Phosphofructokinase activities within the order Spirochaetales and the characterisation of the pyrophosphate-dependent phosphofructokinase from Spirochaeta thermophila

The subtype of phosphofructokinase activity, either ATP-, ADP- or pyrophosphate-dependent, present in members of three genera from the Spirochaetales was investigated. The individual species/strains examined included Spirochaeta alkalica, S. asiatica, S. halophila, S. isovalerica, S. litoralis, S. zuelzerae, S. thermophila, two thermophilic spirochetes, Treponema bryantii, T. denticola, paragraph signT. pectinovorum, Leptospira biflexa and L. interrogans. All of the Spirochaeta strains, regardless of their phenotype, possessed primarily a pyrophosphate-dependent phosphofructokinase. In contrast, T. bryantii, T. denticola and L. biflexa had predominantly an ATP-dependent activity, whereas no activity was detected in T. pectinovorum or paragraph signL. interrogans. The results suggest that pyrophosphate-dependent phosphofructokinase activity may be a reliable phenotypic marker for the genus Spirochaeta and that there are potentially interesting differences in how the catabolism of saccharides is controlled among members of genera within the Spirochaetales. The pyrophosphate-dependent phosphofructokinase from S. thermophila strain RI 19.B1 was purified (303-fold) to homogeneity and biochemically characterised. The S. thermophila enzyme displayed hyperbolic kinetics with respect to both the forward and reverse cosubstrates and was not significantly affected by traditional activators or inhibitors of phosphofructokinase. The biochemical characterisation represents the first spirochete phosphofructokinase to be described.     

Medium for the Quantitative Recovery of Members of the Genera Pasteurella and Brucella

A modification of peptic digest-starch (PDS) medium for the quantitative enumeration of Pasteurella tularensis is described. This modification (PDM), in which the concentration of L-cysteine was decreased and in which Brilliant Green was substituted for the penicillin added to the original medium, was evaluated and was found satisfactory for the recovery of P. tularensis, P. pestis, and members of the genus Brucella, from freshly harvested and stored cultures as well as aerosolized organisms produced therefrom. With weakly virulent strains of P. tularensis, such as the Jap 4 and LV strains, it was found that PDM was not adequate.     

Antiserum to the 33,000-dalton periplasmic-flagellum protein of "Treponema phagedenis" reacts with other treponemes and Spirochaeta aurantia

"Treponema phagedenis" periplasmic flagella (PF) have two major protein bands at molecular weights of 33,000 and 39,800 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (R. J. Limberger and N. W. Charon, J. Bacteriol. 166:105-112, 1986). By use of Western blotting and a polyclonal antiserum directed toward the 33,000-molecular-weight PF protein, cell lysates of 12 species of spirochetes were surveyed for reactivity. Eight species of Treponema as well as Spirochaeta aurantia were positive. The results suggest that epitopes residing on the 33,000-molecular-weight PF protein of "T. phagedenis" are evolutionarily well conserved among the spirochetes.     

Identification of Molecular Variants in Mitochondrial DNAs of Members of the Genera Beauveria, Verticillium, Paecilomyces, Tolypocladium, and Metarhizium

A set of five mitochondrial (mt) probes derived from a strain of Beauveria bassiana was used to evaluate the similarity of mtDNAs from 15 additional isolates of this fungus and five genera of other entomopathogenic fungi. The probes and genes encoded for (shown in parentheses) were pBbmtE2 (NADI, ATP6), pBbmtE3 (ATP6, small rRNA [srRNA]), pBbmtE4 (srRNA, CO3, NAD6), pBbSE1 (NAD6, tRNA^{Val, Ile, Ser, Trp, Pro}, large rRNA [lrRNA]), and pBbXS1 (lrRNA). The probes produced identical hybridization patterns in EcoRI-digested DNA from nearly all isolates of B. bassiana and Beauveria caledonica. Similar patterns were also observed with Beauveria densa. The isolates of B. caledonica and B. densa DNAs could be differentiated from each other and from B. bassiana on the basis of a HindIII digestion and probing with pBbmtE3. Probe pBbmtE2 produced either a 5.0-kb or a 4.1-kb band in all of the B. bassiana isolates. This observation was used to categorize the mtDNA of B. bassiana into two types, designated A and B. Hybridization of the five probes produced distinct banding patterns in Beauveria brongniartii, Tolypocladium cylindrosporum, Tolypocladium nivea, Metarhizium anisopliae, Verticillium lecanii, and Paecilomyces farinosus. Hybridizations carried out with multiple probes simultaneously present produced unique patterns which characterized the B. bassiana group from all other fungi tested. These results are discussed in terms of how mtDNA polymorphisms in B. bassiana may relate to natural population structures, mt transmission in deuteromycetes, and the use of mtDNA polymorphisms in structural analysis of mtDNA.     

