Inhibition of LDL oxidation by flavonoids in relation to their structure and calculated enthalpy

Twenty flavonoid compounds of five different subclasses were selected, and the relationship of their structure to the inhibition of low-density lipoprotein (LDL) oxidation in vitro was investigated. The most effective inhibitors, by either copper ion or 2,2'-azobis (2-amidino-propane) dihydrochloride (AAPH) induction, were flavonols and/or flavonoids with two adjacent hydroxyl groups at ring B. In the presence of the later catechol group, the contribution of the double bond and the carbonyl group at ring C was negligible. Isoflavonoids were more effective inhibitors than other flavonoid subclasses with similar structure. Substituting ring B with hydroxyl group(s) at 2' position resulted in a significantly higher inhibitory effect than by substituting ring A or ring B at other positions. The type of LDL inducer had no effect in flavonoids with catechol structure. Calculated heat of formation data (deltadeltaH(f)) revealed that the donation of a hydrogen atom from position 3 was the most likely result, followed by that of a hydroxyl from ring B. Position 3 was favored only in the presence of conjugated double bonds between ring A to ring B. This study makes it possible to assign the contribution of different functional groups among the flavonoid subclasses to in vitro inhibition of LDL oxidation.     

Mutations in the chloroplast inner envelope protein TIC100 impair and repair chloroplast protein import and impact retrograde signaling

Chloroplast biogenesis requires synthesis of proteins in the nucleocytoplasm and the chloroplast itself. Nucleus-encoded chloroplast proteins are imported via multiprotein translocons in the organelle’s envelope membranes. Controversy exists around whether a 1-MDa complex comprising TIC20, TIC100, and other proteins constitutes the inner membrane TIC translocon. The Arabidopsis thaliana cue8 virescent mutant is broadly defective in plastid development. We identify CUE8 as TIC100. The tic100^cue8 mutant accumulates reduced levels of 1-MDa complex components and exhibits reduced import of two nucleus-encoded chloroplast proteins of different import profiles. A search for suppressors of tic100^cue8 identified a second mutation within the same gene, tic100^soh1, which rescues the visible, 1 MDa complex-subunit abundance, and chloroplast protein import phenotypes. tic100^soh1 retains but rapidly exits virescence and rescues the synthetic lethality of tic100^cue8 when retrograde signaling is impaired by a mutation in the GENOMES UNCOUPLED 1 gene. Alongside the strong virescence, changes in RNA editing and the presence of unimported precursor proteins show that a strong signaling response is triggered when TIC100 function is altered. Our results are consistent with a role for TIC100, and by extension the 1-MDa complex, in the chloroplast import of photosynthetic and nonphotosynthetic proteins, a process which initiates retrograde signaling.

Complementary mutations in TIC100 of the chloroplast inner envelope membrane cause reductions or corrective improvements in chloroplast protein import, and highlight a signaling role.

IN A NUTSHELLBackground: Plants harvest energy from the sun and CO₂ from the air and convert them into the energy-rich molecules they, and eventually us, are made of. Plants do this, photosynthesis, in bodies called chloroplasts inside their cells. Chloroplasts, made of protein and membrane material, were, before plants evolved, free-living bacteria, but the synthesis of most of their proteins occurs outside them, using information carried by the cell’s nuclear DNA, so most proteins have to be brought into developing chloroplasts, across the double membrane surrounding them, through dedicated, selective channels, formed by TOC (outer) and TIC (inner envelope) proteins. The identity of those channels matters as it helps determine versions of chloroplasts suited for particular environments. Which TIC proteins constitute the inner envelope channel has been a matter of controversy.Question: A mutant Arabidopsis plant called cue8 is slow-to-green (young leaves begin almost white) and shows delayed chloroplast and plant development. We looked for the molecular identity of the CUE8 gene. We also caused further mutations in this mutant and searched whether any corrected the defects in cue8.Findings: We found the mutated gene causing the cue8 defects is the TIC100 gene. This is one essential component of the “TIC 1-MDa complex,” one of the two versions of the TIC import complex under debate. That complex is made of several proteins, all present at reduced levels in cue8. In laboratory assays in which proteins are imported into isolated chloroplasts, cue8 performed worse than normal plants for a photosynthetic and a housekeeping chloroplast protein. A corrective, “suppressor” mutant was identified, and it carried a second mutation in TIC100, one physically complementary to the first one. Both the single and the double (suppressed) mutant still were slow-to-green, which evidences a signaling role for import defects to the nucleus, making photosynthetic genes active or not.Next steps: Surprisingly the grasses, including the cereals, have one core protein of the TIC 1 MDa complex but not the rest (including TIC100). We don’t know how their TIC channels operate. We also need to learn how the information on the defect in protein import, which occurs at the chloroplast envelope, is relayed to the cell’s nucleus (but we do have some clues).     

A GABAergic inhibitory microcircuit controlling cholinergic outflow to the airways.

