Molecular analyses of a Lesch-Nyhan syndrome mutation (hprt Montreal) by use of T-lymphocyte cultures

Summary The frequency of hprt mutants in peripheral blood T-lymphocytes of two putative Lesch-Nyhan individuals and their parents was determined by a cell cloning assay to quantify the frequency of thioguanine-resistant mutants. The results confirmed the Lesch-Nyhan diagnosis and demonstrated that the mother has an elevated mutant frequency consistent with being heterozygous for an hprt mutation. Mass cultures of T-lymphocytes from both the children and their mother, as well as cultures of hprt mutant clones from the mother, were employed as sources of mRNA for cDNA sequence analysis. These hprt mutants show a single base substitution (TC transition) at position 170 (exon 3). The predicted amino acid change is the substitution of threonine for methionine₅₆. We have designated this new Lesch-Nyhan mutation hprt _Montreal. The use of T-lymphocyte cultures allows rapid sequence analyses of hprt mutations, as well as family studies to define the origin of a particular mutation.     

Identification of Integrin α3 as a New Substrate of the Adenovirus E4orf6/E1B 55-Kilodalton E3 Ubiquitin Ligase Complex

The human adenovirus E4orf6 and E1B55K proteins promote viral replication by targeting several cellular proteins for degradation. The E4orf6 product has been shown by our group and others to form an E3 ubiquitin ligase complex that contains elongins B and C and cullin family member Cul5. E1B55K associates with this complex, where it is believed to function primarily to introduce bound substrates for degradation via proteasomes. In addition to p53, its first known substrate, the E4orf6/E1B 55-kDa complex (E4orf6/E1B55K) was shown to promote the degradation of Mre11 and DNA ligase IV; however, additional substrates are believed to exist. This notion is strengthened by the fact that none of these substrates seems likely to be associated with additional functions shown to be mediated by the E4orf6-associated E3 ubiquitin ligase complex, including export of late viral mRNAs and blockage of export of the bulk cellular mRNAs from the nucleus. In an attempt to identify new E4orf6/E1B55K substrates, we undertook a proteomic screen using human p53-null, non-small-cell lung carcinoma H1299 cells expressing either E4orf6 protein alone or in combination with E1B55K through infection by appropriate adenovirus vectors. One cellular protein that appeared to be degraded by E1B55K in combination with the E4orf6 protein was a species of molecular mass ∼130 kDa that was identified as the integrin α3 subunit (i.e., very late activation antigen 3 alpha subunit). Preliminary analyses suggested that degradation of α3 may play a role in promoting release and spread of progeny virions.Viruses are well known to promote replication by inhibiting or enhancing endogenous cellular machinery or, in some cases, by reprogramming key cellular pathways. Human adenoviruses have developed effective ways to modulate the immune response, apoptosis, double-strand break repair, mRNA export, and translation to optimize virus replication and the spreading of progeny virions. The expression of adenovirus E1A proteins stabilizes p53 and induces apoptosis (8, 33); however, this effect is reversed in infected cells by the action of two early products: the E1B 55-kDa (E1B55K) and E4orf6 proteins (35, 36). We and others have shown that these proteins act through the formation of an E3 ubiquitin ligase complex analogous to the SCF and VBC complexes but which contains, in addition to elongins B and C and the RING protein Rbx1, the cullin family member Cul5 (18, 41, 43). This E4orf6-mediated E3 ligase complex blocks p53-induced apoptosis (35, 36) by promoting the ubiquitination of p53, followed by its degradation by proteasomes (41, 43). E4orf6 protein mediates the assembly of the complex by its interaction with elongin C through its three BC boxes (11, 41, 43). E1B55K, which appears to associate with the E4orf6 protein only when present in the ligase complex (4), is thought to function as a substrate recognition factor that brings substrates to the complex because, although both E4orf6 and E1B55K bind p53 independently, interaction of E1B55K with p53 is essential for the efficient degradation of p53 (41, 48). In addition to protecting infected cells from early lysis via p53-induced apoptosis, the E4orf6/E1B55K ligase complex performs other functions essential for virus replication. Two other substrates of the complex have been identified: a member of the MRN DNA repair complex, Mre11, and the central component of the nonhomologous end-joining DNA repair system, DNA ligase IV (2, 56). Degradation of both of these proteins prevents viral genome concatenation, which interferes with the packaging of viral DNA into virions (2, 56). E1B55K binds to p53, Mre11, and DNA ligase IV and has been demonstrated to colocalize with p53 and Mre11 in perinuclear cytoplasmic bodies termed aggresomes (1, 2, 32). More recently, we and others have obtained results that suggest that the E4orf6-associated E3 ligase complex regulates viral and cellular mRNA export (5, 66). The Cul5-based ligase activity was shown to be essential for selective viral mRNA export and the block