Leaching of Pyrite by Acidophilic Heterotrophic Iron-Oxidizing Bacteria in Pure and Mixed Cultures

Seven strains of heterotrophic iron-oxidizing acidophilic bacteria were examined to determine their abilities to promote oxidative dissolution of pyrite (FeS₂) when they were grown in pure cultures and in mixed cultures with sulfur-oxidizing Thiobacillus spp. Only one of the isolates (strain T-24) oxidized pyrite when it was grown in pyrite-basal salts medium. However, when pyrite-containing cultures were supplemented with 0.02% (wt/vol) yeast extract, most of the isolates oxidized pyrite, and one (strain T-24) promoted rates of mineral dissolution similar to the rates observed with the iron-oxidizing autotroph Thiobacillus ferrooxidans. Pyrite oxidation by another isolate (strain T-21) occurred in cultures containing between 0.005 and 0.05% (wt/vol) yeast extract but was completely inhibited in cultures containing 0.5% yeast extract. Ferrous iron was also needed for mineral dissolution by the iron-oxidizing heterotrophs, indicating that these organisms oxidize pyrite via the “indirect” mechanism. Mixed cultures of three isolates (strains T-21, T-23, and T-24) and the sulfur-oxidizing autotroph Thiobacillus thiooxidans promoted pyrite dissolution; since neither strains T-21 and T-23 nor T. thiooxidans could oxidize this mineral in yeast extract-free media, this was a novel example of bacterial synergism. Mixed cultures of strains T-21 and T-23 and the sulfur-oxidizing mixotroph Thiobacillus acidophilus also oxidized pyrite but to a lesser extent than did mixed cultures containing T. thiooxidans. Pyrite leaching by strain T-23 grown in an organic compound-rich medium and incubated either shaken or unshaken was also assessed. The potential environmental significance of iron-oxidizing heterotrophs in accelerating pyrite oxidation is discussed.     

Spermatozoal ultrastructure in the spiny lobster Jasus novaehollandiae Holthuis, 1963 (Palinuridae,Palinura, Decapoda)

The spermatozoal ultrastructure of the spiny lobster Jasus novaehollandiae is most similar to that in other investigated palinurans and, in particular, to the spermatozoa of Panulirus species. Shared characters include the globular nucleus penetrated by the bases of three or more microtubular arms; an anteriorly situated cytoplasmic zone with mitochondria and conspicuous lamellar bodies; a complex, four-zoned acrosomal vesicle (however, lacking the crystalline region present in Panulirus) with a homogeneous region; a scroll region; a flocculent region; and a region of periacrosomal material that forms finger-like involutions into the flocculent region. The related scyllarid slipper lobsters (Scyllarus and Thenus) possess spermatozoa with acrosome morphology similar to that of Jasus, but the sperm is generally more flattened, numerous radiating acrosome fins are present, and the microtubular arms (in Scyllarus) are cytoplasmic in origin and not nuclear. Sperm morphology provides preliminary evidence in support of the hypothesis of two independent lines of evolution in the Palinuridae but investigation into additional taxa within this group is required. J. Morphol. 236:117-126, 1998. © 1998 Wiley-Liss, Inc.     

The Ultrastructure of the Spermatozoa of Three Species of Myobatrachid Frogs (Anura,Amphibia) with Phylogenetic Considerations

