Apocarotenoids in the sexual interaction of Phycomyces blakesleeanus

A simple genetic test allowed us to carry out the first systematic study of the apocarotenoids in the Mucorales. We have identified 13 apocarotenoids in the culture media of the fungus Phycomyces blakesleeanus (Mucoromycota, Mucorales). Three of these compounds were novel apocarotenoids: (2S,8R,E)-8,14-epoxycyclofarnesa-4,6,9-triene-2,11-diol (6), (2S,6E,8E)-cyclofarnesa-4,6,8-triene-2,10,11-triol (7), and its 6Z isomer (8). Four of the remaining compounds have been reported previously from this fungus and six from other Mucorales. All of them belong to three families, the 18-carbon trisporoids, the 15-carbon cyclofarnesoids, and the 7-carbon methylhexanoids, derived from the three fragments that result when β-carotene is cleaved at its 11',12' and 12,13 double bonds. The apocarotenoids were more varied and more abundant in mated cultures of strains of opposite sex than in single cultures. The presence of acetate in the medium blocked the production of many apocarotenoids while having little effect on the concentrations of the remaining ones.     

MODIFIED ACTION SPECTRA OF PHOTOGEOTROPIC EQUILIBRIUM IN Phycomyces blakesleeanus MUTANTS WITH DEFECTS IN GENES madA, madB, madC, and madH

Abstract— Action spectra of photogeotropic equilibrium were measured for behavioral mutants of Phycomyces blakesleeanus with defects in the genes madB, madC and madH as well as for a double mutant defective in the genes madA and madC. The action spectra of strains C109 (madB), LI (madC) and L52 (madA madC) all lack the broad near-ultraviolet peak which extends from 347 to 386 nm in the wild type; the peaks at 414 and 491 nm are also missing in these mutants. The double mutant L52 (madA madC) shows a novel broad peak at 477 nm; the relative quantum effectiveness of L52 at 477 nm is 10 times higher than in LI (madC119). These properties of the double mutant L52 (madA madC) suggest steric interaction of the madA and the madC gene products in the photoreceptor complex. For the hypertropic mutant L84 (madH) the action spectrum and absolute sensitivity are similar to those for wild type. These results confirm and extend previous findings that multiple photoreceptors are mediating phototropism in P. blakesleeanus.     

RESOLUTION OF SPECTRAL AND LIFETIME HETEROGENEITY OF TRYPTOPHAN FLUORESCENCE IN BACTERIORHODOPSIN

Abstract— The picosecond time-resolved fluorescence decay of bacteriorhodopsin (BR) was analyzed by the maximum entropy method. Results showed five distributions of lifetimes indicating at least five decay components. A wavelength-dependent study of emission decay of BR was carried out in the wavelength region from 310 to 390 nm. The decay at each wavelength was resolvable into four decay components by the discrete exponential analysis. The three short lifetime components (100 ± 20 ps, 400 ± 50 ps and 1.0 ± 0.1 ns) were independent of wavelength, whereas the longest lifetime component was wavelength dependent (varying from 4.1 ns at 310 nm to 5.7 ns at 390 nm). These results are inconsistent with the existing model of associating the fluorescence of bacteriorhodopsin with two or four lifetime components. An attempt is made to associate the five decay components with the emitting tryptophans of BR.     

FLUORESCENCE LIFETIME OF ACRIDINE ORANGE IN SODIUM DODECYL SULFATE PREMICELLAR SOLUTIONS

Abstract— The absorption and fluorescence spectra, and the fluorescence lifetime of acridine orange (AO) were measured in a wide range of the sodium dodecyl sulfate (SDS) concentration below and above the critical micelle concentration (cmc). The fluorescence consisted of two components with different lifetimes; short (<3 ns) and long (>3 ns). The short and long lifetime components are attributed to the AO monomer and dimer associated with detergent, respectively. The lifetime of the dimer increased with increasing the SDS concentration just below the cmc. It decreased suddenly to a constant value just above the cmc. The lifetime of the monomer showed only a slight increase in the concentration range of SDS employed.     

