The accuracy of magnetic resonance phase velocity measurements in stenotic flow

Nuclear magnetic resonance (MR) can be used to measure velocities in fluid flow using the technique of phase velocity mapping. Advantages of MR velocimetry include the simultaneous mapping of the entire flow field through a non-contacting, magnetic window. The phase velocity mapping technique assumes that velocity is constant over the measurement time (typically around 10 ms). For many fluid flows, this assumption is not valid. The current study showed that MR phase velocity measurements of velocity through stenotic flow can be in error by over 100% immediately upstream and downstream of the stenosis throat and by 20% far downstream of the throat in comparison with laser Doppler anemometer measurements taken at the same location. Highly turbulent flow also led to significant errors in velocity measurement. These errors can be attributed to several sources including low signal-to-noise ratio, additional phase shifts due to non-constant velocities, and non-stationary transit-time effects. Velocity measurement errors could be reduced to under 30% at all measurement locations through the use of MR sequences with high signal-to-noise ratios, low echo times, and thick slices.     

Simultaneous biodegradation of chlorobenzene and toluene by a Pseudomonas strain.

Pseudomonas sp. strain JS6 grows on a wide range of chloro- and methylaromatic substrates. The simultaneous degradation of these compounds is prevented in most previously studied isolates because the catabolic pathways are incompatible. The purpose of this study was to determine whether strain JS6 could degrade mixtures of chloro- and methyl-substituted aromatic compounds. Strain JS6 was maintained in a chemostat on a minimal medium with toluene or chlorobenzene as the sole carbon source, supplied via a syringe pump. Strain JS6 contained an active catechol 2,3-dioxygenase when grown in the presence of chloroaromatic compounds; however, in cell extracts, this enzyme was strongly inhibited by 3-chlorocatechol. When cells grown to steady state on toluene were exposed to 50% toluene-50% chlorobenzene, 3-chlorocatechol and 3-methylcatechol accumulated in the medium and the cell density decreased. After 3 h, the enzyme activities of the modified ortho ring fission pathway were induced, the metabolites disappeared, and the cell density returned to previous levels. In cell extracts, 3-methylcatechol was degraded by both catechol 1,2- and catechol 2,3-dioxygenase. Strain JS62, a catechol 2,3-dioxygenase mutant of JS6, grew on toluene, and ring cleavage of 3-methylcatechol was catalyzed by catechol 1,2-dioxygenase. The transient metabolite 2-methyllactone was identified in chlorobenzene-grown JS6 cultures exposed to toluene. These results indicate that strain JS6 can degrade mixtures of chloro- and methylaromatic compounds by means of a modified ortho ring fission pathway.     

Chlorinated biphenyl mineralization by individual populations and consortia of freshwater bacteria

Comparative studies were performed to investigate the contribution of microbial consortia, individual microbial populations, and specific plasmids to chlorinated biphenyl biodegradation among microbial communities from a polychlorinated biphenyl-contaminated freshwater environment. A bacterial consortium, designated LPS10, was shown to mineralize 4-chlorobiphenyl (4CB) and dehalogenate 4,4'-dichlorobiphenyl. The LPS10 consortium involved three isolates: Pseudomonas testosteroni (LPS10A), which mediated the breakdown of 4CB and 4,4'-dichlorobiphenyl to 4-chlorobenzoic acid; an isolate tentatively identified as an Arthrobacter sp. (LPS10B), which mediated 4-chlorobenzoic acid degradation; and Pseudomonas putida bv. A (LPS10C), whose role in the consortium has not been determined. None of these isolates contained detectable plasmids or sequences homologous to the 4CB-degradative plasmid pSS50. A freshwater isolate, designated LBS1C1, was found to harbor a 41-megadalton plasmid that was related to the 35-megadalton plasmid pSS50, and this isolate was shown to mineralize 4CB. In chemostat enrichments with biphenyl and 4CB as primary carbon sources, the LPS10 consortium was found to outcomplete bacterial populations harboring plasmids homologous to pSS50. These results demonstrate that an understanding of the biodegradative capacity of individual bacterial populations as well as interacting populations of bacteria must be considered in order to gain a better understanding of polychlorinated biphenyl biodegradation in the environment.     

