Additional confirmation of successful cell suspension fractionation of metastatic axillary node tissue.

We present herein an extension to our recently developed and published method termed "Fractionation of Nodal Cell Suspension" (FNCS). The method enables efficient subcellular fractionation into nuclear (N) and cytosolic (C) compartments of extremely fibrous and problematic metastatic axillary lymph node (mALN) tissue, using the entire nodule. For the purpose of the present study, a case of invasive lobular breast cancer (BC) patient with pT2N3aMx status and defined primary tumor markers (ERα 8, PR-B 8, and HER2 score 0) was available. Initially, the mALN tissue of this patient was analyzed by immunohistochemistry (IHC), and a positive correlation of nodal ERα, PR-B and HER2 biomarkers to those of the primary tumor was obtained. Subsequently, the mALN was FNCS fractionated into N and C, and Western blot (WB) analysis demonstrated a single band for ERα, PR-B and nuclear loading control (HDAC1) in nuclear, but not in the cytosolic compartments, confirming the efficiency of our fractionation protocol. At the same time, HER2 bands were not observed in either compartment, in accordance with HER2 negativity determined by IHC in both primary tumor and mALN tissue. In conclusion, by confirming the nuclear expression of ERα and PR-B biomarkers in metastatic loci, we demonstrate the purity of the FNCS-generated compartments - the protocol that offers a reliable tool for further analysis of nuclear versus cytosolic content in downstream analysis of novel biomarkers in the whole mALN of BC patients.

Unveiling the agonistic properties of Preyssler-type Polyoxotungstates on purinergic P2 receptors.

The Preyssler-type polyoxotungstate ({P5W30}) belongs to the family of polyanionic metal-oxides formed by group V and VI metal ions, such as V, Mo and W, commonly known as polyoxometalates (POMs). POMs have demonstrated inhibitory effect on a significant number of ATP-binding proteins in vitro. Purinergic P2 receptors, widely expressed in eukaryotic cells, contain extracellularly oriented ATP-binding sites and play many biological roles with health implications. In this work, we use the immortalized mouse hippocampal neuronal HT-22 cells in culture to study the effects of {P5W30} on the cytosolic Ca2+ concentration. Changes in cytosolic Ca2+ concentration were monitored using fluorescence microscopy of HT-22 cells loaded with the fluorescent Ca2+ indicator Fluo3. 31P-Nuclear magnetic resonance measurements of {P5W30} indicate its stability in the medium used for cytosolic Ca2+ measurements for over 30 min. The findings reveal that addition of {P5W30} to the extracellular medium induces a sustained increase of the cytosolic Ca2+ concentration within minutes. This Ca2+ increase is triggered by extracellular Ca2+ entry into the cells and is dose-dependent, with a half-of-effect concentration of 0.25 ± 0.05 µM {P5W30}. In addition, after the {P5W30}-induced cytosolic Ca2+ increase, the transient Ca2+ peak induced by extracellular ATP is reduced up to 100% with an apparent half-of-effect concentration of 0.15 ± 0.05 µM {P5W30}. Activation of metabotropic purinergic P2 receptors affords about 80% contribution to the increase of Fluo3 fluorescence elicited by {P5W30} in HT-22 cells, whereas ionotropic receptors contribute, at most, with 20%. These results suggest that {P5W30} could serve as a novel agonist of purinergic P2 receptors.

The canonical antiviral protein oligoadenylate synthetase 1 elicits antibacterial functions by enhancing IRF1 translation.

An important property of the host innate immune response during microbial infection is its ability to control the expression of antimicrobial effector proteins, but how this occurs post-transcriptionally is not well defined. Here, we describe a critical antibacterial role for the classic antiviral gene 2'-5'-oligoadenylate synthetase 1 (OAS1). Human OAS1 and its mouse ortholog, Oas1b, are induced by interferon-γ and protect against cytosolic bacterial pathogens such as Francisella novicida and Listeria monocytogenes in vitro and in vivo. Proteomic and transcriptomic analysis showed reduced IRF1 protein expression in OAS1-deficient cells. Mechanistically, OAS1 binds and localizes IRF1 mRNA to the rough endoplasmic reticulum (ER)-Golgi endomembranes, licensing effective translation of IRF1 mRNA without affecting its transcription or decay. OAS1-dependent translation of IRF1 leads to the enhanced expression of antibacterial effectors, such as GBPs, which restrict intracellular bacteria. These findings uncover a noncanonical function of OAS1 in antibacterial innate immunity.

