Inorganic sulfur turnover in oligohaline estuarine sediments

Inorganic sulfur turnover was examined in oligohaline (salinity < 2 g kg^-1) Chesapeake Bay sediments during the summer. Cores incubated for < 3 hr exhibited higher sulfate reduction (SR) rates (13–58 mmol m^-2 d^-1) than those incubated for 3–8 hr (3–8 mmol m^-2 d^-1). SR rates (determined with³⁵SO ₄ ^2- ) increased with depth over the top few cm to a maximum at 5 cm, just beneath the boundary between brown and black sediment. SR rates decreased below 5 cm, probably due to sulfate limitation (sulfate < 25 μM). Kinetic experiments yielded an apparent half-saturating sulfate concentration (K_s) of 34 μM, ≈ 20-fold lower than that determined for sediments from the mesohaline region of the estuary. Sulfate loss from water overlying intact cores, predicted on the basis of measured SR rates, was not observed over a 28-hr incubation period. Reduction of³⁵SO ₄ ^2- during diffusion experiments with intact core segments from 0–4 and 5–9 cm horizons was less than predicted by non-steady state diagenetic models based on³⁵SO ₄ ^2- reduction in whole core injection experiments. The results indicate that net sulfate flux into sediments was an order of magnitude lower than the gross sulfur turnover rate. Solid phase reduced inorganic sulfur concentrations were only 2–3 times less than those in sediments from the mesohaline region of the Bay, despite the fact that oligohaline bottom water sulfate concentrations were 10-fold lower. Our results demonstrate the potential for rapid SR in low salinity estuarine sediments, which are inhabited by sulfate-reducing bacteria with a high affinity for sulfate, and in which sulfide oxidation processes replenish the pore water sulfate pool on a time scale of hours.     

Maximum likelihood estimation of the heterogeneity of substitution rate among nucleotide sites

This paper presents a maximum likelihood approach to estimating the variation of substitution rate among nucleotide sites. We assume that the rate varies among sites according to an invariant+gamma distribution, which has two parameters: the gamma parameter alpha and the proportion of invariable sites theta. Theoretical treatments on three, four, and five sequences have been conducted, and computer program have been developed. It is shown that rho = (1 + theta alpha)/(1 + alpha) is a good measure for the rate heterogeneity among sites. Extensive simulations show that (1) if the proportion of invariable sites is negligible, i.e., theta = 0, the gamma parameter alpha can be satisfactorily estimated, even with three sequences; (2) if the proportion of invariable sites is not negligible, the heterogeneity rho can still be suitably estimated with four or more sequences; and (3) the distances estimated by the proposed method are almost unbiased and are robust against violation of the assumption of the invariant + gamma distribution.      

Nasal Immunization of Mice with Human Papillomavirus Type 16 Virus-Like Particles Elicits Neutralizing Antibodies in Mucosal Secretions

To specifically induce a mucosal antibody response to purified human papillomavirus type 16 (HPV16) virus-like particles (VLP), we immunized female BALB/c mice orally, intranasally, and/or parenterally and evaluated cholera toxin (CT) as a mucosal adjuvant. Anti-HPV16 VLP immunoglobulin G (IgG) and IgA titers in serum, saliva, and genital secretions were measured by enzyme-linked immunosorbent assay (ELISA). Systemic immunizations alone induced HPV16 VLP-specific IgG in serum and, to a lesser extent, in genital secretions but no secretory IgA. Oral immunization, even in the presence of CT, was inefficient. However, three nasal immunizations with 5 μg of VLP given at weekly intervals to anesthetized mice induced high (>10⁴) and long-lasting (>15 weeks) titers of anti-HPV16 VLP antibodies in all samples, including IgA and IgG in saliva and genital secretions. CT enhanced the VLP-specific antibody response 10-fold in serum and to a lesser extent in saliva and genital secretions. Nasal immunization of conscious mice compared to anesthetized mice was inefficient and correlated with the absence of uptake of a marker into the lung. However, a 1-μg VLP systemic priming followed by two 5-μg VLP intranasal boosts in conscious mice induced both HPV16 VLP-specific IgG and IgA in secretions, although the titers were lower than in anesthetized mice given three intranasal immunizations. Antibodies in serum, saliva, and genital secretions of immunized mice were strongly neutralizing in vitro (50% neutralization with ELISA titers of 65 to 125). The mucosal and systemic/mucosal HPV16 VLP immunization protocols that induced significant titers of neutralizing IgG and secretory IgA in mucosal secretions in mice may be relevant to genital HPV VLP-based human vaccine trials.     

