The eIF4E RNA regulon promotes the Akt signaling pathway

Eukaryotic initiation factor 4E (eIF4E) promotes cellular proliferation and can rescue cells from apoptotic stimuli such as serum starvation. However, the mechanisms underlying apoptotic rescue are not well understood. In this study, we demonstrate that eIF4E overexpression leads to enhanced survival signaling through Akt and that eIF4E requires Akt1 to rescue serum-deprived fibroblasts. Furthermore, a mutant form of eIF4E (W73A), which is messenger RNA (mRNA) export competent but does not promote translation, rescues cells as readily as wild-type eIF4E. We show that eIF4E mediates Akt activation via up-regulation of Nijmegen breakage syndrome 1 (NBS1), a phosphoinositide-3 kinase-Akt pathway upstream activator. Additionally, eIF4E coordinately up-regulates the expression of downstream effectors of the Akt pathway, thereby amplifying Akt signaling effects. A negative regulator of eIF4E, the promyelocytic leukemia protein (PML), suppresses Akt activation and apoptotic rescue. These PML activities likely arise, at least in part, through its inhibition of eIF4E-mediated NBS1 mRNA export. In summary, eIF4E coordinately regulates gene expression to potentiate Akt activation, an activity required for apoptotic rescue.     

Heparin-induced cis- and trans-dimerization modes of the thrombospondin-1 N-terminal domain

Through its interactions with proteins and proteoglycans, thrombospondin-1 (TSP-1) functions at the interface of the cell membrane and the extracellular matrix to regulate matrix structure and cellular phenotype. We have previously determined the structure of the high affinity heparin-binding domain of TSP-1, designated TSPN-1, in association with the synthetic heparin, Arixtra. To establish that the binding of TSPN-1 to Arixtra is representative of the association with naturally occurring heparins, we have determined the structures of TSPN-1 in complex with heparin oligosaccharides containing eight (dp8) and ten (dp10) subunits, by x-ray crystallography. We have found that dp8 and dp10 bind to TSPN-1 in a manner similar to Arixtra and that dp8 and dp10 induce the formation of trans and cis TSPN-1 dimers, respectively. In silico docking calculations partnered with our crystal structures support the importance of arginine residues in positions 29, 42, and 77 in binding sulfate groups of the dp8 and dp10 forms of heparin. The ability of several TSPN-1 domains to bind to glycosaminoglycans simultaneously probably increases the affinity of binding through multivalent interactions. The formation of cis and trans dimers of the TSPN-1 domain with relatively short segments of heparin further enhances the ability of TSP-1 to participate in high affinity binding to glycosaminoglycans. Dimer formation may also involve TSPN-1 domains from two separate TSP-1 molecules. This association would enable glycosaminoglycans to cluster TSP-1.     

Intraductal papillary neoplasms of bile duct. A distinct entity like its counterpart in pancreas

To recognize the new entity-intraductal papillary neoplasia of bile duct in liver, the authors reviewed the clinical records of sixteen patients, analyzed the microscopic features, and selected immunohistochemical reactivity (cytokeratins and mucins) that might correlate with classification. Ten patients were male and six were female, with a mean age of 58 years (range, 21-73 years). According to their cell phenotypes, these papillary tumors were classified as intestinal type (6 cases), pancratobiliary type (4 cases), gastric type (5 cases) and oncocytic type (1 case). Most were located in the left hepatic duct and accompanied with bile duct dilatation (10 cases). Eight showed minimal expansile invasion into the ductal wall and eight were noninvasive. Five patients were treated with a hepatectomy, three underwent segmental resections, and one underwent a left hepatic lobectomy. One patient died of unrelated causes 6 years after operation, and another died of postoperative complications. The remaining 7 patients are alive and disease free 1-5 years after surgery. Because of its distinct clinical, pathological features and a favorable prognosis can be expected after complete surgical resection, we suggested that intraductal papillary neoplasia should be distinguished from other types of peripheral cholangiocarcinoma, as a distinct entity, like its counterparts in the pancreas. Neoexpressed and overexpressed mucins are of clinical value as a marker for supportive diagnosis, prognosis or monitoring therapy.     

Effects of chromium picolinate on oxidative damage in primary piglet hepatocytes

Chromium picolinate is a popular nutritional supplement whose safety has been questioned because of the potential risk of oxidative DNA damage. To investigate this possibility, a dose-dependent study was performed in piglet hepatocyte cultures in which low (8 microM), medium (200 microM), and high (400 microM) doses of chromium picolinate were tested and compared to untreated controls. After 48 h incubation, there were no significant differences in the levels of intracellular reactive oxygen species, medium lactate dehydrogenase activity, and comet indicators between the three experimental groups and controls (p > 0.05). In the 8 microM-treated group, the intracellular malondialdehyde content was significantly decreased relative to controls (p < 0.05). All of the studied parameters showed a dose-dependent increase that was statistically significant between the low and high doses (p < 0.05). These results suggest that: (1) chromium picolinate may affect the oxidative status of piglet hepatocytes; (2) the appropriate dose (approximately physiological concentration) of chromium picolinate can inhibit lipid peroxidation, and (3) high doses of chromium picolinate have no significant effects on oxidative damage in piglet hepatocytes, but the existing evidence also imply that exposure to a higher dose appears to be unwarranted.     

