Two soluble glycosyltransferases glycosylate less efficiently in vivo than their membrane bound counterparts

Many Golgi glycosyltransferases are type II membrane proteins which are cleaved to produce soluble forms that are released from cells. Cho and Cummings recently reported that a soluble form of alpha1, 3- galactosyltransferase was comparable to its membrane bound counterpart in its ability to galactosylate newly synthesized glycoproteins (Cho,S.K. and Cummings,R.D. (1997) J. Biol. Chem., 272, 13622-13628). To test the generality of their findings, we compared the activities of the full length and soluble forms of two such glycosyltransferases, ss1,4 N-Acetylgalactosaminyltransferase (GM2/GD2/ GA2 synthase; GalNAcT) and beta galactoside alpha2,6 sialyltransferase (alpha2,6-ST; ST6Gal I), for production of their glycoconjugate products in vivo . Unlike the full length form of GalNAcT which produced ganglioside GM2 in transfected cells, soluble GalNAcT did not produce detectable GM2 in vivo even though it possessed in vitro GalNAcT activity comparable to that of full length GalNAcT. When compared with cells expressing full length alpha2,6-ST, cells expressing a soluble form of alpha2,6-ST contained 3-fold higher alpha2,6-ST mRNA levels and secreted 7-fold greater alpha2,6-ST activity as measured in vitro , but in striking contrast contained 2- to 4-fold less of the alpha2,6-linked sialic acid moiety in cellular glycoproteins in vivo . In summary these results suggest that unlike alpha1,3-galactosyltransferase the soluble forms of these two glycosyltransferases are less efficient at glycosylation of membrane proteins and lipids in vivo than their membrane bound counterparts.      

Polyribosome formation and protein synthesis in imbibed but dormant lettuce seeds

Dormancy is maintained in Grand Rapids lettuce (Lactuca sativa) seeds imbibed on water in darkness at 25 C. Polyribosome formation and protein synthesis occur early in the imbibition phase and considerable polysomal material is also present after 24 and 48 hours, even though the seeds have failed to germinate. Incorporation of labeled leucine into protein following a 24-hour preincubation period shows that these polysomes are active in protein synthesis.     

Distance,environmental and substrate factors impacting recovery of bryophyte communities after harvesting

Aims

Bryophyte re‐colonization after disturbance is largely governed by environmental conditions within disturbed forests. In particular, distance to a forest edge is an important predictor of bryophyte community re‐colonization, through either direct constraints, such as dispersal limitation, or indirectly by altering environmental conditions. This study examines a range of factors – environmental, distance to an edge, substrate specific environment or local‐level environment – to determine which are important in the re‐colonization of bryophyte communities after forest harvesting. As bryophyte communities vary with the particular substrate inhabited, responses were examined across four substrates (rock, exposed roots, ground and CWD).

Location

Tasmanian southern forests, Australia.

Methods

Bryophyte composition was examined on four substrates (ground, coarse wood debris, exposed roots, rocks) within three ages (~7, ~27 and ~45 years post‐disturbance) of harvested wet eucalypt forest. Re‐colonization success of bryophyte communities was determined by comparing communities in regeneration forest to mature forest communities using axis scores from one‐dimensional constrained ordination. The importance of various environmental conditions for re‐colonization success was then modelled. Finally, path analysis was used to determine whether the impact of distance to a forest edge was meditated through its effects on key environmental variables.

Results

Multiple environmental factors impacted re‐colonization of mature bryophyte communities. Local‐level conditions such as microclimate (temperature, humidity and VPD) and LAI were the most important in determining re‐colonization across substrates. Path analysis showed that distance to a forest edge had a significant impact on re‐colonization success, but only a relatively small part of this was mediated through its impact on environmental factors.

Conclusions

Bryophyte re‐colonization is driven by a combination of microclimate conditions and factors related to distance from a forest edge (most likely dispersal distance). While some substrate‐specific factors impact bryophyte re‐colonization success, the consistent impact of local environmental factors across substrates suggests that harvesting management strategies that develop more ‘mature’ microclimate conditions and increase proximity to nearby mature forest patches will be beneficial for all bryophytes communities. As bryophyte re‐colonization was correlated with temporally dynamic environmental conditions, we suggest that forest age needs to be considered in future work.     

