Oxidation-reduction potential dependence of the interaction of cytochromes,bacteriochlorophyll and carotenoids at 77°K in chromatophores of Chromatium D and Rhodopseudomonas gelatinosa

1. A technique for assay of light-induced reactions at 77°K as a function of oxidation-reduction potential has been developed.     

Genetic diversity and genetic relationship of Caragana microphylla, Caragana davazamcii and Caragana korshinskii on the Inner Mongolia Plateau, China

C. microphylla, C. davazamcii and C. korshinskii exhibit a geographical replacement series from east to west on the Inner Mongolia Plateau. Currently, there is still a debate about the taxonomic and genetic relationship among these 3 species. We studied the genetic diversity and genetic relationship among these 3 species by analyzing DNA samples of individual plants from within 10 populations with random amplified polymorphic DNA (RAPD) markers. We identified 678 RAPD loci in total, of which all were polymorphic (PPB = 100%). There were 41 unique loci (6.05%). In general, a trend presented that the genetic diversity of these species decreased from east to west. Further, the genetic diversity was significantly negatively correlated with the local annual mean temperature. Analysis of molecular variation (AMOVA) showed that the genetic variation among these 3 species was only 6.08% of the total genetic variation. Between the species, the genetic variation was insignificant (P = 0.9961). The proportion of genetic variation among populations within each species was 11.90% (P < 0.001) of the total genetic variation, and the total genetic variation mainly existed within the populations (82.02%). Estimated with Shannon's index, genetic differentiation within the populations (Hpop/Hsp) was 0.8013, the coefficient of gene differentiation (Gst) was 0.1603, and the gene flow index (Nm) was 2.6192. This, thus, indicates that there is relatively high gene flow among these populations, and that these 3 species are crossbreeding. The genetic diversity level and the population distribution pattern showed geographic continuity to some extent.     

Electron spin resonance study of the role of nitrosyl-heme in the activation of guanylate cyclase by nitrosoguanidine and related agonists.

One major component of lens plasma membrane is a glycoprotein that SDS-polyacrylamide gel electrophoresis shows to possess an apparent molecular weight of 26,000. When this protein is solubilized in low ionic strength buffers containing SDS, and heated to 100° for 1 to 3 min prior to electrophoresis, conversion into high molecular weight aggregate results. The heat lability of this protein is greatly enhanced if it solubilized and heated in buffers containing 0.1 M NaCl. At this ionic strength, incubation for 3 h at 38° results in conversion of 20% of the protein into high melecular weight aggregates. Most other membrane proteins isolated from lens membrane are insensitive to heat treatment. It is concluded that temperature and ionic strength must be recorded and controlled carefully when using SDS-polyacrylamide gel electrophoresis to study this membrane protein.     

Theoretical analysis of the molecular motion of spin labels in membranes. ESR spectra of labeled Bacillus subtilis membranes

Electron spin resonance (ESR) spectral line shapes are calculated for a nitroxide radical undergoing rapid motion within a cone. The analysis is correct to second order, and explicit expression are derived for the hyperfine splittings and g-values by averaging both the secular and pseudosecular terms within the Hamiltonian. The simulated spectra are found to closely resemble those observed experimentally over a wide range of temperatures for stearic acid spin labels in cytoplasmic membranes of Bacillus subtilis. The present approach offers a simple, yet realistic way of interpreting spectra of nitroxide spin labels such as fatty acids and steroids when the motion is anisotropic.     

Relationship of occurrence of Fruhstorferiola tonkinensis Will. and plant communities on riversides in northern Guangdong Province, China

