Two copies of the human apolipoprotein C-I gene are linked closely to the apolipoprotein E gene

The gene for human apolipoprotein (apo) C-I was selected from human genomic cosmid and lambda libraries. Restriction endonuclease analysis showed that the gene for apoC-I is located 5.5 kilobases downstream of the gene for apoE. A copy of the apoC-I gene, apoC-I', is located 7.5 kilobases downstream of the apoC-I gene. Both genes contain four exons and three introns; the apoC-I gene is 4653 base pairs long, the apoC-I' gene 4387 base pairs. In each gene, the first intron is located 20 nucleotides upstream from the translation start signal; the second intron, within the codon of Gly-7 of the signal peptide region; and the third intron, within the codon for Arg39 of the mature plasma protein coding region. The upstream apoC-I gene encodes the known apoC-I plasma protein and differs from the downstream apoC-I' gene in about 9% of the exon nucleotide positions. The most important difference between the exons results in a change in the codon for Gln-2 of the signal peptide region, which introduces a translation stop signal in the downstream gene. Major sequence differences are found in the second and third introns of the apoC-I and apoC-I' genes, which contain 9 and 7.5 copies, respectively, of Alu family sequences. The apoC-I gene is expressed primarily in the liver, and it is activated when monocytes differentiate into macrophages. In contrast, no mRNA product of the apoC-I' gene can be detected in any tissue, suggesting that it may be a pseudogene. The similar structures and the proximity of the apoE and apoC-I genes suggest that they are derived from a common ancestor. Furthermore, they may be considered to be constituents of a family of seven apolipoprotein genes (apoE, -C-I, -C-II, -C-III, -A-I, -A-II, and -A-IV) that have a common evolutionary origin.     

Phosphorylation of smooth muscle caldesmon by calmodulin-dependent protein kinase II. Identification of the phosphorylation sites

Smooth muscle caldesmon was phosphorylated by smooth muscle calmodulin-dependent protein kinase II. The extent of phosphorylation obtained was 5.65 mol of phosphate/mol of caldesmon. Phosphorylated protein was subjected to the complete trypsin proteolysis and the produced phosphopeptides were purified by C-8 reverse phase chromatography. Nine phosphopeptides were isolated and by amino acid sequence analysis, eight phosphorylation sites were identified. According to the published amino acid sequence of chicken gizzard caldesmon (Bryan, J., Imai, M., Lee, R., Moore, P., Cook, R. G., and Lin, W.-G. (1989) J. Biol. Chem. 264, 13873-13879), these sites were serine 26, serine 59, serine 73, threonine 469, serine 475, serine 587, serine 620, and serine 726. The time course of phosphorylation of these sites was also measured and it was concluded that the first site was serine 73, the second site was serine 26, the third site was serine 726, and the fourth site was serine 587. The preferred phosphorylation sites were located in the amino terminus myosin binding domain whereas slower phosphorylation occurred in the carboxyl terminus actin/calmodulin domain.     

Differing rates of cholesterol absorption among inbred mouse strains yield differing levels of HDL-cholesterol

Inbred strains of mice with differing susceptibilities to atherosclerosis possess widely varying plasma HDL levels. Cholesterol absorption and lipoprotein formation were compared between atherosclerosis-susceptible, low-HDL C57BL6/J mice and atherosclerosis-resistant, high-HDL FVBN/J mice. [³H]cholesterol and triglyceride appeared in the plasma of FVB mice gavaged with cholesterol in olive oil at a much higher rate than in C57 mice. The plasma cholesterol was found almost entirely as HDL-cholesterol in both strains. Inhibition of lipoprotein catabolism with Tyloxapol revealed that the difference in the rate of [³H]cholesterol appearance in the plasma was due entirely to a greater rate of chylomicron secretion from the intestine of the FVB mice. Lipid absorption into the 2nd quarter of the small intestine is greater in the FVB mice and indicates that this region may contain the factors that give rise to the differences in absorption observed between the two mouse strains. Additionally, ad libitum feeding prior to cholesterol gavage accentuates the absorption rate differences compared with fasting. The resultant remodeling of the increased levels of chylomicron in the plasma may contribute to increased plasma HDL. Intestinal gene expression analysis reveals several genes that may play a role in these differences, including microsomal triglyceride transfer protein and ABCG8.     