Preliminary evaluation of non-native rainbow trout (Oncorhynchus mykiss) impact on the Cederberg ghost frog (Heleophryne depressa) in South Africa’s Cape Fold Ecoregion

We evaluated the impact of non-native rainbow trout Oncorhynchus mykiss on a population of endemic Cedarberg ghost frog Heleophryne depressa in the upper Krom River (Olifants-Doring River Catchment, Cape Fold Ecoregion). We compared H. depressa abundance (using kick-sampling and underwater video analysis) and environmental conditions between sites above and below a waterfall that marks the upper distribution limit of O. mykiss. Heleophryne depressa abundance was significantly greater above the waterfall than that below it, and, because there was no significant difference in measured environmental variables, O. mykiss presence is identified as the most likely explanation for the observed decrease in H. depressa abundance.     

P2Y<Subscript>2</Subscript> and P2Y<Subscript>6</Subscript> receptor activation elicits intracellular calcium responses in human adipose-derived mesenchymal stromal cells

Adipose tissue contains self-renewing multipotent cells termed mesenchymal stromal cells. In situ, these cells serve to expand adipose tissue by adipogenesis, but their multipotency has gained interest for use in tissue regeneration. Little is known regarding the repertoire of receptors expressed by adipose-derived mesenchymal stromal cells (AD-MSCs). The purpose of this study was to undertake a comprehensive analysis of purinergic receptor expression. Mesenchymal stromal cells were isolated from human subcutaneous adipose tissue and confirmed by flow cytometry. The expression profile of purinergic receptors was determined by quantitative real-time PCR and immunocytochemistry. The molecular basis for adenine and uracil nucleotide-evoked intracellular calcium responses was determined using Fura-2 measurements. All the known subtypes of P2X and P2Y receptors, excluding P2X2, P2X3 and P2Y₁₂ receptors, were detected at the mRNA and protein level. ATP, ADP and UTP elicited concentration-dependent calcium responses in mesenchymal cells (N?=?7–9 donors), with a potency ranking ADP (EC₅₀ 1.3 ± 1.0 μM)?>?ATP (EC₅₀ 2.2 ± 1.1 μM)?=?UTP (3.2 ± 2.8 μM). Cells were unresponsive to UDP (<?30 μM) and UDP-glucose (<?30 μM). ATP responses were attenuated by selective P2Y₂ receptor antagonism (AR-C118925XX; IC₅₀ 1.1 ± 0.8 μM, 73.0?±?8.5% max inhibition; N?=?7 donors), and UTP responses were abolished. ADP responses were attenuated by the selective P2Y₆ receptor antagonist, MRS2587 (IC₅₀ 437 ± 133nM, 81.0?±?8.4% max inhibition; N?=?6 donors). These data demonstrate that adenine and uracil nucleotides elicit intracellular calcium responses in human AD-MSCs with a predominant role for P2Y₂ and P2Y₆ receptor activation. This study furthers understanding about how human adipose-derived mesenchymal stromal cells can respond to external signalling cues.     

Insertion of leader peptidase into the thylakoid membrane during synthesis in a chloroplast translation system

The mechanisms of targeting and insertion of chloroplast-encoded thylakoid membrane proteins are poorly understood. In this study, we have used a translation system isolated from chloroplasts to begin to investigate these mechanisms. The bacterial membrane protein leader peptidase (Lep) was used as a model protein because its targeting and insertion mechanisms are well understood for Escherichia coli and for the endoplasmic reticulum. Lep could thus provide insight into the functional homologies between the different membrane systems. Lep was efficiently expressed in the chloroplast translation system, and the protein could be inserted into thylakoid membranes with the same topology as in E. coli cytoplasmic membranes, following the positive-inside rule. Insertion of Lep into the thylakoid membrane was stimulated by the trans-thylakoid proton gradient and was strongly inhibited by azide, suggesting a requirement for SecA activity. Insertion most likely occurred in a cotranslational manner, because insertion could only be observed if thylakoid membranes were present during translation reactions but not when thylakoid membranes were added after translation reactions were terminated. To halt the elongation process at different stages, we translated truncated Lep mRNAs without a stop codon, resulting in the formation of stable ribosome nascent chain complexes. These complexes showed a strong, salt-resistant affinity for the thylakoid membrane, implying a functional interaction of the ribosome with the membrane and supporting a cotranslational insertion mechanism for Lep. Our study supports a functional homology for the insertion of Lep into the thylakoid membrane and the E. coli cytoplasmic membrane.     

