A chemically synthesized radiolabeled signal peptide: design, preparation, and biological evaluation of an iodinated analog of preproparathyroid hormone

The chemically synthesized signal peptide (native-sequence signal peptide) of preproparathyroid hormone exhibits signal sequence-like activity by inhibiting the translocation/processing of precursor proteins to their mature forms in an in vitro translation system. In order to prepare a biologically functional radiolabeled form of this peptide, we undertook structure-function studies of the native-sequence signal peptide. Since conventional iodination of peptides is performed under oxidizing conditions, chemical design efforts were focused on the oxidation-labile residues, methionine and cysteine, present in the native sequence. Substitution of the three methionines with norleucine and the single cysteine with alanine yielded a surfur-free analog, [Nle-(-25), Nle-(-21),Nle-(-18),Ala-(-14),D-Tyr-(+1)]pre-proPTH-(-29-+1)amide, which is resistant to oxidation and active in the inhibition of processing assay. An interaction between the signal region and one of the components of the intracellular secretory apparatus, signal recognition particle (SRP), was demonstrated: iodinated sulfur-free analog was cross-linked (using the homo-bifunctional reagent disuccinimidyl suberate) to the 54 kilodalton (kDa) subunit of SRP. The 68 kDa and 72 kDa subunits of SRP were also labeled, but to a lesser extent, by the iodinated peptide.     

The SecDFyajC domain of preprotein translocase controls preprotein movement by regulating SecA membrane cycling.

Escherichia coli preprotein translocase comprises a membrane-embedded hexameric complex of SecY, SecE, SecG, SecD, SecF and YajC (SecYEGDFyajC) and the peripheral ATPase SecA. The energy of ATP binding and hydrolysis promotes cycles of membrane insertion and deinsertion of SecA and catalyzes the movement of the preprotein across the membrane. The proton motive force (PMF), though not essential, greatly accelerates late stages of translocation. We now report that the SecDFyajC domain of translocase slows the movement of preprotein in transit against both reverse and forward translocation and exerts this control through stabilization of the inserted form of SecA. This mechanism allows the accumulation of specific translocation intermediates which can then complete translocation under the driving force of the PMF. These findings establish a functional relationship between SecA membrane insertion and preprotein translocation and show that SecDFyajC controls SecA membrane cycling to regulate the movement of the translocating preprotein.     

Interleukin 1 induces multinucleation and bone-resorbing activity of osteoclasts in the absence of osteoblasts/stromal cells

Interleukin-1 (IL-1) is one of the most potent bone-resorbing factors involved in bone loss associated with inflammation. We previously reported that IL-1 prolonged the survival of multinucleated osteoclast-like cells (OCLs) formed in cocultures of murine osteoblasts/stromal cells and bone marrow cells via the prevention of spontaneously occurring apoptosis. It was reported that macrophage colony-stimulating factor (M-CSF/CSF-1) prolongs the survival of OCLs without the help of osteoblasts/stromal cells. The present study was conducted to determine whether IL-1 also directly induces the multinucleation and activation of OCLs. Mononuclear osteoclast-like cells (prefusion osteoclasts; pOCs) were purified using the "disintegrin" echistatin from cocultures of murine osteoblastic cells (MB 1.8 cells) and bone marrow cells. Both IL-1 and M-CSF prolonged the survival and induced the multinucleation of pOCs through their respective receptors. However, actin ring formation (a functional marker of osteoclasts) by multinucleated cells was observed in the pOC cultures treated with IL-1, but not those treated with M-CSF. We previously reported that enriched multinucleated OCLs as well as pOCs placed on bone/dentine slices formed few resorption pits, but their pit-forming activity was greatly increased by the addition of osteoblasts/stromal cells. Here, pit-forming activity of both pOCs and enriched OCLs placed on dentine slices was induced by adding IL-1, even in the absence of osteoblasts/stromal cells. M-CSF failed to induce pit-forming activity in pOC and enriched OCL cultures. These results indicate that IL-1 induces the multinucleation and bone-resorbing activity of osteoclasts even in the absence of osteoblasts/stromal cells.     

Triplet Excitons in Highly Efficient Solar Cells Based on the Soluble Small Molecule p‐DTS(FBTTh2)2

Triplet exciton formation in neat 7,7‐(4,4‐bis(2‐ethylhexyl)‐4H‐silolo[3,2‐b:4,5‐b′] dithiophene‐2,6‐diyl)bis(6‐fluoro‐4‐(5′‐hexyl‐[2,2′‐bithiophen]‐5‐yl)benzo[c][1,2,5]thiadiazole) (p‐DTS(FBTTh₂)₂) and blends with [6,6]‐Phenyl C₇₀ butyric acid methyl ester (PC₇₀BM), with and without the selective solvent additive 1,8‐diiodooctane, is investigated by means of spin sensitive photoluminescence measurements. For all three material systems, a significant amount of long living triplet excitons is detected, situated on the p‐DTS(FBTTh₂)₂ molecules. The characteristic zero‐field splitting parameters for this state are identified to be D = 42 mT (1177 MHz) and E = 5 mT (140 MHz). However, no triplet excitons located on PC₇₀BM are detectable. Using electrically detected spin resonance, the presence of these triplet excitons is confirmed even at room temperature, highlighting that triplet excitons form during solar cell operation and influence the photocurrent and photovoltage. Surprisingly, the superior performing blend is found to have the largest triplet population. It is concluded, that the formation of triplet excitons from charge transfer states via electron back transfer has no crucial impact on device performance in p‐DTS(FBTTh₂)₂:PC₇₀BM based solar cells.     