Comparison of the Biological and Biochemical Activities of Several Members of the Alphaherpesvirus ICP0 Family of Proteins

Immediate-early protein ICP0 of herpes simplex virus type 1 (HSV-1) is an E3 ubiquitin ligase of the RING finger class that is required for efficient lytic infection and reactivation from latency. Other alphaherpesviruses also express ICP0-related RING finger proteins, but these have limited homology outside the core RING domain. Existing evidence indicates that ICP0 family members have similar properties, but there has been no systematic comparison of the biochemical activities and biological functions of these proteins. Here, we describe an inducible cell line system that allows expression of the ICP0-related proteins of bovine herpes virus type 1 (BHV-1), equine herpesvirus type 1 (EHV-1), pseudorabies virus (PRV), and varicella-zoster virus (VZV) and their subsequent functional analysis. We report that the RING domains of all the proteins have E3 ubiquitin ligase activity in vitro. The BHV-1, EHV-1, and PRV proteins complement ICP0-null mutant HSV-1 plaque formation and induce derepression of quiescent HSV-1 genomes to levels similar to those achieved by ICP0 itself. VICP0, the ICP0 expressed by VZV, was found to be extremely unstable, which limited its analysis in this system. We compared the abilities of the ICP0-related proteins to disrupt ND10, to induce degradation of PML and Sp100, to affect key components of the interferon signaling pathway, and to interfere with induction of interferon-stimulated genes. We found that the property that correlated most closely with their biological activities was the ability to preclude the recruitment of cellular ND10 proteins to sites closely associated with incoming HSV-1 genomes and early replication compartments.The members of the alphaherpesvirus subfamily are characterized by their ability to establish life-long latent infections in neuronal tissues after the primary infection. Although certain core genes are conserved in all herpesviruses of all subfamilies, there are also genes that are characteristic of particular subfamilies. Among these are the genes that encode the ICP0-related proteins of the alphaherpesviruses, of which the most widely studied is ICP0 of herpes simplex virus type 1 (HSV-1). The interest in ICP0 stems from its biological roles in stimulating lytic infection and reactivation from latency (for reviews, see references 17, 18 and 33). Members of the ICP0 family of proteins are characterized by the presence of a RING finger domain near their N termini, a zinc-stabilized fold that in many other proteins confers E3 ubiquitin ligase activity (43). This has proved to be true of ICP0 (3), and the available evidence indicates that other members of the ICP0 family have similar biochemical functions (13, 61). Although a number of ICP0-related alphaherpesvirus proteins have been studied in a variety of contexts, notably those expressed by bovine herpesvirus 1 (BHV-1), equine herpes virus 1 (EHV-1), pseudorabies virus (PRV), and varicella-zoster virus (VZV), there has been no systematic comparison of their abilities to complement ICP0 null mutant HSV-1 or to induce derepression of quiescent HSV-1 genomes.This paper describes a comparative study of the ICP0-related proteins expressed by the viruses listed above. In terms of nomenclature, the proteins expressed by BHV-1 and EHV-1 have been named BICP0 and EICP0, so although other names have been used for the PRV and VZV proteins (such as EP0 and orf61, respectively), we have adopted the names PICP0 and VICP0 for this study. Previous work found that, like ICP0 itself, all four proteins activate gene expression in reporter assays in a RING finger-dependent manner (4, 5, 8, 29, 38, 41, 45, 51, 54, 59, 64, 75, 76, 78). VICP0 and EICP0 also complement, at least partially, ICP0 null mutant HSV-1 (15, 48, 53, 54). BHV-1, EHV-1, PRV, and VZV mutants in which the ICP0-related genes have been deleted have been isolated and found to have reduced replication efficiencies, as expected by analogy with ICP0 null mutant HSV-1 (2, 7, 11, 12, 30, 46, 74, 77).A prominent property of ICP0 is its localization to and disruption of cellular nuclear substructures known as ND10 or promyelocytic leukemia (PML) nuclear domains. Interactions between ND10 and BICP0, EICP0, PICP0, and VICP0 have also been observed, with various consequences for ND10 integrity (47, 60, 63). Whereas ICP0 achieves ND10 disruption through induction of the degradation of PML and SUMO-modified forms of Sp100 (21, 60), EICP0 appears less efficient than ICP0 in inducing PML degradation (60) while VICP0 is inactive (47). While it is likely that all the ICP0 family members discussed here have RING finger-mediated E3 ubiquitin ligase activity (61), the only other protein for which this has been confirmed is BICP0 (13).The similarities between these members of the ICP0 family of proteins and their apparent differences prompted us to investigate in more detail the properties of these proteins in order to determine which of their properties correlate most closely with biological functions in complementing ICP0 null mutant HSV-1. In addition, there was no existing evidence on whether the related proteins could, like ICP0, induce derepression of gene expression from quiescent HSV-1 genomes. We have taken two approaches to these issues. The first is the use of an inducible cell line system that has been used to study ICP0 itself (24, 26). Although inducible cell line systems have been described for VICP0 and BICP0 (53, 69), much of the work described in the current study is novel. The second approach is in vitro analysis of the E3 ubiquitin ligase activities of the isolated RING finger domains of the proteins. The major findings of the study are the following: (i) that all the proteins studied are active in E3 ubiquitin ligase assays; (ii) that VICP0 is extremely unstable, compromising comparative functional analysis in this system; (iii) that BICP0, EICP0, and PICP0 complement to various degrees the plaque-forming defect of ICP0 null mutant HSV-1; (iv) that these three proteins also efficiently stimulate derepression of gene expression from quiescent HSV-1 genomes; (v) that none of the ICP0 family members impedes interferon (IFN)-induced expression of IFN-stimulated genes (ISGs) or affects the stability of important components of the IFN signaling system (namely STAT1, STAT2, and IRF3); (vi) that BICP0, EICP0, and PICP0 cause some disruption of ND10 integrity and have various effects on PML and Sp100 abundance; and (vii) that the property of the proteins that correlated most closely with their stimulation of ICP0 null mutant HSV-1 infection and derepression of quiescent genomes is their ability to inhibit the recruitment of PML and other ND10 proteins to sites associated with parental HSV-1 genomes and early replication compartments.     