Plant communities on riversides in northern Guangdong Province were investigated with all-survey method, where a kind of native grasshopper Fruhstorferiola tonkinensis Will. broke out severely. 108 plant species belonging to 43 families were identified, and some plant indexes in those plant communities including their name, average height, congregation index, total coverage rate, phenological phase and biod were noted. In addition, plant comparability, plant abundance, quantity character and attribute symbol of dominant species in plant communities on riversides were studied. The results showed that the arrangement and distribution of vegetations on riversides offered a favorable habitat for F. tonkinensis. Moreover, the density of first instar nymphs of F. tonkinensis in plant communities was calculated. The results of partial correlation analysis (PCA) about the relationship between plant communities and occurrence of F. tonkinensis showed that Pterocarya stenoptera C. DC. was the most palatable plant to F. tonkinensis, presenting a very significantly positive correlation between coverage rate of P. stenoptera and occurrence of F. tonkinensis. The partial correlation coefficient (PCC) was 0.856072 (F=32.92012, P=0.0001). Bambusa flexuosa was a non-preferable plant of F. tonkinensis, presenting a significantly negative correlation between them, and the partial correlation coefficient was −0.607920 (F=0.16173, P=0.0211). Those results would help us to propose some approaches for the ecology control of F. tonkinensis on riversides in northern Guangdong Province.     

Electron paramagnetic resonance study of the oxidation of N-methyl-substituted aromatic amines catalyzed by hemeproteins

The oxidation of N-mono- and dimethyl-substituted toluidines and aniline by H₂O₂, catalyzed by horseradish peroxidase or metmyoglobin, produces organic free radicals, detectable by electron paramagnetic resonance spectroscopy at room temperature. The radical cation of N,N-dimethyl-p-toluidine was conclusively identified, but the other resolved EPR signals were assigned to radical cations of radical dimerization products, e.g., N,N,N′,N′-tetramethylbenzidine formed from N,N-dimethylaniline. The N-demethylase activities of metmyoglobin were found to be uniformly smaller than those of horseradish peroxidase, consistent with the much faster reaction of the latter hemeprotein with H₂O₂. Detection of the monomeric radical cation of N,N-demethyl-p-toluidine correlated with the largest rate of N-demethylation among this class of compounds. These findings emphasize the importance of radical stability (provided, for example, by the para methyl substituent) on subsequent competing reactions of the radical cation of the N-methyl substrate, i.e., one-electron oxidation leading to formaldehyde release or radical dimerization, which becomes more probable for the less stable radical intermediates. Attempts were made to correlate these results with data obtained for the $\text{O}{}_2\text{NADPH}\text{-supported}$ oxidation of these same substrates by liver microsomal cytochrome P-450. However, pronounced differences in physical state and kinetic properties of this heterogeneous, membrane-associated microsomal hemeprotein and the soluble “model” hemeprotein systems precluded firm conclusions concerning a radical mechanism of N-demethylation monooxygenase activities of microsomal fractions.     

Resolved difference spectra of redox centers involved in photosynthetic electron flow in Rhodopseudomonas capsulata and rhodopseudomonas sphaeroides

1. In Rhodopseudomonas sphaeroides the Q_x absorption band of the reaction center bacteriochlorophyll dimer which bleaches on photo-oxidation is both blue-shifted and has an increased extinction coefficient on solubilisation of the chromatophore membrane with lauryldimethylamine-N-oxide. These effects may be attributable in part to the particle flattening effect.2. The difference spectrum of photo-oxidisable c type cytochrome in the chromatophore was found to have a slightly variable peak position in the α-band (λ_max at 551–551.25 nm); this position was always red-shifted in comparison to that of isolated cytochrome c₂ (λ_max at 549.5 ± 0.5 nm). The shift in wavelength maximum was not due to association with the reaction center protein. A possible heterogeneity in the c-type cytochromes of chromatophores is discussed.3. Flash-induced difference spectra attributed to cytochrome b were resolved at several different redox potentials and in the presence and absence of antimycin. Under most conditions, one major component, cytochrome b₅₀ appeared to be involved. However, in some circumstances, reduction of a component with the spectral characteristics of cytochrome b_?90 was observed.4. Difference spectra attributed to (BChl)₂, Q^?_II, c type cytochrome and cytochrome b₅₀ were resolved in the Soret region for Rhodopseudomonas capsulata.5. A computer-linked kinetic spectrophotometer for obtaining automatically the difference spectra of components functioning in photosynthetic electron transfer chains is described. The system incorporates a novel method for automatically adjusting and holding the photomultiplier supply voltage.     

