Positively-charged semi-tunnel is a structural and surface characteristic of polyphosphate-binding proteins: an in-silico study.

Phosphate is essential for all major life processes, especially energy metabolism and signal transduction. A linear phosphate polymer, polyphosphate (polyP), linked by high-energy phosphoanhydride bonds, can interact with various proteins, playing important roles as an energy source and regulatory factor. However, polyP-binding structures are largely unknown. Here we proposed a putative polyP binding site, a positively-charged semi-tunnel (PCST), identified by surface electrostatics analyses in polyP kinases (PPKs) and many other polyP-related proteins. We found that the PCSTs in varied proteins were folded in different secondary structure compositions. Molecular docking calculations revealed a significant value for binding affinity to polyP in PCST-containing proteins. Utilizing the PCST identified in the ß subunit of PPK3, we predicted the potential polyP-binding domain of PPK3. The discovery of this feature facilitates future searches for polyP-binding proteins and discovery of the mechanisms for polyP-binding activities. This should greatly enhance the understanding of the many physiological functions of protein-bound polyP and the involvement of polyP and polyP-binding proteins in various human diseases.

Ischemia preconditioning protects astrocytes from ischemic injury through 14-3-3γ.

Stroke is a leading cause of death and disability, and new strategies are required to reduce neuronal injury and improve prognosis. Ischemia preconditioning (IPC) is an intrinsic phenomenon that protects cells from subsequent ischemic injury and might provide promising mechanisms for clinical treatment. In this study, primary astrocytes exhibited significantly less cell death than control when exposed to different durations of IPC (15, 30, 60, or 120 min). A 15-min duration was the most effective IPC to protect astrocytes from 8-hr-ischemia injury. The protective mechanisms of IPC involve the upregulation of protective proteins, including 14-3-3γ, and attenuation of malondialdehyde (MDA) content and ATP depletion. 14-3-3γ is an antiapoptotic intracellular protein that was significantly upregulated for up to 84 hr after IPC. In addition, IPC promoted activation of the c-Jun N-terminal kinase (JNK), extracellular signal-related kinase (ERK)-1/2, p38, and protein kinase B (Akt) signaling pathways. When JNK was specifically inhibited with SP600125, the upregulation of 14-3-3γ induced by IPC was almost completely abolished; however, there was no effect on ATP or MDA levels. This suggests that, even though both energy preservation and 14-3-3γ up-regulation were turned on by IPC, they were controlled by different pathways. The ERK1/2, p38, and Akt signaling pathways were not involved in the 14-3-3γ upregulation and energy preservation. These results indicate that IPC could protect astrocytes from ischemia injury by inducing 14-3-3γ and by alleviating energy depletion through different pathways, suggesting multiple protection of IPC and providing new insights into potential stroke therapies.

Astrocytic exportin-7 responds to ischemia through mediating LKB1 translocation from the nucleus to the cytoplasm.

The superfamily of importin-ß-related proteins is the largest class of nuclear transport receptors and can be generally divided into importins and exportins according to their transport directions. Eleven importins and seven exportins have been identified, and the expression patterns of both classes are important for their functions in nucleocytoplasmic transport activities. This study demonstrates that all of the importins (importin-ß; transportin-1, -2, and -3; and importin-4, -5, -7, -8, -9, -11, and -13) and all the exportins (exportin-1, -2, -4, -5, -6, -7, and -t) are differentially expressed in the cerebral cortex, cerebellum, hippocampus, and brainstem and in primary cultures of cerebral cortical astrocytes and neurons. For astrocytes, we observed that different importins and exportins displayed different expression changes during 0-6 hr of ischemia treatment, especially an increase of both the mRNA and the protein of exportin-7. Immunostaining showed that exportin-7 accumulated inside the nucleus and around the nuclear envelope. In addition, we noticed an increased cytoplasmic distribution of one of the cargo proteins of exportin-7, LKB1, an important element in maintaining energy homeostasis. This increased cytoplasmic distribution was accompanied by an increased expression of exportin-7 under ischemia in astrocytes. We demonstrate that exportin-7 responds to ischemia in astrocytes and that this response involves translocation of LKB1, a protein that plays important roles during metabolic stress, from the nucleus to the cytoplasm.

Traumatic scratch injury in astrocytes triggers calcium influx to activate the JNK/c-Jun/AP-1 pathway and switch on GFAP expression.

Astrocyte activation is a hallmark of central nervous system injuries resulting in glial scar formation (astrogliosis). The activation of astrocytes involves metabolic and morphological changes with complex underlying mechanisms, which should be defined to provide targets for astrogliosis intervention. Astrogliosis is usually accompanied by an upregulation of glial fibrillary acidic protein (GFAP). Using an in vitro scratch injury model, we scratched primary cultures of cerebral cortical astrocytes and observed an influx of calcium in the form of waves spreading away from the wound through gap junctions. Using the calcium blocker BAPTA-AM and the JNK inhibitor SP600125, we demonstrated that the calcium wave triggered the activation of JNK, which then phosphorylated the transcription factor c-Jun to facilitate the binding of AP-1 to the GFAP gene promoter to switch on GFAP upregulation. Blocking calcium mobilization with BAPTA-AM in an in vivo stab wound model reduced GFAP expression and glial scar formation, showing that the calcium signal, and the subsequent regulation of downstream signaling molecules, plays an essential role in brain injury response. Our findings demonstrated that traumatic scratch injury to astrocytes triggered a calcium influx from the extracellular compartment and activated the JNK/c-Jun/AP-1 pathway to switch on GFAP expression, identifying a previously unreported signaling cascade that is important in astrogliosis and the physiological response following brain injury.

