Destruction of photosystem I iron-sulfur centers of spinach andAnacystis nidulans by mercurials

Several mercurials destroyed Photosystem I (PSI) Fe−S centers in thylakoids and PSI particles from spinach and fromAnacystis nidulans as revealed by EPR measurement and acid-labile sulfide determination. Of the mercurials tested, HgCl₂ was the most effective, followed by phenylmercuric acetate (PMA), Mersalyl and pCMB in the order of decreasing effectiveness. Fe−S centers in thylakoids were much more labile than those in PSI particles. InA. nidulans thylakoids, Center B was more susceptible than Center A and X to PMA. P700 was less susceptible to PMA than these centers. For 50% inactivation of Fe−S centers inA. nidulans thylakoids, about 0.4 mM PMA was required for Center B, and about 1 mM was required for Center A and X. These differential susceptibilities of Fe−S centers were more pronounced with HgCl₂ than with the other three mercurials.     

Interaction of the protein components of 5-oxoprolinase. Substrate-dependent enzyme complex formation

5-Oxo-L-prolinase from Pseudomonas putida is composed of two reversibly dissociable proteins: Component A catalyzes 5-oxoproline-dependent cleavage of ATP, but does not catalyze the decyclization of 5-oxoproline; Component B is required for the coupling of ATP cleavage to ring-opening of 5-oxoproline to form glutamate (Seddon, A. P., Li, L., and Meister, A. (1984) J. Biol. Chem. 259, 8091-8094). We describe here the purifications of Components A and B to apparent homogeneity and the interactions between these two proteins. The cellular content of Component B activity is significantly greater than that of Component A. By gel filtration, Component A is a hexamer; but in the presence of substrates, it is a dimer. Component B can exist as an aggregate, an octamer, or a tetramer, depending upon the conditions used. Gel filtration of a mixture of Components A and B in the presence of substrates gives a unique protein species that exhibits 5-oxoprolinase activity. The Mr of this Component A-Component B complex indicates that it probably has an A2-B2 structure. The molar ratio of Component A to Component B in the complex was determined to be 1:1 by the continuous variation method (Job). Titrations of each component by the other suggest that phosphorylated 5-oxoproline-bound Component A is the entity that interacts with Component B. These studies indicate that the binding of phosphorylated 5-oxoproline-bound Component A to Component B to form a complex proceeds by a cooperative type mechanism. This is supported by the observed shifts of the intersection points of the Job curves (see Appendix).     

Electron spin resonance studies of the bound iron-sulfur centers in Photosystem I. Photoreduction of center A occurs in the absence of center B

The Photosystem I acceptor system of a subchloroplast particle from spinach was investigated by optical and electron spin resonance (ESR) spectroscopy following graduated inactivation of the bound iron-sulfur proteins by urea/ferricyanide solution. The chemical analysis of iron and sulfur and the ESR properties of centers A, B and X are consistent with the participation of three iron-sulfur centers in Photosystem I. A differential decrease in centers A, B and X is observed under conditions that induce S^2? →S⁰ conversion in the bound iron-sulfur proteins. Center B is shown to be the most susceptible, while center ‘X’ is the least susceptible component to oxidative denaturation. Stepwise inactivation experiments suggest that electron transport in Photosystem I does not occur sequentially from X→B→A, since there is quantitative photoreduction of center A in the absence of center B. We propose that center A is directly reduced by X; thus, X may serve as a branch point for parallel electron flow through centers A and B.     

Complete amino acid sequence of theGlycera dibranchiata monomer hemoglobin Component IV: Structural implications

