Cost analysis of seedling supply systems adapted for mechanized tree planting: a case study from southern Sweden

Because today's tree planting machines do a good job silviculturally, the Nordic forest sector is interested in finding ways to increase the planting machines' productivity. Faster seedling reloading increases machine productivity, but that solution might require investments in specially designed seedling packaging. The objective of our study was to compare the cost-efficiency of cardboard box concepts that increase the productivity of tree planting machines with that of today's two most common seedling packaging systems in southern Sweden. We modelled the total cost of these five different seedling packaging systems using data from numerous sources including manufacturers, nurseries, contractors, and forest companies. Under these southern Swedish con-ditions, the total cost of cardboard box concepts that increase the productivity of intermittently advancing tree planting machines was higher than the cost of the cultivation tray system (5-49% in the basic scenario). However, the conceptual packaging system named ManBox_fast did show promise, especially with increasing primary transport distances and increased planting machine productivities and hourly costs. Thus, our results show that high seedling packing density is of fundamental importance for cost-efficiency of cardboard box systems designed for mechanized tree planting. Our results also illustrate how different factors in the seedling supply chain affect the cost-efficiency of tree planting machines. Consequently, our results underscore that the key development factor for mechanized tree planting in the Nordic countries is the development of cost-efficient seedling handling systems between nurseries and planting machines

Testing the Potential for Predictive Modeling and Mapping and Extending Its Use as a Tool for Evaluating Management Scenarios and Economic Valuation in the Baltic Sea (PREHAB)

Peer reviewe

THE JOURNAL OF BIOLOGICAL CHEMISTRY Sulfated N-Linked Oligosaccharides in Mammalian Cells I. COMPLEX-TYPE CHAINS WITH SIALIC ACIDS AND O-SULFATE ESTERS*

