Spindel und kinetochoren in der Mitose und Meiose der Baumwollwanze Dysdercus intermedius (Heteroptera)

In spermatogonial mitoses of Dysdercus intermedius, chromosomal spindle microtubules insert in discrete kinetochores. The latter consist of two electron dense plates of about 0,3 m diameter located at opposite sites at the poleward surfaces of the daughter chromatids. The evidence presented permits the interpretation that a localized kinetochore owes its layered substructure to successive binding sites for kinetochore material at the ends of bundled microtubules. A working hypothesis, based upon different distributions of genetic loci for kinetochore material in species with localized and diffuse kinetochores is proposed. In Dysdercus, holokinetic chromosome behavior in mitotic anaphase movement is not due to truly diffuse kinetochores but to chromosomal interconnections which are established at prometaphase. Such interconnections are absent in the first meiotic division when each bivalent is enclosed by a sheath of endoplasmic reticulum. Spindle microtubules penetrate through terminal openings in this sheath and end singly in dense spots of kinetochore material.     

Surface antigens of Paramecium primaurelia : Membrane-bound and soluble forms

The surface antigens of Paramecium constitute a family of high molecular weight (ca 300 kD) iso-proteins whose alternative expression, adjusted to environmental conditions, involves both intergenic and interallelic exclusion. Since the surface antigen molecules had previously been shown to play a key role in the control of their own expression, it seemed important to compare the structural particularities of different surface antigens: the G and D antigens of P. primaurelia expressed at different temperatures, and which are coded by two unlinked loci. Here we demonstrate that in all cases a given surface antigen presents two biochemically distinct basic forms: a soluble form recovered from ethanolic extraction of whole cells, and a membrane-bound form recovered from ciliary membranes solubilized by detergent. The membrane-bound form differs from the soluble one by its mobility on SDS gels and by an electrophoretic mobility shift in the presence of anionic or cationic detergents. Furthermore, two 40-45 kD polypeptides sharing common determinants with soluble antigens were found exclusively in ethanolic extracts but not in ciliary membranes: the cross-reactivity of these light polypeptides with ethanol-extracted antigens could be demonstrated only after beta-mercaptoethanol treatment. Immunological comparisons between allelic and non-allelic soluble antigens demonstrate that allelic antigens share a great number of surface epitopes, most of which are not accessible in vivo, while non-allelic antigens appear to share essentially sequence-antigenic determinants. The significance of these results is discussed in relation to the mechanism of antigenic variation.     

Interaction of cholesterol,phospholipid and apoprotein in high density lipoprotein recombinants

To examine the effect of incorporation of cholesterol into high density lipoprotein (HDL) recombinants, multilamellar liposomes of ³H cholesterol/¹⁴C dimyristoyl phosphatidylcholine were incubated with the total apoprotein (apoHDL) and principal apoproteins (apoA-1 and apoA-2) of human plasma high density lipoprotein. Soluble recombinants were separated from unreacted liposomes by centrifugation and examined by differential scanning calorimetry and negative stain electron microscopy. At 27°C, liposomes containing up to approx. 0.1 mol cholesterol/mol dimyristoyl phosphatidylcholine (DMPC) were readily solubilized by apoHDL, apoA-1 or apoA-2. However, the incorporation of DMPC and apoprotein into lipoprotein complexes was markedly reduced when liposomes containing a higher proportion of cholesterol were used. For recombinants prepared from apoHDL, apoA-1 or apoA-2, the equilibrium cholesterol content of complexes was approx. 45% that of the unreacted liposomes. Electron microscopy showed that for all cholesterol concentrations, HDL recombinants were predominantly lipid bilayer discs, approx. $\text{160 × 55}\text{A}\text{?}$. Differential scanning calorimetry of cholesterol containing recombinants of DMPC/cholesterol/apoHDL or DMPC/cholesterol/apoA-1 showed, with increasing cholesterol content, a linear decrease in the enthalpy of the DMPC gel to liquid crystalline transition, extrapolating to zero enthalpy at 0.15 cholesterol/DMPC. The enthalpy values were markedly reduced compared to control liposomes, where the phospholipid transition extrapolated to zero enthalpy at approx. 0.45 cholesterol/DMPC. The calorimetric and solubility studies suggest that in high density lipoprotein recombinants cholesterol is excluded from 55% of DMPC molecules bound in a non-melting state by apoprotein.     

