Characterizing bisexual Artemia populations by isoelectric focusing

Isoelectric focusing (IEF) of protein extracts, -Esterase and Phosphoglucose isomerase, from groups of Artemia adults from different bisexual populations were studied. Both gave clear separation between the Old and New World species. The second was more polymorphic, allowing a discrimination among Mediterranean populations.     

Critical considerations for the development of potency tests for therapeutic applications of mesenchymal stromal cell-derived small extracellular vesicles

Mesenchymal stromal/stem cells (MSCs) have been widely tested against many diseases, with more than 1000 registered clinical trials worldwide. Despite many setbacks, MSCs have been approved for the treatment of graft-versus-host disease and Crohn disease. However, it is increasingly clear that MSCs exert their therapeutic functions in a paracrine manner through the secretion of small extracellular vesicles (sEVs) of 50–200 nm in diameter. Unlike living cells that can persist long-term, sEVs are non-living and non-replicative and have a transient presence in the body. Their small size also renders sEV preparations highly amenable to sterilization by filtration. Together, acellular MSC-sEV preparations are potentially safer and easier to translate into the clinic than cellular MSC products. Nevertheless, there are inherent challenges in the development of MSC-sEV drug products. MSC-sEVs are products of living cells, and living cells are sensitive to changes in the external microenvironment. Consequently, quality control metrics to measure key identity and potency features of MSC-sEV preparations have to be specified during development of MSC-sEV therapeutics. The authors have previously described quantifiable assays to define the identity of MSC-sEVs. Here the authors discuss requirements for prospective potency assays to predict the therapeutic effectiveness of the drug substance in accordance with International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines. Although potency assays should ideally reflect the mechanism of action (MoA), this is challenging because the MoA for the reported efficacy of MSC-sEV preparations against multiple diseases of diverse underlying pathology is likely to be complex and different for each disease and difficult to fully elucidate. Nevertheless, robust potency assays could be developed by identifying the EV attribute most relevant to the intended biological activity in EV-mediated therapy and quantifying the EV attribute. Specifically, the authors highlight challenges and mitigation measures to enhance the manufacture of consistent and reproducibly potent sEV preparations, to identify and select the appropriate EV attribute for potency assays despite a complex “work-in-progress” MoA and to develop assays likely to be compliant with regulatory guidance for assay validation.     

The biofilm formation defect of a Bacillus subtilis flotillin‐defective mutant involves the protease FtsH

Biofilm formation in Bacillus subtilis requires the differentiation of a subpopulation of cells responsible for the production of the extracellular matrix that structures the biofilm. Differentiation of matrix‐producing cells depends, among other factors, on the FloT and YqfA proteins. These proteins are present exclusively in functional membrane microdomains of B. subtilis and are homologous to the eukaryotic lipid raft‐specific flotillin proteins. In the absence of FloT and YqfA, diverse proteins normally localized to the membrane microdomains of B. subtilis are not functional. Here we show that the absence of FloT and YqfA reduces the level of the septal‐localized protease FtsH. The flotillin homologues FloT and YqfA are occasionally present at the midcell in exponentially growing cells and the absence of FloT and YqfA negatively affects FtsH concentration. Biochemical experiments indicate a direct interaction between FloT/YqfA and FtsH. Moreover, FtsH is essential for the differentiation of matrix producers and hence, biofilm formation. This molecular trigger of biofilm formation may therefore be used as a target for the design of new biofilm inhibitors. Accordingly, we show that the small protein SpoVM, known to bind to and inhibit FtsH activity, inhibits biofilm formation in B. subtilis and other distantly related bacteria.     

Late steps of egg cell differentiation are accelerated by pollination in Zea mays L.

 Egg cells were analysed cytologically during the female receptivity period in maize (Zea mays L., line A 188). Three classes of egg cell were distinguished: type A – small, non-vacuolated cells with a central nucleus; type B – larger cells with small vacuoles surrounding the perinuclear cytoplasm located in the middle of the cell; type C – big cells with a large apical vacuole and the mid-basal perinuclear cytoplasm. The less-dense cytoplasm of the vacuolated egg cells usually contained numerous cup- or bell-shaped mitochondria. The three egg types appear to correspond to three late stages of egg cell differentiation. The frequencies of each of the three egg types were monitored in developing maize ears before and after pollination. In young ears, with the silks just extending out of the husks, small A-type cells were found in about 86% of ovules. Their frequency decreased to about 58% at the optimum silk length, remained unchanged in non-pollinated ears, and fell to 16% at the end of the female receptivity period. However, after pollination and before fertilisation the frequency of these cells decreased to about 33%, and the larger vacuolated egg cells (types B and C) prevailed. At various stages of the receptivity period, pollination accelerated changes in the egg population, increasing the number of ovules bearing larger, vacuolated egg cells. Experiments with silk removal demonstrated that putative pollination signals act immediately after pollen deposition and are not species-specific. Received: 5 February 1999 / Accepted: 28 August 1999     

Modulation of Adherence and Chemotaxis of Macrophages by Norepinephrine. Influence of Ageing

We evaluated thein vitro effect of norepinephrine (NE), over the range of concentrations between 10^-12 M and 10^-3 M, on adherence (to plastic surfaces) and chemotaxis (in a Boyden chamber) of peritoneal macrophages from BALB/c mice of different ages: young (12 weeks), adult (22 weeks), mature (48 weeks) and old (72 weeks). Increased adherence was induced by 10^-12 M of NE in macrophages from young, adult, mature and old mice. Also, 10^-9 M stimulated adherence in old animals, 10^-5 M in mature mice, and 10^-3 M in both young and old mices. With respect to chemotaxis, the low concentration of NE (10^-12 M) was stimulatory only in young and adult animals, higher concentrations (10^-5 M and 10^-7 M) were inhibitory for macrophages from mature and old animals, and the highest concentration of NE (10^-3M) stimulated this capacity of macrophages only in young and mature animals. The conclusion is that while the mobility of macrophages to the focus of infection (i.e. chemotaxis) is stimulated by low concentrations of NE (10^-12, M) only in young-adult animals, this neurotransmitter induces a decline in this capacity in mature and old mice at high concentrations (10^-5 M - 10^-7 M). Also, macrophages from old animals have lost the capacity to respond to pharmacological (10^-3 M) concentrations of NE. The lower capacity of response to NE by macrophages from old animals possibly contributes to immunosenescence.     

