BMP-2 functions independently of SHH signaling and triggers cell condensation and apoptosis in regenerating axolotl limbs

Background  

Axolotls have the unique ability, among vertebrates, to perfectly regenerate complex body parts, such as limbs, after amputation. In addition, axolotls pattern developing and regenerating autopods from the anterior to posterior axis instead of posterior to anterior like all tetrapods studied to date. Sonic hedgehog is important in establishing this anterior-posterior axis of limbs in all tetrapods including axolotls. Interestingly, its expression is conserved (to the posterior side of limb buds and blastemas) in axolotl limbs as in other tetrapods. It has been suggested that BMP-2 may be the secondary mediator of sonic hedgehog, although there is mounting evidence to the contrary in mice. Since BMP-2 expression is on the anterior portion of developing and regenerating limbs prior to digit patterning, opposite to the expression of sonic hedgehog, we examined whether BMP-2 expression was dependent on sonic hedgehog signaling and whether it affects patterning of the autopod during regeneration.     

Rates of degradation of glycoproteins from normal and regenerating rat livers: A study using double isotopes

The average decay rates (half-lives) of mixed glycoproteins were measured using double isotopes of fucose and glucosamine and compared to those of mixed overall proteins measured with leucine and NaH¹⁴CO₃ in whole homogenates and plasma membranes from normal and regenerating rat livers. A large reutilization of leucine was observed under both normal and regenerating conditions. Fucose seems to be recycling most predominantly in regenerating liver, whereas glucosamine was found to be very little if not at all reutilized under both conditions. Comparison of the results obtained with NaH¹⁴CO₃ and glucosamine demonstrated that glycoproteins from normal liver homogenate are degraded at a faster rate than mixed proteins. Contrary to that of mixed proteins, the half-life of glycoproteins remains unchanged during liver regeneration, and the use of glucosamine revealed that the degradation of plasma membrane glycoproteins is identical to that found in whole homogenate under both normal and regenerating conditions. Finally, the relative degradation rates of fractionated plasma membrane proteins and glycoproteins were evaluated under the same conditions. During liver regeneration some readjustments are observed in the relative degradation rates of individual species which suggest that the synthesis and degradation of the various surface membrane glycoproteins proceed at rates that are controlled independently.     

Activity of antioxidant enzymes and secondary metabolites during in vitro regeneration of Sterculia urens

The changes in the activities of antioxidant enzymes and amounts of proteins, phenols, and flavonoids in regenerating and non-regenerating calli during organogenesis of Sterculia urens were monitored. Maximum growth of calli and the most efficient regeneration of shoots occurred on Murashige and Skoog (MS) medium supplemented with 0.5 mg dm^?3 benzylaminopurine (BAP) + 2 or 4 mg dm^?3 naphtalene acetic acid (NAA). Peroxidase (POD), catalase, and superoxide dismutase activities increased in the regenerating calli but decreased in the non-regenerating calli. Six POD isoenzymes were detected. Protein content decreased in the non-regenerating calli and increased significantly during regeneration of shoots from callus. Total phenols and flavonoids increased in the non regenerating calli. SDS-PAGE analysis revealed a role of many proteins in organogenesis.     

Typing of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> of fish and human diarrhoeal origin by outer membrane proteins and lipopolysaccharides

A study was undertaken to discriminate the strains of Aeromonas hydrophila isolated from fish and diarrhoeal samples by SDS-PAGE analysis of outer membrane proteins (OMPs) and lipopolysaccharides (LPSs). Common bands at 47 kDa positions for OMPs and at 31–38 kDa for LPSs were observed. No strain of A. hydrophila from clinical or fish samples was found identical in either OMPs or LPSs profile.     

