Characterization of two novel yeast strains used in mediated biosensors for wastewater

After isolation from a pulp mill wastewater treatment facility, two yeast strains, designated SPT1 and SPT2, were characterized and used in the development of mediated biochemical oxygen demand (BOD) biosensors for wastewater. 18S rRNA gene sequence analysis revealed a one nucleotide difference between the sequence of SPT1 and those of Candida sojae and Candida viswanthii. While SPT2 had the highest overall homology to Pichia norvegensis, at only 73.5%, it is clearly an ascomycete, based on BLAST comparisons and phylogenetic analyses. Neighbor-joining dendrograms indicated that SPT1 clustered with several Candida spp., and that SPT2 clustered with Starmera spp., albeit as a very deep branch. Physiological tests, microscopic observations, and fatty acid analysis confirmed that SPT1 and SPT2 are novel yeast strains. Physiological tests also indicated that both strains had potential for use in mediated biosensors for estimation of BOD in wastewater. The lower detection limits of SPT1- and SPT2-based K3Fe(CN)6-mediated biosensors for a pulp-mill effluent were 2 and 1 mg BOD/L, respectively. Biosensor-response times for effluents from eight different pulp mills were in the range of 5 min. Reliability and sensitivity of the SPT1- and SPT2-based biosensors were good, but varied with the wastewater.     

Identification and mapping of the QTL for aluminum tolerance introgressed from the new source, <Emphasis Type="SmallCaps">ORYZA RUFIPOGON</Emphasis> Griff., into indica rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

This study was conducted to identify and map the quantitative trait locus (QTL) controlling Al tolerance in rice using molecular markers. A population of 171 F(6) recombinant inbred lines (RILs) derived from the cross of Oryza sativa (IR64), the Al susceptible parent, and Oryza rufipogon, the Al tolerant parent, was evaluated for Al tolerance using a nutrient solution with and without 40 ppm of active Al(+3). A genetic map, consisting of 151 molecular markers covering 1,755 cM with an average distance of 11.6 cM between loci, was constructed. Nine QTLs were dentified including one for root length under non-stress conditions (CRL), three for root length under Al stress (SRL) and five for relative root length (RRL). O. rufipogon contributed favorable alleles for each of the five QTLs for RRL, which is a primary parameter for Al tolerance, and individually they explained 9.0-24.9% of the phenotypic variation. Epistatic analysis revealed that CRL was conditioned by an epistatic effect, whereas SRL and RRL were controlled by additive effects. Comparative genetic analysis showed that QTLs for RRL, which mapped on chromosomes 1 and 9, appear to be consistent among different rice populations. Interestingly, a major QTL for RRL, which explained 24.9% of the phenotypic variation, was found on chromosome 3 of rice, which is conserved across cereal species. These results indicate the possibilities to use marker-assisted selection and pyramiding QTLs for enhancing Al tolerance in rice. Positional cloning of such QTLs introgressed from O. rufipogon will provide a better understanding of the Al tolerance mechanism in rice and the evolutionary genetics of plant adaptation to acid-soil conditions across cereal species.     

Spontaneous gallstone formation in deer mice: interaction of cholesterol,bile acids,and dietary fiber

A study of the physiologic and ecologic factors involved in a spontaneous seasonal gallstone cycle of deer mice (Peromyscus maniculatus gambelii) was conducted at the Tulelake National Wildlife Refuge (California, USA) from March 1991 to June 1992. The specific hypothesis examined was whether or not seasonal increases in dietary fiber intake provides the necessary conditions for a solubility defect, or supersaturation mechanism, resulting in precipitation of cholesterol gallstones. Results indicated that in addition to the seasonal gallstone prevalence cycle, these deer mice exhibit significant seasonal cycling in serum cholesterol, serum bile acids, fecal bile acids, and diet composition. These physiologic and dietary cycles were phase-advanced 3 mo over the gallstone prevalence cycle, indicating an approximate 3 mo time period for gallstone formation under field conditions. Further, seasonal dietary fiber (plant material and seeds) was positively correlated with both serum cholesterol and the fecal bile acids. This suggests that in wild deer mice, variations in dietary fiber may significantly affect the resorption of bile acids, thereby providing a potential physiologic and nutritional mechanism for spontaneous cholesterol gallstone formation.     

Role of mass spectrometry in the purification of peptides and proteins

Experiments were carried out to evaluate the fractionation of proteins and peptides according to mass. Model mixtures were separated by either reversed-phase or ion-exchange chromatography with mass spectrometry-compatible mobile phase additives. Fraction collection was triggered by the mass/charge ratio of each one of the components of the mixture. Chromatography was additionally monitored with a UV-Vis detector in order to compare the new technique with generally accepted in separations. The results indicated that adequate purification is achieved by this new technique. Fraction collection triggered by changes in the mass/charge ratio reduces sample handling and analysis time. This study demonstrates the utility of mass-directed fractionation of peptides and proteins when mass spectrometry-compatible mobile phase additives are used.     