The Order Euplotida (Ciliophora): Taxonomy, with Division of Euplotes into Several Genera

ABSTRACT. The order Euplotida represents a monophyletic order of five families of hypotrich ciliates united by morphology, stomatogenesis, ultrastructure, cyst structure, and behavior. A review of variability of ciliation and nuclei among the 14 genera suggests that lines of evolution may have involved both the loss of cirri and nuclear simplification. We present a binary key to genera in the families Aspidiscidae ( Aspidisca and Euplotaspis ), Certesiidae n. fam. ( Certesia ), Gastrocirrhidae ( Cytharoides, Euplotidium , and Gas-trocirrhus ), Uronychiidae ( Diophryopsis, Diophrys, Paradiophrys , and Uronychia ), and Euplotidae. The latter family contains species formerly in the genus Euplotes. Based primarily on cortical structure, endosymbionts, data from morphometric analysis, and ecology, we recognize four different groups. The first group of species remains in Euplotes with Euplotes charon as type. We place a second group of species into the genus Moneuplotes Jankowski 1979 with Moneuplotes vannus (Müller, 1786) as type. We erect two new genera: Euplotoides n. g. and Euplotopsis n. g. with Euplotoides patella (Müller, 1773) n. comb. and Euplotopsis affinis (Dujardin, 1841) n. comb. as type species respectively. We discuss possible phylogenetic relationships within the order.     

