The mevalonate pathway is important for the biosynthesis of isoprenoids such as geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate, as well as cholesterol. It has been reported that treatment with 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor ameliorates glomerular injury in several experimental models of progressive glomerular disease. However, the effect of HMG-CoA reductase inhibitor on mesangial cell function has not been fully understood. This investigation was performed to elucidate the role of a mevalonate metabolite(s) in mesangial cell proliferation and extracellular matrix accumulation.

Deoxyribonuclease I (DNase I) was purified from the hen pancreas to electrophoretic homogeneity using six-step column chromatography. The purified enzyme showed a molecular mass of about 33 kDa and maximum activity at pH 7.0. It required divalent cations, Mg2+ and Ca2+, for its activity and was inhibited by EDTA, EGTA and an antibody specific to the purified enzyme but not by G-actin. A 1066-bp cDNA encoding hen DNase I was constructed from the total RNA of a hen pancreas using a combination of the reverse transcriptase-polymerase chain reaction and rapid amplification of cDNA ends methods, followed by sequencing. The cDNA was expressed in Escherichia coli, and the recombinant polypeptide exhibited significant enzyme activity. The mature hen DNase I protein was found to consist of 262 amino acids. In human and bovine DNase I four amino acid residues, Glu-13, Tyr-65, Val-67 and Ala-114 are involved in actin binding, whereas in the hen DNase I these positions were occupied by Asp, Phe, Ser and Phe, respectively. A survey of the DNase I distribution in 15 hen tissues showed that the pancreas had the highest levels of both DNase I enzyme activity and DNase I gene expression. The results of our phylogenetic and immunological analyses indicate that the hen DNase I is not closely related to the mammalian enzymes. This is the first report in which has been described the results of molecular, biochemical and immunological analyses on hen DNase I.

Central blockade of mineralocorticoid receptors (MRs) or angiotensin II type 1 receptors (AT1Rs) attenuates aldosterone (aldo)-salt induced hypertension. We examined the role of the subfornical organ (SFO), aldo synthesized locally in the brain, and MR and AT1R specifically in the paraventricular nucleus (PVN) in aldo-salt hypertension. Wistar rats were treated with subcutaneous aldo (1 μg/h) plus saline as drinking fluid, and gene expression was assessed by real-time qPCR. Other sets of rats received chronic intra-cerebroventricular (icv) infusion of aldo synthase (AS) inhibitor FAD286, MR blocker eplerenone or vehicle, electrolytic or sham lesions of the SFO, or intra-PVN infusion of AAV-MR-siRNA or AAV-AT1aR-siRNA. Infusion of aldo had no effect on 11βHSD2, MR and AT1R mRNA in different nuclei but increased CYP11B2 mRNA in the SFO, and serum and glucocorticoid-kinase 1 (Sgk1) and epithelial sodium channel (ENaC) γ subunit mRNA in the SFO and supraoptic nucleus (SON). MR-siRNA decreased both MR and AT1R mRNA in the PVN by ∼ 60%, but AT1aR-siRNA only decreased AT1R mRNA. SFO lesion, blockade of brain AS or MR, or knockdown of MR or AT1R in the PVN similarly attenuated aldosterone-induced saline intake by ∼ 50% and hypertension by ∼ 70%. These results suggest that an increase in circulating aldosterone may via MR and AT1R in the SFO increase local aldosterone production in hypothalamic nuclei such as the SON and PVN, and via MR enhance AT1R signaling in the PVN. This central aldosterone-MR-AT1R neuro-modulatory pathway appears to play a major role in the progressive hypertension.

The integral selectivity characteristic of the blood brain barrier (BBB) limits therapeutic options for many neurologic diseases and disorders. Currently, very little is known about the mechanisms that govern the dynamic nature of the BBB. Recent reports have focused on the development and application of human brain organoids developed from neuro-progenitor cells. While these models provide an excellent platform to study the effects of disease and genetic aberrances on brain development, they may not model the microvasculature and BBB of the adult human cortex. To date, most in vitro BBB models utilize endothelial cells, pericytes and astrocytes. We report a 3D spheroid model of the BBB comprising all major cell types, including neurons, microglia and oligodendrocytes, to recapitulate more closely normal human brain tissue. Spheroids show expression of tight junctions, adherens junctions, adherens junction-associated proteins and cell specific markers. Functional assessment using MPTP, MPP+ and mercury chloride indicate charge selectivity through the barrier. Junctional protein distribution was altered under hypoxic conditions. Our spheroid model may have potential applications in drug discovery, disease modeling, neurotoxicity and cytotoxicity testing.