The structures of sulfated N-linked oligosaccharides have been reported for a few specific proteins. We recently demonstrated that such oligosaccharides occur in many different types of tissue culture cell lines 16 SAa2+(3)6Ga1@1+4GlcNAcj3l+R where R is the remainder of the underlying oligosaccharide, and SA is sialic acid. In addition to these molecules, a more highly charged group of sulfated N-linked oligosaccharides sharing structural features with glycosaminoglycans was found in CPAE cells, but not in CHO cells. These are described in the following paper (Sundblad, G., Holojda, S., ROUX, L., Varki * This research was supported by Grant CA38701 from the United States Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $Postdoctoral trainee supported by Grant HL07107-12. Recipient of an Established Investigatorship of the American Heart Association. 1 Recipient of Senior Faculty Research Award FRA-295 from the American Cancer Society. To whom correspondence should be addressed. Various types of macromolecules are sulfated in mammalian cells. These include lipids (l), steroid sulfates (2), proteins (tyrosine O-sulfate) (3), proteoglycans (4), and glycoproteins. The sulfated oligosaccharides of glycoproteins can be bound to the peptide via either O-glycosidic (5-7) or N-glycosidic linkages. The presence of sulfate in N-linked oligosaccharides has been reported in several proteins including viral glycoproteins (8,9), thyroid plasma membrane proteins (lo), basement membrane components (ll), the low density lipoprotein receptor (12), ovalbumin (13), pituitary hormones (14, 15), brain glycopeptides Most of the oligosaccharides were enzymatically released and contained both sulfate esters and phosphodiesters. The major sulfated monosaccharide was Man-6-SOr. ' Green et al. (21) have elucidated the structure of the sulfated N-linked oligosaccharides of bovine lutropin, in which one or two PGalNAc-4-so4 residues terminate an otherwise typical biantennary chain. In the case of ovalbumin, Man-4-S04 was found on hybrid-type oligosaccharides (13). GlcNAc-S04 residues have been reported in endothelial cells (22, 23) and in thyroid proteins (10). Previous work from this laboratory has shown that sulfation of N-linked oligosaccharides is more widespread in eukaryotic cell lines than heretofore suspected (24). In this study we have improved the labeling, release, and recovery of such molecules from several mammalian cell lines. The released oligosaccharides consist of two general classes. The first class is described in detail in this report and the second in the next paper (25). A specific protein that carries oligosaccharides of both kinds is described in the following paper (26). EXPERIMENTAL PROCEDURES AND RESULTS~ Choice of Cell Lines for Further Study-We used the approach described in the Miniprint Section to label, release, The abbreviations used are: Man, mannose; CPAE, bovine pulmonary artery endothelial cells; CHO, Chinese hamster ovary cells; CHO 761, Chinese hamster ovary mutant 761 cells; a-MEM, modified Eagle's medium a; SLIM, sulfate label incorporation medium; TM, tunicamycin; PNGaseF, peptide:N-glycosidase F FCS, fetal calf serum; PBS, phosphate-buffered saline; ConA, concanavalin A; RCA-1, Ricinus communis agglutinin-1; L-PHA, phytohemagglutinin-Ld; endo, endo-j3-N-acetylglucosaminidase; SA, sialic acid (type not determined); BSA, bovine serum albumin; AUN, Arthrobacter ureafaciens neuraminidase; NDVN, Newcastle disease virus neuraminidase; SDS, sodium dodecyl sulfate. Portions of this paper (including "Experimental Procedures," part of "Results," and Figs. 1 and 2) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press. filtration column. Released oligosaccharides are defined as those which appear in the included volume (S200R). To rule out nonspecific breakdown of macromolecules a control incubation was always carried out under identical conditions in the absence of added enzyme and similarly analyzed by gel filtration. Substantially less radioactivity appeared in the included volume in the sham-treated controls Several other conclusions can be drawn from the experiments described in Analysis of Sulfated N-Linked Oligosaccharides by Ion Exchange Chromatography-We next examined the behavior of the 35S04/[3H]Man-labeled oligosaccharides from CHO 761 and CPAE cells on QAE-Sephadex ion exchange columns in a manner analogous to that previously described for phosphorylated N-linked oligosaccharides (31, 32). In both cell lines, TABLE I Effect of tunicamycin and different media on the incorporation of PHIleucine and 3sS0, into CPAE macromolecules and PNGmeF-released oligosaccharides CPAE cells were cultured in replicate dishes in complete modified Eagle's medium a (a-MEM) until nearly confluent. Selected dishes were then incubated with tunicamycin for 0.5 h. Medium from all dishes was then removed and cells labeled with 10 pCi of [3H]leucine and the indicated amounts of 36s04 (Experiment 1,l.O mCi; Experiments 2 and 3,O.l mCi) in modified Eagle's medium a or sulfate label incorporation medium (SLIM). Tunicamycin treatment was continued in those dishes which had been previously incubated with the drug. Cells were harvested, lysed, subjected to S-200 gel filtration, and the percentage of the isotopes appearing in the S200VoA from each experiment determined. This material (representing macromolecules) was then treated with PNGaseF and reapplied to the same gel filtration column. The percent of total radioactivity released by PNGaseF (S200R) was then determined. Only selected data are presented in this table for the 3 pg/ml tunicamycin concentration. See "Experimental Procedures" for details. almost all of the 35s04 bound and eluted with varying concentrations of NaCl Thus, the sulfated molecules under study can be operationally defined as belonging to two different classes. In both CPAE and CHO 761 cells there is a group of sulfated sialylated molecules which elute from QAE-Sephadex between 20 and 400 mM NaCl. These are defined as Class I molecules. The second, defined as Class 11, includes very highly charged sulfated species which elute above 400 mM NaCl and do not shift significantly after neuraminidase treatment. There is some overlap between the two classes at the 400 mM NaCl elution step. The relative amount of material eluting at 400 mM NaCl also varied between preparations (see In separate experiments, we compared the elution profiles of these molecules from Sephadex G-50 columns. As demonstrated in Types of Sulfate Esters Present on CPAE Class I Oligosaccharides-Nitrous acid deamination, which specifically cleaves N-sulfate esters, did not release any of the 35S04 label from these oligosaccharides. Mild acid hydrolysis sufficient to remove N-sulfate esters also did not release any 35S01. However, all of the "SO4 label could be released as free bariumprecipitable radioactivity by acid hydrolysis under conditions that release 0-sulfate esters. Distributwn of Negative Charges on CPAE Class I Oligosaccharides-The 35S04-labeled Class I molecules should be admixed with