of cellular mRNA export from the nucleus (66), thus contributing to the shutoff of cellular protein synthesis initiated by L4-100K (20). The actual substrates of the complex responsible for regulating mRNA export are currently unknown.As discussed in detail below, our efforts to identify substrates of the E4orf6/E1B55K complex led us to consider a member of the integrin family as a potential substrate. Integrins are members of a family of surface receptors that function in several ways through the formation of cell-extracellular matrices and cell-cell interactions (reviewed in references 21, 26, and 63). Integrins are typically composed of two transmembrane glycoproteins forming heterodimers of α and β subunits each of approximately 80 to 150 kDa. There are at least 18 α subunits and 8 β subunits in mammals that can dimerize in limited combinations to form more than 20 functionally distinct integrins with different ligand specificities. Integrin heterodimers function as transmembrane receptors that link external factors to intracellular signaling pathways. In addition to roles in cell adhesion, these communication events are implicated in a large range of cellular processes, including proliferation, differentiation, translation, migration, and apoptosis. Some of these processes depend on the intracellular trafficking pathways of the integrins (reviewed in references 9, 24, 40, and 44), including the long-loop recycling pathway in which integrins present in clathrin-coated endosomes move first to the perinuclear recycling center, where some accumulate, including the β1 integrin subunit (31), before returning to the plasma membrane. The integrin α3β1 is a member of the β1 integrin subfamily in which the α3 subunit (VLA-3a) is coupled to the β1 subunit to form the very late activation antigen (VLA-3 or CD49c) (21, 59, 60). α3β1 is expressed in a wide range of tissues in which it binds a variety of extracellular matrix substrates, including fibronectin, collagen, thrombospondin 1, and laminins 1, 5, 8, 10, and 11 (13). These associations allow the integrin α3β1 to fill its primary role in cell adhesion. α3β1 also participates in intercellular adhesion through several protein-protein interactions (10, 27, 53, 55, 58), making it a major contributor in the regulation of cellular adhesion.Human adenovirus type 5 (Ad5) particles interact with cell surface receptors to facilitate internalization into target cells. In the high-affinity interacting model (reviewed in reference 29), the viral fiber knob polypeptide binds the coxsackie adenovirus receptor (CAR) protein on the surface of cells as the primary cell binding event (primary receptor). The penton base polypeptide then binds a cell surface integrin (secondary receptor), leading to entry of the capsid into the cell by a process termed receptor-mediated endocytosis or clathrin-mediated endocytosis. Several types of integrins have been identified as being used by Ad5 to mediate virus internalization: αMβ1, αMβ2, αVβ1, αVβ3, αVβ5, and α5β1 (22, 30, 49, 65). Salone et al. have shown that α3β1 serves as an alternative cellular receptor for adenovirus serotype 5 (49). It promotes entry of the virus into cells, transduction of DNA, and mediates adenovirus infection in both CAR-positive and CAR-negative cell lines. Thus, in addition to functions related to cell adhesion, integrin α3β1 plays an important role in the adenovirus infection cycle.To identify new targets for degradation by the E4orf6/E1B55K ubiquitin ligase, we used a proteomic screen covering most cellular proteins to look for any polypeptide that exhibited a significant decrease in amount following the coexpression from appropriate adenovirus vectors of the E4orf6 protein and E1B55K. This screen revealed several interesting candidates, including integrin α3, a species of 130 kDa that also was found to be reduced in wild-type (wt) virus infection. The degradation of α3 was seen to be dependent on the Cul5-based ligase complex driven by E4orf6 and E1B55K. We also found evidence that the E4orf6/E1B55K ligase complex appears to be involved in cell detachment from the extracellular matrix, a function that could play a role in virus spread.     

Ice premelting during differential scanning calorimetry

Premelting at the surface of ice crystals is caused by factors such as temperature, radius of curvature, and solute composition. When polycrystalline ice samples are warmed from well below the equilibrium melting point, surface melting may begin at temperatures as low as -15 degrees C. However, it has been reported (. Biophys. J. 65:1853-1865) that when polycrystalline ice was warmed in a differential scanning calorimetry (DSC) pan, melting began at about -50 degrees C, this extreme behavior being attributed to short-range forces. We show that there is no driving force for such premelting, and that for pure water samples in DSC pans curvature effects will cause premelting typically at just a few degrees below the equilibrium melting point. We also show that the rate of warming affects the slope of the DSC baseline and that this might be incorrectly interpreted as an endotherm. The work has consequences for DSC operators who use water as a standard in systems where subfreezing runs are important.     

