Leukocyte Adhesion—an Integrated Molecular Process at the Leukocyte Plasma Membrane

Leukocyte adhesion is of pivotal functional importance, because most leukocyte functions depend on cell–cell contact. It must be strictly controlled, both at the level of specificity and strength of interaction, and therefore several molecular systems are involved. The most important leukocyte adhesion molecules are the selectins, the leukocyte-specific ₂-integrins and the intercellular adhesion molecules. The selectins induce an initial weak contact between cells, whereas firm adhesion is achieved through integrin–intercellular adhesion molecular binding. Although studies during the past twenty years have revealed several important features of leukocyte adhesion much is still poorly understood, and further work dealing with several aspects of adhesion is urgently needed. In this short essay, we review some recent developments in the field.     

Diversity of fungus‐growing termites (Macrotermes) and their fungal symbionts (Termitomyces) in the semiarid Tsavo Ecosystem,Kenya

Fungus‐growing termites of the subfamily Macrotermitinae together with their highly specialized fungal symbionts (Termitomyces) are primary decomposers of dead plant matter in many African savanna ecosystems. The termites provide crucial ecosystem services also by modifying soil properties, translocating nutrients, and as important drivers of plant succession. Despite their obvious ecological importance, many basic features in the biology of fungus‐growing termites and especially their fungal symbionts remain poorly known, and no studies have so far focused on possible habitat‐level differences in symbiont diversity across heterogeneous landscapes. We studied the species identities of Macrotermes termites and their Termitomyces symbionts by excavating 143 termite mounds at eight study sites in the semiarid Tsavo Ecosystem of southern Kenya. Reference specimens were identified by sequencing the COI region from termites and the ITS region from symbiotic fungi. The results demonstrate that the regional Macrotermes community in Tsavo includes two sympatric species (M. subhyalinus and M. michaelseni) which cultivate and largely share three species of Termitomyces symbionts. A single species of fungus is always found in each termite mound, but even closely adjacent colonies of the same termite species often house evolutionarily divergent fungi. The species identities of both partners vary markedly between sites, suggesting hitherto unknown differences in their ecological requirements. It is apparent that both habitat heterogeneity and disturbance history can influence the regional distribution patterns of both partners in symbiosis.     

Modification of cellulose fiber surfaces by use of a lipase and a xyloglucan endotransglycosylase

A strategy for the modification of cellulose fiber surfaces was developed that used the ability of Candida antarctica lipase B (CALB) to acylate carbohydrates with high regioselectivity, combined with the transglycosylating activity of the Populus tremula x P. tremuloides xyloglucan endotransglycosylase 16A (PttXET16A). Xyloglucan oligosaccharides (XGOs) prepared from tamarind xyloglucan were acylated with CALB as a catalyst and vinyl stearate or gamma-thiobutyrolactone as acyl donors to produce carbohydrate molecules with hydrophobic alkyl chains or reactive sulfhydryl groups, respectively. The modified XGOs were shown to act as glycosyl acceptors in the transglycosylation reaction catalyzed by PttXET16A and could therefore be incorporated into high M(r) xyloglucan chains. The resulting xyloglucan molecules exhibited a high affinity for cellulose surfaces, which enabled the essentially irreversible introduction of fatty acid esters or thiol groups to cellulose fibers.     

Body mass prediction from stature and bi-iliac breadth in two high latitude populations, with application to earlier higher latitude humans

Previous studies have indicated that body mass can be estimated from stature and bi-iliac (maximum pelvic) breadth with reasonable accuracy in modern humans, supporting the use of this method to estimate body mass in earlier human skeletal samples. However, to date the method has not been tested specifically on high latitude individuals, whose body form in some ways more closely approximates that of earlier higher latitude humans (i.e., large and broad-bodied). In this study, anthropometric data for 67 Alaskan Inupiat and 54 Finnish adults were used to test the stature/bi-iliac body mass estimation method. Both samples are very broad-bodied, and the Finnish sample is very tall as well. The method generally works well in these individuals, with average directional biases in body mass estimates of 3% or less, except in male Finns, whose body masses are systematically underestimated by an average of almost 9%. A majority of individuals in the total pooled sample have estimates to within +/-10% of their true body masses, and more than three-quarters have estimates to within +/-15%. The major factor found to affect directional bias is shoulder to hip breadth (biacromial/bi-iliac breadth). Male Finns have particularly wide shoulders, which may in part explain their systematic underestimation. New body mass estimation equations are developed that include the new data from this study. When applied to a sample of earlier (late middle Pleistocene to early Upper Paleolithic) higher latitude skeletal specimens, differences between previous and new body estimates are small (less than 2%). However, because the Finns significantly extend the range of morphological variation beyond that represented in the original world-wide reference sample used in developing the method, thereby increasing its generality, it is recommended that these new formulas be used in subsequent body mass estimations.     

