Skeletal Muscle Electrical Stimulation Prevents Progression of Disuse Muscle Atrophy via Forkhead Box O Dynamics Mediated by Phosphorylated Protein Kinase B and Peroxisome Proliferator-Activated Receptor gamma Coactivator-1alpha.

Although electrical muscle stimulation (EMS) of skeletal muscle effectively prevents muscle atrophy, its effect on the breakdown of muscle component proteins is unknown. In this study, we investigated the biological mechanisms by which EMS-induced muscle contraction inhibits disuse muscle atrophy progression. Experimental animals were divided into a control group and three experimental groups: immobilized (Im; immobilization treatment), low-frequency (LF; immobilization treatment and low-frequency muscle contraction exercise), and high-frequency (HF; immobilization treatment and high-frequency muscle contraction exercise). Following the experimental period, bilateral soleus muscles were collected and analyzed. Atrogin-1 and Muscle RING finger 1 (MuRF-1) mRNA expression levels were significantly higher for the experimental groups than for the control group but were significantly lower for the HF group than for the Im group. Peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) mRNA and protein expression levels in the HF group were significantly higher than those in the Im group, with no significant differences compared to the Con group. Both the Forkhead box O (FoxO)/phosphorylated FoxO and protein kinase B (AKT)/phosphorylated AKT ratios were significantly lower for the Im group than for the control group and significantly higher for the HF group than for the Im group. These results, the suppression of atrogin-1 and MuRF-1 expression for the HF group may be due to decreased nuclear expression of FoxO by AKT phosphorylation and suppression of FoxO transcriptional activity by PGC-1alpha. Furthermore, the number of muscle contractions might be important for effective EMS.

Correlative near-infrared light and cathodoluminescence microscopy using Y2O3:Ln, Yb (Ln = Tm, Er) nanophosphors for multiscale, multicolour bioimaging.

This paper presents a new correlative bioimaging technique using Y2O3:Tm, Yb and Y2O3:Er, Yb nanophosphors (NPs) as imaging probes that emit luminescence excited by both near-infrared (NIR) light and an electron beam. Under 980 nm NIR light irradiation, the Y2O3:Tm, Yb and Y2O3:Er, Yb NPs emitted NIR luminescence (NIRL) around 810 nm and 1530 nm, respectively, and cathodoluminescence at 455 nm and 660 nm under excitation of accelerated electrons, respectively. Multimodalities of the NPs were confirmed in correlative NIRL/CL imaging and their locations were visualized at the same observation area in both NIRL and CL images. Using CL microscopy, the NPs were visualized at the single-particle level and with multicolour. Multiscale NIRL/CL bioimaging was demonstrated through in vivo and in vitro NIRL deep-tissue observations, cellular NIRL imaging, and high-spatial resolution CL imaging of the NPs inside cells. The location of a cell sheet transplanted onto the back muscle fascia of a hairy rat was visualized through NIRL imaging of the Y2O3:Er, Yb NPs. Accurate positions of cells through the thickness (1.5 mm) of a tissue phantom were detected by NIRL from the Y2O3:Tm, Yb NPs. Further, locations of the two types of NPs inside cells were observed using CL microscopy.

A low-angle laser light scattering study of the association behavior of a major membrane protein of Rhodospirillum rubrum chromatophore at various concentrations of sodium dodecyl sulfate where polypeptides derived from water-soluble globular proteins are solubilized monomerically.

The major membrane protein of Rhodospirillum rubrum chromatophore could be solubilized in the presence of free sodium dodecyl sulfate (SDS) in concentration above 0.8 mM. At this concentration, the protein was highly associated to give a weight-averaged molecular weight as high as one million as determined by the low-angle laser light scattering technique. With the increase of free SDS concentration, the aggregates were progressively dissociated to give a molecular weight of 8300 at the critical micelle concentration of SDS. Three protein polypeptides derived from typical water-soluble globular proteins, bovine serum albumin, ovalbumin and beta-lactoglobulin, were found to be solubilized monomerically even at 0.8 mM free SDS. The results obtained suggest that there is substantial difference in the mode of solubilization between polypeptides derived from intrinsic membrane proteins and those from water-soluble globular proteins.