Subcellular targeting strategies for protein and peptide delivery.

Cytosolic delivery of proteins and peptides provides opportunities for effective disease treatment, as they can specifically modulate intracellular processes. However, most of protein-based therapeutics only have extracellular targets and are cell-membrane impermeable due to relatively large size and hydrophilicity. The use of organelle-targeting strategy offers great potential to overcome extracellular and cell membrane barriers, and enables localization of protein and peptide therapeutics in the organelles. Although progresses have been made in the recent years, organelle-targeted protein and peptide delivery is still challenging and under exploration. We reviewed recent advances in subcellular targeted delivery of proteins/peptides with a focus on targeting mechanisms and strategies, and highlight recent examples of active and passive organelle-specific protein and peptide delivery systems. This emerging platform could open a new avenue to develop more effective protein and peptide therapeutics.

Potential Mechanism and Involvement of P120-Catenin in the Malignant Biology of Glioma.

Objective: This study analyzed the influence of p120-catenin (CTNND1) on the malignant characteristics of glioma and elucidated the potential underlying mechanism. Methods: The p120 expression level was assessed in the brain tissues of 42 glioma patients and 10 patients with epilepsy by using the immunohistochemical method. Meanwhile, quantitative PCR technology was employed to assess the expression of P120 in the brain tissues of 71 glioma patients and 13 epilepsy patients. LN229, U251, and U87 glioma cells were used for in vitro analysis and categorized into four treatment groups: siRNA-BC group (no RNA sequence was transfected), siRNA-NC group (transfected control RNA sequences with no effect), and siRNA-1 and siRNA-2 groups (two p120-specific interfering RNA transfection). p120 expression in these treatment groups was quantified by western blotting assay. The migratory and invasive capabilities of glioma cells were studied by wound healing assay and Transwell invasion assay, respectively, under different treatment conditions. MTT assay and cell cycle and apoptosis assay were used to determine glioma cell proliferation and apoptosis, respectively. Enzyme-labeled assay was performed to measure intracellular calcium ion concentration. Immunofluorescence assay was performed for determining microtubule formation and glioma cell distribution. Results: Brain tissues of the glioma group exhibited a remarkable increase in the p120 expression level as compared to brain tissues of the nontumor group (P < 0.05). Furthermore, a strong positive correlation was noted between the malignancy degree in glioma brain tissues and p120 expression in Western blotting (r = 0.906, P = 0.00) and QT-PCR (F=830.6, P＜0.01). Compared to the BC and NC groups, the siRNA transfection groups showed a significant suppression in p120 expression in glioma cells (P < 0.05), with a marked attenuation in the invasive, migratory, and proliferative capabilities of glioma cells as well as an increase in apoptotic potential (P < 0.05). Enzyme-labeled assay showed a remarkable increase in calcium concentration in glioma cells after siRNA treatment. Immunofluorescence assay revealed that the microtubule formation ability of glioma cells reduced after siRNA treatment. Conclusion: p120 has a pivotal involvement in facilitating glioma cell invasion and proliferation by potentially modulating these processes through its involvement in microtubule formation and regulation of intracellular calcium ion levels.

CYTOSOLIC INVERTASE2 regulates flowering and reactive oxygen species-triggered programmed cell death in tomato.

Cytosolic invertase (CIN) in plants hydrolyzes sucrose into fructose and glucose, influencing flowering time and organ development. However, the underlying molecular mechanisms remain elusive. Through expressional, genetic, and histological analyses, we identified a substantially role of SlCIN2 (localized in mitochondria) in regulating flowering and pollen development in tomato (Solanum lycopersicum). The overexpression of SlCIN2 resulted in increased hexose accumulation and decreased sucrose and starch content. Our findings indicated that SlCIN2 interacts with Sucrose transporter2 (SlSUT2) to inhibit the sucrose transport activity of SlSUT2, thereby suppressing sucrose content in flower buds and delaying flowering. We found that higher levels of glucose in SlCIN2-overexpressing anthers result in the accumulation of abscisic acid (ABA) and reactive oxygen species (ROS), thereby disrupting programmed cell death (PCD) in anthers and delaying the end of tapetal degradation. Exogenous sucrose partially restored fertility in SlCIN2-overexpressing plants. This study revealed the mechanism by which SlCIN2 regulates pollen development and demonstrated a strategy for creating sugar-regulated gene male sterility lines for tomato hybrid seed production.

