Synthesis and biological activity of N-Acylated ornithine analogues of daptomycin

N-Acylated ornithine analogues of daptomycin were synthesized and tested for their antibacterial efficacy.     

Orderly order in protein intrinsic disorder distribution: disorder in 3500 proteomes from viruses and the three domains of life

Intrinsically disordered proteins and intrinsically disordered protein regions are highly abundant in nature. However, the quantitative and qualitative measures of protein intrinsic disorder in species with known genomes are still not available. Furthermore, although the correlation between high fraction of disordered residues and advanced species has been reported, the details of this correlation and the connection between the disorder content and proteome complexity have not been reported as of yet. To fill this gap, we analysed entire proteomes of 3484 species from three domains of life (archaea, bacteria and eukaryotes) and from viruses. Our analysis revealed that the evolution process is characterized by distinctive patterns of changes in the protein intrinsic disorder content. We are showing here that viruses are characterized by the widest spread of the proteome disorder content (the percentage of disordered residues ranges from 7.3% in human coronavirus NL63 to 77.3% in Avian carcinoma virus). For several organisms, a clear correlation is seen between their disorder contents and habitats. In multicellular eukaryotes, there is a weak correlation between the complexity of an organism (evaluated as a number of different cell types) and its overall disorder content. For both the prokaryotes and eukaryotes, the disorder content is generally independent of the proteome size. However, disorder shows a sharp increase associated with the transition from prokaryotic to eukaryotic cells. This suggests that the increased disorder content in eukaryotic proteomes might be used by nature to deal with the increased cell complexity due to the appearance of the various cellular compartments.     

Thermal preferences of hatchling saltwater crocodiles (Crocodylus porosus) in response to time of day,social aggregation and feeding

Three month old hatchling Crocodylus porosus with data loggers in their stomachs were placed in thermal gradients, in isolation (N=16) and in groups of 4 (N=8 groups; 32 individuals). Mean T_b and variation in T_b (SD) was not different whether individual crocodiles in isolation were fasted or fed, or if individuals were housed in isolation (I) or in groups (G). However, individuals in isolation (N=16) maintained slightly lower T_bs than those in groups (N=32) during the early morning (06:00–11:00 h). The overall mean T_b recorded for fasted individuals in the isolated and group treatments (N=48) was 30.9±2.3 °C SD, with 50% of T_bs (T_set) between 29.4 °C and 32.6 °C, and a voluntary maximum and minimum of 37.6 °C and 23.2 °C respectively. During the day (11:00–17:00 h), individuals in isolation and in groups selected the warmer parts of the gradient on land, where they moved little. Outside of this quiescent period (QP), activity levels were much higher and they used the water more. There was a strong diurnal cycle for fasted individuals in isolation and in groups, with T_b during the QP (31.9±2.09 °C; N=48) significantly higher than during the non-quiescent period (NQP: 30.6±2.31 °C). Thermal variation (SD) in T_b was relatively stable throughout the day, with the highest variation at around dusk and early evening (18:00–20:00 h), which coincided with a period of highest activity. The diurnal activity cycle appears innate, and may reflect the need to engage in feeding activity at the water's edge in the early evening, despite ambient temperatures being cooler, with reduced activity and basking during the day. If so, preferred T_b may be more accurately defined as the mean T_b during the QP rather than the NQP. Implications for the thermal environment best suited for captive C. porosus hatchlings are discussed.     

EphA signaling promotes actin-based dendritic spine remodeling through slingshot phosphatase

Actin cytoskeletal remodeling plays a critical role in transforming the morphology of subcellular structures across various cell types. In the brain, restructuring of dendritic spines through actin cytoskeleletal reorganization is implicated in the regulation of synaptic efficacy and the storage of information in neural circuits. However, the upstream pathways that provoke actin-based spine changes remain only partly understood. Here we show that EphA receptor signaling remodels spines by triggering a sequence of events involving actin filament rearrangement and synapse/spine reorganization. Rapid EphA signaling over minutes activates the actin filament depolymerizing/severing factor cofilin, alters F-actin distribution in spines, and causes transient spine elongation through the phosphatases slingshot 1 (SSH1) and calcineurin/protein phosphatase 2B (PP2B). This early phase of spine extension is followed by synaptic reorganization events that take place over minutes to hours and involve the relocation of pre/postsynaptic components and ultimately spine retraction. Thus, EphA receptors utilize discrete cellular and molecular pathways to promote actin-based structural plasticity of excitatory synapses.     