Assessment study on the use of the low angle laser light scattering technique for the estimation of molecular weight of the polypeptide forming a complex with sodium dodecyl sulfate.

Molecular weights of the protein moieties of several complexes formed between sodium dodecyl sulfate and protein polypeptides of known molecular weights could be correctly estimated by the low angle laser light scattering technique (Kaye, W., Havlik, A.J. and MacDaniel, J.B. (1971) Polymer Lett. 9, 695-699). Since the surfactant can solubilize membrane proteins indiscriminately, the technique seems to be promising for the unique determination of molecular weights of membrane proteins.

Heterologous expression of clostridial hydrogenase in the Cyanobacterium synechococcus PCC7942.

The Clostridium pasteurianum hydrogenase I has been expressed in the cyanobacterium Synechococcus PCC7942. The Shine-Dalgarno sequence of the structural gene encoding hydrogenase I from C. pasteurianum was changed to that of the cat (chloramphenicol acetyltransferase) gene. The hydrogenase gene was cloned downstream of a strong promoter, isolated from Synechococcus PCC7942, with the cat gene as a reporter gene. Expression of clostridial hydrogenase was confirmed by Western and Northern blot analyses in Synechococcus and Escherichia coli, whereas in vivo/in vitro measurements and activity staining of soluble proteins separated on non-denaturing polyacrylamide gels revealed functional expression of hydrogenase only in cyanobacterial cells. The changed Shine-Dalgarno sequence appeared to be essential for the functional expression of clostridial hydrogenase in Synechococcus, but had no influence on the expression and activity of clostridial hydrogenase expressed in E. coli.

Partitioning of triphenylalkylphosphonium homologues in gel bead-immobilized liposomes: chromatographic measurement of their membrane partition coefficients.

Unilamellar liposomes of small or large size, SUVs and LUVs, respectively, were stably immobilized in the highly hydrophilic Sepharose 4B or Sephacryl S-1000 gel beads as a membrane stationary phase for immobilized liposome chromatography (ILC). Lipophilic cations of triphenylmethylphosphonium and tetraphenylphosphonium (TPP+) have been used as probes of the membrane potential of cells. Interaction of TPP+ and triphenylalkylphosphonium homologues with the immobilized liposomal membranes was shown by their elution profiles on both zonal and frontal ILC. Retardation of the lipophilic cations on the liposome gel bed was increased as the hydrophobicity of the cations increased, indicating the partitioning of lipophilic cations into the hydrocarbon region of the membranes. The cations did not retard on the Sepharose or Sephacryl gel bed without liposomes, confirming that the cations only interact with the immobilized liposomes. Effects of the solute concentration, flow rate, and gel-matrix substance on the ILC were studied. The stationary phase volume of the liposomal membranes was calculated from the volume of a phospholipid molecule and the amount of the immobilized phospholipid, which allowed us to determine the membrane partition coefficient (KLM) for the lipophilic cations distributed between the aqueous mobile and membrane stationary phases. The values of KLM were generally increased with the hydrophobicity of the solutes increased, and were higher for the SUVs than for the LUVs. The ILC method described here can be applied to measure membrane partition coefficients for other lipophilic solutes (e.g., drugs).

Redox properties of an H-subunit-depleted photosynthetic reaction center from Rhodopseudomonas viridis.