The globin derived from the monomer Component IV hemoglobin of the marine annelid,Glycera dibranchiata, has been completely sequenced, and the resulting information has been used to create a structural model of the protein. The most important result is that the consensus sequence of Component IV differs by 3 amino acids from a cDNA-predicted amino acid sequence thought earlier to encode the Component IV hemoglobin. This work reveals that the histidine (E7), typical of most heme-containing globins, is replaced by leucine in Component IV. Also significant is that this sequence is not identical to any of the previously reportedGlycera dibranchiata monomer hemoglobin sequences, including the sequence from a previously reported crystal structure, but has high identity to all. A three-dimensional structual model for monomer Component IV hemoglobin was constructed using the published 1.5 å crystal structure of a monomer hemoglobin fromGlycera dibranchiata as a template. The model shows several interesting features: (1) a Phe31 (B10) that is positioned in the active site; (2) a His39 occurs in an interhelical region occupied by Pro in 98.2% of reported globin sequences; and (3) a Met41 is found at a position that emerges from this work as a previously unrecognized heme contact.Abbreviations used GMHX the holo-protein (including b-type heme, Glycera dibranchiata monomer hemoglobin Component X (X=2, 3, or 4) - GMGX the apo-protein, or globin, Glycera dibranchiata monomer globin derived from Component X (X=2, 3, or 4) - rec-gmg the globin derived from a recombinant holoprotein of a Glycera dibranchiata monomer hemoglobin, rec-gmh, whose sequence has been inferred from an isolated cDNA insert - CB label refers to peptides generated from cyanogen bromide cleavage of GMG4 - HPLC high-performance liquid chromatography - T label refers to peptides generated from trypsin digests of GMG4 - Mb myoglobin - MCS monomer hemoglobin crystal structure from Glycera dibranchiata. H, N-terminal sequence of GMG4 - SWMb sperm whale myoglobin     

Complete amino acid sequence of theGlycera dibranchiata monomer hemoglobin Component IV: Structural implications

The globin derived from the monomer Component IV hemoglobin of the marine annelid,Glycera dibranchiata, has been completely sequenced, and the resulting information has been used to create a structural model of the protein. The most important result is that the consensus sequence of Component IV differs by 3 amino acids from a cDNA-predicted amino acid sequence thought earlier to encode the Component IV hemoglobin. This work reveals that the histidine (E7), typical of most heme-containing globins, is replaced by leucine in Component IV. Also significant is that this sequence is not identical to any of the previously reportedGlycera dibranchiata monomer hemoglobin sequences, including the sequence from a previously reported crystal structure, but has high identity to all. A three-dimensional structual model for monomer Component IV hemoglobin was constructed using the published 1.5 å crystal structure of a monomer hemoglobin fromGlycera dibranchiata as a template. The model shows several interesting features: (1) a Phe31 (B10) that is positioned in the active site; (2) a His39 occurs in an interhelical region occupied by Pro in 98.2% of reported globin sequences; and (3) a Met41 is found at a position that emerges from this work as a previously unrecognized heme contact.     

Trapping of an intermediate in the reaction catalyzed by 5-oxoprolinase

Bacterial 5-oxoprolinase is composed of two protein components: Component A, which catalyzes 5-oxoproline-dependent ATP-hydrolysis and Component B, which couples the hydrolysis of ATP with the decyclization of 5-oxoproline to form glutamate (Seddon, A. P., Li, L., and Meister, A. (1984) J. Biol. Chem. 259, 8091-8094). Studies on this unusual enzyme system have led to evidence that an intermediate is formed by Component A. Application of the isotope-trapping method demonstrated an activated 5-oxoproline intermediate, whose formation requires ATP, Mg2+, and Component A. The amount of ATP-dependent trapping was close to the number of enzyme active sites. The intermediate formed by Component A was shown to be reducible by potassium borohydride to proline in low yield; when Component B was added, the formation of proline was abolished. Treatment of reaction mixtures containing Component A, 5-oxoproline, and [gamma-32P] ATP with diazomethane led to appearance of a 32P-labeled compound (found on thin layer chromatography), whose formation was significantly reduced when Component B was present. The new compound, which is labile, breaks down to form dimethyl[32P]phosphate. The total amount of dimethyl[32P]phosphate formed after breakdown is close to the number of active sites of Component A. The data are consistent with the conclusion that a phosphorylated form of 5-oxoproline is formed by Component A and suggest that Component B is required for conversion of this intermediate to glutamate.     

