The speed of soil carbon throughput in an upland grassland is increased by liming

In situ (13)C pulse labelling was used to measure the temporal and spatial carbon flow through an upland grassland. The label was delivered as (13)C-CO(2) to vegetation in three replicate plots in each of two treatments: control and lime addition. Harvests occurred over a two month period and samples were taken along transects away from the label delivery area. The (13)C concentration of shoot, root, bulk soil, and soil-respired CO(2) was measured. There was no difference in the biomass and (13)C concentration of shoot and root material for the control and lime treatments meaning that the amount of (13)C-CO(2) assimilated by the vegetation and translocated below ground was the same in both treatments. The (13)C concentration of the bulk soil was lower in the lime treatment than in the control and, conversely, the (13)C concentration of the soil-respired CO(2) was higher in the lime. Unlike the difference in bulk soil (13)C concentration between treatments, the difference in the (13)C concentration of the soil-respired CO(2) was obvious only at the delivery site and primarily within 1 d after labelling. An observed increase in the abundance of mycorrhizal fungi in the lime treatment was a possible cause for this faster carbon throughput. The potential key role of mycorrhizas in the soil carbon cycle is discussed. The importance of a better understanding of soil processes, especially biological ones, in relation to the global carbon cycle and environmental change is highlighted.     

Expression and characterization of GSK-3 mutants and their effect on beta-catenin phosphorylation in intact cells

Glycogen synthase kinase-3 (GSK-3) is a serine-threonine kinase that is involved in multiple cellular signaling pathways, including the Wnt signaling cascade where it phosphorylates beta-catenin, thus targeting it for proteasome-mediated degradation. Unlike phosphorylation of glycogen synthase, phosphorylation of beta-catenin by GSK-3 does not require priming in vitro, i.e. it is not dependent on the presence of a phosphoserine, four residues C-terminal to the GSK-3 phosphorylation site. Recently, a means of dissecting GSK-3 activity toward primed and non-primed substrates has been made possible by identification of the R96A mutant of GSK-3beta. This mutant is unable to phosphorylate primed but can still phosphorylate unprimed substrates (Frame, S., Cohen, P., and Biondi R. M. (2001) Mol. Cell 7, 1321-1327). Here we have investigated whether phosphorylation of Ser(33), Ser(37), and Thr(41) in beta-catenin requires priming through prior phosphorylation at Ser(45) in intact cells. We have shown that the Arg(96) mutant does not induce beta-catenin degradation but instead stabilizes beta-catenin, indicating that it is unable to phosphorylate beta-catenin in intact cells. Furthermore, if Ser(45) in beta-catenin is mutated to Ala, beta-catenin is markedly stabilized, and phosphorylation of Ser(33), Ser(37), and Thr(41) in beta-catenin by wild type GSK-3beta is prevented in intact cells. In addition, we have shown that the L128A mutant, which is deficient in phosphorylating Axin in vitro, is still able to phosphorylate beta-catenin in intact cells although it has reduced activity. Mutation of Tyr(216) to Phe markedly reduces the ability of GSK-3beta to phosphorylate and down-regulate beta-catenin. In conclusion, we have found that the Arg(96) mutant has a dominant-negative effect on GSK-3beta-dependent phosphorylation of beta-catenin and that targeting of beta-catenin for degradation requires prior priming through phosphorylation of Ser(45).     

Competing pathways determine fibril morphology in the self-assembly of beta2-microglobulin into amyloid

Despite its importance in biological phenomena, a comprehensive understanding of the mechanism of amyloid formation remains elusive. Here, we use atomic force microscopy to map the formation of beta2-microglobulin amyloid fibrils with distinct morphologies and persistence lengths, when protein concentration, pH and ionic strength are varied. Using the resulting state-diagrams, we demonstrate the existence of two distinct competitive pathways of assembly, which define an energy landscape that rationalises the sensitivity of fibril morphology on the solution conditions. Importantly, we show that semi-flexible (worm-like) fibrils, which form rapidly during assembly, are kinetically trapped species, formed via a non-nucleated pathway that is explicitly distinct from that leading to the formation of the relatively rigid long-straight fibrils classically associated with amyloid. These semi-flexible fibrils also share an antibody epitope common to other protein oligomers that are known to be toxic species linked to human disease. The results demonstrate the heterogeneity of amyloid assembly, and have important implications for our understanding of the importance of oligomeric states in amyloid disease, the origins of prion strains, and the development of therapeutic strategies.     

Evidence that MEK1 positively promotes interhomologue double-strand break repair

During meiosis there is an imperative to create sufficient crossovers for homologue segregation. This can be achieved during repair of programmed DNA double-strand breaks (DSBs), which are biased towards using a homologue rather than sister chromatid as a repair template. Various proteins contribute to this bias, one of which is a meiosis specific kinase Mek1. It has been proposed that Mek1 establishes the bias by creating a barrier to sister chromatid repair, as distinct from enforcing strand invasion with the homologue. We looked for evidence that Mek1 positively stimulates strand invasion of the homologue. This was done by analysing repair of DSBs induced by the VMA1-derived endonuclease (VDE) and flanked by directly repeated sequences that can be used for intrachromatid single-strand annealing (SSA). SSA competes with interhomologue strand invasion significantly more successfully when Mek1 function is lost. We suggest the increase in intrachromosomal SSA reflects an opportunistic default repair pathway due to loss of a MEK1 stimulated bias for strand invasion of the homologous chromosome. Making use of an inhibitor sensitive mek1-as1 allele, we found that Mek1 function influences the repair pathway throughout the first4–5 h of meiosis. Perhaps reflecting a particular need to create bias for successful interhomologue events before chromosome pairing is complete.     

