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Biomedical subjects

M M Robinson

Publications and source records attributed to M M Robinson.

At least 19 recordsLinked to original sources
PubMed2005

Mapping and functional characterization of the TAF11 interaction with TFIIA.

TFIIA interacts with TFIID via association with TATA binding protein (TBP) and TBP-associated factor 11 (TAF11). We previously identified a mutation in the small subunit of TFIIA (toa2-I27K) that is defective for interaction with TAF11. To further explore the functional link between TFIIA and TAF11, the toa2-I27K allele was utilized in a genetic screen to isolate compensatory mutants in TAF11. Analysis of these compensatory mutants revealed that the interaction between TAF11 and TFIIA involves two distinct regions of TAF11: the highly conserved histone fold domain and the N-terminal region. Cells expressing a TAF11 allele defective for interaction with TFIIA exhibit conditional growth phenotypes and defects in transcription. Moreover, TAF11 imparts changes to both TFIIA-DNA and TBP-DNA contacts in the context of promoter DNA. These alterations appear to enhance the formation and stabilization of the TFIIA-TBP-DNA complex. Taken together, these studies provide essential information regarding the molecular organization of the TAF11-TFIIA interaction and define a mechanistic role for this association in the regulation of gene expression in vivo.

Gene Expression Regulation, Fungal↗
PubMed2001

Transcriptional activity of the TFIIA four-helix bundle in vivo.

TFIIA contributes to transcription initiation by stabilizing the TBP-TATA interaction and by mediating the response to transcriptional activators and inhibitors. TFIIA contains a six-stranded beta-sheet domain and a four-helix bundle. The beta-domain makes functional contacts with DNA and TBP. The role of the four-helix bundle was investigated using a structure-based model of this domain (called 4HB). 4HB adopts a highly stable, helical fold, consistent with its structure in the context of TFIIA. Like TBP and other intact transcription factors, 4HB is able to activate transcription in vivo when artificially recruited to a promoter via a heterologous DNA-binding domain. Thus, in addition to making important contacts with TBP and DNA via the beta-domain, TFIIA makes other specific, functional contacts with the transcriptional machinery via the four-helix bundle. Proteins 2001;43:227-232.

Animals↗
PubMed2001

Resistance of cattle to scrapie by the oral route.

Early epidemiological information indicated that bovine spongiform encephalopathy (BSE) originated from scrapie in sheep. The question arose if scrapie in North America would induce a BSE-like disease in cattle. Six years ago, we reported that brain tissue from sheep with scrapie caused a neurologic disease when injected directly into the brains of cattle, but the disease induced was different from BSE as it occurs in the United Kingdom and Europe. Here, we report that cattle fed raw brain or meat and bone meal and tallow prepared from sheep with scrapie remained normal for 8 years after exposure. This work indicates that cattle are highly resistant to North American scrapie by the oral route.

Animals↗
PubMed2000

Development and evaluation of a micropipette tip washing system.

A tip washing system has been developed for use with disposable polypropylene micropipette tips of different sizes. The primary hardware components of the system are constructed from polyvinyl chloride (PVC) and Plexiglas. The system relies on a low concentration of detergent, gravity or aspirator-assisted feed and flush of minimal volumes of distilled/deionized water, and air or oven drying of the tips. Comparative analyses of new versus washed tips in FAO/IAEA radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA) protocols indicated that disposable polypropylene micropipette tips of various sizes (e.g. 5-300 and 50-1000 microl) can be washed and reused at least ten times without introducing detectable variability into the results of either assay, if the tips are properly washed and dried using this system. Recovery cost of the system can be achieved after approximately eight reuses of 1000 tips, excluding technician time.

Enzyme-Linked Immunosorbent Assay↗
PubMed2000

Evaluation of antigen-coating procedures of enzyme-linked immunosorbent assay method for detection of trypanosomal antibodies.

