Uptake of amino acids and thymidine during the first cell cycle of synchronized hamster cells

Summary The net total uptake of four amino acids (valine, leucine, lysine and methionine) used at concentrations required for growth, and of thymidine at tracer concentrations, has been studied during the first cell cycle of an asparagine-dependent strain of transformed BHK cells synchronized by asparagine starvation. The rate of the total uptake of the amino acids, the free pool of the amino acids taken up, and the rate of their incorporation into protein at the cell cycle. The increase in these parameters during the cell cycle was not linear. The uptake of thymidine started before the onset of DNA synthesis and proceeded linearly beyond the peak of the S phase. The rate of accumulation of thymidine into the acid-soluble fraction also increased during the S phase, apart from a tendency to plateau off at the peak of this phase. It reached a second plateau towards the end of the cell cycle, and then declined slightly. Evidence is presented which suggests that the total quantity of protein synthesized during the cell cycle is more than the newly synthesized protein present in the cells at the end of the cell cycle; this indicates degradation and/or secretion of a substantial proportion of the newly synthesized protein. The total protein synthesized at different time points in the cell cycle appeared to contain different proportions of the amino acids used.     

The Heat-Resistant Agglutinin Family Includes a Novel Adhesin from Enteroaggregative Escherichia coli Strain 60A

Heat-resistant agglutinin 1 (Hra1) is an accessory colonization factor of enteroaggregative Escherichia coli (EAEC) strain 042. Tia, a close homolog of Hra1, is an invasin and adhesin that has been described in enterotoxigenic E. coli. We devised a PCR-restriction fragment length polymorphism screen for the associated genes and found that they occur among 55 (36.7%) of the enteroaggregative E. coli isolates screened, as well as lower proportions of enterotoxigenic, enteropathogenic, enterohemorrhagic, and commensal E. coli isolates. Overall, 25%, 8%, and 3% of 150 EAEC strains harbored hra1 alone, tia alone, or both genes, respectively. One EAEC isolate, 60A, produced an amplicon with a unique restriction profile, distinct from those of hra1 and tia. We cloned and sequenced the full-length agglutinin gene from strain 60A and have designated it hra2. The hra2 gene was not detected in any of 257 diarrheagenic E. coli isolates in our collection but is present in the genome of Salmonella enterica serovar Heidelberg strain SL476. The cloned hra2 gene from strain 60A, which encodes a predicted amino acid sequence that is 64% identical to that of Hra1 and 68% identical to that of Tia, was sufficient to confer adherence on E. coli K-12. We constructed an hra2 deletion mutant of EAEC strain 60A. The mutant was deficient in adherence but not autoaggregation or invasion, pointing to a functional distinction from the autoagglutinin Hra1 and the Tia invasin. Hra1, Tia, and the novel accessory adhesin Hra2 are members of a family of integral outer membrane proteins that confer different colonization-associated phenotypes.     

Inhibition of Cdc42 activity extends lifespan and decreases circulating inflammatory cytokines in aged female C57BL/6 mice

Cdc42 is a small RhoGTPase regulating multiple functions in eukaryotic cells. The activity of Cdc42 is significantly elevated in several tissues of aged mice, while the Cdc42 gain‐of‐activity mouse model presents with a premature aging‐like phenotype and with decreased lifespan. These data suggest a causal connection between elevated activity of Cdc42, aging, and reduced lifespan. Here, we demonstrate that systemic treatment of aged (75‐week‐old) female C57BL/6 mice with a Cdc42 activity‐specific inhibitor (CASIN) for 4 consecutive days significantly extends average and maximum lifespan. Moreover, aged CASIN‐treated animals displayed a youthful level of the aging‐associated cytokines IL‐1β, IL‐1α, and INFγ in serum and a significantly younger epigenetic clock as based on DNA methylation levels in blood cells. Overall, our data show that systemic administration of CASIN to reduce Cdc42 activity in aged mice extends murine lifespan.     

Understanding the binding interaction between phenyl boronic acid P1 and sugars: determination of association and dissociation constants using S–V plots,steady‐state spectroscopic methods and molecular docking

Continuous monitoring of glucose and sugar sensing plays a vital role in diabetes control. The drawbacks of the present enzyme‐based sugar sensors have encouraged the investigation into alternate approaches to design new sensors. The popularity of fluorescence sensors is due to their ability to bind reversibly to compounds containing diol. In this study we investigated the binding ability of phenyl boronic acid P1 for monosaccharides and disaccharides (sugars) in aqueous medium at physiological pH 7.4 using steady‐state fluorescence and absorbance. P1 fluorescence was quenched due to formation of esters with sugars. Absorbance and fluorescence measurements led to results that indicated that the sugars studied could be ordered in terms of their affinity to P1, as stated: sucrose > lactose > galactose > xylose > ribose > arabinose. In each case, the slope of modified Stern–Volmer plots was nearly 1, indicating the presence of only a single binding site in boronic acids for sugars. Docking studies were carried out using Schrodinger Maestro v.11.2 software. The binding affinity of phenyl boronic acid P1 with periplasmic protein (PDB ID 2IPM and 2IPL) was estimated using GlideScore.     