Interferon regulatory factor-4 binding protein (IBP) is a novel upstream activator of Rho GTPases. Our previous studies have shown that ectopic expression of IBP was correlated with malignant behaviors of human breast cancer cells, and invasive human breast cancer had high expression of IBP that promoted the proliferation of these cells. However, it remains unknown whether autophagy inhibition contributes to IBP-mediated tumorigenesis. In this study, we for the first time, reported that upregulation of IBP expression significantly suppressed the autophagy of breast cancer cells, and downregulation of IBP expression markedly induced autophagy of these cells. Further investigation revealed that IBP effectively counteracted autophagy by directly activating mammalian target of rapamycin complex 2 (mTORC2) and upregulating phosphorylation of Akt on ser473 and FOXO3a on Thr32. Moreover, IBP-mediated suppression of autophagy was dependent on mTORC2/Akt/FOXO3a signaling pathway. Finally, our results demonstrated that IBP-mediated breast cancer cell growth in vitro and in vivo was strongly correlated with suppression of mTORC2-dependent autophagy. These findings suggest that the anti-autophagic property of IBP has an important role in IBP-mediated tumorigenesis, and IBP may serve as an attractive target for treatment of breast cancer.

The relationship between the chemical structure and the mode of action of piperacillin-analogues (PIPC-analogues) against Escherichia coli and Klebsiella pneumoniae were investigated. The antibacterial activity of PIPC-analogues increased with an increase in the number of carbon atoms at the N-4 position of 2,3-dioxopiperazine. Their mode of action is discussed on the basis of the results of studies on outer membrane permeability, stability to beta-lactamase and binding affinity to penicillin-binding proteins (PBPs). The outer membrane permeability and stability to beta-lactamase were hardly affected by the chain length of the alkyl group at the N-4 position. On the other hand, the affinity to PBPs, especially to PBP 3, became stronger with increase of the number of carbon atoms at N-4 position. These results suggest that increased affinity to PBPs is the main reason for the increased antibacterial activity of the PIPC-analogues reported here.

The post-discharge period is an extremely vulnerable period for patients, particularly for those discharged from inpatient children and adolescent mental health services (CAMHS). Poor discharge practices and discontinuity of care can put children and youth at heightened risk for readmission, among other adverse outcomes. However, there is limited understanding of the structure and effectiveness of interventions to facilitate discharges from CAMHS. As such, a scoping review was conducted to identify the literature on discharge interventions. This scoping review aimed to describe key components, designs, and outcomes of existing discharge interventions from CAMHS. Nineteen documents were included in the final review. Discharge interventions were extracted and summarized for pre-discharge, post-discharge, and bridging elements. Results of this scoping review found that intervention elements included aspects of risk assessment, individualized care, discharge preparation, community linkage, psychoeducation, and follow-up support. Reported outcomes of discharge interventions were also extracted and included positive patient and caregiver satisfaction, improved patient health outcomes, and increased cost effectiveness. Literature on discharge interventions from inpatient CAMHS, while variable in structure, consistently underscore the role of such interventions in minimizing patient and family vulnerability post-discharge. However, findings are limited by inadequate reporting and heterogeneity across studies. There is a need for further research into the design, implementation, and evaluation of interventions to support successful discharges from inpatient child and adolescent mental health care.

Visceral hypersensitivity in irritable bowel syndrome (IBS) is still poorly understood, despite that chronic abdominal pain is the most common symptoms in IBS patients. To study effects of BK channels on visceral hypersensitivity in IBS rats and the underlying mechanisms, IBS rats were established by colorectal distention (CRD) in postnatal rats. The expression of large-conductance calcium and voltage-dependent potassium ion channels (BK channels) of the thoracolumbar spinal cord was examined in IBS and control rats. The effects of BK channel blockade on visceral hypersensitivity were evaluated. The interaction of BK channels and N-methyl-D-aspartate acid (NMDA) receptors was explored, and synaptic transmission at superficial dorsal horn (SDH) neurons of the thoracolumbar spinal cord was recorded by whole-cell patch clamp in IBS rats.

