Characteristics of mutagenesis and repair following UV irradiation in the methylotrophic bacterium Pseudomonas methanolica

The methylotrophic bacterium Pseudomonas methanolica was shown to be very resistant to the bactericidal and mutagenic action of UV irradiation. The activity of reparation processes after UV irradiation was also detected as well as a weak photoreactivating activity in P. methanolica. The decrease in the survival rate of irradiated cells under the action of caffeine and acriflavine, reparation inhibitors, is indicative of the activity of the excision reparation systems and, possibly, the recombination branch of postreplicative reparation. No activity of the inducible reparation system was found. It has been concluded that the elevated resistance of P. methanolica cells to the bactericidal and mutagenic action of short-wavelength UV irradiation is associated with the activity of the reparation systems.     

The ultrastructure of the uterine epithelium of the pig during the estrous cycle and early pregnancy

Summary In the compound eye of the moth Antheraea polyphemus, three types of visual pigments were found in extracts from the retina and by microspectrophotometry in situ. The absorption maxima of the receptor pigment P and the metarhodopsin M are at (1) P 520–530 nm, M 480–490 nm; (2) P 460–480 nm, M 530–540 nm; (3) P 330–340 nm, M 460–470 nm. Their localization was investigated by electron microscopy on eyes illuminated with different monochromatic lights. Within the tiered rhabdom, constituted of the rhabdomeres of nine visual cells, the basal cell contains a blue-and the six medial cells have a greenabsorbing pigment. The two distal cells of most ommatidia also have the blue pigment; only in the dorsal region of the eye, these cells contain a UV-absorbing pigment, which constitutes a portion of only 5% of the visual pigment content within the entire retina. The functional significance of this distribution is discussed.     

Propionibacterium jensenii produces the polyene pigment granadaene and has hemolytic properties similar to those of Streptococcus agalactiae

The red polyene pigment granadaene was purified and identified from Propionibacterium jensenii. Granadaene has previously been identified only in Streptococcus agalactiae, where the pigment correlates with the hemolytic activity of the bacterium. A connection between hemolytic activity and the production of the red pigment has also been observed in P. jensenii, as nonpigmented strains are nonhemolytic. The pigment and hemolytic activity from S. agalactiae can be extracted from the bacterium with a starch extraction solution, and this solution also extracts the pigment and hemolytic activity from P. jensenii. A partial purification of the hemolytic activity was achieved, but the requirement for starch to preserve its activity made the purification unsuccessful. Partially purified hemolytic fractions were pigmented, and the color intensity of the fractions coincided with the hemolytic titer. The pigment was produced in a soluble form when associated with starch, and the UV-visual spectrum of the extract gave absorption peaks of 463 nm, 492 nm, and 524 nm. The pigment could also be extracted from the cells by a low-salt buffer, but it was then aggregated. The purification of the pigment from P. jensenii was performed, and mass spectrometry and nuclear magnetic resonance analysis revealed that P. jensenii indeed produces granadaene as seen in S. agalactiae.     

THE YELLOW PIGMENT CELLS OF HYDROBIA ULVAE (PENNANT) (MOLLUSCA : PROSOBRANCHIA)

The distribution of yellow pigment cells in the veliger larvaeand post-metamorphic snails of Hydrobia ulvae has been recorded.The number of cells increases with the size of the snail andthe pigment is characteristic of the foot of the veliger larva,and the foot, mantle, tentacles and penis of adult snails. Thecells contain numerous, double membrane-bound vesicles and havea highly granular cytoplasm. The absorption spectra of acetoneand methanolic HC1 extracts show single peaks at 337 and 392nm respectively, while chloroform extracts show peaks at 249,274 and 283 nm with an inflexion at 293 nm. The pigment hasa pale green fluorescence in ultraviolet light. The resultsof feeding and starvation experiments using larval and post-metamorphicsnails lead to the hypothesis that the pigment is a waste productof metabolism which is stored in the vesicles of the cells (Received 3 January 1979;     

