Location of nucleolar organizers in animal and plant chromosomes by means of an improved N-banding technique

With an improved N-banding technique, the location of nucleolar organizing region was determined in 27 kinds of material including mammals, a marsupial, birds, amphibians, fishes, an insect and plants. In most cases the N-bands were clearly located on certain specific regions of chromosomes, such as the secondary constriction, satellite, centromere, telomere and heterochromatic segment, while in some species they were detected as very minute bodies distributed over many chromosomes. From the available cytological and biochemical data it was suggested that the N-bands represent certain structural non-histone proteins specifically linked to nucleolar organizers in various eukaryotic chromosomes.     

Protein and cellular engineering with unnatural amino acids

Proteins are the central functional constituents in all living organisms ranging from viruses, bacteria, yeast, and plants to mammals. All of these biopolymers that are formed by natural biosynthetic pathways are composed of a genetically determined sequence of the 20 so-called natural amino acids. The physical and chemical properties of proteins are a reflection of the side chains of each of the component amino acids. However, for some purposes it would be very desireable to have amino acids with side chains of various selected physical chemical properties, such as a keto group, a crosslinker, or a NMR probe group, incorporated into the protein. Although chemical and biochemical methods for modifying amino acid moieties in proteins have been achieved, recent successes in incorporating unnatural amino acids in vivo open entirely new avenues for determining protein functions in vivo and for the creation of unnatural proteins with novel functionalities. Several examples by employing the novel activity of unnatural amino acids have shown significant roles in both basic research and biotechnology.     

Pseudomonas evades immune recognition of flagellin in both mammals and plants

The building blocks of bacterial flagella, flagellin monomers, are potent stimulators of host innate immune systems. Recognition of flagellin monomers occurs by flagellin-specific pattern-recognition receptors, such as Toll-like receptor 5 (TLR5) in mammals and flagellin-sensitive 2 (FLS2) in plants. Activation of these immune systems via flagellin leads eventually to elimination of the bacterium from the host. In order to prevent immune activation and thus favor survival in the host, bacteria secrete many proteins that hamper such recognition. In our search for Toll like receptor (TLR) antagonists, we screened bacterial supernatants and identified alkaline protease (AprA) of Pseudomonas aeruginosa as a TLR5 signaling inhibitor as evidenced by a marked reduction in IL-8 production and NF-κB activation. AprA effectively degrades the TLR5 ligand monomeric flagellin, while polymeric flagellin (involved in bacterial motility) and TLR5 itself resist degradation. The natural occurring alkaline protease inhibitor AprI of P. aeruginosa blocked flagellin degradation by AprA. P. aeruginosa aprA mutants induced an over 100-fold enhanced activation of TLR5 signaling, because they fail to degrade excess monomeric flagellin in their environment. Interestingly, AprA also prevents flagellin-mediated immune responses (such as growth inhibition and callose deposition) in Arabidopsis thaliana plants. This was due to decreased activation of the receptor FLS2 and clearly demonstrated by delayed stomatal closure with live bacteria in plants. Thus, by degrading the ligand for TLR5 and FLS2, P. aeruginosa escapes recognition by the innate immune systems of both mammals and plants.     

