Photooxidation and light-induced transport of phenazine methosulfate in chromatophores of purple bacteria

The light-induced interaction of phenazine methosulfate (PMS) with chromatophores of the purple bacteria Rhodospirillum rubrum and Rhodopseudomonas sphaeroides was studied, using an ion-specific electrode. Illumination caused an initial rapid increase in the concentration of methylphenazinium cation (MP+) and a subsequent slow (1-3 min) decrease of the MP+ concentration to a low steady level. The rapid phase of the light-induced MP+ concentration change is specifically enhanced by ascorbate. The slow phase (uptake of MP+ from the medium) is stimulated on addition of valinomycin, which is known to collapse the membrane potential of energized chromatophores, and is partly inhibited by NH4Cl, which enhances the membrane potential in chromatophores. The light-induced uptake of MP+ is sharply stimulated by dibromothymoquinone. It is concluded that the initial rapid increase of the MP+ concentration in the outer medium results from the oxidation of the reduced PMS by photooxidized reaction centers. The slow decrease of the external MP+ concentration is due to active transport of MP+ into the internal space of the chromatophores via a mechanism of a chemiosmotic type. The accumulation of MP+ is directly mediated by the redox reactions of PMS at the outer and inner surfaces of the photosynthetic membrane, which are involved in cyclic electron transport.     

Molecular characterization of proteins in protein-body membrane that disappear most rapidly during transformation of protein bodies into vacuoles

During the post-germination growth of seeds, protein bodies fuse with one another and are converted to a central vacuole. To investigate this transition, protein-body membranes from dry seeds of pumpkin (Cucurbita sp.) were prepared and their protein components characterized. Five major proteins (designated MP23, MP27, MP28, MP32 and MP73) were detected in the protein-body membranes. A cDNA clone encoding both MP27 and MP32 has been isolated. The deduced precursor polypeptide was composed of a hydrophobic signal sequence, MP27 and MP32, in that order. A putative site of cleavage between MP27 and MP32 was located on the COOH-terminal side of asparagine 278, an indication that the post-translational cleavage may occur by the action of a vacuolar processing enzyme that converts proprotein precursors of seed proteins into the mature forms. Immunoelectron microscopic analysis showed that MP27 and MP32 were associated with protein-body membrane of dry pumpkin seeds. Among the five membrane proteins, MP27 and MP32 disappeared most rapidly during seedling growth. The degradation of MP27 and MP32 starts just after seed germination and proceeds in parallel with the transformation of the protein bodies into a vacuole.     

Developmental changes in plasmodesmata in transgenic tobacco expressing the movement protein of tobacco mosaic virus

Summary Cell-to-cell communication in plants occurs through plasmodesmata, cytoplasmic channels that traverse the cell wall between neighboring cells. Plasmodesmata are also exploited by many viruses as an avenue for spread of viral progeny. In the case of tobacco mosaic virus (TMV), a virally-encoded movement protein (MP) enables the virus to move through plasmodesmata during infection. We have used thin section electron microscopy and immunocytochemistry to examine the structure of plasmodesmata in transgenic tobacco plants expressing the TMV MP. We observed a change in structure of the plasmodesmata as the leaves age, both in control and MP expressing [MP(+)] plants. In addition, the plasmodesmata of older cells of MP(+) plants accumulate a fibrous material in the central cavity. The presence of the fibers is correlated with the ability to label plasmodesmata with anti-MP antibodies. The developmental stage of leaf tissue at which this material is observed is the stage at which an increase in the size exclusion limit of the plasmodesmata can be measured in MP(+) plants. Using cell fractionation and aqueous phase partitioning studies, we identified the plasma membrane and cell wall as the compartments with which the MP stably associates. The nature of the interaction between the MP and the plasma membrane was studied using sodium carbonate and Triton X-100 washes. The MP behaves as an integral membrane protein. Identifying the mechanism by which the MP associates with plasma membrane and plasmodesmata will lead to a better understanding of how the MP alters the function of the plasmodesmata.Abbreviations MP movement protein - TMV tobacco mosaic virus     

Caveolin-1β promotes the production of active human microsomal glutathione S-transferase in induced intracellular vesicles in Spodoptera frugiperda 21 insect cells