Recovery of Humic-Reducing Bacteria from a Diversity of Environments

To evaluate which microorganisms might be responsible for microbial reduction of humic substances in sedimentary environments, humic-reducing bacteria were isolated from a variety of sediment types. These included lake sediments, pristine and contaminated wetland sediments, and marine sediments. In each of the sediment types, all of the humic reducers recovered with acetate as the electron donor and the humic substance analog, 2,6-anthraquinone disulfonate (AQDS), as the electron acceptor were members of the family Geobacteraceae. This was true whether the AQDS-reducing bacteria were enriched prior to isolation on solid media or were recovered from the highest positive dilutions of sediments in liquid media. All of the isolates tested not only conserved energy to support growth from acetate oxidation coupled to AQDS reduction but also could oxidize acetate with highly purified soil humic acids as the sole electron acceptor. All of the isolates tested were also able to grow with Fe(III) serving as the sole electron acceptor. This is consistent with previous studies that have suggested that the capacity for Fe(III) reduction is a common feature of all members of the Geobacteraceae. These studies demonstrate that the potential for microbial humic substance reduction can be found in a wide variety of sediment types and suggest that Geobacteraceae species might be important humic-reducing organisms in sediments.     

The cDNA cloning and molecular evolution of reptile and pigeon lactate dehydrogenase isozymes

The cDNAs encoding lactate dehydrogenase isozymes LDH-A (muscle) and LDH-B (heart) from alligator and turtle and LDH-A, LDH-B, and LDH-C (testis) from pigeon were cloned and sequenced. The evolutionary relationships among vertebrate LDH isozymes were analyzed. Contrary to the traditional belief that the turtle lineage branched off before the divergence between the lizard/alligator and bird lineages, the turtle lineage was found to be clustered with either the alligator lineage or the alligator-bird clade, while the lizard lineage was found to have branched off before the divergence between the alligator/turtle and bird lineages. The pigeon testicular LDH-C isozyme was evidently duplicated from LDH-B (heart), so it is not orthologous to the mammalian testicular LDH-C isozymes.      

Bias-corrected paralinear and LogDet distances and tests of molecular clocks and phylogenies under nonstationary nucleotide frequencies

The statistical properties of the paralinear and LogDet distances under nonstationary nucleotide frequencies were studied. First, we developed formulas for correcting the estimation biases of the paralinear and LogDet distances, i.e., the bias-corrected distance is estimated by dc = d - 2var(d), where d and var(d) are the estimated distance and sampling variance, respectively. The performances of these formulas and the formulas for sampling variances were examined by computer simulation. Second, we developed a method for estimating the variance- covariance matrix of paralinear distances, so that statistical tests of DNA phylogenies can be conducted in the nonstationary case. Third, a new LogDet-based method for testing the molecular clock hypothesis was developed under nonstationary nucleotide frequencies.      

Dual methylation pathways in lignin biosynthesis

Caffeoyl-coenzyme A (CoA) O-methyltransferase (CCoAOMT) has been proposed to be involved in an alternative methylation pathway of lignin biosynthesis. However, no direct evidence has been available to confirm that CCoAOMT is essential for lignin biosynthesis. To understand further the methylation steps in lignin biosynthesis, we used an antisense approach to alter O-methyltransferase (OMT) gene expression and investigated the consequences of this alteration. We generated transgenic tobacco plants with a substantial reduction in CCoAOMT as well as plants with a simultaneous reduction in both CCoAOMT and caffeic acid O-methyltransferase (CAOMT). Lignin analysis showed that the reduction in CCoAOMT alone resulted in a dramatic decrease in lignin content. The reduction in CCoAOMT also led to a dramatic alteration in lignin composition. Both guaiacyl lignin and syringyl lignin were reduced in the transgenic plants. However, guaiacyl lignin was preferentially reduced, which resulted in an increase in the S/G (syringl/guaiacyl) ratio. We have also analyzed lignin content and composition in transgenic plants having a simultaneous reduction in both CCoAOMT and CAOMT. The reduction in both OMTs resulted in a further decrease in total lignin content. This is in sharp contrast to the effect that resulted from the reduction in CAOMT alone, which only decreased the syringl lignin unit without a reduction in overall lignin content. These results unequivocally demonstrate that methylation reactions in lignin biosynthesis are catalyzed by both CCoAOMT and CAOMT.