Methylene Blue as a Cerebral Metabolic and Hemodynamic Enhancer

By restoring mitochondrial function, methylene blue (MB) is an effective neuroprotectant in many neurological disorders (e.g., Parkinson’s and Alzheimer’s diseases). MB has also been proposed as a brain metabolic enhancer because of its action on mitochondrial cytochrome c oxidase. We used in vitro and in vivo approaches to determine how MB affects brain metabolism and hemodynamics. For in vitro, we evaluated the effect of MB on brain mitochondrial function, oxygen consumption, and glucose uptake. For in vivo, we applied neuroimaging and intravenous measurements to determine MB’s effect on glucose uptake, cerebral blood flow (CBF), and cerebral metabolic rate of oxygen (CMRO₂) under normoxic and hypoxic conditions in rats. MB significantly increases mitochondrial complex I–III activity in isolated mitochondria and enhances oxygen consumption and glucose uptake in HT-22 cells. Using positron emission tomography and magnetic resonance imaging (MRI), we observed significant increases in brain glucose uptake, CBF, and CMRO₂ under both normoxic and hypoxic conditions. Further, MRI revealed that MB dramatically increased CBF in the hippocampus and in the cingulate, motor, and frontoparietal cortices, areas of the brain affected by Alzheimer’s and Parkinson’s diseases. Our results suggest that MB can enhance brain metabolism and hemodynamics, and multimetric neuroimaging systems offer a noninvasive, nondestructive way to evaluate treatment efficacy.     

Phylogeny and taxonomy of species in the Grosmannia serpens complex

Grosmannia serpens was first described from pine in Italy in 1936 and it has been recorded subsequently from many countries in both the northern and southern hemispheres. The fungus is vectored primarily by root-infesting bark beetles and has been reported to contribute to pine-root diseases in Italy and South Africa. The objective of this study was to consider the identity of a global collection of isolates not previously available and using DNA sequence-based comparisons not previously applied to most of these isolates. Phylogenetic analyses of the ITS2-LSU, actin, beta-tubulin, calmodulin and translation elongation factor-1 alpha sequences revealed that these morphologically similar isolates represent a complex of five cryptic species. Grosmannia serpens sensu stricto thus is redefined and comprises only isolates from Italy including the ex-type isolate. The ex-type isolate of Verticicladiella alacris was shown to be distinct from G. serpens, and a new holomorphic species, G. alacris, is described. The teleomorph state of G. alacris was obtained through mating studies in the laboratory, confirming that this species is heterothallic. Most of the available isolates, including those from South Africa, USA, France, Portugal and some from Spain, represent G. alacris. The remaining three taxa, known only in their anamorph states, are described as the new species Leptographium gibbsii for isolates from the UK, L. yamaokae for isolates from Japan and L. castellanum for isolates from Spain and the Dominican Republic.     

State of water in extremely halophilic bacteria: Heat of dilution ofHalobacterium halobium

Summary Heat of dilution ofHalobacterium halobium was measured when thick pastes of the bacteria, harvested throughout a complete growth cycle, were lysed by mixing with 40 times their volume of water in a microcalorimeter. A series of comparative measurements was made with pastes of bacteria previously disrupted by freezing and thawing but otherwise identical to the pastes of whole bacteria. The frozen-thawed pastes gave endothermic values some 18% greater than those obtained with intact bacteria; the difference was highly significant. Evidence was obtained that the mechanical component of bursting did not contribute to the difference between whole and lysed bacteria. On the other hand, when a correction was applied for heat of mixing of intracellular salts with extracellular NaCl, such as occurs when the bacteria lyse, the difference between whole and disrupted organisms was largely eliminated from exponential phase halobacteria but not from those harvested in stationary phase. It is concluded that there is no evidence, as reflected in heat of dilution, of abnormal solution properties of the cytosol of young halobacteria, which are rich in potassium. On the other hand, and paradoxically, some doubt remains about stationary phase organisms whose cytosol has a much higher Na⁺ content (and Na/K⁺ ratio) than the cytosol of exponential phase bacteria.     

Enantiomer-Selective Photo-Induced Reaction of Protonated Tryptophan with Disaccharides in the Gas Phase

In order to investigate chemical evolution in interstellar molecular clouds, enantiomer-selective photo-induced chemical reactions between an amino acid and disaccharides in the gas phase were examined using a tandem mass spectrometer containing an electrospray ionization source and a cold ion trap. Ultraviolet photodissociation mass spectra of cold gas-phase noncovalent complexes of protonated tryptophan (Trp) enantiomers with disaccharides consisting of two d-glucose units, such as d-maltose or d-cellobiose, were obtained by photoexcitation of the indole ring of Trp. NH₂CHCOOH loss via cleavage of the C_α–C_β bond in Trp induced by hydrogen atom transfer from the NH₃ ⁺ group of a protonated Trp was observed in a noncovalent heterochiral H⁺(l-Trp)(d-maltose) complex. In contrast, a photo-induced chemical reaction forming the product ion with m/z 282 occurs in homochiral H⁺(d-Trp)(d-maltose). For d-cellobiose, both NH₂CHCOOH elimination and the m/z 282 product ion were observed, and no enantiomer-selective phenomena occurred. The m/z 282 product ion indicates that the photo-induced C-glycosylation, which links d-glucose residues to the indole moiety of Trp via a C–C bond, can occur in cold gas-phase noncovalent complexes, and its enantiomer-selectivity depends on the structure of the disaccharide.