Stereochemical aspects of the metabolism of the isomeric methylcyclohexanols and methylcyclohexanones

1. The seven isomeric optically inactive forms of methylcyclohexanol (i.e. 1-, and cis- and trans-2-, -3- and -4-) are excreted by rabbits mainly as glucuronides of the thermodynamically more stable forms of the alcohols. The eighth isomer, cyclohexylmethanol, however, undergoes aromatization in vivo, giving rise to benzoic acid and hippuric acid. The (±)-2-, (±-3- and 4-methylcyclohexanones are reduced in the rabbit and excreted mainly as the glucuronides of the thermodynamically more stable forms of the corresponding methylcyclohexanols. 2. Racemic cis- and trans-2-methylcyclohexanol and 2-methylcyclohexanone are all excreted as conjugated trans-2-methylcyclohexanol. However, when the (±)-cis-alcohol or the (±)-ketone is fed, (+)-trans-2-methylcyclohexanol is excreted, whereas when the (±)-trans-alcohol is fed it is excreted as the (±)-trans-alcohol. 3. Racemic cis- and trans-3-methylcyclohexanol and 3-methylcyclohexanone are all excreted as conjugated racemic cis-3-methylcyclohexanol. cis- and trans-4-Methylcyclohexanol and 4-methylcyclohexanone are all excreted as conjugated trans-4-methylcyclohexanol. 4. The metabolic differences between the various methylcyclohexanols are explicable in terms of their conformations and of Vennesland's (1958) hypothesis of the role of NADH in dehydrogenation reactions.     

Mutations in the Gene Encoding the Ancillary Pilin Subunit of the Streptococcus suis srtF Cluster Result in Pili Formed by the Major Subunit Only

Pili have been shown to contribute to the virulence of different Gram-positive pathogenic species. Among other critical steps of bacterial pathogenesis, these structures participate in adherence to host cells, colonization and systemic virulence. Recently, the presence of at least four discrete gene clusters encoding putative pili has been revealed in the major swine pathogen and emerging zoonotic agent Streptococcus suis. However, pili production by this species has not yet been demonstrated. In this study, we investigated the functionality of one of these pili clusters, known as the srtF pilus cluster, by the construction of mutant strains for each of the four genes of the cluster as well as by the generation of antibodies against the putative pilin subunits. Results revealed that the S. suis serotype 2 strain P1/7, as well as several other highly virulent invasive S. suis serotype 2 isolates express pili from this cluster. However, in most cases tested, and as a result of nonsense mutations at the 5′ end of the gene encoding the minor pilin subunit (a putative adhesin), pili were formed by the major pilin subunit only. We then evaluated the role these pili play in S. suis virulence. Abolishment of the expression of srtF cluster-encoded pili did not result in impaired interactions of S. suis with porcine brain microvascular endothelial cells. Furthermore, non-piliated mutants were as virulent as the wild type strain when evaluated in a murine model of S. suis sepsis. Our results show that srtF cluster-encoded, S. suis pili are atypical compared to other Gram-positive pili. In addition, since the highly virulent strains under investigation are unlikely to produce other pili, our results suggest that pili might be dispensable for critical steps of the S. suis pathogenesis of infection.     

Unravelling the Wolbachia evolutionary role: the reprogramming of the host genomic imprinting

Environmental factors can induce significant epigenetic changes that may also be inherited by future generations. The maternally inherited symbiont of arthropods Wolbachia pipientis is an excellent candidate as an ‘environmental’ factor promoting trans-generational epigenetic changes: by establishing intimate relationships with germ-line cells, epigenetic effects of Wolbachia symbiosis would be manifested as a ‘maternal effect’, in which infection of the mother modulates the offspring phenotype. In the leafhopper Zyginidia pullula, Wolbachia feminizes genetic males, leaving them as intersexes. With the exception of male chitinous structures that are present in the last abdominal segment, feminized males display phenotypic features that are typical of females. These include ovaries that range from a typical histological architecture to an altered structure. Methylation-sensitive random amplification of polymorphic DNA profiles show that they possess a female genomic imprint. On the other hand, some rare feminized males bear testes instead of ovaries. These specimens possess a Wolbachia density approximately four orders of magnitude lower than feminized males with ovaries and maintain a male genome—methylation pattern. Our results indicate that Wolbachia infection disrupts male imprinting, which dramatically influences the expression of genes involved in sex differentiation and development, and the alteration occurs only if Wolbachia exceeds a density threshold. Thus, a new Wolbachia''s role as an environmental evolutionary force, inducing epigenetic trans-generational changes, should now be considered.     