Chemical Studies on the Cell Walls of Leptospira biflexa Strain Urawa and Treponema pallidum Strain Reiter

The preparation and chemical poperties of the cell walls of Leptospira biflexa Urawa and Treponema pallidum Reiter are described. Both cell walls are composed mainly of polysaccharides and peptidoglycans. The data of chemical analysis indicate that the cell wall of L. biflexa Urawa contains rhamnose, arabinose, xylose, mannose, galactose, glucose and unidentified sugars as neutral sugars, and alanine, glutamic acid, α,ε-diaminopimelic acid, glucosamine and muramic acid as major amino acids and amino sugars. As major chemical constituents of the cell wall of T. pallidum Reiter, rhamnose, arabinose, xylose, mannose, galactose, glucose, alanine, glutamic acid, ornithine, glycine, glucosamine and muramic acid have been detected. The chemical properties of protein and polysaccharide fractions prepared from the cells of T. pallidum Reiter were also partially examined.     

Lipids and fatty acids of Leptospira interrogans serovar copenhageni virulent strain Shibaura.

Lipids and fatty acids of Leptospira interrogans serovar copenhageni virulent strain Shibaura were analyzed by thin-layer chromatography, gas-liquid chromatography, gas-mass spectrometry and infrared absorption spectrometry. The virulent cells possessed a characteristic lipid pattern consisting of free fatty acid (FFA) (41.8%), one major unidentified phospholipid (14.8%), phosphatidylethanolamine (PE) (12.9%), cholesteryl ester (CE) (9.3%), lysophosphatidylethanolamine (LPE) (4.9%) and diphosphatidyl-glycerol (DPG) (1.1%). Various fatty acids such as hexadecanoic (26.9%), hexadecenoic (15.4%), octadecenoic (26.5%) and octadecadienoic (27.4%) acids were detected in the FFA. The fatty acid composition of the major unidentified phospholipid distinctly differed from those of other lipids including PE, LPE, DPG and CE, and comprised mainly tetradecadienoic (53.6%), tetradecatrienoic (14.0%) and octadecanoic (13.8%) acids. This phospholipid with a large amount of polyunsaturated fatty acids with chain lengths of 14 carbon atoms was detected only in the lipids of the virulent cells.     

Immunological Comparisons of Chymotrypsin Inhibitor I among Several Genera of the Solanaceae

Microcomplement fixation was employed to compare the immunological differences that occur between purified inhibitor I from potato tubers, the four purified protomers that comprise it, and inhibitor I from tuber and leaf extracts. Total inhibitors of chymotrypsin and trypsin in leaves of seven genera of the Solanaceae were identified by enzymatic assay. In leaves of three genera, Solanum, Lycopersicum, and Datura, chymotrypsin inhibitor I was identified immunologically. In petals of all seven genera inhibitor I was also identified immunologically. With the microcomplement fixation technique inhibitor I from leaf or petal extracts of eight Solanaceae genera were compared. An immunological relationship of inhibitor I among seven of these genera was established.     