Overexpression of the acidic dehydrin WCOR410 improves freezing tolerance in transgenic strawberry leaves

Progress in freezing tolerance (FT) improvement through plant breeding approaches has met with little success in the last 50 years. Engineering plants for greater FT through plant transformation is one possible way to reduce the damage caused by freezing. Here, we report an improvement of the selection procedure and the transfer of the wheat Wcor410a acidic dehydrin gene in strawberry. The encoded protein has previously been shown to be associated with the plasma membrane, and its level of accumulation has been correlated with the degree of FT in different wheat genotypes. The WCOR410 protein was expressed in transgenic strawberry at a level comparable with that in cold-acclimated wheat. Freezing tests showed that cold-acclimated transgenic strawberry leaves had a 5 degrees C improvement of FT over wild-type or transformed leaves not expressing the WCOR410 protein. However, no difference in FT was found between the different plants under non-acclimated conditions, suggesting that the WCOR410 protein needs to be activated by another factor induced during cold acclimation. These data demonstrate that the WCOR410 protein prevents membrane injury and greatly improves FT in leaves of transgenic strawberry. A better understanding of the limiting factors allowing its activation may open up the way for engineering FT in different plant organs, and may find applications for the cryopreservation of human tissues and organs.     

The cytokine and chemokine expression profile of nucleus pulposus cells: implications for degeneration and regeneration of the intervertebral disc

Introduction

The aims of these studies were to identify the cytokine and chemokine expression profile of nucleus pulposus (NP) cells and to determine the relationships between NP cell cytokine and chemokine production and the characteristic tissue changes seen during intervertebral disc (IVD) degeneration.

Methods

Real-time q-PCR cDNA Low Density Array (LDA) was used to investigate the expression of 91 cytokine and chemokine associated genes in NP cells from degenerate human IVDs. Further real-time q-PCR was used to investigate 30 selected cytokine and chemokine associated genes in NP cells from non-degenerate and degenerate IVDs and those from IVDs with immune cell infiltrates (‘infiltrated’). Immunohistochemistry (IHC) was performed for four selected cytokines and chemokines to confirm and localize protein expression in human NP tissue samples.

Results

LDA identified the expression of numerous cytokine and chemokine associated genes including 15 novel cytokines and chemokines. Further q-PCR gene expression studies identified differential expression patterns in NP cells derived from non-degenerate, degenerate and infiltrated IVDs. IHC confirmed NP cells as a source of IL-16, CCL2, CCL7 and CXCL8 and that protein expression of CCL2, CCL7 and CXCL8 increases concordant with histological degenerative tissue changes.

Conclusions

Our data indicates that NP cells are a source of cytokines and chemokines within the IVD and that these expression patterns are altered in IVD pathology. These findings may be important for the correct assessment of the ‘degenerate niche’ prior to autologous or allogeneic cell transplantation for biological therapy of the degenerate IVD.     

Above‐ and belowground linkages in Sphagnum peatland: climate warming affects plant‐microbial interactions

Peatlands contain approximately one third of all soil organic carbon (SOC). Warming can alter above‐ and belowground linkages that regulate soil organic carbon dynamics and C‐balance in peatlands. Here we examine the multiyear impact of in situ experimental warming on the microbial food web, vegetation, and their feedbacks with soil chemistry. We provide evidence of both positive and negative impacts of warming on specific microbial functional groups, leading to destabilization of the microbial food web. We observed a strong reduction (70%) in the biomass of top‐predators (testate amoebae) in warmed plots. Such a loss caused a shortening of microbial food chains, which in turn stimulated microbial activity, leading to slight increases in levels of nutrients and labile C in water. We further show that warming altered the regulatory role of Sphagnum‐polyphenols on microbial community structure with a potential inhibition of top predators. In addition, warming caused a decrease in Sphagnum cover and an increase in vascular plant cover. Using structural equation modelling, we show that changes in the microbial food web affected the relationships between plants, soil water chemistry, and microbial communities. These results suggest that warming will destabilize C and nutrient recycling of peatlands via changes in above‐ and belowground linkages, and therefore, the microbial food web associated with mosses will feedback positively to global warming by destabilizing the carbon cycle. This study confirms that microbial food webs thus constitute a key element in the functioning of peatland ecosystems. Their study can help understand how mosses, as ecosystem engineers, tightly regulate biogeochemical cycling and climate feedback in peatlands     

Effect of fertilizer levels on grain yield,incidence and severity of downy mildew in pearl millet

The effect of various levels of nitrogen (0.0, 30.0, 60.0, 120.0) and phosphorus (0.0, 6.5, 13.0, 36.0) on the incidence and severity of downy mildew of pearl millet and yield of two pearl millet varieties (Zango and GB8375) were studied under field conditions in 2000 and 2001 respectively. Both nitrogen and phosphorus significantly increased incidence and severity of the disease in the two varieties. Grain yield and 1000 grain weight of the varieties also increased with nitrogen and phosphorus levels.