Stability of positional identity of axolotl blastema cells in vitro

Summary Previous grafting experiments have demonstrated that cells from non-contiguous positions within developing and regenerating limbs differ in a property referred to as positional identity. The goal of this study was to determine how long the positional identity of axolotl limb blastema cells is stable during culture in vitro. We have developed an assay for posterior positional properties such that blastema cells can be cultured and then grafted into anterior positions in host blastemas, to determine if they can stimulate supernumerary digit formation. We report that posterior blastema cells are able to maintain their positional identities for at least a week in culture. In addition, we observed that blastema cells are able to rapidly degrade collagenous substrates in vitro, a property that apparently distinguishes them from limb cells of other vertebrates. These results provide information regarding the time boundaries within which the positional properties of blastema cells can be studied and manipulated in vitro. Correspondence to: S.V. Bryant     

Expression and purification of two major outer membrane proteins from <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis>

Vibrio alginolyticus, a Gram-negative bacterium, is one of Vibrio pathogens common to human and marine animals. Outer membrane proteins of bacteria play an important role during infection and induction of host immune response. In present research, two outer membrane protein genes (OmpK and OmpW) of V. alginolyticus were cloned and expressed. The open reading frames of OmpK and OmpW contain 846 bp and 645 bp, respectively, the mature proteins consist of 261 and 193 amino acid residues. At the signal peptides positions −3 to −1, the amino acids were V-M-A in OmpK and V-F-A in OmpW, which consistented with the observed sequence V-X-A of the signal peptides of transmembrane OMP. The alignment analysis indicated that both proteins were highly conserved, which could serve as surface antigens for vaccine candidates. SDS-PAGE indicated two genes over-expressed in E. coli BL21 (DE3). By affinity chromatography on Ni²⁺-nitriloaceate resin, the recombinant proteins were purified from inclusion bodies. Western blot analysis revealed that both proteins had immunoreactivity, which provided a base for further study on the evaluation of diagnostication and vaccine candidates.     

Membrane Vesicles: A Common Feature in the Extracellular Matter of Cold-Adapted Antarctic Bacteria

Many Gram-negative, cold-adapted bacteria from the Antarctic environment produce large amounts of extracellular matter, which has potential biotechnology applications. We examined the ultrastructure of extracellular matter from five Antarctic bacteria (Shewanella livingstonensis NF22^T, Shewanella vesiculosa M7^T, Pseudoalteromonas sp. M4.2, Psychrobacter fozii NF23^T, and Marinobacter guineae M3B^T) by transmission electron microscopy after high-pressure freezing and freeze substitution. All analyzed extracellular matter appeared as a netlike mesh composed of a capsular polymer around cells and large numbers of membrane vesicles (MVs), which have not yet been described for members of the genera Psychrobacter and Marinobacter. MVs showed the typical characteristics described for these structures, and seemed to be surrounded by the same capsular polymer as that found around the cells. The analysis of MV proteins from Antarctic strains by SDS-PAGE showed different banding profiles in MVs compared to the outer membrane, suggesting some kind of protein sorting during membrane vesicle formation. For the psychrotolerant bacterium, S. livingstonensis NF22^T, the growth temperature seemed to influence the amount and morphology of MVs. In an initial attempt to elucidate the functions of MVs for this psychrotolerant bacterium, we conducted a proteomic analysis on membrane vesicles from S. livingstonensis NF22^T obtained at 4 and 18°C. At both temperatures, MVs were highly enriched in outer membrane proteins and periplasmic proteins related to nutrient processing and transport in Gram-negative bacteria suggesting that MVs could be related with nutrient sensing and bacterial survival. Differences were observed in the expression of some proteins depending on incubation temperature but further studies will be necessary to define their roles and implications in the survival of bacteria in the extreme Antarctic environment.     

Regenerating neurons

We have been studying the phosphorylation of proteins of both normal and regenerating superior cervical ganglia of the rat. Here we report the incorporation of radioactive phosphate into proteins of ganglia homogenates incubated with³²P-labeled ATP under various conditions at day 3 after postganglionic axotomy. The proteins were analyzed by two-dimensional electrophoresis followed by autoradiography. Incubation in the presence of Ca²⁺ or Ca²⁺ plus cyclic AMP produced only about 20 spots corresponding to distinctly labeled proteins. This number was reduced to about five under EGTA plus cyclic AMP conditions, whereas the presence of EGTA alone suppressed the phosphorylation reaction almost totally. All these proteins fell within the narrow pI range of 4–6, whereby no qualitative differences between regenerating and control cases were observed. However, the growth-associated protein, variously designated GAP-43, B-50, F-1, and pp-46, had enhanced levels of phosphate incorporation in regenerating ganglia compared to controls. Injury also caused consistently higher levels of phosphorylation of proteins running in the position of α- and β-tubulin. Since these three proteins are major constitutents of regenerating axons, these results suggest that the changes in their phosphorylation induced by injury may be involved in the regulation of their transport.     