The heterologous expression in Spodoptera frugiperda 21 (Sf21) insect cells of the β isoform of canine caveolin-1 (caveolin-1β), using a baculovirus-based vector, resulted in intracellular vesicles enriched in caveolin-1β. We investigated whether these vesicles could act as membrane reservoirs, and promote the production of an active membrane protein (MP) when co-expressed with caveolin-1β. We chose hMGST1 (human microsomal glutathione S-transferase 1) as the co-expressed MP. It belongs to the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) family of integral MPs, and, as a phase II detoxification enzyme, it catalyzes glutathione conjugation of lipophilic drugs present in the lipid membranes. In addition to its pharmaceutical interest, its GST activity can be conveniently measured. The expression of both MPs were followed by Western blots and membrane fractionation on density gradient, and their cell localization by immunolabeling and transmission electron microscopy. We showed that caveolin-1β kept its capacity to induce intracellular vesicles in the host when co-expressed with hMGST1, and that hMGST1 is in part addressed to these vesicles. Remarkably, a fourfold increase in the amount of active hMGST1 was found in the most enriched membrane fraction, along with an increase of its specific activity by 60% when it was co-expressed with caveolin-1β. Thus, heterologously expressed caveolin-1β was able to induce cytoplasmic vesicles in which a co-expressed exogenous MP is diverted and sequestered, providing a favorable environment for this cargo.     

MP38 contains the membrane-embedded domain of the lens fiber gap junction protein MP70

A 70-kDa lens membrane polypeptide (MP70) is a specific component of the fiber gap junctions. The C-terminal portion of MP70 is removed by age-related proteolytic processing, leaving an N-terminal 38-kDa polypeptide (MP38) in the membrane. Membrane association and topology of MP70 and of its processed form MP38 have been studied by hydrophobic labeling with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine and phenyl isothio[14C]cyanate. Membrane-embedded segments have been identified. They are localized in the N-terminal 30-kDa portion of MP70 and MP38. The C-terminal 40-kDa portion of MP70 appears to be exposed entirely at the cytoplasmic side of the junctional membranes. Hence, potentially poreforming peptide segments in the MP70 molecule are conserved upon age-related processing to MP38.     

Molecular portrait of lens gap junction protein MP70

A 70-kDa membrane protein (MP70) is a component of the lens fiber gap junctions. Its membrane topology and its N-terminal sequence are similar to those of the connexin family of proteins. Some features of MP70 containing fiber gap junctions are, however, distinct from gap junctions in other mammalian tissues: (i) Lens connexons form crystalline arrays only after cleavage of junctional proteins in vitro. These hexagonal arrays have a periodicity of 13.6 nm which is significantly larger than the 8- 9-nm spacing of liver and heart gap junctions. (ii) Lens fiber gap junctions dissociate in low concentrations of nonionic detergent and this provides an avenue to purify MP70 directly from a membrane mixture. Isolated MP70 in the form of 17 S structures has an appearance consistent with connexon pairs. (iii) The C-terminal half of MP70 is cleaved in situ by a lens endogenous calcium-dependent protease. The processed from MP38 remains in the membrane and is abundant in the central region of the lens. A testable hypothesis for MP70 function is presented.     

cAMP-dependent protein kinase phosphorylates gap junction protein in lens cortex but not in lens nucleus

MP70 (a 70 kDa membrane protein) is a component of the gap junctions of the young fibre cells in the lens outer cortex. In the older fibres deeper in the mammalian lens (lens nucleus), MP70 is processed to MP38 by cleavage and removal of the carboxy terminal half. It is shown here that cortical MP70, and its derivative MP64, can be phosphorylated with cAMP-dependent protein kinase. In contrast, MP38 from the lens nucleus is not phosphorylated by the enzyme. Proteolytic processing and this lens region specific phosphorylation are relevant for the future development of functional assays for lens gap junctions.     

