The mechanism of formation, structure and physiological relevance of covalent hemoglobin attachment to the erythrocyte membrane

Covalent hemoglobin binding to membranes leads to band 3 (AE1) clustering and the removal of erythrocytes from the circulation; it is also implicated in blood storage lesions. Damaged hemoglobin, with the heme being in a redox and oxygen-binding inactive hemichrome form, has been implicated as the binding species. However, previous studies used strong non-physiological oxidants. In vivo hemoglobin is constantly being oxidised to methemoglobin (ferric), with around 1% of hemoglobin being in this form at any one time. In this study we tested the ability of the natural oxidised form of hemoglobin (methemoglobin) in the presence or absence of the physiological oxidant hydrogen peroxide to initiate membrane binding. The higher the oxidation state of hemoglobin (from Fe(III) to Fe(V)) the more binding was observed, with approximately 50% of this binding requiring reactive sulphydryl groups. The hemoglobin bound was in a high molecular weight complex containing spectrin, ankyrin and band 4.2, which are common to one of the cytoskeletal nodes. Unusually, we showed that hemoglobin bound in this way was redox active and capable of ligand binding. It can initiate lipid peroxidation showing the potential to cause cell damage. In vivo oxidative stress studies using extreme endurance exercise challenges showed an increase in hemoglobin membrane binding, especially in older cells with lower levels of antioxidant enzymes. These are then targeted for destruction. We propose a model where mild oxidative stress initiates the binding of redox active hemoglobin to the membrane. The maximum lifetime of the erythrocyte is thus governed by the redox activity of the cell; from the moment of its release into the circulation the timer is set

Effect of Cistanches Herba Aqueous Extract on Bone Loss in Ovariectomized Rat

To assess the ability of traditional Chinese medicine Cistanches Herba extract (CHE) to prevent bone loss in the ovariectomized (OVX) rat, Cistanches Herba extract (CHE) was administered intragastrically to the rats. Female rats were anesthetized with pentobarbital sodium (40 mg kg−1, i.p.), and their ovaries were removed bilaterally. The rats in the sham-operated group were anesthetized, laparotomized, and sutured without removing their ovaries. After 1 week of recovery from surgery, the OVX rats were randomly divided into three groups and orally treated with H2O (OVX group) or CHE (100 or 200 mg kg−1 daily) for 3 months. The sham-operated group (n = 8) was orally treated with H2O. After 3 months, the total body bone mineral density (BMD), bone mineral content (BMC), Bone biomechanical index, blood mineral levels and blood antioxidant enzymes activities were examined in sham-operated, ovariectomized and Cistanches Herba extract treated rats. Results showed that Cistanches Herba extract treatment significantly dose-dependently enhanced bone mineral density (BMD), bone mineral content (BMC), maximum load, displacement at maximum load, stress at maximum load, load at auto break, displacement at auto break, and stress at auto break, and blood antioxidant enzymes activities, decreased blood Ca, Zn and Cu levels compared to the OVX group. This experiment demonstrates that the administration of Cistanches Herba extract to ovariectomized rats reverses bone loss and prevents osteoporosis

High expression of CXCR4 may predict poor survival in resected pancreatic adenocarcinoma

Chemokines and their receptors are involved in tumourigenicity and clinicopathological significance of chemokines receptor expression in pancreatic adenocarcinoma (PA) is not fully understood. This study was conducted to determine patients' outcome according to the expressions of CXCR4, CXCR7 and HIF-1α after resection of PA. Immunohistochemistry for CXCR4, CXCR7 and HIF-1α expressions as well as cell proliferative index (Ki-67) was conducted in 71 resected (R0) PA and their 48 related lymph nodes (LN) using tissue microarray. CXCR4 and CXCR7 expressions were positively correlated to HIF-1α suggesting a potential role of HIF-1α in CXCR4 and CXCR7 transcription activation. Patients with CXCR4high tumour expression had shorter OS than those with low expression (median survival: 9.7 vs 43.2 months, P=0.0006), a higher risk of LN metastases and liver recurrence. In multivariate analysis, high CXCR4 expression, LN metastases and poorly differentiated tumour are independent negative prognosis factors. In a combining analysis, patients with CXCR4low/CXCR7low tumour had a significantly shorter DFS and OS than patients with a CXCR7high/CXCR4high tumour. CXCR4 in resected PA may represent a valuable prognostic factor as well as an attractive target for therapeutic purpose