anionic molecules carrying various combinations of other negative residues such as sialic acids and phosphate groups. Following neuraminidase treatment of 35SOo,/[3H] Man-labeled oligosaccharides, 33% of the 3H label remained bound to the QAE-Sephadex columns To better quantify the relative ratios of the various anionic molecules we, therefore, prepared oligosaccharides metabolically labeled with only 35S04 and introduced a 3H label into the reducing termini of the oligosaccharides by treatment with [3H]NaBH4 (see "Experimental Procedures" for details). Such double-labeled Class I oligosaccharides were then subjected to various treatments such as mild acid, alkaline phosphatase, neuraminidase, dimethyl sulfoxide solvolysis, or a combination of these procedures and applied to QAE-Sephadex columns. The contribution to total negative charge by each of the various anions can be inferred from the amount of 3H label rendered neutral after removal of that group. Initially, 28% of 'H label from intact untreated oligosaccharides was neutral. After treatment with mild acid (which cleaves sialic acids and "uncovers" phosphodiesters), this percentage increased to 78%. Somewhat less release was obtained with neuraminidase (65% 3H appeared in the neutral region). This could be explained by the presence of neuraminidase-resistant sialic acids (e.g. unusual linkages or modifications). Treatment of total oligosaccharides with alkaline phosphatase did not greatly increase 3H in the neutral fraction (28-33%), indicating that approximately 5% of total 3H-labeled oligosaccharides contained phosphomonoesters only. Because phosphate groups may also occur as mild acid-sensitive phosphodiesters with outer GlcNAc (32) or Glc (41) residues, another aliquot was treated with mild acid followed by alkaline phosphatase. The result obtained after these treatments was not different from that observed with mild acid alone (78-79%), indicating that only 1% or less of total 3H-labeled oligosaccharides contain &esterified phosphate groups. Solvolysis of pyridinium salts of sulfated oligosaccharides in dimethyl sulfoxide has previously been shown to release sulfate residues without affecting the underlying high mannose oligosaccharide structure (18). As shown in The proportion of total negative charge contributed by each anion was calculated as described in the legend to 8883 Treatment % of total [aH] ters, and a negligible amount of total negative charges was due to phosphodiesters. These rough estimations do not of course take into account the overlap caused by mixed molecules that contain sialic acids and phosphate groups (described in Ref. 32) nor those with sialic acids and sulfate esters (described above). We next attempted to fractionate the mixture of 36S04-labeled oligosaccharides by gradient elution from QAE-Sephadex (31) or high pressure liquid chromatography anion exchange columns (36). However, it was not possible to identify and cleanly separate a single major labeled species from this highly complex mixture of sulfated sialylated molecules. We, therefore, chose to continue the structural analysis on the mixture of Class I oligosaccharides. Lectin Affinity Chromatography of Class I Oligosacchurides-Unreduced CPAE Class I oligosaccharides labeled in 35S04 alone were obtained as shown in Very little 36S04-labeled material (1.6%) was specifically bound to and eluted from ConA-Sepharose with either 10 mM a-Me-Glc or 100 mM a-Me-Man (not shown). This indicates that CPAE cells contain few sulfated biantennary or high mannose chains. The failure of endo H to release 36S04 from CPAE macromolecules (data not shown) is also consistent with the lack of sulfated high mannose chains. Of course, sulfate residues could prevent the binding of such underlying oligosaccharides to the lectin or the action of the enzyme. In contrast to the behavior of 36S04-labeled CPAE Class I oligosaccharides on ConA-Sepharose, a substantial portion of these molecules was retarded by L-PHA-agarose Taken together, the results from lectin affinity chromatography indicate that sulfated CPAE Class I oligosaccharides are predominantly tri-and tetra-antennary chains that are extensively sialylated and contain subterminal 0-galactose residues. Since sulfate esters could be interfering with the binding of lectins to the underlying oligosaccharide, all of these results probably represent the minimal values for such binding. Determination of Sialic Acid Glycosidic Linkage-In order to determine if sialylation of sulfated Class I oligosaccharides preferentially occurs via a 2 4 3 or a 2 4 linkages to @-galactose, oligosaccharides metabolically labeled with [3H]Man and %04 were treated with mild acid, Arthrobacter ureafaciens neuraminidase (AUN), or Newcastle disease virus neuraminidase (NDVN), and then analyzed by QAE-Sephadex anion exchange chromatography. NDVN specifically cleaves a2+3 linkages (43), whereas AUN cleaves both a2-3 and a 2 4 linkages (44). In the experiment illustrated in Identification of the Sulfated Monosaccharide-Intact 3SS04-labeled Class I oligosaccharides were treated with various combinations of mild acid, @-galactosidase, and @-hex- Sulfated N-Linked Oligosaccharides. I 8885 osaminidase A. Mild acid was used to ensure complete release of sialic acids. No free sulfate was released under these conditions (data not shown). The 0-hexosaminidase A was used at low pH, since it has been reported to cleave intact GlcNAc-6-S04 residues from oligosaccharides under these conditions (45). Treated or control samples were then analyzed by gel filtration on Sephadex G-25 in 0.1 M acetic acid. These results are consistent with the following structure Class I oligosaccharides Intact 35S04-labeled Class I oligosaccharides, which had been depleted of SDS and desalted, were treated with various combinations of sequential mild acid, @-galactosidase, and @-hexosaminidase A at low pH (see "Experimental Procedures" for details). Samples were then subjected to gel filtration on Sephadex G-25 in 0.1 M acetic acid. The percentage of radioactivity appearing in the included volume at the elution position of [3H]glucitol-6-S04 is indicated as percent released. The true negative control (a) was not sham-incubated. All other samples which lacked one or more of the treatments were shamincubated. Treatment 6" 9 8 10 7 6 9 44 for some of the antennae on these oligosaccharides. Digestion with Other Enzymes-No low molecular weight labeled material was released by heparinase, heparitinase, or chondroitinase ABC, indicating the absence of heparin, chondroitin, or dermatan sulfate chains (data not shown). Endo-8-galactosidase also did not release low molecular weight material, indicating that extended type 2 chains (Gal(31-4GlcNAc)" with occasional GlcNAc-6-SOd residues were not present. DISCUSSION We have used an approach that allows the release and isolation of radiochemically pure 35S04-labeled N-linked oligosaccharides from cells. However, it is important to discuss artifacts that could provide alternate explanations for the apparently released