Research was undertaken to improve the antigen-coating step of indirect enzyme-linked immunosorbent assay (ELISA) method through the use of polystyrene 96-well plates precoated with antigenically stabile crude trypanosomal antigens. The plates were precoated with antigens, air dried and sealed before being packed in plastic bags with silica gel desiccant packets. Such plates stored at +4 and +37 degrees C provided an assay performance, which was superior to that of plates freshly coated with antigens from a frozen stock. Antigen-precoated plates consistently proved stable after storage up to +50 degrees C for at least 1 year. The accuracy of the assay was not affected, i.e. trypanosomal antibody-positive sera were clearly discriminated from trypanosomal antibody-negative negative sera. In contrast, lyophilized trypanosomal antigens lacked stability on storage at +37 degrees C for longer than 1 month. It was concluded that the routine use of antigen precoated polystyrene plates for the enzyme immunoassay technique will contribute to improved assay robustness at an acceptable diagnostic proficiency. The modified coating procedure will also provide an improved quality assurance and standardization procedure for the assay, which is required to allow the reliable detection of trypanosomal antibodies and comparison of data from different laboratories.

Animals↗
PubMed2000

Detection of Trypanosoma congolense antibodies with indirect ELISAs using antigen-precoated microtitre plates.

The study reports the performance of four indirect enzyme-linked immunosorbent assays (ELISAs) for antibody (AB) detection using microtitre plates which were precoated with native or heat/detergent denatured antigens (AGs) from Trypanosoma congolense (T.c.) and T. vivax (T.v.), and stored for between 1 to 206 days at +37 degrees C. Bovine serum samples were obtained by sequential bleeding of 3-months old T.c.-infected bulls and their uninfected cohorts, as well as by a single bleeding of uninfected adult cattle. The first day of AB detection, and observations on samples after this (defined as estimated ELISA sensitivity), depended on the cut-off value in the specific ELISAs. Cut-off values from pre- and early post-infection samples of individual animals demonstrated a seroconversion in all ELISAs on average after 10-15 days post-infection (dpi). The AB detection was delayed in the T.c. native and denatured AG-based ELISAs using cut-off points from uninfected cohort cattle (16.5 dpi, 19.3 dpi) and the adult cattle population (22.1 dpi, 25.0 dpi). The T.v. AG-based ELISAs however lacked crossreactiviy to T.c. ABs. The estimated sensitivity of each T.c. AG-based ELISA was above 96% throughout, but significantly lower for the T.c. native AG-based ELISA (91.1%) when the adult cattle derived cut-off point was used (p<0.01). The sensitivity of the phase contrast buffy coat technique was similar to the T.c. AG-based ELISAs, but significantly lower when the T.c. denatured AG-based ELISA was used at the adult cattle derived cut-off point (p<0.05). The implications of the results and future research aspects on ELISAs to detect trypanosomal ABs and AGs are discussed.

Animals↗
PubMed1999

Interactions of the systemic and brain renin-angiotensin systems in the control of drinking and the central mediation of pressor responses.

Most of the biological actions of the circulating (a.k.a., the systemic or blood-borne) renin-angiotensin system require the generation of the octapeptide angiotensin (ANG) II from the decapeptide ANG I. In the case of circulating ANG I, the lungs are generally considered the major site for this conversion. The present experiments explored the possibility that under conditions of marked elevations of blood-borne ANG I, the generation of ANG II takes place within brain-associated target tissues, most notably circumventricular organs (CVOs) that lack a blood-brain barrier. The first important result of these experiments demonstrates that intracerebroventricular (i.c.v.) infusion of the converting enzyme inhibitor, captopril, completely blocks the drinking response and significantly attenuates the pressor response produced by systemically infused ANG I. This result indicates that under physiological/pathophysiological conditions associated with large elevations of circulating ANG I, an important part of the biological responses derived from blood-borne ANG may result from local conversion of ANG I to ANG II within specific brain target tissues which have high concentrations of converting enzyme. This local conversion process provides an important mechanism that would act to reinforce the "classic" conversion process which takes place in the lungs thereby delivering more ANG II immediately to central target receptors. The second important finding from these studies showed that drinking produced by systemically infused ANG II was not attenuated by an i.c.v. dose of captopril which was effective in blocking a comparable dipsogenic response induced by i.v. ANG I. This observation suggests that drinking induced by systemic ANG II does not require an intact metabolic cascade within the brain for the formation of ANG II (or ANG II-like effector peptide) from ANG I.