Recently, we reported that a H-subunit-depleted photosynthetic reaction center (RC-H) was purified from purple nonsulfer photosynthetic bacterium Rhodopseudomonas viridis (Rps. viridis) using a strong detergent sodium alkyl ether sulfate. We compared the redox properties of a native photosynthetic reaction center (RC) and RC-H of Rps. viridis. In RC-H prepared by our method, secondary quinone (QB) was removed while primary quinone (QA) was retained. Absorption spectrum of RC-H was similar to that of RC. After reconstitution of ubiquinone 10 into QB sites, RC-H showed electron transfer activity that was the same as that for native RC. This is the first report about the redox properties of RC-H of Rps. viridis.

Stabilization of liposomal membranes by thermozeaxanthins: carotenoid-glucoside esters.

Thermozeaxanthins (TZS) are novel carotenoid-glucoside esters existing in the cell membranes of the thermophilic bacterium, Thermus thermophilus. The effect of TZS on membrane permeability was studied by measuring the leakage of the fluorescent dye from calcein-entrapped large unilamellar liposomes (LUVs). The LUVs were composed of a small portion (0.2-1.0 mol%) of TZS and phosphatidylcholine (PC) of various length and saturation degree of hydrocarbon chains. Incorporation of TZS in egg PC LUVs stabilized the liposomes in the temperature range from 30 to 80 degrees C, as only 2.6% of the entrapped calcein leaked out in contrast to 10% release from the egg PC liposomes without TZS. LUVs composed of dipalmitoylphosphatidylcholine (DPPC) or dioleoylphosphatidylcholine (DOPC) were stabilized by the incorporation of TZS at a temperature below 30 degrees C. Inclusion of TZS in LUVs composed of dimyristoylphosphatidylcholine, whose hydrocarbon chains are shorter than both DPPC and DOPC, did not stabilize the liposomes. About 90% of the entrapped dye was lost indicating defects of the liposomal membranes. Matching of the lipid bilayer thickness with the molecular length of TZS in the bilayers is discussed.

Analysis of the structure and expression of the chicken gene encoding a homolog of the human RREB-1 transcription factor.

Ras proteins are involved in a number of signal transduction pathways including the mitogen-activated kinase cascade. Activated MAPKs translocate to the nucleus and phosphorylate transcription factors such as c-myc, TCF and AP-1. Recently, a Ras-responsive element binding transcription factor, RREB-1, was cloned from a human medullary thyroid carcinoma cell line. RREB-1 is a zinc finger protein that binds to a Ras-responsive element in the promoter of the human calcitonin gene. We report the cloning of the chicken homologue to human RREB-1. Amino-acid alignment demonstrates that chicken and human RREB-1 are 53% identical and 69% similar. Genomic southern analysis indicates that chicken rreb-1 is a single-copy gene in the chicken genome. We demonstrate that chicken and human rreb-1 display the same tissue distribution, being expressed in all tissues examined except the brain. Interestingly, chicken RREB-1 has an extended N-terminus and contains 16 zinc fingers of the TFIIIA subclass, in comparison to human RREB-1 which was reported to contain only four zinc fingers. The size discrepancy between the two predicted gene products is further discussed. An unusual structural feature of RREB-1 is the widely spaced arrangement of the zinc fingers.

Isolation of a membrane protein from R rubrum chromatophores and its abnormal behavior in SDS-polyacrylamide gel electrophoresis due to a high binding capacity for SDS.

A membrane protein insoluble in water was isolated by gel chromatography in the presence of 0.1% sodium dodecyl sulfate (SDS) from chromatophores of a photosynthetic bacterium, Rhodospirillum rubrum. This is one of the major membrane proteins of the chromatophore. The protein was found to bind about four grams of SDS per gram, a value which is more than twice the amount generally observed with protein polypeptides derived from water-soluble globular proteins. The electrophoretic behavior of the complex between the membrane protein and SDS is abnormal due to this high capacity for binding SDS. Estimation of the molecular weight of this protein by SDS-polyacrylamide gel electrophoresis was thus impossible. Such an anomaly in SDS binding is unlikely to be restricted to the particular membrane protein described in this paper. The possibility of such a deviation from standard behavior in the interaction with SDS should be taken into consideration in studies of other membrane proteins, since SDS is often used both in analytical and preparative procedures.