GABA is the main inhibitory neurotransmitter that participates in the regulation of cholinergic outflow to the airways. We have tested the hypothesis that a monosynaptic GABAergic circuit modulates the output of airway-related vagal preganglionic neurons (AVPNs) in the rostral nucleus ambiguus by using a dual-labeling electron microscopic method combining immunocytochemistry for glutamic acid decarboxylase (GAD) with retrograde tracing from the trachea. We also determined the effects of blockade of GABAA receptors on airway smooth muscle tone. The results showed that retrogradely labeled AVPNs received a significant GAD-immunoreactive (GAD-IR) terminal input. Out of a pooled total of 3,161 synaptic contacts with retrogradely labeled somatic and dendritic profiles, 20.2% were GAD-IR. GAD-IR terminals formed significantly more axosomatic synapses than axodendritic synapses (P < 0.02). A dense population of GABAergic synaptic contacts on AVPNs provides a morphological basis for potent physiological effects of GABA on the excitability of AVPNs. GAD-IR terminals formed exclusively symmetric synaptic specializations. GAD-IR terminals were significantly larger (P < 0.05) in both length and width than unlabeled terminals synapsing on AVPNs. Therefore, the structural characteristics of certain nerve terminals may be closely correlated with their function. Pharmacological blockade of GABAA receptors within the rostral nucleus ambiguus increased activity of putative AVPNs and airway smooth muscle tone. We conclude that a tonically active monosynaptic GABAergic circuit utilizing symmetric synapses regulates the discharge of AVPNs.     

Novel inner membrane retention signals in Pseudomonas aeruginosa lipoproteins

The ultimate membrane localization and function of most of the 185 predicted Pseudomonas aeruginosa PAO1 lipoproteins remain unknown. We constructed a fluorescent lipoprotein, CSFP^OmlA-ChFP, by fusing the signal peptide and the first four amino acids of the P. aeruginosa outer membrane lipoprotein OmlA to the monomeric red fluorescent protein mCherry (ChFP). When cells were plasmolyzed with 0.5 M NaCl, the inner membrane separated from the outer membrane and formed plasmolysis bays. This permits the direct observation of fluorescence in either the outer or inner membrane. CSFP^OmlA-ChFP was shown to localize in the outer membrane by fluorescence microscopy and immunoblotting analysis of inner and outer membrane fractions. The site-directed substitution of the amino acids at positions +2, +3, and +4 in CSFP^OmlA-ChFP was performed to test the effects on lipoprotein localization of a series of amino acid sequences selected from a panel of predicted lipoproteins. We confirmed Asp⁺² and Lys⁺³ Ser⁺⁴ function as inner membrane retention signals and identified four novel inner membrane retention signals: CK⁺² V⁺³ E⁺⁴, CG⁺² G⁺³ G⁺⁴, CG⁺² D⁺³ D⁺⁴, and CQ⁺² G⁺³ S⁺⁴. These inner membrane retention signals are found in 5% of the 185 predicted P. aeruginosa lipoproteins. Full-length chimeras of predicted lipoproteins PA4370 and PA3262 fused to mCherry were shown to reside in the inner membrane and showed a nonuniform or patchy distribution in the membrane. The optical sectioning of cells producing PA4370^CGDD-ChFP and PA3262^CDSQ-ChFP by confocal microscopy improved the resolution and indicated a helix-like localization pattern in the inner membrane. The method described here permits the in situ visualization of lipoprotein localization and should work equally well for other membrane-associated proteins.     

Catecholaminergic microcircuitry controlling the output of airway-related vagal preganglionic neurons.

In this study, we have investigated the ultrastructure and function of the catecholaminergic circuitry modulating the output of airway-related vagal preganglionic neurons (AVPNs) in ferrets. Immunoelectron microscopy was employed to characterize the nature of catecholaminergic innervation of AVPN at the ultrastructural level. In addition, immunofluorescence was used to examine the expression of the alpha(2A)-adrenergic receptor (alpha(2A)-AR) on AVPNs, and norepinephrine release within the rostral nucleus ambiguous (rNA) was measured by using microdialysis. Physiological experiments were performed to determine the effects of stimulation of the noradrenergic locus coeruleus (LC) cell group on airway smooth muscle tone. The results showed that 1) catecholaminergic nerve endings terminate in the vicinity of identified AVPNs but very rarely form axosomatic or axodendritic synapses with the AVPNs that innervate the extrathoracic trachea; 2) AVPNs express the alpha(2A)-AR; 3) LC stimulation-induced norepinephrine release within the rNA region was associated with airway smooth muscle relaxation; and 4) blockade of alpha(2A)-AR on AVPNs diminished the inhibitory effects of LC stimulation on airway smooth muscle tone. It is concluded that a noradrenergic circuit originating within the LC is involved in the regulation of AVPN activity within the rNA, and stimulation of the LC dilates the airways by the release of norepinephrine and activation of alpha(2A)-AR expressed by AVPNs, mainly via volume transmission.     

