Holographic optogenetic stimulation of patterned neuronal activity for vision restoration.

When natural photoreception is disrupted, as in outer-retinal degenerative diseases, artificial stimulation of surviving nerve cells offers a potential strategy for bypassing compromised neural circuits. Recently, light-sensitive proteins that photosensitize quiescent neurons have generated unprecedented opportunities for optogenetic neuronal control, inspiring early development of optical retinal prostheses. Selectively exciting large neural populations are essential for eliciting meaningful perceptions in the brain.

Cellular Resolution Panretinal Imaging of Optogenetic Probes Using a Simple Funduscope.

Purpose: To acquire and characterize cellular-resolved in vivo fluorescence images of optogenetic probes expressed in rodent retinal ganglion cells, by adapting a low-cost and simple fundus system based on a topical endoscope.

Methods: A custom endoscope-based fundus system was constructed (adapted from the design of Paques and colleagues). Bright field and fluorescence images were acquired from head-fixed transgenic mice expressing Channelrhodopsin2-eYFP, and Sprague Dawley rats virally transfected with the optogenetic probe GCaMP3.

Transcriptional control of axonal guidance and sorting in dorsal interneurons by the Lim-HD proteins Lhx9 and Lhx1.

Background: Lim-HD proteins control crucial aspects of neuronal differentiation, including subtype identity and axonal guidance. The Lim-HD proteins Lhx2/9 and Lhx1/5 are expressed in the dorsal spinal interneuron populations dI1 and dI2, respectively. While they are not required for cell fate acquisition, their role in patterning the axonal trajectory of dI1 and dI2 neurons remains incompletely understood.

The source of heterogeneity in the heme vicinity of ferricytochrome c.

Heterogeneity in the heme vicinity of ferricytochrome c was reported to be detectable by a split of the NMR signal of the heme methyl 3 group [P.D. Burns and G.

Direct voltammetric observation of redox driven changes in axial coordination and intramolecular rearrangement of the phenylalanine-82-histidine variant of yeast iso-1-cytochrome c.

Direct square-wave and cyclic voltammetric electrochemical examination of the yeast iso-1-cytochrome c Phe82His/Cys102Ser variant revealed the intricacies of redox driven changes in axial coordination, concomitant with intramolecular rearrangement. Electrochemical methods are ideally suited for such a redox study, since they provide a direct and quantitative visualization of specific dynamic events. For the iso-1-cytochrome c Phe82His/Cys102Ser variant, square-wave voltammetry showed that the primary species in the reduced state is the Met80-Fe2+-His18 coordination form, while in the oxidized state the His82-Fe3+-His18 form predominates.

The nature of the thermal equilibrium affecting the iron coordination of ferric cytochrome c.

In cytochrome c, ligation of the heme iron by the methionine-80 sulfur plays a major role in determining the structure and the thermodynamic stability of the protein. In the ferric state, this bond is reversibly broken by moderately acid or alkaline pH's (pK's 2.5 and 9.

The role of histidines 26 and 33 in the structural stabilization of cytochrome c.

Comparative studies of the importance of the two histidines of rat cytochrome c that are not ligands of the heme iron, for the stability of the protein, were carried out by site-directed mutagenesis. Histidine 26 was substituted by valine and the resulting effects on the stability of the Met-80-sulfur to heme iron bond to changes in pH and temperature, and of the global stability of the protein to unfolding in urea solutions, were measured. It is suggested that the loss of the hydrogen bond between the His-26 imidazole and the backbone amide of Asn-31 caused the observed decreases in local stability; and that, in addition, the elimination of the hydrogen bond between this imidazole and the carbonyl of Pro-44 resulted in an increase of the mobility of the lower loop (residues 41-47) on the right side of the protein and of its distance from the middle loop (residues 26-31), probably leading to greater hydration of the interior right side of the molecule.

A chemical modification of cytochrome-c lysines leading to changes in heme iron ligation.

Although 13 lysines of horse cytochrome c are invariant, and three more are extremely conserved, the modification of their side-chain epsilon-amino groups by beta-thiopropionylation caused important changes in protein properties for only three of them; lysines 72,73 and 79. Optical spectroscopy, electron and nuclear paramagnetic resonance, electron spin echo envelope modulation, and molecular weight studies, as well as the unique features of their reaction with cytochrome-c oxidase, indicate that in the oxidized state the modification of these lysines resulted in equilibria between two different states of iron ligation: the native state, in which the metal is coordinated by the methionine-80 sulfur, and a new state in which this ligand is displaced by the sulfhydryl groups of the elongated side chains. The reduction potentials of the TP Lys-72 and the TP Lys-79 derivatives were 201 and 196 millivolt, respectively, indicating that the equilibria favored the sulfhydryl ligated state by 1.

Myopathy, lactic acidosis, and sideroblastic anemia: a new syndrome.

We describe 2 sibs (brother and sister) with myopathy, sideroblastic anemia, lactic acidosis, mental retardation, microcephaly, high palate, high philtrum, distichiasis, and micrognathia. Very low levels of cytochromes a, b, and c were detected in the patients' muscle mitochondria. Deposition of iron within the mitochondria of bone marrow erythroblasts was observed on electron microscopy.

Effects of mutating Asn-52 to isoleucine on the haem-linked properties of cytochrome c.

Asn-52 of rat cytochrome c and baker's yeast iso-1-cytochrome c was changed to isoleucine by site-directed mutagenesis and the mutated proteins expressed in and purified from cultures of transformed yeast. This mutation affected the affinity of the haem iron for the Met-80 sulphur in the ferric state and the reduction potential of the molecule. The yeast protein, in which the sulphur-iron bond is distinctly weaker than in vertebrate cytochromes c, became very similar to the latter: the pKa of the alkaline ionization rose from 8.