Bioremediation of nitroexplosive wastewater by an yeast isolate <Emphasis Type="Italic">Pichia sydowiorum</Emphasis> MCM Y-3 in fixed film bioreactor

Nitroexplosives are essential for security and defense of the nation and hence their production continues. Their residues and transformed products, released in the environment are toxic to both terrestrial and aquatic life. This necessitates remediation of wastewaters containing such hazardous chemicals to reduce threat to human health and environment. Bioremediation technologies using microorganisms become the present day choice. High Melting Explosive (HMX) is one of the nitroexplosives produced by nitration of hexamine using ammonium nitrate and acetic anhydride and hence the wastewater bears high concentration of nitrate and acetate. The present investigation describes potential of a soil isolate of yeast Pichia sydowiorum MCM Y-3, for remediation of HMX wastewater in fixed film bioreactor (FFBR). The flask culture studies showed appreciable growth of the organism in HMX wastewater under shake culture condition within 5–6 days of incubation at ambient temperature (28 ± 2°C). The FFBR process operated in both batch and continuous mode, with Hydraulic Retention Time (HRT) of 1 week resulted in 50–55% removal in nitrate, 70–88% in acetate, 50–66% in COD, and 28–50% in HMX content. Continuous operation of the reactor showed better removal of nitrate as compared to that in the batch operation, while removal of acetate and COD was comparable in both the modes of operation of the reactor. Insertion of baffles in the reactor increased efficiency of the reactor. Thus, FFBR developed with baffles and operated in continuous mode will be beneficial for bioremediation of high nitrate and acetate containing wastewater using the culture of P. sydowiorum.     

Monitored releases of Rhinoncomimus latipes (Coleoptera: Curculionidae), a biological control agent of mile-a-minute weed (Persicaria perfoliata), 2004–2008

Mile-a-minute weed, Persicaria perfoliata (L.) H. Gross, is an invasive annual vine of Asian origin that has developed extensive monocultures, especially in disturbed open areas in the Mid-Atlantic region of the United States. A host-specific Asian weevil, Rhinoncomimus latipes Korotyaev, was approved for release in North America in 2004, and weevils have been reared at the New Jersey Department of Agriculture Beneficial Insect Laboratory since then. By the end of 2007 more than 53,000 weevils had been reared and released, mostly in New Jersey, but also in Delaware, Maryland, Pennsylvania, and West Virginia. The beetles established at 63 out of 65 sites (96.9%) where they were released between 2004 and 2007, with successful releases consisting of as few as 200 weevils. Weevils were recorded at 30 additional non-release sites in New Jersey, where they had dispersed at an average rate of 4.3 km/year. Standardized monitoring of fixed quadrats was conducted in paired release and control sites at eight locations. Significant differences in mile-a-minute weed populations in the presence and absence of weevils were found at three locations, with reduction in spring densities to 25% or less of what they had been at the start within 2–3 years at release sites, while weed densities at control sites were largely unchanged. Mile-a-minute weed populations at a fourth site were similarly reduced at the release site, but without control data for comparison due to rapid colonization of the paired control site. At the other four locations, all on islands, mile-a-minute weed populations were reduced at both release and control sites without large weevil populations developing, apparently due to environmental conditions such as late frost and extreme drought.     

Detection and Quantification of Functional Genes of Cellulose- Degrading,Fermentative, and Sulfate-Reducing Bacteria and Methanogenic Archaea