Angiotensin I↗
PubMed1999

Identification of unacceptable background caused by non-specific protein adsorption to the plastic surface of 96-well immunoassay plates using a standardized enzyme-linked immunosorbent assay procedure.

A standardized enzyme-linked immunosorbent assay (ELISA) was used to examine the capacity of immunoassay plates to prevent non-specific protein binding under blocking conditions. Data from 16 types of 96-well microtitre plate from seven commercial sources, are described. Plates were evaluated with respect to their capacity to adsorb a conjugated antibody in diluent buffer containing non-ionic detergent Tween 20 (0.05%) and skimmed milk proteins (5%). Plates with an absorbance value of > or = 0.05, in not more than one well, were defined as within acceptable limits. Major problems were seen in high binding gamma-irradiated polystyrene plates, from all sources, where only < or = 30% of plates were acceptable. These showed high, randomly distributed, non-specific binding, with some wells showing absorbance values > 2.0. Similar results were obtained when high binding plates were repeatedly gamma-irradiated, and after gamma-irradiation of low binding polystyrene plates. For high binding, non- gamma-irradiated polystyrene plates, approximately 70% of plates were acceptable. Better results (86-100% acceptability) were observed for all low binding polystyrene plates. Only one source in three provided acceptable, low binding, polyvinylchloride plates. This paper confirms a widely held view that non-specific binding to certain plates could be a serious factor in both the development and application of ELISAs. Therefore, the test protocol described is proposed as an additional quality control method for certifying ELISA plates by commercial companies.

Adsorption↗
PubMed1998

Veterinary diagnostic laboratories in developing countries: the challenge of credibility.

The problems facing veterinary diagnostic laboratories in developing countries range from simple problems, such as limited funds to keep good personnel or obtain equipment, supplies, reagents or training, to the more complex problems of designing and executing appropriate sample collection schemes for disease surveillance of evaluating the performance characteristics of a new diagnostic assay. While many developing countries are addressing these problems independently and a number of international and national organisations provide various forms of external support, the Office International des Epizooties (OIE) must continue in its critical role to provide guidelines for the most appropriate diagnostic assays for trade purposes and the development of the primary reference reagents for these assays. In addition, the OIE should take the lead in development of quality management guidelines for veterinary diagnostic testing laboratories. Without these guidelines and standards, diagnostic laboratories in developing countries will have difficulty in gaining the credibility necessary to help improve the positions of their countries in the international trade of livestock and livestock commodities.

Animal Diseases↗
PubMed1996

Transmissible encephalopathies and biopharmaceutical production.

The use of post-mortem tissues as sources for the production of biologicals, vaccines and feedstuffs has led to the transmission or generation of transmissible encephalopathies in some recipients. For example, the use of pituitary-derived human growth hormone and gonadotropins has resulted in the transmission of Creutzfeldt-Jakob disease to other humans [1], the use of formalin-inactivated sheep brain as a source for louping ill vaccine led to the transmission of scrapie to over 1,000 sheep from one vaccine lot [2], and the use of rendered products from ruminant carcasses in the domestic animal food chain led to the emergence and epizootic of bovine spongifrom encephalopathy in the United Kingdom [3]. Infection with transmissible encephalopathies by iatrogenic or other mechanisms is difficult to predict or control. The characteristics of these pathogens do not permit easy detection, clearance, or inactivation in routine biopharmaceutical production environments.

Animals↗
PubMed1995

Experimental infection of cattle with the agents of transmissible mink encephalopathy and scrapie.

