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B E Boyes

Publications and source records attributed to B E Boyes.

At least 19 recordsLinked to original sources
PubMed1995

Improved reversed-phase high performance liquid chromatography columns for biopharmaceutical analysis.

Reversed-phase high performance liquid chromatography continues to grow in importance for the analysis of peptides and proteins in biomolecular and pharmaceutical research. The mobile-phase conditions for separation of proteins and peptides are essentially fixed. Most separations of these solutes are conducted with shallow gradients using aqueous buffers modified with acetonitrile. Therefore, changing selectivity in peptide and protein separations is often best accomplished by a change in bonded-phase chemistry of the column (e.g. by using CN- or C3-bonded phases rather than C8 or C18). In general, however, short-chain bonded phases are more unstable and irreproducible than bonded phases having longer chains, due to increased susceptibility to hydrolysis as hydrophobicity of the bonded phase decreases. These problems are minimized using bonded phases that are protected through use of sterically bulky side-groups (StableBond Technology). This paper describes a series of comparisons between traditional polymeric bonded-phase silica columns and sterically protected, highly purified, silica stationary phases. These studies compare lot-to-lot reproducibility, stability of the bonded phase, selectivity effects between bonded phases, and operational advantages that can be obtained by high temperature operation using a series of stable bonded phases.

Chemical Phenomena↗
PubMed1994

Temperature as a variable in reversed-phase high-performance liquid chromatographic separations of peptide and protein samples. II. Selectivity effects observed in the separation of several peptide and protein mixtures.

Changes in band spacing as a function of temperature and/or gradient steepness were investigated for four peptide or protein samples. Reversed-phase HPLC in a gradient mode was used to separate tryptic digests of tissue plasminogen activator and calmodulin. Additionally, a synthetic peptide mixture and a storage protein sample from wheat were studied. Simultaneous changes in gradient steepness and temperature were found to provide considerable control over band spacing and sample resolution. The effects of temperature and gradient steepness on selectivity in these systems appear to be complementary. Simultaneous optimization of both temperature and gradient steepness thus represents a powerful and convenient means of controlling band spacing and separation. Because of the complexity of these sample chromatograms, computer simulation proved to be a useful tool in both interpreting these experiments and in optimizing final separations.

Calmodulin↗
PubMed1992

Identification and characterization of a large human brain gene whose expression is increased in Alzheimer disease.

A cDNA clone that was isolated from a human substantia innominata cDNA library is described. By Northern hybridization analysis, a 15.5 kilobase (kb) transcript was identified by this clone in RNA samples from several brain regions, but not in RNA samples from white matter, liver or placenta. Hybridization to human genomic DNA revealed a pattern indicative of a single copy gene. DNA sequence analysis showed 3.0 kb of 3' untranslated region with no significant open reading frame. An additional cDNA clone, representing a section of an alternate form of this transcript, was isolated that contained an additional 1.5 kb at the 3' end. Using a nuclease protection assay, the expression of this gene was found to be increased by 30% in Alzheimer disease temporal cortex RNA samples compared to temporal cortex RNA samples from normal controls, but to be at equivalent levels in Alzheimer disease, as compared to normal control, substantia innominata RNA samples. This assay also showed that this gene was expressed at 3.5-fold higher levels in normal substantia innominata than in normal cerebellum. In situ hybridization analysis showed that the transcript could be detected in cerebellar neurons.

Aged↗
PubMed1991

Cytomegalovirus infection of the developing brain alters catecholamine and indoleamine metabolism.

Tissue concentrations of noradrenaline (NA), serotonin (5-HT), dopamine (DA) and selected metabolites were measured in the spinal cord, cerebellum, cerebral cortex and caudate-putamen of developing mice following intraventricular inoculation with murine cytomegalovirus (MCMV) on postnatal day 10. MCMV-infected animals exhibited transient signs of neurological impairment, including apparent hypertonicity of hindlimb extensors and abnormal gait, beginning on days 14-16 and continuing for 3-5 days. At the onset of neurological impairment, tissue concentrations of NA were significantly reduced in the spinal cord (20%), cerebellum (32%) and cerebral cortex (40%) of infected animals. Levels of 5-HT were significantly increased in the caudate-putamen (50%), while 5-hydroxyindoleacetic acid (5-HIAA) was increased in both the spinal cord (94%) and caudate-putamen (65%). The ratio of 5-HIAA/5-HT, which is frequently used as an estimate of turnover of 5-HT, was significantly increased in the spinal cord (90%) at the onset of neurological impairment. In the caudate-putamen of MCMV-infected animals, there were significant increases in the tissue levels of DA (37%), homovanillic acid (HVA, 41%) and 3,4-dihydroxyphenylacetic acid (DOPAC, 34%). All neurochemical parameters were normal in the MCMV-infected animals by postnatal day 70, approximately 50 days after the resolution of neurological signs. These results indicate transient alterations in monoamine metabolism in the developing nervous system during the pathogenesis of cytomegalovirus-induced movement and postural disorders.

