Horizontal Gene Transfer Contributed to the Evolution of Extracellular Surface Structures: The Freshwater Polyp Hydra Is Covered by a Complex Fibrous Cuticle Containing Glycosaminoglycans and Proteins of the PPOD and SWT (Sweet Tooth) Families

The single-cell layered ectoderm of the fresh water polyp Hydra fulfills the function of an epidermis by protecting the animals from the surrounding medium. Its outer surface is covered by a fibrous structure termed the cuticle layer, with similarity to the extracellular surface coats of mammalian epithelia. In this paper we have identified molecular components of the cuticle. We show that its outermost layer contains glycoproteins and glycosaminoglycans and we have identified chondroitin and chondroitin-6-sulfate chains. In a search for proteins that could be involved in organising this structure we found PPOD proteins and several members of a protein family containing only SWT (sweet tooth) domains. Structural analyses indicate that PPODs consist of two tandem β-trefoil domains with similarity to carbohydrate-binding sites found in lectins. Experimental evidence confirmed that PPODs can bind sulfated glycans and are secreted into the cuticle layer from granules localized under the apical surface of the ectodermal epithelial cells. PPODs are taxon-specific proteins which appear to have entered the Hydra genome by horizontal gene transfer from bacteria. Their acquisition at the time Hydra evolved from a marine ancestor may have been critical for the transition to the freshwater environment.     

Disrupting two Arabidopsis thaliana xylosyltransferase genes results in plants deficient in xyloglucan, a major primary cell wall component

Xyloglucans are the main hemicellulosic polysaccharides found in the primary cell walls of dicots and nongraminaceous monocots, where they are thought to interact with cellulose to form a three-dimensional network that functions as the principal load-bearing structure of the primary cell wall. To determine whether two Arabidopsis thaliana genes that encode xylosyltransferases, XXT1 and XXT2, are involved in xyloglucan biosynthesis in vivo and to determine how the plant cell wall is affected by the lack of expression of XXT1, XXT2, or both, we isolated and characterized xxt1 and xxt2 single and xxt1 xxt2 double T-DNA insertion mutants. Although the xxt1 and xxt2 mutants did not have a gross morphological phenotype, they did have a slight decrease in xyloglucan content and showed slightly altered distribution patterns for xyloglucan epitopes. More interestingly, the xxt1 xxt2 double mutant had aberrant root hairs and lacked detectable xyloglucan. The reduction of xyloglucan in the xxt2 mutant and the lack of detectable xyloglucan in the xxt1 xxt2 double mutant resulted in significant changes in the mechanical properties of these plants. We conclude that XXT1 and XXT2 encode xylosyltransferases that are required for xyloglucan biosynthesis. Moreover, the lack of detectable xyloglucan in the xxt1 xxt2 double mutant challenges conventional models of the plant primary cell wall.     

Performances, meat quality and boar taint of castrates and entire male pigs fed a standard and a raw potato starch-enriched diet

