Evidence that CD147 modulation of beta-amyloid (Abeta) levels is mediated by extracellular degradation of secreted Abeta

Cerebral deposition of beta-amyloid (Abeta) peptides is a pathological hallmark of Alzheimer disease. Intramembranous proteolysis of amyloid precursor protein by a multiprotein gamma-secretase complex generates Abeta. Previously, it was reported that CD147, a glycoprotein that stimulates production of matrix metalloproteinases (MMPs), is a subunit of gamma-secretase and that the levels of secreted Abeta inversely correlate with CD147 expression. Here, we show that the levels and localization of CD147 in fibroblasts, as well as postnatal expression and distribution in brain, are distinct from those of integral gamma-secretase subunits. Notably, we show that although depletion of CD147 increased extracellular Abeta levels in intact cells, membranes isolated from CD147-depleted cells failed to elevate Abeta production in an in vitro gamma-secretase assay. Consistent with an extracellular source that modulates Abeta metabolism, synthetic Abeta was degraded more rapidly in the conditioned medium of cells overexpressing CD147. Moreover, modulation of CD147 expression had no effect on epsilon-site cleavage of amyloid precursor protein and Notch1 receptor. Collectively, our results demonstrate that CD147 modulates Abeta levels not by regulating gamma-secretase activity, but by stimulating extracellular degradation of Abeta. In view of the known function of CD147 in MMP production, we postulate that CD147 expression influences Abeta levels by an indirect mechanism involving MMPs that can degrade extracellular Abeta.     

Hypoxia regulates cross-talk between Syk and Lck leading to breast cancer progression and angiogenesis

Hypoxia is a key parameter that controls tumor angiogenesis and malignant progression by regulating the expression of several oncogenic molecules. The nonreceptor protein-tyrosine kinases Syk and Lck play crucial roles in the signaling mechanism of various cellular processes. The enhanced expression of Syk in normal breast tissue but not in malignant breast carcinoma has prompted us to investigate its potential role in mammary carcinogenesis. Accordingly, we hypothesized that hypoxia/reoxygenation (H/R) may play an important role in regulating Syk activation, and Lck may be involved in this process. In this study, we have demonstrated that H/R differentially regulates Syk phosphorylation and its subsequent interaction and cross-talk with Lck in MCF-7 cells. Moreover, Syk and Lck play differential roles in regulating Sp1 activation and expressions of melanoma cell adhesion molecule (MelCAM), urokinase-type plasminogen activator (uPA), matrix metalloproteinase-9 (MMP-9), and vascular endothelial growth factor (VEGF) in response to H/R. Overexpression of wild type Syk inhibited the H/R-induced uPA, MMP-9, and VEGF expression but up-regulated MelCAM expression. Our data also indicated that MelCAM acts as a tumor suppressor by negatively regulating H/R-induced uPA secretion and MMP-9 activation. The mice xenograft study showed the cross-talk between Syk and Lck regulated H/R-induced breast tumor progression and further correlated with the expressions of MelCAM, uPA, MMP-9, and VEGF. Human clinical specimen analysis supported the in vitro and in vivo findings. To our knowledge, this is first report that the cross-talk between Syk and Lck regulates H/R-induced breast cancer progression and further suggests that Syk may act as potential therapeutic target for the treatment of breast cancer.     

Resonance Raman spectroscopic investigation of the light-harvesting chromophore in escherichia coli photolyase and Vibrio cholerae cryptochrome-1

