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M A Harrison

Publications and source records attributed to M A Harrison.

At least 37 records · Page 2Linked to original sources
PubMed1997

The vacuolar H+-ATPase: a universal proton pump of eukaryotes.

The vacuolar H+-ATPase (V-ATPase) is a universal component of eukaryotic organisms. It is present in the membranes of many organelles, where its proton-pumping action creates the low intra-vacuolar pH found, for example, in lysosomes. In addition, there are a number of differentiated cell types that have V-ATPases on their surface that contribute to the physiological functions of these cells. The V-ATPase is a multi-subunit enzyme composed of a membrane sector and a cytosolic catalytic sector. It is related to the familiar FoF1 ATP synthase (F-ATPase), having the same basic architectural construction, and many of the subunits from the two display identity with one another. All the core subunits of the V-ATPase have now been identified and much is known about the assembly, regulation and pharmacology of the enzyme. Recent genetic analysis has shown the V-ATPase to be a vital component of higher eukaryotes. At least one of the subunits, i.e. subunit c (ductin), may have multifunctional roles in membrane transport, providing a possible pathway of communication between cells. The structure of the membrane sector is known in some detail, and it is possible to begin to suggest how proton pumping is coupled to ATP hydrolysis.

Animals↗
PubMed1996

Interaction of dibutyltin-3-hydroxyflavone bromide with the 16 kDa proteolipid indicates the disposition of proton translocation sites of the vacuolar ATPase.

The organotin complex dibutyltin-3-hydroxyflavone bromide [Bu2Sn(of)Br] has been shown to bind to the 16 kDa proteolipid of Nephrops norvegicus, either in the form of the native protein or after heterologous expression in Saccharomyces and assembly into a hybrid vacuolar H(+)-ATPase. Titration of Bu2Sn(of)Br against the 16 kDa proteolipid results in a marked fluorescence enhancement, consistent with binding to a single affinity site on the protein. Vacuolar ATPase-dependent ATP hydrolysis was also inhibited by Bu2Sn(of)Br, with the inhibition constant correlating well with dissociation constants determined for binding of Bu2Sn(of)Br complex to the proteolipid. The fluorescence enhancement produced by interaction of probe with proteolipid can be back-titrated by dicyclohexylcarbodiimide (DCCD), which covalently modifies Glu140 on helix-4 of the polypeptide. Expression of a mutant proteolipid in which Glu140 was changed to a glycine resulted in assembly of a vacuolar ATPase which was inactive in proton pumping and which had reduced ATPase activity. Co-expression studies with this mutant and wild-type proteolipids suggest that proton pumping can only occur in a vacuolar ATPase containing exclusively wild-type proteolipid. The fluorescent enhancement of affinity of Bu2Sn(of)Br for the mutant proteolipid was not significantly altered, with the organotin complex having no effect on residual ATPase activity. Interaction of the probe with mutant proteolipid was unaffected by DCCD. These data suggest an overlap in the binding sites of organotin and DCCD, and have implications for the organization and structure of proton-translocating pathways in the facuolar H(+)-ATPase.

Adenosine Triphosphate↗
PubMed1996

Risk of Clostridium botulinum type E toxin production in blue crab meat packaged in four commercial-type containers.

The aim of this investigation was to determine if a risk of Clostridium botulinum growth and toxin production existed in four different packaged crabmeat products. Freshly picked blue crab meat was inoculated with 10(3) to 10(4) spores per g of a mixed pool of four strains of C. botulinum type E (Beluga, Minnesota, G21-5, and 070). The lump crabmeat was packaged in four different packaging containers: (i) 12-oz copolymer polyethylene cups currently used by most crab processors; (ii) 12-oz copolymer polyethylene cups with heat-shrink, tamper-evident low-density polypropylene seals; (iii) 8-oz copolymer polyethylene cups with easy-open aluminum ends: and (iv) 8-oz copolymer polypropylene cups with integral tamper-evident pull-tabs. The packages were stored at either 4 degrees C for 21 days or 10 degrees C for 15 days. Storage at 10 degrees C was used to simulate temperature abuse. The mouse bioassay was used to detect the presence of C. botulinum toxin. Psychotrophic and anaerobic populations were enumerated and were found to increase with time regardless of packaging type. No botulinum toxin was detected in any of the four packaging types stored at 4 degrees C or 10 degrees C throughout the entire storage period.