On the polarity of cellulose in the cell wall of Valonia

The orientation of the triclinic phase of cellulose in the cell wall of Valonia ventricosa J. Agardh was investigated by X-ray- and electron-diffraction analysis. In addition to the well-documented uniplanar-axial organization of the cell wall which requires that the a ^* axis should be always perpendicular to the wall surface, the direction of this axis was also found to be pointing outward from the plasma membrane side of the wall. This unidirectionality was persistent throughout the various layers that constitute the cell wall and also for the three microfibrillar orientations that occur in Valonia cell walls. The unidirectionality of the a ^* axis indicates, in particular, that the Valonia cellulose microfibrils are not twisted along their axis. These observations are consistent with a cellulose biosynthetic scheme where a close association exists between terminal-complex orientations and those of the cellulose microfibrils. In this context, the unidirectionality of the a ^* axis of cellulose seems to be related to the restricted mobility of the terminal complexes which are able to slide in the plasma membrane but not to rotate along their long axis.Abbreviations TC terminal complex This work was initiated during a visit of J.F.R at Grenoble in the framework of a France-Québec exchange program. J.S. was recipient of a CNRS fellowship. The diagram in Fig. 8 was kindly drawn for us by Miss Yukie Saito from the Department of Forest Products, the University of Tokyo.     

Selective Reconstitution of the Tonoplast H+-ATPase of the Crassulacean-Acid Metabolism Plant Kalanchoë daigremontiana

IA detergent removal technique was used to reconstitute solubilized tonoplast proteins of mesophyll cells of the CAM plant Kalanchoë daigremontiana into phosphatidylcholine liposomes. The proteoliposomes were able to hydrolyse ATP and to pump protons across the vesicle membrane. Both activities were inhibited by nitrate, an inhibitor of V-type ATPases. Freeze-fracture micrographs confirmed the incorporation of membrane proteins into liposomes. Increase of specific ATP-hydrolysis activity compared to solubilized tonoplast proteins and SDS-PAGE analysis of reconstituted proteins in comparison with the polypeptide pattern of the purified tonoplast H⁺-ATPase from the same plant source indicated a highly selective reconstitution of the tonoplast H⁺-ATPase.     

Peripheral nervous system myelin assembly in vitro: perturbation by the ionophore monensin

Abstract: Sciatic nerves from 13-day-old rats were incubated in vitro with [³⁵S]methionine in the presence or absence of 0.22 μM monensin and total paniculate and myelin fractions prepared. The total particulate was further subfractionated by continuous density gradient centrifugation, after which the maximal specific activities of three marker enzymes, 2′,3′-cyclic nucleotide phospho-diesterase (myelin), 5′-nucleotidase (plasma membrane), and cerebroside sulphotransferase were recovered at 0.72, 0.82, and 0.92 M sucrose, respectively. The radiolabelled proteins present in the gradient subtractions were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography, and bands corresponding to the P₀ and myelin basic proteins were identified by co-migration with unlabelled myelin marker proteins on both one-dimensional SDS-PAGE and two-dimensional nonequilibrium isoelectric focussing/SDS-PAGE systems. Following a 90-min incubation with [³⁵S]methionine, newly synthesized myelin basic proteins were recovered in fractions between 0.5 and 0.7 M sucrose; this distribution was unaltered by monensin. In contrast, the distribution of newly synthesized P₀ protein across the gradients was influenced by monensin: a bimodal distribution across the control gradients with peaks of recovery of 0.60 and 0.82 M sucrose was altered to give a single peak at an intermediate density of 0.72 M sucrose. The total proportions of newly synthesized P₀ and myelin basic proteins (MBP) present across the entire gradients were calculated from the fluorograms, and the ratio was found to be 2.8 P₀: (LBP + SBP), in both the presence and absence of the ionophore. However, only 70% and 50% of the control levels of MBP and P₀ were recovered with a purified myelin fraction after incubation with monensin. The results are discussed with reference to different intracellular transport processes for the P₀ glycoprotein and the MBP within the Schwann cell, and also to the differential compartmentation of the sites of synthesis and membrane export within the Golgi body.     

