Discovery of multiple IGS haplotypes within genotypes of Puccinia striiformis

In a search for specific molecular markers for population analysis of Puccinia striiformis f. sp. tritici, the ribosomal DNA (rDNA) intergenic spacer (IGS) 1 region (rDNA-IGS1, between the 28S and the 5S rDNA genes) was amplified, cloned, and sequenced. It was found to exhibit multiple bands and length polymorphism. Surprisingly, single isolates were found to possess between three to five different IGS1 haplotypes. Bands were cloned and sequenced, and two highly variable regions (α and β) were found between conserved regions, with repeat units interspersed in both types of regions. There were 14 different repeat units, and these were sometimes grouped further into four combinations of repeat units, with a few individual nucleotides (A or C) inserted between the repeats. Among three geographically dispersed isolates, the variable region α was divided into eight types, and the variable region β was divided into two types based on repeat units. Most of the 14 repeat units were shared by the variable and the conserved regions. Among the three isolates, there were a total of 12 IGS1 haplotypes, but some of these were shared between isolates such that there were only eight unique haplotypes. The occurrence of multiple haplotypes within single isolates may be useful for analyzing the population structure, tracking the origin of annual epidemics and providing insights into evolutionary biology of this pathogen.     

Recombinant human albumin supports single cell cloning of CHO cells in chemically defined media

Biologic drugs, such as monoclonal antibodies, are commonly made using mammalian cells in culture. The cell lines used for manufacturing should ideally be clonal, meaning derived from a single cell, which represents a technically challenging process. Fetal bovine serum is often used to support low cell density cultures, however, from a regulatory perspective, it is preferable to avoid animal‐derived components to increase process consistency and reduce the risk of contamination from adventitious agents. Chinese hamster ovary (CHO) cells are the most widely used cell line in industry and a large number of serum‐free, protein‐free, and fully chemically defined growth media are commercially available, although these media alone do not readily support efficient single cell cloning. In this work, we have developed a simple, fully defined, single‐cell cloning media, specifically for CHO cells, using commercially available reagents. Our results show that a 1:1 mixture of CD‐CHO? and DMEM/F12 supplemented with 1.5 g/L of recombinant albumin (Albucult®) supports single cell cloning. This formulation can support recovery of single cells in 43% of cultures compared to 62% in the presence of serum. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Life cycle of Chattonella marina (Raphidophyceae) inferred from analysis of microsatellite marker genotypes

Red tides of Chattonella spp. have caused continuous damage to Japanese aquaculture, however, the life cycle of this organism remains incompletely understood. To further investigate this matter, we assessed genotypes at 14 microsatellite markers in three varieties of Chattonella marina, viz., C. marina var. antiqua, C. marina var. marina, and C. marina var. ovata, to establish whether Chattonella undergoes asexual diploidization or sexual reproduction. After genotyping 287 strains of C. marina, all but one of these strains was shown to be heterozygous for at least some loci, and thus, in the diploid state, suggesting that Chattonella strains undergo sexual reproduction. In addition, we performed single‐cell amplification on ‘small cells’ that are derived from vegetative cells under dark and low‐nutrient conditions. The results indicated the existence of two types of small cells. The ‘Small cell Type 1’ was found to be heterozygous, genotypically equivalent to the vegetative cells, and is therefore diploid. These small cells may change to resting cells (cysts) directly. The ‘Small cell Type 2’ was homozygous at all analyzed loci, suggesting that these small cells are haploid and may be derived by meiosis. As fusion between small cells has previously been observed, the ‘Small cell Type 2’ may be the gamete of Chattonella. We present a construct of the full life cycle of Chattonella marina based on our own and previous results.     

Protein trafficking rates assessed by quantum dot quenching with bromocresol green

Quantum dots are bright, photostable fluorophores used extensively to investigate biological processes. In this study, we report that bromocresol green (BCG) at low micromolar concentrations rapidly, efficiently and reversibly quenches the fluorescence of commercial quantum dots having a wide range of functionalities. The broad utility of BCG quenching of quantum dots in cell biology is showed in quantitative assays of trafficking of the β(2) -adrenergic receptor (β(2) AR) and the cystic fibrosis transmembrane conductance regulator (CFTR).     