Insight into the role of cetaceans in the life cycle of the tetraphyllideans (Platyhelminthes: Cestoda)

Four types of tetraphyllidean larvae infect cetaceans worldwide: two plerocercoids differing in size, ‘small’ (SP) and ‘large’ (LP), and two merocercoids referred to as Phyllobothrium delphini and Monorygma grimaldii. The latter merocercoid larvae parasitize marine mammals exclusively and exhibit a specialised cystic structure. Adult stages are unknown for any of the larvae and thus the role of cetaceans in the life cycle of these species has been a long-standing problem. The SP and LP forms are thought to be earlier stages of P. delphini and M. grimaldii that are presumed to infect large pelagic sharks that feed on cetaceans. A molecular analysis of the D2 variable region of the large subunit ribosomal DNA gene based on several individuals of each larval type collected from three Mediterranean species of cetaceans showed consistent and unique molecular signatures for each type regardless of host species or site of infection. The degree of divergence suggested that LP, P. delphini and M. grimaldii larvae may represent separate species, whereas SP may be conspecific with M. grimaldii. In all host species, individuals of SP accumulated in the gut areas in which the lymphoid tissue was especially developed. We suggest therefore that these larvae use the lymphatic system to migrate to the abdominal peritoneum and mesenteries where they develop into forms recognizable as M. grimaldii. The plerocercoid stage of P. delphini remains unknown. In a partial phylogenetic tree of the Tetraphyllidea, all larvae formed a clade that included a representative of the genus Clistobothrium, some species of which parasitize sharks such as the great white which is known to feed on cetaceans. A bibliographic examination of tetraphyllidean infections in marine mammals indicated that these larvae are acquired mostly offshore. In summary, the evidence suggests that cetaceans play a significant role in the life cycle of these larvae. In addition, it seems clear that cetaceans act as natural intermediate hosts for P. delphini and M. grimaldii, as within these hosts they undergo development from the plerocercoid stage to the merocercoid stage. Because tetraphyllidean species use fish, cephalopods and other marine invertebrates as intermediate hosts, the inclusion of cetaceans in the life cycle would have facilitated their transmission to apex predators such as the large, lamnid sharks. The biological significance of infections of LP in cetaceans is unclear, but infections do not seem to be accidental as such larvae show high prevalence and abundance as well as a high degree of site specificity, particularly in the anal crypts and bile ducts.     

Prepatterning the Drosophila notum: the three genes of the iroquois complex play intrinsically distinct roles

The Drosophila thorax exhibits 11 pairs of large sensory organs (macrochaetes) identified by their unique position. Remarkably precise, this pattern provides an excellent model system to study the genetic basis of pattern formation. In imaginal wing discs, the achaete-scute proneural genes are expressed in clusters of cells that prefigure the positions of each macrochaete. The activities of prepatterning genes provide positional cues controlling this expression pattern. The three homeobox genes clustered in the iroquois complex (araucan, caupolican and mirror) are such prepattern genes. mirror is generally characterized as performing functions predominantly different from the other iroquois genes. Conversely, araucan and caupolican are described in previous studies as performing redundant functions in most if not all processes in which they are involved. We have addressed the question of the specific role of each iroquois gene in the prepattern of the notum and we clearly demonstrate that they are intrinsically different in their contribution to this process: caupolican and mirror, but not araucan, are required for the neural patterning of the lateral notum. However, when caupolican and/or mirror expression is reduced, araucan loss of function has an effect on thoracic bristles development. Moreover, the overexpression of araucan is able to rescue caupolican loss of function. We conclude that, although retaining some common functionalities, the Drosophila iroquois genes are in the process of diversification. In addition, caupolican and mirror are required for stripe expression and, therefore, to specify the muscular attachment sites prepattern. Thus, caupolican and mirror may act as common prepattern genes for all structures in the lateral notum.     

