Cloning and characterization of the gene encoding 1-cyclohexenylcarbonyl coenzyme A reductase from Streptomyces collinus.

We report the cloning of the gene encoding the 1-cyclohexenylcarbonyl coenzyme A reductase (ChcA) of Streptomyces collinus, an enzyme putatively involved in the final reduction step in the formation of the cyclohexyl moiety of ansatrienin from shikimic acid. The cloned gene, with a proposed designation of chcA, encodes an 843-bp open reading frame which predicts a primary translation product of 280 amino acids and a calculated molecular mass of 29.7 kDa. Highly significant sequence similiarity extending along almost the entire length of the protein was observed with members of the short-chain alcohol dehydrogenase superfamily. The S. collinus chcA gene was overexpressed in Escherichia coli by using a bacteriophage T7 transient expression system, and a protein with a specific ChcA activity was detected. The E. coli-produced ChcA protein was purified and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels as the enoyl-coenzyme A reductase protein prepared from S. collinus. The enzyme demonstrated the ability to catalyze, in vitro, three of the reductive steps involved in the formation of cyclohexanecarboxylic acid. An S. collinus chcA mutant, constructed by deletion of a genomic region comprising the 5' end of chcA, lost the ChcA activity and the ability to synthesize either cyclohexanecarboxylic acid or ansatrienin. These results suggest that chcA encodes the ChcA that is involved in catalyzing multiple reductive steps in the pathway that provides the cyclohexanecarboxylic acid from shikimic acid.     

Spatial refuges and associational defenses promote harmful blooms of the alga Caulerpa sertularioides onto coral reefs

Extreme population fluctuations, or outbreaks, are driven by interacting processes that are often more complex than isolated changes in consumer or resource control. Blooms of the macroalga Caulerpa sertularioides in the eastern tropical Pacific overgrew and killed reef-building corals, with blooms onto reefs corresponding to cool La Niña phases of inter-decadal fluctuations of the El Niño–Southern Oscillation. We quantified factors responsible for the maintenance of C. sertularioides patches in off-reef areas, namely an associational mutualism with an epiphytic cyanobacteria (Lyngbya majuscula), coupled with spatial refuges at the scales of individual thalli and habitat. Maintenance of near reef algal populations with a strong response to nutrient addition showed that these populations were primed to bloom onto reefs in response to enhanced nutrient delivery, such as those potentially associated with La Niña conditions. However, our experiments demonstrated that no single factor related to consumer or resource control was likely to stimulate bloom formation in isolation. Rather, we propose a novel model of reef bloom formation where off-reef blooms are sustained by processes reducing consumer control, and then bloom onto reefs through an interaction between increased allochthonous nutrient input and an uncoupling of consumer control by an association with epiphytic cyanobacteria.     

Production rates of prostaglandin F, 6-keto-PGF1α and thromboxane B2 by perifused human endometrium

Human endometrium obtained from fresh hysterectomy specimens was perifused for 7 hr in 95% O₂/5% CO₂ at 37°C. The phase of the menstrual cycle was determined by histological examination. The concentrations of PGF, 6-keto-PGF_1α and TxB₂ in 20 min fractions of the perifusion medium were measured by radioimmunoassay and production rates were calculated in terms of dry weight of tissue. Biphasic patterns of production were observed; high initial values fell to about 20% at 2 hr and then increased to relatively stable values at about 4 hr which were maintained for the next 2 hr. During this latter period, production rates in endometria taken at different phases of the cycle differed markedly from each other; the production rates of PGF in secretory and early proliferative endometria were low (15.8 ± 2.6, mean ± SEM and 67.2 ± 8.3 ng/min/g respectively) whereas they were high in late proliferative and premenstrual endometria (188.0 ± 16.7 and 196.4 ± 16.9 ng/min/g respectively). The patterns of production of 6-keto-PGF_1α and TxB₂ were similar to those of PGF but the absolute values were much lower (<10%). We conclude that the observed rates of production of prostaglandins by perifused human endometrium are consistent with synthesis being stimulated either by estrogen or withdrawal of hormonal support and being inhibited by progesterone.     