Comparison of Mucosal,Subcutaneous and Intraperitoneal Routes of Rat Leptospira Infection

Leptospirosis is a zoonosis found worldwide that is caused by a spirochete. The main reservoirs of Leptospira, which presents an asymptomatic infection, are wild rodents, including the brown rat (Rattus norvegicus). Experimental studies of the mechanisms of its renal colonization in rats have previously used an intraperitoneal inoculation route. However, knowledge of rat-rat transmission requires the use of a natural route of inoculation, such as a mucosal or subcutaneous route. We investigated for the first time the effects of subcutaneous and mucosal inoculation routes compared to the reference intraperitoneal route during Leptospira infection in adult rats. Infection characteristics were studied using Leptospira renal isolation, serology, and molecular and histological analyses. Leptospira infection was asymptomatic using each inoculation route, and caused similar antibody production regardless of renal colonization. The observed renal colonization rates were 8 out of 8 rats, 5 out of 8 rats and 1 out of 8 rats for the intraperitoneal, mucosal and subcutaneous inoculation routes, respectively. Thus, among the natural infection routes studied, mucosal inoculation was more efficient for renal colonization associated with urinary excretion than the subcutaneous route and induced a slower-progressing infection than the intraperitoneal route. These results can facilitate understanding of the infection modalities in rats, unlike the epidemiological studies conducted in wild rats. Future studies of other natural inoculation routes in rat models will increase our knowledge of rat-rat disease transmission and allow the investigation of infection kinetics.     

Neutral Lipids in the Study of Relationships of Members of the Family Micrococcaceae

The organisms studied were those of the family Micrococcaceae which cannot participate in genetic exchange with Micrococcus luteus and those whose biochemical and physiological characteristics appear to bridge the genera Staphylococcus and Micrococcus. The hydrocarbon compositions of M. luteus ATCC 4698 and Micrococcus sp. ATCC 398 were shown to be similar to those previously reported for many M. luteus strains, consisting of isomers of branched monoolefins in the range C25 to C31. However, Micrococcus sp. ATCC 398 differed somewhat by having almost all C29 isomers (approximately 88% of the hydrocarbon composition). Micrococcus spp. ATCC 401 and ATCC 146 and M. roseus strains ATCC 412, ATCC 416, and ATCC 516 contained the same type of hydrocarbon patterns, but the predominant hydrocarbons were within a lower distribution range (C23 to C27), similar to Micrococcus sp. ATCC 533 previously reported. The chromatographic profile and carbon range of the hydrocarbons of an atypical strain designated M. candicans ATCC 8456 differed significantly from the hydrocarbon pattern presented above. The hydrocarbons were identified as branched and normal olefins in the range C16 to C22. Studies of several different strains of staphylococci revealed that these organisms do not contain readily detectable amounts of aliphatic hydrocarbons. The members of the family Micrococcaceae have been divided into two major groups based on the presence or absence of hydrocarbons. With the exception of M. candicans ATCC 8456, this division corresponded to the separation of these organisms according to their deoxyribonucleic acid compositions.     

Taxonomy of the Cupressaceae: Subfamilies,Tribes and Genera

Cupressaceae and Taxodiaceae have recently been merged under the earlier name Cupressaceae s.I. by many authors, as the two families are similar in a number of morpho logical characters. Sciadopitys S. et Z., which has often been treated as a morphologically isolated member of the Taxodiaceae, has recently been considered as a monotypic family, Sciadopityaceae. The Cupressaceae s.s. may be reorganized into two subfamilies. The Cu pressoideae is composed of genera with the uppermost cone-scales infertile and can be divided into four tribes: Cnpresseae, including Cupressus, X Cupressocyparis, Charnaecyparis and Fokeinia;Thujopsideae, including Thuja, Thujopsis and Platycladusl Junipereae, including Juniperus and Microbiota; and Tetraclineae, including Calocedrus and Tetraclinis. The Callitroideae is composed of genera with the uppermost cone-scales fertile and can be divided into three tribes: Actinostrobeae, including Actinostrobus, Callitris, Fitzroya and Neocallitropsis; Widdringtoneae, including Pilgerodendron, Diselma and Widdringtonia ; Libocedreae, including Libocedrus, Papuacedrus and Austrocedrus. Five geographical distribution patterns are recognized in the 21 genera of Cupressaceae. (a) One genus, X Cupressocyparis, is a natural hybrid derived from selections in England; (b) Two genera, Cupressus and Juniperus, are distributed in Africa, Europe, Asia and North America; (c) Three genera, Thuja, Chamaecyparis, and Calocedrus, are disjnnctly distributed in Eastem Asia and North America; (d) Five genera, Actinostrobus, Callitris, Libocedrus, Papuacedrus and Widdringtonia, have limited distribution; and (e) The other 10 genera, which are monotypic, are restricted to narrow areas except Plotycladus. Three centers of genera diversity are identified in the Cupressaceae, i. e Eastern Asia with nine genera, southwestern North America with five genera, and Australia and its adjacent islands in the east　with six genera, including New Zealand,. Tasmania, New Caledonia, and New Guinea. Other important areas are western Mediterranean with three genera and Chile and Argentinawith three genera.     

