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PubMed1978

The ATPase activity of subfragment-1 from the hypertrophied heart.

Myosin and subfragment-1 were prepared from rabbit hearts hypertrophied secondary to pulmonary artery constriction. The Ca2+ -stimulated ATPase activity was reduced while the potassium/EDTA-stimulated ATPase activity was unchanged in both the myosin and subfragment 1 (S-1) from hypertrophied hearts. When hypertrophy myosin was mixed with an equal quantity of control myosin, the ATPase activity of the mixed protein fell halfway between control and hypertrophy values. Similar results were obtained with control and hypertrophy S-1. The actin-stimulated ATPase activity of hypertrophy S-1 was slightly depressed but unlike hypertrophy myosin this depression was not significant when compared to normal S-1. This suggests that papain cleavage may have removed part of the conformational difference that exists between control and hypertrophy myosins.

Actins↗

PubMed1985

Immunological quantitation of tyrosinase from wild-type and albino mutant mice.

The relationship between gene dosage, enzyme activity, and level of immunologically cross-reacting material (CRM) was examined in mammalian tyrosinase (EC 1.14.18.1) by rocket immunoelectrophoresis. Skin extracts from mice heterozygous (C/c) and homozygous (c/c) for the albino locus contain 46% and 0% of CRM, respectively, as compared with wild-type (C/C) animals. Enzyme activity and CRM level were directly proportional in these genotypes, suggesting that the albino locus controls the quantity of tyrosinase produced in melanocytes.

Animals↗

PubMed1980

An effective and reliable computerised system for storage and retrieval of references to scientific publications.

A suite of programs has been implemented to facilitate the storage and retrieval of references to published works in a typical scientific research establishment. The retrieval process is governed by searches for specified combinations of keywords, author names, individual references, and lengths of time that references have been held on file. The system currently caters for approximately 1290 journals together with abstracts, reports, supplements, symposia, proceedings and books. Five years experience has proved the system to be reliable and effective, and it requires only 1.5 technician-days per week to operate it and to input about 25 000 articles annually. This system fulfills a requirement felt by many specialised institutions in providing localised control over quantities of information which are too large to be processed manually.

Bibliographies as Topic↗

PubMed1993

Quantitative measurement of calretinin and beta-actin mRNA [correction of mRNAIN] in rat brain micropunches without prior isolation of RNA.

A microdissection technique for quantitation of neurochemicals in discrete brain nuclei has been applied to quantitative measurement of mRNA. The method permits quantitation of low abundance mRNA from submilligram amounts of tissue (10-500 micrograms protein). Discrete nuclei and other regions of the brain are solubilized in concentrated guanidine thiocyanate solution, mRNA is directly hybridized with riboprobes, and detected with a ribonuclease protection assay. This method eliminates the necessity for RNA isolation from solid tissue. No assumptions regarding RNA recovery are necessary since tissue specimens are solubilized, hybridized and treated with ribonuclease in a single tube. We have determined the mRNA levels of calretinin, a predominantly neuron-specific calcium binding protein in microdissected nuclei and other regions of rat brain. For interassay comparison, measurement of sample protein and beta-actin mRNA permits normalization and quantitation in terms of these internal controls. The quantity of calretinin mRNA ranged from 281 +/- 35 fg/micrograms protein in the thalamic paraventricular nucleus to 2.3 +/- 0.5 fg/micrograms protein for the cerebral cortex. The calretinin/beta-actin ratios ranged from 79.9 +/- 9.3% to 1.3 +/- 0.1%, respectively. The combination of microdissection techniques with a lysate RNase protection assay: (1) establishes this technique as quantitative for detection of high and low abundance mRNAs from microdissected brain specimens; (2) bypasses the inefficiencies and uncertainties associated with isolating RNA; and (3) enables large numbers of determinations from discrete brain nuclei to be analyzed in 2 to 3 days.

Actins↗

PubMed2005

Methoxypoly(ethylene glycol)-conjugated carboxypeptidase A for solid tumor targeting: part I: synthesis and characterization.