Rational design of lipid nanoparticles: overcoming physiological barriers for selective intracellular mRNA delivery.

This review introduces the typical delivery process of messenger RNA (mRNA) nanomedicines and concludes that the delivery involves a at least four-step SCER cascade and that high efficiency at every step is critical to guarantee high overall therapeutic outcomes. This SCER cascade process includes selective organ-targeting delivery, cellular uptake, endosomal escape, and cytosolic mRNA release. Lipid nanoparticles (LNPs) have emerged as a state-of-the-art vehicle for in vivo mRNA delivery. The review emphasizes the importance of LNPs in achieving selective, efficient, and safe mRNA delivery. The discussion then extends to the technical and clinical considerations of LNPs, detailing the roles of individual components in the SCER cascade process, especially ionizable lipids and helper phospholipids. The review aims to provide an updated overview of LNP-based mRNA delivery, outlining recent innovations and addressing challenges while exploring future developments for clinical translation over the next decade.

Nitrogen inhibition of nitrogenase activity involves the modulation of cytosolic invertase in soybean nodule.

Legume symbiotic nitrogen fixation (SNF) is suppressed by inorganic N in the soil. High N inhibition of nitrogenase activity is associated with the deprivation of carbon allocation and metabolism in nodules. However, the underlying molecular mechanisms remain unclear. Here, we identify GmCIN1 which encodes a cytosolic invertase, as a gateway for the N-tuning of sucrose utilization in nodules. GmCIN1 is enriched in mature soybean nodules and its expression is regulated by nitrogen status. The knockout of GmCIN1 using genome editing partially mimicks the inhibitory effects of N on nitrogenase activity and sugar content and the impact of high N on nodule transcriptomes. This indicates that GmCIN1 partially mediates the high N inhibition of nodule activity. Moreover, ChIP-qPCR and EMSA reveal that SNAP1/2 transcription factors directly bind to the GmCIN1 promoter. In addition, SNAP1/2 may be involved in the repression of GmCIN1 expression in mature nodules at high N concentrations. Our findings provide insights into the involvement of the transcriptional tuning of C metabolism genes by N-signaling modulators in the N-induced inhibition of nitrogenase activity.

Cytosolic acidification and oxidation are the toxic mechanisms of SO2 in Arabidopsis guard cells.

SO2/H2SO3 can damage plants. However, its toxic mechanism has still been controversial. Two models have been proposed, cytosolic acidification model and cellular oxidation model. Here, we assessed the toxic mechanism of H2SO3 in three cell types of Arabidopsis thaliana, mesophyll cells, guard cells and petal cells. The sensitivity of guard cells of CHLORIDE CHANNEL a (CLCa)-knockout mutants to H2SO3 was significantly lower than those of wildtype plants. Expression of other CLC genes in mesophyll cells and petal cells were different from guard cells. Treatment with antioxidant, disodium 4,5-dihydroxy-1,3-benzenedisulfonate (tiron), increased the median lethal concentration (LC50) of H2SO3 in guard cells indicating the involvement of cellular oxidation, while the effect was negligible in mesophyll cells and petal cells. These results indicate that there are two toxic mechanisms of SO2 to Arabidopsis cells: cytosolic acidification and cellular oxidation, and the toxic mechanism may vary among cell types.

Inhibition of PLA2G4A attenuated valproic acid- induced lysosomal membrane permeabilization and restored impaired autophagic flux: Implications for hepatotoxicity.