Secretome profiling of Propionibacterium freudenreichii reveals highly variable responses even among the closely related strains

This study compared the secretomes (proteins exported out of the cell) of Propionibacterium freudenreichii of different origin to identify plausible adaptation factors. Phylosecretomics indicated strain‐specific variation in secretion of adhesins/invasins (SlpA, InlA), cell‐wall hydrolysing (NlpC60 peptidase, transglycosylase), protective (RpfB) and moonlighting (DnaK, GroEL, GaPDH, IDH, ENO, ClpB) enzymes and/or proteins. Detailed secretome comparison suggested that one of the cereal strains (JS14) released a tip fimbrillin (FimB) in to the extracellular milieu, which was in line with the electron microscopy and genomic analyses, indicating the lack of surface‐associated fimbrial‐like structures, predicting a mutated type‐2 fimbrial gene cluster (fimB‐fimA‐srtC2) and production of anchorless FimB. Instead, the cereal strain produced high amounts of SlpB that tentatively mediated adherent growth on hydrophilic surface and adherence to hydrophobic material. One of the dairy strains (JS22), producing non‐covalently bound surface‐proteins (LspA, ClpB, AraI) and releasing SlpA and InlA into the culture medium, was found to form clumps under physiological conditions. The JS22 strain lacked SlpB and displayed a non‐clumping and biofilm‐forming phenotype only under conditions of increased ionic strength (300 mM NaCl). However, this strain cultured under the same conditions was not adherent to hydrophobic support, which supports the contributory role of SlpB in mediating hydrophobic interactions. Thus, this study reports significant secretome variation in P. freudenreichii and suggests that strain‐specific differences in protein export, modification and protein–protein interactions have been the driving forces behind the adaptation of this bacterial species.     