Plasma membrane proteins associated with undifferentiated and embryonicDaucus carota tissue

Summary Membranes and membrane proteins from undifferentiated cells and torpedo-stage embryos were compared. A comparison of marker enzyme profiles on linear sucrose gradients showed that the membrane vesicles obtained from 14-day-old embryos were consistently less dense than those obtained from undifferentiated carrot cells. The density of the endoplasmic reticulum, for instance, was 1.10g/cm³ in embryos and 1.12g/cm² in undifferentiated cells. Proteins and glycoproteins from endoplasmic reticulum-, Golgi apparatus-, and plasma membrane-enriched fractions were compared from undifferentiated carrot cells with 14-day-old embryos by 2D SDS-PAGE. When these two tissues were compared, extensive qualitative and quantitative changes in the steady-state endomembrane and plasma membrane proteins were observed. The plasma membrane was examined further by labeling the plasma membrane proteins with sulfosuccinimidylbiotin. Using this specific label, plasma membrane proteins of 54 kD, 41 kD, 16 kD, and 15 kD were found to be uniquely associated with the embryonic state. Conversely, a 70 kD protein and a 45 kD glycoprotein were found to be associated with only undifferentiated cells. These results demonstrate that proteins of the plasma membrane exhibit distinct changes as a result of somatic embryogenesis in carrot.Abbreviations conA concanavilin A - 2,4-D 2,4-dichlorophenoxyacetic acid - p protein - gp glycoprotein - kD kilodalton - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

von Willebrand factor D and EGF domains is an evolutionarily conserved and required feature of blastemas capable of multitissue appendage regeneration

Regenerative ability varies tremendously across species. A common feature of regeneration of appendages such as limbs, fins, antlers, and tails is the formation of a blastema—a transient structure that houses a pool of progenitor cells that can regenerate the missing tissue. We have identified the expression of von Willebrand factor D and EGF domains (vwde) as a common feature of blastemas capable of regenerating limbs and fins in a variety of highly regenerative species, including axolotl (Ambystoma mexicanum), lungfish (Lepidosiren paradoxa), and Polpyterus (Polypterus senegalus). Further, vwde expression is tightly linked to the ability to regenerate appendages in Xenopus laevis. Functional experiments demonstrate a requirement for vwde in regeneration and indicate that Vwde is a potent growth factor in the blastema. These data identify a key role for vwde in regenerating blastemas and underscore the power of an evolutionarily informed approach for identifying conserved genetic components of regeneration.     

Differential Labeling of Components in Human Erythrocyte Membranes Associated with the Transport of Glucose

The irreversible inhibition of glucose transport by 1-fluoro-2,4-dinitrobenzene (FDNB) has been used to identify membrane proteins possibly associated with glucose transport in human crythrocytes. D-Glucose was shown to enhance significantly the rate of FDNB inhibition of transport when present during the reaction, whereas cytochalasin B (CB) and D-maltose retarded this FDNB inhibition of transport. This modulation of the inhibition reaction formed the basis for a double isotopic differential labeling technique using [¹⁴C]- and [³H]FDNB followed by SDS-polyacrylamide gel electrophoresis to distinguish transport-associated polypeptides from bulk membrane dinitrophenylated proteins.

Reactions in the presence of CB or maltose revealed the presence of a differentially labeled polypeptide(s), with a molecular weight of approximately 60,000-65,000 daltons. This effect could in part be reversed in the presence of D-glucose but not L-glucose. Reactions in the presence of D-glucose resulted in two regions of differential labeling. One region was around 200,000 daltons and the other corresponded to a 90,000-dalton band.

Extraction of membrane proteins with p-chloromercuribenzene sulfonate resulted in no loss of the 60,000-dalton peak, indicating that this labeled polypeptide(s) was firmly anchored in the hydrophobic core of the membrane.

These results indicate that as many as three membrane polypeptides are differentially labeled by FDNB under conditions strongly associated with the inhibition of the glucose transport system and may be involved in the regulation of glucose transport.     

