Monitoring yeast spindles in the fluorescence microscope

Formation of the complete spindles during the budding process of Saccharomyces uvarum was investigated by fluorescence microscopy of protoplasted cells. Protoplasts were treated with anti-tubulin antibodies and DAPI, a fluorescent dye staining DNA. Thus, both chromatin and spindles could be visualized. Duplication as well as formation of separated spindle pole bodies during the different stages of budding are documented, demonstrating the occurrence and behaviour of microtubules during yeast cell cycle.     

Changes of acid solubility and fluoride content of the enamel surface in children consuming fluoridated milk

Changes of the acid solubility and fluoride content of the surface layer of the enamel of the first incisor of seventy-nine 9 to 10-year-old children living in a closed community were measured by the method of enamel biopsy at the 6th and 12th months of the consumption of fluoridated milk. Half-year after the beginning of consumption of fluoridated milk the acid solubility decreased significantly, while one-year-long consumption led to significant increase of the fluoride content of the enamel surface.     

Identification of the phosphorylated β-tubulin isotype in differentiated neuroblastoma cells

The tubulin molecule consists of an - and a β-subunit, each of which exists in several isotypic forms. It has been previously shown that one of the isotypes of neuroblastoma β-tubulin is phosphorylated at a serine residue in vivo [(1985) J. Cell Biol. 100, 764–774]. Here we identify the phosphorylated isotype as β₂ (type III). Moreover, the large size of the phosphorylated tryptic peptide and sequence comparisons of vertebrate β-tubulins suggest that one of the two serines in positions 444 and 446 is the phosphorylated residue. Our results raise the possibility that β₂-tubulin differs functionally from the other β-tubulin isotypes.     

Dominance and recessiveness at the protein level in mutant x wildtype crosses in Sacchaomyces cerevisiae

Summary The is ₁-locus of the yeast Saccharomyces cerevisiae is the structural gene for threonine dehydratase. is ₁-mutants require isoleucine for growth and do not have active threonine dehydratase.Interallelic complementation is frequent among is ₁-mutants. This is indicative for an aggregate or multimeric structure of yeast threonine dehydratase.Complementing and non-complementing mutants were crossed to wildtype. Properties of threonine dehydratase were assayed in crude extracts of the resulting heterozygotes.Specific activities varied considerably between full wildtype activity and a level about 10% of that. The apparent Michaelis constants were increased in many heterozygotes. This effect was probably due to the aggregation of both mutant and wildtype subunits to form a hybrid threonine dehydratase with reduced substrate affinity in addition to pure wildtype enzyme. This notion is supported by the observation in one heterozygote of two enzyme fractions with increased Michaelis constants in addition to a wildtype-like fraction.The possible formation of hybrid enzymes with normal, reduced or no activity is considered to blur gene dosage relations.A given pair of alleles in a heterozygous cell can generate a new type of enzyme with properties not encountered in the corresponding two homozygous cells. This situation is not accounted for by the classical concepts of dominant-recessive or intermediate behaviour, because the difference between the heterozygotes and the homozygotes is not necessarily only quantitativ but also qualitative.We dedicate this publication to Prof. Dr. C. Auerbach on occasion of her official retirement in admiration for her pioneer work and many contribution to genetics.     

Professor Dr. Geoffrey Burnstock (1929–2020)

Purinergic Signalling -     

An Automatable Hydrogel Culture Platform for Evaluating Efficacy of Antibody‐Based Therapeutics in Overcoming Chemoresistance

The microenvironment plays a major role in conferring chemoresistance to cancer cells. In order to better inform clinical response to chemoresistance, preclinical models that recapitulate its hallmark features are needed to enable screening for resistance‐specific therapeutic targets. A novel platform for seeding cancer cells in 3D hydrogels is presented utilizing derivatives of chitosan and alginate that, critically, is amenable to high throughput screening: cell seeding in hydrogels, media changes, dosing of anticancer compounds, and cell viability assays are all automated using a standard and commercially available liquid handling robot. Culture in these hydrogels elicits resistance in ovarian, lung, and prostate cancer cells to treatment by doxorubicin and paclitaxel. In correlation, proteomics analysis of SKOV3 cells cultured in 3D reveals enrichment of proteins associated with extreme drug resistance including HMOX1 and ALDH2. Subsequently, therapeutic antibodies targeted to tumor‐associated antigens upregulated in 3D cultures are shown to have higher efficacy compared to 2D cultures. Collectively, this automated 3D cell culture platform provides a powerful tool with utility in identification of drugs that may overcome chemoresistance.     

Evolutionary significance of the variation in acoustic communication of a cryptic nocturnal primate radiation (Microcebus spp.)

Acoustic phenotypic variation is of major importance for speciation and the evolution of species diversity. Whereas selective and stochastic forces shaping the acoustic divergence of signaling systems are well studied in insects, frogs, and birds, knowledge on the processes driving acoustic phenotypic evolution in mammals is limited. We quantified the acoustic variation of a call type exchanged during agonistic encounters across eight distinct species of the smallest‐bodied nocturnal primate radiation, the Malagasy mouse lemurs. The species live in two different habitats (dry forest vs. humid forest), differ in geographic distance to each other, and belong to four distinct phylogenetic clades within the genus. Genetically defined species were discriminated reliably on the phenotypic level based on their acoustic distinctiveness in a discriminant function analysis. Acoustic variation was explained by genetic distance, whereas differences in morphology, forest type, or geographic distance had no effect. The strong impact of genetics was supported by a correlation between acoustic and genetic distance and the high agreement in branching pattern between the acoustic and molecular phylogenetic trees. In sum, stochastic factors such as genetic drift best explained acoustic diversification in a social communication call of mouse lemurs.