鳜牙齿形态结构及与不同种属间比较观察

牙齿是肉食性鱼类重要的摄食器官.为探究鳜(Siniperca chuatsi)牙齿形态结构,采用解剖镜观察了鳜牙齿分布、形态与数量.并比较其与大眼鳜(S.kneri)、斑鳜(S.scherzeri)及中国少鳞鳜(Coreoperca whiteheadi)牙齿差异.采用茜素红染色、组织切片、扫描电镜、X射线能谱及红外光...     

Wound-induced Accumulation of Trypsin Inhibitor Activities in Plant Leaves: Survey of Several Plant Genera

Asexual embryogenesis in Daucus carota L. `Queen Anne's Lace' callus was suppressed by Ethephon, ethylene, and 2,4-dichlorophenoxyacetic acid (2,4-D). The Ethephon effect could be attributed to volatile and nonvolatile substances. The volatile component was probably entirely ethylene. Ethylene was liberated in the cultures in direct proportion to Ethephon added to the medium. Autoclaving of Ethephon caused a substantial decrease of measurable ethylene. Continuous exposure of callus to 5 μl/l ethylene depressed somatic cell embryogenesis, but not markedly. Depression of embryogenesis by 2,4-D was unrelated to ethylene evolution.     

Comparison of peptidase, glycosidase and esterase activities of oral and non-oral Treponema species

The enzyme profiles of 20 oral and non-oral Treponema strains were investigated using an API ZYM Complete Research kit. The test included 10 2-naphthyl derivatives of fatty acids, 20 p-nitrophenol derivatives of carbohydrates and 60 2-naphthylamide derivatives of amino acids and peptides. The oral Treponema species investigated were T. denticola, T. vincentii and T. Pectinovorum. The non-oral species examined were T. phagedenis, T. hyodysenteriae and intestinal spirochaetes of human and chicken origin. Esterase activities on C5 to C10 fatty acids were common among different Treponema species. Glycosidase activities were infrequently observed in T. vincentii, T. pectinovorum and T. phagedenis Reiter strain. Arabinosidase, lactosidase and xylosidase activity was observed in the T. hyodysenteriae strains but alpha-L-fucosidase activity was found only in T. denticola and T. phagedenis. More exo- and endo-peptidase activities were found in T. denticola than in other species. The enteropathogenic T. hyodysenteriae isolates had a very low proteolytic profile. Dipeptidyl prolyl amidase activity was observed in all species except in the T. phagedenis Reiter strain and the avian intestinal spirochaetes. The enzyme profiles did not discriminate between oral and non-oral Treponema species.     

Comparison of the Lipids of Intracellular and Extracellular Rabies Viruses

The lipid composition of both intracellular and extracellular forms of the ERA strain of rabies virus grown in BHK/21 cells was determined. The lipids from purified preparations of both intracellular and extracellular virus yielded 57 and 58% neutral lipid, respectively. The phospholipids of the intracellular and extracellular virus constituted 43 and 42%, respectively. Triglyceride and cholesterol appear to be the major neutral lipids, whereas sphingomyelin, phosphatidylethanolamine, and phosphatidylcholine comprise the major bulk of phospholipid in both virus types. The molar ratio of cholesterol to phospholipid was 0.87 (intracellular) and 0.92 (extracellular). On the basis of the data presented, it is reasonable to assume that the lipids of both intracellular and extracellular rabies virus are similar.