Valproic acid (VPA) has broad efficacy against several seizures but causes liver injury limiting its prolonged clinical use. Some studies have demonstrated that VPA-induced hepatotoxicity is characterized by microvesicular hepatic steatosis. However, novel detailed mechanisms to explain VPA-induced hepatic steatosis and experimentally rigorously validated protective agents are still lacking. In this study, 8-week-old C57BL/6J mice were gavaged with VPA (500 mg/kg/d) for 4 weeks to establish an in vivo model of VPA-induced chronic liver injury. Quantitative proteomic and non-targeted lipidomic analyses were performed to explore the underlying mechanisms of VPA-induced hepatotoxicity. As a result, VPA-induced hepatotoxicity is associated with impaired autophagic flux, which is attributed to lysosomal dysfunction. Further studies revealed that VPA-induced lysosomal membrane permeabilization (LMP), allows soluble lysosomal enzymes to leak into the cytosol, which subsequently led to impaired lysosomal acidification. A lower abundance of glycerophospholipids and an increased abundance of lysophospholipids in liver tissues of mice in the VPA group strongly indicated that VPA-induced LMP may be mediated by the activation of phospholipase PLA2G4A. Metformin (Met) acted as a potential protective agent attenuating VPA-induced liver dysfunction and excessive lipid accumulation. Molecular docking and cellular thermal shift assays demonstrated that Met inhibited the activity of PLA2G4A by directly binding to it, thereby ameliorating VPA-induced LMP and autophagic flux impairment. In conclusion, this study highlights the therapeutic potential of targeting PLA2G4A-mediated lysosomal dysfunction in VPA-induced hepatotoxicity.

Unveiling compositional images of specific proteins in individual cells by LA-ICP-MS: Labelling with ruthenium red and metal nanoclusters.

BACKGROUND: Recent biological studies have demonstrated that changes can occur in the cellular genome and proteome due to variations in cell volume. Therefore, it is imperative to take cell volume into account when analyzing a target protein. This consideration becomes especially critical in experimental models involving cells subjected to different treatments. Failure to consider cell volume could obscure the studied biological phenomena or lead to erroneous conclusions. However, quantitative imaging of proteins within cells by LA-ICP-MS is limited by the lack of methods that provide the protein concentration (protein mass over cell volume) rather than just protein mass within individual cells. RESULTS: The combination of a metal tagged immunoprobe with ruthenium red (RR) labelling enables the simultaneous analysis of a specific protein and the cell volume in each cell analyzed by LA-ICP-(Q)MS. The results indicate that the CYP1B1 concentration exhibits a quasi-normally distribution in control ARPE-19 cells, whereas AAPH-treated cells reveal the presence of two distinct cell groups, responding and non-responding cells to an in vitro induced oxidative stress. The labelling of the membrane with RR and the measurement of Ru mass in each cell by LA-ICP-MS offers higher precision compared to manually delimitation of the cell perimeter and eliminates the risk of biased information, which can be prone to inter-observer variability. The proposed procedure is fast and minimizes errors in cell area assignment and offers the possibility to carry out a faster data treatment approach if just relative volumes are compared, which can be advantageous for specific applications. SIGNIFICANCE AND NOVELTY: This work presents an innovative strategy to directly study the distribution and concentration of proteins within individual cells by LA-ICP-MS. This method employs ruthenium red as a cell volume marker and Au nanoclusters (AuNCs) tagged immunoprobes to label the protein of interest. Furthermore, the proposed labelling strategy enables rapid data processing, allowing for the calculation of relative concentrations and thus facilitating the comparison across large datasets. As a proof-of-concept, the concentration of the CYP1B1 protein was quantified in ARPE-19 cells under both control and oxidative stress conditions.

N,N-Disubstituted 4-sulfamoylbenzoic Cid Derivatives as Inhibitors of Cytosolic Phospholipase A2α: Synthesis, Aqueous Solubility, and Activity in a Vesicle and a Whole Blood Assay.