Noncellular nonspecific defence mechanisms of fish

Fish tissues and body fluids contain naturally occurring proteins or glycoproteins of non-immunoglobulin (Ig) nature that react with a diverse array of environmental antigens and may confer an undefined degree of natural immunity to fish. They consist of microbial growth inhibitory compounds that include “acute phase” proteins such as transferrins, caeruloplasmin, and metallothionein. Their action is simply to chelate metal ions and deprive bacteria and other parasites of essential inorganic ion sources. Both serum and cellular interferons are found in fish, and this anti-virus protein has been demonstrated mainly in salmonids during viral disease studies. Enzyme-inhibitors (α2-macroglobulin and other α-globulins) thus far detected in fish appear to be antibacterial proteinases, and are involved in the inhibition of extracellular proteases secreted by fish pathogens. Fish also possess a variety of relatively specific lytic molecules that cause cell lysis, and some of these materials are hydrolase enzymes (lysozyme, chitinase, chitobiase) whose main actions are against bacteria and fungi. In addition, mucus contains trypsin-like proteinases which destroy gram-negative bacteria. Nonspecific lysins and agglutinins against erythrocytes and other cellular antigens are found in serum, eggs, and skin mucus. The lysins, including toxins, some of which are bacteriolytic in activity, are, in their mode of action, natural or spontaneous, antibody-independent and noncomplement-mediated. In contrast, specific hemolytic antibodies (Ig), which complex with antigens, bind complement, and cause complement-mediated immune lysis, are reported to exist. The agglutinins are generally reactive toward certain sugar residues on erythrocyte or bacterial cell walls, and in most cases act as lectins or lectin-like molecules. Natural lysins and agglutinins behave in a similar way as antigen-induced antibodies or Igs, but exhibit a high degree of cross-reactions, due to the occurrence of similar carbohydrate determinants on many types of microbial cell surface. As with mammals, both C-type (calcium-dependent) and S-type (thiol-dependent) lectins are present in fish. They more resemble invertebrate lectins than those of higher animals. Fish lectins appear to play antibacterial or antifungal roles and in some instances seem to be involved in egg-sperm fusion, polyspermy prevention, and embryo development. Natural, non-Ig precipitins (e.g. α-precipitin and C-reactive proteins) are found largely, but not exclusively, in fish serum and precipitate with simple monosaccharides or long chain polysaccharides of certain stereochemistry and glycosidic linkages. Their functions remain unknown, but C-reactive protein is induced following stress-induction and exposure to inflammatory agents. Many of the above mentioned “defence” substances are present in skin mucus and possess the capacity to react with potentially infective microorganisms including parasites. Mucus thus acts as an immediate defence barrier to invasion and/or colonisation of pathogens.     

Biogenesis and functions of lipid droplets in plants: Thematic Review Series: Lipid Droplet Synthesis and Metabolism: from Yeast to Man

The compartmentation of neutral lipids in plants is mostly associated with seed tissues, where triacylglycerols (TAGs) stored within lipid droplets (LDs) serve as an essential physiological energy and carbon reserve during postgerminative growth. However, some nonseed tissues, such as leaves, flowers and fruits, also synthesize and store TAGs, yet relatively little is known about the formation or function of LDs in these tissues. Characterization of LD-associated proteins, such as oleosins, caleosins, and sterol dehydrogenases (steroleosins), has revealed surprising features of LD function in plants, including stress responses, hormone signaling pathways, and various aspects of plant growth and development. Although oleosin and caleosin proteins are specific to plants, LD-associated sterol dehydrogenases also are present in mammals, and in both plants and mammals these enzymes have been shown to be important in (steroid) hormone metabolism and signaling. In addition, several other proteins known to be important in LD biogenesis in yeasts and mammals are conserved in plants, suggesting that at least some aspects of LD biogenesis and/or function are evolutionarily conserved.     

ATP-dependent Clp Proteases in Photosynthetic Organisms— A Cut Above the Rest!

Proteases are critical regulatory factors for many metabolic cellular processes as well as being vital for degrading proteins damaged during environmental stresses. Many of those responsible for targeted protein degradation require the hydrolysis of ATP, and one class that has attracted much attention recently are the Clp proteases. They are among the best characterized proteases to date, and were the first shown to rely on an ATPase regulatory subunit possessing molecular chaperone activity, which functions both within the proteolytic complex and independently. A range of Clp proteins has been identified from many different bacteria and eukaryotes, with by far the greatest number and diversity of forms in oxygenic photobionts such as cyanobacteria and higher plants. Functionally, Clp proteins have also evolved into one of the more critical proteolytic enzymes within photobionts, and it is now somewhat of a paradox that we currently know least about Clp protease functions in the photosynthetic organisms, where they have their most important roles. This discrepancy is now being addressed, with studies on Clp protein in cyanobacteria and, in an increasing number, in higher plants.     