Cellulose degradation, fermentation, sulfate reduction, and methanogenesis are microbial processes that coexist in a variety of natural and engineered anaerobic environments. Compared to the study of 16S rRNA genes, the study of the genes encoding the enzymes responsible for these phylogenetically diverse functions is advantageous because it provides direct functional information. However, no methods are available for the broad quantification of these genes from uncultured microbes characteristic of complex environments. In this study, consensus degenerate hybrid oligonucleotide primers were designed and validated to amplify both sequenced and unsequenced glycoside hydrolase genes of cellulose-degrading bacteria, hydA genes of fermentative bacteria, dsrA genes of sulfate-reducing bacteria, and mcrA genes of methanogenic archaea. Specificity was verified in silico and by cloning and sequencing of PCR products obtained from an environmental sample characterized by the target functions. The primer pairs were further adapted to quantitative PCR (Q-PCR), and the method was demonstrated on samples obtained from two sulfate-reducing bioreactors treating mine drainage, one lignocellulose based and the other ethanol fed. As expected, the Q-PCR analysis revealed that the lignocellulose-based bioreactor contained higher numbers of cellulose degraders, fermenters, and methanogens, while the ethanol-fed bioreactor was enriched in sulfate reducers. The suite of primers developed represents a significant advance over prior work, which, for the most part, has targeted only pure cultures or has suffered from low specificity. Furthermore, ensuring the suitability of the primers for Q-PCR provided broad quantitative access to genes that drive critical anaerobic catalytic processes.The gene encoding the 16S small ribosomal subunit has served as a highly suitable target for studying bacterial species. When one obtains 16S rRNA gene sequence information, it is sometimes possible to infer function from an identical match to a well-characterized pure culture. More commonly, however, the similarity to pure cultures is low, and/or the highest similarities correspond to 16S rRNA gene sequences identified without isolation or phenotypic characterization. In either case, care must be taken, because distinct phenotypes [e.g., dissimilatory Fe(III) reduction, chlorate reduction] are found in microorganisms with highly similar (e.g., 99.5%) 16S rRNA gene sequences (1). In addition, 16S rRNA gene surveys of broad phylogenetic groups can be time-, labor-, and cost-intensive. For example, it is estimated that the 16S rRNA gene-based detection of all recognized lineages of sulfate-reducing bacteria (SRB) would require approximately 132 16S rRNA gene-targeted microarray probes (32).A more-direct approach for the study of microbes that span phylogenetic groups is to target them as a physiologically coherent guild by using specific genetic markers (functional genes) for the functions of interest. Functional genes have been successfully targeted in bioremediation studies to investigate microbial populations responsible for the degradation of various contaminants. Some examples include the use of the large alpha subunit of benzylsuccinate synthase to monitor anaerobic hydrocarbon-degrading bacteria (5), the monitoring of ars genes for the identification and quantification of arsenic-metabolizing bacteria (45), and the detection of catechol 1,2-dioxygenase in aromatic-hydrocarbon-degrading Rhodococcus spp. (48). In the field of mine drainage/metal remediation, functional genes have been used to target SRB (17, 26), but the methods have suffered both from a lack of broad specificity for SRB and from the inability to distinguish SRB from sulfur-oxidizing bacteria (SOB). A general challenge to the functional-gene approach has been the relative lack of characterization and unavailability of target sequences. As a consequence, the primer sets that are available tend to be more relevant to pure cultures than to complex environmental samples.Microbial communities in natural and engineered anaerobic environments that utilize cellulose as the primary carbon source, such as those in rumina (56), termite guts (54), decomposing wood (7), sulfate-reducing and methanogenic sediments (9, 22), wetlands (28), and sulfate-reducing bioreactors (26), are particularly challenging to characterize. 16S rRNA gene-based studies have revealed the complexity of these microbial communities and their high levels of phylogenetic and functional diversity. In such anaerobic environments, mineralization of complex organic matter occurs through the concerted action of a variety of microorganisms. Primary fermenters, such as cellulose degraders, break down the complex molecules and ferment the hydrolysis products. Secondary fermenters also ferment the hydrolysis products. When sulfate is available, SRB utilize the fermentation products as carbon and energy sources. In addition, methanogens can also utilize some of the fermentation products. In many cases, functionally important members, such as SRB, are present only as a small fraction of the community (36, 38), making them difficult to detect by use of 16S rRNA gene-targeted fingerprinting methods. Furthermore, the phylogenetic diversity of cellulose degraders, fermenters, and SRB prevents their quantification using a small number of 16S rRNA gene-targeted probes.In this study, degenerate PCR primers were developed, validated, and demonstrated for the amplification of key functional groups in anaerobic environments possessing genes encoding glycoside hydrolases of families 5 (collectively designated cel5 in this study) and 48 (collectively designated cel48 in this study) (cellulose degradation), the alpha subunit of iron hydrogenase (hydA) (fermentation), dissimilatory sulfite reductase (dsrA) (sulfate reduction), and methyl coenzyme M reductase (mcrA) (methanogenesis). This work is particularly novel considering that the vast majority of existing methods are suitable only for pure cultures, especially in the cases of cel5, cel48, and hydA (21, 44, 47). Thus, the approach provides access to uncultured and unsequenced markers, a critical feature for the study of key anaerobic processes in complex environments. Specificity was also enhanced where possible, notably in the case of dsrA, for which existing primers either do not distinguish SRB from SOB (14, 17) or have good alignment with only a narrow range of SRB (31, 52). Finally, all primers in this study were designed and validated for quantitative PCR (Q-PCR), in order to provide valuable quantitative functional information about complex anaerobic communities. The approach is demonstrated on mine drainage remediation systems and is expected to be of broad value to a variety of fields, including advancing the understanding of biohydrogen production, global carbon cycling, and other important biogeochemical processes.