Production of Modified Vaccinia Ankara Virus by Intensified Cell Cultures: A Comparison of Platform Technologies for Viral Vector Production

Modified Vaccinia Ankara (MVA) virus is a promising vector for vaccination against various challenging pathogens or the treatment of some types of cancers, requiring a high amount of virions per dose for vaccination and gene therapy. Upstream process intensification combining perfusion technologies, the avian suspension cell line AGE1.CR.pIX and the virus strain MVA-CR19 is an option to obtain very high MVA yields. Here the authors compare different options for cell retention in perfusion mode using conventional stirred-tank bioreactors. Furthermore, the authors study hollow-fiber bioreactors and an orbital-shaken bioreactor in perfusion mode, both available for single-use. Productivity for the virus strain MVA-CR19 is compared to results from batch and continuous production reported in literature. The results demonstrate that cell retention devices are only required to maximize cell concentration but not for continuous harvesting. Using a stirred-tank bioreactor, a perfusion strategy with working volume expansion after virus infection results in the highest yields. Overall, infectious MVA virus titers of 2.1–16.5 × 10⁹ virions/mL are achieved in these intensified processes. Taken together, the study shows a novel perspective on high-yield MVA virus production in conventional bioreactor systems linked to various cell retention devices and addresses options for process intensification including fully single-use perfusion platforms.     

Endemism,palaeoendemism and migration: the case for the ‘European endemic’, Mallomonas intermedia

The Synurophyceae is a well-supported clade of ecologically important heterokont algae found largely in freshwater planktonic habitats worldwide, whose members have cell coverings consisting of species-specific siliceous scales overlapped in a highly organized manner. Many synurophytes have been described as endemic and are found only in specific regions of the world. A thriving population of the European endemic, Mallomonas intermedia, was discovered in a remote desert pond situated in the Virgin Valley, Nevada, USA and in a stratigraphic sequence from the middle Eocene fossil locality known as Horsefly in British Columbia, Canada. Both North American finds were closely compared with populations from Europe, confirming the identifications. Before these discoveries, this species was recorded from numerous waterbodies exclusively in Europe, but was lacking from hundreds of sites examined from other continents. Its presence in western North America during the warm middle Eocene confirms that historically this species had a significantly wider distribution and may be best classified as a palaeoendemic. Additional species uncovered from a second fossil locality that are closely related to M. intermedia further support the presence of this lineage in North America during the Eocene. The living population in northern Nevada presents an enigma. Does this remote desert population represent a remnant population that has gone undetected until now, or is it a recent arrival from an unknown region by an unknown vector?     

Expressed mutational load increases toward the edge of a species’ geographic range

There is no general explanation for why species have restricted geographic distributions. One hypothesis posits that range expansion or increasing scarcity of suitable habitat results in accumulation of mutational load due to enhanced genetic drift, which constrains population performance toward range limits and further expansion. We tested this hypothesis in the North American plant, Arabidopsis lyrata. We experimentally assessed mutational load by crossing plants of 20 populations from across the entire species range and by raising the offspring of within- and between-population crosses at five common garden sites within and beyond the range. Offspring performance was tracked over three growing seasons. The heterosis effect, depicting expressed mutational load, was increased in populations with heightened genomic estimates of load, longer expansion distance or long-term isolation, and a selfing mating system. The decline in performance of within-population crosses amounted to 80%. Mutation accumulation due to past range expansion and long-term isolation of populations in the area of range margins is therefore a strong determinant of population-mean performance, and the magnitude of effect may be sufficient to cause range limits.     

Longevity in response to lowered insulin signaling requires glycine N‐methyltransferase‐dependent spermidine production

Reduced insulin/IGF signaling (IIS) extends lifespan in multiple organisms. Different processes in different tissues mediate this lifespan extension, with a set of interplays that remain unclear. We here show that, in Drosophila, reduced IIS activity modulates methionine metabolism, through tissue‐specific regulation of glycine N‐methyltransferase (Gnmt), and that this regulation is required for full IIS‐mediated longevity. Furthermore, fat body‐specific expression of Gnmt was sufficient to extend lifespan. Targeted metabolomics showed that reducing IIS activity led to a Gnmt‐dependent increase in spermidine levels. We also show that both spermidine treatment and reduced IIS activity are sufficient to extend the lifespan of Drosophila, but only in the presence of Gnmt. This extension of lifespan was associated with increased levels of autophagy. Finally, we found that increased expression of Gnmt occurs in the liver of liver‐specific IRS1 KO mice and is thus an evolutionarily conserved response to reduced IIS. The discovery of Gnmt and spermidine as tissue‐specific modulators of IIS‐mediated longevity may aid in developing future therapeutic treatments to ameliorate aging and prevent disease.     

Best practices for authenticating cell lines

Experiments using cell cultures are only valid to the extent that the cell culture is a true model system for the biological system being investigated. To assure that a cell line is and remains an appropriate biological model, its identity, purity, ploidy, and phenotype must be maintained. These characteristics comprise and determine the authenticity of a cell line. Routine monitoring of the cell line through microscopic examination of morphology can help to determine authenticity, as can the determination of phenotypic status. Assays designed to confirm cell identity and ploidy and freedom from cross-contaminating cell types may need to be performed at certain times, as such information may not be obtained through morphologic and phenotypic examinations alone. The best practices associated with establishing cell line authenticity are described in this article.