Control of the unidirectional topological orientation of a cross-linked complex composed of the bacterial photosynthetic reaction center and horse heart cytochrome c reconstituted into proteoliposomes.

Control of the unidirectional topological orientation was achieved for a cross-linked complex composed of the bacterial photosynthetic reaction center and horse heart cytochrome c (RC/cyt c) reconstituted into proteoliposomes. Using the method of Ueno et al. [Ueno et al. (1995) Mater. Sci. Eng. C3, 1-6], we prepared RC/cyt c by conjugating cyt c to the H-subunit of RC of Rhodobacter sphaeroides R-26 using a bifunctional cross-linking reagent, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), as previously reported. The freeze-thaw method was used to incorporate RC/cyt c into liposomes that contained dipalmitoyl-L-alpha-phosphatidylcholine and dipalmitoyl-L-alpha-phosphatidylglycerol (1:9). The topological orientation of RC/cyt c in the proteoliposomes was determined using three methods: (i) release of the cyt c moiety from the proteoliposomes by cleaving the disulfide bond in the linker residue, (ii) electron transfer from free cyt c outside the proteoliposomes to the RC moiety, and (iii) photo-induced membrane potential of RC- and RC/cyt c-reconstituted proteoliposomes. The results indicated that about 90% of the RC/cyt c in proteoliposomes was oriented with the H-subunit exposed on the outside of the liposomes, whereas only about 60% of the RC in proteoliposomes had this orientation. Thus, we successfully controlled the unidirectional topological orientation of the RC moiety in liposomes using the RC/cyt c complex.

Detection of porphyrin using a short peptide immobilized on a surface plasmon resonance sensor chip.

In this paper the development and feasibility of a novel detection system for a low molecular weight chemical, in which a peptide was utilized as a binding molecule, are described. Surface plasmon resonance (SPR) apparatus was used as a transducer. The porphyrin binding peptide, PSP2, was used as a model peptide ligand, while a porphyrin derivative, H(2)TMpyP, was used as a model low-molecular-weight chemical. PSP2 was covalently immobilized onto the SPR sensor chip and SPR measurement using the PSP2-immobilized chip for various concentrations of porphyrin was carried out. H(2)TMpyP was detectable in the range from 100 ng ml(-1) to 10 microg ml(-1) with a linear correlation and good precision and the PSP2-immobilized chip could be regenerated within 1 min after measurement in this system. From comparison of the detection manners of three porphyrin derivatives, the ability of a short peptide to discriminate between differences in molecular structure was demonstrated. Moreover, the self-assembled monolayer (SAM) of PSP2 was successfully prepared on the gold substrate and H(2)TMpyP could be detected using the PSP2-SAM chip.

Pavlovian conditioning of discriminatively elicited eyeblink responses with short onset latency attributable to lengthened interstimulus intervals.

Conditioned eyeblink responses were obtained in cats by pairing click (CS) with glabella tap (US) and electrical stimulation of the hypothalamus (HS). Hiss was used as a discriminative stimulus (DS). Onset latencies of conditioned responses (CRs) of 20-56 ms were obtained by using an interstimulus interval (ISI) of 570-10 ms between CS and US-HS. Longer latency (90-320 ms) blink CRs were obtained with ISIs of 340-240 and 340-10 ms. The timing of associatively learned movements has been thought to increase with lengthening of the intervals between CS and US presentation. The production of shorter latency CRs of this type by lengthening the ISI is a novel result and one unexpected from widely held beliefs.

Layer-by-layer assembly of metal-mediated multiporphyrin arrays.

Two types of multiporphyrin arrays, mediated by PdCl4(2-) complex ions at the air-water interface, were alternately transferred onto solid supports to form three-dimensional organized multilayers by a layer-by-layer method.