INVARIANT PROPERTIES OF ABSORPTION PROFILES IN SPORANGIOPHORES OF Phycomyces blakesleeanus UNDER BALANCING BILATERAL ILLUMINATION

Abstract— Absorption spectra and spectra of the refractive indices were separately measured for cytoplasm and vacuole of Phycomyces blakesleeanus in the wavelengths domains 300 to 750 nm and 250 to 750 nm respectively. These data were then used to simulate absorption profiles for different orientations of the photoreceptor for measured action spectra for phototropic balance. The photoreceptor was assumed to be a flavin. Using the reference wavelengths 394, 450 and 507 nm as an example it is shown that the absorption profiles have, independent of the photoreceptor orientation, invariant properties. Under bilateral balancing illumination the symmetry of the absorption profiles is conserved and the total amount of energy absorbed at proximal and distal sides is an invariant under a symmetry transformation front to the rear side.     

Altered flavin patterns in photobehavioral mutants of Phycomyces blakesleeanus.

Flavins were extracted from sporangiophores of the lower fungus Phycomyces blakesleeanus and identified by HPLC with fluorescence detection. In the wild-type strain NRRL1555 they were found to be present at the following concentrations: riboflavin (5.5 x 10(-6) M), flavin mononucleotide (FMN) (4.0 x 10(-6) M) and flavin adenine dinucleotide (1.4 x 10(-6) M). The HPLC elution profiles of the wild type were compared to a set of behavioral mutants (genotype mad) with specific defects in their light-transduction pathway. The photoreceptor mutants C109 (madB), C111 (madB) and L1 (madC) had normal amounts of flavins. The most prominent changes were found in single mutants with a defective madA gene which contained about 25% of riboflavin and about 10% of FMN and FAD normally found in the wild type. A hypertropic mutant with a defective madH gene contained instead 80% of riboflavin and 120% of FMN and FAD. The double mutant L52 (madA madC) and the triple mutant L72 (madA madB madC) had normal amounts of FAD and FMN. This indicates that the madC mutation, which itself causes loss of light sensitivity and which affects the near-UV/blue-light receptor (Galland and Lipson, 1985, Photochem. Photobiol. 41, 331-335) functions as a restorer of the flavin content in a genetic madA background. The double mutant L51 (madA madB) had about 40% of FMN and FAD, suggesting that the madB mutation functions as a partial restorer of flavin content. The photogravitropic thresholds (450 nm) reported for the wild type and the madA and madH mutants were positively correlated to the endogeneous concentrations of FMN and FAD.(ABSTRACT TRUNCATED AT 250 WORDS)     

Action Spectra for Photoinhibition of Sexual Development in Phycomyces blakesleeanus

The sexual development of the fungus Phycomyces is inhibited by light. The action spectra for this photoinhibitory effect were determined for 48 h continuous exposure between 350 and 700 nm wavelengths during the mating process. Effective wavelengths were shorter than 490 nm, but the most effective wavelengths depended on the stage of sexual development. In early stages of progametangium formation, the major peaks appeared near 360 nm with small shoulders at 410 nm, but in later stages, after gametangium formation, only single peaks were detected in the UVA range (350–390 nm). Low-fluence irradiation in the later stage, however, revealed inhibitory effectiveness at 370–410 nm, implying the existence of a dual photoresponse and multiple regulatiory systems in the mating process of Phycomyces.     

A FLUORESCENCE LIFETIME ASSAY OF A MEMBRANE-BOUND FLAVIN FROM CORN COLEOPTILES

Abstract— The fluorescence lifetime of a partially-purified membrane fraction containing flavin and b-type cytochrome, presumably on the same protein, has been described. Possible implications of the blue light photoreactivity of the flavin as an assay, on the basis of its short fluorescence lifetime (about 1.37 ns), has been discussed.     

