Anti-apoptotic genes, bag-1 and bcl-2, enabled hybridoma cells to survive under treatment for arresting cell cycle

Hybridoma 2E3-O cells were transfected with bcl-2 alone or with bcl-2 and bag-1 in combination. The bcl-2/bag-1 transfectant survived maintaining viability above 75% for almost 5 days when the cells were treated with excess (30 mM) thymidine for arresting cell cycle, whereas the mock transfectant survived for only 2 days, and the bcl-2 alone transfectant lived for 4 days. Owing to this extended viable culture period, the bcl-2/bag-1 transfectant produced twofold amount of antibody in comparison with the mock transfectant in non-proliferating state prepared by the excess thymidine treatment. When their proliferation was arrested by serum limitation, the bcl-2/bag-1 transfectant and the bcl-2 alone transfectant survived for 3 days maintaining viability above 75% while the mock transfectant survived only 1 day. The bcl-2/bag-1 transfectans produced the antibody at the rate three times as high as the bcl-2 alone transfectant and the mock transfectant in non-proliferating state established by serum limitation. Such genetic engineering of hybridoma cells for improving survival in the non-proliferating state will be useful for using nutrients in culture medium efficiently to produce antibody, since nutrients could be diverted from cell proliferation to antibody production in such non-proliferating viable cell culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Dosage suppressors of pds1 implicate ubiquitin-associated domains in checkpoint control

In budding yeast, anaphase initiation is controlled by ubiquitin-dependent degradation of Pds1p. Analysis of pds1 mutants implicated Pds1p in the DNA damage, spindle assembly, and S-phase checkpoints. Though some components of these pathways are known, others remain to be identified. Moreover, the essential function of Pds1p, independent of its role in checkpoint control, has not been elucidated. To identify loci that genetically interact with PDS1, we screened for dosage suppressors of a temperature-sensitive pds1 allele, pds1-128, defective for checkpoint control at the permissive temperature and essential for viability at 37 degrees C. Genetic and functional interactions of two suppressors are described. RAD23 and DDI1 suppress the temperature and hydroxyurea, but not radiation or nocodazole, sensitivity of pds1-128. rad23 and ddi1 mutants are partially defective in S-phase checkpoint control but are proficient in DNA damage and spindle assembly checkpoints. Therefore, Rad23p and Ddi1p participate in a subset of Pds1p-dependent cell cycle controls. Both Rad23p and Ddi1p contain ubiquitin-associated (UBA) domains which are required for dosage suppression of pds1-128. UBA domains are found in several proteins involved in ubiquitin-dependent proteolysis, though no function has been assigned to them. Deletion of the UBA domains of Rad23p and Ddi1p renders cells defective in S-phase checkpoint control, implicating UBA domains in checkpoint signaling. Since Pds1p destruction, and thus checkpoint regulation of mitosis, depends on ubiquitin-dependent proteolysis, we propose that the UBA domains functionally interact with the ubiquitin system to control Pds1p degradation in response to checkpoint activation.     

Bias Assessment of General Chemistry Analytes using Commutable Samples

Harmonisation of reference intervals for routine general chemistry analytes has been a goal for many years. Analytical bias may prevent this harmonisation. To determine if analytical bias is present when comparing methods, the use of commutable samples, or samples that have the same properties as the clinical samples routinely analysed, should be used as reference samples to eliminate the possibility of matrix effect. The use of commutable samples has improved the identification of unacceptable analytical performance in the Netherlands and Spain. The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) has undertaken a pilot study using commutable samples in an attempt to determine not only country specific reference intervals but to make them comparable between countries. Australia and New Zealand, through the Australasian Association of Clinical Biochemists (AACB), have also undertaken an assessment of analytical bias using commutable samples and determined that of the 27 general chemistry analytes studied, 19 showed sufficiently small between method biases as to not prevent harmonisation of reference intervals. Application of evidence based approaches including the determination of analytical bias using commutable material is necessary when seeking to harmonise reference intervals.     

When males and hermaphrodites coexist: a review of androdioecy in animals

Androdioecy (populations consisting of males and hermaphrodites)is a rare mating system in plants and animals: up to 50 plantsand only 36 animals have been described as being androdioecious,with most of the latter being crustaceans. To date, a thoroughcomparative analysis of androdioecy in animals has not beenundertaken. Herein we present such an analysis. Androdioecyhas only been extensively surveyed in 2 animal taxa: the nematodeCaenorhabditis and the clam shrimp Eulimnadia. The other majortaxon having androdioecious species is the Cirripedia (barnacles),but there are only limited studies on androdioecy in this group.In animals, androdioecy is found either in species that havemorphologically and ecologically distinct sexes (that is, hermaphroditesand small, "complemental" males) that are derived from hermaphroditicancestors (that is, the barnacles) or in species that have similarly-sizedmales and hermaphrodites that have been derived from dioeciousancestors (the remaining androdioecious species). We suggestthat the barnacles have evolved a sexual specialization in theform of these complemental males that can more efficiently usethe constrained habitats that these barnacles often experience.For the remaining species, we suggest that androdioecy has evolvedas a response to reproductive assurance in species that experienceepisodic low densities. Additionally, we hypothesize that thedevelopment of mechanisms allowing reproductive assurance inspecies with a number of sexually differentiated traits is mostlikely to result in androdioecy rather than gynodioecy (mixturesof females and hermaphrodites), and that these species may bedevelopmentally constrained to stay androdioecious rather thanbeing capable of evolving into populations solely consistingof efficient, self-compatible hermaphrodites. We conclude bysuggesting several areas in need of further study to understandmore completely the evolution and distribution of this interestingmating system in animals.     