Animals↗
PubMed1995

The first putative transmembrane helix of the 16 kDa proteolipid lines a pore in the Vo sector of the vacuolar H(+)-ATPase.

The 16 kDa proteolipid is the major component of the vacuolar H(+)-ATPase membrane sector, responsible for proton translocation. Expression of a related proteolipid from the arythropod Nephrops norvegicus in a Saccharomyces strain in which the VMA3 gene for the endogenous proteolipid has been disrupted results in restored vacuolar H(+)-ATPase function. We have used this complementation system, coupled to cysteine substitution mutagenesis and protein chemistry, to investigate structural features of the proteolipid. Consecutive cysteines were introduced individually into putative transmembrane segment 1 of the proteolipid, and at selected sites in extramembranous regions and in segment 3 and 4. Analysis of restored vacuolar H(+)-ATPase function showed that segment 1 residues sensitive to mutation to cysteine were clustered on a single face, but only if the segment was helical. Only residues insensitive to mutation could be covalently modified by the cysteine-specific reagent fluorescein 5-maleimide. A cysteine introduced into segment 3 was the only residue accessible to a relatively hydrophobic reagent, suggesting accessibility to the lipid phase. Analysis of disulphide bond formation between introduced cysteines indicates that the first transmembrane alpha-helices of each monomer are adjacent to each other at the centre of the proteolipid multimeric complex. The data are consistent with a model in which the fluorescein maleimide-accessible face of helix I lines a pore at the centre of a hexameric complex formed by the proteolipid, with the mutationally sensitive face oriented into the protein core. The implications for ion-transport function in this family of proteins are discussed in the context of this structural model.

Animals↗
PubMed1994

Functional properties of a hybrid vacuolar H(+)-ATPase in Saccharomyces cells expressing the Nephrops 16-kDa proteolipid.

The hydrophobic 16-kDa polypeptide which forms gap-junction-like structures in the crustacean Nephrops norvegicus is a member of a highly conserved family of proteolipids involved in a variety of membrane transport functions in eukaryotic cells. This family also includes the product of the Saccharomyces cerevisiae VMA3 gene which encodes an integral membrane component of the vacuolar membrane H(+)-ATPase. The cDNA for the Nephrops proteolipid complements a mutation in the yeast VMA3 gene, resulting in assembly of a hybrid H(+)-ATPase comprising yeast catalytic subunits and Nephrops integral membrane components. The hybrid vacuolar ATPase was capable of ATP hydrolysis which was coupled to proton translocation and showed inhibitor binding and enzymological properties similar to those of wild-type V-ATPases (Km for ATP, 0.4 mM), suggesting that both yeast and crustacean proteolipids share conserved structure at regions of protein interaction. To facilitate isolation of the Nephrops proteolipid by affinity chromatography on a Ni(2+)-binding support, six C-terminal histidine residues were added to the proteolipid. This modification did not prohibit assembly into the hybrid H(+)-ATPase, although the resultant enzyme did have a markedly elevated Km (1.8 mM). The membrane-bound Vo sector of the ATPase was isolated by the affinity-chromatography procedure and reconstituted into synthetic vesicles. This complex was found to be impermeable to small cations in the absence of catalytic ATPase subunits either in situ in the vacuolar membrane or in the reconstituted system. The functional significance of this impermeability and the structure/function relationships between proteolipids from different sources are discussed.

Adenosine Triphosphate↗
PubMed1994

Possible relationship of succinate dehydrogenase and fatty acid synthetase activities to Aspergillus parasiticus (NRRL 5139) growth and aflatoxin production.