Corticotropin-releasing Factor Receptor 2 Mediates Sex-Specific Cellular Stress Responses

Although females suffer twice as much as males from stress-related disorders, sex-specific participating and pathogenic cellular stress mechanisms remain uncharacterized. Using corticotropin-releasing factor receptor 2–deficient (Crhr2^−/− ) and wild-type (WT) mice, we show that CRF receptor type 2 (CRF₂) and its high-affinity ligand, urocortin 1 (Ucn1), are key mediators of the endoplasmic reticulum (ER) stress response in a murine model of acute pancreatic inflammation. Ucn1 was expressed de novo in acinar cells of male, but not female WT mice during acute inflammation. Upon insult, acinar Ucn1 induction was markedly attenuated in male but not female Crhr2^−/− mice. Crhr₂^−/− mice of both sexes show exacerbated acinar cell inflammation and necrosis. Electron microscopy showed mild ER damage in WT male mice and markedly distorted ER structure in Crhr2^−/− male mice during pancreatitis. WT and Crhr2^−/− female mice showed similarly distorted ER ultrastructure that was less severe than distortion seen in Crhr2^−/− male mice. Damage in ER structure was accompanied by increased ubiquitination, peIF2, and mistargeted localization of vimentin in WT mice that was further exacerbated in Crhr2^−/− mice of both sexes during pancreatitis. Exogenous Ucn1 rescued many aspects of histological damage and cellular stress response, including restoration of ER structure in male WT and Crhr2^−/−mice, but not in females. Instead, females often showed increased damage. Thus, specific cellular pathways involved in coping and resolution seem to be distinct to each sex. Our results demonstrate the importance of identifying sex-specific pathogenic mechanisms and their value in designing effective therapeutics.     

Differential Modulation of TREM2 Protein during Postnatal Brain Development in Mice

During postnatal development, microglia, the resident innate immune cells of the central nervous system are constantly monitoring the brain parenchyma, cleaning the cell debris, the synaptic contacts overproduced and also maintaining the brain homeostasis. In this context, the postnatal microglia need some control over the innate immune response. One such molecule recently described to be involved in modulation of immune response is TREM2 (triggering receptor expressed on myeloid cells 2). Although some studies have observed TREM2 mRNA in postnatal brain, the regional pattern of the TREM2 protein has not been described. We therefore characterized the distribution of TREM2 protein in mice brain from Postnatal day (P) 1 to 14 by immunostaining. In our study, TREM2 protein was expressed only in microglia/macrophages and is developmentally downregulated in a region-dependent manner. Its expression persisted in white matter, mainly in caudal corpus callosum, and the neurogenic subventricular zone for a longer time than in grey matter. Additionally, the phenotypes of the TREM2+ microglia also differ; expressing CD16/32, MHCII and CD86 (antigen presentation markers) and CD68 (phagocytic marker) in different regions as well as with different intensity till P7. The mannose receptor (CD206) colocalized with TREM2 only at P1–P3 in the subventricular zone and cingulum, while others persisted at low intensities till P7. Furthermore, the spatiotemporal expression pattern and characterization of TREM2 indicate towards its other plausible roles in phagocytosis, progenitor’s fate determination or microglia phenotype modulation during postnatal development. Hence, the increase of TREM2 observed in pathologies may recapitulate their function during postnatal development, as a better understanding of this period may open new pathway for future therapies.     

Plasticity in male mating behavior modulates female life history in fruit flies

In many species, intense male-male competition for the opportunity to sire offspring has led to the evolution of selfish reproductive traits that are harmful to the females they mate with. In the fruit fly, Drosophila melanogaster, males modulate their reproductive behavior based on the perceived intensity of competition in their premating environment. Specifically, males housed with other males subsequently transfer a larger ejaculate during a longer mating compared to males housed alone. Although the potential fitness benefits to males from such plasticity are clear, its effects on females are mostly unknown. Hence, we tested the long-term consequences to females from mating with males with distinct social experiences. First, we verified that competitive experience influences male mating behavior and found that males housed with rivals subsequently have shorter mating latencies and longer mating durations. Then, we exposed females every other day for 20 days to males that were either housed alone or with rivals, and subsequently measured their fitness. We found that females mated to males housed with rivals produce more offspring early in life but fewer offspring later in life and have shorter lifespans but similar intrinsic population growth rates. These results indicate that plasticity in male mating behavior can influence female life histories by altering females’ relative allocation to early versus late investment in reproduction and survival.