Mitochondrial transcription factor A (TFAM) shapes metabolic and invasion gene signatures in melanoma.

Mitochondria are central key players in cell metabolism, and mitochondrial DNA (mtDNA) instability has been linked to metabolic changes that contribute to tumorigenesis and to increased expression of pro-tumorigenic genes. Here, we use melanoma cell lines and metastatic melanoma tumors to evaluate the effect of mtDNA alterations and the expression of the mtDNA packaging factor, TFAM, on energetic metabolism and pro-tumorigenic nuclear gene expression changes. We report a positive correlation between mtDNA copy number, glucose consumption, and ATP production in melanoma cell lines. Gene expression analysis reveals a down-regulation of glycolytic enzymes in cell lines and an up-regulation of amino acid metabolism enzymes in melanoma tumors, suggesting that TFAM may shift melanoma fuel utilization from glycolysis towards amino acid metabolism, especially glutamine. Indeed, proliferation assays reveal that TFAM-down melanoma cell lines display a growth arrest in glutamine-free media, emphasizing that these cells rely more on glutamine metabolism than glycolysis. Finally, our data indicate that TFAM correlates to VEGF expression and may contribute to tumorigenesis by triggering a more invasive gene expression signature. Our findings contribute to the understanding of how TFAM affects melanoma cell metabolism, and they provide new insight into the mechanisms by which TFAM and mtDNA copy number influence melanoma tumorigenesis.

An extrusion strategy for the FeMo cofactor from nitrogenase. Towards synthetic iron-sulfur proteins.

Iron-sulfur clusters are ubiquitous in biological systems, facilitating functions such as electron transfer (rubredoxins, ferredoxins, rieske centres), isomerization (aconitase) and small molecule activation such as dinitrogen reduction (nitrogenases). Of global importance and recently particular interest, is the iron-sulfur-containing iron-molybdenum cofactor (FeMoco) cluster that achieves the biological reduction of dinitrogen under mild conditions. This biologically unique cluster has proved difficult to investigate due to its extreme air sensitivity and the instability of the cluster's structural integrity, outside the protective protein matrix. Here, we report a model iron-sulfur cluster (Roussins black salt (NH(4))[Fe(4)S(3)(NO)(7)]) that has been used to achieve the first example of a metal cluster (guest) embedded within a pseudo-protein, cyclodextrin (host). The product formed is supramolecular, that is, it contained no covalent bonds and was stabilized by predominantly entropy effects. Formation of a 1 : 1 complex between the host and the guest was established for the iron-sulfur cluster with either seven- or eight-membered cyclodextrins (beta- or gamma-cyclodextrin). A range of techniques was used to characterize the new complexes in both the solid and solution states. Electrospray mass spectra indicated the presence of parent ions of the host-guest complexes and electrochemistry was also used to define the redox behavior of the complexes. The iron-sulfur clusters were significantly more stable in the presence of the host cyclodextrin, as revealed by a negative shift for the reduction potential for the host-guest product. Using the beta-cyclodextrin as host, the reduction potential of the iron-sulfur cluster shifted more negative by 60 mV; the effect was even more dramatic for the larger gamma-cyclodextrin where the reduction potential for the cluster was shifted by 90 mV more negative than the 'unbound' [Fe(4)S(3)(NO)(7)]- cluster. This is the first example of a metal cluster, stabilized as a supramolecular complex in a 'host' environment outside of a covalently bonded protein matrix. Creating such stable environments for metal cofactors or clusters that otherwise spontaneously degrade or are catalytically inactive outside the protein matrix could have enormous practical value. Specific implications for the development of extrusion methods for FeMoco from nitrogenase are enormous, with previously difficult, high-energy molecular transformations, such as dinitrogen to ammonia, now more realistically accessible.

Design and fabrication of high-efficiency beam splitters and beam deflectors for integrated planar micro-optic systems.

High-frequency gratings with rectangular-groove profiles are used to generate high-efficiency beam splitters and beam deflectors. The effects of the grating design parameters, i.e., period, groove depth, duty cycle, number of phase levels, and polarization state (TE and TM) of the incoming signal, are considered. The case of the binary beam splitter grating is analyzed by using rigorous electromagnetic grating analysis. Fabrication techniques are presented in which three different lithographic techniques are considered (optical contact, deep-UV stepper reduction, and electron-beam direct write). Experimental results of 97% efficiency for the beam splitter grating and up to 80% for the beam deflector grating are reported.

A prospective random trial of home uterine activity monitoring in pregnancies at increased risk of preterm labor.

In a prospective trial we enrolled 157 women at increased risk of preterm birth, randomly assigning women in a ratio of 1:2 to receive either frequent (greater than or equal to 5 days/wk) nursing contact, education in preterm labor symptoms, and self-palpation of uterine activity (group E, n = 50), or daily nursing contact, preterm labor education, and the Term Guard home uterine activity monitor (group EM, n = 107). Comparison of the rate of preterm birth, the incidence of preterm labor and successful tocolysis, and the mean birth weight and gestational age revealed no significant differences and suggested that beneficial effects previously attributed to monitored contraction data may in fact be the result of frequent (five or more times per week) nursing contact and careful attention to preterm labor symptoms and perceived contractions.