Cell-to-cell and phloem-mediated transport of potato virus X. The role of virions

Movement-deficient potato virus X (PVX) mutants tagged with the green fluorescent protein were used to investigate the role of the coat protein (CP) and triple gene block (TGB) proteins in virus movement. Mutants lacking either a functional CP or TGB were restricted to single epidermal cells. Microinjection of dextran probes into cells infected with the mutants showed that an increase in the plasmodesmal size exclusion limit was dependent on one or more of the TGB proteins and was independent of CP. Fluorescently labeled CP that was injected into epidermal cells was confined to the injected cells, showing that the CP lacks an intrinsic transport function. In additional experiments, transgenic plants expressing the PVX CP were used as rootstocks and grafted with nontransformed scions. Inoculation of the PVX CP mutants to the transgenic rootstocks resulted in cell-to-cell and systemic movement within the transgenic tissue. Translocation of the CP mutants into sink leaves of the nontransgenic scions was also observed, but infection was restricted to cells close to major veins. These results indicate that the PVX CP is transported through the phloem, unloads into the vascular tissue, and subsequently is transported between cells during the course of infection. Evidence is presented that PVX uses a novel strategy for cell-to-cell movement involving the transport of filamentous virions through plasmodesmata.     

The movement protein of cucumber mosaic virus traffics into sieve elements in minor veins of nicotiana clevelandii

The location of the 3a movement protein (MP) of cucumber mosaic virus (CMV) was studied by quantitative immunogold labeling of the wild-type 3a MP in leaves of Nicotiana clevelandii infected by CMV as well as by using a 3a-green fluorescent protein (GFP) fusion expressed from a potato virus X (PVX) vector. Whether expressed from CMV or PVX, the 3a MP targeted plasmodesmata and accumulated in the central cavity of the pore. Within minor veins, the most extensively labeled plasmodesmata were those connecting sieve elements and companion cells. In addition to targeting plasmodesmata, the 3a MP accumulated in the parietal layer of mature sieve elements. Confocal imaging of cells expressing the 3a-GFP fusion protein showed that the 3a MP assembled into elaborate fibrillar formations in the sieve element parietal layer. The ability of 3a-GFP, expressed from PVX rather than CMV, to enter sieve elements demonstrates that neither the CMV RNA nor the CMV coat protein is required for trafficking of the 3a MP into sieve elements. CMV virions were not detected in plasmodesmata from CMV-infected tissue, although large CMV aggregates were often found in the parietal layer of sieve elements and were usually surrounded by 3a MP. These data suggest that CMV traffics into minor vein sieve elements as a ribonucleoprotein complex that contains the viral RNA, coat protein, and 3a MP, with subsequent viral assembly occurring in the sieve element parietal layer.     

Fine structure DNA mapping studies of the chromosomal region harboring the genetic defect in neurofibromatosis type 1

To better map the location of the von Recklinghausen neurofibromatosis (NF1) gene, we have characterized a somatic cell hybrid designated 7AE-11. This microcell-mediated, chromosome-transfer construct harbors a centromeric segment and a neo-marked segment from the distal long arm of human chromosome 17. We have identified 269 cosmid clones with human sequences from a 7AE-11 library and, using a panel of somatic cell hybrids with a total of six chromosome 17q breakpoints, have mapped 240 of these clones on chromosome 17q. The panel included a hybrid (NF13) carrying a der(22) chromosome that was isolated from an NF1 patient with a balanced translocation, t(17;22) (q11.2;q11.2). Fifty-three of the cosmids map into a region spanning the NF13 breakpoint, as defined by the two closest flanking breakpoints (17q11.2 and 17q11.2-q12). RFLP clones from a subset of these cosmids have been mapped by linkage analysis in normal reference families, to localize the NF1 gene more precisely and to enhance the potential for genetic diagnosis of this disorder. The cosmids in the NF1 region will be an important resource for testing DNA blots of large-fragment restriction-enzyme digests from NF1 patient cell lines, to detect rearrangements in patients' DNA and to identify the 17;22 NF1 translocation breakpoint.