Photolyases and cryptochromes are flavoproteins that belong to the class of blue-light photoreceptors. They usually bind two chromophores: flavin adenine dinucleotide (FAD), which forms the active site, and a light-harvesting pigment, which is a 5,10-methenyltetrahydrofolate polyglutamate (MTHF) in most cases. In Escherichia coli photolyase (EcPhr), the MTHF cofactor is present in substoichiometric amounts after purification, while in Vibrio cholerae cryptochrome-1 (VcCry1) the MTHF cofactor is bound more strongly and is present at stoichiometric levels after purification. In this paper, we have used resonance Raman spectroscopy to monitor the effect of loss of MTHF on the protein-FAD interactions in EcPhr and to probe the protein-MTHF interactions in both EcPhr and VcCry1. We find that removal of MTHF does not perturb protein-FAD interactions, suggesting that it may not affect the physicochemical properties of FAD in EcPhr. Our data demonstrate that the pteridine ring of MTHF in EcPhr has different interactions with the protein matrix than that of MTHF in VcCry1. Comparison to solution resonance Raman spectra of MTHF suggests that the carbonyl of its pteridine ring in EcPhr experiences stronger hydrogen bonding and a more polar environment than in VcCry1, but that hydrogen bonding to the pteridine ring amine hydrogens is stronger in VcCry-1. These differences in hydrogen bonding may account for the higher binding affinity of MTHF in VcCry1 compared to EcPhr.     

Expression and functional features of NaCT, a sodium-coupled citrate transporter, in human and rat livers and cell lines

In this article, we report on the expression and function of a Na(+)-coupled transporter for citrate, NaCT, in human and rat liver cell lines and in primary hepatocytes from the rat liver. We also describe the polarized expression of this transporter in human and rat livers. Citrate uptake in human liver cell lines HepG2 and Huh-7 was obligatorily dependent on Na+. The uptake system showed a preference for citrate over other intermediates of the citric acid cycle and exhibited a Michaelis constant of approximately 6 mM for citrate. The transport activity was stimulated by Li+, and the activation was associated with a marked increase in substrate affinity. Citrate uptake in rat liver cell line MH1C1 was also Na+ dependent and showed a preference for citrate. The Michaelis constant for citrate was approximately 10 microM. The transport activity was inhibited by Li+. Primary hepatocytes from the rat liver also showed robust activity for Na+-coupled citrate uptake, with functional features similar to those described in the rat liver cell line. Immunolabeling with a specific anti-NaCT antibody showed exclusive expression of the transporter in the sinusoidal membrane of hepatocytes in human and rat livers. This constitutes the first report on the expression and function of NaCT in liver cells.     

Synchronous modulation of cell surface lectin and its receptor in a homologous cell population: a novel mechanism of cellular regulation

Testicular immotile sperm undergo maturation during epididymal transit when these cells pass through caput, corpus, and cauda-epididymal regions. Maturing goat spermatozoa specifically at the distal corpus epididymal stage show head-to-head autoagglutination when incubated in vitro in a modified Ringer's solution. Here, we show the biochemical mechanism of autoagglutination event and its functional significance. A lectin-like molecule located on sperm surface specifically interacts with its receptor of the neighboring homologous cells to cause autoagglutination. Lectin is a Ca++-dependent galactose-specific protein. Failure of the pre- and post-distal corpus sperm to show autoagglutination is due to lack of lectin-like molecule and its receptors, respectively. Maturing sperm at distal corpus stage acquire lectin-like molecule followed by sharp disappearance of its receptor, and this event is synchronously associated with the initiation of sperm forward motility that is essential for fertilization in vivo. Lectin and its receptor isolated from sperm plasma membrane showed high efficacy for blocking autoagglutination phenomenon. The data are consistent with the view that synchronous modulation of homologous cell surface lectin and their receptors constitutes a novel mechanism for cellular regulation by generating waves of signals by manipulating lectin-sugar-dependent "self-talk" and cell-cell "cross-talk".     

Adrenocorticotrophin secreting cells in the hypophysis of the brown spiny mouseMus platythrix (Bennett)

Adrenocorticotrophin secreting cells are identified in the hypophysis of the brown spiny mouseMus platythrix by conventional methods of light microscopy. Quantitative data showed that certain smaller acidophilic cells in thepars distalis, under conditions provoking their hypersecretion such as unilateral adrenalectomy and metopirone treatment, increase in number and size from the pre-existing corticotrophs. There is no evidence for the transmigration of these cells from the chromophobes, basophils or any other cell type. Thepars intermedia revealed two types of cells of which the type II cells are histochemically identical to adrenocorticotrophin secreting cells of thepars distalis     

Characterization of Salmonella typhi OmpC and OmpF porins engineered with HIV‐gp41 epitope on the surface loops