Cattle are susceptible to experimental infection with the Stetsonville isolate of the transmissible mink encephalopathy (TME) agent. To determine if they are susceptible to other TME isolates, two groups of calves were inoculated intracerebrally with homogenate of mink brain containing the Hayward isolate or the Blackfoot isolate. For comparison, a third group was inoculated with a brain homogenate from a steer infected with the Stetsonville isolate in its primary cattle passage and a fourth group was inoculated with a pool of brain homogenate from three cattle experimentally infected with a sheep and goat scrapie agent in its primary cattle passage. Clinical signs of neurological disease appeared in each steer of every group between 15 and 25 months after inoculation. An encephalopathy characterized by severe spongiform change and pronounced astrocytosis occurred in the three groups inoculated with the TME agent. In contrast, the neurohistological changes in the steers inoculated with the cattle-passaged scrapie agent were slight and subtle. Analysis of the octapeptide repeat region of the bovine protease-resistant protein (PrP) gene showed that variations in incubation period, clinical signs, and neurohistological changes were unrelated to the homozygous or heterozygous condition of six or six/five octapeptide repeats.

Animals↗
PubMed1994

Intracerebral transmission of scrapie to cattle.

To determine if sheep scrapie agent(s) in the United States would induce a disease in cattle resembling bovine spongiform encephalopathy, 18 newborn calves were inoculated intracerebrally with a pooled suspension of brain from 9 sheep with scrapie. Half of the calves were euthanatized 1 year after inoculation. All calves kept longer than 1 year became severely lethargic and demonstrated clinical signs of motor neuron dysfunction that were manifest as progressive stiffness, posterior paresis, general weakness, and permanent recumbency. The incubation period was 14-18 months, and the clinical course was 1-5 months. The brain from each calf was examined for lesions and for protease-resistant prion protein. Lesions were subtle, but a disease-specific isoform of the prion protein was present in the brain of all calves. Neither signs nor lesions were characteristic of those for bovine spongiform encephalopathy.

Animals↗
PubMed1994

SCID mouse spleen does not support scrapie agent replication.

BALB/c and severe combined immunodeficiency (SCID) mice were inoculated intracerebrally or intraperitoneally with scrapie agent strain ME7 to examine the role of functional lymphocytes and follicular dendritic cells in splenic infectivity and PrPSc accumulation. Intracerebrally inoculated BALB/c and SCID mice developed the clinical signs and microscopic lesions characteristic of scrapie. Spleens from terminally affected BALB/c mice contained PrPSc which was detectable by immunoblot analysis; SCID mouse spleens did not contain detectable PrPSc. SCID mouse spleens collected during the first 90 days after intraperitoneal infection contained neither infectivity nor PrPSc.

Amino Acid Sequence↗
PubMed1994

Experimental infection of mink with bovine spongiform encephalopathy.

To determine whether the aetiological agent of bovine spongiform encephalopathy (BSE) is pathogenic for mink, standard dark mink were inoculated with coded homogenates of bovine brain from the U.K. Two homogenates were from cows affected with BSE. The third was from a cow that came from a farm with no history of having had BSE or having been fed ruminant-derived, rendered by-products, the proposed vehicle for introduction of the BSE agent. Each homogenate was inoculated intracerebrally into separate groups of mink and a pool of the three was fed to a fourth group. Signs of neurological disease appeared in mink an average of 12 months after intracerebral inoculation and 15 months after feeding. Decreased appetite, lethargy and mild to moderate pelvic limb ataxia were the predominant clinical signs, quite unlike the classic clinical picture of transmissible mink encephalopathy (TME). Microscopic changes in brain sections of most affected mink were those of a scrapie-like spongiform encephalopathy. Vacuolar change in grey matter neuropil was accompanied by prominent astrocytosis. Varying greatly in severity from one mink to another, the degenerative changes occurred in the cerebral cortex, dorsolateral gyri of the frontal lobe, corpus striatum, diencephalon and brainstem. Although resembling TME, the encephalopathy was distinguishable from it by less extensive changes in the cerebral cortex, by more severe changes in the caudal brainstem and by sparing of the hippocampus. The results of this study extend the experimental host range of the BSE agent and demonstrate for the first time the experimental oral infection of mink with a transmissible spongiform encephalopathy agent from a naturally infected ruminant species.