In Europe there is increasing concern about the common practice of surgical castration of piglets without anaesthesia. One possible alternative to completely avoid castration is entire male pig production. Thus, the objective of the study was to compare the growth performance, carcass characteristics, organ weights, meat quality traits, fat score and boar taint compounds in the adipose tissue of group-penned entire male pigs and castrates. Furthermore, the effect of raw potato starch (RPS) fed for 7 days prior to slaughter was determined. Pigs (n = 36) were blocked by BW into 12 blocks (3 littermates/block) and assigned to three experimental groups: surgical castrates (C); entire males (EM); and entire males offered RPS (30 g RPS/100 g diet) for 7 days prior to slaughter (EM+). Pigs had ad libitum access to the feed from 22 to 107 kg, individual feed intake was recorded daily and BW once a week. Entire males grew slower (EM: 771, EM+: 776 v. C: 830 g/day; P < 0.01), consumed less feed (EM: 1.87, EM+: 1.89 v. C: 2.23 kg/day; P < 0.01) and were more efficient (feed conversion ratio: EM: 2.42, EM+: 2.44 v. C: 2.69 kg/kg; P < 0.01) than C. Compared to C, carcass dressing percentage was lower (EM: 79.4, EM+: 79.4 v. C: 81.6%; P < 0.01) and percentage of valuable cuts was higher (EM: 57.3, EM+: 56.5 v. 52.6%; P < 0.01) in entire males. The hearts (EM: 426, EM+: 425 v. C: 378 g), kidneys (EM: 387, EM+: 378 v. C: 311 g), bulbourethral (EM: 200, EM+: 195 v. C: 7 g) and salivary glands (EM: 99, EM+: 94 v. C: 42 g) were heavier (P < 0.001) in entire males than in C. Meat quality traits did not (P > 0.05) differ among experimental groups but the adipose tissue was more unsaturated in entire males than in C as indicated by the higher fat scores (EM: 69.1, EM+: 67.2 v. C: 63.6; P < 0.01). Feeding RPS reduced (P = 0.04) the skatole tissue concentrations (expressed in μg/g lipid) in EM+ (0.22) compared to EM (0.85), whereas androstenone and indole levels were not (P 0.60) affected (EM: 1.7 and 0.10, EM+: 2.0 and 0.09, respectively). Although the current results confirmed the high efficiency of entire males compared to castrates, the observed high androstenone levels represent a major challenge to implement entire males production.     

Activity-dependent shedding of the NMDA receptor glycine binding site by matrix metalloproteinase 3: a PUTATIVE mechanism of postsynaptic plasticity

Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs). Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS.     

High-throughput functional assessment of polysaccharide-active enzymes using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry as exemplified on plant cell wall polysaccharides

Despite a wealth of sequence information on genes encoding carbohydrate-active enzymes (e.g., transferases, esterases, hydrolases), very few of these enzymes have been described in detail, particularly regarding substrate specificities. A facile and rapid method for the characterization of substrate specificities of polysaccharide-active enzymes that uses matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS) has been developed. This method has been applied to characterize a xyloglucan fucosyltransferase and a pectin methyl-esterase. Reactions were performed in liquid phase, and aliquots of the reaction mixtures were spotted on a polyvinylidene fluoride (PVDF) membrane. Reaction products were precipitated onto the membrane and cleaned by treatment with an ethanol-water mixture. Subsequently, the reaction products were hydrolyzed by specific endoglycanases, and the resulting oligosaccharides were directly analyzed onto the PVDF membrane by MALDI-TOF MS. The new method is amenable to high-throughput analysis and, thus, constitutes an emerging avenue to rapidly fill the gap in our knowledge of the specificities of polysaccharide-active enzymes.     

The nucleus together with the cytosol generates patterns of specific cellular calcium signatures in tobacco suspension culture cells

Plant cell suspension cultures respond to osmotic changes by alterations in levels of free cellular calcium. Using the aequorin recombinant method, we have measured the spatial and temporal characteristics of calcium signatures in the nucleus and the cytosol of BY-2 tobacco suspension cells challenged with hypo- or hyper-osmotic shock. We show here that the nuclear compartment contributes together with the cytosol to produce calcium signal patterns that discriminate hypo- from hyper-osmotic treatments, i.e. turgor from tension. We also demonstrate that calcium responses in the nucleus and the cytosol are differentially modulated by the strength and the nature of hyper-osmotic treatments. We conclude that qualitative and quantitative changes in the parameters of an external stimulus such as osmotic changes are converted into calcium signatures, distinctive in their temporal and subcellular characteristics, involving both the nucleus and the cytosol. Our results illustrate the versatility of calcium signaling in plant cells. In addition to the physiological 'address' of the cell, the compartmentation of the calcium signal is probably an important parameter in encoding response specificity.     

