Purification of cytochrome a 1 c 1 from Nitrobacter agilis and characterization of nitrite oxidation system of the bacterium

Cytochrome a ₁ c ₁ was highly purified from Nitrobacter agilis. The cytochrome contained heme a and heme c of equimolar amount, and its reduced form showed absorption peaks at 587, 550, 521, 434 and 416 nm. Molecular weight per heme a of the cytochrome was estimated to be approx. 100,000–130,000 from the amino acid composition. A similar value was obtained by determining the protein content per heme a. The cytochrome molecule was composed of three subunits with molecular weights of 55,000, 29,000 and 19,000, respectively. The 29 kd subunit had heme c.Hemes a and c of cytochrome a ₁ c ₁ were reduced on addition of nitrite, and the reduced cytochrome was hardly autoxidizable. Exogenously added horse heart cytochrome c was reduced by nitrite in the presence of cytochrome a ₁ c ₁; K _m values of cytochrome a ₁ c ₁ for nitrite and N. agilis cytochrome c were 0.5 mM and and 6 M, respectively. V _max was 1.7 mol ferricytochrome c reduced/min·mol of cytochrome a ₁ c ₁ The pH optimum of the reaction was about 8. The nitrite-cytochrome c reduction catalyzed by cytochrome a ₁ c ₁ was 61% and 88% inhibited by 44M azide and cyanide, respectively. In the presence of 4.4 mM nitrate, the reaction was 89% inhibited. The nitrite-cytochrome c reduction catalysed by cytochrome a ₁ c ₁ was 2.5-fold stimulated by 4.5 mM manganous chloride. An activating factor which was present in the crude enzyme preparation stimulated the reaction by 2.8-fold, and presence of both the factor and manganous ion activated the reaction by 7-fold.Cytochrome a ₁ c ₁ showed also cytochrome c-nitrate reductase activity. The pH optimum of the reaction was about 6. The nitrate reductase activity was also stimulated by manganous ions and the activating factor.     

In vitro hexagonal assembly of lipopolysaccharide of Escherichia coli K-12

We examined Escherichia coli K-12 lipopolysaccharide (LPS), which is known to be an R-form LPS, for its ability to form a hexagonal lattice structure in vitro. The LPS from E. coli K-12 strain JE1011 did not form a hexagonal lattice structure when it was precipitated by addition of two volumes of 10 mM MgCl2-ethanol, but it did form such a structure when it was electrodialyzed and then converted to the magnesium or calcium salt form. The lattice constant of the magnesium salt form was 15.2 +/- 0.3 nm and that of the calcium salt form 18.5 +/- 0.3 nm. Since prior treatment of the LPS with proteinase K in the presence of sodium dodecyl sulfate did not affect its capability of hexagonal assembly, the lattice formation by the LPS does not require the presence of proteins.     

Effects of temperature shift on growth rate of Escherichia coli BB at lower glucose concentration

Although a shift in nutritinal conditins brings about transient unbalanced growth in normally grown Escherichia coli, a shift in temperature without changing the nutritional conditions results in immediate adaptation to the new conditions. However, when a medium contained an insufficient amount of nutrient, such as glucose, a temperature shift caused a lag time in temperature shiftup was primarily determined by the postshift temperature. These situatins were quite similar to those observed in nutrient shiftup, but a growth profile during the lag time was more distorted than that found in the nutrient shiftup. The transient unbalanced growth appeared to be caused by a difference in physiological states of bacteria, as expressed by macromolecule content per cell characterized by the pre and postshift environments, and was capable of expressing theoretically its profile and duration according to the model of Cooper and Helmstetter. On the other hand, the shiftdown in temperature in the presence of a limiting concentration of glucose caused extraordinarily long lag time, and transient cessation of cell division during that period. This response was unable to explain by the Cooper and Helmstetter model. In contrast to the temperature shiftup, the duration of lag time in the shiftdown was expresed as functions of the poshift temperature and the difference in physiological states of the pre- and postshift environments.     

Oxidation of hydroxylamine to nitrite catalyzed by hydroxylamine oxidoreductase purified fromNitrosomonas europaea

The oxidation of hydroxylamine to nitrite, which had been catalyzed by hydroxylamine oxidoreductase purified fromNitrosomonas europaea, was studied. The enzyme oxidized hydroxylamine almost completely to nitrite under aerobic conditions if sufficient amount of cytochromec or ferricyanide was added and the reaction was performed in phosphate buffer. Even under anaerobic conditions, hydroxylamine was oxidized to nitrite by the enzyme, but nitrite, once formed, disappeared when the reaction was continued for more than several minutes.     

