Changes in the selected hematological parameters and gill Na/K ATPase activity of juvenile crucian carp Carassius auratus during elevated ammonia exposure and the post-exposure recovery

The increase in concentration of ammonia in lake water during the degradation of algal blooms may last for several weeks and thus cause chronic toxicity to aquatic organisms. The purpose of this study was to assess the chronic toxicity of ammonia on the selected hematological parameters and gill Na⁺/K⁺ ATPase activity of juvenile crucian carp Carassius auratus during elevated ammonia exposure and the post-exposure recovery. Juvenile crucian carp were exposed in different ammonia solutions for 45 days and then immediately transferred to pristine freshwater to initiate a 15-day recovery period. Results showed sub-lethal ammonia significantly deters growth and a 15-day recovery period was not sufficient for the fish to compensate for the loss of growth. The fish exhibited a continuous decrease in red blood cell (RBC), the total hemoglobin (Hb), and gill Na⁺/K⁺ ATPase activity as the concentration of NH₃-N increased. After the 15-day recovery period, RBC, Hb, and gill Na⁺/K⁺ ATPase activity had recovered to similar levels as the controls.     

COPS5 Protein Overexpression Increases Amyloid Plaque Burden,Decreases Spinophilin-immunoreactive Puncta,and Exacerbates Learning and Memory Deficits in the Mouse Brain

Brain accumulation of neurotoxic amyloid β (Aβ) peptide because of increased processing of amyloid precursor protein (APP), resulting in loss of synapses and neurodegeneration, is central to the pathogenesis of Alzheimer disease (AD). Therefore, the identification of molecules that regulate Aβ generation and those that cause synaptic damage is crucial for future therapeutic approaches for AD. We demonstrated previously that COPS5 regulates Aβ generation in neuronal cell lines in a RanBP9-dependent manner. Consistent with the data from cell lines, even by 6 months, COPS5 overexpression in APΔE9 mice (APΔE9/COPS5-Tg) significantly increased Aβ40 levels by 32% (p < 0.01) in the cortex and by 28% (p < 0.01) in the hippocampus, whereas the increases for Aβ42 were 37% (p < 0.05) and 34% (p < 0.05), respectively. By 12 months, the increase was even more robust. Aβ40 levels increased by 63% (p < 0.001) in the cortex and by 65% (p < 0.001) in the hippocampus. Similarly, Aβ42 levels were increased by 69% (p < 0.001) in the cortex and by 71% (p < 0.011) in the hippocampus. Increased Aβ levels were translated into an increased amyloid plaque burden both in the cortex (54%, p < 0.01) and hippocampus (64%, p < 0.01). Interestingly, COPS5 overexpression increased RanBP9 levels in the brain, which, in turn, led to increased amyloidogenic processing of APP, as reflected by increased levels of sAPPβ and decreased levels of sAPPα. Furthermore, COPS5 overexpression reduced spinophilin in both the cortex (19%, p < 0.05) and the hippocampus (20%, p < 0.05), leading to significant deficits in learning and memory skills. Therefore, like RanBP9, COPS5 also plays a pivotal role in amyloid pathology in vivo.     

Desmoglein 2 is a receptor for adenovirus serotypes 3, 7, 11 and 14

We have identified desmoglein-2 (DSG-2) as the primary high-affinity receptor used by adenoviruses Ad3, Ad7, Ad11 and Ad14. These serotypes represent key human pathogens causing respiratory and urinary tract infections. In epithelial cells, adenovirus binding of DSG-2 triggers events reminiscent of epithelial-to-mesenchymal transition, leading to transient opening of intercellular junctions. This opening improves access to receptors, for example, CD46 and Her2/neu, that are trapped in intercellular junctions. In addition to complete virions, dodecahedral particles (PtDds), formed by excess amounts of viral capsid proteins, penton base and fiber during viral replication, can trigger DSG-2-mediated opening of intercellular junctions as shown by studies with recombinant Ad3 PtDds. Our findings shed light on adenovirus biology and pathogenesis and may have implications for cancer therapy.     

Chemokine (C-C motif) ligand 2 (CCL2) in sera of patients with type 1 diabetes and diabetic complications

Background

Chemokine (C-C motif) ligand 2 (CCL2), commonly known as monocyte chemoattractant protein-1 (MCP-1), has been implicated in the pathogenesis of many diseases characterized by monocytic infiltration. However, limited data have been reported on MCP-1 in type 1 diabetes (T1D) and the findings are inconclusive and inconsistent.

