Cytostatic action of methylmercuric chloride on mammalian duodenal cells

Adult male mice of the ICR/Swiss Webster strain received a single intragastric administration of methylmercuric chloride 1,000 ppm, at dose levels of 5,10,15,20,25 and 30 mg/kg of body weight. The animals were killed six hours later. Tissue samples from the duodenum were fixed in 10% neutral buffered formalin for light microscopy. Chromosome clumping was observed in dividing cells at all dose levels, resembling a C-mitotic effect. It would lead to reduced mitotic cell formation on account of the subsequent lysis of the arrested metaphases. The cytostatic effect was brought about by the inactivation of the microtubule spindle fiber polymerization mechanism induced by methylmercuric chloride. There was a direct positive correlation between the varying dose levels of methylmercury and the proportion of cells arrested in metaphase in the crypts of the duodenum.     

The glycosylation of human myelin basic protein at threonines 95 and 98 occurs sequentially

Human myelin basic protein (MBP) was glycosylated by the enzyme, UDP-GalNAc:polypeptide N-acetylgalactosaminyl transferase (EC 2.4.2.41). A maximum of 1.7 mol of GalNAc was transferred to basic protein on threonines 95 and 98 of the protein. Proton NMR studies of basic protein glycosylated with 0.48-1.7 mol of GalNAc/mol of MBP showed that the order of addition to the two threonine residues is not random but sequential. The Thr-95 resonances shifted downfield, followed by the downfield shift of the Thr-98 resonances with increasing glycosylation. Since this peptide segment of the molecule is highly structured, conformational factors are probably responsible for this directed addition.     

Reversible deactivation of beta-lactamase by quinacillin. Extent of the conformational change in the isolated transitory complex.

Conditions have been established where the deactivation of the beta-lactamase from Staphylococcus aureus PC1 by the penicillin substrate, quinacillin, is close to complete but fully reversible. The temperature-dependence of the rate of re-activation indicated a half-life of about 170 min for the deactivated state at 0 degrees C. Measurement of the relative viscosity of mixtures of enzyme and quinacillin at 8.4 degrees C ruled out any significant difference in shape or solvation between the deactivated and the normal enzyme. C.d. measurements of the deactivated protein, separated from excess quinacillin, showed that the quinacillin side-chain chromophore was bound in an asymmetric environment. The ellipticity associated with the bound quinacillin chromophore decreased with the same first-order rate constant as that for reappearance of enzyme activity. These findings support the accumulation of a deactivated state that contains bound quinacillin or a derivative. Quinacillin caused a 3-fold increase in the rate of 3H exchange-out (at a rate that was low compared with that for the substantially unfolded or expanded protein). However, there was rapid exchange-out of about 50 3H atoms on addition of 1 M-urea to the deactivated enzyme, whereas the same concentration had no effect on the exchange-out of 3H from native enzyme. The interpretation that quinacillin increases the susceptibility of the native state to unfolding in the presence of urea is supported by the demonstration that SO4(2)- ions decreased the rate and extent of deactivation but had no effect on the rate of re-activation, as predicted from the observation that SO4(2)- ions, in competition with urea, stabilize the native state relative to the partially unfolded state H [Mitchinson & Pain (1985) J. Mol. Biol. 184, 331-342].     

Effect of lead on blood protoporphyrin levels of a group of ring teal ducks (Callonetta leucophrys)

This research determined the relationships between blood lead level and zinc protoporphyrin (ZnPROTO), protoporphyrin IX (PROTO), and free erythrocyte protoporphyrin (FEP) levels in a group of 18 ring teal ducks. Blood samples were drawn from two groups of ring teal ducks as part of the routine health maintenance program of the New York Zoological Park. One group of six teals had been exposed to what is believed to be lead-contaminated dust, while the second group of twelve teals was unexposed. Blood samples were analyzed for lead by flameless atomic absorption and for protoporphyrins by fluorescence. Blood lead level and log blood lead level had positive correlations with each of the protoporphyrins: the logarithmic correlations were better than the nonlogarithmic correlations, and PROTO correlated better than ZnPROTO. With one exception, PROTO levels were higher than ZnPROTO levels. The results suggest that PROTO, FEP, or ZnPROTO could serve as a biological indicator of lead poisoning in ring teal ducks.     

Insulin secretion and protein phosphorylation in PKC-depleted islets of Langerhans.

Protein kinase C (PKC)-dependent phosphorylation of endogenous substrates was measured in electrically permeabilised rat islets of Langerhans. The PKC-activating phorbol ester, 4 beta-phorbol myristate acetate (PMA), caused a slow but prolonged increase in insulin secretion from permeabilised islets, which was accompanied by increased 32P incorporation into several islet proteins of apparent M.W. 30-50 kDa. Depletion of islet PKC by prolonged exposure to PMA abolished subsequent secretory and phosphorylating responses to the phorbol ester. However, PKC-depleted islets did not show diminished responses to glucose, suggesting that PKC-mediated phosphorylation of these proteins is not essential for nutrient-induced insulin secretion.     