THE [14C]DEOXYGLUCOSE METHOD FOR THE MEASUREMENT OF LOCAL CEREBRAL GLUCOSE UTILIZATION: THEORY,PROCEDURE, AND NORMAL VALUES IN THE CONSCIOUS AND ANESTHETIZED ALBINO RAT1

Abstract— A method has been developed for the simultaneous measurement of the rates of glucose consumption in the various structural and functional components of the brain in vivo. The method can be applied to most laboratory animals in the conscious state. It is based on the use of 2-deoxy-D-[¹⁴C]glucose ([¹⁴C]DG) as a tracer for the exchange of glucose between plasma and brain and its phosphorylation by hexokinase in the tissues. [¹⁴C]DG is used because the label in its product, [¹⁴C]deoxyglucose-6-phosphate, is essentially trapped in the tissue over the time course of the measurement. A model has been designed based on the assumptions of a steady state for glucose consumption, a first order equilibration of the free [¹⁴C]DG pool in the tissue with the plasma level, and relative rates of phosphorylation of [¹⁴C]DG and glucose determined by their relative concentrations in the precursor pools and their respective kinetic constants for the hexokinase reaction. An operational equation based on this model has been derived in terms of determinable variables. A pulse of [¹⁴C]DG is administered intravenously and the arterial plasma [¹⁴C]DG and glucose concentrations monitored for a preset time between 30 and 45min. At the prescribed time, the head is removed and frozen in liquid N₂-chilled Freon XII, and the brain sectioned for autoradiography. Local tissue concentrations of [¹⁴C]DG are determined by quantitative autoradiography. Local cerebral glucose consumption is calculated by the equation on the basis of these measured values. The method has been applied to normal albino rats in the conscious state and under thiopental anesthesia. The results demonstrate that the local rates of glucose consumption in the brain fall into two distinct distributions, one for gray matter and the other for white matter. In the conscious rat the values in the gray matter vary widely from structure to structure (54-197 μmol/100 g/min) with the highest values in structures related to auditory function, e.g. medial geniculate body, superior olive, inferior colliculus, and auditory cortex. The values in white matter are more uniform (i.e. 33–40 μmo1/100 g/min) at levels approximately one-fourth to one-half those of gray matter. Heterogeneous rates of glucose consumption are frequently seen within specific structures, often revealing a pattern of cytoarchitecture. Thiopental anesthesia markedly depresses the rates of glucose utilization throughout the brain, particularly in gray matter, and metabolic rate throughout gray matter becomes more uniform at a lower level.     

A copper protein and a cytochrome bind at the same site on bacterial cytochrome c peroxidase

Pseudoazurin binds at a single site on cytochrome c peroxidase from Paracoccus pantotrophus with a K(d) of 16.4 microM at 25 degrees C, pH 6.0, in an endothermic reaction that is driven by a large entropy change. Sedimentation velocity experiments confirmed the presence of a single site, although results at higher pseudoazurin concentrations are complicated by the dimerization of the protein. Microcalorimetry, ultracentrifugation, and (1)H NMR spectroscopy studies in which cytochrome c550, pseudoazurin, and cytochrome c peroxidase were all present could be modeled using a competitive binding algorithm. Molecular docking simulation of the binding of pseudoazurin to the peroxidase in combination with the chemical shift perturbation pattern for pseudoazurin in the presence of the peroxidase revealed a group of solutions that were situated close to the electron-transferring heme with Cu-Fe distances of about 14 A. This is consistent with the results of (1)H NMR spectroscopy, which showed that pseudoazurin binds closely enough to the electron-transferring heme of the peroxidase to perturb its set of heme methyl resonances. We conclude that cytochrome c550 and pseudoazurin bind at the same site on the cytochrome c peroxidase and that the pair of electrons required to restore the enzyme to its active state after turnover are delivered one-by-one to the electron-transferring heme.     

Physiological, structural, and immunological characterization of leaf and chloroplast development in cotton

