SALTANT PRODUCTION BY WAVE LENGTHS OF VISIBLE AND LONG ULTRAVIOLET MONOCHROMATIC IRRADIATION,AND A COMPARISON WITH SALTANTS PRODUCED BY SHORT WAVE LENGTHS OF MONOCHROMATIC ULTRAVIOLET IRRADIATION IN THE FUNGUS CHAETOMIUM GLOBOSUM

1. Saltants have been produced in the fungus Chaetomium globosum by longer wave lengths than previously reported—by 365 mµ and by a visible line 404 mµ. 2. Absence at these wave lengths of the K saltant, which is so abundant at short wave lengths, is marked. 3. Ratio of percentage irradiated spores germinating to control spores germinating decreases from 83 per cent at 265 mµ, a short ultraviolet wave length, to 57 per cent at 404 mµ, a visible violet wave length.     

THE PIGMENT-PROTEIN COMPOUND IN PHOTOSYNTHETIC BACTERIA : II. THE ABSORPTION CURVES OF PHOTOSYNTHIN FROM SEVERAL SPECIES OF BACTERIA

Absorption curves have been obtained in the spectral region of 450 to 900 mµ for the water soluble cell juice of four species of photosynthetic bacteria, Spirillum rubrum (strain S1), Rhodovibrio sp. (strain Gaffron), Phaeomonas sp. (strain Delft), and Streptococcus varians (strains C11 and orig.). These curves all show maxima at 790 and 590 mµ due to bacteriochlorophyll, whose highest band, however, occurs at 875, 855, or 840 mµ depending on the species. The bacteria that appear red rather than brown have a band at 550 mµ due to a carotinoid pigment. An absolute absorption curve of bacteriophaeophytin has maxima at 530 and 750 mµ. The extraction of cell juice by supersonic vibration does not change the position of the absorption bands or of the light absorbing capacity of the pigment.     

Photoreversal of nuclear and cytoplasmic effects of short ultraviolet radiation on Paramecium caudatum

1. Irradiation with three short ultraviolet (UV) wave lengths, 226, 233, and 239 mµ rapidly immobilizes Paramecium caudatum, the dosage required being smaller the shorter the wave length. 85 per cent of paramecia immobilized with wave length 226 mµ recover completely. Recovery from immobilizing doses is less the longer the wave length. 2. Irradiation continued after immobilization kills the paramecia in a manner which is markedly different for very short (226, 233, and 239 mµ) and longer (267 mµ) wave lengths. 3. An action spectrum for immobilization in P. caudatum was determined for the wave lengths 226, 233, 239, 248, and 267 mµ, and found to resemble the absorption of protein and lipide in the wave length region below 248 mµ. Addition of these data to those of Giese (1945 b) gives an action spectrum resembling the absorption by albumin-like protein. 4. Division of P. caudatum is delayed by doses of wave lengths 226, 233, and 239 mµ which cause immobilization, the longest wave length being most effective. 5. Immobilization at any of the wave lengths tested (226, 233, 239, 248, 267 mµ) is not photoreversible when UV-treated paramecia are concurrently illuminated. 6. Division delay resulting from immobilizing doses of 226, 233, and 239 mµ is photoreversible by exposure to visible light concurrently with the UV. 7. Division delay induced by exposure to wave length 267 mµ is reduced by exposure to visible light applied concurrently with UV or immediately afterwards. 8. The data suggest that the shortest UV wave length tested (226 mµ) affects the cytoplasm selectively, because it is absorbed superficially as indicated by unilateral fluorescence in UV. Consequently it immobilizes paramecia rapidly but has little effect on the division rate because little radiation reaches the nucleus. 9. The data support the view that nuclear effects of UV are readily photoreversed but cytoplasmic effects are not.     