Proteomics of Arabidopsis redox proteins in response to methyl jasmonate

Protein redox regulation is increasingly recognized as an important switch of protein activity in yeast, bacteria, mammals and plants. In this study, we identified proteins with potential thiol switches involved in jasmonate signaling, which is essential for plant defense. Methyl jasmonate (MeJA) treatment led to enhanced production of hydrogen peroxide in Arabidopsis leaves and roots, indicating in vivo oxidative stress. With monobromobimane (mBBr) labeling to capture oxidized sulfhydryl groups and 2D gel separation, a total of 35 protein spots that displayed significant redox and/or total protein expression changes were isolated. Using LC–MS/MS, the proteins in 33 spots were identified in both control and MeJA-treated samples. By comparative analysis of mBBr and SyproRuby gel images, we were able to determine many proteins that were redox responsive and proteins that displayed abundance changes in response to MeJA. Interestingly, stress and defense proteins constitute a large group that responded to MeJA. In addition, many cysteine residues involved in the disulfide dynamics were mapped based on tandem MS data. Identification of redox proteins and their cysteine residues involved in the redox regulation allows for a deeper understanding of the jasmonate signaling networks.     

Production of outer surface protein A by Borrelia burgdorferi during transmission from infected mammals to feeding ticks is insufficient to trigger OspA seroconversion

The Lyme disease spirochete, Borrelia burgdorferi, produces two outer surface lipoproteins, OspA and OspB, that are essential for colonization of tick vectors. Both proteins are highly expressed during transmission from infected mammals to feeding ticks and during colonization of tick midguts, but are repressed when bacteria are transmitted from ticks to mammals. Humans and other infected mammals generally do not produce antibodies against either protein, although some Lyme disease patients do seroconvert and produce antibodies against OspA for unknown reasons. We hypothesized that, if such patients had been fed upon by additional ticks, bacteria moving from the patients' bodies to the feeding ticks would have produced OspA and OspB proteins, which then led to immune system recognition and antibody production. This hypothesis was tested by analyzing immune responses of infected mice following feedings by additional Ixodes scapularis ticks. However, results of the present studies demonstrate that expression of OspA and OspB by B. burgdorferi during transmission from infected mammals to feeding ticks does not trigger seroconversion.     

Proteomics of Arabidopsis redox proteins in response to methyl jasmonate

Protein redox regulation is increasingly recognized as an important switch of protein activity in yeast, bacteria, mammals and plants. In this study, we identified proteins with potential thiol switches involved in jasmonate signaling, which is essential for plant defense. Methyl jasmonate (MeJA) treatment led to enhanced production of hydrogen peroxide in Arabidopsis leaves and roots, indicating in vivo oxidative stress. With monobromobimane (mBBr) labeling to capture oxidized sulfhydryl groups and 2D gel separation, a total of 35 protein spots that displayed significant redox and/or total protein expression changes were isolated. Using LC–MS/MS, the proteins in 33 spots were identified in both control and MeJA-treated samples. By comparative analysis of mBBr and SyproRuby gel images, we were able to determine many proteins that were redox responsive and proteins that displayed abundance changes in response to MeJA. Interestingly, stress and defense proteins constitute a large group that responded to MeJA. In addition, many cysteine residues involved in the disulfide dynamics were mapped based on tandem MS data. Identification of redox proteins and their cysteine residues involved in the redox regulation allows for a deeper understanding of the jasmonate signaling networks.     

Mechanisms of lipid-body formation

Most organisms transport or store neutral lipids as lipid bodies - lipid droplets that usually are bounded by specific proteins and (phospho)lipid. Neutral-lipid bodies vary considerably in their morphology and are associated with an extremely diverse range of proteins. However, the mechanisms by which they are generated in plants, animals and microorganisms appear to share many common features: lipid bodies probably arise from microdomains of the endoplasmic reticulum (or the plasma membrane in prokaryotes) that contain lipid-biosynthesis enzymes, and their synthesis and size appear to be controlled by specific protein components.     