Inactivation of the serine proteinase operon (proMCD) of Staphylococcus warneri M: Serine proteinase and cysteine proteases are involved in the autolysis

Unlike other members of coagulase negative staphylococci (CNS), strain warneri has proMCD operon, a homologue of sspABC proteinase operon of S. aureus. The proM and proC encode serine glutamyl endopeptidase and cysteine protease respectively, whereas proD directs homologue of SspC, putative cytoplasmic inhibitor which protects the host bacterium from premature activation of SspB. We determined whole nucleotide sequence of proMCD operon of S. warneri M, succeeded in expression of these genes, and investigated their functions by gene inactivation and complementation experiments. In gelatin zymography of the culture supernatant, a 20-kDa band corresponding to PROC cysteine protease was detected. By Western blotting, PROD was also confirmed in the cytoplasmic protein fraction. PROC and PROD showed significant similarity to SspB and SspC of S. aureus (73% and 58%, respectively). Inactivation mutants of proMCD, proCD and proD genes were established, separately. In the proMCD mutant, degradation/processing of extracellular proteins was drastically reduced, suggesting that PROM was responsible for the cleavage of extracellular proteins. By the proD mutation, the growth profile was not affected, and secretion of PROC was retained. Extracellular protein profiles of the proCD and proD mutants were not so different each other, but autolysin profiles were slightly dissimilar, around 39–48 kDa and 20 kDa bands in zymogram. Experiments in buffer systems showed that autolysis was significantly diminished in proMCD mutant, and was promoted by addition of purified PROM. The proC gene was cloned into a multicopy plasmid, and introduced into the proMCD mutant. Compared with the wild type, autolysis of the proC-complemented strain was definitely enhanced by addition of purified PROM. These results suggested that PROM and PROC affected the coccal autolysis, through processing of the autolysin.     

Storage-excretion of metallic cations in the adult housefly, Musca domestica

The objective of this investigation was to elucidate further the phenomenon of storage-excretion of metallic cations in the housefly. The sites for deposition of zinc, calcium and copper have been identified in this study. The metal intake of the flies was altered by raising one group on sucrose and tap water while in the experimental groups sucrose was supplemented with either 0.05% zinc sulphate, 0.05% calcium phosphate or 0.03% copper sulphate. There were no significant differences in the average life spans of the flies in different groups indicating physiological tolerance to the higher mineral intake. The Malpighian tubules, the midgut and the remainder of the body were analyzed for mineral content in houseflies ranging from 1 to 25 days posteclosion by atomic absorption spectrophotometry. There was a progressive age-associated increase in the total metal content of the flies with age. Zinc and calcium were primarily stored in the Malpighian tubules whereas copper was sequestered in the midgut. Microscopic examination of the epithelial cells of the Malpighian tubules and the midgut revealed a corresponding age-associated increase in mineralized concretions.     

Both chlorophylls a and d are essential for the photochemistry in photosystem II of the cyanobacteria, Acaryochloris marina

We have measured the flash-induced absorbance difference spectrum attributed to the formation of the secondary radical pair, P⁺Q⁻, between 270 nm and 1000 nm at 77 K in photosystem II of the chlorophyll d containing cyanobacterium, Acaryochloris marina. Despite the high level of chlorophyll d present, the flash-induced absorption difference spectrum of an approximately 2 ms decay component shows a number of features which are typical of the difference spectrum seen in oxygenic photosynthetic organisms containing no chlorophyll d. The spectral shape in the near-UV indicates that a plastoquinone is the secondary acceptor molecule (Q_A). The strong C-550 change at 543 nm confirms previous reports that pheophytin a is the primary electron acceptor. The bleach at 435 nm and increase in absorption at 820 nm indicates that the positive charge is stabilized on a chlorophyll a molecule. In addition a strong electrochromic band shift, centred at 723 nm, has been observed. It is assigned to a shift of the Qy band of the neighbouring accessory chlorophyll d, Chl_D1. It seems highly likely that it accepts excitation energy from the chlorophyll d containing antenna. We therefore propose that primary charge separation is initiated from this chlorophyll d molecule and functions as the primary electron donor. Despite its lower excited state energy (0.1 V less), as compared to chlorophyll a, this chlorophyll d molecule is capable of driving the plastoquinone oxidoreductase activity of photosystem II. However, chlorophyll a is used to stabilize the positive charge and ultimately to drive water oxidation.     

