free of low molecular weight components. The activity for reducing DHAA was approximately equal in the various supernatants; resuspended pellets had little activity. The active component has several properties of a protein, including being precipitated by solid ammonium sulfate addition to the tissue extract; most activity appeared in the 40-80% saturated fraction. The activity was stable up to a temperature of 80 degrees C, but was lost at 95 degrees C. The protein was digested by trypsin. The results suggest that a cytosolic component of cerebrum regenerates ascorbic acid in a step that preferentially uses GSH and NADPH as reducing cofactors. At least one form of DHAA reductase exists in brain.

Animals↗
PubMed1993

Evolution of the antibody response to human papillomavirus type 11 (HPV-11) in patients with condyloma acuminatum according to treatment response.

To evaluate the variation over time of seroreactivity to human papillomavirus type 11 (HPV-11) according to disease outcome, we selected a sample of 42 condyloma acuminatum patients from a group of subjects enrolled in a placebo-controlled trial of three alpha-interferon preparations administered parenterally for the treatment of condyloma acuminatum. This sample included 14 subjects who were cured by the end of follow-up (cured group) and 28 subjects who were not (failed group). For each individual, the first and last sera collected in the study were tested with an intact HPV-11 virion-based enzyme linked immunosorbent assay (ELISA). The sera of 20 nuns with no lifetime sexual exposure served as controls. The median optical density (OD) value of the first serum samples (as well as that of the last samples) from the patients, 0.155, was higher than that of the control sera, 0.073 (P = 2 x 10(-4)). Sensitivity of the assay was 50%. To test if evolution of seroreactivity in the seropositive patients was related to disease outcome after treatment, we examined the average percentage of daily change in OD between the two serum collections. The median OD in the cured group (n = 7) dropped by 0.05% a day whereas in the failed group (n = 11) it increased by 0.07% a day, a highly statistically significant difference (P = 0.006). It is concluded that changes in the seroreactivity to HPV-11 virions are related to outcome of condyloma acumination after treatment. Therefore, improved serological assays may eventually contribute to the monitoring of HPV disease activity.

Adult↗
PubMed1993

Propagation of human papillomavirus type 11 in human xenografts using the severe combined immunodeficiency (SCID) mouse and comparison to the nude mouse model.

We report propagation of human papillomavirus (HPV) type 11 in human xenografts in the severe combined immunodeficiency (SCID) mouse and compare this new animal model for HPV infection to the previously established athymic nude mouse model. HPV-11-infected foreskin fragments grafted under the renal capsule of SCID mice formed large epithelial cysts that had the histologic and immunocytochemical features of HPV infection. This infection was successfully passaged to nude mice. Viral particles that reacted to an antibody directed to HPV-11 virions were identified from samples recovered from the SCID and nude mice. Viral DNA sequence analysis confirmed that the passaged virus was HPV-11. In a comparative experiment of the nude mouse and SCID mouse models, the latter produced HPV-11-infected xenografts that were larger and more often positive for HPV by immunocytochemistry and presence of viral mRNA than those propagated in the former model. Finally, we observed that growth of HPV-11-infected foreskin fragments in the SCID mouse model is not restricted to the kidney as in the nude mouse, but also can occur in the subcutis and the peritoneum.

Animals↗
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↗