Human RECQL5 overcomes thymidine-induced replication stress

Accurate DNA replication is essential to genome integrity and is controlled by five human RecQ helicases, of which at least three prevent cancer and ageing. Here, we have studied the role of RECQL5, which is the least characterised of the five human RecQ helicases. We demonstrate that overexpressed RECQL5 promotes survival during thymidine-induced slowing of replication forks in human cells. The RECQL5 protein relocates specifically to stalled replication forks and suppresses thymidine-induced RPA foci, CHK1 signalling, homologous recombination and γH2AX activation. It is unlikely that RECQL5 promotes survival through translesion synthesis as PCNA ubiquitylation is also reduced. Interestingly, we also found that overexpressing RECQL5 relieves cells of the cell cycle arrest normally imposed by thymidine, but without causing mutations. In conclusion, we propose that RECQL5 stabilises the replication fork allowing replication to overcome the effects of thymidine and complete the cell cycle.     

Relaxin-3 and receptors in the human and rhesus brain and reproductive tissues

Evidence suggests that relaxin-3 may have biological functions in the reproductive and central nervous systems. To date, however, relaxin-3 biodistribution has only been investigated in the mouse, rat, pig and teleost fish. Characterizing relaxin-3 gene structure, expression patterns, and function in non-human primates and humans is critical to delineating its biological significance. Experiments were performed to clone the rhesus macaque orthologues of the relaxin-3 peptide hormone and its cognitive receptors (RXFP1 and RXFP4). An investigation of rhesus relaxin-3 bioactivity and RXFP1 binding properties was also performed. Next we sought to investigate relaxin-3 immunoreactivity in human and rhesus macaque tissues. Immunohistofluorescence staining for relaxin-3 in the brain, testis, and prostate indicated predominant immunostaining in the ventral and dorsal tegmental nuclei, interstitial space surrounding the seminiferous tubules, and prostatic stromal cells, respectively. Further, in studies designed towards exploring biological functions, we observed neuroprotective actions of rhesus relaxin-3 on human neuronal cell cultures. Taken together, this study broadens the significance of relaxin-3 as a peptide involved in both neuronal cell function and reproductive tissues in primates.     

Presence of arbuscular mycorrhizas in typically ectomycorrhizal host species from Cameroon and New Zealand

 Ectomycorrhizas (EcM) and arbuscular mycorrhizas (AM) were screened for in saplings of 14 EcM tree species from the N'Dupé and Korup National Park rainforests, SW Cameroon, belonging to Caesalpiniaceae and Uapacaceae. The pattern of EcM and AM colonisation of a dual mycorrhizal species from this rainforest (Uapaca staudtii, Uapacaceae) was compared with dual EcM/AM colonisation of Leptospermum scoparium (Myrtaceae) from New Zealand. Both species were collected in a range of habitats. EcM and AM colonisation differed among species in the Korup National Park rainforest: 12 species belonging to the Caesalpiniaceae (Amherstieae) were consistently EcM, and AM structures occurred occasionally in six of them; two other species belonging to Caesalpiniaceae (Afzelia bipindensis) and Uapacaceae (U. staudtii) were dual mycorrhizal with variable levels of colonisation by both EcM and AM fungi. EcM and AM dual colonisation varied with both habitat and identity of the partners. The presence of EcM fungi in most of the root samples of U. staudtii and a negative relationship between AM and EcM colonisation within the same root system suggested a greater EcM affinity of this species. In contrast, most root samples of L. scoparium were colonised by AM, but only a few by EcM. Genuine dual EcM/AM associations in root samples of U. staudtii where the two mycorrhizal types co-occurred could be attributed to an AM-EcM succession. However, differences between predicted and observed frequencies of genuine dual EcM/AM associations in several samples of both U. staudtii and L. scoparium indicated that other factors influenced dual EcM/AM associations. The results of this study showed the importance of the identity of the host species in determining the pattern of dual EcM and AM colonisation. Accepted: 18 September 1998     

Coincident regulation of PKCdelta in human platelets by phosphorylation of Tyr311 and Tyr565 and phospholipase C signalling

PKC (protein kinase C)d plays a complex role in platelets, having effects on both positive and negative signalling functions. It is phosphorylated on tyrosine residues in response to thrombin and collagen, and it has recently been shown that Tyr311 is phosphorylated in response to PAR (protease-activated receptor) 1 and PAR4 receptor activation. In the present study, we show that Tyr311 and Tyr565 are phosphorylated in response to thrombin, and have examined the interplay between phosphorylation and the classical lipid-mediated activation of PKCd. Phosphorylation of both Tyr311 and Tyr565 is dependent on Src kinase and PLC (phospholipase C) activity in response to thrombin. Importantly, direct allosteric activation of PKCd with PMA also induced phosphorylation of Tyr311 and Tyr565, and this was dependent on the activity of Src kinases, but not PLC. Membrane recruitment of PKCd is essential for phosphorylation of this tyrosine residue, but tyrosine phosphorylation is not required for membrane recruitment of PKCd. Both thrombin and PMA induce recruitment of PKCd to the membrane, and for thrombin, this recruitment is a PLC-dependent process. In order to address the functional role of tyrosine residue phosphorylation of PKCd, we demonstrate that phosphorylation can potentiate the activity of the kinase, although phosphorylation does not play a role in membrane recruitment of the kinase. PKCd is therefore regulated in a coincident fashion, PLC-dependent signals recruiting it to the plasma membrane and by phosphorylation on tyrosine residues, potentiating its activity.