Study on structure-property-reactivity-function relationship of human neuronal growth inhibitory factor (hGIF)

Human metallothionein-3 (hMT3), also named as human neuronal growth inhibitory factor (hGIF), can inhibit the outgrowth of embryonic cortical neurons in the presence of brain extracts. In order to systematically study the structure-property-reactivity-function relationship of hGIF, our laboratory designed a series of mutants and studied their structure, property, reactivity and functions by a series of chemical and biological tools including UV spectroscopy, CD spectroscopy, NMR, chemical reaction and primary neuronal culture assays. In summary, we concluded that the bioactivity of hGIF was regulated by multiple factors, including the ⁶CPCP⁹ motif, an additional threonine insert at sequence position 5, domain-domain interactions, the structure and stability of the metal-thiolate cluster and the linker. Our studies provide more and more evidences which revealed that the bioactivity of hGIF is mainly related to the essential metal release and its characteristic conformation.     

Knocking down Wnt9a mRNA levels increases cellular proliferation

Wnts are secreted lipid-modified signaling proteins. Activation of Wnt signalling in many tissues has also been associated with cancer. In many eukaryotes, expression of nuclear-encoded mRNA can be strongly inhibited by the presence of a small double-stranded RNA corresponding to exon sequences in the mRNA. In this study we used pAVU6+27 vectors, which have SalI and XbaI clone sites, to construct the siRNA expression vectors for human Wnt9a. Two kinds of small interfering RNA inserts were designed, synthesized and visually tested for efficacy by in situ hybridization, the results demonstrated that in the cells, transfected with U6+27 cassettes with anti-Wnt9a hairpin siRNA inserts, dramatically reduced Wnt9a signals were observed as compared to the untransfected cells. The results of flow cytometry analysis showed that the cell proliferation was promoted after lowering expression of the human Wnt9a in MCF-7 cells by RNAi, but was inhibited after over-expression of human Wnt9a. These results suggests the expression level of human Wnt9a in MCF-7 that breast cancer may play a role in adjusting the rate of cellular proliferation.     

C3G regulates cortical neuron migration, preplate splitting and radial glial cell attachment

Neuronal migration is integral to the development of the cerebral cortex and higher brain function. Cortical neuron migration defects lead to mental disorders such as lissencephaly and epilepsy. Interaction of neurons with their extracellular environment regulates cortical neuron migration through cell surface receptors. However, it is unclear how the signals from extracellular matrix proteins are transduced intracellularly. We report here that mouse embryos lacking the Ras family guanine nucleotide exchange factor, C3G (Rapgef1, Grf2), exhibit a cortical neuron migration defect resulting in a failure to split the preplate into marginal zone and subplate and a failure to form a cortical plate. C3G-deficient cortical neurons fail to migrate. Instead, they arrest in a multipolar state and accumulate below the preplate. The basement membrane is disrupted and radial glial processes are disorganised and lack attachment in C3G-deficient brains. C3G is activated in response to reelin in cortical neurons, which, in turn, leads to activation of the small GTPase Rap1. In C3G-deficient cells, Rap1 GTP loading in response to reelin stimulation is reduced. In conclusion, the Ras family regulator C3G is essential for two aspects of cortex development, namely radial glial attachment and neuronal migration.     

Optical protein sensor for detecting cancer markers in saliva

A surface immobilized optical protein sensor has been utilized to detect Interleukin-8 (IL-8) protein, an oral cancer marker, and can reach limit of detection (LOD) at 1.1pM in buffer without using enzymatic amplification. Only after applying enzymatic amplification to increase the signal level by a few orders of magnitude, ELISA can reach the LOD of 1pM level. We then develop the confocal optics based sensor for further reducing the optical noise and can extend the LOD of the surface immobilized optical protein sensor two orders in magnitude. These improvements have allowed us to detect IL-8 protein at 4.0fM in buffer. In addition, these sensitive LODs were achieved without the use of enzymatic signal amplification, such that the simplified protocol can further facilitate the development of point-of-care devices. The ultra sensitive optical protein sensor presented in this paper has a wide number of applications in disease diagnoses. Measurements for detecting biomarkers in clinical sample are much more challenging than the measurements in buffer, due to high background noise contributed by large collections of non-target molecules. We used clinical saliva samples to validate the functionality of the optical protein sensor. Clinical detection of disease-specific biomarkers in saliva offers a non-invasive, alternative approach to using blood or urine. Currently, the main challenge of using saliva as a diagnostic fluid is its inherently low concentration of biomarkers. We compare the measurements of 40 saliva samples; half from oral cancer patients and half from a control group. The data measured by the optical protein sensor is compared with the traditional Enzyme-Linked Immunosorbant Assay (ELISA) values to validate the accuracy of our system. These positive results enable us to proceed to using confocal optical protein sensor to detect other biomarkers, which have much lower concentrations.