Animals↗
PubMed1989

Strategies for the identification of novel brain specific genes affected in Alzheimer disease.

The pathological changes that occur in Alzheimer disease (AD) brain lead to a large loss of various classes of neurons and the production of novel proteinaceous elements such as neuritic plaques and neurofibrillary tangles. For the neuronal loss to occur and these elements to arise, there must be a disturbance in the expression or regulation of genes that code for proteins required for normal cell maintenance, or perhaps even for the expression of genes unique to AD. We describe the construction of a cDNA library from the human substantia innominata and strategies for isolating genes that are expressed differentially between brain regions and which may be affected by AD. Some of the results obtained using these strategies and a preliminary description of a novel brain specific mRNA of 15.5kb, whose expression is increased in AD affected temporal cortex, are presented.

Alzheimer Disease↗
PubMed1989

Increased uric acid in the developing brain and spinal cord following cytomegalovirus infection.

Tissue concentrations of uric acid were determined in the spinal cord, cerebellum, caudate-putamen, and cerebral cortex of developing mice following intraventricular inoculation with murine cytomegalovirus (MCMV) on postnatal day 10. Transient signs of neurological impairment were observed in MCMV-infected animals beginning on days 13-16 and continuing until days 19-21. At the onset of neurological impairment, uric acid concentrations in tissues from infected animals were 17-60-fold greater than in control animals. On postnatal day 70, 60 days after inoculation and 40 days after resolution of neurological signs, uric acid levels were still two- to threefold greater in infected animals. Histological examination revealed signs of focal ischemia in the cerebral and cerebellar cortices of MCMV-infected mice only at the onset of neurological impairment, with ischemic cell changes in some pyramidal neurons of the cerebral cortex. These results indicate that uric acid may be a sensitive marker of persistent vascular pathology resulting from cytomegalovirus infection of the developing nervous system.

Aging↗
PubMed1988

Methylmercury-induced movement and postural disorders in developing rat: regional analysis of brain catecholamines and indoleamines.

Subcutaneous administration of methylmercury (MeHg) to rats during early postnatal development resulted in movement and postural disorders by day 22-24. Tissue concentrations of norepinephrine (NE), serotonin (5-HT), dopamine (DA) and selected metabolites were measured in the cerebral cortex, spinal cord and caudate-putamen at the onset of neurological impairment and at two subclinical stages of toxicity. In the cerebral cortex there was a significant increase in tissue concentrations of 5-HT (54-81%) and 5-hydroxyindoleacetic acid (HIAA, 133-178%) at the onset of neurological impairment. Similar increases were detected in the spinal cord for 5-HT (19-43%) and HIAA (98-123%) as well as an increase in the concentration of NE (42-51%). In the caudate-putamen there were significant increases in the concentrations of NE (98-116%), HIAA (108-124%) and DA (28-29%) with a significant decrease in the concentration of 3,4-dihydroxyphenylacetic acid (DOPAC, 20-27%); however, tissue levels of homovanillic acid (HVA) did not change significantly. Many of these changes were detected at subclinical stages of MeHg toxicity. The ratio of HIAA/5-HT, which is frequently used as an estimate of turnover for 5-HT, was significantly increased in all 3 tissues at the onset of neurological impairment (38-94%) and at one subclinical stage (47-114%). The ratio of (DOPAC + HVA)/DA was significantly decreased in caudate-putamen at all 3 stages of toxicity (18-40%). These changes indicate altered metabolism in aromatic amine systems in the developing central nervous system during the pathogenesis of MeHg-induced movement and postural disorder.

Aging↗
PubMed1988

Separation of large DNA restriction fragments on a size-exclusion column by a nonideal mechanism.