THE PHOTOTROPIC SENSITIVITY OF PHYCOMYCES AS RELATED TO WAVE-LENGTH

Under the circumstances of experimentation described, the sporangiophores of Phycomyces are found to be most sensitive to stimulation by light in the violet between 400 and 430 mµ. Toward the red, sensitivity falls to nearly zero near 580 mµ, while in the near ultra-violet around 370 mµ, sensitivity is still high. The previous experiments of Blaauw had placed the point of greatest sensitivity some 80 mµ nearer the red end of the spectrum. Because of the known presence in the sporangiophores of Phycomyces of "accessory" pigments, care must be taken in identifying such results with the absorption spectrum of the photosensitive substance.     

The spectral sensitivities of the dorsal ocelli of cockroaches and honeybees; an electrophysiological study

The spectral sensitivities of the dorsal ocelli of cockroaches (Periplaneta americana, Blaberus craniifer) and worker honeybees (Apis mellifera) have been measured by electrophysiological methods. The relative numbers of quanta necessary to produce a constant size electrical response in the ocellus were measured at various wave lengths between 302 and 623 mµ. The wave form of the electrical response (ERG) of the dark-adapted roach ocellus depends on the intensity but not the wave length of the stimulating light. The roach ocellus appears to possess a single photoreceptor type, maximally sensitive about 500 mµ. The ERG's of bee ocelli are qualitatively different in the ultraviolet and visible regions of the spectrum. The bee ocellus has two types of photoreceptor, maximally sensitive at 490 mµ and at about 335 to 340 mµ. The spectral absorption of the ocellar cornea of Blaberus craniifer was measured. There is no significant absorption between 350 and 700 mµ.     

A Genetic Analysis of the Functional Interactions within Mycobacterium tuberculosis Single-Stranded DNA Binding Protein

Single-stranded DNA binding proteins (SSBs) are vital in all organisms. SSBs of Escherichia coli (EcoSSB) and Mycobacterium tuberculosis (MtuSSB) are homotetrameric. The N-terminal domains (NTD) of these SSBs (responsible for their tetramerization and DNA binding) are structurally well defined. However, their C-terminal domains (CTD) possess undefined structures. EcoSSB NTD consists of β1-β1′-β2-β3-α-β4-β45₁-β45₂-β5 secondary structure elements. MtuSSB NTD includes an additional β-strand (β6) forming a novel hook-like structure. Recently, we observed that MtuSSB complemented an E. coli Δssb strain. However, a chimeric SSB (mβ4-β5), wherein only the terminal part of NTD (β4-β5 region possessing L₄₅ loop) of EcoSSB was substituted with that from MtuSSB, failed to function in E. coli in spite of its normal DNA binding and oligomerization properties. Here, we designed new chimeras by transplanting selected regions of MtuSSB into EcoSSB to understand the functional significance of the various secondary structure elements within SSB. All chimeric SSBs formed homotetramers and showed normal DNA binding. The mβ4-β6 construct obtained by substitution of the region downstream of β5 in mβ4-β5 SSB with the corresponding region (β6) of MtuSSB complemented the E. coli strain indicating a functional interaction between the L₄₅ loop and the β6 strand of MtuSSB.     

The Photosensitive Retinal Pigment System of Gekko gekko

Retinal extracts of Gekko gekko were found to contain two retinene₁ photopigments, one with maximum absorption at about 521 mµ, the second with a maximum in the region of 478 mµ. These pigments were assayed by the method of partial bleaching and their spectral characteristics studied by examining their difference spectra. The 478 mµ pigment was present in the extracts as 8 per cent of the total photopigment concentration. The two pigment systems were shown to be biochemically independent and to have different properties. Unlike the 521 mµ pigment, for example, the 478 mµ pigment was found to resist the action of NH₂OH and, within the cells, to be unaffected by sucrose solutions. These solutions destroyed or altered the 521 system so that extracts of sucrose-treated retinae were found to contain significantly less 521 photopigment. In digitonin solution the 521 pigment was unaffected by sucrose treatment. Both pigments were extracted from separated, washed outer segments and so are considered to be visual pigments. This dual system accounts for the spectral sensitivity of this gecko as determined by Denton. A search was made, but no evidence was secured for the presence of a photopigment absorbing at longer wavelengths. Electoretinographic data suggest, however, that an elevated sensitivity at longer wavelengths occurs in some geckos so that a continued search is justified for a third photopigment.     