Genomic Relatedness of Xanthomonas campestris Strains Causing Diseases of Citrus

Xanthomonas campestris strains that cause disease in citrus were compared by restriction endonuclease analysis of DNA fragments separated by pulsed-field gel electrophoresis and by DNA reassociation. Strains of X. campestris pv. citrumelo, which cause citrus bacterial spot, were, on average, 88% related to each other by DNA reassociation, although these strains exhibited diverse restriction digest patterns. In contrast, strains of X. campestris pv. citri groups A and B, which cause canker A and canker B, respectively, had relatively homogeneous restriction digest patterns. The groups of strains causing these three different citrus diseases were examined by DNA reassociation and were found to be from 55 to 63% related to one another. Several pathovars of X. campestris, previously shown to cause weakly aggressive symptoms on citrus, ranged from 83 to 90% similar to X. campestris pv. citrumelo by DNA reassociation. The type strain of X. campestris pv. campestris ranged from 30 to 40% similar in DNA reassociation experiments to strains of X. campestris pv. citrumelo and X. campestris pv. citri groups A and B. Whereas DNA reassociation quantified the difference between relatively unrelated groups of bacterial strains, restriction endonuclease analysis distinguished between closely related strains.     

Discovery of Streptococcus pneumoniae Serotype 6 Variants with Glycosyltransferases Synthesizing Two Differing Repeating Units

Streptococcus pneumoniae is a persistent, opportunistic commensal of the human nasopharynx and is the leading cause of community-acquired pneumonia. It expresses an anti-phagocytic capsular polysaccharide (PS). Genetic variation of the capsular PS synthesis (cps) locus is the molecular basis for structural and antigenic heterogeneity of capsule types (serotypes). Serogroup 6 has four known members (6A–6D) with distinct serologic properties, homologous cps loci, and structurally similar PSs. cps of serotypes 6A/6B have wciNα, encoding α-1,3-galactosyltransferase, whereas serotypes 6C/6D have wciNβ encoding α-1,3-glucosyltransferase. Two atypical serogroup 6 isolates (named 6X11 and 6X12) have been discovered recently in Germany. Flow cytometric studies using monoclonal antibodies show that 6X11 has serologic properties of 6B/6D, whereas 6X12 has 6A/6C. NMR studies of their capsular PSs revealed that 6X11 and 6X12 have two different repeating units with a distribution of ∼40:60 6B:6D and 75:25 6A:6C PS, respectively. Sequencing of the wciNα gene in 6X12 and 6X11 revealed single and double nucleotide substitutions, respectively, resulting in the amino acid changes A150T and D38N. Substitution of alanine with threonine at position 150 in a 6A strain was associated with hybrid serologic and chemical profiles like 6X12. The hybrid serotypes represented by 6X12 and 6X11 strains are now named serotypes 6F and 6G. Single amino acid changes in cps genes encoding glycosyltransferases can alter substrate specificities, permit biosynthesis of heterogeneous capsule repeating units, and result in new hybrid capsule types that may differ in their interaction with the immune system of the host.     

A note on the presence of B chromosome in the small Japanese field mouse,Apodemus argenteus,in central Honshu,Japan

Previously, many studies have revealed the presence of B chromosomes in wild mouse taxa of the genus Apodemus (Rodentia, Muridae). In one of the Apodemus species, A. argenteus, which is endemic to Japan, it is known that B chromosomes were confirmed only in individuals (2n = 46 + B chromosome) from Hokkaido, Japan. There is no report of the presence of B chromosomes from other localities in the Japanese Islands. In this study, we analyzed the chromosomal constitutions of 43 individuals of A. argenteus from three localities in Honshu, Japan. A total of three individuals from central Honshu showed 2n = 47, and each individual carried a dot-like B chromosome. In addition, these B chromosome features were analyzed by differential staining methods, and the C- and QM-banding patterns of the B chromosomes were identical to those of the X chromosomal heterochromatic region showing the delayed-fluorescent response. Thus, it is considered that these B chromosomes would be derived from the heterochromatin of the X chromosomes, as reported in previously published papers.     