Fatty acid synthetase (FAS) activity measured over time corresponded to aflatoxin B1 biosynthesis by Aspergillus parasiticus grown in minimal salts sucrose medium. Succinate dehydrogenase (SDH) activity, our primary metabolism indicator, decreased as FAS activity increased demonstrating that as primary metabolism slows, secondary metabolism and subsequently aflatoxin production begins. Fungal biomass, as measured by chitin, increased up to day 13 then stabilized. Calcium, potassium, magnesium, manganese, zinc, and a combination of these minerals were tested to determine their effect in culture on FAS and SDH activities. Cultures grown in broth supplemented with zinc had greater FAS activity and produced more aflatoxin B1 when compared to the unsupplemented control. To determine if enzyme activity in a complex substrate is altered due to mineral composition, peanuts were cultivated with gypsum (calcium sulfate) supplementation. The peanuts grown had higher calcium content but less zinc. All peanuts grown in gypsum treated fields had less aflatoxin produced on them when compared to unsupplemented peanuts. Also, FAS activity was lower and chitin content was less when compared to the unsupplemented control peanuts. The FAS activity observed in these experiments indirectly suggests that the FAS complex may be responsible for producing the precursor for aflatoxin synthesis. However, additional information is needed to validate this hypothesis.

Aflatoxin B1↗
PubMed1994

The effects of masking on vibrotactile temporal summation in the detection of sinusoidal and noise signals.

Thresholds for the detection of vibrotactile signals of varied duration applied to the thenar eminence were measured in the absence of and in the presence of a masking stimulus. Signals were 250- and 500-Hz sinusoids and noise bursts with bandwidth limited to 250-1000 Hz. The masking stimulus was either a 250-Hz sinusoid, which was presented in phase with the signal when it was sinusoidal, or noise. Changes in threshold as a function of changes in signal duration were found which were predicted accurately from Zwislocki's theory [J. Acoust. Soc. Am. 32, 1046-1060 (1960)] of temporal summation when the signal was detected by the Pacinian channel, but not when it was detected by a non-Pacinian channel (NP). However, when either the signal or the masking stimulus or both were noise, NP thresholds were affected by changes in signal duration. Only when the signal and masking stimuli were both sinusoids were NP thresholds independent of signal duration. It is concluded that signal duration effects in the NP channel are not due to temporal integration, but rather to increases in information about the signal content provided to the subject as exposure duration is increased.

Acoustic Stimulation↗
PubMed1994

Partial purification of a cyanobacterial membrane protein with amino terminal sequence similarity to the N-methylphenylalanine pilins.

Cyanobacterial pilin was extracted from Synechococcus 6301 membranes using a detergent mixture comprising 1% Triton X-100, 1% Thesit and 0.5% dodecyl beta-D-maltoside. Partial purification of pilin from the crude extract was achieved by a single-step purification applying the Rotofor isoelectric focusing system. Up to 100-fold purification of pilin from the crude extract was achieved in a single run. SDS-PAGE analysis showed Synechococcus 6301 pilin migration with an apparent molecular weight of 11 kDa. The amino terminal sequence of the first 28 amino acid residues was identified. Alignment of the predicted sequence showed a 60-80% identity with amino terminal sequences of pilins from pathogenic gram-negative bacteria (enterobacteria). The apparent mass of Synechococcus 6301 pilin was, however, lower. The amino terminus of Synechococcus 6301 pilin, as with other pilins, has a high content of hydrophobic amino acids.

Amino Acid Sequence↗
PubMed1992

Protein phosphorylation and Mg2+ influence light harvesting and electron transport in chloroplast thylakoid membrane material containing only the chlorophyll-a/b-binding light-harvesting complex of photosystem II and photosystem I.

