Protein folding within the cell is influenced by controlled rates of polypeptide elongation.

Previous studies have proposed that specific translational pauses have evolved to promote protein folding inside the cell by temporally separating the folding of specific regions of some polypeptide chains during their synthesis. Here we show that this is the case for a bifunctional protein in Saccharomyces cerevisiae. The yeast TRP3 gene contains a translational pause comprising ten contiguous non-preferred codons within its second functional domain (indoleglycerol phosphate synthase). Site-directed mutagenesis was used to remove this translational pause by increasing the codon bias of the region without changing the amino acid sequence of the protein (to create the gene TRP3pr: pause replaced). The TRP3pr gene was able to complement a trp3:: URA3 null mutation in yeast. No significant differences in the doubling times of TRP3 or TRP3pr yeast transformants were observed during growth at 25 degrees C, 30 degrees C or 37 degrees C, or in the presence of sublethal concentrations of the analogue, 5-methyltryptophan. However, further analysis of TRP3 and TRP3pr transformants revealed that the removal of the translational pause causes a 1.5-fold decrease in indoleglycerol phosphate synthase activity per TRP3 mRNA. This observation which is statistically significant (P < 0.05) and reproducible, suggests that translational pausing promotes the correct intracellular folding of the TRP3 protein.     

Isolation of soluble elastin from lathyritic chicks. Comparison to tropoelastin from copper deficient pigs.

Tropoelastin was isolated from the aortas of chicks rendered lathyritic by treatment with beta-aminopropionitrile. The soluble elastin was judged homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis and possessed an estimated molecular weight of 70000. Automated sequential analysis revealed that the N-terminal region of the chick tropoelastin is very homologous to tropoelastin isolated from copper-deficient piglets. N-terminal analysis of a trypsin digest of chick tropoelastin showed that tyrosine frequently is found adjacent to lysine residues. This positioning of tyrosine residues may be significant in terms of a possible regulatory role in elastin cross-link formation.     

Functional changes in human leukemic cell line HL-60. A model for myeloid differentiation

Polar solvents induce terminal differentiation in the human promyelocytic leukemia cell line HL-60. The present studies describe the functional changes that accompany the morphologic progression from promyelocytes to bands and poly-morphonuclear leukocytes (PMN) over 9 d of culture in 1.3 percent dimethylsulfoxide (DMSO). As the HL-60 cells mature, the rate of O(2-) production increase 18-fold, with a progressive shortening of the lag time required for activation. Hexosemonophosphate shunt activity rises concomitantly. Ingestin of paraffin oil droplets opsonized with complement or Ig increases 10-fold over 9 d in DMSO. Latex ingestion per cell by each morphologic type does not change significantly, but total latex ingestion by groups of cells increases with the rise in the proportion of mature cells with greater ingestion capacities. Degranulation, as measured by release of β-glucuronidase, lysozyme, and peroxidase, reaches maximum after 3-6 d in DMSO, then declines. HL-60 cells contain no detectable lactoferrin, suggesting that their secondary granules are absent or defective. However, they kill staphylococci by day 6 in DMSO. Morphologically immature cells (days 1-3 in DMSO) are capable of O(2-) generation, hexosemonophosphate shunt activity, ingestion, degranulation, and bacterial killing. Maximal performance of each function by cells incubated in DMSO for longer periods of time is 50-100 percent that of normal PMN. DMSO- induced differentiation of HL-60 cells is a promising model for myeloid development.     

Influence of applied electrical fields on yeast and hyphal growth of Candida albicans

Yeast cells of Candida albicans which had been attached to polylysine-coated microscope slides were induced to form buds or germ tubes in the presence of external electrical fields. The sites of budding and germ tube formation and the growth of germ tubes and hyphal branches were polarized preferentially towards the cathode. Buds were not converted to pseudohyphae or germ tubes by the field and the field had no effect on the positioning of nuclei or septa in the yeast cell or germ tube. Buds were less polarized than germ tubes at any given applied voltage. The polarization of buds reached a peak at an electrical field of 12 mV per cell. Polarization of germ tubes was biphasic, increasing rapidly with increasing field strengths up to 5 mV per cell, and then more slowly in stronger fields. An electrical field was only required for a fraction of the time taken for germ tubes to start to form, so cells retained a memory of experiencing an electrical field which influenced the selection of sites of evagination. Increasing the electrical field delayed the time of germ tube evagination and inhibited the rate of germ tube extension. Unlike previous findings with other filamentous fungi, germ tubes grew unidirectionally towards the cathode for extended periods and did not deviate to a perpendicular orientation. This result suggests that the septal pore of the filamentous form may have high electrical resistance and would act as an effective barrier to solute transport between intercalary compartments.     

Computing approximate standard errors for genetic parameters derived from random regression models fitted by average information REML

Approximate standard errors (ASE) of variance components for random regression coefficients are calculated from the average information matrix obtained in a residual maximum likelihood procedure. Linear combinations of those coefficients define variance components for the additive genetic variance at given points of the trajectory. Therefore, ASE of these components and heritabilities derived from them can be calculated. In our example, the ASE were larger near the ends of the trajectory.     