Propionibacterium jensenii Produces the Polyene Pigment Granadaene and Has Hemolytic Properties Similar to Those of Streptococcus agalactiae

The red polyene pigment granadaene was purified and identified from Propionibacterium jensenii. Granadaene has previously been identified only in Streptococcus agalactiae, where the pigment correlates with the hemolytic activity of the bacterium. A connection between hemolytic activity and the production of the red pigment has also been observed in P. jensenii, as nonpigmented strains are nonhemolytic. The pigment and hemolytic activity from S. agalactiae can be extracted from the bacterium with a starch extraction solution, and this solution also extracts the pigment and hemolytic activity from P. jensenii. A partial purification of the hemolytic activity was achieved, but the requirement for starch to preserve its activity made the purification unsuccessful. Partially purified hemolytic fractions were pigmented, and the color intensity of the fractions coincided with the hemolytic titer. The pigment was produced in a soluble form when associated with starch, and the UV-visual spectrum of the extract gave absorption peaks of 463 nm, 492 nm, and 524 nm. The pigment could also be extracted from the cells by a low-salt buffer, but it was then aggregated. The purification of the pigment from P. jensenii was performed, and mass spectrometry and nuclear magnetic resonance analysis revealed that P. jensenii indeed produces granadaene as seen in S. agalactiae.     

Spectral differentiation of blue opsins between phylogenetically close but ecologically distant goldfish and zebrafish

Zebrafish and goldfish are both diurnal freshwater fish species belonging to the same family, Cyprinidae, but their visual ecological surroundings considerably differ. Zebrafish are surface swimmers in conditions of broad and shortwave-dominated background spectra and goldfish are generalized swimmers whose light environment extends to a depth of elevated short wavelength absorbance with turbidity. The peak absorption spectrum (lambdamax) of the zebrafish blue (SWS2) visual pigment is consistently shifted to short wavelength (416 nm) compared with that of the goldfish SWS2 (443 nm). Among the amino acid differences between the two pigments, only one (alanine in zebrafish and serine in goldfish at residue 94) was previously known to cause a difference in absorption spectrum (14-nm lambdamax shift in newt SWS2). In this study, we reconstructed the ancestral SWS2 pigment of the two species by applying likelihood-based Bayesian statistics and performing site-directed mutagenesis. The reconstituted ancestral photopigment had a lambdamax of 430 nm, indicating that zebrafish and goldfish achieved short wavelength (-14 nm) and long wavelength (+13 nm) spectral shifts, respectively, from the ancestor. Unexpectedly, the S94A mutation resulted in only a -3-nm spectral shift when introduced into the goldfish SWS2 pigment. Nearly half of the long wavelength shift toward the goldfish pigment was achieved instead by T116L (6 nm). The S295C mutation toward zebrafish SWS2 contributed to creating a ridge of absorbance around 400 nm and broadening its spectral sensitivity in the short wavelength direction. These results indicate that the evolutionary engineering approach is very effective in deciphering the process of functional divergence of visual pigments.     

Intracellular distribution of lysosomal enzymes in the mouse pigment mutants pale ear and pallid

Summary The size distribution of lysosomes was determined in kidney proximal tubule cells of two mouse pigment mutants, pale ear and pallid, which have an increase in kidney lysosomal enzyme content caused by a decreased rate of secretion of lysosomal enzymes into urine. Both mutations have larger lysosomes when compared with normal mice. However, neither mutant contains the giant lysosomes (up to 11 micron diameter) common to the well-characterized beige mutant, which has a kidney secretory defect similar to the pale ear and pallid mutants. Subcellular distribution studies, performed by the osmotic shock technique, likewise suggested differences among the pigment mutants. A very high content of soluble enzyme, indicative of lysosomal fragility during homogenization, was found in extracts from the beige mutation. By comparison, the percent of soluble enzyme became progressively lower in extracts of the pallid and pale ear mutants and was lowest in extracts from normal mice. All 3 pigment mutants had normal concentrations of osmotically resistant membrane-bound lysosomal enzymes. This indicates that the excess, non-secreted, lysosomal enzyme in all three pigment mutants likely is present in classical lysosomal organelles rather than in other non-lysosomal subcellular membrane fractions. The results also illustrate that mammalian mutants which exhibit decreased lysosomal secretory rates can have strikingly different effects on morphology of lysosomes.Supported in Part by National Science Foundation Grant PCM 77-24804. E. K. N. was supported in part by United States Public Health Service Grant GM 007093-03.     