The Gillette Site (39ST23), Oahe Reservoir,South Dakota

Abstract

Fieldwork at the Gillette Site primarily cons is ted of the partial excavation ofthreecircular houses, across section of a fortification ditch, and the removal of a buriaL The latest occupation, Component A, is identified as a manifestation of the Stanley or Snake Butte focus, dating from about 1700 to 1800 A. D. Component B represents a circular house tradition probably belonging to an earlier period in the Coalescent Tradition than does Component A. The limited data from Component C implies a village occupation of an indeterminate cultural affiliation earlier than Component B.     

Host defense in cryptococcosis. II. Cryptococcosis in the nude mouse.

In the homozygous state, mice carrying the “nude” (nu) gene are hairless (nude), lack a thymus and have profound deficiency of cell-mediated immunity. Cryptococcosis was studied in BALB/c and Swiss mice, each strain carrying the nu gene. The purpose was to determine the interactions of the nu gene and mouse strain in terms of susceptibility to Cryptococcosis. Mice of both strains could be sensitized to produce delayed-type hypersensitivity reactions to cryptococcal extract in the heterozygous nu/X state, but not in the nu/nu state. Nu/X Swiss mice were more resistant than nu/X BALB/c mice to infection with a highly virulent strain (B) of Cryptococcus neoformans. However, nu/nu BALB/c and nu/nu Swiss mice were both highly susceptible to the same microorganism. Challenge with another cryptococcal strain (A) of much lower virulence for nu/X mice killed 100% of BALB/c and Swiss nu/nu mice. These studies indicate that thymus-dependent immune functions are critical determinants of host resistance to murine Cryptococcosis.     

Morphological Variation among 23 Xiphinema americanum Populations

Morphometrics of 23 United States populations of Xiphinema americanum sensu lato, sharing the characteristics of an offset lip region and conoid tail, were examined and analyzed statistically by canonical discriminant analysis (CDA). Specimens were collected from Arkansas, Georgia, Tennessee, Mississippi, Florida, Oklahoma, California, and North Dakota. Eleven measurements and body ratios obtained from female specimens were used in the analysis. Xiphinema americanum, X. bricolensis, X. californicum, X. citricolum, X, intermedium, X. tarjanense, and X. thornei, and one undescribed species were identified among the 23 populations. Three groups -- X. americanum-group, X. californicum-group, and X. intermedium-group (X. intermedium and X. tarjanense) -- were formed and four populations belonging to four different species were separated consistently from these groups in CDA scatterplots of the 23 populations. Composition of the groups was somewhat related to the geographical origins of the populations in the groups. A population from California had morphometrics intermediate between X. americanum and X. californicum. Separation between the X. americanum-group and X. californicum-group in the CDA scatterplots was not as distinct as that between them and the X. intermedium-group or between any of the three groups and the four single outlying populations.     

Mapping of recognition sites for the restriction endonuclease from Escherichia coli K12 on bacteriophage PM2 DNA.

Several mutations in gene B of phage S13 appear to shorten the B protein by elimination of an N-terminal fragment, without destroying the B protein function. The shortened B protein resulting from each of these mutations can block the unique DNA-nicking properties of the S13 gene A protein. Because of the block in gene A function, normal gene B protein may have a function in phage DNA synthesis in addition to its known role in catalyzing capsid assembly.From gel electrophoresis the mutant B protein is estimated to be shorter than the normal S13 B protein by 1720 ± 70 daltons and is therefore believed to be an internal reinitiation fragment. The reinitiated fragments are functional and are made in about twice the amount of the normal B protein.The phage mutants which yield the reinitiation fragments are double mutants, each phage containing the same gene B nonsense mutation and each appearing to contain a different compensating gene B mutation. Various data support the assumption that the compensating mutations are frame-shifts, including the fact that suppression does not restore the normal-sized B protein. The reinitiation is assumed to occur at a pre-existing out-of-phase initiator codon, near the nonsense triplet; the correct reading frame would then be restored by each of the several different compensating mutations.The position of the normal S13 B protein in the gel electrophoresis pattern has been located both by elimination and shifting of the B peak, using appropriate amber mutants. The molecular weight of the S13 B protein is about 17,200, and is 2100 daltons less than the B protein of phage φX174; the S13 B protein can nevertheless substitute for the φX 174 B protein. Thus substantial portions of the B protein can be deleted without destroying its function.     