In vivo efficacy of novel anticancer agents has been hindered by the inability to deliver effective concentrations of drugs to tumors. The use of macromolecules such as antibodies and polymers for enzyme delivery to tumors has revealed that catalyzing the conversion of a nontoxic prodrug into its cytotoxic form can generate an effective level of cytotoxic agents at tumor sites. This study primarily focuses on the synthesis and characterization of methoxypoly(ethylene glycol)-modified carboxypeptidase A (CPA) for solid tumor targeting. The molecular weight of CPA has been successfully altered from 35 to 40-50 kDa via attachment of a defined number of mPEG moieties. Relatively pure mPEG-CPA conjugates containing one, two, and three mPEG chains were obtained at preparative scale quantities through controlled PEGylation followed by fractionation that involved size-exclusion chromatography. An enhancement in kinetic properties including k(cat) and k(cat)/K(m) towards hippuryl-L-phenylalanine (hipp-L-phe) was observed in mPEG-CPA conjugates. An increase in the V(m) appeared to be responsible for this enhancement. The attachment of mPEG to CPA substantially improved the stability of the enzyme with respect to the specific peptidase activity toward the model substrate. This finding is particularly important in the development of a novel CPA/methotrexate-alpha-peptide system in solid tumor chemotherapy.

Animals↗

PubMed2006

Permeability predictions for sand-clogged Portland cement pervious concrete pavement systems.

Pervious concrete is an alternative paving surface that can be used to reduce the nonpoint source pollution effects of stormwater runoff from paved surfaces such as roadways and parking lots by allowing some of the rainfall to permeate into the ground below. This infiltration rate may be adversely affected by clogging of the system, particularly clogging or covering by sand in coastal areas. A theoretical relation was developed between the effective permeability of a sand-clogged pervious concrete block, the permeability of sand, and the porosity of the unclogged block. Permeabilities were then measured for Portland cement pervious concrete systems fully covered with extra fine sand in a flume using simulated rainfalls. The experimental results correlated well with the theoretical calculated permeability of the pervious concrete system for pervious concrete systems fully covered on the surface with sand. Two different slopes (2% and 10%) were used. Rainfall rates were simulated for the combination of direct rainfall (passive runoff) and for additional stormwater runoff from adjacent areas (active runoff). A typical pervious concrete block will allow water to pass through at flow rates greater than 0.2 cm/s and a typical extra fine sand will have a permeability of approximately 0.02 cm/s. The limit of the system with complete sand coverage resulted in an effective system permeability of approximately 0.004 cm/s which is similar to the rainfall intensity of a 30 min duration, 100-year frequency event in the southeastern United States. The results obtained are important in designing and evaluating pervious concrete as a paving surface within watershed management systems for controlling the quantity of runoff.

Facility Design and Construction↗

PubMed2001

Modification of fatty acids changes the flavor volatiles in tomato leaves.

Expression of the yeast Delta9 desaturase gene in tomato (Lycopersicon esculentum Mill.) resulted in changes in the profiles of fatty acids in tomato leaves. Transgenic leaves displayed a dramatic increase in cis-Delta9 16:1, which only existed in a small quantity in control leaves. Also higher, but not as dramatic, were 18:1 and 16:3 fatty acids. Several fatty acids, viz. 16:0, 18:0, and 18:3 declined in transgenic leaves. Changes in fatty acids were accompanied by changes in certain volatile compounds derived from fatty acids. On a percentage basis, most notable increases (>3-fold) were 1-hydroxy-2-butanone, 1-penten-3-ol, heptanal, 3-hexen-1-ol, 2-octanol,cis-3-hexenal, hexanal and 2-nonenal. Several flavor compounds not known to be biochemically derived from fatty acids, viz. 2-ethyl-furan, 5-ethyl-2-[5H]-furanone, eugenol, and 2-ethylthiophene also showed sharp increases in transgenic leaves.

Fatty Acids↗

PubMed2000

Biological effects of ultrasound: development of safety guidelines. Part I: personal histories.