The Role of Carbonium Ions in Color Reception

It is a fundamental property of conjugated systems to accept a proton or Lewis acid and form a stable carbonium ion. Polyenes that are protonated or add Lewis acids in this manner undergo substantial red shifts. For example, vitamin A₁ acetate absorbs at 350 mµ in neutral and at 650 mµ in acidic benzene solution. The fundamental basis for absorption of polyene systems was described in detail in quantum mechanical terms. Applying the carbonium ion treatment to the visual chromophores retinal₁ and retinal₂ gives a very satisfactory explanation why these polyenes can be made to absorb in the visual region. Furthermore, by proper placement of the Lewis acid several absorption maxima can be gained from the carbonium ions which result. This treatment can be applied to explain experimental results. Individual cones from the frog are now known to absorb at 455, 537, and 625 mµ. If the value for the green cone (537 mµ) is used to calculate the V_o value in Kuhn's equation, the other two wave lengths may then be calculated. The calculated values are 460 and 600 mµ; this is in good agreement with the results from experiment.     

Kinetics of Phosphomevalonate Kinase from Saccharomyces cerevisiae

The mevalonate-based isoprenoid biosynthetic pathway is responsible for producing cholesterol in humans and is used commercially to produce drugs, chemicals, and fuels. Heterologous expression of this pathway in Escherichia coli has enabled high-level production of the antimalarial drug artemisinin and the proposed biofuel bisabolane. Understanding the kinetics of the enzymes in the biosynthetic pathway is critical to optimize the pathway for high flux. We have characterized the kinetic parameters of phosphomevalonate kinase (PMK, EC 2.7.4.2) from Saccharomyces cerevisiae, a previously unstudied enzyme. An E. coli codon-optimized version of the S. cerevisiae gene was cloned into pET-52b+, then the C-terminal 6X His-tagged protein was expressed in E. coli BL21(DE3) and purified on a Ni²⁺ column. The K_M of the ATP binding site was determined to be 98.3 µM at 30°C, the optimal growth temperature for S. cerevisiae, and 74.3 µM at 37°C, the optimal growth temperature for E. coli. The K_M of the mevalonate-5-phosphate binding site was determined to be 885 µM at 30°C and 880 µM at 37°C. The V_max was determined to be 4.51 µmol/min/mg enzyme at 30°C and 5.33 µmol/min/mg enzyme at 37°C. PMK is Mg²⁺ dependent, with maximal activity achieved at concentrations of 10 mM or greater. Maximum activity was observed at pH = 7.2. PMK was not found to be substrate inhibited, nor feedback inhibited by FPP at concentrations up to 10 µM FPP.     

A new photosensitive pigment of the euryhaline teleost,Gillichthys mirabilis

Retinal extracts have been prepared from dark-adapted mudsuckers by treatment of retinal tissue or of isolated outer segments of the visual cells with digitonin solution. The extracts were examined spectrophotometrically and found to absorb light maximally between the wave lengths of 488 and 510 mµ, depending on the proportion of yellow impurities and light-sensitive pigment present. This photosensitive pigment was shown to be homogeneous by partial bleaching of the extracts with monochromatic light of various wave lengths from 390 to 660 mµ. The mudsucker pigment was specifically demonstrated not to be a mixture of rhodopsin and porphyropsin; the adequacy of the method used to analyze such mixtures was shown by performing a control experiment with an artificial mixture of bullfrog rhodopsin and carp porphyropsin. Comparison of the hydroxylamine difference spectrum and of the absorption maximum of the purest retinal extract located the mudsucker photosensitive pigment maximum at 512 ± 1 mµ. Extraction of retinal tissue with a fat solvent after exposure to white light gave a preparation which after the addition of antimony chloride reagent developed the absorption band maximal near 664 mµ, which is characteristic of retinene₁. If an hour intervened between exposure of the retinal tissue to light and extraction of the carotenoid, the antimony trichloride test gave a color band maximal at 620 mµ, characteristic of vitamin A₁. No evidence of retinene₂ or vitamin A₂ was obtained. The euryhaline mudsucker has, therefore, a photosensitive retinal pigment with an absorption maximum halfway between the peaks of rhodopsins and of porphyropsins and belonging to the retinene₁ system characteristic of rhodopsins. The pigment is therefore named a retinene₁ pigment 512 of the mudsucker, Gillichthys mirabilis. It is uncertain whether this type of photosensitive pigment will be found in other euryhaline fishes.     