Ascorbic acid partly antagonizes resveratrol mediated heme oxygenase-1 but not paraoxonase-1 induction in cultured hepatocytes - role of the redox-regulated transcription factor Nrf2

<p>Abstract</p> <p>Background</p> <p>Both resveratrol and vitamin C (ascorbic acid) are frequently used in complementary and alternative medicine. However, little is known about the underlying mechanisms for potential health benefits of resveratrol and its interactions with ascorbic acid.</p> <p>Methods</p> <p>The antioxidant enzymes heme oxygenase-1 and paraoxonase-1 were analysed for their mRNA and protein levels in HUH7 liver cells treated with 10 and 25 μmol/l resveratrol in the absence and presence of 100 and 1000 μmol/l ascorbic acid. Additionally the transactivation of the transcription factor Nrf2 and paraoxonase-1 were determined by reporter gene assays.</p> <p>Results</p> <p>Here, we demonstrate that resveratrol induces the antioxidant enzymes heme oxygenase-1 and paraoxonase-1 in cultured hepatocytes. Heme oxygenase-1 induction by resveratrol was accompanied by an increase in Nrf2 transactivation. Resveratrol mediated Nrf2 transactivation as well as heme oxygenase-1 induction were partly antagonized by 1000 μmol/l ascorbic acid.</p> <p>Conclusions</p> <p>Unlike heme oxygenase-1 (which is highly regulated by Nrf2) paraoxonase-1 (which exhibits fewer ARE/Nrf2 binding sites in its promoter) induction by resveratrol was not counteracted by ascorbic acid. Addition of resveratrol to the cell culture medium produced relatively low levels of hydrogen peroxide which may be a positive hormetic redox-signal for Nrf2 dependent gene expression thereby driving heme oxygenase-1 induction. However, high concentrations of ascorbic acid manifold increased hydrogen peroxide production in the cell culture medium which may be a stress signal thereby disrupting the Nrf2 signalling pathway.</p

A late role for a subset of neurogenic genes to limit sensory precursor recruitments in Drosophila embryos

In Drosophila , mutations in a class of genes, the neurogenic genes, produce an excess of neurons. This neural hyperplasia has been attributed to the formation of more than the normal number of neuronal precursor cells at the expense of epidermal cells. In order to find out whether the neurogenic genes only act at this intial step of neurogenesis, we studied the replication pattern of the sensory organ precursor cells by monitoring BrdU incorporation in embryos mutant for Notch ( N ), Delta ( Dl ), mastermind ( mam ), almondex ( amx ), neuralized ( neu ), big brain ( bib ) and the Enhancer of split -Complex ( E ( spl )- C ). Using temperature sensitive alleles of two of the neurogenic genes, DI and N , we also induced an acute increase of replicating sensory precursors by shifting briefly to the restricted temperature. We have found that the loss of function of all the seven neurogenic loci that were tested causes an increase in replicating sensory precursor cells, consistent with the model that these neurogenic genes normally participate in the process of restricting the number of neuronal precursors. Whereas the temporal pattern of replication appeared normal in mutants of five of the seven neurogenic loci, in N and mam embryos replicating PNS cells are present beyond the time when they normally undergo replication. Experiments with colchicine suggest that many of these late replicating cells may be newly emerging precursors and probably not additional cell divisions of already recruited precursors. Thus, different neurogenic genes may be required over different periods of time for the specification of sensory precursor cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47511/1/427_2004_Article_BF00188736.pd