Porins form trimers in the outer membrane and help transport nutrients and waste products across the bacterial cell membrane. Porin loops are suitable candidates as display systems due to their high immunogenicity and presentation at the bacterial cell surface. In this study, Salmonella typhi porins (OmpC and OmpF) engineered with the Kennedy peptide from gp41 of HIV were characterised. The chimeric OmpC carrying the Kennedy peptide in loop7 did not trimerise, whereas the chimeric OmpF with the epitope in loop5 formed trimers and also was recognised by the antibodies in the HIV patient serum. The results suggest that chimeric S. typhi OmpF may be taken further as a potential candidate to develop as an epitope display system. Proteins 2017; 85:657–664. © 2016 Wiley Periodicals, Inc.     

The microtubule depolymerizing agent naphthazarin induces both apoptosis and autophagy in A549 lung cancer cells

Naphthazarin (DHNQ, 5,8-dihydroxy-l,4-naphthoquinone) is a naturally available 1,4-naphthoquinone derivatives. In this study, we focused on elucidating the cytotoxic mechanism of naphthazarin in A549 non-small cell lung carcinoma cells. Naphthazarin reduced the A549 cell viability considerably with an IC₅₀ of 16.4 ± 1.6 μM. Naphthazarin induced cell death in a dose- and time-dependent manner by activating apoptosis and autophagy pathways. Specifically, we found naphthazarin inhibited the PI3K/Akt cell survival signalling pathway, measured by p53 and caspase-3 activation, and PARP cleavage. It also resulted in an increase in the ratio of Bax/Bcl2 protein levels, indicating activation of the mitochondrial apoptotic pathway. Similarly naphthazarin triggered LC3II expression and induced autophagic flux in A549 cells. We demonstrated further that naphthazarin is a microtubule inhibitor in cell-free system and in A549 cells. Naphthazarin treatment depolymerized interphase microtubules and disorganised spindle microtubules and the majority of cells arrested at the G₂/M transition. Together, these data suggest that naphthazarin, a microtubule depolymerizer which activates dual cell death machineries, could be a potential novel chemotherapeutic agent.     

Combinatorial determination of sequence specificity for nanomolar DNA-binding hairpin polyamides

Development of sequence-specific DNA-binding drugs is an important pharmacological goal, given the fact that numerous existing DNA-directed chemotherapeutic drugs rely on the strength and selectivity of their DNA interactions for therapeutic activity. Among the DNA-binding antibiotics, hairpin polyamides represent the only class of small molecules that can practically bind any predetermined DNA sequence. DNA recognition by these ligands depends on their side-by-side amino acid pairings in the DNA minor groove. Extensive studies have revealed that these molecules show extremely high affinity for sequence-directed, minor groove interaction. However, the specificity of such interactions in the presence of a large selection of sequences such as the human genome is not known. We used the combinatorial selection method restriction endonuclease protection, selection, and amplification (REPSA) to determine the DNA binding specificity of two hairpin polyamides, ImPyPyPy-gamma-PyPyPyPy-beta-Dp and ImPyPyPy-gamma-ImPyPyPy-beta-Dp, in the presence of more than 134 million different sequences. These were verified by restriction endonuclease protection assays and DNase I footprinting analysis. Our data showed that both hairpin polyamides preferentially selected DNA sequences having consensus recognition sites as defined by the Dervan pairing rules. These consensus sequences were rather degenerate, as expected, given that the stacked pyrrole-pyrrole amino acid pairs present in both polyamides are unable to discriminate between A.T and T.A base pairs. However, no individual sequence within these degenerate consensus sequences was preferentially selected by REPSA, indicating that these hairpin polyamides are truly consensus-specific DNA-binding ligands. We also discovered a preference for overlapping consensus binding sites among the sequences selected by the hairpin polyamide ImPyPyPy-gamma-PyPyPyPy-beta-Dp, and confirmed by DNase I footprinting that these complex sites provide higher binding affinity. These data suggest that multiple hairpin polyamides can cooperatively bind to their highest-affinity sites.