Differential protein-DNA interactions at the promoter and enhancer regions of developmentally regulated U4 snRNA genes.

In the chicken genome there are two closely-linked genes, U4B and U4X, that code for different sequence variants of U4 small nuclear RNA (snRNA). Both genes are expressed with nearly equal efficiency in the early embryo, but U4X gene expression is specifically down-regulated relative to U4B as development proceeds. At the present time, little is known about the mechanisms that regulate differential expression of snRNA genes. We have now identified a novel chicken factor, PPBF, that binds sequence-specifically in vitro to the proximal regulatory region of the U4X gene, but not to the proximal region of the U4B gene. PPBF is itself regulated during development and may therefore be a key factor involved in differentially regulating U4X gene transcription relative to U4B. The U4X and U4B enhancers contain distinct sequence variants of two essential motifs (octamer and SPH). The Oct-1 transcription factor binds with similar affinities to both the U4X and U4B octamer motifs. However, a second essential snRNA enhancer-binding protein, SBF, has a 20- to 30-fold lower affinity for the SPH motif in the U4X enhancer than for the homologous SPH motif in the U4B enhancer. A potential role therefore exists for SBF, as well as PPBF, in the preferential down-regulation of the U4X RNA gene during chicken development.

Identification of a SPH element in the distal region of a human U6 small nuclear RNA gene promoter and characterization of the SPH binding factor in HeLa cell extracts.

Vertebrate small nuclear RNA (snRNA) gene promoters contain a distal, enhancer-like region that is composed of an octamer motif adjacent to at least one other element. Here we show that a human U6 snRNA distal region contains a SPH motif previously found in several chicken snRNA gene enhancers and the 5'-flanking region of vertebrate selenocysteine tRNA genes. SPH binding factor (SBF) was detected in either chicken or HeLa cell extracts that could bind SPH elements in a species-independent manner. Both human and chicken SBF required divalent cation to bind effectively to DNA. DNase I footprinting experiments indicated that human SBF specifically protected the human U6 SPH element. Furthermore, a SBF polypeptide of approximately 85 kDa was detected in both HeLa and chicken extracts following ultraviolet light-mediated cross-linking to human U6 or chicken U4 SPH elements. A part of the human U6 SPH element was quite sensitive to mutation, as demonstrated by both specific protein binding and transcription assays. From these data it is apparent that the distal regions of some RNA polymerase III- and RNA polymerase II-transcribed small RNA promoters are virtually identical in composition, and their mechanisms of transcriptional activation are possibly quite similar.

Effect of liposome type and membrane fluidity on drug-membrane partitioning analyzed by immobilized liposome chromatography.

Immobilized liposome chromatography (ILC) has been proven to be a useful method for the study or rapid screening of drug-membrane interactions. To obtain an adequate liposomal membrane phase for ILC, unilamellar liposomes were immobilized in gel beads by avidin-biotin binding. The retardation of 15 basic drugs on the liposome column could be converted to membrane partitioning coefficients, K(LM). The effects of small or large unilamellar liposomes and multilamellar liposomes on the drug-membrane partitioning were compared. The K(LM) values for both small and large liposomes were similar, but higher than those for the multilamellar liposomes. The basic drugs showed stronger partitioning into negatively charged liposomes than into either neutral liposomes or positively charged liposomes. The membrane fluidity of the immobilized liposomes was modulated by incorporating cholesterol into the liposomal membranes, by changing the acyl chain length and degree of unsaturation of the phospholipids, and by changing the temperature for ILC runs. Our data show that K(LM) obtained using ILC correlated well with those reported by batch studies using free liposomes. It is concluded that negatively charged or cholesterol-containing large unilamellar liposomes are suitable models for the ILC analysis of drug-membrane interactions.

Effects of gastrectomy on motility, perfusion pressure, and caerulein-induced relaxation of sphincter of Oddi in dogs.