Working memory (WM) tasks may increase or decrease the interference effect of concurrently performed cognitive control tasks. However, the neural oscillatory correlates of this modulation effect of WM on the Stroop task are still largely unknown. In the present study, behavioral and electroencephalographic (EEG) data were recorded from 32 healthy participants during their performance of the single Stroop task and the same task with a concurrent WM task. We observed that the Stroop interference effect represented in both response times (RTs) and theta-band event-related spectral perturbation (ERSP) magnitude reduced under the dual-task condition compared with the single-task condition. The reduction of interference in theta-band ERSP was further positively correlated with interference reduction in RTs, and was mainly explained by the source in the left middle frontal gyrus. In conclusion, the present study suggests that the effect of concurrent WM tasks on the reduction of the Stroop interference effect can be indexed by EEG oscillations in theta-band rhythm in the centro-frontal regions and this modulation was mediated by the reduced cognitive control under the concurrent WM task.

Epithelial-to-mesenchymal transition (EMT) is a crucial process for the invasion and metastasis of epithelial tumors. However, the molecular mechanisms underlying this transition are poorly understood. In this study, we demonstrate that interferon regulatory factor 4 binding protein (IBP) regulates EMT and the motility of breast cancer cells through Rac1, RhoA and Cdc42 signaling pathways. We found that increased expression of IBP was associated with the progression of breast cancer and that IBP protein levels were significantly elevated in matched distant metastases. High IBP levels also predict shorter overall survival of breast cancer patients. Furthermore, the forced expression of IBP decreased the expression of the epithelial marker E-cadherin but increased the mesenchymal markers in breast cancer cells. In contrast, silencing IBP in metastatic breast tumor cells promoted a shift toward an epithelial morphology concomitant with increased expression of E-cadherin and decreased expression of mesenchymal markers. IBP silencing also reduced the expression of EMT-inducing transcription factors (Snail, Slug, ZEB1 and ZEB2). Moreover, we identified a role for IBP in endogenous EMT induced by epidermal growth factor (EGF) and deletion of IBP attenuated EGF receptor (EGFR) signaling in breast cancer cells. Furthermore, IBP regulates the migration, invasion and matrix metalloprotease production in breast cancer cells as well as actin cytoskeleton rearrangement and the activation of GTP-Rac1, GTP-RhoA and GTP-Cdc42. Taken together, our findings demonstrate an oncogenic property for IBP in promoting the metastatic potential of breast cancer cells.

The bed nucleus of the stria terminalis (BNST) is a center of integration for limbic information and valence monitoring. The BNST, sometimes referred to as the extended amygdala, is located in the basal forebrain and is a sexually dimorphic structure made up of between 12 and 18 sub-nuclei. These sub-nuclei are rich with distinct neuronal subpopulations of receptors, neurotransmitters, transporters and proteins. The BNST is important in a range of behaviors such as: the stress response, extended duration fear states and social behavior, all crucial determinants of dysfunction in human psychiatric diseases. Most research on stress and psychiatric diseases has focused on the amygdala, which regulates immediate responses to fear. However, the BNST, and not the amygdala, is the center of the psychogenic circuit from the hippocampus to the paraventricular nucleus. This circuit is important in the stimulation of the hypothalamic-pituitary-adrenal axis. Thus, the BNST has been largely overlooked with respect to its possible dysregulation in mood and anxiety disorders, social dysfunction and psychological trauma, all of which have clear gender disparities. In this review, we will look in-depth at the anatomy and projections of the BNST, and provide an overview of the current literature on the relevance of BNST dysregulation in psychiatric diseases.

In recent years, there are an increasing number of proteomics studies that investigated the alterations in the protein expression relevant to human diseases but none for stroke. We, therefore, attempted such a study in a paradigm of focal cerebral ischemia in rat. Rats were subjected to cerebral ischemia by unilateral occlusion of the middle cerebral artery. Global protein analysis was performed after 24h on the lesioned and sham-control cerebral cortex using two-dimensional gel electrophoresis. Protein spots with more than a 3-fold change in intensity were identified by mass spectrometry. Middle cerebral artery occlusion (MCAO) caused infarct volume of 18-22% predominantly in the cortex of the lesioned hemisphere. Two-dimensional gel electrophoresis resolved about 1500 protein spots of which only 12 were significantly upregulated by 3-46-fold. Three spots were identified to be dihydropyrimidinase-related protein 2 (DRP-2, also known as collapsin response mediator protein 2 (CRMP-2) or turned on after division, 64 kD protein (TOAD-64)). The spots varied in pI values only and this may reflect different phosphorylation status of the same protein. Two spots were identified as spectrin alpha II chain (rat fragment, also known as alpha-fodrin or non-erythroid alpha chain, SPNA-2); and one spot each for heat shock cognate protein 70 pseudogene 1 (HSC70-ps1, also known as heat shock protein 8 pseudogene 1), and tropomodulin 2 (Tmod2). The upregulation of protein expression was corroborated by observed upregulation of mRNA expression. The remaining five spots were not identified satisfactorily. As DRP-2, spectrin, and Tmod2 are involved in axonal and neurite growth as well as synaptic plasticity and maturation, the presently observed upregulation of the expression of these proteins may indicate active neuroregeneration and repair at 24h after the induction of cerebral ischemia.