A chemical dichotomy in phytoalexin induction within the tribe vicieae of the leguminosae

Using either leaf or cotyledon tissues, the phytoalexin response of over 60 representative members of the genera Lathyrus, Lens, Pisum and Vicia was determined. Twenty-nine Vicia species produced furanoacetylenes, as did the two Lens species examined. By contrast, Pisum and Lathyrus failed to produce any of these phytoalexins, but formed pisatin instead. Thus, pisatin was formed as the major phytoalexin in 29 of 31 Lathyrus species tested, representing 10 sections within the genus. This dichotomy in phytoalexin response is discussed in relation to the taxonomy of the tribe Vicieae.     

Manual and expert annotation of the nearly complete genome sequence of Staphylococcus sciuri strain ATCC 29059: A reference for the oxidase-positive staphylococci that supports the atypical phenotypic features of the species group

Staphylococcus sciuri is considered to be one of the most ancestral species in the natural history of the Staphylococcus genus that consists of 48 validly described species. It belongs to the basal group of oxidase-positive and novobiocin-resistant staphylococci that diverged from macrococci approximately 250 million years ago. Contrary to other groups, the S. sciuri species group has not developed host-specific colonization strategies. Genome analysis of S. sciuri ATCC 29059 provides here the first genetic basis for atypical traits that would support the switch between the free-living style and the infective state in animals and humans. From among the most remarkable features, it was noticed in this extensive study that there were a number of phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTS), almost twice as many as any other staphylococci, and the co-occurrence of mevalonate and non-mevalonate pathways for isoprenoid synthesis. The sequenced strain was devoid of the main virulence factors present in Staphylococcus aureus, although it exhibited numerous heme and iron acquisition systems, as well as crt and aldH genes necessary for gold pigment synthesis. The sensing and signaling networks, exemplified by a large and typical repertoire of two-component regulatory systems and a complete panel of master regulators, such as agr, rex, mgrA, rot, sarA and sarR genes, depict the background in which S. aureus virulence genes were later acquired. An additional sigma factor, a distinct set of electron transducer elements and many gene operons similar to those found in Bacillus spp. would constitute the most visible remnant links with Bacillaceae organisms.     

Halophilic bacteria are colonizing the exhibition areas of the Capuchin Catacombs in Palermo,Italy

The Capuchin Catacombs of Palermo, Italy, contain over 1800 mummies dating from the 16th to 20th centuries AD. Their environment is not conducive to the conservation of the remains due to, among other factors, water infiltration, which is producing salt efflorescences on the walls. A multiphasic approach was applied to investigate the halophilic microbiota present in the Catacombs. Enrichment cultures were conducted on media containing different NaCl concentrations, ranging from 3 to 20 %. For screening of the strains, the following two PCR-based methods were used and compared: fluorescence internal transcribed spacer PCR (f-ITS) and random amplification of polymorphic DNA (RAPD) analyses. Results derived from RAPD profiles were shown to be slightly more discriminative than those derived from f-ITS. In addition, the proteolytic and cellulolytic abilities were screened through the use of plate assays, gelatin agar and Ostazin Brilliant Red H-3B (OBR-HEC), respectively. Many of the strains isolated from the wall samples displayed proteolytic activities, such as all strains belonging to the genera Bacillus, Virgibacillus and Arthrobacter, as well as some strains related to the genera Oceanobacillus, Halobacillus and Idiomarina. In addition, many of the strains isolated from materials employed to stuff the mummies showed cellulolytic activities, such as those related to species of the genera Chromohalobacter and Nesterenkonia, as well as those identified as Staphylococcus equorum and Halomonas sp. Furthermore, many of the strains were pigmented ranging from yellow to a strong pink color, being directly related to the discoloration displayed by the materials.     

New Ichneumonidae (Hymenoptera) from the Upper Cretaceous ambers of the Taimyr Peninsula

Two new genera and four new species of Ichneumonidae are described from the Upper Cretaceous ambers of the Taimyr Peninsula: Agapia sukatchevae gen. et sp. nov., Agapteron popovi gen. et sp. nov., Eubaeus abdominalis sp. nov., and Urotryphon baikurensis sp. nov. New detailed diagnoses are provided for the genera Urotryphon and Eubaeus. The genera Catachora, Urotryphon, and Eubaeus, previously placed in the subfamily Tryphoninae, are transferred to the subfamily Labenopimplinae, as well as the new genera Agapia and Agapteron. Possible causes of the miniaturization in ichneumonid wasps in the Cretaceous are discussed.     

