Unique techniques for cell analysis utilizing mithramycin and flow microfluorometry.

Cell cycle analysis through utilization of the mithramycin/flow microfluorometric technique combines simplicity (one-step cell preparation) with speed (results available within 20 min after removal of a sample from a culture). Furthermore, the technique is useful in many situations in which standard ³H-thymidine autoradiography and mitotic accumulation are unsatisfactory. Examples are provided which demonstrate a continuous monitoring of experiments in progress, including analysis of populations devoid of cells in S or M phases, analysis of arrested or slowly progressing populations resulting from exposure to toxic agents, detection of abnormalities in mitosis such as nondisjunction and polyploidization, and localization within the cell cycle of dying cells in cultures exposed to toxic agents.     

Use of DiO-C5-3 to improve Hoechst 33342 uptake, resolution of DNA content, and survival of CHO cells

Chinese hamster cells (line CHO) stained with either 9 microM Hoechst 33342 (HO) alone or in combination with the membrane potential fluorochrome DiO-C5-3 (DiO) were analyzed using uv laser powers between 25 and 500 mW and sorted for determination of survival by a colony formation assay. The combination of HO-DiO increased fluorescence twofold and provided coefficients of variation (CVs) as low as 3.0% under conditions where viability of cells, even at 500 mW excitation, was unaffected. HO-stained cells yielded CVs of about 8.5% and survivals of approximately 90% under similar analytical conditions. At laser powers of 25 mW, CV values for HO-DiO-stained populations were 4.0% compared to 9.4% for HO-stained cells. Results with another membrane potential dye, rhodamine 123 (R 123), in combination with HO showed no improvement compared to HO-stained cells. No preferential, cell cycle phase-specific killing was observed in either the HO- or HO-DiO-stained populations. CVs of human skin diploid fibroblasts stained with HO or with HO-DiO were comparable over the entire laser power range; however, percentage survival was slightly higher for the HO-DiO-stained populations when analyzed and sorted at the higher power (400-500 mW) range. Long-term cultures of sorted CHO-K1 subpopulations, differing in DNA ploidy, were established from HO-DiO-stained cells. Advantages of this new staining procedure include improved DNA content resolution (low CV values) and the potential use of less expensive FCM uv laser systems coupled with less perturbing excitation powers.     

Sperm competition and the evolution of sperm design in mammals

Background  

The influence of sperm competition upon sperm size has been a controversial issue during the last 20 years which remains unresolved for mammals. The hypothesis that, when ejaculates compete with rival males, an increase in sperm size would make sperm more competitive because it would increase sperm swimming speed, has generated contradictory results from both theoretical and empirical studies. In addition, the debate has extended to which sperm components should increase in size: the midpiece to accommodate more mitochondria and produce more energy to fuel motility, or the principal piece to generate greater propulsion forces.     

The translation initiation complex eIF3 in trypanosomatids and other pathogenic excavates – identification of conserved and divergent features based on orthologue analysis

Background

The initiation of translation in eukaryotes is supported by the action of several eukaryotic Initiation Factors (eIFs). The largest of these is eIF3, comprising of up to thirteen polypeptides (eIF3a through eIF3m), involved in multiple stages of the initiation process. eIF3 has been better characterized from model organisms, but is poorly known from more diverged groups, including unicellular lineages represented by known human pathogens. These include the trypanosomatids (Trypanosoma and Leishmania) and other protists belonging to the taxonomic supergroup Excavata (Trichomonas and Giardia sp.).

