T lymphocyte activation and the cellular form of the prion protein, PrPc.
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Animals↗
Biomedical subjects
K L Brown
Publications and source records attributed to K L Brown.
T-lymphocyte activation and the cellular form of the prion protein.
The transmissible spongiform encephalopathies are neurodegenerative disorders which include Creutzfeldt-Jakob disease in humans, and scrapie and bovine spongiform encephalopathy in animals. A major component of the infectious agent responsible for these diseases is considered to be a post-translationally modified form of a host-encoded glycoprotein PrPc, termed PrPSc. While PrPc is abundantly expressed in tissues of the central nervous system (CNS), little is known about its normal function. The expression of PrPc is not restricted to the CNS, as this protein can also be detected in the lymphoid tissues of mice and sheep. In this report we demonstrate that resting murine splenic lymphocytes express PrPc protein on their cell membranes. Furthermore, expression of PrPc was significantly enhanced following in vitro stimulation with the non-specific T-cell mitogen concanavalin A (Con A). Genetically engineered mice with an inactive PrPc gene (PrP-/- mice), were utilized to investigate the involvement of PrPc in lymphocyte activation. Experiments revealed that the Con A-induced proliferation of lymphocytes from PrP-/- mice was significantly reduced to approximately 50-80% that of wild-type (PrP+/+) mice 48 hr post-stimulation. These findings demonstrate an important role for PrPc in extra-neuronal tissues and suggest that PrPc is a lymphocyte surface molecule that participates in T-cell activation.
Animals↗
Severely combined immunodeficient (SCID) mice resist infection with bovine spongiform encephalopathy.
Following combined intraperitoneal and intracerebral injection with bovine spongiform encephalopathy (BSE) cow brain homogenate, SCID mice show a resistance to infection in comparison with immunocompetent CB20 mice. BSE occurred in only five out of 22 challenged SCID mice, with a mean incubation period of 573 days, whereas all the CB20 mice developed the disease with a mean incubation period of 456 days. In contrast, previous studies have shown that intracerebral infection of SCID mice with a mouse-passaged scrapie strain, ME7, produces 100% incidence of disease but no replication of infectivity in spleen. The results with BSE suggest that there is little or no direct infection of the CNS in interspecies transmissions, but that processing or replication of infectivity in peripheral lymphoid tissues may facilitate subsequent spread of infection to the CNS.
Animals↗
Assaying phage-borne peptides by phage capture on fibrinogen or streptavidin.
There is no simple and efficient method for assaying phage isolated from libraries without having to resort to PEG purification of the phage, or to the biotinylation or other labelling of the target molecule. We report here a method for producing 'bifunctional' phage that express two types of peptide; one peptide, fused to pVIII, will bind to immobilized fibrinogen, allowing capture of the phage out of culture supernatants; this allows the other peptide, fused to pIII or pVIII to be assayed by simple ELISA. This system has also been developed for the capture of phage bearing a streptavidin-binding peptide. The bifunctional phage are produced by bacterial cells bearing a plasmid that expresses pVIII fused either to the fibrinogen-binding peptide or to the streptavidin-binding one. Thus, when these cells are infected with a phage clone or pool to be assayed, phage will be produced whose 'capture-peptide' is produced from the plasmid and whose 'assay-peptide' is produced from the phage genome. We show here that, by this method, bifunctional phage can be produced that will bind to immobilized streptavidin or fibrinogen.
Antibody Affinity↗
Delayed gastric emptying and decreased antral contractility in normal premenopausal women compared with men.