Altered pterin patterns in photobehavioral mutants of Phycomyces blakesleeanus.

Pterins were extracted with methanol from sporangiophores of the lower fungus Phycomyces blakesleeanus and separated and identified by high performance liquid chromatography (HPLC) with fluorescence detection. The following pterins were found and identified for the wild-type strain NRRL1555: carboxypterin (6.7 x 10(-6) M), neopterin (4.2 x 10(-7) M), xanthopterin (5.3 x 10(-6) M), biopterin (3.9 x 10(-7) M), pterin (9.1 x 10(-7) M), and 6,7-dimethylpterin (1.2 x 10(-6) M). The HPLC elution profiles of the wild type were compared to a set of phototropism mutants (genotype mad) with specific defects in the light-transduction pathway. The mutant profiles were qualitatively similar to those of the wild type. Quantitative differences were, however, discerned for madA, madC, and madH mutants. The madA mutation was associated with increased amounts of biopterin and 6,7-dimethylpterin and a reduction of neopterin, pterin, xanthopterin, and unidentified pterins eluting at 14-18 min. The stimulatory effect of the madA mutation on biopterin and 6,7-dimethylpterin appears to be compensated by a secondary mutation (pde) which is responsible for the loss of 75% of adenosine 3',5'-cyclic monophosphate (cAMP)-phosphodiesterase activity. In a madA pde double mutant the amounts of biopterin and 6,7-dimethylpterin fell below the wild-type level. These results suggest that an increased level of endogenous cAMP represses the biosynthesis of these pterins. The madC mutation increased the amounts of biopterin and xanthopterin and that of the unidentified pterins which could be derivatized to carboxypterin. Single madB mutations had, compared to the wild type, two times higher amounts of biopterin and two times lower amounts of neopterin.(ABSTRACT TRUNCATED AT 250 WORDS)     

三元若丹明激光染料荧光寿命研究

利用单光子计数技术测试了新型三元若丹明激光染料在不同溶剂中的荧光寿命、荧光光谱及其寿命的实验数据.实验表明,所研究的三元若丹明染料存在着有效的分子内能量传递过程,这些过程使得激光染料的荧光量子效率及光稳定性明显改善.     

FLUORESCENCE LIFETIME STUDY OF THE DENATURATION OF RIBONUCLEASE-A

Abstract. Fluorescence quantum yield and lifetime measurements of the tyrosine residues in ribonuclease-A (RNase) were used to study the conformational changes involved in the denaturation of the enzyme. Measurements were done on RNase and on selectively acetylated RNase in the native, the partly denatured (reductive cleavage of S-S bridges or treatment with 8 M urea) and in the fully denatured state. The data were interpreted to mean that the opening of the S-S bridges causes large parts of the enzyme chain to unfold while leaving a hydrophobic region; including one of the tyrosine residues, intact. The biological activity of RNase is destroyed by this unfolding. Urea apparently does penetrate the protein coil but does not greatly affect the RNase structure since some of its biological activity is still retained. The opening of the S-S bridges in the presence of urea destroys the native conformation (and biological activity) completely leaving the protein in the form of an uncoiled polypeptide chain. It is suggested which parts of the protein structure might be affected by partial denaturation.     

胭脂红酸的荧光发射寿命及荧光猝灭

以蒸馏水中的糖原悬浮液为参照物，测定了胭脂红酸的荧光发射寿命，τ＝０．０９３±０．０１０ｎｓ．研究了ＯＨ－和Ｉ－对胭脂红酸荧光的猝灭，后者遵守Ｓｔｅｒｎ－Ｖｏｌｍｅｒ方程，其猝灭常数ＫＱ＝１．２７±０．０９Ｌ·ｍｏｌ￣（－１），前者则不是遵守此方程的简单荧光猝灭，这可能是由于胭脂红酸中酚基的存在降低了最低激发态能量的原因。     