A Rhodobacter sphaeroides puf L, M and X deletion mutant and its complementation in trans with a 5.3 kb puf operon shuttle fragment

A Rhodobacter sphaeroides puf L, M and X deletion mutant was constructed using interposon mutagenesis. The puf L, M and X genes were replaced with a kanamycin resistance cartridge isolated from the transposon Tn5. The deletion strain PUFLMX 21 did not grow photoheterotrophically and was resistant to kanamycin. Southern blot analysis of genomic DNA from the deletion strain confirmed that the kanamycin resistance was inserted specifically into the puf operon and that the L, M and X genes were deleted. A spontaneous carotenoid mutant of PUFLMX was selected and was found to accumulate primarily neurosporene. Spectroscopic analysis of chromatophores isolated from the deletion strain showed normal B875 and B800-850 expression providing further evidence that the photosynthetic minus phenotype was not the result of insertional inactivation of the promoter region of the puf operon, or the puf Q region. The deletion strain could be returned to the photosynthetic plus phenotype by complementation in trans with a 5.3 kb puf operon shuttle fragment, although the generation time of the complemented strain was 30% longer than wild type.     

Electron-spin resonance studies of the bound iron-sulfur centers in Photosystem I II. Correlation of P-700 triplet production with urea/ferricyanide inactivation of the iron-sulfur clusters

Photosystem I charge separation in a subchloroplast particle isolated from spinach was investigated by electron spin resonance (ESR) spectroscopy following graduated inactivation of the bound iron-sulfur centers by urea-ferricyanide treatment. Previous work demonstrated a differential decrease in iron-sulfur centers A, B and X which indicated that center X serves as a branch point for parallel electron flow through centers A and B (Golbeck, J.H. and Warden, J.T. (1982) Biochim. Biophys. Acta 681, 77–84). We now show that during inactivation the disappearance of iron-sulfur centers A, B, and X correlates with the appearance of a spin-polarized triplet ESR signal with |D| = 279·10⁻⁴ cm⁻¹ and |E| = 39·10⁻⁴ cm⁻¹. The triplet resonances titrate with a midpoint potential of +380 ± 10 mV. Illumination of the inactivated particles results in the generation of an asymmetric ESR signal with g = 2.0031 and ΔH_pp = 1.0 mT. Deconvolution of the P-700⁺ contribution to this composite resonance reveals the spectrum of the putative primary acceptor species, A₀, which is characterized by g = 2.0033 ± 0.0004 and ΔH_pp = 1.0 ± 0.2 mT. The data presented in this report do not substantiate the participation of the electron acceptor A₁ in PS I electron transport, following destruction of the iron-sulfur cluster corresponding to center X. We suggest that A₁ is closely associated with center X and that this component is decoupled from the electron-transport path upon destruction of center X. The inability to photoreduce A₁ in reaction centers lacking a functional center X may result from alteration of the reaction center tertiary structure by the urea-ferricyanide treatment or from displacement of A₁ from its binding site.     

Identification of Group B Streptococci by Immunofluorescence Staining

Gamma globulin fractions of rabbit antisera prepared with whole cell vaccines of group B types Ia, Ib, II, and III and labeled with fluorescein isothiocyanate stained group B streptococci type specifically. Type Ic cells, which contain the Ia polysaccharide antigen of type Ia and the Ic protein antigen of type Ib, were specifically stained by both Ia and Ib conjugates. A group B conjugate pool (B pool) that contained one conjugate specific for each group B type at its predetermined titer gave positive fluorescent-antibody (FA) reactions (4+ intensity) with group B stock strains and negative FA reactions (less than 2+ intensity) with stock strains of streptococcal groups A, C through H, and K through U, viridans streptococci, Streptococcus pneumoniae, Staphylococcus aureus, Neisseria gonorrhoeae, and representative Enterobacteriaceae. Examination of 883 clinical isolates submitted to the Streptococcus Laboratory (Center for Disease Control, Atlanta, Ga.) for identification revealed a 99.1% agreement between FA and culture-precipitin methods. All 305 group B streptococci identified by culture-precipitin and six nonhemolytic group B streptococci missed initially by culture tests were identified correctly by FA. Results of cultural and FA methods in a double-blind study of 99 vaginal swabs agreed on 96 of 99 strains. Three nonhemolytic group B streptococci were identified first by FA and later confirmed by culture-precipitin tests.     