The significance of denaturant titrations of protein stability: a comparison of rat and baker's yeast cytochrome c and their site-directed asparagine-52-to-isoleucine mutants.

The residue asparagine-52 of rat cytochrome c and baker's yeast iso-1-cytochrome c was mutated to isoleucine by site-directed mutagenesis, and the unfolding of the wild-type and mutant proteins in urea or guanidinium chloride solutions was studied. Whereas the yeast mutant cytochrome unfolded in 4-7 M urea with a rate constant (k) of 1.7 x 10(-2) s-1, the rat mutant protein unfolded with k = 5.

Relationship between local and global stabilities of proteins: site-directed mutants and chemically-modified derivatives of cytochrome c.

The methionine 80 sulfur-heme iron bond of rat cytochrome c, whose stability is decreased by mutating the phylogenetically invariant residue proline 30 to alanine and increased when tyrosine 67 is changed to phenylalanine, recovers its wild-type characteristics when both substitutions are performed on the same molecule. Titrations with urea, analyzed according to the heteropolymer theory [Alonso, D. O.

Degradation of native human hemoglobin following hemolysis by Prevotella loescheii.

Prevotella loescheii PK1295 can grow on native hemoglobin as a source of heme. Supernatants of P. loescheii cultures hemolysed human erythrocytes and degraded native hemoglobin.

Mastoparan induces hypothermia in mice.

The polypeptide mastoparan, isolated from the venom of the Oriental Hornet, Vespa orientalis, induces hypothermia in white mice 15 minutes after its intraperitoneal injection. The hypothermic effect is induced by mastoparan obtained from different hornet and wasp venoms. The normal murine core temperature is lowered by mastoparan from 38 degrees C to as far as 33 degrees C.

The reactivity of cytochrome c with soft ligands.

The spectral changes caused by binding soft ligands to the cytochrome c iron and their correlation to ligand affinities support the hypothesis that the iron-methionine sulfur bond of this heme protein is enhanced by delocalization of the metal t2g electrons into the empty 3d orbitals of the ligand atom. These findings also explain the unique spectrum of cytochrome c in the far red.

Changing the invariant proline-30 of rat and Drosophila melanogaster cytochromes c to alanine or valine destabilizes the heme crevice more than the overall conformation.

Drosophila melanogaster and rat cytochromes c in which proline-30 was converted to alanine or valine were expressed in a strain of baker's yeast, Saccharomyces cerevisiae, where they sustained aerobic growth. The mutations had no significant effect on the spectra or redox potentials but altered drastically the stability of the bond between the methionine-80 sulfur and the heme iron, as judged by four criteria: (i) the alkaline pKa values of the 695-nm band of the ferric form of the mutant proteins decreased by almost 1 pH unit as compared to the wild types; (ii) the acid pKa values increased by 0.5 to 1.

Isolation and properties of a soluble fraction of Paracentrotus lividus sea urchin eggs responsible for the calcium-induced oxidative burst.

The system responsible for the oxidative burst (OB) activity in Paracentrotus lividus eggs is different from those described earlier for other sea urchin species. The OB in P. lividus is associated with a soluble fraction resulting from centrifugation at 150,000 g.

Structural significance of an internal water molecule studied by site-directed mutagenesis of tyrosine-67 in rat cytochrome c.

The tyrosine-67 to phenylalanine mutated rat cytochrome c is similar to the unmutated protein in its spectral, reduction potential, and enzymic electron-transfer properties. However, the loss of the 695-nm band, characteristic of the ferric form of the normal low-spin physiologically active configuration, occurs 1.2 pH units higher on the alkaline side and 0.

The binding characteristics of the cytochrome c iron.

A comparison of the binding properties of myoglobin and cytochrome c shows that the latter, in the reduced state, has an unusually large affinity for ligands, including thioethers. This explains the outstanding stability of the methionine-iron bond of ferrous cytochrome c, and results from the intrinsic ability of the cytochrome c iron to delocalize its electrons into orbitals of the sixth axial ligand.

Isolation and properties of the soluble c-type cytochromes of the dinoflagellate Peridinium cinctum.

Four soluble cytochromes of the c type were isolated from the freshwater dinoflagellate Peridinium cinctum collected from Lake Kinneret, Israel. Cytochrome c with alpha-band maximum at 550 nm in the reduced state had a molecular mass of 10,200 Da, pI 7.4, and Em of 278 m V.

Stepwise modification of the electrostatic charge of cytochrome c. Effects on protein conformation and oxidation-reduction properties.

Horse heart cytochrome c was progressively maleylated, and fractions containing increasing numbers of modified lysines were obtained. The 695 nm band was present in derivatives containing up to 14 maleylated residues. Circular dichroic spectra showed minor changes beginning with 8 substituted lysines; in derivatives with 14 or more maleylated lysines, circular dichroism indicated total disruption of the native conformation.

1H-NMR studies of the coordination geometry at the heme iron and the electronic structure of the heme group in cytochrome c-552 from Euglena gracilis.

The 1H-NMR lines of heme c in reduced and oxidized cytochrome c-552 from Euglena gracilis were individually assigned and the coordination geometry of the axial ligands was investigated. The electronic structure of the heme and the chirality of the axially bound methionine were found to be of the same type as in mammalian cytochrome c, but different from cytochrome c-551 from Pseudomonas aeruginosa. These results provide additional support for a previously proposed correlation between the chirality of attachment of the axial methionine and the electronic wave functions in oxidized cytochromes of the c type.