OBJECTIVES: The effects of gender on gastric emptying have not been fully elucidated. The aims of this study were to determine how gender affects gastric emptying and to see whether any of the observed differences in gastric emptying correlate with alterations in antral motility as measured by dynamic antral scintigraphy (DAS), cutaneous electrogastrography (EGG), and antroduodenal manometry (ADM). METHODS: Nine normal women [age 27.9 +/- 2.2 (mean +/- SEM) yr] in the first 10 days of the menstrual cycle and 13 normal men (age 27.5 +/- 1.7 yr) underwent simultaneous gastric emptying scintigraphy, DAS, EGG, and ADM. After an overnight fast and placement of an ADM catheter and EGG electrodes, a 60-min fasting recording was obtained, followed by ingestion of a 99m Tc-labeled solid meal. Measurements for all modalities were acquired every 10-15 min for 180 min. RESULTS: The gastric T1/2 was longer in women than in men [102 +/- 18 min vs. 71 +/- 4 (mean +/- SEM) min, p < 0.05]. A comparison of the gastric emptying pattern in women with that in men revealed no difference in proximal gastric emptying and lag phase, but in women the terminal slope of emptying was decreased compared with that in men (p < 0.05). The contractility measured in mid-antrum by DAS was significantly lower for women (p < 0.05). A decrease was also seen in the strength of contractions as measured by ADM. CONCLUSIONS: These data demonstrate that gastric emptying of solid food in normal young, premenopausal women is slower than in age-matched men, even during the first 10 days of the menstrual cycle. The findings suggest that the delay is due primarily to altered distal gastric motor function. This hypothesis was corroborated by finding decreased antral contractility as recorded by both dynamic antral scintigraphy and ADM. This study demonstrates the need to use appropriate control values to evaluate symptomatic female patients.
Adolescent↗
Differential effects of diet and obesity on high and low affinity sulfonylurea binding sites in the rat brain.
The brain contains neurons which alter their firing rates when ambient glucose concentrations change. An ATP-sensitive K+ (Katp) channel on these neurons closes and increases cell firing when ATP is produced by intracellular glucose metabolism. Binding of the antidiabetic sulfonylurea drugs to a site linked to this channel has a similar effect. Here rats with a propensity to develop diet-induced obesity (DIO) or to be diet-resistant (DR) when fed a diet moderately high in fat, energy and sucrose (HE diet) had low and high affinity sulfonylurea binding assessed autoradiographically with [3H]glyburide in the presence or absence of Gpp(NH)p. Before HE diet exposure, chow-fed DIO- and DR-prone rats were separated by their high vs. low 24 h urine NE levels. In DR-prone rats, low affinity [3H]glyburide binding sites comprised up to 45% of total binding with highest concentrations in the hypothalamus and amygdala. But DIO-prone rats had few or no low affinity binding sites throughout the forebrain. High affinity [3H]glyburide binding was similar between phenotypes. When rats developed DIO after 3 months on HE diet, their low affinity binding increased slightly. DR rats fed the HE diet gained the same amount of weight as chow-fed controls but their low affinity binding sites were reduced to DIO levels and both were significantly lower than chow-fed controls. By contrast, high affinity [3H]glyburide binding was increased in DR rats throughout the forebrain so that it significantly exceeded that in both DIO and chow-fed control rats. These studies demonstrate a significant population of low affinity sulfonylurea binding sites throughout the forebrain which, along with high affinity sites, are regulated as a function of both weight gain phenotype and diet composition.
ATP-Binding Cassette Transporters↗
Replication of scrapie in spleens of SCID mice follows reconstitution with wild-type mouse bone marrow.
SCID mice are resistant to intraperitoneal infection with 10(3) and 10(4) intracerebral ID50 units of ME7 scrapie agent whereas they develop disease after intracerebral challenge. However, higher doses introduced, by intraperitoneal or subcutaneous routes, produce disease. Immunocompetent mice of the same strain (CB20) developed scrapie following either intracerebral or intraperitoneal infection. Bioassay of spleens from SCID mice infected with 10(-1) dilutions of ME7 scrapie by intraperitoneal, intracerebral or abdominal subcutaneous injection showed traces or low levels of infectivity in spleen. However, subcutaneous injection beneath the skin of the neck failed to infect the spleen. CB20 bone marrow reconstitution of SCID mice resulted in the regeneration of a normal lymphoid architecture in the spleen. Spleens from these reconstituted mice, infected intracerebrally with a 10(-1) dilution of ME7 contained high levels of infectivity. These results suggest that the ability to replicate scrapie agent in spleen or lymphoid tissue depends on the restoration of normal lymphoid structure and in particular the presence of differentiated follicular dendritic cells. The possibility that SCID mice can select minor strains of scrapie which are normally unrecognized in cloned ME7 is discussed.
Animals↗
Hemorrhagic complications of large volume abdominal paracentesis.