Cytochrome spectrum of an obligate anaerobe, Eubacterium lentum.

An obligately anaerobic bacterium, Eubacterium lentum, was shown to contain cytochromes a, b, and c and a carbon monoxide-binding pigment. Extracts of cells grown with hemin gave a typical absorption spectrum for cytochrome c with maxima at 424, 525, and 553 nm. Extracts from cells grown in the absence of hemin also had an absorption peak corresponding to cytochrome b (562 nm) in their reduced versus oxidized spectrum. Extraction of hemes and formation of pyridine hemochromes allowed quantitation of protoheme IX and heme c. Large amounts of cytochrome c masked the presence of cytochrome b in cells grown in medium containing hemin. When cells were grown in the presence of 50 mM nitrate, cytochrome A (606 nm) was detected. In anaerobic extracts of cells grown either with or without nitrate, cytochromes b and c were reduced by formate and oxidized by NO3. Cytochrome a appeared to be partially oxidized by NO3 and completely oxidized by air.     

Retinal pigments and carotenoids of the light-shading "spectacles" of the greenling Hexagrammus octogrammus]

Three light-sensitive pigments having lambdamax of 480, 505 and 540 nm which contain retinal as a chromophore were found in the digitonine extracts from the retina of H. octogrammus. In summer time, only one pigment (lambdamax equals 480 nm) was found, whereas during autumn and winter periods the other two pigments (lambdamax equals 505 and 540 nm) could be also observed together with the first one. The lambdamax 480 pigment is easily degraded when being exposed to light, although it is resistant to the effect of hydroxylamine. The other two pitments are less sensitive to the light, but are readily bleached by hydroxylamine. The yellow-orange coloured cells of the light-shading "spectacles" contain a mixture of beta-carotenoids. When extracted by petroleum ether, these beta-carotenoids display lambdamax at 425, 445 and 476 nm. Column chromatography on aluminium oxide revealed 6 fractions in the extracted carotenoids: light-yellow, dark-yellow, brown, reddish-brown, pink and pinkish ones. In the range from yellow to pink fractions, the contribution of the lambdamax 475 nm band increases, while that of two other ones-decreases.     

红龙草红色素稳定性的研究

分析了pH值、温度、光、过氰化氢、亚硫酸钠、Vc、葡萄糖、蔗糖和苯甲酸钠等对红龙草（Altemanthera dentate ‘Ruliginosa’）红色素稳定性的影响。结果表明,红龙草红色素对热的耐受性较强,但耐光照和耐氧化性较差,且还原剂亚硫酸钠对其也有微弱的影响;在不同的pH值条件下,其吸收峰没有改变,最大吸收波长为530nm;Vc和蔗糖对该色素没有破坏作用,并有一定的护色效果;葡萄糖和苯甲酸钠对该色素也无明显影响。     

Inhibition of nicotinoprotein (NAD+-containing) alcohol dehydrogenase by trans-4-(N,N-dimethylamino)-cinnamaldehyde binding to the active site

Ethanol oxidation by nicotinoprotein alcohol dehydrogenase (np-ADH) from the bacterium Amycolatopsis methanolica is inhibited by trans-4-(N,N-dimethylamino)-cinnamaldehyde through direct binding to the catalytic zinc ion in a substrate-like geometry. This binding is accompanied by a characteristic red shift of the aldehyde absorbance from 398 nm to 467 nm. Np-ADH is structurally related to mammalian ADH class I, and a model of np-ADH shows how the cinnamaldehyde derivative can be accommodated in the active site of the nicotinoprotein, correlating the structural and enzymological data.     