A material containing only photosystem I (PSI) and the chlorophyll-a/b-binding light-harvesting complex of PSII (LHC-II) has been isolated from the chloroplast thylakoid membrane by solubilization with Triton X-100. Fluorescence spectroscopy shows that, within the material, LHC-II is coupled to PSI for excitation-energy transfer and that this coupling is decreased by the presence of Mg2+, which also decreased PSI electron transport specifically at limiting light intensity. Inclusion of phosphorylated LHC-II within the material did not alter its structure, but gave decreased energy transfer to PSI and inhibition of electron transport which was independent of light intensity, implying effects of phosphorylation on both light harvesting and directly on electron transport. Inclusion of Mg2+ within the phosphorylated material gave decreased energy transfer, but slightly increased PSI electron transport. A cation-induced direct promotion of PSI electron transport was also observed in isolated PSI particles. The PSI/LHC-II material represents a model system for examining protein interactions during light-state adaptations and the possibility that LHC-II can contribute to the antenna of PSI in light state 2 in vivo is discussed.

Chlorophyll↗
PubMed1992

Benesh Movement Notation. A tool to record observational assessment.

This article describes the introduction into physiotherapy practice of Benesh Movement Notation, a method for recording observations of patients' posture and movement sequences. Assessments of the technique show it to be a reliable and practical tool for clinical use.

Documentation↗
PubMed1991

Photosynthetic electron transport controls nitrogen assimilation in cyanobacteria by means of posttranslational modification of the glnB gene product.

A glnB gene is identified in the cyanobacterium Synechococcus sp. PCC 7942, and its gene product is found to be covalently modified as a result of imbalance in electron transfer in photosynthesis, where photosystem II is favored over photosystem I. The gene was cloned and sequenced and found to encode a polypeptide of 112 amino acid residues, whose sequence shows a high degree of similarity to the Escherichia coli regulatory protein, PII. In E. coli, PII is involved in signal transduction in transcriptional and post-translational regulation of nitrogen assimilation. Increase in ammonium ion concentration is shown to decrease covalent modification of the Synechococcus PII protein, as in enteric bacteria. We therefore propose that the photosynthetic electron transport chain may regulate the pathway of nitrogen assimilation in cyanobacteria by means of posttranslational, covalent modification of the glnB gene product. The existence of the glnB gene in different strains of cyanobacteria is demonstrated and its implications are discussed.

Amino Acid Sequence↗
PubMed1991

Cyanobacterial thylakoid membrane proteins are reversibly phosphorylated under plastoquinone-reducing conditions in vitro.

Reversible, light-dependent protein phosphorylation was observed in isolated thylakoid membranes of the cyanobacterium Synechococcus 6301. A polypeptide of 15 kDa in particular was phosphorylated under plastoquinone-reducing conditions and was not phosphorylated under plastoquinone-oxidising conditions. Phosphorylation and dephosphorylation reactions involving this and several other membrane polypeptides showed sensitivity to inhibitors of protein kinases and phosphatases. Changes in phosphorylation state correlated with changes in low temperature fluorescence emission characteristic of changes in excitation energy distribution between the photosystems. The 15 kDa phosphopolypeptide is likely to be involved directly in light state adaptations in cyanobacteria.

Cyanobacteria↗
PubMed1990

Modification of a glnB-like gene product by photosynthetic electron transport in the cyanobacterium Synechococcus 6301.

Covalent modification of a 13 kDa soluble-phase protein occurs during adaptation of cells of the cyanobacterium Synechococcus 6301 (mutant AN112) to light specifically absorbed by photosystem II. This adaptation is accompanied by functional changes indicative of altered excitation energy distribution between the photosystems. The 13 kDa protein is identified by solid-phase N-terminal sequencing as a protein related to PII, the glnB gene product of E. coli. In E. coli, the PII protein undergoes uridylylation and acts as a regular of glutamine synthetase at both the post-translational and transcriptional levels. The implications of modification of a transcriptional regulator by photosynthetic electron transport are discussed.

Amino Acid Sequence↗
PubMed1990

Occupational asthma and specific IgE to a diazonium salt intermediate used in the polymer industry.