The incidence of hemorrhagic complications from large volume paracentesis in patients with cirrhosis and portal hypertension is unknown. We have reviewed the cases of 179 outpatients undergoing large volume paracentesis at our institution during a 1-yr period. Of these 179 patients, four developed severe hemorrhagic complications requiring hospital admission and blood transfusion. Three of these patients developed intraperitoneal hemorrhage, one of which was localized to the paracentesis puncture site. One patient experienced an abdominal wall hematoma, localized by ultrasound. The symptoms and signs of hemorrhage became evident anywhere from hours up to 1 wk after completion of the paracentesis procedure. The mechanism of delayed hemorrhage is not known but may relate to the the rupture of large intra-abdominal venous collaterals in these patients. The literature does not support a correlation between degree of coagulopathy or thrombocytopenia and risk of bleeding in this setting. To promote early detection of this potentially life-threatening complications, a mechanism should exist for close outpatient follow-up of patients after large volume paracentesis.
Abdomen↗
Widespread and lateralization effects of acute traumatic brain injury on norepinephrine turnover in the rat brain.
Norepinephrine (NE) has been implicated in recovery of function following traumatic brain injury (TBI). While bilateral decrease in brain NE turnover occur at 6-24 h after TBI, it is unknown what effects unilateral TBI might have on brain NE turnover the first few minutes after injury. Her male Sprague-Dawley rats had unilateral confusions of either the right or left somatosensory cortex produced by an air between piston. At 30 min after TBI, brain NE turnover was assessed by measuring the ratio of 3-methoxy-4 hydroxyphenylglycol (MHPG) to NE levels in various brain regions. Both right and left TBI produced 32-103% increases in NE turnover at the injury site and in the ipsilateral cerebral cortex surrounding, rostral and caudal to the injury as compared to the contralateral, uninjured site or to the homologous sites in uninjured controls. NE turnover was also altered selectively in some brain areas not affected by right TBI. Left TBI decreased NE turnover by 29% in the frontal cortex contralateral to the injury and by 24% bilaterally in the hypothalamus while increasing locus coeruleus NE turnover by 72% compared to uninjured controls. Thus, unilateral cortical TBI produced predominantly ipsilateral increases in cortical NE turnover but variable, bilateral changes in NE turnover in subcortical areas which were dependent upon the side of injury. These subcortical differences may explain some of the lateralized effects of cortical injury on post-injury behavior.
Animals↗
The role of anti-sigma factors in gene regulation.
Despite the isolation of an anti-sigma factor over 20 years ago, it is only recently that the concept of an anti-sigma factor emerged as a general mechanism of transcriptional regulation in prokaryotic systems. Anti-sigma factors bind to sigma factors and inhibit their transcriptional activity. Studies on the mechanism of action of anti-sigma factors has shed new light on the regulation of gene expression in bacteria, as the anti-sigma factors add another layer to transcriptional control via negative regulation. Their cellular roles are as diverse as FIgM of Salmonella typhimurium, which can be exported to sense the structural state of the flagellar organelle, to SpoIIAB of Bacillus subtilis participating in the switch from one cell type to another during the process of sporulation. Additionally, the bacteriophage T4 uses an anti-sigma factor to sabotage the Escherichia coli E.sigma 70 RNA polymerase in order to direct exclusive transcription of its own genes. Cross-linking, co-immunoprecipitations, and co-purification indicate that the anti-sigma factors directly interact with their corresponding sigma factor to negatively regulate transcription. In B. subtilis, anti anti-sigma factors regulate anti-sigma factors by preventing an anti-sigma factor from interacting with its cognate sigma factors, thereby allowing transcription to occur.
Amino Acid Sequence↗
Increased potency and binding of mazindol to putative brain anorectic receptors in obesity-prone rats.
A class of sodium-sensitive, low affinity binding sites in the brain recognizes [3H]mazindol (MAZ). Competition for [3H]MAZ binding at these sites correlates with the anorectic potency of various phenethylamine drugs suggesting that these might be anorectic binding sites. Here [3H]MAZ binding, in the absence of sodium, was assessed by quantitative receptor autoradiography in rat brain. Binding was saturable, widespread and heterogenous with Kd = 3-229 microM and Bmax = 0.64-21.9 nmol/mg protein in various brain areas. By saturation studies, highest binding was in the somatosensory cortex, central amygdalar nucleus and bed nucleus of the stria terminalis. Hypothalamic subnuclei had intermediate and the piriform cortex had low binding. Rats were identified as prone to develop (DIO-prone) or resist (DR-prone) diet-induced obesity by their low vs. high 24 h urine norepinephrine excretion, respectively. While similar in body weight and basal 30 min intake of 4% sucrose, DIO-prone rats had 28% greater inhibition of sucrose intake by 3 mg/kg MAZ, i.p. (86 +/- 5%) than DR-prone rats (67 +/- 6%; P = 0.05). DIO-prone rats also had 23-55% higher levels of 10 nM [3H]MAZ binding in various hypothalamic and amygdalar nuclei, the somatosensory, piriform and gustatory cortices and thalamus. Given their greater sensitivity the highest dose of MAZ used and their higher binding of MAZ to putative brain anorectic receptors, DIO-prone rats might have a deficiency of an endogenous satiety factor which could predispose them to develop obesity when challenged with high energy, high sucrose diets.