Membrane Protein Location-Dependent Regulation by PI3K (III) and Rabenosyn-5 in Drosophila Wing Cells

The class III phosphatidylinositol-3 kinase (PI3K (III)) regulates intracellular vesicular transport at multiple steps through the production of phosphatidylinositol-3-phosphate (PI(3)P). While the localization of proteins at distinct membrane domains are likely regulated in different ways, the roles of PI3K (III) and its effectors have not been extensively investigated in a polarized cell during tissue development. In this study, we examined in vivo functions of PI3K (III) and its effector candidate Rabenosyn-5 (Rbsn-5) in Drosophila wing primordial cells, which are polarized along the apical-basal axis. Knockdown of the PI3K (III) subunit Vps15 resulted in an accumulation of the apical junctional proteins DE-cadherin and Flamingo and also the basal membrane protein β-integrin in intracellular vesicles. By contrast, knockdown of PI3K (III) increased lateral membrane-localized Fasciclin III (Fas III). Importantly, loss-of-function mutation of Rbsn-5 recapitulated the aberrant localization phenotypes of β-integrin and Fas III, but not those of DE-cadherin and Flamingo. These results suggest that PI3K (III) differentially regulates localization of proteins at distinct membrane domains and that Rbsn-5 mediates only a part of the PI3K (III)-dependent processes.     

Alterations in Membrane Protein-Profile during Cold Treatment of Alfalfa

Changes in pattern of membrane proteins during cold acclimation of alfalfa have been examined. Cold acclimation for 2 to 3 days increases membrane protein content. Labeling of membrane proteins in vivo with [³⁵S]methionine indicates increases in the rate of incorporation as acclimation progresses. Cold acclimation induces the synthesis of about 10 new polypeptides as shown by SDS-PAGE and fluorography of membrane proteins labeled in vivo.     

Aluminum‐dependent dynamics of ion transport in Arabidopsis: specificity of low pH and aluminum responses

Low‐pH and Al³⁺ stresses are the major causes of poor plant growth in acidic soils. However, there is still a poor understanding of plant responses to low‐pH and Al³⁺ toxicity. Low‐pH or combined low‐pH and Al³⁺ stress was imposed in order to measure rhizosphere pH, ion fluxes, plasma membrane potential and intracellular H⁺ concentration in distal elongation and mature zones (MZs) along the longitudinal axis of Arabidopsis thaliana roots. Low‐pH stress facilitated H⁺ influx into root tissues and caused cytoplasmic acidification; by contrast, combined low‐pH/Al³⁺ treatment either decreased H⁺ influx in the distal elongation zone (DEZ) or induced H⁺ efflux in the MZ, leading to cytoplasmic alkalinization in both zones. Low‐pH stress induced an increase in rhizosphere pH in the DEZ, whereas combined low‐pH/Al³⁺ stress resulted in lower rhizosphere pH in both root zones compared with the low‐pH treatment alone. Low‐pH stress facilitated K⁺ efflux; the presence of Al³⁺ diminished K⁺ efflux or favored K⁺ influx into root tissues. In both zones, low‐pH treatment induced plasma membrane (PM) depolarization, which was significantly diminished (P≤ 0.05) when combined stresses (low‐pH/100 µM Al³⁺) were imposed. After 60 min of exposure, low pH caused PM depolarization, whereas low pH/100 µM Al³⁺ caused PM hyperpolarization. Thus, low pH and Al³⁺ toxicity differentially affect root tissues and, consequently, the rhizosphere, which might underpin the differential mechanisms of plant adaptation to these abiotic stresses.     

Proteomic analysis of regenerating mouse liver following 50% partial hepatectomy

Background

Although 70% (or 2/3) partial hepatectomy (PH) is the most studied model for liver regeneration, the hepatic protein expression profile associated with lower volume liver resection (such as 50% PH) has not yet been reported. Therefore, the aim of this study was to determine the global protein expression profile of the regenerating mouse liver following 50% PH by differential proteomics, and thereby gaining some insights into the hepatic regeneration mechanism(s) under this milder but clinically more relevant condition.