Analysis of an equivalent electric circuit of the hepatocyte during stopping and restoration of the blood flow

An analysis of an electric scheme of hepatocyte under conditions of stopping and restoring the blood flow taking into account the morphological and biochemical characteristics of the normal and injured liver was carried out. It is shown that the fast phase of restoring the membrane potential (MP) when restoring the blood flow following ischemia is stipulated by restoring extracellular concentrations of potential-generating ions; and slow restoring of the normal liver MP is connected with the phase of potassium efflux hyperpolarization. The injured liver MP stability to ischemia is higher because of the transmembrane ion gradient decrease before ischemia and next effect of depolarisation of passive currents weakening under conditions of closed extracellular space, and total ATPase activity decrease, i.e. a more economical ATP expenditure by the cell at ischemia.     

Viability and membrane potential analysis of Bacillus megaterium cells by impedance flow cytometry

Single cell analysis is an important tool to gain deeper insights into microbial physiology for the characterization and optimization of bioprocesses. In this study a novel single cell analysis technique was applied for estimating viability and membrane potential (MP) of Bacillus megaterium cells cultured in minimal medium. Its measurement principle is based on the analysis of the electrical cell properties and is called impedance flow cytometry (IFC). Comparatively, state-of-the-art fluorescence-based flow cytometry (FCM) was used to verify the results obtained by IFC. Viability and MP analyses were performed with cells at different well-defined growth stages, focusing mainly on exponential and stationary phase cells, as well as on dead cells. This was done by PI and DiOC(2)(3) staining assays in FCM and by impedance measurements at 0.5 and 10 MHz in IFC. In addition, transition growth stages of long-term cultures and agar plate colonies were characterized with both methods. FCM and IFC analyses of all experiments gave comparable results, quantitatively and qualitatively, indicating that IFC is an equivalent technique to FCM for the study of physiological cell states of bacteria.     

Design and characterization of MP0250, a tri-specific anti-HGF/anti-VEGF DARPin® drug candidate

MP0250 is a multi-domain drug candidate currently being tested in clinical trials for the treatment of cancer. It comprises one anti-vascular endothelial growth factor-A (VEGF-A), one anti-hepatocyte growth factor (HGF), and two anti-human serum albumin (HSA) DARPin® domains within a single polypeptide chain. While there is first clinical validation of a single-domain DARPin® drug candidate, little is known about DARPin® drug candidates comprising multiple domains. Here, we show that MP0250 can be expressed at 15 g/L in soluble form in E. coli high cell-density fermentation, it is stable in soluble/frozen formulation for 2 years as assessed by reverse phase HPLC, it has picomolar potency in inhibiting VEGF-A and HGF in ELISA and cellular assays, and its domains are simultaneously active as shown by surface plasmon resonance. The inclusion of HSA-binding DARPin® domains leads to a favorable pharmacokinetic profile in mouse and cynomolgus monkey, with terminal half-lives of ～ 30 hours in mouse and ～ 5 days in cynomolgus monkey. MP0250 is thus a highly potent drug candidate that could be particularly useful in oncology. Beyond MP0250, the properties of MP0250 indicate that multi-domain DARPin® proteins can be valuable next-generation drug candidates.     

Structural studies of lens fiber junction protein MP26 by cyanogen bromide cleavage

The lens fiber-cell plasma membrane MP26 from chick, bovine, and human lenses yielded identical cyanogen bromide peptide maps, confirming the essential conservation of structure in the junction protein of vertebrate lens fiber cells. Immunoblot analyses of the cyanogen bromide peptide maps of human lens MP26 and of its age-dependent proteolytic product MP22 confirmed that MP22 is a derivative of MP26. The findings in this study are the first consistent with the positioning of the methionine residues in lens MP26 as predicted by its cDNA-derived sequence.     

Interaction between the movement protein of barley yellow dwarf virus and the cell nuclear envelope: role of a putative amphiphilic alpha-helix at the N-terminus of the movement protein