Copy number variation-based polymorphism in a new pseudoautosomal region 3 (PAR3) of a human X-chromosome-transposed region (XTR) in the Y chromosome

A 3.5-Mb region of the X chromosome underwent duplication and transposition to the Y chromosome ~5–6 Mya. This X-transposed-region (XTR) originated at Xq21.3 and was inserted at Yp11.2. The two locations have 98.78 % homology and a high concentration of tandem repeats. In whole-genome scans of ten large families with dyslexic members, we identified transposed blocks comprising >102 kb of the Yp11.2 region in its homologous region at Xq21.3 in three females from three different families. Although recombination is known to be limited only to the pseudoautosomal regions (PARs) of the X and Y chromosomes, we report allelic unequal recombination between the XTR region Yp11.2 and Xq21.3, indicating the presence of a new PAR, which we named PAR3. This PAR3 region was also found in 2 % of the general population. An additional layer of justification could be provided from six other dyslexic cases which harbored duplications and deletions in the same Xq21.3 and Yp11.2 regions through allelic unequal recombination.     

PEATmoss (Physcomitrella Expression Atlas Tool): a unified gene expression atlas for the model plant Physcomitrella patens

Physcomitrella patens is a bryophyte model plant that is often used to study plant evolution and development. Its resources are of great importance for comparative genomics and evo‐devo approaches. However, expression data from Physcomitrella patens were so far generated using different gene annotation versions and three different platforms: CombiMatrix and NimbleGen expression microarrays and RNA sequencing. The currently available P. patens expression data are distributed across three tools with different visualization methods to access the data. Here, we introduce an interactive expression atlas, Physcomitrella Expression Atlas Tool (PEATmoss), that unifies publicly available expression data for P. patens and provides multiple visualization methods to query the data in a single web‐based tool. Moreover, PEATmoss includes 35 expression experiments not previously available in any other expression atlas. To facilitate gene expression queries across different gene annotation versions, and to access P. patens annotations and related resources, a lookup database and web tool linked to PEATmoss was implemented. PEATmoss can be accessed at https://peatmoss.online.uni-marburg.de     

Integration of high-throughput analytics and cell imaging enables direct early productivity and product quality assessment during Chinese Hamster ovary cell line development for a complex multi-subunit vaccine antigen

Mammalian cell line generation typically includes stable pool generation, single cell cloning and several rounds of clone selection based on cell growth, productivity and product quality criteria. Individual clone expansion and phenotype-based ranking is performed initially for hundreds or thousands of mini-scale cultures, representing the major operational challenge during cell line development. Automated cell culture and analytics systems have been developed to enable high complexity clone selection workflows; while ensuring traceability, safety, and quality of cell lines intended for biopharmaceutical applications. Here we show that comprehensive and quantitative assessment of cell growth, productivity, and product quality attributes are feasible at the 200–1,200 cell colony stage, within 14 days of the single cell cloning in static 96-well plate culture. The early cell line characterization performed prior to the clone expansion in suspension culture can be used for a single-step, direct selection of high quality clones. Such clones were comparable, both in terms of productivity and critical quality attributes (CQAs), to the top-ranked clones identified using an established iterative clone screening approach. Using a complex, multi-subunit antigen as a model protein, we observed stable CQA profiles independently of the cell culture format during the clonal expansion as well as in the batch and fed-batch processes. In conclusion, we propose an accelerated clone selection approach that can be readily incorporated into various cell line development workstreams, leading to significant reduction of the project timelines and resource requirements.     

Phosphofructokinase relocalizes into subcellular compartments with liquid-like properties in vivo

Although much is known about the biochemical regulation of glycolytic enzymes, less is understood about how they are organized inside cells. We systematically examine the dynamic subcellular localization of glycolytic protein phosphofructokinase-1/PFK-1.1 in Caenorhabditis elegans. We determine that endogenous PFK-1.1 localizes to subcellular compartments in vivo. In neurons, PFK-1.1 forms phase-separated condensates near synapses in response to energy stress from transient hypoxia. Restoring animals to normoxic conditions results in cytosolic dispersion of PFK-1.1. PFK-1.1 condensates exhibit liquid-like properties, including spheroid shapes due to surface tension, fluidity due to deformations, and fast internal molecular rearrangements. Heterologous self-association domain cryptochrome 2 promotes formation of PFK-1.1 condensates and recruitment of aldolase/ALDO-1. PFK-1.1 condensates do not correspond to stress granules and might represent novel metabolic subcompartments. Our studies indicate that glycolytic protein PFK-1.1 can dynamically form condensates in vivo.