In Planta Localization of Stilbenes within Picea abies Phloem

Phenolic stilbene glucosides (astringin, isorhapontin, and piceid) and their aglycons commonly accumulate in the phloem of Norway spruce (Picea abies). However, current knowledge about the localization and accumulation of stilbenes within plant tissues and cells remains limited. Here, we used an innovative combination of novel microanalytical techniques to evaluate stilbenes in a frozen-hydrated condition (i.e. in planta) and a freeze-dried condition across phloem tissues. Semiquantitative time-of-flight secondary ion-mass spectrometry imaging in planta revealed that stilbenes were localized in axial parenchyma cells. Quantitative gas chromatography analysis showed the highest stilbene content in the middle of collapsed phloem with decreases toward the outer phloem. The same trend was detected for soluble sugar and water contents. The specimen water content may affect stilbene composition; the glucoside-to-aglycon ratio decreased slightly with decreases in water content. Phloem chemistry was correlated with three-dimensional structures of phloem as analyzed by microtomography. The outer phloem was characterized by a high volume of empty parenchyma, reduced ray volume, and a large number of axial parenchyma with porous vacuolar contents. Increasing porosity from the inner to the outer phloem was related to decreasing compactness of stilbenes and possible secondary oxidation or polymerization. Our results indicate that aging-dependent changes in phloem may reduce cell functioning, which affects the capacity of the phloem to store water and sugar, and may reduce the defense potential of stilbenes in the axial parenchyma. Our results highlight the power of using a combination of techniques to evaluate tissue- and cell-level mechanisms involved in plant secondary metabolite formation and metabolism.The bark of conifers has anatomically and chemically integrated defense strategies that are either constitutive (i.e. continuously produced) or inducible (i.e. activated as a response to insect or pathogen attack; Krokene, 2015). Many defense traits exist in both forms (Franceschi et al., 2005). For example, axial phloem parenchyma cells (or polyphenolic parenchyma) are critical in conifer bark defense. These cells regularly form in Pinaceae during annual phloem formation (Franceschi et al., 1998, 2000; Krekling et al., 2000; Jyske et al., 2015) but also are produced on invasion (Franceschi et al., 2005; Krokene, 2015). In Norway spruce (Picea abies) phloem, axial parenchyma forms distinctive, continuous tangential sheets across conducting (i.e. noncollapsed) and nonconducting (i.e. collapsed) tissue.Pioneering studies using microscopy with different dye agents and autofluorescence showed that the large vacuole is a special feature of the axial phloem parenchyma that contains phenolic substances (i.e. phenolic bodies; Franceschi et al., 1998). Microscopic imaging techniques also showed that polyphenolic content is highly dynamic (Franceschi et al., 1998, 2000, 2005) and changes seasonally (Krekling et al., 2000). Within the last 5 years, progress in laser microdissection (LMD) has facilitated the sampling of individual tissues and cells, providing information about the exact chemical composition of phenolic content. Li et al. (2012) used LMD to show that the axial parenchyma is the main site of phenolic accumulation in spruce bark, including that of stilbene compounds.Stilbenes are secondary metabolites that are composed of two phenol moieties linked by a C₂ bridge. These compounds are derived from the phenylpropanoid pathway, in which the last steps of biosynthesis are catalyzed by stilbene synthase (Chong et al., 2009). There is increasing interest in these antioxidant, antibacterial, and antiinflammatory compounds for use in healthy human diets, therapeutic approaches, and as protective agents in materials sciences (Shibutani et al., 2004; Metsämuuronen and Siren, 2014; Reinisalo et al., 2015; Hedenström et al., 2016; Sirerol et al., 2016). The tetrahydroxystilbene glucosides trans-astringin (3,3ʹ,4ʹ,5-tetrahydroxystilbene 3-O-β-d-glucoside) and trans-isorhapontin (3,4ʹ,5-trihydroxy-3ʹ-methoxystilbene 3-O-β-d-glucoside) are the most abundant constitutive stilbene compounds of Norway spruce, while the trihydroxystilbene glucoside trans-piceid (resveratrol 3-O-β-glucoside) and stilbene aglycons (i.e. without the sugar moiety) are less abundant. Stilbene synthesis in spruce probably proceeds through the formation of resveratrol (i.e. aglycon of piceid) followed by further modifications (i.e. hydroxylation, O-methylation, and O-glycosylation) to yield tetrahydroxystilbene glucosides (Hammerbacher et al., 2011). Stilbenes are assumed to provide protection against a wide variety of environmental stressors (Franceschi et al., 2005; Witzell and Martin, 2008; Chong et al., 2009). Stilbenes appear to contribute to antifungal defense in spruce (Hammerbacher et al., 2011, 2013). The fungal inoculation of spruce bark with the blue-stain fungus Endoconidiophora polonica (previously named Ceratocystis polonica; de Beer et al., 2014) causes astringin levels to decrease, in parallel with increasing dimeric stilbene glucoside levels in the LMD-isolated axial phloem parenchyma (Li et al., 2012) or increasing levels of corresponding aglycons in bulk tissue (Viiri et al., 2001). During the annual formation of phloem in Norway spruce, the accumulation of stilbene glucosides inside the newest, LMD-isolated phloem ring is preceded by the formation and cellular development of a new band of axial parenchyma (Jyske et al., 2015). These observations strongly indicate that the inducible and constitutive stilbene compounds of spruce phloem are both stored and synthesized in the axial parenchyma.New mass spectrometry imaging techniques provide significant improvements in the mapping of plant metabolites (Briggs and Seah, 1993; Vickerman and Briggs, 2001; Burrell et al., 2007; Cha et al., 2008; Lee et