The open reading frame 4 (ORF 4) gene product of barley yellow dwarf virus (BYDV) may act as a movement protein (MP) by assisting the transport of viral genomic RNA across the nuclear envelope (NE) of host plant cells. To investigate interactions between BYDV MP and the NE, wild-type and mutant open reading frame (ORF 4)-green fluorescent protein (GFP) fusion cistrons were expressed in insect cells. A fusion protein expressed by the wild-type ORF 4-GFP cistron associated with the NE and caused protrusions from its surface. The fusion protein expressed by the mutant ORF 4-GFP cistron lacked a putative amphiphilic alpha-helix at its N-terminus and although associating with the NE, showed decreased levels of protrusions. A peptide homologue of this putative alpha-helix induced an increase of 7 degrees C in the phase transition temperature of dimyrystoyl phosphatidylserine (DMPS) membranes, accompanied by a decrease in membrane fluidity, but exhibited no significant interaction with either dimyristoyl phosphatidylcholine (DMPC) or dimyristoyl phosphatidylethanolamine (DMPE) membranes. These results strongly support the view that BYDV MP may interact with the NE to help transport viral genomic RNA into the nuclear compartment. This function of BYDV MP appears to involve protrusions on the surface of the NE and may require the presence of an N-terminal amphiphilic alpha-helix, which is speculated to destabilize membranes, thereby assisting the entry of BYDV-GAV into the nuclear compartment.     

Gross and cellular analysis of 6-mercaptopurine-induced cleft palate in hamster

The present study analyzes the morphological, histochemical, and ultrastructural aspects of the pathogenesis of 6-mercaptopurine (6MP)-induced cleft palate in hamster fetuses. Gross and light microscopic observations indicated that 6MP stunts the growth of vertical palatal shelves and thus induces cleft palate. Ultrastructural analysis showed that, in contrast to controls, 6MP-induced alterations were first seen in the mesenchymal cells 24 hr after drug administration. The initial alterations were characterized by swelling of the nuclear membrane. During the next 12 hr, lysosomes were seen first in the mesenchymal cells and then in the cells of the medial edge epithelium (MEE) of the developing palatal primordia. The appearance of lysosomes was temporally abnormal and was interpreted as a sublethal response to 6MP treatment. Subsequently, the nuclear alterations and the lysosomes diminished; and 48 hr after 6MP administration, they were absent from the palatal tissues. Ninety hours after 6MP administration, unlike the controls (in which the palatal shelves were already fused), changes were seen at the epithelial-mesenchymal interface in the developing cleft palatal shelves. These changes were characterized by breakdown of the basal lamina and epithelial-mesenchymal contacts. Eventually, at term, the MEE of the vertical shelf stratified. It was suggested that 6MP affected cytodifferentiation in the palatal tissues during the critical phase of early vertical shelf development and thereby induced cleft palate.     

Outer Membrane Protein F Stabilised with Minimal Amphipol Forms Linear Arrays and LPS-Dependent 2D Crystals

Amphipols (APol) are polymers which can solubilise and stabilise membrane proteins (MP) in aqueous solutions. In contrast to conventional detergents, APol are able to keep MP soluble even when the free APol concentration is very low. Outer membrane protein F (OmpF) is the most abundant MP commonly found in the outer membrane (OM) of Escherichia coli. It plays a vital role in the transport of hydrophilic nutrients, as well as antibiotics, across the OM. In the present study, APol was used to solubilise OmpF to characterize its interactions with molecules such as lipopolysaccharides (LPS) or colicins. OmpF was reconstituted into APol by the removal of detergents using Bio-Beads followed by size-exclusion chromatography (SEC) to remove excess APol. OmpF/APol complexes were then analysed by SEC, dynamic light scattering (DLS) and transmission electron microscopy (TEM). TEM showed that in the absence of free APol–OmpF associated as long filaments with a thickness of ~6 nm. This indicates that the OmpF trimers lie on their sides on the carbon EM grid and that they also favour side by side association. The formation of filaments requires APol and occurs very rapidly. Addition of LPS to OmpF/APol complexes impeded filament formation and the trimers form 2D sheets which mimic the OM. Consequently, free APol is undoubtedly required to maintain the homogeneity of OmpF in solutions, but ‘minimum APol’ provides a new phase, which can allow weaker protein–protein and protein–lipid interactions characteristic of native membranes to take place and thus control 1D–2D crystallisation.     

MP20, the second most abundant lens membrane protein and member of the tetraspanin superfamily,joins the list of ligands of galectin-3

Background  

Although MP20 is the second most highly expressed membrane protein in the lens its function remains an enigma. Putative functions for MP20 have recently been inferred from its assignment to the tetraspanin superfamily of integral membrane proteins. Members of this family have been shown to be involved in cellular proliferation, differentiation, migration, and adhesion. In this study, we show that MP20 associates with galectin-3, a known adhesion modulator.     