Methods

In this study, MCP-1 was measured in the sera from 2,472 T1D patients and 2,654 healthy controls using a Luminex assay. The rs1024611 SNP in the promoter region of MCP-1 was genotyped for a subset of subjects (1764 T1D patients and 1323 controls) using the TaqMan-assay.

Results

Subject age, sex or genotypes of MCP-1 rs1024611SNP did not have a major impact on serum MCP-1 levels in either healthy controls or patients. While hemoglobin A1c levels did not have a major influence on serum MCP-1 levels, the mean serum MCP-1 levels are significantly higher in patients with multiple complications (mean = 242 ng/ml) compared to patients without any complications (mean = 201 ng/ml) (p = 3.5×10⁻⁶). Furthermore, mean serum MCP-1 is higher in controls (mean = 261 ng/ml) than T1D patients (mean = 208 ng/ml) (p<10⁻²³). More importantly, the frequency of subjects with extremely high levels (>99^th percentile of patients or 955 ng/ml) of serum MCP-1 is significantly lower in the T1D group compared to the control group (odds ratio = 0.11, p<10⁻³³).

Conclusion

MCP-1 may have a dual role in T1D and its complications. While very high levels of serum MCP-1 may be protective against the development of T1D, complications are associated with higher serum MCP-1 levels within the T1D group.     

Novel and essential subunits in the 300-kilodalton nuclear cap binding complex of Trypanosoma brucei

One of the unique aspects of RNA processing in trypanosomatid protozoa is the presence of a cap 4 structure (m7Gpppm2(6)AmpAmpCmpm3Um) at the 5' end of all mRNAs. The cap 4 becomes part of the mRNA through trans-splicing of a 39-nucleotide-long sequence donated by the spliced leader RNA. Although the cap 4 modifications are required for trans-splicing to occur, the underlying mechanism remains to be determined. We now describe an unconventional nuclear cap binding complex (CBC) in Trypanosoma brucei with an apparent molecular mass of 300 kDa and consisting of five protein components: the known CBC subunits CBP20 and importin-alpha and three novel proteins that are only present in organisms featuring a cap 4 structure and trans-splicing. Competitive binding studies are consistent with a specific interaction between the CBC and the cap 4 structure. Downregulation of several individual components of the T. brucei CBC by RNA interference demonstrated an essential function at an early step in trans-splicing. Thus, our studies are consistent with the CBC providing a mechanistic link between cap 4 modifications and trans-splicing.     

Molecular analysis of the O-antigen gene cluster of Escherichia coli O86:B7 and characterization of the chain length determinant gene (wzz)

Escherichia coli O86:B7 has long been used as a model bacterial strain to study the generation of natural blood group antibody in humans, and it has been shown to possess high human blood B activity. The O-antigen structure of O86:B7 was solved recently in our laboratory. Comparison with the published structure of O86:H2 showed that both O86 subtypes shared the same O unit, yet each of the O antigens is polymerized from a different terminal sugar in a different glycosidic linkage. To determine the genetic basis for the O-antigen differences between the two O86 strains, we report the complete sequence of O86:B7 O-antigen gene cluster between galF and hisI, each gene was identified based on homology to other genes in the GenBank databases. Comparison of the two O86 O-antigen gene clusters revealed that the encoding regions between galF and gnd are identical, including wzy genes. However, deletion of the two wzy genes revealed that wzy in O86:B7 is responsible for the polymerization of the O antigen, while the deletion of wzy in O86:H2 has no effect on O-antigen biosynthesis. Therefore, we proposed that there must be another functional wzy gene outside the O86:H2 O-antigen gene cluster. Wzz proteins determine the degree of polymerization of the O antigen. When separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the lipopolysaccharide (LPS) of O86:B7 exhibited a modal distribution of LPS bands with relatively short O units attached to lipid A-core, which differs from the LPS pattern of O86:H2. We proved that the wzz genes are responsible for the different LPS patterns found in the two O86 subtypes, and we also showed that the very short type of LPS is responsible for the serum sensitivity of the O86:B7 strain.     

Conserved structural and functional control of N-methyl-D-aspartate receptor gating by transmembrane domain M3