This study describes sensitization to a diazonium salt intermediate, diazonium tetrafluoroborate (DTFB), produced during the manufacture of a fluorine polymer precursor. Most of the workers exposed to DTFB powder complained of respiratory and mucosal irritation. However, some of these individuals had symptoms typical of occupational asthma. Clinical and immunologic studies, including bronchial provocation testing and specific IgE measurements, were performed on two individuals with asthmatic symptoms associated with exposure to DTFB. These studies confirmed a diagnosis of occupational asthma. In an investigation of 43 other exposed workers in two separate factories handling the diazonium compound, specific IgE antibodies to DTFB-human serum albumin conjugate were found in 20% of individuals. There was a good correlation between specific IgE and exposure-related respiratory symptoms, which suggests an IgE-mediated pathogenesis. DTFB-human serum albumin conjugates were characterized by electrophoretic techniques, and it was found that conjugates prepared at pH 10 elicited optimum IgE binding in RAST. In vitro cross-linking of albumin by DTFB was demonstrated but had no effect on RAST binding. Our findings on the effect of pH and polymerization on IgE binding support proposed mechanisms for in vivo conjugation and provide information on the antigenic determinants important in IgE recognition of hapten-carrier protein conjugates.

Antibody Formation↗
PubMed1990

Response of the Photosynthetic Apparatus in Dunaliella salina (Green Algae) to Irradiance Stress.

The response of the photosynthetic apparatus in the green alga Dunaliella salina, to irradiance stress was investigated. Cells were grown under physiological conditions at 500 millimoles per square meter per second (control) and under irradiance-stress conditions at 1700 millimoles per square meter per second incident intensity (high light, HL). In control cells, the light-harvesting antenna of photosystem I (PSI) contained 210 chlorophyll a/b molecules. It was reduced to 105 chlorophyll a/b in HL-grown cells. In control cells, the dominant form of photosystem II (PSII) was PSII(alpha)(about 63% of the total PSII) containing >250 chlorophyll a/b molecules. The smaller antenna size PSII(beta) centers (about 37% of PSII) contained 135 +/- 10 chlorophyll a/b molecules. In sharp contrast, the dominant form of PSII in HL-grown cells accounted for about 95% of all PSII centers and had an antenna size of only about 60 chlorophyll a molecules. This newly identified PSII unit is termed PSII(gamma). The HL-grown cells showed a substantially elevated PSII/PSI stoichiometry ratio in their thylakoid membranes (PSII/PSI = 3.0/1.0) compared to that of control cells (PSII/PSI = 1.4/1.0). The steady state irradiance stress created a chronic photoinhibition condition in which D. salina thylakoids accumulate an excess of photochemically inactive PSII units. These PSII units contain both the reaction center proteins and the core chlorophyll-protein antenna complex but cannot perform a photochemical charge separation. The results are discussed in terms of regulatory mechanism(s) in the plant cell whose function is to alleviate the adverse effect of irradiance stress.

Journal Article↗
PubMed1989

Auxin asymmetry during gravitropism by tomato hypocotyls.

Gravitropic asymmetry of auxin was observed in hypocotyls of tomato (Lycopersicon esculentum Mill.) soon after horizontal placement: the ratio of apically supplied [3H]IAA collected from the lower sides to that from the upper sides was about 1.4 between 5 and 10 minutes. This was adequately early to account for the beginning of curvature. The auxin asymmetry ratio rose to about 2.5 between 20 and 25 minutes, and to 3.5 during the main phase of curvature. This compares reasonably well with the roughly 3.9 ratio for elongation on the lower side to elongation on the upper side that is the basis for the curvature. These data extend evidence that the Went-Cholodny theory for the mediation of tropisms is valid for dicot stems. Also consistent with the theory, an auxin asymmetry ratio of 2.5 was observed when wrong-way gravitropic curvature developed following application of a high level of auxin. In addition to reversing the asymmetry of elongation, the large supplement of auxin resulted in lower net elongation. Previous data established that ethylene is not involved in this decrease of growth as a function of increasing level of auxin.

Gravitropism↗