label. PNGaseF) were performed in every case. These showed little (1-2% or less) of the radioactivity eluting in the region where released material would be expected to appear. However, in cell lines from which a small fraction (6% or less) of the 35s04 label is released by PNGaseF, such trace contaminants could represent a significant proportion of the "released material." We, therefore, confined our detailed studies to cell lines in which >lo% of the 36s04 label was specifically released by PNGaseF. (c) If a trace of undetected protease is still present in the PNGaseF preparation itself, nonspecific release could occur during the enzyme incubation which would not be seen in the control. However, the absence of significant degradation of whole cell [3H]leucine label in the presence of PNGaseF indicates that this cannot be occurring. ( d ) Tunicamycin, a drug which inhibits N-linked glycosylation, greatly reduced the amount of 35S04 incorporated in these molecules, under conditions where there was no significant inhibition of protein synthesis During the course of these investigations of sulfated oligosaccharides, we have also improved our previous methods (24) for the labeling and isolation of these molecules. The use of homogeneous PNGaseF rather than a mixture of two enzymes (PNGaseF/endo F) is desirable because this glycosidase has a wider substrate specificity than endo F and because it cleaves at the GlcNAc/Asn bond rather than within the chitobiosyl linkage (30). We have also found that gel filtration with Sephacryl S-200 rather than Sephadex G-50 allows the display of released species not seen with the latter column. For unknown reasons, the released oligosaccharides also tend to appear in two major peaks on the S-200 column, with the first containing almost all of the anionic oligosaccharides grouped together. This allows the convenient isolation of this group of molecules, which can then be fractionated by other means. The structural integrity of the sulfated oligosaccharides was also monitored throughout their isolation and characterization. None of the procedures generated free sulfate, as determined by the absence of 36S label in appropriate regions of the gel filtration columns and the absence of barium-precipitable 35s04 radioactivity. We also found that labeling in cysteine-free sulfate-free low methionine medium has the advantage of increasing the total amount of radiolabeled material. However, it did not adversely affect the level of protein synthesis nor the proportion of the sulfate label which was released by the PNGaseF digestion during a 6-h labeling period. Recently, several workers have shown that lowering the exogenous sulfate concentration may alter the composition and the degree of sulfation of some macromolecules (46,47). Thus, we cannot rule out qualitative alterations in the types of sulfated oligosaccharides produced under the different labeling conditions. The identity of the "so4 radioactivity remaining in void volume material after treatment with PNGaseF (S200VoB) is unknown but should include glycosaminoglycans, sulfolipids, sulfated 0-linked oligosaccharides, or tyrosine-sulfate residues of proteins. However, it is also possible that they include sulfated N-linked oligosaccharides of unknown structure that are resistant to PNGaseF or are so large that they still run in this region after release from proteins. Our structural studies of the sulfated CPAE Class I oligosaccharides indicate that these molecules bear 0-sulfate monoesters on otherwise typical complex-type oligosaccharide chains with sialic acids at the nonreducing termini. The exact number of negative charges cannot be ascertained in each case, because it may not be appropriate to extrapolate data on QAE-Sephadex elution previously obtained with phosphorylated high mannose-type chains (32) to these larger complex-type molecules. However, taken together, the results indicate that these molecules may carry from two to six negative charges that can be contributed to by any combination of sialic acids and sulfate esters. Another structural feature that these chains share with typical complex-type N-linked chains is the ability to interact with certain lectins. Since Galp1-3GlcNAc residues bind very poorly to RCA-1-agarose? the lectin affinity studies indicate that most, if not all, of the galactose residues are in 61-4 linkage. The L-PHA binding indicates the presence of the Gal@l-4GlcNAc@-Man sequence and the likelihood of the Gal@ldGlcNAc@1-6Man sequence and/or bissecting GlcNAc residues on the @-linked mannose (33, 34, 42). Both the terminal (sialic acid) and subterminal (@-galactose) monosaccharides typical of complex chains are present on these molecules. Treatment with neuraminidases suggests that while the total oligosaccharides contain sialic acids in both a2+3 and a 2 4 linkages, the sulfated chains are highly enriched in a24-linked residues. However, these data are inconsistent with the recent report of Green and Baenziger (42) that L-PHA-agarose cannot bind oligosaccharides completely substituted with a24-linked sialic acids. It is possible that their data obtained with reduced oligosaccharides cannot be extrapolated to the unreduced oligosaccharides studied here. An alternate explanation is that the sulfate esters selectively inhibit NDVN and not AUN. A third possibility is that there are in fact several terminal @-galactoside residues which mediate binding to L-PHA, but whose binding to RCA-1 is selectively affected by sulfate esters. Identification of the sulfated monosaccharide(s) was based on the following empirical and theoretical considerations. The acid hydrolysis kinetics indicated that the monosaccharide(s) was sulfated on a primary hydroxyl group. SA and Gal were unlikely candidates because they were susceptible to conventional enzymatic cleavage. In those tri-or tetra-antennary chains which bind to L-PHA-agarose, only one a-linked Man would have C6 free for sulfation; however, each oligosaccharide must accommodate from one to four sulfate esters. Thus, GlcNAc-6-S04 residues seemed the most probable sulfated monosaccharide. The sequential removal of SA, Gal, and GlcNAc-6-S04 confirmed this hypothesis for at least a third of the sulfate esters. Since the @-galactosidase appears to be inhibited to some extent by neighboring sulfate residues the estimated percentage of sulfate in GlcNAc-6-SOI is a minimal one. The possibility that sulfate is also esterified to C6 of an R. Cummings, personal communication. Sulfated N-Linked Oligosaccharides. I 8887 a-mannose or to C6 of core GlcNAc residues cannot be excluded at this time. Previous structural analysis of phosphorylated oligosaccharides was greatly aided by the availability of a nonspecific alkaline phosphatase (31,32). Unfortunately a corresponding nonspecific oligosaccharide sulfatase is not available. On the other hand, sulfate esters will block the activity of most exoglycosidases. We have taken advantage of the fact that (3-hexosaminidase A (at low pH) is an exception to this rule (45). We did observe (data