On-line monitoring of cell growth and cytotoxicity using electric cell-substrate impedance sensing (ECIS)

An on-line and continuous technique based on electric cell-substrate impedance sensing (ECIS) was developed for measuring the concentration and time response function of fibroblastic V79 cells exposed to mercury chloride and 1,3,5-trinitrobenzene (TNB). Attachment, spreading and proliferation of V79 fibroblastic cells cultured on a microarray of small gold electrodes precoated with fibronectin were detected as resistance changes. The response function was derived to reflect the resistance change as a result of cell attachment, spreading, mitosis and cytotoxicity effect. Exposure of V79 cells to mercury chloride or TNB led to alterations in cell behavior, and therefore, chemical cytotoxicity was easily screened by measuring the response function of the attached and spread cells in the presence of inhibitor. The half inhibition concentration, the required concentration to achieve 50% inhibition, was obtained from the response function to provide information about cytotoxicity during the course of the assay. A simple mathematical model was developed to describe the responses of ECIS that were related to the attachment, spreading, and proliferation of V79 fibroblastic cells. The novel results of this paper are mainly characterized by the systematic study of several parameters including the cell number, detection limit, sensor sensitivity, and cytotoxicity, and they may motivate further research and study of ECIS sensors.     

Using matK sequence data to unravel the phylogeny of Casuarinaceae

Casuarinaceae are a Gondwanic family with a unique combination of morphological characters not comparable to any other family. Until recently, the 96 species in the family were classified in a single genus, Casuarina s.l. A recent morphological revision of the family resulted in the splitting of Casuarina s.l. into four genera-Allocasuarina, Casuarina s.s., Ceuthostoma, and Gymnostoma. This study uses matK sequence data from 76 species of Casuarinaceae and eight outgroup taxa to examine the phylogenetic structure within the Casuarinaceae. The study demonstrates the monophyly of the four genera and examines the relationships within the family; it tests the validity of the infra-generic subdivision of Allocasuarina; it discovers geography-based infra-generic subdivisions within Gymnostoma and Casuarina; and, finally, provides a molecular framework on which to trace the evolution of xeromorphy in the Casuarinaceae.     

Autoimmune islet destruction in spontaneous type 1 diabetes is not beta-cell exclusive

Pancreatic islets of Langerhans are enveloped by peri-islet Schwann cells (pSC), which express glial fibrillary acidic protein (GFAP) and S100beta. pSC-autoreactive T- and B-cell responses arise in 3- to 4-week-old diabetes-prone non-obese diabetic (NOD) mice, followed by progressive pSC destruction before detectable beta-cell death. Humans with probable prediabetes generate similar autoreactivities, and autoantibodies in islet-cell autoantibody (lCA) -positive sera co-localize to pSC. Moreover, GFAP-specific NOD T-cell lines transferred pathogenic peri-insulitis to NOD/severe combined immunodeficient (NOD/SCID) mice, and immunotherapy with GFAP or S100beta prevented diabetes. pSC survived in rat insulin promoter Iymphocytic choriomeningitis virus (rip-LCMV) glycoprotein/CD8+ T-cell receptor(gp) double-transgenic mice with virus-induced diabetes, suggesting that pSC death is not an obligate consequence of local inflammation and beta-cell destruction. However, pSC were deleted in spontaneously diabetic NOD mice carrying the CD8+/8.3 T-cell receptor transgene, a T cell receptor commonly expressed in earliest islet infiltrates. Autoimmune targeting of pancreatic nervous system tissue elements seems to be an integral, early part of natural type 1 diabetes.     

An aging-related cell surface NADH oxidase (arNOX) generates superoxide and is inhibited by coenzyme Q

This report describes a novel ECTO-NOX protein with an oscillating activity having a period length of ca. 26 min encountered with buffy coat fractions and sera of aged individuals (70–100 years) that generates superoxide as measured by the reduction of ferricytochrome c. The oscillating, age-related reduction of ferricytochrome c is sensitive to superoxide dismutase, is inhibited by coenzyme Q and is reduced or absent from sera of younger individuals (20–40 years). An oscillating activity with a regular period length is a defining characteristic of ECTO-NOX proteins (a group of cell surface oxidases with enzymatic activities that oscillate). The period length of ca. 26 min is longer than the period length of 24 min for the usual constitutive (CNOX) ECTO-NOX proteins of the cell surface and sera which neither generate superoxide nor reduce ferricytochrome c. The aging-related ECTO-NOX protein (arNOX) provides a mechanism to transmit cell surface oxidative changes to surrounding cells and circulating lipoproteins potentially important to atherogenesis. Additionally, the findings provide a rational basis for the use of dietary coenzyme Q to retard aging-related arterial lesions.     

The color loci of mice--a genetic century

Color loci in mammals are those genetic loci in which mutations can affect pigmentation of the hair, skin, and/or eyes. In the mouse, over 800 phenotypic alleles are now known, at 127 identified color loci. As the number of color loci passed 100 only recently, we celebrate this 'century' with an overview of these loci, especially the 59 that have been cloned and sequenced. These fall into a number of functional groups representing melanocyte development and differentiation, melanosomal components, organelle biogenesis, organelle transport, control of pigment-type switching, and some systemic effects. A human ortholog has been identified in all cases, and the majority of these human genes are found to be loci for human disorders, often affecting other body systems as well as pigmentation. We expect that a significant number of color loci remain to be identified. Nonetheless, the large number known already provide a treasury of resources for reconstruction of the mechanisms, at the subcellular, cellular and tissue levels, that produce a functional pigmentary system and contribute to the normal development and functioning of many other organ systems. The mutant mice also provide valuable models for the study of human disease.