Structural, mechanistic and clinical aspects of MRP1

The cDNA encoding ATP-binding cassette (ABC) multidrug resistance protein MRP1 was originally cloned from a drug-selected lung cancer cell line resistant to multiple natural product chemotherapeutic agents. MRP1 is the founder of a branch of the ABC superfamily whose members (from species as diverse as plants and yeast to mammals) share several distinguishing structural features that may contribute to functional and mechanistic similarities among this subgroup of transport proteins. In addition to its role in resistance to natural product drugs, MRP1 (and related proteins) functions as a primary active transporter of structurally diverse organic anions, many of which are formed by the biotransformation of various endo- and xenobiotics by Phase II conjugating enzymes, such as the glutathione S-transferases. MRP1 is involved in a number of glutathione-related cellular processes. Glutathione also appears to play a key role in MRP1-mediated drug resistance. This article reviews the discovery of MRP1 and its relationships with other ABC superfamily members, and summarizes current knowledge of the structure, transport functions and relevance of this protein to in vitro and clinical multidrug resistance.     

Tobacco plastidial thioredoxins as modulators of recombinant protein production in transgenic chloroplasts

Thioredoxins (Trxs) are small ubiquitous disulphide proteins widely known to enhance expression and solubility of recombinant proteins in microbial expression systems. Given the common evolutionary heritage of chloroplasts and bacteria, we attempted to analyse whether plastid Trxs could also act as modulators of recombinant protein expression in transgenic chloroplasts. For that purpose, two tobacco Trxs (m and f) with different phylogenetic origins were assessed. Using plastid transformation, we assayed two strategies: the fusion and the co-expression of Trxs with human serum albumin (HSA), which was previously observed to form large protein bodies in tobacco chloroplasts. Our results indicate that both Trxs behave similarly as regards HSA accumulation, although they act differently when fused or co-expressed with HSA. Trxs-HSA fusions markedly increased the final yield of HSA (up to 26% of total protein) when compared with control lines that only expressed HSA; this increase was mainly caused by higher HSA stability of the fused proteins. However, the fusion strategy failed to prevent the formation of protein bodies within chloroplasts. On the other hand, the co-expression constructs gave rise to an absence of large protein bodies although no more soluble HSA was accumulated. In these plants, electron micrographs showed HSA and Trxs co-localization in small protein bodies with fibrillar texture, suggesting a possible influence of Trxs on HSA solubilization. Moreover, the in vitro chaperone activity of Trx m and f was demonstrated, which supports the hypothesis of a direct relationship between Trx presence and HSA aggregates solubilization in plants co-expressing both proteins.     

Molecular mechanisms of ammonium transport and accumulation in plants

The integral membrane proteins of the ammonium transporter (AMT/Rh) family provide the major route for shuttling ammonium (NH(4)(+)/NH(3)) across bacterial, archaeal, fungal and plant membranes. These proteins are distantly related to the Rh (rhesus) glycoproteins, which are absent in higher plants, but are present in many species, including bacteria and mammals. It appears that the large nitrogen requirement of plants resulted in unique strategies to acquire, capture and/or release ammonium. The biological function of plant ammonium transporters will be discussed and compared to other AMT/Rh proteins.     

Detection of and Response to Signals Involved in Host-Microbe Interactions by Plant-Associated Bacteria

Diverse interactions between hosts and microbes are initiated by the detection of host-released chemical signals. Detection of these signals leads to altered patterns of gene expression that culminate in specific and adaptive changes in bacterial physiology that are required for these associations. This concept was first demonstrated for the members of the family Rhizobiaceae and was later found to apply to many other plant-associated bacteria as well as to microbes that colonize human and animal hosts. The family Rhizobiaceae includes various genera of rhizobia as well as species of Agrobacterium. Rhizobia are symbionts of legumes, which fix nitrogen within root nodules, while Agrobacterium tumefaciens is a pathogen that causes crown gall tumors on a wide variety of plants. The plant-released signals that are recognized by these bacteria are low-molecular-weight, diffusible molecules and are detected by the bacteria through specific receptor proteins. Similar phenomena are observed with other plant pathogens, including Pseudomonas syringae, Ralstonia solanacearum, and Erwinia spp., although here the signals and signal receptors are not as well defined. In some cases, nutritional conditions such as iron limitation or the lack of nitrogen sources seem to provide a significant cue. While much has been learned about the process of host detection over the past 20 years, our knowledge is far from being complete. The complex nature of the plant-microbe interactions makes it extremely challenging to gain a comprehensive picture of host detection in natural environments, and thus many signals and signal recognition systems remain to be described.     