Photoreactivation in vitro of ultraviolet-inactivated Hemophilus influenzae transforming factor

Hemophilus influenzae—transforming DNA, which has been inactivated by ultra-violet radiation, is reactivated by visible light in the presence of a cell-free extract of Escherichia coli B. The time rate of reactivation is increased by increasing the E. coli extract concentration, the temperature, and the intensity of illumination. Only DNA containing an ultraviolet-damaged genetic marker exhibits increased transforming activity after treatment with the photoreactivating system. The reactivating capacity of the extract remains in the top supernatant after centrifugation at 110,000 x g for 1 hour and is not present in the pellet. This capacity is destroyed by heating to 90°C. for 1 minute. The active system of the E. coli extract is separable into dialyzable, heat-stable and non-dialyzable, heat-labile fractions. The dialyzable fraction contains at least one component which limits the maximum degree of recovery attained.     

Particle Deposition in a Child Respiratory Tract Model: In Vivo Regional Deposition of Fine and Ultrafine Aerosols in Baboons

To relate exposure to adverse health effects, it is necessary to know where particles in the submicron range deposit in the respiratory tract. The possibly higher vulnerability of children requires specific inhalation studies. However, radio-aerosol deposition experiments involving children are rare because of ethical restrictions related to radiation exposure. Thus, an in vivo study was conducted using three baboons as a child respiratory tract model to assess regional deposition patterns (thoracic region vs. extrathoracic region) of radioactive polydisperse aerosols ([d16–d84], equal to [0.15 µm–0.5 µm], [0.25 µm–1 µm], or [1 µm–9 µm]). Results clearly demonstrated that aerosol deposition within the thoracic region and the extrathoraic region varied substantially according to particle size. High deposition in the extrathoracic region was observed for the [1 µm–9 µm] aerosol (72%±17%). The [0.15 µm–0.5 µm] aerosol was associated almost exclusively with thoracic region deposition (84%±4%). Airborne particles in the range of [0.25 µm–1 µm] showed an intermediate deposition pattern, with 49%±8% in the extrathoracic region and 51%±8% in the thoracic region. Finally, comparison of baboon and human inhalation experiments for the [1 µm–9 µm] aerosol showed similar regional deposition, leading to the conclusion that regional deposition is species-independent for this airborne particle sizes.     

Binding of aminoglycoside antibiotics to helix 69 of 23S rRNA

Aminoglycosides antibiotics negate dissociation and recycling of the bacterial ribosome’s subunits by binding to Helix 69 (H69) of 23S rRNA. The differential binding of various aminoglycosides to the chemically synthesized terminal domains of the Escherichia coli and human H69 has been characterized using spectroscopy, calorimetry and NMR. The unmodified E. coli H69 hairpin exhibited a significantly higher affinity for neomycin B and tobramycin than for paromomycin (K_ds = 0.3 ± 0.1, 0.2 ± 0.2 and 5.4 ± 1.1 µM, respectively). The binding of streptomycin was too weak to assess. In contrast to the E. coli H69, the human 28S rRNA H69 had a considerable decrease in affinity for the antibiotics, an important validation of the bacterial target. The three conserved pseudouridine modifications (Ψ1911, Ψ1915, Ψ1917) occurring in the loop of the E. coli H69 affected the dissociation constant, but not the stoichiometry for the binding of paromomycin (K_d = 2.6 ± 0.1 µM). G1906 and G1921, observed by NMR spectrometry, figured predominantly in the aminoglycoside binding to H69. The higher affinity of the E. coli H69 for neomycin B and tobramycin, as compared to paromomycin and streptomycin, indicates differences in the efficacy of the aminoglycosides.     