BACKGROUND: Cytosolic phospholipase A2α (cPLA2α) is the key enzyme that initiates the arachidonic acid cascade through which proinflammatory lipid mediators can be formed. Therefore, cPLA2α is considered an interesting target for the development of anti-inflammatory drugs. Although several effective inhibitors of the enzyme have been developed, none of them has yet reached clinical application. OBJECTIVE: Recently, we have prepared new 4-sulfamoylbenzoic acid derivatives based on a cPLA2α inhibitor found in a ligand-based virtual screening. The most effective of these compounds were now subjected to further variations in which the substitution pattern on the sulfamoyl nitrogen atom was changed. METHODS: The new compounds were tested in vitro in a vesicle assay for cPLA2α inhibition as well as for their water solubility, metabolic stability, and selectivity towards related enzymes. In addition, they were evaluated ex vivo in a whole blood assay in which metabolites of the arachidonic acid cascade formed after activation of cPLA2α were quantified using a combined online dilution/online solid phase extraction HPLC-MS method. RESULTS: Inhibitors with submicromolar inhibitory in vitro potency were found with favourable water solubility and selectivity. However, their efficacy did not match that of the highly effective, known, structurally related cPLA2a inhibitor giripladib, which was also tested as a reference. One advantage of some of the new compounds compared to giripladib was their significantly improved water solubility. When analyzing the substances in the ex vivo whole blood assay, it was found that the obtained inhibition data correlated better with the in vivo results when the phorbol ester 12-Otetradecanoylphorbol-13-acetate was used for activation of the enzyme in the blood cells instead of the calcium ionophore A23187. CONCLUSION: New compounds with good activity towards cPLA2α and reasonable physicochemical properties were identified. Overall, the results obtained could be helpful in the development of clinically applicable inhibitors of this enzyme.

GRminusRD: A Sensitive Assay to Detect Activation Processes at the Plasma Membrane in Living Cells.

Activation processes at the plasma membrane have been studied with life-cell imaging using GFP fused to a protein that binds to a component of the activation process. In this way, PIP3 formation has been monitored with CRAC-GFP, Ras-GTP with RBD-Raf-GFP, and Rap-GTP with Ral-GDS-GFP. The fluorescent sensors translocate from the cytoplasm to the plasma membrane upon activation of the process. Although this translocation assay can provide very impressive images and movies, the method is not very sensitive, and amount of GFP-sensor at the plasma membrane is not linear with the amount of activator. The fluorescence in pixels at the cell boundary is partly coming from the GFP-sensor that is bound to the activated membrane and partly from unbound GFP-sensor in the cytosolic volume of that boundary pixel. The variable and unknown amount of cytosol in boundary pixels causes the low sensitivity and nonlinearity of the GFP-translocation assay. Here we describe a method in which the GFP-sensor is co-expressed with cytosolic-RFP. For each boundary pixels, the RFP fluorescence is used to determine the amount of cytosol of that pixel and is subtracted from the GFP fluorescence of that pixel yielding the amount of GFP-sensor that is specifically associated with the plasma membrane in that pixel. This GRminusRD method using GFP-sensor/RFP is at least tenfold more sensitive, more reproducible, and linear with activator compared to GFP-sensor alone.

Immunostimulatory nucleic acid nanoparticles (NANPs) augment protective osteoblast and osteoclast type I interferon responses to Staphylococcus aureus.

Recalcitrant staphylococcal osteomyelitis may be due, in part, to the ability of Staphylococcus aureus to invade bone cells. However, osteoclasts and osteoblasts are now recognized to shape host responses to bacterial infection and we have recently described their ability to produce IFN-ß following S. aureus infection and limit intracellular bacterial survival/propagation. Here, we have investigated the ability of novel, rationally designed, nucleic acid nanoparticles (NANPs) to induce the production of immune mediators, including IFN-ß, following introduction into bone cells. We demonstrate the successful delivery of representative NANPs into osteoblasts and osteoclasts via endosomal trafficking when complexed with lipid-based carriers. Their delivery was found to differentially induce immune responses according to their composition and architecture via discrete cytosolic pattern recognition receptors. Finally, the utility of this nanoparticle technology was supported by the demonstration that immunostimulatory NANPs augment IFN-ß production by S. aureus infected bone cells and reduce intracellular bacterial burden.

Deafness causing neuroplastin missense variants fail to promote plasma membrane Ca2+-ATPase levels and Ca2+ transient regulation in brain neurons.