Double-strand DNA (dsDNA) restriction fragments were chromatographed on the DuPont Bioseries GF-250 column. Two anomolous chromatographic properties were observed. (1) A triphasic dependence of retention on dsDNA chain length was observed. Small DNA fragments (less than 500 base pairs) displayed typical size exclusion, intermediate size DNA (800-5000 base pairs) eluted in the void volume, and larger DNA fragments were increasingly retained. (2) The void volume for nucleic acids was less than that for large polypeptides. The retention of moderately large DNA fragments increased linearly as the square root of the chain length over the range 5.5 to 50 kilobase pairs (ca. 3-30 X 10(6) Mr). A number of eluant manipulations were carried out in order to examine the mechanism by which the larger DNA fragments were being retained and separated. Evidence was not obtained to support either ion exchange or reverse phase as the retention mechanism. The usefulness of such a column for molecular biological manipulations is illustrated by the rapid isolation of homogeneous viral DNA fragments resected from their cloning vectors with restriction endonucleases.

Base Composition↗
PubMed1988

Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson's and Alzheimer's disease brains.

We detected large numbers of HLA-DR-positive reactive microglia (macrophages), along with Lewy bodies and free melanin, in the substantia nigra of all cases studied with Parkinson's disease (5) and parkinsonism with dementia (PD) (5). We found similar, but less extensive, pathology in the substantia nigra of six of nine cases of dementia of the Alzheimer type (DAT) but in only one of 11 age-matched nonneurologic cases. All dementia cases with a premortem diagnosis of DAT or PD showed large numbers of HLA-DR-positive reactive microglia and significant plaque and tangle counts in the hippocampus, as well as reduced cortical choline acetyltransferase activity. One of 11 nondemented controls showed mild evidence of similar cortical pathology. These data indicate that HLA-DR-positive reactive microglia are a sensitive index of neuropathologic activity. They suggest a frequent coexistence of DAT- and Parkinson-type pathology in elderly patients.

Aged↗
PubMed1987

Altered metabolism of [18F]-6-fluorodopa in the hooded rat following inhibition of catechol-O-methyltransferase with U-0521.

[18F]-6-Fluoro-L-DOPA ([18F]DOPA), a tracer for cerebral dopamine in studies utilizing positron emission tomography (PET), is rapidly metabolized by catechol-O-methyltransferase (COMT) in the periphery following intravenous injection to carbidopa-pretreated humans and rats. Experiments were performed to determine the effect of pretreatment with 3',4'-dihydroxy-2-methyl-propiophenone (U-0521), a competitive inhibitor of COMT, on [18F]DOPA metabolism in the carbidopa-pretreated hooded rat. U-0521 (25 mg/kg, i.p.), administered 10 min prior to the [18F]DOPA, served to increase the persistence of [18F]DOPA in plasma over a 2-hr period by decreasing the rate of formation of the peripheral metabolite 3-O-methyl-6-fluorotyrosine (Me[18F]DOPA). This compound passes readily into brain and was the sole [18F]DOPA metabolite observed in cortex and cerebellum. U-0521 produced a short-lasting decrease in Me[18F]DOPA levels in these two tissues. In striatum, decreases in Me[18F]DOPA were found to last at least 90 min. Associated with the elevated availability of [18F]DOPA in plasma produced by U-0521 were 50% increases in striatal [18F]dopamine ([18F]DA) levels and 40% increases in the levels of [18F]dihydroxyphenylacetic acid ([18F]DOPAC) at times between 30 and 90 min following [18F]DOPA injection. Increased decarboxylation of [18F]DOPA in the striatum of U-0521-treated rats resulted in heightened radiocontrast between striatum and other cerebral tissues.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1987

The metabolism of [18F]6-fluoro-L-3,4-dihydroxyphenylalanine in the hooded rat.