Animals↗
PubMed1991

Genetic structure of the pol-env region of the caprine arthritis-encephalitis lentivirus genome.

The nucleotide sequence of the pol-env intergenic region of two isolates of caprine arthritis-encephalitis virus (CAEV) was determined. Two open reading frames (orfs) were identified, designated Q and S by homology with visna virus. CAEV orf S is a single exon encoding a deduced 87-amino acid gene product sharing 36 amino acid identities with the visna trans-acting transcriptional activator (Tat). Ten of these identities comprise a conserved CGCRLCNPGW sequence similar to a cysteine-rich domain essential for trans-activation by human immunodeficiency virus Tat. To determine if transcription promoted by the CAEV long terminal repeat (LTR) could be stimulated in CAEV-infected goat synovial membrane cells, a plasmid (pCAE-CAT) expressing bacterial chloramphenicol acetyltransferase (CAT) under control of the CAEV LTR was transfected into uninfected and infected cells. Sixfold enhancement of CAT activity was observed in infected cells using 100 ng of transfected plasmid. To determine if the pol-env region encodes a gene product which trans-activates the CAEV LTR, goat synovial membrane cells were cotransfected with pCAE-CAT and pRSV-1.9, a plasmid expressing the pol-env region under control of the Rous sarcoma virus LTR. Results indicated that the CAEV genome encodes a tat gene product attributable to orf S.

Amino Acid Sequence↗
PubMed1990

Organ-specific modification of the dose-response relationship of scrapie infectivity.

The dose-response relationships of scrapie strain 263K-infected hamster brain and spleen homogenates were compared to determine if intracerebral end-point titrations of infectivity in these homogenates were measures of the same pathogenic phenomenon. Analysis of the dose-response curves indicated that the average increase in incubation period per 10-fold dilution (i.e., the dilution kinetics) of brain infectivity was significantly different from that of spleen infectivity. This difference contradicted the assumption that the same pathogen or pathogenic mechanisms were responsible for producing disease in each titration. Therefore, the end-point titrations and infectivity titers of the brain and spleen homogenates were measures of two different phenomena. Subsequent passage of a scrapie-infected spleen homogenate demonstrated that the dose-response relationship of scrapie infectivity in this agent-host system was dependent on the organ titrated, not the tissue source or inoculation route of previous passage.

Animals↗
PubMed1988

Effect of arterial pressure on drinking and urinary responses to angiotensin II.

To investigate the relationship between angiotensin II (ANG II) and mean arterial pressure (MAP) in the control of drinking in rats, we infused ANG II intravenously at constant rates (either 50 or 100 ng.kg-1.min-1 for 90 min) and varied MAP by intravenous injections of diazoxide (5-20 mg/kg). Rats were pretreated with captopril to block the endogenous synthesis of ANG II. When given alone, low and high doses of ANG II increased MAP approximately 30 and 50 mmHg, respectively. The low but not the high dose significantly increased water intake above control levels. Both doses caused such a large diuresis and natriuresis that the net effect was fluid loss. Reducing MAP toward normal greatly increased the drinking response to the high but not the low dose of ANG II and reduced the urinary solute and water loss to both doses. These results support the hypothesis that water intake and net fluid gain are inhibited when MAP is above normal. When MAP was reduced below normal in rats given constant infusions of ANG II the amount of water drunk and net fluid gain was proportional to the dose of ANG II but not the dose of diazoxide, the degree of hypotension, or urinary losses. This is consistent with previous reports that ANG II is essential for the drinking response to hypotension. Furthermore, it demonstrates that ANG II is not merely permissive but probably the signal controlling water intake when arterial pressure is reduced below normal.

Angiotensin II↗