AQP5 is differentially regulated in astrocytes during metabolic and traumatic injuries.

Water movement plays vital roles in both physiological and pathological conditions in the brain. Astrocytes are responsible for regulating this water movement and are the major contributors to brain edema in pathological conditions. Aquaporins (AQPs) in astrocytes play critical roles in the regulation of water movement in the brain. AQP1, 3, 4, 5, 8, and 9 have been reported in the brain. Compared with AQP1, 4, and 9, AQP3, 5, and 8 are less studied. Among the lesser known AQPs, AQP5, which has multiple functions identified outside the central nervous system, is also indicated to be involved in hypoxia injury in astrocytes. In our study, AQP5 expression could be detected both in primary cultures of astrocytes and neurons, and AQP5 expression in astrocytes was confirmed in 1- to 4-week old primary cultures of astrocytes. AQP5 was localized on the cytoplasmic membrane and in the cytoplasm of astrocytes. AQP5 expression was downregulated during ischemia treatment and upregulated after scratch-wound injury, which was also confirmed in a middle cerebral artery occlusion model and a stab-wound injury model in vivo. The AQP5 increased after scratch injury was polarized to the migrating processes and cytoplasmic membrane of astrocytes in the leading edge of the scratch-wound, and AQP5 over-expression facilitated astrocyte process elongation after scratch injury. Taken together, these results indicate that AQP5 might be an important water channel in astrocytes that is differentially expressed during various brain injuries.

Nucleic acid-based diagnostics for infectious diseases in public health affairs.

Infectious diseases, mostly caused by bacteria and viruses but also a result of fungal and parasitic infection, have been one of the most important public health concerns throughout human history. The first step in combating these pathogens is to get a timely and accurate diagnosis at an affordable cost. Many kinds of diagnostics have been developed, such as pathogen culture, biochemical tests and serological tests, to help detect and fight against the causative agents of diseases. However, these diagnostic tests are generally unsatisfactory because they are not particularly sensitive and specific and are unable to deliver speedy results. Nucleic acid-based diagnostics, detecting pathogens through the identification of their genomic sequences, have shown promise to overcome the above limitations and become more widely adopted in clinical tests. Here we review some of the most popular nucleic acid-based diagnostics and focus on their adaptability and applicability to routine clinical usage. We also compare and contrast the characteristics of different types of nucleic acid-based diagnostics.

Comprehensive serial analysis of gene expression of the cervical transcriptome.

BACKGROUND: More than half of the approximately 500,000 women diagnosed with cervical cancer worldwide each year will die from this disease. Investigation of genes expressed in precancer lesions compared to those expressed in normal cervical epithelium will yield insight into the early stages of disease. As such, establishing a baseline from which to compare to, is critical in elucidating the abnormal biology of disease. In this study we examine the normal cervical tissue transcriptome and investigate the similarities and differences in relation to CIN III by Long-SAGE (L-SAGE). RESULTS: We have sequenced 691,390 tags from four L-SAGE libraries increasing the existing gene expression data on cervical tissue by 20 fold. One-hundred and eighteen unique tags were highly expressed in normal cervical tissue and 107 of them mapped to unique genes, most belong to the ribosomal, calcium-binding and keratinizing gene families. We assessed these genes for aberrant expression in CIN III and five genes showed altered expression. In addition, we have identified twelve unique HPV 16 SAGE tags in the CIN III libraries absent in the normal libraries. CONCLUSION: Establishing a baseline of gene expression in normal cervical tissue is key for identifying changes in cancer. We demonstrate the utility of this baseline data by identifying genes with aberrant expression in CIN III when compared to normal tissue.

Transferrin receptor 2 (TfR2) and HFE mutational analysis in non-C282Y iron overload: identification of a novel TfR2 mutation.

Hereditary hemochromatosis (HH) is classically associated with a Cys282Tyr (C282Y) mutation of the HFE gene. Non-C282Y HH is a heterogeneous group accounting for 15% of HH in Northern Europe. Pathogenic mutations of the transferrin receptor 2 (TfR2) gene have been identified in 4 Italian pedigrees with the latter syndrome. The goal of this study was to perform a mutational analysis of the TfR2 and HFE genes in a cohort of non-C282Y iron overload patients of mixed ethnic backgrounds. Several sequence variants were identified within the TfR2 gene, including a homozygous missense change in exon 17, c2069 A-->C, which changes a glutamine to a proline residue at position 690. This putative mutation was found in a severely affected Portuguese man and 2 family members with the same genotype. In summary, pathologic TfR2 mutations are present outside of Italy, accounting for a small proportion of non-C282Y HH.