al., 2012; Bjarnholt et al., 2014; Aoki et al., 2016). To elucidate the synthesis, distribution, and metabolism of secondary plant metabolites, it is essential to gather positional information about them in a living state, as pretreatment of specimens, such as drying, may change the distribution and concentration features of soluble chemicals (Metzner et al., 2008; Li et al., 2012; Kuroda et al., 2013). In this study, we used a unique system of time-of-flight secondary ion mass spectrometry and scanning electron microscopy connected with a cryo-shuttle (cryo-TOF-SIMS/SEM) to study the localization and accumulation patterns of stilbenes within cells and tissues of phloem. This system has been developed to study chemical distributions at high-spatial resolution (1 µm) directly from the surfaces of plant specimens in a frozen-hydrated state (i.e. in planta) representing living tissues (Kuroda et al., 2013; Aoki et al., 2016). Time-of-flight secondary ion mass spectrometry (TOF-SIMS) directly detects organic and inorganic compounds on the specimen surface over a broad mass-to-charge ratio (m/z) range by mass spectrometry with high chemical sensitivity. Specimen surface morphology is visualized by the detection of total secondary ion content. The quality of cellular integrity may be further observed by scanning electron microscopy connected with a cryo-shuttle (cryo-SEM) imaging of the frozen surface of the same specimen. The cryo-TOF-SIMS/SEM system has still rarely been applied to the analysis of plant physiology (Metzner et al., 2008, 2010; Iijima et al., 2011; Kuroda et al., 2013; Aoki et al., 2016).Mass spectrometer imaging techniques consist of an ionizer and a mass analyzer. In the TOF-SIMS system, secondary ion mass spectrometry is used as an ionizer and time-of-flight as a mass analyzer. In another mainstream imaging mass spectrometry technique, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), matrix-assisted laser desorption/ionization is used as ionizer. Compared with TOF-SIMS, MALDI-MS is more quantitative and has high-M_r acceptance, but the resolution of MALDI-MS is not high enough for cell-level detection (Aoki et al., 2016). Instead, the spatial resolution of TOF-SIMS is superior to focus on cell functions. The disadvantage of TOF-SIMS is that the ionization and fragmentation phenomenon may be affected by the matrix effect, causing some degree of uncertainty. However, when time-of-flight secondary ion mass spectrometry connected with a cryo-shuttle (cryo-TOF-SIMS) is used in combination with quantitative gas chromatography, it is very powerful to study the positional and temporal distributions of metabolites within living plants.To complement TOF-SIMS analysis, we applied quantitative chemical microanalysis methods to study the amounts of stilbene glucosides and to correlate those with the amounts of total extractives, monosaccharides and disaccharides, and water across phloem and bark. The methods include tangential cryo-sectioning of tissues and their chemical microanalysis by gas chromatography with flame-ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS).To combine the chemical information with phloem morphology, the cellular and subcellular features of the axial phloem parenchyma were analyzed by three-dimensional (3D) synchrotron radiation microtomography (µCT). µCT is a prominent tool that has gained popularity for 3D analysis of xylem structure and physiology (Brodersen, 2013; Cochard et al., 2015), but only recently has it been applied to the 3D analysis of phloem (Jyske et al., 2015). This method offers advantages over traditional light microscopic approaches, as high-throughput data at the submicrometer level can be produced from significantly larger tissue volumes. The data allow for representative volumetric analysis of cellular distributions along with 3D visualization of subcellular features.In this study, we used a novel combination of cutting-edge techniques to analyze in parallel (1) in planta cellular localization and accumulation of stilbene glucosides across phloem and bark by semiquantitative cryo-TOF-SIMS/SEM; (2) tissue-level quantitative amounts of stilbene glucosides, total extractives, and monosaccharides and disaccharides across phloem and bark by tangential cryo-sectioning and GC-FID and GC-MS; (3) 3D cell abundance distributions across phloem and bark by µCT; and (4) variation in water content across phloem and bark (Fig. 1).Open in a separate windowFigure 1.Schematic presentation of the specimen structure and preparation for different analyses. Sample blocks were taken from living tree stem (A) or stem discs (B) at 1.3 m on the stem. The blocks (C) containing outer bark (periderm), phloem, cambium, and part of the outermost xylem ring (D; transverse view of phloem and bark) were further divided into subblocks (1–3; C and E). Subblocks 1 and 2 were quick frozen, and subblock 3 was fixed chemically. Subblock 1 was used for the direct chemical mapping of stilbenes across the phloem from the cambium to the outer bark (i.e. semiquantitative analysis of stilbene localization and accumulation across transverse and radial surfaces [purple] of the tissue block by TOF-SIMS; E-1). To obtain quantitative data on the amounts of stilbenes, other extractives, and carbohydrates across phloem and bark, tangential cryo-sections (250 or 450 µm each; cut slices illustrated with purple in E-2) were cut across subblock 2 and directed for chemical microanalysis by GC-FID (E-2). Subblock 3 was divided into four to six zones, and from each zone, small cuboids (illustrated with purple in E-3) were cut and directed for morphological analysis of phloem by phase-contrast µCT (E-3). Water content across the phloem and bark was analyzed from separate fresh blocks, which were further cut tangentially into thin sections. Black arrows indicate the radial direction from the cambium toward the outer bark. Purple areas show the analyzed locations of each subblock (E). Note that schematic drawings are not to scale.