Biosynthesis of a chlorophyllide b-like pigment in phenanthroline-treated Chlamydomonas reinhardtii y-1

Incubation of degreened Chlamydomonas reinhardtii y-1 cells in the dark with m-phenanthroline induced de novo synthesis of a chlorophyllide b-like pigment. The rate of synthesis of this pigment in the dark was greater than that of total chlorophyll in illuminated cells. Most of the newly synthesized pigment was excreted into the culture medium. The product was extracted from the medium as the metal-free pheophorbide, which had a fluorescence excitation maximum at 428 +/- 1 nm and an emission maximum at 657 +/- 1 nm (E428F657) in ethyl acetate (E427F657 in diethyl ether). Three pheophorbide species were extracted from the medium of green cells treated in the dark, a minor component with a spectrum (E410F670) identical to demetallated chlorophyll a, and two major species with spectral values of E428F657 and E433F657. The latter, predominant form had a spectrum identical to demetallated chlorophyll b, which was purified from the algal cells. E428F657 and E433F657 reacted with hydroxylamine and Girard's T-reagent, which caused a shift in the fluorescence emission maximum to 668 nm. Pheophytin b, which contains an aldehyde group, exhibited an identical spectral shift when treated in the same way, but pheophytin a or porphyrin biosynthetic intermediates did not. Proton NMR analysis of the E428F657 chlorin produced by yellow cells treated with m-phenanthroline confirmed the presence of an aldehydic proton. Chelating and nonchelating phenanthroline analogs equally stimulated synthesis of this product.     

Modulation of enzyme activity in azaguanine-resistant V79 cells selected by chronic drug exposure

Exposure of V79 cells to azaguanine (7-21 microM for 2-7 weeks) had little effect on growth or plating efficiency but resulted in gradual acquisition of resistance to 8-azaguanine (AZ) and 6-thioguanine (TG) and loss of ability to grow in HAT. The rate of evolution of the resistant phenotype was dependent on the concentration and duration of exposure to AZ. The increase in proportion of resistant cells was paralleled by a rise in phosphatase activity (pH optimum 7.0-7.5) expressed by intact cells and this preceded the fall in HGPRT activity. Elevated phosphatase activity and a resistant phenotype were stably expressed in clones isolated and cultured in the absence of AZ. Hypoxanthine guanine phosphoribosyl transferase (HGPRT) activity in cell extracts of three resistant clones ranged from 18 to 43% of wild-type levels but was unaltered with respect to substrate affinity and electrophoretic mobility. Mg2+-dependent activity dephosphorylated inosine 5'monophosphate (IMP), guanine 5'monophosphate (GMP), adenosine-5-monophosphate (AMP) and p-nitrophenylphosphate (PNPP) and was also elevated with respect to wild-type levels in resistant cell extracts. Purine nucleoside phosphorylase levels were similar in sensitive and resistant cell extracts. Cross-sensitivity studies with other purine analogues suggest that the elevated phosphatase activity does not contribute to the resistant phenotype. No karyotypic changes were observed in the resistant cell lines.     

Toxicity of mercaptoethanol to mutant strains of the yeast Pichia methanolica growing on different carbon sources

Addition of beta-mercaptoethanol at a concentration of 2-3 mM to media containing methanol, glucose, or yeast extract caused a 50% inhibition of the growth of wild-type yeast Pichia methanolica; mercaptoethanol at a concentration of 0.7 to 25 mM inhibited the growth of the mutant strain ecr1. The mutation mth1 of P. methanolica repressed its ability to consume methanol and was accompanied by the loss of alcohol oxidase (EC 1.1.3.13) activity. beta-Mercaptoethanol restored the ability of mth1 mutant cells to grow on methanol and stimulated their growth under derepression conditions. The growth effect of beta-mercaptoethanol during derepression was accompanied by partial restoration of alcohol oxidase activity.     