Role of P30 in replication and spread of TMV

The P30 movement protein (MP) of tobacco mosaic virus is essential for distribution of sites of replication within infected cells and for cell–cell spread of infection. MP is an integral membrane protein and in early and mid-stages of infection causes severe disruption of the cortical endoplasmic reticulum (ER). MP also associates with microtubules, and in late stages is targeted for degradation by the 26S proteosome. During these stages, the ER regains its normal pre-infection configuration. Viral RNA is associated with ER and microtubules in the presence of MP. The MP is phosphorylated and mutation of the phosphorylated amino acid reduced association of MP with the ER, plasmodesmata, and microtubules, and altered the stability of the MP. The nature of the association of MP with vRNA and ER and microtubules, and the role of phosphorylation of MP in each of these functions, if any, remains to be determined.     

Membrane potential of muscle fibers exposed to corticotropin and hydrocortisone]

The effect of a single intraperitoneal administration of corticotropin (1 unit) and hydrocortisone (5 mg) per 100 g of a body weight on the membrane potential (MP) as well as on the response rate of miniature end plate potentials (RRMEPP) of musculus soleus fibres of various polarization levels has been investigated in rats. It is shown that administration of corticotropin does not change the MP value, while that of hydrocortisone elicits its increase at the low initial polarization level of the muscle fibre membrane and its decrease at the high level. Hydrocortisone administration does not change the MP value at normal levels of fibre polarization. Corticotropin having been administered, RRMEPP of fibres both with high MP levels and with low ones has increased. Fibres with normal polarization also show a tendency to increase. Administration of hydrocortisone has induced a substantial increase of RRMEPP in fibres with high polarization levels within 45 min, while PRMEPP of fibres with normal polarization levels increased within first 5 min., and that of fibres with low levels of polarization remained unchanged.     

小麦不同蘖位叶片出生的两段线性模式及其品种、播期效应

叶片出生动态是小麦生长发育进程及其协调状况的重要表现,研究发现,小科叶片出生与播后累积ＧＤＤ（ｆｇｒｏｗｉｎｇ ｄｅｇｒｅｅ ｄａｙｓ ａｆｔｅｒ ｓｏｗｉｎｇ）的关系遵循两段（阶段Ⅰ快于阶段Ⅱ）线性模式,护颖分化期为两段模式的分界点,这一规律在正常发育的冬性和春性品种的７主茎及分蘖中表现一致,冬性品种播期１（９月３０日）、播期３（３月２日）的主茎及冬、春性品种各播期的Ｔ３分蘖,因生长发育异常而”     

Structure prediction of membrane proteins

There is a large gap between the number of membrane protein (MP) sequences and that of their decoded 3D structures, especially high-resolution structures, due to difficulties in crystal preparation of MPs. However, detailed knowledge of the 3D structure is required for the fundamental understanding of the function of an MP and the interactions between the protein and its inhibitors or activators. In this paper, some computational approaches that have been used to predict MP structures are discussed and compared.     

Use of tetrazolium salts for electron transport studies in meningopneumonitis. I. Reduced nicotinamide adenine dinucleotide system

Allen, Emma G. (Downstate Medical Center, Brooklyn, N.Y.). Use of tetrazolium salts for electron transport studies in meningopneumonitis. I. Reduced nicotinamide adenine dinucleotide system. J. Bacteriol. 90:1505-1512. 1965.-Purified preparations of meningopneumonitis virus (MP) prepared from allantoic fluids of infected chick embryo reduce several tetrazolium salts in the presence of reduced nicotinamide adenine dinucleotide under both aerobic and anaerobic conditions. The pattern of reduction by MP differs from that seen in normal allantoic membrane homogenates, and is inhibited by several cations but not by KCN, atabrine, amytal, antimycin A, or 2,3-dimercaptopropanol (BAL). The reduction of cytochrome c by purified preparations of MP differs from its reduction of tetrazolium salts in that the cytochrome reaction is completely inhibited by BAL and partially inhibited by amytal, atabrine, and antimycin A. The cytochrome reductase of normal allantoic membrane preparations is completely inhibited by each of these compounds.