Population dose from natural radionuclides in phosphate fertilizers

Summary The natural radionuclide content of mineral phosphate fertilizers has been determined gammaspectrometrically. The investigations comprised ca. 70% of the mineral phosphate fertilizers authorized and used in the Federal Republic of Germany (FRG). At maximum, we found specific activities of 62 nCi U_nat/kg, 23 nCi²²⁶Ra/kg, 1.6 nCi Th_nat/kg and 262 nCi⁴⁰K/kg. The mean values, weighted by the percentual agricultural consumption of the main phosphate fertilizer groups in 1973/74 and related to the phosphate content, amounted to 58, 40, 2, and 584 nCi/kg P₂O₅ for U_nat,²²⁶Ra, Th_nat, and⁴⁰K respectively. This resulted in an annual distribution due to phosphate fertilizing of about 3.9 µCi U_nat, 2.7 µCi²²⁶Ra, 0.1 µCi Th_nat, and 39.9 µCi⁴⁰K per ha of arable or pasture land in 1973/74 on the average. From these values the air dose rates over agricultural areas have been estimated under extreme conservative assumptions resulting in an additional external exposure of members of the population of 0.02 mrd/a on the average and 0.4 mrd/a in the region of highest phosphate fertilizing intensity. If it is assumed that radium contained in phosphate fertilizers were completely accumulated in the soils during the last 80 years, this value would be raised to 0.3 mrd/a on the average. The occupational external radiation exposure due to natural radionuclides contained in phosphate fertilizers was estimated to be 0.1 mrd/a on the average and 2.3 mrd/a at maximum for persons working in agriculture. These estimates show that natural radionuclides in phosphate fertilizers contribute but very little to the mean terrestrial radiation exposure of the population which is 50 to 55 mrd/a in Germany. Only for the small group of persons working in fertilizer production plants or storehouses a significant increase of the external radiation exposure has to be expected which could reach a doubling of the mean natural exposure value.Dedicated to Prof. Dr. H. Muth on the occasion on his 60th birthday     

Evaluation of growth rate in adhering cell cultures using a simple colorimetric method

In this paper we present a simple colorimetric method for evaluating cell growth in adhering cell cultures. This technique is based on the staining of basophilic cellular compounds (mainly nucleic acids) with methylene blue. To check its reliability, we have compared the quantity of dye fixed by the cells with the number of cells released from the culture substratum by trypsinization, and with the total cellular protein content determined by the Lowry's method. In these experiments we have used human skin fibroblasts and human epithelial cells derived from the epithelium of the full-term umbilical cord. Like other alternative methods, the methylene blue colorimetric technique can be used in 96-well microplates and allows the rapid collection of large amounts of data. As an illustration, we report on the selection of optimal composition of culture medium for human skin fibroblasts.     

Base dynamics of nitroxide-labeled thymidine analogues incorporated into (dA-dT)n by DNA polymerase I from Escherichia coli

Nitroxide-labeled thymidine substrates (dL) for Escherichia coli DNA polymerase I (pol I) were used to synthesize spin-labeled alternating double-stranded copolymers with (dA-dT)n as a template. All dL substrates use an alkane or alkene tether substituted into the 5-position of the pyrimidine ring to link a five- or six-membered ring nitroxide to the pyrimidine base. The kinetics of dL incorporation show some tether dependence with respect to tether length and tether geometry. The electron spin resonance (ESR) spectra of (dA-dT,dL)n duplexes directly formed by polymerization with pol I are compared with the ESR spectra of (dA)n(dT,dL)n duplexes, which are obtained after annealing of nitroxide-labeled single strands with complementary unlabeled single strands. The ESR spectra indicate that nitroxide-labeled analogues with tethers short enough to let the nitroxide ring reside in the major groove are excellent reporter groups for monitoring hybridization. A small difference between the ESR line shapes of the alternating duplexes (dA-dT,dL)n and the homopolymer duplexes (dA)n(dT,dL)n containing the same dL is detectable, suggesting the presence of subtle differences in the base dynamics between both systems. Computer simulation of the ESR spectra of the (dA-dT,dL)n duplexes was successful with the same motional model reported earlier [Kao, S.-C., & Bobst, A.M. (1985) Biochemistry 24, 5465-5469]. The thymidine motion arising from tilting and torsion of base pairs and base twisting in (dA-dT)n is similar to that in (dA)n(dT)n and is of the order of 4 ns.