Results

An in depth bioinformatic search was carried out to recover the full content of eIF3 subunits from the available genomes of L. major, T. brucei, T. vaginalis and G. duodenalis. The protein sequences recovered were then submitted to homology analysis and alignments comparing them with orthologues from representative eukaryotes. Eleven putative eIF3 subunits were found from both trypanosomatids whilst only five and four subunits were identified from T. vaginalis and G. duodenalis, respectively. Only three subunits were found in all eukaryotes investigated, eIF3b, eIF3c and eIF3i. The single subunit found to have a related Archaean homologue was eIF3i, the most conserved of the eIF3 subunits. The sequence alignments revealed several strongly conserved residues/region within various eIF3 subunits of possible functional relevance. Subsequent biochemical characterization of the Leishmania eIF3 complex validated the bioinformatic search and yielded a twelfth eIF3 subunit in trypanosomatids, eIF3f (the single unidentified subunit in trypanosomatids was then eIF3m). The biochemical data indicates a lack of association of the eIF3j subunit to the complex whilst highlighting the strong interaction between eIF3 and eIF1.

Conclusions

The presence of most eIF3 subunits in trypanosomatids is consistent with an early evolution of a fully functional complex. Simplified versions in other excavates might indicate a primordial complex or secondary loss of selected subunits, as seen for some fungal lineages. The conservation in eIF3i sequence might indicate critical functions within eIF3 which have been overlooked. The identification of eIF3 subunits from distantly related eukaryotes provides then a basis for the study of conserved/divergent aspects of eIF3 function, leading to a better understanding of eukaryotic translation initiation.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1175) contains supplementary material, which is available to authorized users.     

Accumulation of the insecticidal crystal protein of Bacillus thuringiensis subsp. kurstaki in post-exponential-phase Bacillus subtilis

A gene from Bacillus thuringiensis subsp. kurstaki that codes for a Lepidoptera-specific insecticidal toxin (delta-endotoxin) was engineered for expression in Bacillus subtilis. A low-copy-number plasmid vector that replicates in Escherichia coli and B. subtilis was constructed to transform B. subtilis with gene fusions first isolated and characterized in E. coli. Naturally occurring promoter sequences from B. subtilis (43, veg, ctc, and spoVG) were inserted upstream from the plasmid-borne structural gene. In the most prolific case, when the sporulation-specific spoVG promoter was fused to the heterologous toxin gene, the toxin product accumulated during postexponential growth to greater than 25% of the total cell protein. However, the resulting specific activity of the insecticidal toxin product was not commensurate with the abundance of the protein.     

DIMETHYL SULFOXIDE-INDUCED REVERSION OF SEVERAL FEATURES OF POLYOMA TRANSFORMED BABY HAMSTER KIDNEY CELLS (BHK-21) : Alterations in Growth and Morphology

Cells of a polyoma virus transformed clonal line (Cl-I) of baby hamster kidney fibroblasts (BHK-21) were grown in medium containing 2 percent dimethylsulfoxide (DMSO). Unlike the untransformed BHK-21 cells, Cl-I cells adapted to replication in the presence of DMSO, and they exhibited a rapidly reversible phenotypic reversion of a number of properties characteristic of the transformed state. Restoration of density dependent growth inhibition with accumulation of cells in the G₁ phase of the cell cycle occurred and was associated with restoration of contact dependent behavior and with reversion of histological and ultrastructural features towards those which characterize untransformed cells. Concomitantly, Cl-I cells grown in 2 percent DMSO lost the ability to form colonies in semisolid medium. The data presented suggest that DMSO alters the expression of cellular functions which were altered as a result of viral transformation and which may be involved in cell tumorigenicity.     

Plasma membrane-associated cysteine proteinases in human and animal tumors

The ability of tumor cells to invade into and through normal tissue during the metastatic cascade has been attributed to tumor-associated degradative enzymes including proteinases of the metallo, serine and cysteine classes. Work from several laboratories has established that the cysteine proteinases cathepsins L and B are released from tumor cells, primarily as latent precursor forms. In addition, a cathepsin B-like cysteine proteinase has been shown to be associated with the plasma membrane fraction of several animal and human tumors. This form of the enzyme retains activity under physiologic (or pathologic) conditions including at neutral pH and in the presence of low Mr inhibitors. Since we have established that cathepsin B can degrade the basement membrane attachment glycoprotein laminin, we speculate that plasma membrane-associated cathepsin B may participate in focal dissolution of the basement membrane during tumor cell extravasation.