THE RATE OF CO2 ASSIMILATION BY PURPLE BACTERIA AT VARIOUS WAVE LENGTHS OF LIGHT

1. The relative absorption spectrum of the pigments in their natural state in the photosynthetic bacterium Spirillum rubrum is given from 400 to 900 mµ. The position of the absorption maxima in the live bacteria due to each of the pigments is: green pigment, 420, 590, 880; red pigment, 490, 510, 550. 2. The relative absorption spectrum of the green pigment in methyl alcohol has been determined from 400 to 900 mµ. Bands at 410, 605, and 770 mµ were found. 3. The wave length sensitivity curve of the photosynthetic mechanism has been determined and shows maxima at 590 and about 900 mµ. 4. It is concluded that the green bacteriochlorophyll alone and not the red pigment can act as a light absorber for photochemical CO₂ reduction.     

Delayed light production by blue-green algae,red algae,and purple bacteria

1. Blue-green algae, red algae, and purple bacteria all show the emission of delayed light. 2. The action spectra for the production of delayed light by three species of blue-green algae have one broad band with a peak at 620 mµ. 3. The action spectrum for production of delayed light by the red algae has one peak at 550 mµ with a shoulder from 600 to 660 mµ. 4. The emission spectra of the delayed light from both the blue-green and red algae were the same as from the green algae, Chlorella. 5. The action spectra for the production of delayed light by the different species of purple bacteria tested consisted of one or more bands not resolved between 800 and 900 mµ. 6. The emission spectrum of the delayed light from the purple bacteria was largely at wave lengths longer than 900 mµ.     

Structural and Biochemical Studies of a Moderately Thermophilic Exonuclease I from Methylocaldum szegediense

A novel exonuclease, designated as MszExo I, was cloned from Methylocaldum szegediense, a moderately thermophilic methanotroph. It specifically digests single-stranded DNA in the 3ʹ to 5ʹ direction. The protein is composed of 479 amino acids, and it shares 47% sequence identity with E. coli Exo I. The crystal structure of MszExo I was determined to a resolution of 2.2 Å and it aligns well with that of E. coli Exo I. Comparative studies revealed that MszExo I and E. coli Exo I have similar metal ion binding affinity and similar activity at mesophilic temperatures (25–47°C). However, the optimum working temperature of MszExo I is 10°C higher, and the melting temperature is more than 4°C higher as evaluated by both thermal inactivation assays and DSC measurements. More importantly, two thermal transitions during unfolding of MszExo I were monitored by DSC while only one transition was found in E. coli Exo I. Further analyses showed that magnesium ions not only confer structural stability, but also affect the unfolding of MszExo I. MszExo I is the first reported enzyme in the DNA repair systems of moderately thermophilic bacteria, which are predicted to have more efficient DNA repair systems than mesophilic ones.     

Structure of the Red Fluorescence Band in Chloroplasts

Using Weber's method of "matrix analysis" for the estimation of the number of fluorescent species contributing to the emission of a sample, it is shown that the fluorescence¹ band in spinach chloroplast fragments at room temperature originates in two species of chlorophyll a. Emission spectra obtained upon excitation with different wavelengths of light (preferentially absorbed in chlorophyll a or b) are presented. Upon cooling to - 196°C, the fluorescence efficiency increases about twentyfold. Two additional bands, that now appear at 696 and 735 mµ, suggest the participation of four molecular species. Emission spectra observed at different concentrations of chloroplast fragments with excitation in chlorophyll a and b and excitation spectra for different concentrations of chloroplast fragments and measurements at 685 and 760 mµ are presented. Two of the four emission bands may belong to pigment system I and two to system II. The 685, 696, and 738 mµ bands respond differently to temperature changes. In the -196°C to -150°C range, the intensity of the 685 mµ band remains constant, and that of the 696 mµ band decreases twice as fast as that of the 738 mµ band.     