Hearing, the ability to sense sounds, and the processing of auditory information are important for perception of the world. Mice lacking expression of neuroplastin (Np), a type-1 transmembrane glycoprotein, display deafness, multiple cognitive deficiencies, and reduced expression of plasma membrane calcium (Ca2+) ATPases (PMCAs) in cochlear hair cells and brain neurons. In this study, we transferred the deafness causing missense mutations pitch (C315S) and audio-1 (I122N) into human Np (hNp) constructs and investigated their effects at the molecular and cellular levels. Computational molecular dynamics show that loss of the disulfide bridge in hNppitch causes structural destabilization of immunoglobulin-like domain (Ig) III and that the novel asparagine in hNpaudio-1 results in steric constraints and an additional N-glycosylation site in IgII. Additional N-glycosylation of hNpaudio-1 was confirmed by PNGaseF treatment. In comparison to hNpWT, transfection of hNppitch and hNpaudio-1 into HEK293T cells resulted in normal mRNA levels but reduced the Np protein levels and their cell surface expression due to proteasomal/lysosomal degradation. Furthermore, hNppitch and hNpaudio-1 failed to promote exogenous PMCA levels in HEK293T cells. In hippocampal neurons, expression of additional hNppitch or hNpaudio-1 was less efficient than hNpWT to elevate endogenous PMCA levels and to accelerate the restoration of basal Ca2+ levels after electrically evoked Ca2+ transients. We propose that mutations leading to pathological Np variants, as exemplified here by the deafness causing Np mutants, can affect Np-dependent Ca2+ regulatory mechanisms and may potentially cause intellectual and cognitive deficits in humans.

Rational design of diblock copolymer enables efficient cytosolic protein delivery.

Polymer-mediated cytosolic protein delivery demonstrates a promising strategy for the development of protein therapeutics. Here, we propose a new designed diblock copolymer which realizes efficient cytosolic protein delivery both in vitro and in vivo. The polymer contains one protein-binding block composed of phenylboronic acid (PBA) and N-(3-dimethylaminopropyl) (DMAP) pendant units for protein binding and endosomal escape, respectively, followed by the response to ATP enriched in the cytosol which triggers the protein release. The other block is PEG designed to improve particle size control and circulation in vivo. By optimizing the block composition, sequence and length of the copolymer, the optimal one (BP20) was identified with the binding block containing 20 units of both PBA and DMAP, randomly distributed along the chain. When mixed with proteins, the BP20 forms stable nanoparticles and mediates efficient cytosolic delivery of a wide range of proteins including enzymes, toxic proteins and CRISPR/Cas9 ribonucleoproteins (RNP), to various cell lines. The PEG block, especially when further modified with folic acid (FA), enables tumor-targeted delivery of Saporin in vivo, which significantly suppresses the tumor growth. Our results shall inspire the design of novel polymeric vehicles with robust capability for cytosolic protein delivery, which holds great potential for both biological research and therapeutic applications.

ARID1A suppresses R-loop-mediated STING-type I interferon pathway activation of anti-tumor immunity.

Clinical trials have identified ARID1A mutations as enriched among patients who respond favorably to immune checkpoint blockade (ICB) in several solid tumor types independent of microsatellite instability. We show that ARID1A loss in murine models is sufficient to induce anti-tumor immune phenotypes observed in ARID1A mutant human cancers, including increased CD8+ T cell infiltration and cytolytic activity. ARID1A-deficient cancers upregulated an interferon (IFN) gene expression signature, the ARID1A-IFN signature, associated with increased R-loops and cytosolic single-stranded DNA (ssDNA). Overexpression of the R-loop resolving enzyme, RNASEH2B, or cytosolic DNase, TREX1, in ARID1A-deficient cells prevented cytosolic ssDNA accumulation and ARID1A-IFN gene upregulation. Further, the ARID1A-IFN signature and anti-tumor immunity were driven by STING-dependent type I IFN signaling, which was required for improved responsiveness of ARID1A mutant tumors to ICB treatment. These findings define a molecular mechanism underlying anti-tumor immunity in ARID1A mutant cancers.

Cytosolic and Acrosomal pH Regulation in Mammalian Sperm.