Criteria for effective design, construction, and gene knockdown by shRNA vectors

Background  

RNA interference (RNAi) technology is a powerful methodology recently developed for the specific knockdown of targeted genes. RNAi is most commonly achieved either transiently by transfection of small interfering (si) RNA oligonucleotides, or stably using short hairpin (sh) RNA expressed from a DNA vector or virus. Much controversy has surrounded the development of rules for the design of effective siRNA oligonucleotides; and whether these rules apply to shRNA is not well characterized.     

Functional Characterization of Rpn3 Uncovers a Distinct 19S Proteasomal Subunit Requirement for Ubiquitin-Dependent Proteolysis of Cell Cycle Regulatory Proteins in Budding Yeast

By selectively eliminating ubiquitin-conjugated proteins, the 26S proteasome plays a pivotal role in a large variety of cellular regulatory processes, particularly in the control of cell cycle transitions. Access of ubiquitinated substrates to the inner catalytic chamber within the 20S core particle is mediated by the 19S regulatory particle (RP), whose subunit composition in budding yeast has been recently elucidated. In this study, we have investigated the cell cycle defects resulting from conditional inactivation of one of these RP components, the essential non-ATPase Rpn3/Sun2 subunit. Using temperature-sensitive mutant alleles, we show that rpn3 mutations do not prevent the G(1)/S transition but cause a metaphase arrest, indicating that the essential Rpn3 function is limiting for mitosis. rpn3 mutants appear severely compromised in the ubiquitin-dependent proteolysis of several physiologically important proteasome substrates. Thus, RPN3 function is required for the degradation of the G(1)-phase cyclin Cln2 targeted by SCF; the S-phase cyclin Clb5, whose ubiquitination is likely to involve a combination of E3 (ubiquitin protein ligase) enzymes; and anaphase-promoting complex targets, such as the B-type cyclin Clb2 and the anaphase inhibitor Pds1. Our results indicate that the Pds1 degradation defect of the rpn3 mutants most likely accounts for the metaphase arrest phenotype observed. Surprisingly, but consistent with the lack of a G(1) arrest phenotype in thermosensitive rpn3 strains, the Cdk inhibitor Sic1 exhibits a short half-life regardless of the RPN3 genotype. In striking contrast, Sic1 turnover is severely impaired by a temperature-sensitive mutation in RPN12/NIN1, encoding another essential RP subunit. While other interpretations are possible, these data strongly argue for the requirement of distinct RP subunits for efficient proteolysis of specific cell cycle regulators. The potential implications of these data are discussed in the context of possible Rpn3 function in multiubiquitin-protein conjugate recognition by the 19S proteasomal regulatory particle.     

Crystallization and preliminary x-ray diffraction study of cholera toxin B-subunit

Cholera toxin binds to its ganglioside GM1 receptor via its B-subunit, a pentameric assembly of identical subunits (Mr = 11,600). Diffraction quality crystals of cholera toxin B-subunit have been obtained at room temperature by vapor diffusion with polyethylene glycol in the presence of the nonionic detergent beta-octyl glucoside. The crystals have been characterized with x-radiation as monoclinic, space group P21, with unit cell dimensions a = 39.0 A, b = 94.3 A, c = 67.5 A, beta = 96.0 degrees. There are two molecules per unit cell, with one molecule (Mr = 58,000) in each asymmetric unit. Precession photographs (micron = 13 degrees) show that crystals diffract beyond 3.3-A resolution and are stable in the x-ray beam at room temperature for at least 40 h; thus, they can be used to collect three-dimensional crystallographic data.     

2-difluoromethyloestrone 3-O-sulphamate, a highly potent steroid sulphatase inhibitor

Steroid sulphatase is a target enzyme of growing therapeutic importance. The synthesis and in vitro biological evaluation of three novel 2-substituted analogues of oestrone 3-O-sulphamate (EMATE), an established steroid sulphatase inhibitor, are described. One inhibitor, 2-difluoromethyloestrone 3-O-sulphamate (6), was found to have an IC50 of 100 pM and be some 90-fold more potent than EMATE in inhibiting steroid sulphatase activity in a placental microsomal preparation, rendering this agent the most potent steroidal STS inhibitor in vitro reported to date. Lowering of the pKa value of the leaving parent steroid phenol by the 2-difluoromethyl group during irreversible enzyme sulphamoylation most likely facilitates the potent inactivation of steroid sulphatase by (6). However, our preliminary molecular docking studies using the X-ray crystal structure of steroid sulphatase suggest that F.......H interactions between the 2-difluoromethyl group of (6) and hydrogen bond donor residues lining the catalytic site of STS might also contribute to the high potency observed for (6).     

Mapping Chromosomal Genes of SACCHAROMYCES CEREVISIAE Using an Improved Genetic Mapping Method

A triploid (3n) strain of Saccharomyces cerevisiae was constructed carrying a standard marker on each of chromosomes 1 through XVII in the -/+/+ configuration. This is called a "supertriploid." Meiotic spores from this strain (n + approximately n/2) were mated with a haploid (n) carrying an unmapped mutation. Meiotic analysis of each zygote clone (2n + approximately n/2) produced in this way resulted in elimination of an average of 4.2 chromosomes as the possible location of the unmapped marker. The distribution of extra chromosomes in the 2n + approximately n/2) strains was nearly random. Meiotic segregrants of these crosses carrying the unmapped mutation in the -/+ configuration were then crossed with multiply marked haploid strains to further narrow the possible location of the unmapped mutation to a single chromosome. Scoring of markers by complemention tests was simplified by mating spore clones with mixtures of a and alpha strains, each pair carrying the same set of markers. Using this new, more rapid method ("supertriploid mapping"), eight genes required for the maintenance of the killer plasmid were located on the genetic map of S. cerevisiae.