Binding of the Bacillus subtilis LexA protein to the SOS operator

The Bacillus subtilis LexA protein represses the SOS response to DNA damage by binding as a dimer to the consensus operator sequence 5′-CGAACN₄GTTCG-3′. To characterize the requirements for LexA binding to SOS operators, we determined the operator bases needed for site-specific binding as well as the LexA amino acids required for operator recognition. Using mobility shift assays to determine equilibrium constants for B.subtilis LexA binding to recA operator mutants, we found that several single base substitutions within the 14 bp recA operator sequence destabilized binding enough to abolish site-specific binding. Our results show that the AT base pairs at the third and fourth positions from the 5′ end of a 7 bp half-site are essential and that the preferred binding site for a LexA dimer is 5′-CGAACATATGTTCG-3′. Binding studies with LexA mutants, in which the solvent accessible amino acid residues in the putative DNA binding domain were mutated, indicate that Arg-49 and His-46 are essential for binding and that Lys-53 and Ala-48 are also involved in operator recognition. Guided by our mutational analyses as well as hydroxyl radical footprinting studies of the dinC and recA operators we docked a computer model of B.subtilis LexA on the preferred operator sequence in silico. Our model suggests that binding by a LexA dimer involves bending of the DNA helix within the internal 4 bp of the operator.     

Lipases fromRhizomucor miehei andHumicola lanuginosa: Modification of the lid covering the active site alters enantioselectivity

The homologous lipases fromRhizomucor miehei andHumicola lanuginosa showed approximately the same enantioselectivity when 2-methyldecanoic acid esters were used as substrates. Both lipases preferentially hydrolyzed theS-enantiomer of 1-heptyl 2-methyldecanoate (R. miehei:E _S =8.5;H. lanuginosa:E _S =10.5), but theR-enantiomer of phenyl 2-methyldecanoate (E _R =2.9). Chemical arginine specific modification of theR. miehei lipase with 1,2-cyclohexanedione resulted in a decreased enantioselectivity (E _R =2.0), only when the phenyl ester was used as a substrate. In contrast, treatment with phenylglyoxal showed a decreased enantioselectivity (E _S =2.5) only when the heptyl ester was used as a substrate. The presence of guanidine, an arginine side chain analog, decreased the enantioselectivity with the heptyl ester (E _S =1.9) and increased the enantioselectivity with the aromatic ester (E _R =4.4) as substrates. The mutation, Glu 87 Ala, in the lid of theH. lanuginosa lipase, which might decrease the electrostatic stabilization of the open-lid conformation of the lipase, resulted in 47% activity compared to the native lipase, in a tributyrin assay. The Glu 87 Ala mutant showed an increased enantioselectivity with the heptyl ester (E _S =17.4) and a decreased enantioselectivity with the phenyl ester (E _R =2.5) as substrates, compared to native lipase. The enantioselectivities of both lipases in the esterification of 2-methyldecanoic acid with 1-heptanol were unaffected by the lid modifications.     

Genome Dynamics Explain the Evolution of Flowering Time CCT Domain Gene Families in the Poaceae

Numerous CCT domain genes are known to control flowering in plants. They belong to the CONSTANS-like (COL) and PREUDORESPONSE REGULATOR (PRR) gene families, which in addition to a CCT domain possess B-box or response-regulator domains, respectively. Ghd7 is the most recently identified COL gene to have a proven role in the control of flowering time in the Poaceae. However, as it lacks B-box domains, its inclusion within the COL gene family, technically, is incorrect. Here, we show Ghd7 belongs to a larger family of previously uncharacterized Poaceae genes which possess just a single CCT domain, termed here CCT MOTIF FAMILY (CMF) genes. We molecularly describe the CMF (and related COL and PRR) gene families in four sequenced Poaceae species, as well as in the draft genome assembly of barley (Hordeum vulgare). Genetic mapping of the ten barley CMF genes identified, as well as twelve previously unmapped HvCOL and HvPRR genes, finds the majority map to colinear positions relative to their Poaceae orthologues. Combined inter-/intra-species comparative and phylogenetic analysis of CMF, COL and PRR gene families indicates they evolved prior to the monocot/dicot divergence ∼200 mya, with Poaceae CMF evolution described as the interplay between whole genome duplication in the ancestral cereal, and subsequent clade-specific mutation, deletion and duplication events. Given the proven role of CMF genes in the modulation of cereals flowering, the molecular, phylogenetic and comparative analysis of the Poaceae CMF, COL and PRR gene families presented here provides the foundation from which functional investigation can be undertaken.     