After the end of World War II, advances in ultrasound (US) technology brought improved possibilities for medical applications. The first major efforts in this direction were in the use of US to treat diseases. Medical studies were accompanied by experiments with laboratory animals and other model systems to investigate basic biological questions and to obtain better understanding of mechanisms. Also, improvements were made in methods for measuring and controlling acoustical quantities such as power, intensity and pressure. When diagnostic US became widely used, the scope of biological and physical studies was expanded to include conditions for addressing relevant safety matters. In this historical review, a major part of the story is told by 21 investigators who took part in it. Each was invited to prepare a brief personal account of his/her area(s) of research, emphasizing the "early days," but including later work, showing how late and early work are related, if possible, and including anecdotal material about mentors, colleagues, etc.

History, 20th Century↗

PubMed2001

Movement of water and heavy metals (Zn, Cu, Pb and Ni) through sand and sandy loam amended with biosolids under steady-state hydrological conditions.

New guidelines for using biosolids in UK agriculture favour the use of enhanced treated biosolids, such as dried and composted cakes, due to concerns about the potential for transfer of pathogens into the food chain. However, there is a need to ensure that their use is environmentally acceptable and does not increase the risk to potable water supplies or the food chain from other contaminants such as heavy metals and xenobiotic organic chemicals. The objective of this study was to determine whether the use of composted and dried mesophilic anaerobically digested dewatered (MADD) biosolids would increase the risk of heavy metal leaching from cultivated horizons when compared to more conventionally used MADD cake. Three biosolids (MADD sewage sludge cake - fresh, dried and composted) were mixed with a sand (typic quartzipsamments, %OM = 3.0, pH = 6.5) or a sandy loam (typic hapludalf, %OM = 4.8, pH = 7.6) at an application rate equivalent to 250 kg N/ha/y resulting in loadings of approximately Zn: 6 microg, Cu: 2 microg, Pb: 5 microg and Ni: 0.2 microg/g of soil dry weight basis. These amended soils were repacked into columns (0.4 m by 0.1 m internal diameter) and leaching of Zn, Cu, Pb and Ni was investigated following application of two 24 h simulated rainfall events of 4.5 mm/h. Water balance data and the use of conservative tracers (Cl- and Br ) showed that the hydrological regimes of each core were comparable and, thus, unlikely to account for differences in metal leaching observed. Although no significant difference (P = 0.05) was observed between biosolid amended and control soils, those amended with composted sludge consistently gave higher loss of all metals than did the control soils. Total losses of metals from compost amended soil over the two rainfall events were in the ranges, Zn:20.5-58.2, Cu:9.0-30.5, Pb:24.2-51.2 and Ni:16.0-39.8 microg metal/kg amended soil, compared with Zn:16.4-41.1, Cu:6.2-25.3, Pb:16.9-41.7, and Ni:3.7-25.4 microg metal/kg soil from the control soils. Losses of Zn, Cu, Pb and Ni from fresh MADD cake amended soils (19.8-41.3, 3.2-25.8, 21.6-51.6 and 7.6-36.5 microg metal/kg amended soil, respectively) and from dry MADD cake amended soils (10.7-36.7, 1.8-23.8, 21.2-51.2 and 6.8-39.2 microg metal/kg amended soil, respectively) were similar to the controls. Generally, quantities of metals leached followed the order Zn = Pb > Cu > Ni, which was consistent with the levels of metals in the original sludge/soil mixtures. These results suggest that composting or drying MADD biosolids is unlikely to increase the risk of groundwater contamination when compared to the use of MADD cake; therefore, the changes in UK sludge use in agriculture guidelines are satisfactory in this respect.

Agriculture↗

PubMed2002

Expression and characterization of a synthetic protein C activator in Pichia pastoris.