Summary Many of the studies of chloroplast ontogeny in higher plants have utilized suboptimal conditions of light and growth to assess development. In this study, we utilized structural, immunological, and physiological techniques to examine the development of the chloroplast in fieldgrown cotton (Gossypium hirsutum cv. MD 51 ne). Our youngest leaf sample developmentally was completely folded upon itself and about 0.5 cm in length; leaves of this same plastochron were followed for three weeks to the fully expanded leaf. The chloroplasts at the earliest stage monitored had almost all of the lamellae in small, relatively electron-opaque grana, with relatively few thylakoids which were not appressed on at least one surface. During the development of the thylakoids, the membranes increase in complexity, with considerable stroma lamellae development and an increase in the number of thylakoids per granum. Besides the increase in complexity, both the size and numbers of the chloroplast increase during the development of the leaf. Developmental changes in six thylakoid proteins, five stromal proteins, and one peroxisomal protein were monitored by quantitative immunocytochemistry. Even at the earliest stages of development, the plastids are equipped with the proteins required to carry out both light and dark reactions of photosynthesis. Several of the proteins follow three phases of accumulation: a relatively high density at early stages, a linear increase to keep step with chloroplast growth, and a final accumulation in the mature chloroplast. Photosystem-II(PS II)-related proteins are present at their highest densities early in development, with an accumulation of other parts of the photosynthetic apparatus at a latter stage. The early accumulation of PS-II-related proteins correlates with the much lower ratio of chlorophylla tob in the younger leaves and with the changes in fluorescence transients. These data indicate that some of the conclusions on chloroplast development based upon studies of intercalary meristems of monocots or the greening of etiolated plants may not be adequate to explain development of chloroplasts in leaves from apical meristems grown under natural conditions.Abbreviations CF1 chloroplast coupling factor 1 - chl chlorophyll - DAP days after planting - LHC light-harvesting chlorophyll-a/b-binding protein - OEC oxygen-evolving complex of photosystem II - PBS phosphate-buffered saline - PS photosystem - RuBisCo ribulose bisphosphate carboxylase/oxygenase     

Parallel processing of binocular disparity in the cat's retinogeniculocortical pathways

In the cat, parallel streams of information processing have been traced from X-, Y- and W-type retinal ganglion cells to visual cortical areas 17 (X-, Y- and W-type), 18 (Y-type) and 19 (W-type). In the present study we have examined, in the anaesthetized and paralysed adult cat, the role played by X-, Y- and W-subsystems, projecting to areas 17 and 19, in the processing of binocular retinal disparity. The tapetal reflection technique was used to monitor residual eye movements and to provide a map, for each eye, of the retinal blood vessels which could later be compared with retinal wholemounts stained with cresyl violet to reveal the area centralis. The receptive-field disparities of cells recorded from areas 17 and 19 were compared with each other and with reference to the visual axes defined by the area centralis of each eye. Cells of area 19 (receiving W-type input) had horizontal receptive-field disparities that were significantly more divergent than those of the cells in area 17 and 17-18 'border region'. Referred to the area centralis, the mean horizontal receptive-field disparity in area 19 was -0.5 degrees (+/- 0.8 degrees). The mean horizontal receptive-field disparity of area 17 (receiving X-, Y- and W-type input) was convergent with respect to the visual axis at +2 degrees (+/- 0.5 degrees). Finally, the mean horizontal receptive-field disparity of the cells in the 17-18 border region (which receive mainly Y-type input) was even more convergent (2.6 degrees +/- 1.5 degrees) than that of area 17. Binocular interactions of cortical neurons were tested with the Risley biprism technique. Area 19 cells had maximal responses to binocular stimulation when the receptive-field disparities were either close to zero or slightly divergent. In contrast, area 17 cells tended to respond optimally to disparities that were either slightly or strongly convergent. At the level of the lateral geniculate nucleus there were significant differences between the receptive-field disparities inferred from the comparison of receptive-field positions of adjacent neurons recorded on either side of the border between the A and A1 geniculate laminae and those inferred from a similar comparison at the C1-C2 border. The mean horizontal disparities inferred from the interlaminar comparison at the A-A1 border were +2.1 degrees (+/- 0.3 degrees); those inferred from the interlaminar comparison at the C1-C2 border -0.2 (+/- 0.2 degrees) were more divergent.(ABSTRACT TRUNCATED AT 400 WORDS)     

Escherichia coli glycerol kinase. Cloning and sequencing of the glpK gene and the primary structure of the enzyme