Phenotypic heterogeneity is widely observed in cancer cell populations. Here, to probe this heterogeneity, we developed an image-guided genomics technique termed spatiotemporal genomic and cellular analysis (SaGA) that allows for precise selection and amplification of living and rare cells. SaGA was used on collectively invading 3D cancer cell packs to create purified leader and follower cell lines. The leader cell cultures are phenotypically stable and highly invasive in contrast to follower cultures, which show phenotypic plasticity over time and minimally invade in a sheet-like pattern. Genomic and molecular interrogation reveals an atypical VEGF-based vasculogenesis signalling that facilitates recruitment of follower cells but not for leader cell motility itself, which instead utilizes focal adhesion kinase-fibronectin signalling. While leader cells provide an escape mechanism for followers, follower cells in turn provide leaders with increased growth and survival. These data support a symbiotic model of collective invasion where phenotypically distinct cell types cooperate to promote their escape.

Cytokinesis is initiated by the localized assembly of the contractile ring, a dynamic actomyosin structure that generates a membrane furrow between the segregating chromosomal masses to divide a cell into two. Here we show that the stabilization and organization of the cytokinetic furrow is specifically dependent on localized β-actin filament assembly at the site of cytokinesis. β-actin filaments are assembled directly at the furrow by an anillin-dependent pathway that enhances RhoA-dependent activation of the formin DIAPH3, an actin nucleator. DIAPH3 specifically generates homopolymeric filaments of β-actin in vitro. By employing enhancers and activators, cells can achieve acute spatio-temporal control over isoform-specific actin arrays that are required for distinct cellular functions.

In Escherichia coli, the SOS response is induced upon DNA damage and results in the enhanced expression of a set of genes involved in DNA repair and other functions. The initial step, self-cleavage of the LexA repressor, is promoted by the RecA protein which is activated upon binding to single-stranded DNA. In this work, induction of the SOS response by the addition of mitomycin C was found to be prevented by overexpression of the dinI gene. dinI is an SOS gene which maps at 24.6 min of the E.coli chromosome and encodes a small protein of 81 amino acids. Immunoblotting analysis with anti-LexA antibodies revealed that LexA did not undergo cleavage in dinI-overexpressed cells after UV irradiation. In addition, the RecA-dependent conversion of UmuD to UmuD' (the active form for mutagenesis) was also inhibited in dinI-overexpressed cells. Conversely, a dinI-deficient mutant showed a slightly faster and more extensive processing of UmuD and hence higher mutability than the wild-type. Finally, we demonstrated, by using an in vitro reaction with purified proteins, that DinI directly inhibits the ability of RecA to mediate self-cleavage of UmuD.

Accumulating evidence indicates that the Cbl protein plays a negative role in immune receptor signaling; however, the mode of Cbl action in B cell receptor (BCR) signaling still remains unclear. DT40 B cells deficient in Cbl showed enhanced BCR-mediated phospholipase C (PLC)-gamma2 activation, thereby leading to increased apoptosis. A possible explanation for the involvement of Cbl in PLC-gamma2 activation was provided by findings that Cbl interacts via its Src homology 2 (SH2) domain with B cell linker protein (BLNK) after BCR ligation. BLNK is a critical adaptor molecule for PLC-gamma2 tyrosine phosphorylation through its binding to the PLC-gamma2 SH2 domains. As a consequence of the interaction between Cbl and BLNK, the BCR-induced recruitment of PLC-gamma2 to BLNK and the subsequent PLC-gamma2 tyrosine phosphorylation were inhibited. Thus, our data suggest that Cbl negatively regulates the PLC-gamma2 pathway by inhibiting the association of PLC-gamma2 with BLNK.