Amygdala↗
RPS2 of Arabidopsis thaliana: a leucine-rich repeat class of plant disease resistance genes.
Plant disease resistance genes function is highly specific pathogen recognition pathways. PRS2 is a resistance gene of Arabidopsis thaliana that confers resistance against Pseudomonas syringae bacteria that express avirulence gene avrRpt2. RPS2 was isolated by the use of a positional cloning strategy. The derived amino acid sequence of RPS2 contains leucine-rich repeat, membrane-spanning, leucine zipper, and P loop domains. The function of the RPS2 gene product in defense signal transduction is postulated to involve nucleotide triphosphate binding and protein-protein interactions and may also involve the reception of an elicitor produced by the avirulent pathogen.
Amino Acid Sequence↗
Analysis of the Streptomyces coelicolor sigE gene reveals the existence of a subfamily of eubacterial RNA polymerase sigma factors involved in the regulation of extracytoplasmic functions.
sigma E, an RNA polymerase sigma factor of apparent M(r) 28,000, was previously identified by its ability to direct transcription from the P2 promoter of the agarose gene (dagA) of Streptomyces coelicolor. A degenerate oligonucleotide probe, designed from the N-terminal sequence of purified sigma E, was used to isolate the sigma E gene (sigE). The predicted sequence of sigma E shows greatest similarity to sequences of seven other proteins: Myxococcus xanthus CarQ, Pseudomonas aeruginosa AlgU, Pseudomonas syringae HrpL, Escherichia coli sigma E, Alcaligenes eutrophus CnrH, E. coli FecI, and Bacillus subtilis SigX, a protein of unknown function. These eight proteins define a subfamily of eubacterial RNA polymerase factors sufficiently different from other sigma s that, in many cases, they are not identified by standard similarity searching methods. Available information suggests that all of them regulate extracytoplasmic functions and that they function as effector molecules responding to extracytoplasmic stimuli. A. eutrophus CnrH appears to be a plasmid-encoded factor.
Amino Acid Sequence↗
Decontamination studies with the agents of bovine spongiform encephalopathy and scrapie.
Macerates of bovine brain infected with bovine spongiform encephalopathy (BSE) agent, and rodent brain infected with the 263K or ME7 strains of scrapie agent, were subjected to porous-load autoclaving at temperatures between 134 and 138 degrees C for < or = 60 min. Bioassay in rodents showed that none of the regimens produced complete inactivation. Homogenates of BSE-infected bovine brain were exposed for < or = 120 min to solutions of sodium hypochlorite or sodium dichloroisocyanurate containing < or = 16,500 ppm available chlorine. There was no detectable survival of infectivity after the hypochlorite treatments but none of the dichloroisocyanurate solutions produced complete inactivation. Homogenates of BSE-infected bovine brain, and rodent brain infected with the 263K and ME7 strains of scrapie agent, were exposed for < or = 120 min to 1M or 2M sodium hydroxide but no procedure produced complete inactivation of all agents tested.
Animals↗
Spore resistance and ultra heat treatment processes.
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Hot Temperature↗
Heteronuclear nuclear magnetic resonance studies of cobalt corrinoids. 15. The structure of glutathionylcobalamin: a 1H and 13C two-dimensional nuclear magnetic resonance study at 600 MHz.