The glpK gene, which codes for Escherichia coli K-12 glycerol kinase (EC 2.1.7.30, ATP:glycerol 3-phosphotransferase), has been cloned into the HindIII site of pBR322. The gene was contained in a 2.8-kilobase DNA fragment which was obtained from a lambda transducing bacteriophage, lambda dglpK100 (Conrad, C.A., Stearns, G.W., III, Prater, W.E., Rheiner, J.A., and Johnson, J.R. (1984) Mol. Gen. Genet. 195, 376-378). The DNA sequence of 2 kilobases of the cloned HindIII fragment was obtained using the dideoxynucleotide method. The start of the open reading frame for the glpK gene was identified from the N-terminal sequence of the first 22 amino acid residues of the purified enzyme, which was determined by automated Edman degradation. The open reading frame codes for a protein of 502 amino acids and a molecular weight of 56,106 which is in good agreement with the value previously determined by sedimentation equilibrium. The primary structure of the protein as deduced from the gene sequence was corroborated by the isolation and sequencing of four tryptic peptides, which were found to occur at the following amino acid locations: 173-177, 203-211, 279-281, 464-468. The N-terminal sequence of the purified enzyme shows that the enzyme undergoes post-translational processing. Restriction digestion as well as DNA sequencing of the supercoiled plasmid shows that the HindIII fragment is inserted into pBR322 such that the glpK gene is transcribed in a counterclockwise direction. Examination of the upstream DNA sequence reveals two possible promoters of essentially the same efficiency: the P1 promoter of pBR322 and a hybrid promoter which contains both bacterial and pBR322 DNA sequences.     

The role of cytochrome c4 in bacterial respiration. Cellular location and selective removal from membranes.

The cellular location of cytochrome c4 in Pseudomonas stutzeri and Azotobacter vinelandii was investigated by the production of spheroplasts. Soluble cytochrome c4 was found to be located in the periplasm in both organisms. The remaining cytochrome c4 was membrane-bound. The orientation of this membrane-bound cytochrome c4 fraction was investigated by proteolysis of the cytochrome on intact spheroplasts. In P. stutzeri, 78% of the membrane-bound cytochrome c4 could be proteolysed, whilst 82% of the spheroplasts remained intact, suggesting that the membrane-bound cytochrome c4 is on the periplasmic face of the membrane in this organism. Cytochrome c4 was not susceptible to proteolysis on A. vinelandii spheroplasts, in spite of being digestible in the purified state. Cytochrome c5 was shown to have a similar cellular distribution to cytochrome c4. Selective removal of cytochrome c4 from membranes of P. stutzeri was accomplished by the use of sodium iodide and propan-2-ol, with the retention of most of the ascorbate-TMPD (NNN'N'-tetramethylbenzene-1,4-diamine) oxidase activity associated with the membrane. Sodium iodide removed most of the cytochrome c4 from A. vinelandii membranes with retention of 62% of the ascorbate-TMPD oxidase activity. Cytochrome c4 could be returned to the washed membranes, but with no recovery of this enzyme activity. We conclude that cytochrome c4 is not involved in the ascorbate-TMPD oxidase activity associated with the membranes of these two organisms.     

Mutagenic dissection of hemoglobin cooperativity: effects of amino acid alteration on subunit assembly of oxy and deoxy tetramers.

Free energies of oxygen-linked subunit assembly and cooperative interaction have been determined for 34 molecular species of human hemoglobin, which differ by amino acid alterations as a result of mutation or chemical modification at specific sites. These studies required the development of extensions to our earlier methodology. In combination with previous results they comprise a data base of 60 hemoglobin species, characterized under the same conditions. The data base was analyzed in terms of the five following issues. (1) Range and sensitivity to site modifications. Deoxy tetramers showed greater average energetic response to structural modifications than the oxy species, but the ranges are similar for the two ligation forms. (2) Structural localization of cooperative free energy. Difference free energies of dimer-tetramer assembly (oxy minus deoxy) yielded delta Gc for each hemoglobin, i.e., the free energy used for modulation of oxygen affinity over all four binding steps. A structure-energy map constructed from these results shows that the alpha 1 beta 2 interface is a unique structural location of the noncovalent bonding interactions that are energetically coupled to cooperativity. (3) Relationship of cooperativity to intrinsic binding. Oxygen binding energetics for dissociated dimers of mutants strongly indicates that cooperativity and intrinsic binding are completely decoupled by tetramer to dimer dissociation. (4) Additivity, site-site coupling and adventitious perturbations. All these are exhibited by individual-site modifications of this study. Large nonadditivity may be correlated with global (quaternary) structure change. (5) Residue position vs. chemical nature. Functional response is solely dictated by structural location for a subset of the sites, but varies with side-chain type at other sites. The current data base provides a unique framework for further analyses and modeling of fundamental issues in the structural chemistry of proteins and allosteric mechanisms.