Protein C activators are proteases that activate protein C in the mammalian coagulation system. A reptilian protein C activator is a critical component in current functional assays for protein C, its cofactor protein S, as well as for the overall status of the protein C pathway. We have constructed a synthetic gene for a protein C activator, based on a published snake-venom polypeptide sequence. This recombinant protein C activator was expressed in Pichia pastoris as a secreted glycoprotein (ILPCA) using the AOX1 promoter and the alpha-factor signal sequence. A fermentation protocol was developed that produced about 150 mg/L biologically active ILPCA secreted in the fermented broth. A two-step purification scheme was devised to purify ILPCA to approximately 80% purity. The ILPCA produced has an apparent molecular weight of approximately 68 kDa and a deglycosilated molecular weight of 28 kDa. Steady-state kinetic analysis reveals that ILPCA activates purified human protein C with a K(m) of 77 nM and a k(cat) of 0.39 s(-1). In conclusion, ILPCA is a recombinant protein that can be produced reliably and in large quantities under controlled manufacturing conditions, activates protein C, and can be used in coagulation assays as an alternative to native venom preparations.

Animals↗

PubMed2003

Acetaminophen hepatotoxicity.

Unlike the bulk of medications, described in this fascicle, that cause liver injury in humans, acetaminophen is a non-prescription drug that can be purchased in drug stores and supermarkets without a physician's involvement. Death or severe injury is far more likely to occur with its use than with all the other medications considered in this study. Whereas attempts to control the quantity of drug ingested have been made in the United Kingdom and elsewhere in Europe, no comparable moves have taken place in the United States. The Food and Drug Administration claims to have concerns about the situation, however, but has yet to make an effort to more closely regulate the marketing and distribution of the drug. It is to be hoped that this will not be the case by the time the next issue of Drug Hepatotoxicity is scheduled for this series.

Acetaminophen↗

PubMed1998

Regulating energy transfer in the ATP sulfurylase-GTPase system.

ATP sulfurylase, isolated from Escherichia coli K-12, is a GTPase-target complex that catalyzes and links the energetics of GTP hydrolysis to the synthesis of activated sulfate (APS). When the GTP concentration is saturating and held fixed with a regenerating system, the APS reaction reaches a steady state in which its mass ratio is shifted (5.4 x 10(6))-fold toward the product by the hydrolysis of GTP. If GTP is not regenerated, the shift toward the product is transient, producing a pulse-shaped progress curve. The mechanistic basis of this transience is the subject of this paper. The product transient is caused by the binding of GDP to the enzyme which establishes a catalytic pathway that allows the chemical potential that had been transferred to the APS reaction to "leak" into the chemical milieu. The system leaks because the E.GDP complex catalyzes the uncoupled APS reaction. The addition of phosphate to the leaky GDP.E.APS.PPi complex converts it into the central Pi.GDP.E.APS.PPi complex which catalyzes the energy-transfer reaction. Thus, Pi binding directs the system through the coupled mechanism, "plugging" the leak. GMPPNP, which also causes a leak, is used to demonstrate that the mass ratio of the APS reaction can be "tuned" by adjusting flux through the coupled and uncoupled pathways. This energy-coupling mechanism provides a means for controlling the quantity of chemical potential transferred to the APS reaction. This versatile linkage might well be used to the cell's advantage to avoid the toxicity associated with an excess of activated sulfate.

Catalysis↗

PubMed2003

Identification of general linear relationships between activation energies and enthalpy changes for dissociation reactions at surfaces.

The activation energy to reaction is a key quantity that controls catalytic activity. Having used ab inito calculations to determine an extensive and broad ranging set of activation energies and enthalpy changes for surface-catalyzed reactions, we show that linear relationships exist between dissociation activation energies and enthalpy changes. Known in the literature as empirical Brønsted-Evans-Polanyi (BEP) relationships, we identify and discuss the physical origin of their presence in heterogeneous catalysis. The key implication is that merely from knowledge of adsorption energies the barriers to catalytic elementary reaction steps can be estimated.

Journal Article↗

PubMed2006

Novel route to WOx nanorods and WS2 nanotubes from WS2 inorganic fullerenes.