Glutathionylcobalamin (GSCbl), the complex formed between glutathione (GSH, gamma-glutamylcysteinylglycine) and aquacobalamin (H2OCbl), has been implicated as an intermediate in the pathway for the formation of the cobalamin coenzymes. In chemical model studies, GSCbl has been shown to be a substrate for methylcobalamin formation in the presence of S-adenosylmethionine and a thiol reductant. Although GSCbl was first described in 1964, the structure of this compound, particularly the site of GSH coordination, has been unknown. GSCbl was prepared by reacting GSH (5-fold molar excess) with H2OCbl in 0.1 M sodium phosphate (pH 6.5) and was purified by gel-permeation chromatography on a Bio-Gel P2 polyacrylamide column. By use of a combination of homonuclear [homonuclear J-correlated spectroscopy (COSY), homonuclear Hartmann-Hahn spectroscopy (HOHAHA), and absorption-mode nuclear Overhauser effect spectroscopy (NOESY)] and inverse detected heteronuclear [1H-detected heteronuclear multiple-quantum coherence (HMQC) and 1H-detected multiple-bond heteronuclear multiple-quantum coherence (HMBC) spectroscopies] two-dimensional NMR methods at 600 MHz, the complete 1H and 13C NMR spectra of GSCbl have now been assigned. Comparison of the 1H and 13C NMR chemical shifts of the GS moiety of GSCbl to those of free GSH and GS- shows that by far the largest differences occur at the cysteine alpha and beta positions. This result strongly suggests that GSH is coordinated to the cobalt atom in GSCbl via the cysteine sulfur atom.
Carbon Isotopes↗
The inhibition of corrinoid-catalyzed oxidation of mercaptoethanol by methyl iodide: mechanistic implications.
The cobalamin coenzymes (5'-deoxyadenosyl- and methylcobalamin) and their cobinamide counterparts (5'-deoxyadenosyl- and methylcobinamide) catalyze the oxidation of 2-mercaptoethanol to its disulfide with hydrogen peroxide formation under aerobic conditions. The reactions are blocked by methyl iodide. Inhibition by methyl iodide is apparently due to the formation of the trans dialkyl corrinoids: methyl(adenosyl)cobalamin, dimethylcobalamin, methyl(adenosyl)cobinamide, and dimethylcobinamide, respectively. When the reaction system is illuminated with visible light, inhibition is released and a dramatic enhancement in the rate of oxygen consumption occurs. For reactions catalyzed by adenosyl- and methylcobalamin and then inhibited by methyl iodide, the rates observed during photolysis approach those obtained with aquacobalamin. For reactions catalyzed by adenosyl- and methylcobinamide and then inhibited by methyl iodide, the rates observed during photlysis approach those obtained with diaquacobinamide. Thus, both trans axial carbon-cobalt bonds in the putative dialkyl corrinoid are homolyzed during photolysis. In contrast to these results, the catalysis of the aerobic oxidation of 2-mercaptoethanol by aquacobalamin is only weakly inhibited by methyl iodide. This observation suggests that aquacob(II)alamin is produced during the catalysis of this reaction. Superoxide, the anticipated product of the reaction between aquacob(II)alamin and dioxygen, is formed during aquacobalamin-catalyzed 2-mercaptoethanol oxidation since superoxide dismutase decreases the rate of oxygen consumption by 50%. However, the enzyme has no effect on oxygen uptake during reactions catalyzed by cobalamin coenzymes and their cobinamide counterparts. These corrinoid catalysts apparently transfer two electrons to dioxygen from cobalt(I) intermediates formed during the reactions. Nitrogenous bases inhibit corrinoid-catalyzed thiol oxidation by competing with 2-mercaptoethanol for axial-ligand coordination sites on the catalyst. In contrast to the inhibition observed with methyl iodide, visible light has no effect on the inhibition obtained with nitrogenous bases.
Catalysis↗
Transcriptional regulation of the Bacillus thuringiensis subsp. thompsoni crystal protein gene operon.
The two predominant polypeptides of the Bacillus thuringiensis subsp. thompsoni crystal are encoded by the cry40 and cry34 genes. These crystal protein genes are located in an operon. Western analysis (immunoblotting) demonstrated that the operon promoter activity was located in the region upstream of the cry40 gene. The Cry34 protein was expressed only when the upstream promoter region was present. The crystal protein genes are the only cistrons in the operon, and they are expressed during sporulation, with the highest transcript levels detected early in sporulation (1.5 to 3 h after the onset of sporulation). Transcription initiates from two adjacent sites located 84 and 85 bases upstream of the cry40 translational start codon. The B. thuringiensis subsp. thompsoni crystal protein gene operon promoter aligned with other crystal protein gene promoters, which are activated from early to midsporulation and transcribed in vitro by the B. thuringiensis RNA polymerase E sigma 35.
Bacillus thuringiensis↗