An ultrastructural analysis of the development of foetal rat retina transplanted to the occipital cortex, a site lacking appropriate target neurons for optic fibres.

Foetal retina was removed from donor rats at 15 days of gestation and transplanted to the occipital cortex of neonatal host rats. The purpose of this procedure was to examine the development of retinal neurons and photoreceptors, and document synaptic patterns during maturation of the transplanted retina in an environment lacking a normal target for optic axons. Host animals were sacrificed at 5, 10, 15, 20 and 30 days and samples of cortex containing the transplant were subjected to a light and electron microscopic analysis. During early stages of development, (5 days) the retina assumes a radial orientation with the scleral (outer) surface located centrally and the vitreal (inner) surface occupying the periphery. Numerous mitotic figures are found at the centre of the transplant and columns of primitive neuroblasts appear to radiate out from this zone. By 10 to 15 days after transplantation the retinal tissue contains numerous small rosettes each of which displays a histotypic organization with recognizable layers of sensory cells and their centrally-projecting processes, an outer limiting membrane, made up of a network of zonulae adherentes, and a rudimentary outer and inner plexiform layer which delineate the cells of the inner nuclear layer. Ultrastructural analysis of such rosettes confirmed the presence of typical bipolar, amacrine, horizontal and ganglion cells, but revealed that while the plexiform layers were occupied by numerous processes from these neurons, few if any, of these exhibited synaptic vesicles. By 20 to 30 days following transplantation sensory cells have completely differentiated, giving rise to prominent inner and outer segments which display typical cilia, centrioles and basal bodies, together with numerous stacked lamellae of photoreceptors which were contorted, presumably due to growth in an abnormal site. It should be further emphasized that these structures developed in the absence of pigment cells. Synaptic development ensues during this period to form characteristic dyads within the outer and inner plexiform layers. Additionally, clusters of amacrine to amacrine contacts occurred in the inner plexiform layer and were found to be increased relative to other types of junctions. In general, synaptogenesis takes place in the outer and inner plexiform layers and all categories of retinal synapses are established, but the process was found to be significantly delayed in comparison to normal retina at the same stage of development. Quantitative analysis revealed a reduced number of presumptive ganglion cells in proportion to the other categories of neurons. Optic fibres remained small and failed to myelinate. It is suggested that lack of an appropriate target for optic axons induced this alteration and may be indirectly related to the delay in the onset of synaptic development.

Inhibition of axoplasmic transport in the developing visual system of the rat-II, Quantitative analysis of alterations in transport of tritiated proline or fucose.

Developmental alterations in the amount of tritiated proline and fucose incorporated by retinal neurons and transported within axons of the optic nerve to the dorsal lateral geniculate nucleus and superior colliculus were measured at 1, 5, 10 and 15 days postnatal using quantitative autoradiography and liquid scintillation analysis. The amount of axon transport inhibition induced by introacular injections of colchicine (10-4 M-5 X 10-3M) and xylocaine (10-3 M-10-1 M) was also determined by this methodology. Grain counts of retinal autoradiograms obtained from animals of all ages employed in this study demonstrated that [3H] proline is rapidly incorporated by all retinal neurons but becomes increasingly concentrated within the inner nuclear, ganglion cell and optic fiber layers between 2h and 2 days after injection. [3H] fucose is preferentially taken up and concentrated within the plexiform and sensory element layers. Intraocular colchicine administered 24 h prior to isotope injection exhibited no significant effect on incorporation but depressed the amount of activity in the layer of optic fibers. Comparison of the effects of colchicine and xylocaine on axon transport of [3H] proline injected at 3 days of age revealed dose-dependent suppression of transport occurring up to six hr after isotope injection; however, with longer survival periods the effects of xylocaine were no longer significant whereas colchicine maintained suppression of axon transport to 20% of normal for periods of up to 10 days. Additionally, the rate and quantity of 3H] proline transported to the dorsal lateral geniculate nucleus and tectum of 1-15 day old animals was found to be inversely proportional to the age of the animal. Maximally-effective concentrations of colchicine, determined for each age level examined by the previous study to be highest compatible with the viability of the retino-fugal projection, 34 also reduced transport to from 20-40% of normal, being most effective in animals of 1-10 days of age. Autoradiographs of the optic disc demonstrated a colchicine-induced suppression of the amount of label in the exiting optic fibers and similar preparations of the tectum revealed an elimination of activity in the stratum griseum superficialis and stratum opticum which are normally heavily labeled following intraocular [3H] proline or fucose. Following [3H] fucose injection at 10 days of age, isotope appears in the contralateral visual cortex between 3 and 10 days later, indicating a substantial amount of transneuronal transfer of label which appeared to occur in concert with periods of maximal synaptogenesis. Injection of colchicine reduced the amount of label in the cortex to background levels. Concomitantly, autoradiographs of the dorsal lateral geniculate nucleus revealed cytoplasmic labeling of geniculate neurons in control animals but this was eliminated following colchicine injection...