ACTION SPECTRA FOR PHOTOTROPIC BALANCE IN Phycomyces blakesleeanus: DEPENDENCE ON REFERENCE WAVELENGTH and INTENSITY RANGE

Abstract— Action spectra for phototropic balance of Phycomyces blakesleeanus sporangiophores were measured for various reference wavelengths and intensity ranges. Balance action spectra were made at fluence rates of 10^-4 W m^-2 with reference wavelengths of 450 nm, 394 nm, 507 nm, and broadband blue light. For broad-blue light and 450 nm light as references, typical flavin-like action spectra were found with a ma jor peak at 455 nm, a secondary peak at 477 nm, and a minor peak at 383 nm; these peaks are wider for broad blue than for 450 nm light. With the 394 nm reference, there is a major peak at 455 nm, a secondary peak at 477 nm and a minor peak at 394 nm. An action spectrum with 507 nm reference has a major peak at 455 nm and a minor peak at 383 nm, but no peak at 477 nm. A balance action spectrum was made with 450 nm reference light near threshold intensity (2 times 10^-8 W m^-2); there, the 386 nm peak is greatly reduced, while the 455 nm peak is enhanced. The intensity dependence of the 386 nm peak was studied in detail for reference light of 450 nm. We found that the relative quantum efficiency of the 386 nm light increases with the logarithm of the 450 nm fluence rate; in the high intensity range (0.3 W m^-2) the relative quantum efficiency of the 386 nm light is 1.3 and approaches zero at 10^-9 W m^-2. These findings indicate that P. blakesleeanus phototropism is mediated by multiple interacting pigments or by a photochromic photoreceptor.     

FLUORESCENCE SPECTRA IN LUNG WITH PORPHYRIN INJECTION

The fluorescence emission spectra from human bronchial mucosa and tumors, before and after injection of dihematoporphyrin ether/ester, have been measured with an optical multichannel analyzer from 500 to 750 nm. Fluorescence was excited with a violet krypton ion laser (average wavelength 410 nm). The autofluorescence spectra decrease monotonically with increasing wavelength except for a small broad peak near 600 nm. The spectra from tumor sites, after injection of the fluorescent porphyrin, exhibit the characteristic fluorescence emission at 630 and 690 nm, added to the autofluorescence spectrum. The spectra from control or nontumor sites are similar but the magnitude of the component due to the injected porphyrin is smaller than at a tumor site. The magnitude ratio of tumor to control site fluorescence depends on concentration of the porphyrin, tumor thickness, and time after injection. Autofluorescence degrades contrast and thus makes very thin tumors difficult to image. Subtraction of the autofluorescence background is desirable.     

新型双发色团染料荧光光谱及其寿命的研究

有效的染料激光操作需要较高的荧光量子效率,若丹明是在500~700 nm光谱区中一类最重要的激光染料.然而,染料的基态分子和三线态对辐射能量的吸收将会大大降低激光输出效率,再者,由于若丹明类染料在紫外区的吸收系数较小,为了有效吸收泵浦能量(如用XeCI准分子激光,308 nm),就必须使用高浓度染料溶液,在这种情况下,若丹明类染料较小的Stokes位移就势必造成基态分子更大的重复吸收,即造成更大的谐振腔损耗^[1].     

锌酞菁类染料荧光寿命的非瞬态实验估算

合成了一系列的锌酞菁染料。用循环伏安法测定了它们的氧化还原电位, 并测定了用典型的电子受体猝灭它们的荧光动力学数据。按电子转移模式确定了各染料-电子受体对的荧光猝灭速率常数κ_q对电子从染料向受体转移的自由能变化⊿G函数关系。根据测得的氧化还原电位和光谱数据计算出⊿G, 根据函数关系计算出κ_q, 代入到猝灭动力学数据κ_qτ。这样,就用非瞬态的实验估算出染料的荧光寿命r。与已知的实测文献值进行比较, r的大致范围是可信的。