Cell cycle population effects in perturbation studies

Growth condition perturbation or gene function disruption are commonly used strategies to study cellular systems. Although it is widely appreciated that such experiments may involve indirect effects, these frequently remain uncharacterized. Here, analysis of functionally unrelated Saccharyomyces cerevisiae deletion strains reveals a common gene expression signature. One property shared by these strains is slower growth, with increased presence of the signature in more slowly growing strains. The slow growth signature is highly similar to the environmental stress response (ESR), an expression response common to diverse environmental perturbations. Both environmental and genetic perturbations result in growth rate changes. These are accompanied by a change in the distribution of cells over different cell cycle phases. Rather than representing a direct expression response in single cells, both the slow growth signature and ESR mainly reflect a redistribution of cells over different cell cycle phases, primarily characterized by an increase in the G1 population. The findings have implications for any study of perturbation that is accompanied by growth rate changes. Strategies to counter these effects are presented and discussed.     

High-level production of animal-free recombinant transferrin from Saccharomyces cerevisiae

Background

Microorganisms that are exposed to pollutants in the environment, such as metals/metalloids, have a remarkable ability to fight the metal stress by various mechanisms. These metal-microbe interactions have already found an important role in biotechnological applications. It is only recently that microorganisms have been explored as potential biofactories for synthesis of metal/metalloid nanoparticles. Biosynthesis of selenium (Se⁰) nanospheres in aerobic conditions by a bacterial strain isolated from the coalmine soil is reported in the present study.

Results

The strain CM100B, identified as Bacillus cereus by morphological, biochemical and 16S rRNA gene sequencing [GenBank:GU551935.1] was studied for its ability to generate selenium nanoparticles (SNs) by transformation of toxic selenite (SeO₃ ^2-) anions into red elemental selenium (Se⁰) under aerobic conditions. Also, the ability of the strain to tolerate high levels of toxic selenite ions was studied by challenging the microbe with different concentrations of sodium selenite (0.5 mM-10 mM). ESEM, AFM and SEM studies revealed the spherical Se⁰ nanospheres adhering to bacterial biomass as well as present as free particles. The TEM microscopy showed the accumulation of spherical nanostructures as intracellular and extracellular deposits. The deposits were identified as element selenium by EDX analysis. This is also indicated by the red coloration of the culture broth that starts within 2-3 h of exposure to selenite oxyions. Selenium nanoparticles (SNs) were further characterized by UV-Visible spectroscopy, TEM and zeta potential measurement. The size of nanospheres was in the range of 150-200 nm with high negative charge of -46.86 mV.

Conclusions

This bacterial isolate has the potential to be used as a bionanofactory for the synthesis of stable, nearly monodisperse Se⁰ nanoparticles as well as for detoxification of the toxic selenite anions in the environment. A hypothetical mechanism for the biogenesis of selenium nanoparticles (SNs) involving membrane associated reductase enzyme(s) that reduces selenite (SeO₃ ^2-) to Se⁰ through electron shuttle enzymatic metal reduction process has been proposed.     

Biologically relevant effects of mRNA amplification on gene expression profiles

Background  

Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results.     

Tenascin in the human intervertebral disc: alterations with aging and disc degeneration

Our objective for this study was to determine the presence and distribution of tenascin in the human intervertebral disc. The tenascins are a family of extracellular matrix proteins with repeated structural domains homologous to epidermal growth factor, fibronectin type III and the fibrinogens. Little is known about the presence of this protein in the disc. Ten normal human discs donated from subjects newborn to 15 years old, 10 control discs from adult donors aged 24-41 years, and 11 surgical disc specimens from patients aged 26-76 years were examined for immunolocalization of tenascin. In young discs, tenascin was localized throughout the annulus; in the nucleus, localization was confined to pericellular matrix. In adult control and degenerating disc specimens, tenascin in the annulus was localized primarily in pericellular matrix regions encircling either single cells or clusters of disc cells; in rare instances localization was more diffuse in the intraterritorial matrix. In young, healthy disc, tenascin was abundant throughout the annulus. In contrast, degenerating discs in adults showed a localization restricted to the pericellular, and rarely, more restricted intraterritorial matrix. These observations indicate that changes in the amount and distribution of tenascin may have a role in disc aging and degeneration, possibly by modulating fibronectin-disc-cell interactions, and causing alterations in the shape of disc cells.     