Toward visualization of nanomachines in their native cellular environment

The cellular nanocosm is made up of numerous types of macromolecular complexes or biological nanomachines. These form functional modules that are organized into complex subcellular networks. Information on the ultra-structure of these nanomachines has mainly been obtained by analyzing isolated structures, using imaging techniques such as X-ray crystallography, NMR, or single particle electron microscopy (EM). Yet there is a strong need to image biological complexes in a native state and within a cellular environment, in order to gain a better understanding of their functions. Emerging methods in EM are now making this goal reachable. Cryo-electron tomography bypasses the need for conventional fixatives, dehydration and stains, so that a close-to-native environment is retained. As this technique is approaching macromolecular resolution, it is possible to create maps of individual macromolecular complexes. X-ray and NMR data can be ‘docked’ or fitted into the lower resolution particle density maps to create a macromolecular atlas of the cell under normal and pathological conditions. The majority of cells, however, are too thick to be imaged in an intact state and therefore methods such as ‘high pressure freezing’ with ‘freeze-substitution followed by room temperature plastic sectioning’ or ‘cryo-sectioning of unperturbed vitreous fully hydrated samples’ have been introduced for electron tomography. Here, we review methodological considerations for visualizing nanomachines in a close-to-physiological, cellular context. EM is in a renaissance, and further innovations and training in this field should be fully supported. Robert Feulgen Lecture 2009 presented at the 51st symposium of the Society for Histochemistry in Stubai, Austria, October 7–10, 2009.     

Dynein Light Intermediate Chain in Aspergillus nidulans Is Essential for the Interaction between Heavy and Intermediate Chains

Cytoplasmic dynein is a complex containing heavy chains (HCs), intermediate chains (ICs), light intermediate chains (LICs), and light chains (LCs). The HCs are responsible for motor activity. The ICs at the tail region of the motor interact with dynactin, which is essential for dynein function. However, functions of other subunits and how they contribute to the assembly of the core complex are not clearly defined. Here, we analyzed in the filamentous fungus Aspergillus nidulans functions of the only LIC and two LCs, RobA (Roadblock/LC7) and TctexA (Tctex1) in dynein-mediated nuclear distribution (nud). Whereas the deletion mutant of tctexA did not exhibit an apparent nud mutant phenotype, the deletion mutant of robA exhibited a nud phenotype at an elevated temperature, which is similar to the previously characterized nudG (LC8) deletion mutant. Remarkably, in contrast to the single mutants, the robA and nudG double deletion mutant exhibits a severe nud phenotype at various temperatures. Thus, functions of these two LC classes overlap to some extent, but the presence of both becomes important under specific conditions. The single LIC, however, is essential for dynein function in nuclear distribution. This is evidenced by the identification of the nudN gene as the LIC coding gene, and by the nud phenotype exhibited by the LIC down-regulating mutant, alcA-LIC. Without a functional LIC, the HC-IC association is significantly weakened, and the HCs could no longer accumulate at the microtubule plus end. Thus, the LIC is essential for the assembly of the core complex of dynein in Aspergillus.     

Deletion of cscR in Escherichia coli W improves growth and poly-3-hydroxybutyrate (PHB) production from sucrose in fed batch culture

Sucrose has several advantages over glucose as a feedstock for bioprocesses, both environmentally and economically. However, most industrial Escherichia coli strains are unable to utilize sucrose. E. coli W can grow on sucrose but stops growing when sucrose concentrations become low. This is undesirable in fed-batch conditions where sugar levels are low between feeding pulses. Sucrose uptake rates were improved by removal of the cscR gene, which encodes a protein that represses expression of the sucrose utilization genes at low sucrose concentrations. Poly-3-hydroxybutyrate (PHB) was used as a model compound in order to assess the effect of improved sugar utilization on bio-production. In the cscR knockout strain, production from sucrose was improved by 50%; this strain also produced 30% more PHB than the wild-type using glucose. This result demonstrates the feasibility of utilizing sucrose as an industrial feedstock for E. coli-based bioprocesses in high cell density culture.     

Posture shifting after spinal cord injury using functional neuromuscular stimulation--a computer simulation study

The ability for individuals with spinal cord injury (SCI) to affect changes in standing posture with functional neuromuscular stimulation (FNS) was explored using an anatomically inspired musculoskeletal model of the trunk, pelvis and lower extremities (LE). The model tracked trajectories for anteriorly and laterally shifting movements away from erect stance. Forces were applied to both shoulders to represent upper extremity (UE) interaction with an assistive device (e.g., a walker). The muscle excitations required to execute shifting maneuvers with UE forces <10% body-weight (BW) were determined via dynamic optimization. Nine muscle sets were examined to maximize control of shifting posture. Inclusion of the Psoas and External Obliques bilaterally resulted in the least relative UE effort (0.119, mean UE effort = 45.3N ≡ 5.4% BW) for anterior shifting. For lateral shifting, the set including the Psoas and Latissimus Dorsi bilaterally yielded the best performance (0.025, mean UE effort = 27.8 N ≡ 3.3% BW). However, adding the Psoas alone bilaterally competed favorably in overall best performance across both maneuvers. This study suggests suitable activation to specific muscles of the trunk and LE can enable individuals with SCI to alter their standing postures with minimal upper-body effort and subsequently increase reach and standing work volume.