Dual Sensitivities of Cells in Wolf Spider Eyes at Ultraviolet and Visible Wavelengths of Light

Intracellular recordings have been made from visual cells in principal and secondary eyes of in vitro wolf spider preparations. The responses of all cells to all wavelengths of light were graded depolarizations; no hyperpolarizations or nerve discharges were seen. Cells in a secondary eye, the anterior lateral eye, had a maximum sensitivity in the visible at 510 nm and a secondary maximum, or shoulder, of sensitivity in the near ultraviolet at 380 nm. Cells in principal eyes, the anterior median eyes, all responded maximally both in the visible at 510 nm and in the ultraviolet at 360–370 nm or less. However, there was no typical ratio of ultraviolet to visible sensitivities; the differences in log sensitivities (log UV/VIS) varied from 3.3 to -0.5. Each principal eye had a population of cells with different ratios. These populations varied with the time of the year, possibly due to changes in light upon the animals. Chromatic adaptations of cells in anterior median (but not anterior lateral) eyes resulted in small, selective changes in spectral sensitivities, and there was some facilitation of responses from cells repeatedly stimulated. It is concluded that cells of secondary eyes contain only a visual pigment absorbing maximally in the visible, while cells of principal eyes probably contain variable amounts of both this pigment and one absorbing in the ultraviolet as well.     

The physical and morphological properties of the pigment screen in the compound eye of a shrimp (Crustacea)

Summary The pigment cells of the compound eye of the shrimps (Crangon crangon andC. allmani) were studied by electron microscopy (SEM and TEM) and microspectrophotometry. The compound eyes of these species contain light-absorbing and -reflecting pigments contained in granules, located in 5 different cells. The light absorbing pigment granules (light screen) are situated in (1) the distal pigment cells, (2) the retinular cells, (3) the basal pigment cells. The reflecting pigment granules are located in (4) the distal, and (5) the proximal reflecting pigment cells. Another innominate cell type investing the ommatidia contains vacuoles without pigment content. The innominate cell type, and the basal absorbing pigment cell (3) listed above, have not earlier been reported for a crustacean species. Measurements of the spectral absorption on sliced and squashed ommatidia show that all components of the light screen have an increased absorption in the wavelength regions 400–450 nm and 530–570 nm, probably due to xanthommatin and ommin. The spectral absorbancy of the reflecting pigment cells were not determined. Similar cells in other species are known to contain pteridines.We thank Prof. Dr. Langer, Bochum, Germany, for his kind help. The work was supported by funds from the Karolinska Institutet to Doc. G. Struwe, and grant NFR No. 2760-007 to Doc. R. Elofsson.     

Biosynthesis of l-Phenylalanine and l-Tyrosine in the Actinomycete Amycolatopsis methanolica

Auxotrophic mutants of the actinomycete Amycolatopsis methanolica requiring l-Phe or l-Tyr were isolated and identified as strains lacking prephenate dehydratase (strain GH71) or arogenate dehydrogenase (strain GH70), respectively. A. methanolica thus employs single pathways only for the biosynthesis of these aromatic amino acids. Anion-exchange chromatography of extracts revealed two peaks with Phe as well as Tyr aminotransferase (AT) activity (Phe/Tyr ATI and Phe/Tyr ATII) and three peaks with prephenate AT activity (Ppa ATI to Ppa ATIII). Phe/Tyr ATI and Ppa ATI coeluted and appear to function as the A. methanolica branched-chain amino acid AT. Ppa ATII probably functions as the aspartate AT. Mutant studies showed that Phe/Tyr ATII is the dominant AT in l-Phe biosynthesis and in l-Tyr catabolism but not in l-Tyr biosynthesis. Biochemical studies showed that Ppa ATIII is highly specific for prephenate and provided evidence that Ppa ATIII is the dominant AT in l-Tyr biosynthesis.     