WO(x) (2 < x < 3) and WS(2) nanostructures have been widely praised due to applications as catalysts, solid lubricants, field emitters, and optical components. Many methods have been developed to fabricate these nanomaterials; however, most attention was focused on the same dimensional transformation from WO(x) nanoparticles or nanorods to WS(2) nanoparticles or nanotubes. In a solid-vapor reaction, by simply controlling the quantity of water vapor and reaction temperature, we have realized the transformation from quasi-zero-dimensional WS(2) nanoparticles to one-dimensional W(18)O(49) nanorods, and subsequent sulfuration reactions have further converted these W(18)O(49) nanorods into WS(2) nanotubes. The reaction temperature, quantity of water vapor, and pretreatment of the WS(2) nanoparticle precursors are important process parameters for long, thin, and homogeneous W(18)O(49) nanorods growth. The morphologies, crystal structures, and circling transformation mechanisms of sulfide-oxide-sulfide are discussed, and the photoluminescence properties of the resulting nanorods are investigated using a Xe lamp under an excitation of 270 nm.

Journal Article↗

PubMed1981

cis-Interacting genes in the S region of the murine major histocompatibility complex.

Insight into the control of gene expression may be gained by analysing genetic systems marked by both regulatory and structural variants. In such systems one can determine whether a regulatory element controls structural genes on both chromosomes or only on the chromosome to which it is linked. The latter may be detected in individuals heterozygous at both the regulatory and structural loci, in which case the effect of each regulatory allele is seen to be exerted only on the cis-located structural allele. In prokaryotic organisms, the identification of cis interaction of this sort has allowed elucidation of many features of genetic regulation, first for the lac operon and subsequently for a variety of other systems. In higher organisms, however, there have been few opportunities to observe cis-interacting genes. The most thoroughly characterized mammalian system in this regard is the murine beta-glucuronidase locus described by Paigen and his colleagues, in which cis interaction has been shown to occur between two closely linked genetic elements-the beta-glucuronidase structural gene itself and an androgen-activated regulatory gene which controls the quantity of beta-glucuronidase expressed. We report here that cis-interacting genetic elements are also found in the S region of the mouse major histocompatibility complex H-2.

Animals↗

PubMed2000

Desire for alcohol and outcome expectancies as measures of alcohol cue-reactivity in social drinkers.

AIMS: To assess the responsiveness of questionnaire measures of desire to drink and alcohol outcome expectancies. DESIGN: A subjective alcohol cue-reactivity paradigm (alcohol cue: sight, smell, taste) was used with a 2 x 2 between-subject design (n = 88), drink type (soft/alcoholic) and order of assessment (desire-expectancy/expectancy-desire). Covariance analysis controlled for quantity of recent alcohol consumption. SETTING: A quiet alcohol research suite. PARTICIPANTS: Eighty-eight male and female social drinking students, recruited for a 'taste preference' survey. MEASURES: Three composite measures using questionnaire totals and subscales: of desire (DAQ), positive expectancy (AEQ) and negative expectancy (NAEQ). Timeline Follow-back procedure for recent consumption. FINDINGS: Subjective cue-reactivity was found for the DAQ total score and the subscales 'strong intentions and desires' and 'negative reinforcement'. Expectancies did not demonstrate alcohol cue-reactivity. CONCLUSION: The DAQ and subscales are sensitive measures of alcohol cue-reactivity in social drinkers. Potential uses of the subjective cue-reactivity procedure with multi-factorial representations of cue reaction are identified.

Adolescent↗

PubMed2003

Detection of malaria sporozoites by standard ELISA and VecTestTM dipstick assay in field-collected anopheline mosquitoes from a malaria endemic site in Ghana.