Common Functions or Only Phylogenetically Related? The Large Family of PLAC8 Motif-Containing/PCR Genes

PLAC8 motif-containing proteins form a large family and members can be found in fungi, algae, higher plants and animals. They include the PCR proteins of plants. The name giving PLAC8 domain was originally found in a protein residing in the spongiotrophoblast layer of the placenta of mammals. A further motif found in a large number of these proteins including several PCR proteins is the CCXXXXCPC or CLXXXXCPC motif. Despite their wide distribution our knowledge about the function of these proteins is very limited. For most of them two membrane-spanning α-helices are predicted, indicating that they are membrane associated or membrane intrinsic proteins. In plants PLAC8 motif-containing proteins have been described to be implicated in two very different functions. On one hand, it has been shown that they are involved in the determination of fruit size and cell number. On the other hand, two members of this family, AtPCR1 and AtPCR2 play an important role in transport of heavy metals such as cadmium or zinc. Transport experiments and approaches to model the 3_D structure of these proteins indicate that they could act as transporters for these divalent cations by forming homomultimers. In this minireview we discuss the present knowledge about this protein family and try to give an outlook on how to integrate the different proposed functions into a common picture about the role of PLAC8 motif-containing proteins.     

乳酸乳球菌作为黏膜免疫活载体疫苗传递抗原的研究进展

乳酸菌是人和动物肠道内的常见细菌,被公认为安全级(generally recognized as safe,GRAS)微生物。近年来,对于乳酸菌作为宿主菌表达外源蛋白或抗原的研究取得了一定进展。乳酸乳球菌(Lactococcus lactis)是乳酸菌的代表菌种,以其生长迅速、易于操作等优点成为表达外源抗原,作为黏膜免疫活载体疫苗的理想菌株。随着对乳酸乳球菌基因工程的研究逐渐深入,已发展了一系列组成型和诱导型乳酸乳球菌表达系统以及蛋白定位系统。破伤风毒素片段C、布氏杆菌L7/L12蛋白等多种病原微生物抗原已成功在乳酸乳球菌中表达,并已证明部分重组乳酸乳球菌作为黏膜免疫疫苗可以同时刺激局部黏膜免疫应答和系统免疫应答。目前,如何使活载体乳酸乳球菌以最佳方式向黏膜免疫系统提呈抗原继而诱导有效免疫反应是该领域的研究热点,也是巨大挑战。实现外源抗原在乳酸乳球菌中的准确定位及与细胞因子的共表达是未来研究的重要方向之一。乳酸乳球菌作为黏膜免疫活载体疫苗传递外源抗原具有广阔的应用前景。     

Bacterial signalling involving eukaryotic-type protein kinases

Protein Ser, Thr and Tyr kinases play essential roles in signal transduction in organisms ranging from yeast to mammals, where they regulate a variety of cellular activities. During the last few years, a number of genes that encode eukaryotic-type protein kinases have also been identified in four different bacterial species, suggesting that such enzymes are also widespread in prokaryotes. Although many of them have yet to be fully characterized, several studies indicate that eukaryotic-type protein kinases play important roles in regulating cellular activities of these bacteria, such as cell differentiation, pathogenicity and secondary metabolism. A model based on the possible coupling between two-component systems and eukaryotic-type protein kinases is proposed to explain the function of eukaryotic-type protein kinases in bacterial signalling in the light of studies in bacteria, as well as in plants and yeast. These two groups of eukaryotes possess signal-transduction pathways involving both two-component systems and eukaryotic protein kinases.