Sex differences in the influence of mothers on the sociosexual preferences of their offspring

The extent to which "nurture" as opposed to "nature" determines behavior and sociosexual preferences in mammalian species is controversial although most recent interest has focused on genetic determinants. We report here that if sheep and goats are cross-fostered at birth, but raised in mixed-species groups, their play and grooming behavior resembles that of their foster rather than genetic species. There are no sex differences in effects on these behaviors, and other species-specific behavior patterns such as aggression, browsing, climbing, and vocalizations are unaffected. In adulthood, cross-fostered males strongly prefer to socialize and mate with females of their foster mother's species, even if raised with a conspecific of their own species. Castration within 2 days of birth slightly reduces the level of this altered social preference but mating preference following short-term testosterone treatment is the same as for gonadally intact animals. Cross-fostered females also show significant preference for socializing with females and mating with males of their foster mother's species, although this effect is weaker than that in both gonadally intact and castrated males. When cross-fostered animals are placed in flocks containing members of only their genetic species for 3 years, male social and mating preferences for females of their mother's species remain virtually unaffected. Females change to display an exclusive mating preference for members of their genetic species in 1-2 years although they still retain some social interest in female members of their foster species. Thus, there are clear sex differences in the impact of the emotional bond between a mother and her offspring in these mammals. Effects on males are strongest and irreversibly maintained even after altering their social environment, whereas those on females are weaker and mating preferences are clearly adaptable in the face of altered social priorities. These sex differences are presumably caused by pre-, or early postnatal, organizational effects of sex hormones on the brain.     

Chemical communication,sexual selection,and introgression in wall lizards

Divergence in communication systems should influence the likelihood that individuals from different lineages interbreed, and consequently shape the direction and rate of hybridization. Here, we studied the role of chemical communication in hybridization, and its contribution to asymmetric and sexually selected introgression between two lineages of the common wall lizard (Podarcis muralis). Males of the two lineages differed in the chemical composition of their femoral secretions. Chemical profiles provided information regarding male secondary sexual characters, but the associations were variable and inconsistent between lineages. In experimental contact zones, chemical composition was weakly associated with male reproductive success, and did not predict the likelihood of hybridization. Consistent with these results, introgression of chemical profiles in a natural hybrid zone resembled that of neutral nuclear genetic markers overall, but one compound in particular (tocopherol methyl ether) matched closely the introgression of visual sexual characters. These results imply that associations among male chemical profiles, sexual characters, and reproductive success largely reflect transient and environmentally driven effects, and that genetic divergence in chemical composition is largely neutral. We therefore suggest that femoral secretions in wall lizards primarily provide information about residency and individual identity rather than function as sexual signals.     

Murine epidermal growth factor: heterogeneity on high resolution ion-exchange chromatography

We have shown that epidermal growth factor (EGF) purified either by the classical method of Savage and Cohen, or solely by h.p.l.c. techniques can be resolved into two species, EGF alpha and EGF beta. However, despite the apparent purity of such materials, as determined both chromatographically and by amino acid analysis, they failed to give homogeneous products on radioiodination. Analysis by isoelectric focusing on agarose gels followed by transfer to nitrocellulose and silver staining showed that EGF alpha could be further resolved into three sub-species which focused at pH 4.6, 4.3 and 4.1. EGF beta (which also focused at pH 4.6) contained very small amounts of the species with isoelectric points of 4.1 and 4.3, probably due to slight contamination of this preparation by EGF alpha. Preparative separation of the sub-species of EGF alpha was achieved by high performance anion-exchange chromatography at pH 6.5 on a Pharmacia Mono Q column. Radioiodination of these purified sub-species did not produce significant charge heterogeneity. However, two slightly different forms of [125I]EGF alpha 1 (pH 4.6 species) were separable by anion-exchange chromatography on the Mono Q column. All of the EGF species competed for binding to EGF receptors on A431 cells and were active mitogens for BALB/c 3T3 fibroblasts.     