As in most cells, intracellular pH regulation is fundamental for sperm physiology. Key sperm functions like swimming, maturation, and a unique exocytotic process, the acrosome reaction, necessary for gamete fusion, are deeply influenced by pH. Sperm pH regulation, both intracellularly and within organelles such as the acrosome, requires a coordinated interplay of various transporters and channels, ensuring that this cell is primed for fertilization. Consistent with the pivotal importance of pH regulation in mammalian sperm physiology, several of its unique transporters are dependent on cytosolic pH. Examples include the Ca2+ channel CatSper and the K+ channel Slo3. The absence of these channels leads to male infertility. This review outlines the main transport elements involved in pH regulation, including cytosolic and acrosomal pH, that participate in these complex functions. We present a glimpse of how these transporters are regulated and how distinct sets of them are orchestrated to allow sperm to fertilize the egg. Much research is needed to begin to envision the complete set of players and the choreography of how cytosolic and organellar pH are regulated in each sperm function.

Liver-specific Lxr inhibition represses reverse cholesterol transport in cholesterol-fed mice.

BACKGROUND AND AIMS: High density lipoprotein (HDL) exerts an anti-atherosclerotic effect via reverse cholesterol transport (RCT). Several phases of RCT are transcriptionally controlled by Liver X receptors (Lxrs). Although macrophage Lxrs reportedly promote RCT, it is still uncertain whether hepatic Lxrs affect RCT in vivo. METHODS: To inhibit Lxr-dependent pathways in mouse livers, we performed hepatic overexpression of sulfotransferase family cytosolic 2B member 1 (Sult2b1) using adenoviral vector (Ad-Sult2b1). Ad-Sult2b1 or the control virus was intravenously injected into wild type mice and Lxrα/ß double knockout mice, under a normal or high-cholesterol diet. A macrophage RCT assay and an HDL kinetic study were performed. RESULTS: Hepatic Sult2b1 overexpression resulted in reduced expression of Lxr-target genes - ATP-binding cassette transporter G5/G8, cholesterol 7α hydroxylase and Lxrα itself - respectively reducing or increasing cholesterol levels in HDL and apolipoprotein B-containing lipoproteins (apoB-L). A macrophage RCT assay revealed that Sult2b1 overexpression inhibited fecal excretion of macrophage-derived 3H-cholesterol only under a high-cholesterol diet. In an HDL kinetic study, Ad-Sult2b1 promoted catabolism/hepatic uptake of HDL-derived cholesterol, thereby reducing fecal excretion. Finally, in Lxrα/ß double knockout mice, hepatic Sult2b1 overexpression increased apoB-L levels, but there were no differences in HDL levels or RCT compared to the control, indicating that Sult2b1-mediated effects on HDL/RCT and apoB-L were distinct: the former was Lxr-dependent, but not the latter. CONCLUSIONS: Hepatic Lxr inhibition negatively regulates circulating HDL levels and RCT by reducing Lxr-target gene expression.

Alternative localization of HEME OXYGENASE 1 in plant cells regulates cytosolic heme catabolism.

Heme, an organometallic tetrapyrrole, is widely engaged in oxygen transport, electron delivery, enzymatic reactions, and signal transduction. In plants, it is also involved in photomorphogenesis and photosynthesis. HEME OXYGENASE 1 (HO1) initiates the first committed step in heme catabolism, and it has generally been thought that this reaction takes place in chloroplasts. Here, we show that HO1 in both Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) has two transcription start sites (TSSs), producing long (HO1L) and short (HO1S) transcripts. Their products localize to the chloroplast and the cytosol, respectively. During early development or de-etiolation, the HO1L/HO1S ratio gradually increases. Light perception via phytochromes and cryptochromes elevates the HO1L/HO1S ratio in the whole seedling through the functions of ELONGATED HYPOCOTYL 5 (HY5) and HY5 HOMOLOG (HYH) and through the suppression of DE-ETIOLATED 1 (DET1), CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1), and PHYTOCHROME INTERACTING FACTORs (PIFs). HO1L introduction complements the HO1-deficient mutant; surprisingly, HO1S expression also restores the short hypocotyl phenotype and high pigment content and helps the mutant recover from the genomes uncoupled (gun) phenotype. This indicates the assembly of functional phytochromes within these lines. Furthermore, our findings support the hypothesis that a mobile heme signal is involved in retrograde signaling from the chloroplast. Altogether, our work clarifies the molecular mechanism of HO1 TSS regulation and highlights the presence of a cytosolic bypass for heme catabolism in plant cells.