Comparison of the spermatozoa of three genera of limnodynastines (Myobatrachidae: Anura) with those of 42 other species of frogs (in 11 families) previously examined allows the following phylogenetic inferences. The bufonoid neobatrachians (corresponding with the Arcifera of some classifications) form a monophyletic assemblage which is characterized by the possession, unique for the Anura, of a conical perforatorium; the rod-like perforatorium, which is plesiomorphic for tetrapods, is absent. Of the taxa investigated, the myobatrachids appear to be the sister-group of the remaining bufonoids, here termed eubufonoids (leptodactylids, rhinodermatids, hylids, and bufonids). The spermatozoa of the myobatrachids Limnodynastes, Neobatrachus, and Mixophyes are very similar to each other despite extremely varied fertilization biology. Symplesiomorphies for the Anura which they exhibit include the conical acrosome with subacrosomal material, the elongate, cylindrical nucleus, single flagellum and, paralleling this, an undulating membrane which is supported by a longitudinal element, the axial (major) fibre, the latter being accompanied in the midpiece by mitochondria. A myobatrachid synapomorphy appears to be the presence of a periaxial sheath enclosing the axial fibre of the flagellum. Forward extension of the axial fibre into the centriolar fossa in myobatrachids is seen elsewhere only exceptionally (in the bufonid Nectophrynoides). Bufonids, and most other eubufonoids including leptodactylids, differ apomorphically from Limnodynastes, and Neobatrachus in location of the mitochondria at the axonemal end (and in a collar) rather than the axial fibre end of the undulating membrane. In Mixophyes, mitochondria surround the nuclear-axonemal junction in a cytoplasmic droplet and are probably shed with this at maturity.     

Biosynthesis of the angiogenesis inhibitor borrelidin by Streptomyces parvulus Tü4055: insights into nitrile formation

The 18-membered polyketide macrolide borrelidin exhibits a number of important biological activities, including potent angiogenesis inhibition. This has prompted two recent total syntheses as well as the cloning of the biosynthetic gene cluster from Streptomyces parvulus Tü4055. Borrelidin possesses some unusual structural characteristics, including a cyclopentane carboxylic acid moiety at C17 and a nitrile moiety at C12 of the macrocyclic ring. Nitrile groups are relatively rare in nature, and little is known of their biosynthesis during secondary metabolism. The nitrile group of borrelidin is shown here to arise from the methyl group of a methylmalonyl-CoA extender unit incorporated during polyketide chain extension. Insertional inactivation of two genes in the borrelidin gene cluster, borI (coding for a cytochrome P450 monooxygenase) and borJ (coding for an aminotransferase), generated borrelidin non-producing mutants. These mutants accumulated different compounds lacking the C12 nitrile moiety, with the product of the borI-minus mutant (12-desnitrile-12-methyl-borrelidin) possessing a methyl group and that of the borJ-minus mutant (12-desnitrile-12-carboxyl-borrelidin) a carboxyl group at C12. The former but not the latter was converted into borrelidin when biotransformed by an S. parvulus mutant that is deficient in the biosynthesis of the borrelidin starter unit. This suggests that 12-desnitrile-12-methyl-borrelidin is a competent biosynthetic intermediate, whereas the carboxylated derivative is a shunt metabolite. Bioconversion of 12-desnitrile-12-methyl-borrelidin into borrelidin was also achieved in a heterologous system co-expressing borI and borJ in Streptomyces albus J1074. This bioconversion was more efficient when borK, which is believed to encode a dehydrogenase, was simultaneously expressed with borI and borJ. On the basis of these findings, a pathway is proposed for the formation of the nitrile moiety during borrelidin biosynthesis.     

Antiviral cytokines induce hepatic expression of the granzyme B inhibitors, proteinase inhibitor 9 and serine proteinase inhibitor 6

Expression of the granzyme B inhibitors, human proteinase inhibitor 9 (PI-9), or the murine orthologue, serine proteinase inhibitor 6 (SPI-6), confers resistance to CTL or NK killing by perforin- and granzyme-dependent effector mechanisms. In light of prior studies indicating that virally infected hepatocytes are selectively resistant to this CTL effector mechanism, the present studies investigated PI-9 and SPI-6 expression in hepatocytes and hepatoma cells in response to adenoviral infection and to cytokines produced during antiviral immune responses. Neither PI-9 nor SPI-6 expression was detected by immunoblotting in uninfected murine or human hepatocytes. Similarly, human Huh-7 hepatoma cells were found to express only very low levels of PI-9 relative to levels detected in perforin- and granzyme-resistant CTL or lymphokine-activated killer cells. Following in vivo adenoviral infection or in vitro culture with IFN-alphabeta or IFN-gamma, SPI-6 expression was induced in murine hepatocytes. Similarly, after culture with IFN-alpha, induction of PI-9 mRNA and protein expression was observed in human hepatocytes and Huh-7 cells. IFN-gamma and TNF-alpha also induced 4- to 10-fold higher levels of PI-9 mRNA expression in Huh-7 cells, whereas levels of mRNA encoding a related serine proteinase inhibitor, proteinase inhibitor 8, were unaffected by culture of Huh-7 cells with IFN-alpha, IFN-gamma, or TNF-alpha. These findings indicate that cytokines that promote antiviral cytopathic responses also regulate expression of the cytoprotective molecules, PI-9 and SPI-6, in hepatocytes that are potential targets of CTL and NK effector mechanisms.     