Results

Proteins from sham-operated mouse livers and livers regenerating for 24 h after 50% PH were separated by SDS-PAGE and analyzed by nanoUPLC-Q-Tof mass spectrometry. Compared to sham-operated group, there were totally 87 differentially expressed proteins (with 50 up-regulated and 37 down-regulated ones) identified in the regenerating mouse livers, most of which have not been previously related to liver regeneration. Remarkably, over 25 differentially expressed proteins were located at mitochondria. Several of the mitochondria-resident proteins which play important roles in citric acid cycle, oxidative phosphorylation and ATP production were found to be down-regulated, consistent with the recently-proposed model in which the reduction of ATP content in the remnant liver gives rise to early stress signals that contribute to the onset of liver regeneration. Pathway analysis revealed a central role of c-Myc in the regulation of liver regeneration.

Conclusions

Our study provides novel evidence for mitochondria as a pivotal organelle that is connected to liver regeneration, and lays the foundation for further studies on key factors and pathways involved in liver regeneration following 50% PH, a condition frequently used for partial liver transplantation and conservative liver resection.     

ΔμH+ dependency of in vitro protein translocation into Escherichia coli inner-membrane vesicles varies with the signal-sequence core-region composition

Signal sequences frequently contain α-helix-destabilizing amino acids in the hydrophobic core. Nuclear magnetic resonance studies on the conformation of signal sequences in membrane mimetic environments revealed that these residues cause a break in the α-helix. In the precursor of the Escherichia coli outer membrane protein PhoE (pre-PhoE), a glycine residue at position -10 (Gly^?10) is thought to be responsible for the break in the α-helix. We investigated the role of this glycine residue in the translocation process by employing site-directed mutagenesis. SDS-PAGE analysis showed drastic variations in the electrophoretic mobilities of the mutant precursor proteins, suggesting an important role of the glycine residue in determining the conformation of the signal sequence. In vivo, no drastic differences in the translocation kinetics were observed as compared with wild-type PhoE, except when a charged residue (Arg) was substituted for Gly^?10. However, the in vitro translocation of all mutant proteins into inverted inner-membrane vesicles was affected. Two classes of precursors could be distinguished. Translocation of one class of mutant proteins (Ala, Cys and Leu for Gly^?10) was almost independent of the presence of a ΔμH⁺, whereas translocation of the other class of precursors (wild type or Ser) was strongly decreased in the absence of the ΔμH⁺. Apparently, the ΔμH⁺ dependency of in vitro protein translocation varies with the signal-sequence core-region composition. Furthermore, a proline residue at position -10 resulted in a signal sequence that did not prevent the folding of the precursor in an in vitro trimerization assay.     

Selective Solubilization of Membrane Proteins Differentially Labeled by p-Chloromercuribenzenesulfonic Acid in the Presence of Sucrose

Broadbean (Vicia faba L.) leaf discs have been incubated with the slowly permeant thiol reagent [²⁰³Hg]-para-chloromercuribenzenesulfonic acid (PCMBS) in the presence or in the absence of sucrose, and the release of PCMBS-labeled proteins has been monitored in media containing various concentrations of urea, ethyleneglycol-bis-(β-aminoethyl ether)-N, N, N′, N′-tetraacetic acid (EGTA), sodium cholate, sodium dodecyl sulfate, Triton X-100, octylglucoside or (3-[3-cholamidopropyl)-dimethylammonio] 1-propane-sulfonate) (CHAPS). The proteins differentially labeled by PCMBS in the presence of sucrose which, on the basis of previous results, are assumed to include the sucrose carrier, were preferentially solubilized by 1% CHAPS, 1% octylglucoside, or 1% Triton X-100. Other PCMBS-labeled proteins (`background' proteins) could be partially removed by EGTA, urea, or 0.1% cholate. Sequential treatment by 10 mm EGTA and 1% CHAPS was found to give a fraction highly enriched in the differentially labeled proteins. Analysis of the specific activity of microsomal pellets suggests that the results obtained with leaf discs give a good account of what is occurring at the plasma membrane level. These data, which suggest that the proteins differentially labeled by PCMBS in the presence of sucrose are intrinsic membrane proteins, can be used to solubilize these proteins from microsomal fractions.