Notes on the asteraceae of the Southeastern United States

Nomenclatural innovations are validated inAster, Chrysopsis, Eupatorium,Gaillardia, Helianthus, Prenanthes, Silphium, andSolidago. The genusChrysopsis is maintained as distinct fromHeterotheca, andHelianthus smithii Heiser is reduced to synonymy underH. microcephalus Torr. & Gray.     

Studies on the interactions of sperm with the surface of the sea urchin egg

We have examined the relationship between sperm adhesion and fertilization in the cross species insemination of Arbacia punctulata eggs by Strongylocentrotus purpuratus sperm. As previously reported (Kinsey et al., 1980) the addition of S. purpuratus egg jelly results in induction of the acrosome reaction in sperm and significant numbers of S. purpuratus sperm adhere to A. punctulata eggs. However, in the absence of S. purpuratus egg jelly, S. purpuratus sperm fail to bind to A. punctulata eggs. Although at least 200 S. purpuratus sperm bind to an A. punctulata egg in the presence of S. purpuratus jelly, less than 8% of the eggs are fertilized. The adhesion of S. purpuratus sperm meets the same functional criteria as homologous A. punctulata sperm-egg adhesion. Electron microscopy shows that S. purpuratus sperm that have undergone the acrosome reaction adhere to A. punctulata eggs by their bindin-coated acrosomal process in a manner that is morphologically identical to that observed with homologous A. punctulata sperm. We have also compared the ability of S. purpuratus and A. punctulata sperm to fuse and fertilize with A. punctulata eggs after removal of the vitelline layer. Using high levels of sperm of either species, heterologous as well as homologous fertilization is readily detectable. Under these conditions, where stable binding is not demonstrable, there is no difference in the ability of S. purpuratus and A. punctulata sperm to fertilize A. punctulata eggs. These observations suggest that the failure of S. purpuratus sperm to fertilize A. punctulata eggs under normal conditions may be due to their inability to penetrate the vitelline layer so that they can fuse with the egg plasma membrane. In relation to the possible mechanism of vitelline layer penetration, we have also investigated the mode of action of chymostatin, an inhibitor of chymotrypsin that has been reported to inhibit fertilization of sea urchin eggs (Hoshi et al., 1979). Our findings suggest that the fertilization inhibitory activity of chymostatin is not related to its antichymotrypsin activity. Rather, it appears that this inhibition is due to the induction of an abnormal acrosome reaction in sperm that precludes formation of the acrosome process.     

A numerical study of the common narrow-leaved taxa of chenopodium occurring in the Western United States

A numerical taxonomic study was conducted on the common narrow-leaved members of the genusChenopodium that occur in the western United States. As many as six and as few as two species have been recognized. Thirty-five individual popu lations were used as OTU’s, and 35 characters were considered. The data were subjected to cluster (both the weighted-pair group method and the within-group dispersion method) and principal component analysis. The results of both analyses are in general agreement and suggest phenetic relationships that differ from current interpretations in the literature. It is concluded thatC. desiccatum var.leptophylloides is much more closely related phenetically toC. atrovirens (and may not be distinct from it) than it is toC. desiccatum var.desiccatum. Chenopodium hians andC. leptophyllum appear to be phenetically distinct despite the fact that they have sometimes been viewed as conspecific.Cheno podium subglabrum is not very similar toC. leptophyllum, and should probably be treated as a separate species rather than as a variety of the latter. Popu lations referable toC. incognitum form a rather loose cluster that appears some what intermediate between populations ofC. desiccatum var.leptophylloides,C. atrovirens, C. hians, andC. leptophyllum.