Ras-induced cellular events (review)

Ras is a crucial regulator of cell growth in eukaryotic cells. Activated Ras can stimulate signal transduction cascades, leading to cell proliferation, differentiation or apoptosis. It is also one of the most commonly mutated genes in both solid tumours and haematologic neoplasias. In leukaemia and tumours, aberrant Ras signalling can be induced directly by Ras mutation or indirectly by altering genes that associate with Ras or its signalling pathways. A requisite for Ras function is localization to the plasma membrane, which is induced by the post-translational modification farnesylation. Molecules that interfere with this Ras modification have been used as antitumour agents. Ras is emerging as a dual regulator of cell functions, playing either positive or negative roles in the control of proliferation or apoptosis. The diversity of Ras-mediated effects may be related in part to the differential involvement of Ras homologues in distinct cellular processes or to the expanding array of Ras effectors.     

Study of the secondary structure of the C-terminal domain of the antiapoptotic protein bcl-2 and its interaction with model membranes

Bcl-2 is a protein which inhibits programmed cell death. It is associated to many cell membranes such as mitochondrial outer membrane, endoplasmic reticulum, and nuclear envelope, apparently through a C-terminal hydrophobic domain. We have used infrared spectroscopy to study the secondary structure of a synthetic peptide (a 23mer) with the same sequence as this C-terminal domain (residues 217-239) of Bcl-2. The spectrum of this peptide in D(2)O buffer shows an amide I' band with a maximum at 1622 cm(-1), which clearly indicates its tendency to aggregate in aqueous solvent. However, the peptide incorporated in multilamellar phosphatidylcholine membranes shows a totally different spectrum of the amide I' band, with a maximum at 1655 cm(-)(1), indicating a predominantly alpha-helical structure. Addition of the peptide to unilamellar vesicles destabilized them and released encapsulated carboxyfluorescein. Differential scanning calorimetry of dimyristoylphosphatidylcholine multilamellar vesicles in which the peptide was incorporated revealed that increasing concentrations of the peptide progressively broadened the pretransition and the main transition, as is to be expected for a membrane integral molecule. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene in fluid phosphatidylcholine vesicles showed that increasing concentrations of the peptide produced increased polarization values, pointing to an increase in the apparent order of the membrane and indicating that high concentrations of the peptide considerably broaden the phase transition of dimyristoylphosphatidylcholine multilamellar vesicles. Quenching the intrinsic fluorescence of the Tyr-235 of the peptide, by KI, indicated that this aminoacyl residue is highly exposed to aqueous solvent when incorporated in phospholipid vesicles. The results are discussed in terms of their relevance to the proposed topology of insertion of Bcl-2 into biological membranes.     

MATE PREFERENCE FOR A PHENOTYPICALLY PLASTIC TRAIT IS LEARNED,AND MAY FACILITATE PREFERENCE‐PHENOTYPE MATCHING

Fixed, genetically determined, mate preferences for species whose adult phenotype varies with rearing environment may be maladaptive, as the phenotype that is most fit in the parental environment may be absent in the offspring environment. Mate preference in species with polyphenisms (environmentally dependent alternative phenotypes) should therefore either not focus on polyphenic traits, be polyphenic themselves, or learned each generation. Here, we test these alternative hypotheses by first describing a female‐limited seasonal polyphenism in a sexually dimorphic trait in the butterfly Bicyclus anynana, dorsal hindwing spot number (DHSN), and then testing whether male and female mate preferences for this trait exist, and whether they are seasonally polyphenic, or learned. Neither naïve males nor naïve females in either seasonal form exhibited mating preferences for DHSN. However, males, but not females, noticed DHSN variation and learned mate preferences for DHSN. These results suggest that individuals may accommodate environmentally dependent variation in morphological traits via learned mate preferences in each generation, and that learned mate preference plasticity can be sexually dimorphic.     

THE ACTION OF STABILIZING SELECTION,MUTATION, AND DRIFT ON EPISTATIC QUANTITATIVE TRAITS

For a quantitative trait under stabilizing selection, the effect of epistasis on its genetic architecture and on the changes of genetic variance caused by bottlenecking were investigated using theory and simulation. Assuming empirical estimates of the rate and effects of mutations and the intensity of selection, we assessed the impact of two‐locus epistasis (synergistic/antagonistic) among linked or unlinked loci on the distribution of effects and frequencies of segregating loci in populations at the mutation‐selection‐drift balance. Strong pervasive epistasis did not modify substantially the genetic properties of the trait and, therefore, the most likely explanation for the low amount of variation usually accounted by the loci detected in genome‐wide association analyses is that many causal loci will pass undetected. We investigated the impact of epistasis on the changes in genetic variance components when large populations were subjected to successive bottlenecks of different sizes, considering the action of genetic drift, operating singly (D), or jointly with mutation (MD) and selection (MSD). An initial increase of the different components of the genetic variance, as well as a dramatic acceleration of the between‐line divergence, were always associated with synergistic epistasis but were strongly constrained by selection.