The effects of subtotal-gastrectomy (gastrectomy) on the spontaneous motility and caerulein-induced relaxation of the sphincter of Oddi (SO) were investigated in the dog. The spontaneous motility and the response to caerulein of the SO were recorded using perfusion method. The basal perfusion pressure (5.1 +/- 0.5 cmH2O) and the frequency of phasic contractions (6.1 +/- 0.5 cycles/min, c/min) of the SO increased to 8.2 +/- 0.6 cmH2O (p < 0.05) and 9.3 +/- 0.4c/min (p < 0.05) after gastrectomy, respectively. They were observed one month after operation (7.8 +/- 0.5 cmH2O and 9.1 +/- 0.9 c/min, p < 0.05), but did not change by vagotomy with sympathectomy (vagosympathectomy). In the spontaneous motility of the SO, the motility index increased to 143.7 +/- 18.7% (p < 0.05) at 4 hrs and 135.0 +/- 9.1% (p < 0.05) at one month after gastrectomy, but did not increase after vagosympathectomy. Caerulein had an inhibitory effect on the SO motility in the normal animal 48.0 +/- 4.2%). Gastrectomy reversed to the excitatory effect from the inhibitory effect to caerulein at 4 hrs (127.6 +/- 5.3%, p < 0.05) and at one month (126.6 +/- 5.3%, p < 0.05) after operation, but not reversed by vagosympathectomy and sham gastrectomy. The excitatory response to caerulein after gastrectomy was not effected by vagosympathectomy. It is concluded that gastrectomy induced the SO dysfunction, an increase of the perfusion pressure and the frequency of phasic contractions of the SO, and a change of the response to caerulein of the SO. These alterations suggests that one of the mechanisms of the regulation of the SO motility exist as the reflex from the stomach and/or uppermost duodenum through intrinsic nervous pathways.

[Effects of gabexate mesilate (FOY) on the gallbladder, sphincter of Oddi and duodenum of the normal and gastrectomized dogs].

FOY induced a dose-dependent inhibitory response on the gallbladder, sphincter of Oddi and duodenum of normal and gastrectomized dogs, although it induced an excitatory response in some dogs. The inhibitory response was not reduced or terminated by pretreatment with atropine, guanethidine, hexamethonium or/and proglumide. The FOY-induced inhibitory response reversed to the excitatory response in the sphincter of Oddi and duodenum by pretreatment with tetrodotoxin, but not in the gallbladder. These results suggested that the FOY-induced inhibitory response of the sphincter of Oddi and duodenum was caused by stimulation of nonadrenergic noncholinergic inhibitory neurons, not by FOY-induced cholecystokinin secretion. The excitatory response was induced by direct stimulation of their smooth muscles. The inhibitory response of the gallbladder to FOY was induced by direct stimulation of the smooth muscles.

Interactions of chlorophyll a with synthesized peptide in aqueous solution.

The interactions between chlorophyll a and synthesized peptides have been studied using optical spectroscopy. Three 30-residue peptides are designed and synthesized: an amphiphilic peptide without histidine (L), an amphiphilic peptide with histidine (L/H) and a hydrophilic peptide (K/E). These peptide properties thereby allow us to examine the effect of the peptide hydrophobicity and/or histidine residue on pigment-peptide interactions. On mixing with peptides, chlorophyll a has a main absorption band in the Qy region with the maximum at 672 nm. For all three peptides, fluorescence patterns show that at a low concentration of the peptide (0.05 mM) in aqueous solution, the energy is transferred among various forms of the pigment. Only peptide L/H at high concentration (0.5 mM) in solution retains the Qy band of chlorophyll a at 672 nm, and the emission is that typically seen for the monomeric form of the pigment. The aggregation of chlorophyll a is suppressed most strongly in the presence of the peptides L/H. The results suggest that chlorophyll a is ligated to a histidine residue, located in the hydrophobic region of the peptides L/H, and in surrounded or shielded by the peptide alpha-helixes.