Recreational boating degrades vegetation important for fish recruitment

Recreational boating increases globally and associated moorings are often placed in vegetated habitats important for fish recruitment. Meanwhile, assessments of the effects of boating on vegetation, and potential effects on associated fish assemblages are rare. Here, we analysed (i) the effect of small-boat marinas on vegetation structure, and (ii) juvenile fish abundance in relation to vegetation cover in shallow wave-sheltered coastal inlets. We found marinas to have lower vegetation cover and height, and a different species composition, compared to control inlets. This effect became stronger with increasing berth density. Moreover, there was a clear positive relationship between vegetation cover and fish abundance. We conclude that recreational boating and related moorings are associated with reduced cover of aquatic vegetation constituting important habitats for juvenile fish. We therefore recommend that coastal constructions and associated boating should be allocated to more disturbance tolerant environments (e.g. naturally wave-exposed shores), thereby minimizing negative environmental impacts

Data from: A cross-scale trophic cascade from large predatory fish to algae in coastal ecosystems

Trophic cascades occur in many ecosystems, but the factors regulating them are still elusive. We suggest that an overlooked factor is that trophic interactions (TIs) are often scale-dependent and possibly interact across spatial scales. To explore the role of spatial scale for trophic cascades, and particularly the occurrence of cross-scale interactions (CSIs), we collected and analysed food-web data from 139 stations across 32 bays in the Baltic Sea. We found evidence of a four-level trophic cascade linking TIs across two spatial scales: at bay scale, piscivores (perch and pike) controlled mesopredators (three-spined stickleback), which in turn negatively affected epifaunal grazers. At station scale (within bays), grazers on average suppressed epiphytic algae, and indirectly benefitted habitat-forming vegetation. Moreover, the direction and strength of the grazer–algae relationship at station scale depended on the piscivore biomass at bay scale, indicating a cross-scale interaction effect, potentially caused by a shift in grazer assemblage composition. In summary, the trophic cascade from piscivores to algae appears to involve TIs that occur at, but also interact across, different spatial scales. Considering scale-dependence in general, and CSIs in particular, could therefore enhance our understanding of trophic cascades. The data package contains one dataset: - This file contains data on fish, vegetation, epiphytic algae and abiotic conditions at bay and station scale