Die Verarbeitung stationärer optischer Nachrichten im Komplexauge von Limulus

Summary The functional properties of the processing of visual information by the complex eye of Limulus was studied. The spatial distribution of activity that results in the optic nerve when the Limulus eye is exposed to a stationary optical pattern depends upon the transfer characteristics of two subsystems: the dioptric apparatus and the nervous interactions comprising the lateral inhibition system. — The transfer characteristic of the dioptric apparatus is determined by the sensitivity distribution function of single ommatidia. This distribution was measured and found to be approximately of Gauss-function type. The sensitivity falls off to 1/e at a distance of one ommatidium; thus the visual fields of adjacent ommatidia strongly overlap. As a consequence of the overlap, amplitudes of the spatial Fourier components, of which the brightness distribution of the optical surround is made up, are more and more reduced with increasing frequency in the intensity distribution on the receptor mosaic. The amplitude of the spatial frequency 1/=0,25 ( in units of interommatidial distance) is reduced to half of the maximum value, which is attained at zero frequency. It is shown that the amplitude frequency characteristic of the sensitivity distribution function has no zeros, which means that no loss of optical information results from overlap of visual fields. Thus the resolving power of the dioptric apparatus is limited only by the number of receptors per unit area. — The transfer characteristic of the lateral inhibition system in the Limulus eye depends on the distribution of the inhibitory coefficients around the individual receptors. This distribution function was determined from excitatory responses in the optic nerve elicited by a spatial light intensity step function on the receptor mosaic. It is found that this distribution is also Gaussian in form, but decays to 1/e at a distance of eight to nine ommatidia along the major axis of the eye. The average value of the inhibitory coefficients between adjacent ommatidia was found to be 0,025. The amplitude frequency response of the inhibitory system is constant for high spatial frequencies down to 1/=0,1 while amplitudes of lower frequency sinusoids are reduced down to nearly half of the maximum value at frequency zero. The amplitude frequency characteristic of the inhibitory system ensures a one to one correspondence between the intensity distribution on the receptor mosaic and the excitation distribution in the optic nerve. The overall transfer characteristic of the eye is derived from the transfer characteristics of the dioptric apparatus and the inhibitory system. This characteristic is of bandpass type with a maximum amplitude response at a frequency of 1/=0,07. The overall transfer characteristic was independently confirmed in a separate experiment. The nature of the overall transfer characteristic shows that the inhibitory system does not exactly correct for the overlap of the visual fields of single ommatidia, which in principal the system could do if the distributions of inhibitory coefficients and ommatidia sensitivity were equal. The overall transfer characteristic of the Limulus eye garantees a one to one correspondence between patterns in the optical surround and excitation distributions in the optic nerve. — The average values of the inhibitory coefficients derived from these experiments are at least a factor ten smaller than those determined directly by other investigators. Possible explanations of this discrepency are discussed. — In a separate chapter the overall transfer characteristic for eyes submerged in water is described. It was found that this characteristic does not differ from that determined in air for the eye region which was investigated in the experiments. This result is explained by two properties of the eye which are dependent on the refractive index of the surround medium and whose influences cancel each other: the visual fields of ommatidia are reduced under water, while the divergence angles between the optical axes of adjacent ommatidia also diminish.

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This research was supported in part by the United States Air Force under Grant No. AF-EOAR-62-41 and monitored by the European Office, Office of Aerospace Research.     

Neurohormonal control of the mating interval in the male cricket,Gryllus bimaculatus DeGeer

Summary Between two mating acts of the male cricket (Gryllus bimaculatus), spermatophore protrusion (SP) and courtship stridulation (CS), there is a fixed time interval. This interval lasts about 1 h. During the period from SP to CS, the male cricket does not stridulate nor make any type of mating sound (post-spermatophore protrusion silence: PSPS) and tolerates external sensory stimuli. We examined the effects of injections of hemolymph and ganglia extracts on the interval. Extracts obtained from crickets which had just started CS (CS crickets) and those which had finished SP (SP crickets) were effective. The extracts were fractionated by ul trafiltration. Fractions with a molecular weight of less than 1 kdalton affected the length of the PSPS. The fractions from both the hemolymph and the mesothoracic ganglion of CS crickets shortened the PSPS. On the other hand, the fractions from the hemolymph and the brain of SP crickets lengthened the PSPS. We estimated, by gel filtration, the molecular weight of the effective fractions from the mesothoracic ganglion and the brain to be 100–200 daltons. We also examined the effects of biogenic amines on the PSPS. Octopamine shortened the PSPS, whereas serotonin lengthened it. The results in dicate that at least two neurohormones from the brain and the mesothoracic ganglion reciprocally control the elicitation of CS and provide an appropriate interval in the mating sequence of the male cricket. Octopamine and serotonin are possible candidates for these neurohormones.Abbreviations CP copulation - CS courtship stridulation - SP spermatophore protrusion - PSPS post-spermatophore protrusion silence     