Evidence for sequential action of Rab5 and Rab7 GTPases in prevacuolar organelle partitioning

GTPases of the Rab5 and Rab7 families were shown to control vacuolar sorting but their specific subcellular localization is controversial in plants. Here, we show that both the canonical as well as the plant-specific Rab5 reside at the newly discovered 'late prevacuolar compartment' (LPVC) while Rab7 partitions to the vacuolar membrane when expressed at low levels. Higher expression levels of wild-type Rab5 GTPases but not Rab7 lead to dose-dependent inhibition of biosynthetic vacuolar transport. In the case of Ara6, this included aberrant co-localization with markers for earlier post-Golgi compartments including the trans-Golgi network. However, nucleotide-free mutants of all three GTPases (Rha1, Ara6 and Rab7) cause stronger dose-dependent inhibition of vacuolar sorting. In addition, nucleotide-free Rha1 led to a later maturation defect and co-localization of markers for the prevacuolar compartment (PVC) and the LPVC. The corresponding Rab7 mutant strongly inhibited vacuolar delivery without merging of PVC and LPVC markers. Evidence for functional differentiation of the Rab5 family members is underlined by the fact that mutant Rha1 expression can be suppressed by increasing wild-type Rha1 levels while mutant Ara6 specifically titrates the nucleotide exchange factor Vps9. A model describing the sequential action of Rab5 and Rab7 GTPases is presented in the light of the current observations.     

Effects of changes in the shape of the intracellular action potential on the peak-to-peak ratio of single muscle fibre potentials

In situ recording of the intracellular action potential (IAP) of human muscle fibres is not yet feasible, and consequently, knowledge about certain IAP characteristics of these IAPs is still limited. The ratio between the amplitudes of the second and first phases (the so-called peak-to-peak ratio, PPR) of a single fibre action potential (SFAP) is known to be closely related to the IAP profile. The PPR of experimentally recorded SFAPs has been found to be largely independent of changes in the fibre-to-electrode (radial) distance. The main goal of this paper is to analyze the effect of changes in different aspects of the IAP spike on the relationship between PPR and radial distance. Based on this analysis, we hypothesize about the characteristics of IAPs obtained experimentally. It was found that the sensitivity of the SFAP PPR to changes in radial distance is essentially governed by the duration of the IAP spike. Assuming that, for mammals, the duration of the IAP rising phase lies within the range 0.2-0.4 ms, we tentatively suggest that the duration of the IAP spike should be over approximately 0.75 ms, with the shape of the spike strongly asymmetric. These IAP characteristics broadly coincide with those observed in mammal IAPs.     

Existence of memory in membrane channels: analysis of ion current through a voltage‐dependent potassium single channel

Ion current fluctuation of voltage‐dependent potassium channel in LβT2 cells has been investigated by autocorrelation function and DFA (detrended fluctuation analysis) methods. The calculation of the autocorrelation function exponent and DFA exponent of the sample was based on the digital signals or the 0–1 series corresponding to closing and opening of channels after routine evolution, rather than the sequence of sojourn times. The persistent character of the correlation of the time series was evident from the slow decay of the autocorrelation function. DFA exponent α was significantly greater than 0.5. The main outcome has been the demonstration of the existence of memory in this ion channel. Thus, the ion channel current fluctuation provided information about the kinetics of the channel protein. The result suggests the correlation character of the ion channel protein non‐linear kinetics indicates whether the channel is open or not.     

Differential promoter activities of functional haplotypes in the 5′‐flanking region of human Sulfotransferase 1A1

Previously, we reported five common single nucleotide polymorphisms (SNPs), ?624G>C, ?396G>A, ?358A>C, ?341C>G, and ?294T>C, and six common haplotypes (CGACT, GAACT, GGAGC, GGACC, CAACT, and GAACC) in the 5′‐flanking region of the SULT1A1 gene that were associated with altered enzymatic activity. In the present study, we performed in vitro assays to determine the functional impact of these genetic variations on the promoter activity. Dual luciferase reporter assays revealed that these SNPs are located in a negative regulatory fragment of the SULT1A1 gene. Further experiments demonstrated that these SNPs and haplotypes affected promoter activities of SULT1A1. Electrophoretic mobility shift assays showed distinctive binding patterns for the SNPs ‐396G>A and ‐294T>C, due to differential binding affinities of the G/A alleles and the T/C alleles to nuclear proteins extracted from the liver carcinoma cell lines, HepG2 and Huh7. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:422–428, 2012; View this article online at wileyonlinelibrary.com . DOI 10:1002/jbt.21437