Interactions between Sec complex and prepro-alpha-factor during posttranslational protein transport into the endoplasmic reticulum

Posttranslational translocation of prepro-alpha-factor (ppalphaF) across the yeast endoplasmic reticulum membrane begins with the binding of the signal sequence to the Sec complex, a membrane component consisting of the trimeric Sec61p complex and the tetrameric Sec62p/63p complex. We show by photo-cross-linking that the signal sequence is bound directly to a site where it contacts simultaneously Sec61p and Sec62p, suggesting that there is a single signal sequence recognition step. We found no evidence for the simultaneous contact of the signal sequence with two Sec61p molecules. To identify transmembrane segments of Sec61p that line the actual translocation pore, a late translocation intermediate of ppalphaF was generated with photoreactive probes incorporated into the mature portion of the polypeptide. Cross-linking to multiple regions of Sec61p was observed. In contrast to the signal sequence, neighboring positions of the mature portion of ppalphaF had similar interactions with Sec61p. These data suggest that the channel pore is lined by several transmembrane segments, which have no significant affinity for the translocating polypeptide chain.     

An application of Bayesian QTL mapping to early development in double haploid lines of rainbow trout including environmental effects

A Bayesian model and variable dimensional parameter estimation based on Markov chain Monte Carlo was applied to map quantitative trait loci (QTLs) in a doubled haploid mapping population of rainbow trout. To increase power, the analysis was performed using the multiple-QTL model, which simultaneously accounted for all the environmental and genetic main effects that influence the expression of early development life history traits. By doing so we obtained the posterior estimated effects for the environmental factors as well as the number, positions, and the effects for the QTLs. The analyses revealed QTLs for time at hatching, embryonic length and weight at swim-up stage. The posterior expectation of the number of QTLs in different linkage groups shows that at least four QTLs are needed to explain the observed differences in early development between the clonal lines. The Bayesian method effectively combined all the information available to accurately position these QTLs in the rainbow trout genome.     

CBP1 Associates with the Dictyostelium Cytoskeleton and Is Important for Normal Cell Aggregation under Certain Developmental Conditions

In cells of the eukaryotic microorganism Dictyostelium discoideum, at least eight small, four-EF-hand Ca²⁺-binding proteins of unknown function are expressed at specific times during development. One of these proteins, calcium-binding protein 1 (CBP1), first appears just prior to cell aggregation and then is present at relatively constant levels throughout development. To determine a role for CBP1 during development, the protein was used as bait in a yeast two-hybrid screen to reveal putative CBP1-interacting proteins. Two proteins identified in this screen were the actin-binding proteins, protovillin and EF-1α. Using an in vitro binding assay, both of these proteins were found to interact with CBP1 in the absence of Ca²⁺, but the interaction of CBP1 with EF-1α was increased substantially by Ca²⁺. CBP1 was also shown by fluorescence microscopy and by binding assays to associate with the actin cytoskeleton of Dictyostelium cells during development, and these interactions were partially Ca²⁺-dependent. cbpA-null cells grew normally, but under certain developmental conditions, cell aggregation was prolonged and irregular. This defect in aggregation appeared to be related to a general reduction in cell motility rather than to a decrease in the ability of the cells to respond to the chemoattractant cAMP. Together, these results suggest that CBP1 might function to help regulate the reorganization of the Dictyostelium actin cytoskeleton during cell aggregation.