Wnt/β-catenin signaling pathway and thioredoxin-interacting protein (TXNIP) mediate the "glucose sensor" mechanism in metastatic breast cancer-derived cells MDA-MB-231

In this study we investigated the effect of glucose on GSK3β and β-catenin expression and the involvement of the N-linked glycosylation and hexosamine pathways in the Wnt canonical pathway in response to in vitro conditions resembling normoglycemia (5 mmol) and hyperglycemia (20 mmol) in the metastatic breast cancer-derived cell line MDA-MB-231. We also investigated the relationship between this circuitry and the thioredoxin-interacting protein (TXNIP) regulation that seems to be related. MDA-MB-231 cells were grown either in 5 or 20 mM glucose chronically prior to plating. For glucose shift (5/20), cells were plated in 5 mM glucose and shifted to 20 mM at time 0. Both protein and mRNA levels for GSK3β but only the protein expression for β-catenin, were increased in response to high glucose. Furthermore, we assessed the response of GSK3β, β-catenin, and TXNIP to inhibition of the N-linked glycosylation, hexosamine, and Wnt pathways. Wnt signaling pathway activation was validated by specific reporter assay. We show that high levels of glucose regulate mRNA and protein expression of GSK3β, and consequently higher levels of activated β-catenin protein, which locates to the nucleus and is associated with increased levels of cyclin D1 expression. This event coincides with increased level of N-terminal Ser 9 phosphorylation of GSK3β protein. The inhibition of both the hexosamine pathway and N-linked glycosylation along with Wnt signaling pathway by sFRP1 and DKK1 is associated with significant decrease of the protein levels of GSK3β, β-catenin, and TXNIP RNA. Our work illuminates a novel and never described before function of this signaling pathway that relates glucose metabolism with redox regulation mechanism.     

Mapping general anesthetic binding site(s) in human α1β3 γ-aminobutyric acid type A receptors with [³H]TDBzl-etomidate, a photoreactive etomidate analogue

The γ-aminobutyric acid type A receptor (GABA(A)R) is a target for general anesthetics of diverse chemical structures, which act as positive allosteric modulators at clinical doses. Previously, in a heterogeneous mixture of GABA(A)Rs purified from bovine brain, [3H]azietomidate photolabeling of αMet-236 and βMet-286 in the αM1 and βM3 transmembrane helices identified an etomidate binding site in the GABA(A)R transmembrane domain at the interface between the β and α subunits [Li, G. D., et.al. (2006) J. Neurosci. 26, 11599-11605]. To further define GABA(A)R etomidate binding sites, we now use [3H]TDBzl-etomidate, an aryl diazirine with broader amino acid side chain reactivity than azietomidate, to photolabel purified human FLAG-α1β3 GABA(A)Rs and more extensively identify photolabeled GABA(A)R amino acids. [3H]TDBzl-etomidate photolabeled in an etomidate-inhibitable manner β3Val-290, in the β3M3 transmembrane helix, as well as α1Met-236 in α1M1, a residue photolabeled by [3H]azietomidate, while no photolabeling of amino acids in the αM2 and βM2 helices that also border the etomidate binding site was detected. The location of these photolabeled amino acids in GABA(A)R homology models derived from the recently determined structures of prokaryote (GLIC) or invertebrate (GluCl) homologues and the results of computational docking studies predict the orientation of [3H]TDBzl-etomidate bound in that site and the other amino acids contributing to this GABA(A)R intersubunit etomidate binding site. Etomidate-inhibitable photolabeling of β3Met-227 in βM1 by [3H]TDBzl-etomidate and [3H]azietomidate also provides evidence of a homologous etomidate binding site at the β3-β3 subunit interface in the α1β3 GABA(A)R.     

Effect of aging on superovulation efficiency, aneuploidy rates, and sister chromatid cohesion in mice aged up to 15 months

Human eggs are highly aneuploid, with female age being the only known risk factor. Here this aging phenomenon was further studied in Swiss CD1 mice aged between 1 and 15 mo. The mean number of eggs ± SEM recovered from mice following superovulation peaked at 22.5 ± 3.8 eggs/oviduct in 3-mo-old females, decreasing markedly between 6 and 9 mo old, and was only 2.1 ± 0.2 eggs/oviduct by 15 mo. Measurement of aneuploidy in these eggs revealed a low rate, ～3-4%, in mice aged 1 and 3 mo, rising to 12.5% by 9 mo old and to 37.5% at 12 mo. Fifteen-month-old mice had the highest rate of aneuploidy, peaking at 60%. The in situ chromosome counting technique used here allowed us to measure with accuracy the distance between the kinetochores in the sister chromatids of the eggs analyzed for aneuploidy. We observed that this distance increased in eggs from older females, from 0.38 ± 0.01 μm at 1 mo old to 0.82 ± 0.03 μm by 15 mo. Furthermore, in 3- to 12-mo-old females, aneuploid eggs had significantly larger interkinetochore distances than euploid eggs from the same age, and measurements were similar to eggs from the oldest mice. However, the association between aneuploidy and interkinetochore distance was not observed at the oldest, 15-mo age, despite such measurements being maximal. We conclude that in aging CD1 mice, a reduction in the ovulated egg number precedes a rise in aneuploidy and, furthermore, except at very advanced ages, increased interkinetochore distance is associated with aneuploidy.