Antioxidant capacity of a 3-deoxyanthocyanidin from soybean.

Soybean cotyledons directly exposed to UV-C (190-280 nm) contained a colored pigment in those areas of the epidermis directly exposed to UV-C. Ethanolic extracts from UV-C irradiated cotyledons showed a significant peak at 532 nm at pH=10, but not seen at pH=6, successive changes in pH were accompanied by reversible changes in the spectra. The identity of the pigment isolated from soybean cotyledons was established as apigeninidin by comparing the features of standard of a apigeninidin (from sorghum) previously characterized by FAB-MS, UV, HPLC, 1H NMR, and IR spectroscopy. To characterize antioxidant activity of this compound, its ability to scavenge radical species in vitro was tested. In the concentration range tested (up to 200 microg ml (-1)), apigeninidin did not show any scavenger activity towards hydroxyl radical, quinones or NO. However, ascorbyl radical and lipid radicals were effectively quenched in a dose-dependent manner. Overall, UV-C radiation triggers molecular signals that lead in soybean cotyledons to the synthesis and accumulation of an antioxidant pigment, apigeninidin, that shows scavenger activity against ascorbyl and lipid radicals in in vitro studies.     

Chromosome-mediated gene transfer of hydroxyurea resistance and amplification of ribonucleotide reductase activity.

Metaphase chromosomes purified from a hydroxyurea-resistant Chinese hamster cell line were able to transform recipient wild-type cells to hydroxyurea resistance at a frequency of 10(-6). Approximately 60% of the resulting transformant clones gradually lost hydroxyurea resistance when cultivated for prolonged periods in the absence of drug. One transformant was subjected to serial selection in higher concentrations of hydroxyurea. The five cell lines generated exhibited increasing relative plating efficiency in the presence of the drug and a corresponding elevation in their cellular content of ribonucleotide reductase. The most resistant cell line had a 163-fold increase in relative plating efficiency and a 120-fold increase in enzyme activity when compared with the wild-type cell line. The highly hydroxyurea-resistant cell lines had strong electron paramagnetic resonance signals characteristic of an elevated level of the free radical present in the M2 subunit of ribonucleotide reductase. Two-dimensional electrophoresis of cell-free extracts from one of the resistant cell lines indicated that a 53,000-dalton protein was present in greatly elevated quantities when compared with the wild-type cell line. These data suggest that the hydroxyurea-resistant cell lines may contain an amplification of the gene for the M2 subunit of ribonucleotide reductase.     

Light intensity effects on pigment composition and organisation in the green sulfur bacterium Chlorobium tepidum

We have investigated the changes in the pigment composition and organisation of the light-harvesting apparatus of the green sulfur bacterium Chlorobium tepidum growing under different light intensities. Cells grown at lower light intensities had lower exponential growth rates and increased amounts of the main light-harvesting pigments, bacteriochlorophyll c and carotenoids, on a cell protein basis. Absorption spectra of chlorosomes isolated from cells grown at low light intensities revealed a red-shift of up to 8 nm in the Q_y band of bacteriochlorophyll c compared to chlorosomes from high light grown cells. A similar red-shift of up to 4 nm was also observed in the corresponding fluorescence emission peaks. HPLC analysis of pigment extracts showed a correlation between the red-shift and the content of the more alkylated BChl c homologs, which increased as light intensity for growth was lower. Furthermore, analysis of the carotenoid composition in chlorosomes re vealed a conspicuous change in the ratio between chlorobactene and 1, 2-dihydrochlorobactene, which dramatically decreased from 5 to 0.7 in light-limited cultures.