The chemokine,CXCL16, and its receptor,CXCR6, are constitutively expressed in human annulus fibrosus and expression of CXCL16 is up-regulated by exposure to IL-1ß in vitro

Chemokines are an important group of soluble molecules with specialized functions in inflammation. The roles of many specialized chemokines and their receptors remain poorly understood in the human intervertebral disc. We investigated CXCL16 and its receptor, CXCR6, to determine their immunolocalization in disc tissue and their presence following exposure of cultured human annulus fibrosus cells to proinflammatory cytokines. CXCL16 is a marker for inflammation; it also can induce hypoxia-inducible factor 1α (HIF-1α), which is a phenotypic marker of heathy nucleus pulposus tissue. We found CXCL16 and CXCR6 immunostaining in many cells of the annulus portion of the disc. Molecular studies showed that annulus fibrosus cells exposed to IL-1ß, but not TNF-α, exhibited significant up-regulation of CXCL16 expression vs. control cells. There was no significant difference in the percentage of annulus cells that exhibited immunolocalization of CXCL16 in grade I/II, grade III or grade IV/V specimens. The presence of CXCL16 and its receptor, CXCR6, in the annulus in vivo suggests the need for future research concerning the role of this chemokine in proinflammatory functions, HIF-1α expression and disc vascularization.     

A microbore high-performance liquid chromatography strategy for the purification of polypeptides for gas-phase sequence analysis. Structural studies on the murine transferrin receptor

We describe herein the use of reversed-phase high-performance liquid chromatography coupled with the novel application of short (10 cm or less) microbore columns (2 mm internal diameter) to fractionate and purify a number of tryptic peptides generated from approximately 200 pmol purified murine transferrin receptor. The use of reversed-phase microbore columns permits the recovery of submicrogram amounts of purified polypeptides in high yield (greater than 90%) in small eluent volumes (20-60 microliter). In this manner, purified polypeptides can be loaded directly onto the gas-phase sequencer without further manipulation. This procedure avoids sample loss, which frequently occurs with other forms of concentration (e.g. lyophilization, evaporation). The application of second-order-derivative ultraviolet spectroscopy, using a diode array detector, for the analysis of aromatic aminoacid-containing peptides in complex tryptic digests is described. N-terminal amino acid sequence analyses were performed on six tryptic peptides, yielding 105 unique assignments; this corresponds to approximately 14% of the molecule. A comparison of this amino acid sequence information with the primary structure of human transferrin receptor deduced from the mRNA sequence [Nature (Lond.) 311, 675-678 (1984); Cell 39, 267-274 (1984)] reveals, with the exception of one tryptic peptide, a very close sequence homology between the murine and human transferrin receptors.     

Cephenniini with prothoracic glands and internal cavities: new taxa,enigmatic characters and phylogeny of the Cephennomicrus group of genera (Coleoptera,Staphylinidae, Scydmaeninae)

Genera of the Cephennomicrus group of the Cephenniini (Scydmaenitae) are revised, and the following new taxa are described: Trichokrater gen.n. , Trichokrater ekkentros sp.n. (type species of Trichokrater) (Borneo), Cephennococcus gen.n. , Cephennococcus kuchingensis sp.n. (type species of Cephennococcus) (Borneo), Cephennococcus kenyirensis sp.n. (West Malaysia), Cephennococcus crassus sp.n. (Borneo), Cephennococcus minutissimus sp.n. (West Malaysia), Pomphopsilla gen.n. , Pomphopsilla luhya sp.n. (type species of Pomphopsilla) (Kenya) and Pomphopsilla soror sp.n. (Kenya). Unique subcuticular pockets with setose openings on the pronotum of Trurlia and Trichokrater are identified as glandular structures. Enigmatic internal prothoracic cavities are described for the first time in Scydmaeninae (in Cephennococcus, Pomphopsilla and a female of an undescribed genus from Sulawesi); their fine structure and function remain unknown. Parsimony‐based cladistic analysis of the adult morphology of genera of Cephenniini provided robust evidence for a monophyly of the Cephennomicrus group, composed of Cephennomicrus, Cephennula, Lathomicrus, Pomphopsilla, Cephennococcus, Trurlia, Trichokrater and two undescribed Oriental genera known from females only; this distinct and well‐supported lineage is a sister group of Cephennodes + Hlavaciellus. The genus Cephennomicrus represented in the analysis by species belonging to three previously postulated species groups is not monophyletic, and a comprehensive study comprising more taxa is necessary to reclassify this heterogeneous group.