The molecular events controlling glutamate receptor ion channel gating are complex. The movement of transmembrane domain M3 within N-methyl-d-aspartate (NMDA) receptor subunits has been suggested to be one structural determinant linking agonist binding to channel gating. Here we report that covalent modification of NR1-A652C or the analogous mutation in NR2A, -2B, -2C, or -2D by methanethiosulfonate ethylammonium (MT-SEA) occurs only in the presence of glutamate and glycine, and that modification potentiates recombinant NMDA receptor currents. The modified channels remain open even after removing glutamate and glycine from the external solution. The degree of potentiation depends on the identity of the NR2 subunit (NR2A < NR2B < NR2C,D) inversely correlating with previous measurements of channel open probability. MTSEA-induced modification of channels is associated with increased glutamate potency, increased mean single-channel open time, and slightly decreased channel conductance. Modified channels are insensitive to the competitive antagonists D-2-amino-5-phosphonovaleric acid (APV) and 7-Cl-kynurenic acid, as well as allosteric modulators of gating (extracellular protons and Zn(2+)). However, channels remain fully sensitive to Mg(2+) blockade and partially sensitive to pore block by (+)MK-801, (-)MK-801, ketamine, memantine, amantadine, and dextrorphan. The partial sensitivity to (+)MK-801 may reflect its ability to stimulate agonist unbinding from MT-SEA-modified receptors. In summary, these data suggest that the SYTANLAAF motif within M3 is a conserved and critical determinant of channel gating in all NMDA receptors.     

The MEK inhibitor U0126 ameliorates diabetic cardiomyopathy by restricting XBP1's phosphorylation dependent SUMOylation

Background: Chronic diabetes accelerates vascular dysfunction often resulting in cardiomyopathy but underlying mechanisms remain unclear. Recent studies have shown that the deregulated unfolded protein response (UPR) dependent on highly conserved IRE1α-spliced X-box- binding protein (XBP1s) and the resulting endoplasmic reticulum stress (ER-Stress) plays a crucial role in the occurrence and development of diabetic cardiomyopathy (DCM). In the present study, we determined whether targeting MAPK/ERK pathway using MEK inhibitor U0126 could ameliorate DCM by regulating IRE1α-XBP1s pathway.Method: Three groups of 8-week-old C57/BL6J mice were studied: one group received saline injection as control (n=8) and two groups were made diabetic by streptozotocin (STZ) (n=10 each). 18 weeks after STZ injection and stable hyperglycemia, one group had saline treatment while the second group was treated with U0126 (1mg/kg/day), 8 weeks later, all groups were sacrificed. Cardiac function/histopathological changes were determined by echocardiogram examination, Millar catheter system, hematoxylin-eosin staining and western blot analysis. H9C2 cardiomyocytes were employed for in vitro studies.Results: Echocardiographic, hemodynamic and histological data showed overt myocardial hypertrophy and worsened cardiac function in diabetic mice. Chronic diabetic milieu enhanced SUMOylation and impaired nuclear translocation of XBP1s. Intriguingly, U0126 treatment significantly ameliorated progression of DCM, and this protective effect was achieved through enriching XBP1s'' nuclear accumulation. Mechanistically, U0126 inhibited XBP1s'' phosphorylation on S348 and SUMOylation on K276 promoting XBP1s'' nuclear translocation. Collectively, these results identify that MEK inhibition restores XBP1s-dependent UPR and protects against diabetes-induced cardiac remodeling.Conclusion: The current study identifies previously unknown function of MEK/ERK pathway in regulation of ER-stress in DCM. U0126 could be a therapeutic target for the treatment of DCM.     

A protein related to the vaccinia virus cap-specific methyltransferase VP39 is involved in cap 4 modification in Trypanosoma brucei

The spliced-leader (SL) RNA plays a key role in the biogenesis of mRNA in trypanosomes by providing the m(7)G-capped SL sequence to the 5' end of every mRNA. The cap structure of the SL RNA is unique in eukaryotes with 4 nucleotides after the cap carrying a total of seven methyl groups and by convention is referred to as "cap 4". Although the enzymatic machinery for cap addition has been characterized in several organisms, including Trypanosoma brucei, the identification of methyltransferases dedicated to the generation of higher order cap structures has lagged behind, except in viruses. Here we describe T. brucei MT57 (TbMT57), a primarily nuclear polypeptide with structural and functional similarities to vaccinia virus VP39, a bifunctional protein acting at the mRNA 5' end as a cap-specific 2'-O-methyltransferase. Down-regulation by RNAi or genetic ablation of TbMT57 resulted in the accumulation of SL RNA missing 2'-O-methyl groups at positions +3 and +4 and thus bearing a cap 2 rather than a cap 4. Furthermore, competitive binding studies indicated that modifications at the +3 and +4 positions are important for binding to the nuclear cap-binding complex. Genetic ablation of MT57 resulted in viable cells with no apparent defect in SL RNA trans-splicing, suggesting that MT57 is not essential or that trypanosomes have developed alternate mechanisms to counteract the absence of this protein. Interestingly, MT57 homologs are only found in trypanosomatid protozoa that have a cap 4 structure and in poxviruses, of which vaccinia virus is a prototype.