Biofuels and bio-based chemicals from lignocellulose: metabolic engineering strategies in strain development

Interest in developing a sustainable technology for fuels and chemicals has unleashed tremendous creativity in metabolic engineering for strain development over the last few years. This is driven by the exceptionally recalcitrant substrate, lignocellulose, and the necessity to keep the costs down for commodity products. Traditional methods of gene expression and evolutionary engineering are more effectively used with the help of synthetic biology and -omics techniques. Compared to the last biomass research peak during the 1980s oil crisis, a more diverse range of microorganisms are being engineered for a greater variety of products, reflecting the broad applicability and effectiveness of today’s gene technology. We review here several prominent and successful metabolic engineering strategies with emphasis on the following four areas: xylose catabolism, inhibitor tolerance, synthetic microbial consortium, and cellulosic oligomer assimilation.     

Structure and Dynamics of a Compact State of a Multidomain Protein,the Mercuric Ion Reductase

The functional efficacy of colocalized, linked protein domains is dependent on linker flexibility and system compaction. However, the detailed characterization of these properties in aqueous solution presents an enduring challenge. Here, we employ a novel, to our knowledge, combination of complementary techniques, including small-angle neutron scattering, neutron spin-echo spectroscopy, and all-atom molecular dynamics and coarse-grained simulation, to identify and characterize in detail the structure and dynamics of a compact form of mercuric ion reductase (MerA), an enzyme central to bacterial mercury resistance. MerA possesses metallochaperone-like N-terminal domains (NmerA) tethered to its catalytic core domain by linkers. The NmerA domains are found to interact principally through electrostatic interactions with the core, leashed by the linkers so as to subdiffuse on the surface over an area close to the core C-terminal Hg(II)-binding cysteines. How this compact, dynamical arrangement may facilitate delivery of Hg(II) from NmerA to the core domain is discussed.     

Differential impairment of lytic and cytokine functions in senescent human immunodeficiency virus type 1-specific cytotoxic T lymphocytes

Telomere length is abnormally short in the CD8(+) T-cell compartment of human immunodeficiency virus type 1 (HIV-1)-infected persons, likely because of chronic cell turnover. Although clonal exhaustion of CD8(+) cytotoxic T lymphocytes (CTL) has been proposed as a mechanism for loss of antigen-specific responses, the functional consequences of exhaustion are poorly understood. Here we used telomerase transduction to evaluate the impact of senescence on CTL effector functions. Constitutive expression of telomerase in an HIV-1-specific CTL clone results in enhanced proliferative capacity, in agreement with prior studies of other human cell types. Whereas the CTL remain phenotypically normal in terms of antigenic specificity and requirements for proliferation, their cytolytic and antiviral capabilities are superior to those of control CTL. In contrast, their ability to produce gamma interferon and RANTES is essentially unchanged. The selective enhancement of cytolytic function in memory CTL by ectopic telomerase expression implies that loss of this function (but not cytokine production) is a specific consequence of replicative senescence. These data suggest a unifying mechanism for the in vivo observations that telomere lengths are shortened in the CD8(+) cells of HIV-1-infected persons and that HIV-1-specific CTL are deficient in perforin. Telomerase transduction could therefore be a tool with which to explore a potential therapeutic approach to an important pathophysiologic process of immune dysfunction in chronic viral infection.     

Identification of lanthionine synthase C-like protein-1 as a prominent glutathione binding protein expressed in the mammalian central nervous system

Proteomic experiments were performed to identify novel glutathione (GSH) binding proteins expressed in the mammalian central nervous system. Bovine brain lysate was affinity purified using an immobilized glutathione-Sepharose column. Proteins that bound the immobilized glutathione were eluted with free glutathione and identified by one- and two-dimensional electrophoresis coupled with mass spectrometric analysis of tryptic fragments. Major proteins purified by this technique were glutathione S-transferase-mu (GST-mu) and GST-pi and lanthionine synthase C-like protein-1 (LanCL1). LanCL1 is a mammalian homologue of a prokaryotic enzyme responsible for the synthesis of thioether (lanthionine) cross-links within nascent polypeptide chains, yielding macrocyclic proteins with potent microbicidal activity. An antibody against LanCL1 was generated and applied to immunochemical studies of spinal cord tissue from SOD1G93A transgenic mice, a model for amyotrophic lateral sclerosis (ALS), wherein LanCL1 expression was found to be increased at presymptomatic stages of the disease. These results indicate LanCL1 is a glutathione binding protein possibly significant to neurodegenerative disease.