Frequency characteristics of airway and tissue impedances in respiratory diseases

We measured the frequency characteristics (at 10-40 Hz) of airway (Za) and tissue (Zt) impedances in cases of chronic obstructive pulmonary disease [asthmatic bronchitis (AB), chronic pulmonary emphysema (CPE)] and interstitial pneumonitis (IP) by use of an improved random noise oscillation and body box method. The results were then compared with those obtained for normal subjects. The real part of Za was markedly elevated in patients with AB but only slightly elevated in those with CPE. To interpret these data we used an electromechanical analogue including serial inhomogeneity with shunt impedance. From this model we concluded that AB causes both the central and peripheral airway resistances to increase, while CPE brings about a rise mainly in peripheral resistance. In IP patients, only the imaginary part of Zt decreased, which might reflect the decrease in both lung and chest wall compliance. In CPE patients, but not in AB patients, the real part of Zt fell. These data were consistent with the assumption that the decrease in mass per unit volume of lung tissue and hyperinflation of the chest wall in CPE patients might lower the tissue resistances.     

The trend of acquired immunity with live poliovirus vaccine and the effect of revaccination: follow-up of vaccinees for ten years

The persistence of neutralizing antibody (NA) against three types of poliovirus acquired after two doses of trivalent live attenuated poliovirus vaccine (LPV) has been followed up for ten years in individual vaccinees. Sixty-seven children were bled once a year over a five year period following the primary vaccination. More than 80% of them retained NA against all three types of poliovirus. Thirty-two individuals whose NA titres were 1:16 or over for types 1 and 2 and 1:4 or over for type 3 at the fifth year were further followed up for a further five years and it was shown that during this period some of them had a naturally-acquired antibody rise, mostly against type 3 virus. At the sixth to eighth year after the primary vaccination, one further dose of the trivalent vaccine was administered to the children whose NA titres were down to 1:8 or less and the effect of booster vaccination on NA was followed. Other subjects were revaccinated with LPV and their fecal excretion of the vaccine virus was investigated. The results showed that a decrease in serum antibody level could be a good indicator of the local resistance of the alimentary tract and that reinfection could occur if serum NA had decreased to 1:8 or less, which allowed a virus excretion in the stools.     

Effects of crowding on life history parameters of four green leafhoppers,Nephotettix spp. (Homoptera: Cicadellidae)

The effects of intraspecific crowding on the different life history parameters of the three tropical and subtropical green leafhoppers, Nephotettix virescensStål ,N. nigropictusDistant and N. malayanusIshihara etKawase , and one temperate species, N. cincticepsUhler , were studied under 25±1°C using the susceptible Japonica rice variety “Nipponbare”. Crowding during the immature stages of the four species prolonged the nymphal developmental period and resulted to smaller adults with reduced fecundity and shorter life span. Similarly, crowding during the adult stage also shortened the life span of both sexes of adults and decreased the fecundity of females of the four species. The effect of crowding during the adult stage was more adverse than the effect of crowding during the immature stages. N. cincticeps seemed more sensitive to crowding even at lower densities whereas the three tropical species were sensitive mostly at higher densities.     

The complete primary structure of calcineurin A, a calmodulin binding protein homologous with protein phosphatases 1 and 2A

A complementary DNA (cDNA) clone encoding the catalytic subunit of calcineurin (calcineurin A) has been isolated from a rat brain cDNA library. The primary structure of the cDNA consists of 2,337 nucleotides including the entire coding region for 521 amino acids, and the calculated molecular mass is 58,643 Da. The calcineurin A is strikingly homologous to protein phosphatases 1 and 2A, approximately 50% of the amino acids over an internal 250-residue region between residues 78 and 329 being identical. Twenty four amino acid-residue region between residues 391 and 414 shows the consensus structural features for a calmodulin-binding domain. These data suggest that the allosteric character of this chimeric enzyme is generated by gene fusion of two separate protein families.