Molecular Characterization of a Xiphinema hunaniense Population with Morphometric Data of all Four Juvenile Stages

A population of Xiphinema hunaniense Wang and Wu, 1992 with all four juvenile stages was found in the rhizosphere of Pinus sp. in Hangzhou, Zhejiang, China. Morphometrics of 18 females and 35 juveniles of this population are given herein. Detailed morphology and morphometrics of the four juvenile stages are provided. Further comparisons based on morphometrics of the population with previous studies of the females and the first-stage juveniles of X. hunaniense with X. radicicola are given, and morphological variation in X. hunaniense populations are discussed. A revised polytomous key code of Loof and Luc (1990) for X. hunaniense identification is provided, i.e., A1- B4- C4- D4/5- E1- F2(3)- G2- H2-I3- J4- K2- L1. In addition, the sequence of the D2 and D3 expansion region of the 28S rRNA gene was analyzed and compared with sequences of closely related species downloaded from the NCBI database. Cluster analysis of sequences confirmed and supported the species identifications.     

Chemical variability of the volatiles from the leaves of Eugenia protenta McVaugh (Myrtaceae) growing wild in the North of Brazil

Leaf volatiles of eight samples of individual plants of Eugenia protenta from three municipalities in the Northeastern Pará, Brazil, were obtained by hydrodistillation (HD) and investigated by gas chromatography/flame ionization detector (GC/FID) and gas/chromatography/mass spectrometry (GC/MS). Dimethylxanthoxylin, selin-11-en-4α-ol, β-elemene, germacrene D, bicyclogermacrene and δ-cadinene were the principal constituents. The results of the oil compositions were processed by Hierarchical Component Analysis (HCA) allowing the establishment of three groups of essential oils differentiated by the content of dimethylxanthoxylin (Type A), selin-11-en-4α-ol/β-elemene (Type B) and germacrene D accomplished by bicyclogermacrene and δ-cadinene (Type C). Dimethylxanthoxylin is being cited for the first time in the genus Eugenia, however it was previously identified in other genera of the family Myrtaceae (Melaleuca and Austromyrtus).     

Nitrogenase IV. Simple method of purification to homogeneity of nitrogenase components from Azotobacter vinelandii

Extracts of Azotobacter vinelandii have been fractionated by simple techniques to obtain highly purified components of nitrogenase. The yield of each component is greater than 60%. Purified Component I has a specific activity of 1638 nmoles ethylene formed/min per mg protein. The spectrum of Component I exhibits a broad absorption between 300 and 600 nm, with no distinctive peaks or shoulders. Addition of sodium dithionite or exposure to air has no effect on the absorption spectrum. Component I, examined at 4.2 °K has EPR signals at g = 4.2, 3.65 and 2.01. Addition of sodium dithionite does not produce additional resonances nor does it alter the signals already present. Crystals of Component I are dark brown and needle-shaped.Purified Component II has a specific activity of 1815 nmoles ethylene formed/min per mg protein. The absorption spectrum has no peaks or shoulders between 390 and 650 nm. Upon exposure of Component II to air, absorption increases between 400 and 650 nm. Treatment of oxidized Component II with dithionite causes this absorption to fall below that of the native Component II. EPR spectra of Component II has signals at g values of 2.05, 1.94, and 1.88. Upon inactivation by O₂, these signals disappear.Neither component by itself has detectable acetylene-reducing or N₂-fixing activity. The ratio of acetylene reduced to N₂ fixed is 3.86 with different ratios of the components. Both components form aggregated species upon exposure to air. Dithionite does not reverse this effect.