The metabolism of the positron-emitting compound [18F]6-fluoro-L-3,4-dihydroxyphenylalanine (*F-DOPA) was studied in carbidopa-pretreated male hooded rats. Thirty minutes following carbidopa administration (5 mg/kg i.p.), animals received *F-DOPA (500 micrograms/kg; specific activity, 175-230 Ci/mol) as an intrajugular bolus. Blood samples were taken at various times between 5 and 90 min, and the plasma was analyzed by HPLC with gamma counting of fractions. *F-DOPA disappeared rapidly from plasma in concert with the formation of the 3-O-methylated metabolite, Me-*F-DOPA. Animals were killed from 5 to 120 min after injection, and the brains were rapidly dissected. The disappearance of *F-DOPA from both vermis and striatal samples was rapid. Me-*F-DOPA, the sole metabolite observed in the vermis, was the major labeled material in the striatum at greater than or equal to 20 min after injection. Fluorodopamine was an important metabolite in the striatum, making up 25% of total radioactivity at early intervals. Striatal samples also contained fluoro-3,4-dihydroxyphenylacetic acid, which constituted approximately 10% of the total radioactivity, and traces of two radiolabeled compounds, tentatively identified as fluorohomovanillic acid and fluoro-3-methoxytyramine.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1986

Determination of plasma [18F]-6-fluorodopa during positron emission tomography: elimination and metabolism in carbidopa treated subjects.

An investigation of the metabolism of [18F]-6-fluorodopa (FDOPA) given to carbidopa treated subjects for scanning by positron emission tomography (PET) has been carried out by analysis of plasma. Reverse phase ion pair HPLC and alumina extraction were employed to fractionate and identify the [18F]-labelled compounds of plasma over a two hour period. During this time, the plasma levels of both total 18F and FDOPA decreased as a bi-exponential function of time. The rates of 18F, but not FDOPA, elimination were observed to decrease with age. In addition to FDOPA, only one other major peak of radioactivity was resolved by HPLC. Identification of this compound as the O-methylated derivative of FDOPA (MeFDOPA) is based on its shared HPLC elution time with in vitro synthesized O-[methyl-14C]-FDOPA. The ratio of the concentration of MeFDOPA to FDOPA (MeFDOPA/FDOPA) in plasma increased linearly with time, and the slope of this linear relationship decreased with the age of the individual.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1986

Immunohistochemical co-localization of S-100b and the glial fibrillary acidic protein in rat brain.

A rabbit antiserum against purified bovine brain S-100b protein was produced and characterized by immunoassay and immunoblot analysis of electrophoretically resolved soluble brain proteins. Fluorescent immunohistochemistry was conducted in order to determine the cellular localization of the S-100b immunoreactivity. Double immunohistofluorescent experiments on adult rat brain tissue sections with the rabbit antiserum to S-100b and a rat monoclonal antibody to the glial fibrillary acidic protein resulted in immunolabelling of the same cells. This finding determines a strict astroglial localization of the S-100b immunoreactivity. In addition, the immunolabelling of astrocyte perikarya and processes by the S-100b immunohistochemistry is consistent with a cytoplasmic location of S-100b. In contrast, the glial fibrillary acidic protein immunohistochemistry predominantly labeled the fine fibrillary processes of the cells. The present report suggests that S-100b immunohistochemistry is of use for the specific identification and morphological description of astrocytes in the rat brain.

Animals↗
PubMed1982

Physicochemical and optical studies on calcium- and potassium-induced conformational changes in bovine brain S-100b protein.

The brain-specific S-100 protein is a mixture of two components, S-100a and S-100b, with a subunit composition of alpha beta or beta 2, respectively. S-100b, isolated by using hydroxylapatite chromatography in its final purification, is homogeneous by the criteria of gel electrophoresis in the absence and presence of sodium dodecyl sulfate (NaDodSO4) and ultracentrifuge studies. Molecular weight studies by both sedimentation equilibrium in 6 M guanidine hydrochloride and 15% NaDodSO4 gels indicated the subunit molecular weight to be 10 500, and since a molecular weight of 21 000 was obtained in native solvents, the protein exists as a dimer in benign medium. The two subunits are held together by noncovalent forces. The S-100b protein undergoes a conformational change upon binding calcium, as revealed by ultraviolet difference spectroscopy and circular dichroism (CD) studies in the aromatic and far-ultraviolet (UV) range. Far-UV CD studies indicated the apparent helical content drops from approximately 58 to 52% in the presence of Ca2+. The effect of K+ on the protein was antagonistic to Ca2+, and the proteins affinity for calcium was lowered by the presence of K+. The conformational state of the protein is very much dependent upon the metal ions (Ca2+, K+) present, suggesting that changing conformation may be the way S-100 responds to local changes in ionic parameters. Fluorescence studies indicate the presence of an abnormal tyrosine in the protein with the emission maximum centered between 327 and 330 nm when the protein is excited at 280 nm.

Calcium↗