Graph Theoretical Model of a Sensorimotor Connectome in Zebrafish

Mapping the detailed connectivity patterns (connectomes) of neural circuits is a central goal of neuroscience. The best quantitative approach to analyzing connectome data is still unclear but graph theory has been used with success. We present a graph theoretical model of the posterior lateral line sensorimotor pathway in zebrafish. The model includes 2,616 neurons and 167,114 synaptic connections. Model neurons represent known cell types in zebrafish larvae, and connections were set stochastically following rules based on biological literature. Thus, our model is a uniquely detailed computational representation of a vertebrate connectome. The connectome has low overall connection density, with 2.45% of all possible connections, a value within the physiological range. We used graph theoretical tools to compare the zebrafish connectome graph to small-world, random and structured random graphs of the same size. For each type of graph, 100 randomly generated instantiations were considered. Degree distribution (the number of connections per neuron) varied more in the zebrafish graph than in same size graphs with less biological detail. There was high local clustering and a short average path length between nodes, implying a small-world structure similar to other neural connectomes and complex networks. The graph was found not to be scale-free, in agreement with some other neural connectomes. An experimental lesion was performed that targeted three model brain neurons, including the Mauthner neuron, known to control fast escape turns. The lesion decreased the number of short paths between sensory and motor neurons analogous to the behavioral effects of the same lesion in zebrafish. This model is expandable and can be used to organize and interpret a growing database of information on the zebrafish connectome

ppk23-Dependent Chemosensory Functions Contribute to Courtship Behavior in Drosophila melanogaster

Insects utilize diverse families of ion channels to respond to environmental cues and control mating, feeding, and the response to threats. Although degenerin/epithelial sodium channels (DEG/ENaC) represent one of the largest families of ion channels in Drosophila melanogaster, the physiological functions of these proteins are still poorly understood. We found that the DEG/ENaC channel ppk23 is expressed in a subpopulation of sexually dimorphic gustatory-like chemosensory bristles that are distinct from those expressing feeding-related gustatory receptors. Disrupting ppk23 or inhibiting activity of ppk23-expressing neurons did not alter gustatory responses. Instead, blocking ppk23-positive neurons or mutating the ppk23 gene delayed the initiation and reduced the intensity of male courtship. Furthermore, mutations in ppk23 altered the behavioral response of males to the female-specific aphrodisiac pheromone 7(Z), 11(Z)-Heptacosadiene. Together, these data indicate that ppk23 and the cells expressing it play an important role in the peripheral sensory system that determines sexual behavior in Drosophila

Dorsoventral Patterning in Hemichordates: Insights into Early Chordate Evolution

We have compared the dorsoventral development of hemichordates and chordates to deduce the organization of their common ancestor, and hence to identify the evolutionary modifications of the chordate body axis after the lineages split. In the hemichordate embryo, genes encoding bone morphogenetic proteins (Bmp) 2/4 and 5/8, as well as several genes for modulators of Bmp activity, are expressed in a thin stripe of ectoderm on one midline, historically called “dorsal.” On the opposite midline, the genes encoding Chordin and Anti-dorsalizing morphogenetic protein (Admp) are expressed. Thus, we find a Bmp-Chordin developmental axis preceding and underlying the anatomical dorsoventral axis of hemichordates, adding to the evidence from Drosophila and chordates that this axis may be at least as ancient as the first bilateral animals. Numerous genes encoding transcription factors and signaling ligands are expressed in the three germ layers of hemichordate embryos in distinct dorsoventral domains, such as pox neuro, pituitary homeobox, distalless, and tbx2/3 on the Bmp side and netrin, mnx, mox, and single-minded on the Chordin-Admp side. When we expose the embryo to excess Bmp protein, or when we deplete endogenous Bmp by small interfering RNA injections, these expression domains expand or contract, reflecting their activation or repression by Bmp, and the embryos develop as dorsalized or ventralized limit forms. Dorsoventral patterning is independent of anterior/posterior patterning, as in Drosophila but not chordates. Unlike both chordates and Drosophila, neural gene expression in hemichordates is not repressed by high Bmp levels, consistent with their development of a diffuse rather than centralized nervous system. We suggest that the common ancestor of hemichordates and chordates did not use its Bmp-Chordin axis to segregate epidermal and neural ectoderm but to pattern many other dorsoventral aspects of the germ layers, including neural cell fates within a diffuse nervous system. Accordingly, centralization was added in the chordate line by neural-epidermal segregation, mediated by the pre-existing Bmp-Chordin axis. Finally, since hemichordates develop the mouth on the non-Bmp side, like arthropods but opposite to chordates, the mouth and Bmp-Chordin axis may have rearranged in the chordate line, one relative to the other