ArrayIDer: automated structural re-annotation pipeline for DNA microarrays

Background  

Systems biology modeling from microarray data requires the most contemporary structural and functional array annotation. However, microarray annotations, especially for non-commercial, non-traditional biomedical model organisms, are often dated. In addition, most microarray analysis tools do not readily accept EST clone names, which are abundantly represented on arrays. Manual re-annotation of microarrays is impracticable and so we developed a computational re-annotation tool (ArrayIDer) to retrieve the most recent accession mapping files from public databases based on EST clone names or accessions and rapidly generate database accessions for entire microarrays.     

Unrestricted somatic stem cells from human umbilical cord blood grow in serum-free medium as spheres

Background  

Human umbilical cord blood-derived unrestricted somatic stem cells (USSCs), which are capable of multilineage differentiation, are currently under investigation for a number of therapeutic applications. A major obstacle to their clinical use is the fact that in vitro expansion is still dependent upon fetal calf serum, which could be a source of pathogens. In this study, we investigate the capacity of three different stem cell culture media to support USSCs in serum-free conditions; HEScGRO™, PSM and USSC growth medium^ACF. Our findings demonstrate that USSCs do not grow in HEScGRO™ or PSM, but we were able to isolate, proliferate and maintain multipotency of three USSC lines in USSC growth medium^ACF.     

Complete Genome Sequence of the Aerobic Facultative Methanotroph Methylocella silvestris BL2

Methylocella silvestris BL2 is an aerobic methanotroph originally isolated from an acidic forest soil in Germany. It is the first fully authenticated facultative methanotroph. It grows not only on methane and other one-carbon (C₁) substrates, but also on some compounds containing carbon-carbon bonds, such as acetate, pyruvate, propane, and succinate. Here we report the full genome sequence of this bacterium.Methylocella spp. are abundant in acidic soils and wetlands and help attenuate methane emissions from these habitats (2). They are unique in several ways compared to all other known aerobic methanotrophs. Notably, they lack extensive internal membrane systems and also appear to lack the particulate methane monooxygenase (pMMO) enzyme found in all other methanotrophs (6). Instead, they use only a soluble methane monooxygenase (sMMO) for methane oxidation. In addition, Methylocella spp. are not limited like other methanotrophs to growing on one-carbon (C₁) compounds but also utilize a number of multicarbon compounds (3). The genome of Methylocella silvestris BL2 (4) was sequenced, assembled, and annotated by the Joint Genome Institute (U.S. Department of Energy; http://www.jgi.doe.gov/sequencing/strategy.html). A total of 38,459 reads (∼6× coverage), including 32,993 paired-end shotgun Sanger reads, 5,040 Roche 454 reads, and 580 finishing reads were included in the final assembly. Three lanes of Solexa data were used to polish the project.The genome size is 4.3 Mbp. The G+C content is 63%. In total, 3,917 candidate genes were predicted and 99 pseudogenes were found. Functionality was assigned to 67.9% of the genes, while 30.9% of the genes could not be assigned any known function. Based on BLASTP searches against the KEGG (Kyoto Encyclopedia of Genes and Genomes) database, 3,413 out of 3,917 (87.1%) candidate genes have significant similarity to genes from Proteobacteria. Only 11 and 14 genes have best hits to genes from Archaea and Eukarya, respectively. All tRNA-encoding regions were identified, and two identical rRNA operons were found.The absence of any pmoCAB genes encoding a pMMO enzyme that is present in all other genera of methanotrophs is now conclusively verified by the genome sequence. A complete operon encoding sMMO (mmoXYBZDC) was verified, as was a complete operon encoding methanol dehydrogenase (mxaFJGIRSACKLDEH) and all genes necessary for fixation of methane-derived carbon via the serine cycle. Genes encoding key enzymes in both the tetrahydrofolate and the tetrahydromethanopterin-mediated formaldehyde oxidation pathways were found.M. silvestris can grow on two-carbon compounds, particularly acetate. Acetate kinase- and phosphotransacetylase-encoding genes are present, allowing acetate to be fed into the tricarboxylic acid (TCA) cycle. Genes encoding glyoxylate bypass enzymes (i.e., isocitrate lyase and malate synthase) have been identified. This pathway is essential for bacteria when growing on two-carbon compounds (1). The bacterium can also grow on C₃ and C₄ compounds, and a full gene set encoding enzymes of the TCA cycle is present, including genes encoding α-ketoglutarate dehydrogenase, which are lacking in some methanotrophs. Interestingly, a gene cluster encoding di-iron-containing multi-component propane monooxygenase is also present.The genome sequence of M. silvestris is the first genome available for an alphaproteobacterial methanotroph. It joins the gammaproteobacterial methanotroph Methylococcus capsulatus Bath (7) and the verrucomicrobial methanotroph “Methylacidiphilum infernorum” (5). More detailed analyses of the genome as well as comparative analysis with obligate methanotrophs will provide deeper insight into the metabolism of this fascinating bacterium.