Spectral Sensitivity of the Common Prawn, Palaemonetes vulgaris

The vision of Palaemonetes is of particular interest in view of extensive studies of the responses of its chromatophore systems and eye pigments to light. The spectral sensitivity is here examined under conditions of dark adaptation and adaptation to bright colored lights. In each case the relative number of photons per one-fiftieth sec flash needed to evoke a constant peak amplitude (usually 25 or 50 µv) in the electroretinogram (ERG) was measured at various wavelengths throughout the spectrum. The sensitivity is the reciprocal of this number. In dark-adapted animals the spectral sensitivity curve consists of a broad, almost symmetrical band, maximal at about 540 mµ, with a shoulder near 390 mµ. Adaptation to bright red or blue light, left on continuously throughout the measurements, depresses the 540 mµ peak without notably changing its shape or position, implying that only one visual pigment operates in this region. Adaptation to red light, however, spares a violet-sensitive system, so that a high, narrow peak at 390 mµ now dominates the spectral sensitivity function. The 540 and 390 mµ peaks are apparently associated with different visual pigments; and these seem to be segregated in different receptor systems, since the associated ERG's have markedly different time constants. It is suggested that these two sensitivity bands may represent the red- and violet-sensitive components of an apparatus for color differentiation.     

Do Flies Have A Red Receptor?

(1) The compound eye of Musca exhibits characteristics which have heretofore frequently been considered evidence for color receptors: (a) The spectral sensitivity curve has several peaks whose relative heights can be altered by selective adaptation to colored lights, and (b) the shape of the retinal action potential varies with wave length. (2) The action spectrum for the red enhancement of on and off responses is compared with the "red receptor" calculated by Mazokhin-Porshnyakov from colorimetric data obtained in rapid color substitutions. Both have maxima at 615 to 620 mµ and appear to be different expressions of the same phenomenon. (3) A red receptor is absent. The evidence which suggests different types of receptors in the region 500 to 700 mµ can be accounted for by variations in the numbers of receptors stimulated. In red light there is a recruitment of additional ommatidia caused by leakage of long wave lengths through the pigment screen, and this spatial summation potentiates the on and off responses. The principal evidence is: (a) a white eye mutant which has no accessory screening pigments also lacks the peak of sensitivity in the red, even when adapted to violet light; (b) white-eyed flies give identical responses with large on and off effects at all wave lengths from 500 to 700 mµ; and (c) reducing the number of excited ommatidia by decreasing the size of the test spot makes the on and off transients smaller relative to the receptor component.     

Selective Enrichment with a Resuscitation Step for Isolation of Freeze-Injured Escherichia coli O157:H7 from Foods

We studied injury of Escherichia coli O157:H7 cells in 11 food items during freeze storage and methods of isolating freeze-injured E. coli O157:H7 cells from foods. Food samples inoculated with E. coli O157:H7 were stored for 16 weeks at −20°C in a freezer. Noninjured and injured cells were counted by using tryptic soy agar and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Large populations of E. coli O157:H7 cells were injured in salted cabbage, grated radish, seaweed, and tomato samples. In an experiment to detect E. coli O157:H7 in food samples artificially contaminated with freeze-injured E. coli O157:H7 cells, the organism was recovered most efficiently after the samples were incubated in modified E. coli broth without bile salts at 25°C for 2 h and then selectively enriched at 42°C for 18 h by adding bile salts and novobiocin. Our enrichment method was further evaluated by isolating E. coli O157:H7 from frozen foods inoculated with the organism prior to freezing. Two hours of resuscitation at 25°C in nonselective broth improved recovery of E. coli O157:H7 from frozen grated radishes and strawberries, demonstrating that the resuscitation step is very effective for isolating E. coli O157:H7 from frozen foods contaminated with injured E. coli O157:H7 cells.