Molecular cloning,characterization and expression analysis of ATG1 in the silkworm, Bombyx mori

Atg1 is a Serine/Threonine protein kinase that plays a pivotal role in autophagy. A complete coding sequence of ATG1 is not available for the silkworm, Bombyx mori which is a good model for studying the autophagic process.     

Endocrine events during pre-diapause and non-diapause larval-pupal development of the tobacco hornworm, Manduca sexta

The haemolymph ecdysteroid titre and in vitro capacities of prothoracic glands and corpora allata to synthesize ecdysone and juvenile hormone, respectively, during the last-larval instar of diapause-destined (short-day) and non-diapause-destined (long-day) Manduca sexta were investigated. In general, the ecdysteroid titres for both populations of larvae were the same and exhibited the two peaks characteristic of the haemolymph titre during this developmental stage in Manduca. The only difference in the titre occurred between day 7 plus 12 h and day 7 plus 20 h, when the short-day larval titre did not decrease as quickly as the long-day titre. The in vitro synthesis of ecdysone by prothoracic glands of short- and long-day larvae during the pharate pupal phase of the instar were also essentially the same. Activity fluctuated at times which would support the idea that ecdysone synthesis by the glands is a major contributing factor to the changes in the haemolymph ecdysteroid titre. There was one subtle difference in prothoracic gland activity between the two populations, occurring on day 7 plus 2 h. By day 7 plus 10 h, however, rates of ecdysone synthesis by the short- and long-day glands were comparable. This elevated activity of the short-day glands occurred just prior to the period the haemolymph ecdysteroid titre remained elevated in these larvae. The capacities of corpora allata to synthesize juvenile hormone I and III in vitro were not markedly different in long- and short-day last-instar larvae. At the time of prothoracicotropic hormone release in the early pupa, activity of corpora allata from short- and long-day reared animals was low and also essentially the same. There were a few differences in the levels of synthesis at isolated times, but they were not consistent for both homologues. Overall, there are no compelling differences in the fluctuations of ecdysteroids and juvenile hormones between diapause-destined and non-diapause-destined Manduca larvae. Since these hormones do not appear to play any obviously significant role in the induction of pupal diapause in this insect, the photoperiodic induction of diapause in Manduca appears to be a predominantly brain-centred phenomenon not involving endocrine effectors.     

The ultrastructure of the female accessory gland,the cement gland,in the insect Rhodnius prolixus

The cement gland of Rhodnius prolixus is an epidermally derived tubular gland consisting of a distal synthetic region and a proximal muscular duct region. The synthetic region consists of numerous secretory units joined to a central chitinous duct via cuticular ductules. Proteinaceous secretion, synthesized by the goblet-shaped secretory cell, passes through the delicate cuticular lattice of a ductule-end apparatus and out through fine ductules to the central duct. Secretory cells are rich in rough endoplasmic reticulum and mitochondria. Light microscopy, SEM and TEM reveal the delicate lattice-like end apparatus structure, its formation and relationship to the secretory cell. The secretory cell associates via septate junctions with a tubular ductule cell that encloses a cuticle-lined ductule by forming an elaborate septate junction with itself. The ductules are continuous with the cuticle lining of the large central duct that conveys secretion to the proximal area. The proximal muscular duct has a corrugated cuticular lining, a thin epithelium rich in microtubules and thick longitudinal, striated muscles which contract during oviposition, forcing the secretion out. Histochemistry and electrophoresis reveal the secretion as proteinaceous.     

Electron spin resonance study of the role of vitamin E and vitamin C in the inhibition of fatty acid oxidation in a model membrane

The neutral α-tocopheroxyl radicals, generated in monolayers on silica gel containing α-tocopherol and partly autoxidised methyl linoleate at 90°C, were detected and identified by ESR spectroscopy. Addition of ascorbic acid to the monolayer resulted in the complete quenching of the α-tocopheroxyl radical spectrum. This lends support to the view that ascorbate transfers hydrogen to α-tocopheroxyl radicals thus regenerating α-tocopherol.