We compared the VecTestTM dipstick assay for detection of Plasmodium sporozoites in Anopheles vectors of malaria with standard circumsporozoite (CS) microplate ELISA for detection of Plasmodium falciparum circumsporozoite protein (PfCSP) in Anopheles mosquitoes. Mosquitoes were collected from a malaria endemic site (Kassena Nankana district) in northern Ghana. Of 2620 randomly sampled mosquitoes tested, the standard CS-ELISA gave a sporozoite rate of 10.8% compared with 11.2% by VecTestTM, which was not statistically different (P = 0.66). Visual reading of the CS-ELISA results gave a sporozoite rate of 13.4%, which was higher than the other tests (P > 0.05). To allow a more objective evaluation of the sensitivity of the dipstick, an additional 136 known CS-ELISA-positive specimens were analysed. The prevalence of the test (including the additional samples) was 14.6% and 14.7% for CS-ELISA and dipstick, respectively (P > 0.05). The estimated prevalence by visual assessment of the CS-ELISA results was 17.5%. The relative specificity and sensitivity of the VecTestTM dipstick and visually read ELISA were estimated based on the CS-ELISA as a gold standard. The specificities of the dipstick and visual ELISA were high, 98.0% and 96.6%, respectively. However, the sensitivities of the two assays were 88.8% for VecTest and 100% for visual ELISA (P < 0.01). Concordance between VecTest and CS-ELISA was good (kappa = 0.86). Similarly, there was a good concordance between the dipstick and the visually read ELISA (kappa = 0.88). Extrapolating from PfCSP controls (titrated quantities of P. falciparum sporozoites), mean sporozoite loads of CS-ELISA-positive An. gambiae (286 +/- 28.05) and An. funestus (236 +/- 19.32) were determined (P = 0.146). The visual dipstick grades showed high correlation with sporozoite load. The more intense the dipstick colour, the higher the mean sporozoite load (+ = 108, ++ = 207, +++ = 290, r = 0.99, r2 = 1). The VecTest dipstick offers practical advantages for field workers needing rapid and accurate means of detection of sporozoites in mosquitoes.

Animals↗

PubMed2000

Relative contribution of cytochromes P-450 and flavin-containing monoxygenases to the metabolism of albendazole by human liver microsomes.

AIMS: Albendazole (ABZ; methyl 5-propylthio-1H-benzimidazol-2-yl carbamate) is a broad spectrum anthelmintic whose activity resides both in the parent compound and its sulphoxide metabolite (ABS). There are numerous reports of ABZ metabolism in animals but relatively few in humans. We have investigated the sulphoxidation of ABZ in human liver microsomes and recombinant systems. METHODS: The specific enzymes involved in the sulphoxidation of ABZ were determined by a combination of approaches; inhibition with an antiserum directed against cytochrome P450 reductase, the effect of selective chemical inhibitors on ABZ sulphoxidation in human liver microsomes, the capability of expressed CYP and FMO to mediate the formation of ABS, regression analysis of the rate of metabolism of ABZ to ABS in human liver microsomes against selective P450 substrates and regression analysis of the rate of ABS sulphoxidation against CYP expression measured by Western blotting. RESULTS: Comparison of Vmax values obtained following heat inactivation (3min at 45 degrees C) of flavin monoxygenases (FMO), chemical inhibition of FMO with methimazole and addition of an antiserum directed against cytochrome P450 reductase indicate that FMO and CYP contribute approximately 30% and 70%, respectively, to ABS production in vitro. Comparison of CLint values suggests CYP is a major contributor in vivo. A significant reduction in ABZ sulphoxidation (n = 3) was seen with ketoconazole (CYP3 A4; 32-37%), ritonavir (CYP3 A4: 34-42%), methimazole (FMO: 28-49%) and thioacetamide (FMO; 32-35%). Additive inhibition with ketoconazole and methimazole was 69 +/- 8% (n = 3). ABS production in heat - treated microsomes (3 min at 45 degrees C) correlated significantly with testosterone 6beta-hydroxylation (CYP3A4; P < 0.05) and band intensities on Western blots probed with an antibody selective for 3A4 (P < 0.05). Recombinant human CYP3 A4, CYP1A2 and FMO3 produced ABS in greater quantities than control microsomes, with those expressing CYP3A4 producing threefold more ABS than those expressing CYP1A2. Kinetic studies showed the Km values obtained with both CYP3A4 and FMO3 were similar. CONCLUSIONS: We conclude that the production of ABS in human liver is mediated via both FMO and CYP, principally CYP3A4, with the CYP component being the major contributor.

Albendazole↗