Effect of di-n-butylphthalate on the carotenoid synthesis in green plants

Vapour of di- n -butylphthalate in light produces disturbances in the carotenoid synthesis of green plants ( Raphanus, Browallia , a.o.), resulting in chlorophyll deficiency and in extreme cases completely chlorophyll free leaves having a white colour. Absorption spectra of hexane extracts of such leaves, show the presence in higher concentrations than in normal leaves of a pigment with peak positions matching those of phytoene. In such leaves carotenes and xanthophylls are more or less completely lacking. Electron micrographs of white leaves reveal deepgoing changes in the chloroplast structures. The membranes of the grana are irregularly spread over the section areas and the plastoglobules swollen.     

Staphylococcus aureus infection triggers production of neutralizing, V8 protease-specific antibodies

Staphylococcus aureus infection triggers polyclonal B-cell activation. It was sought to further characterize the hypergammaglobulinemia seen in Staphylococcus aureus infection, focusing on the significance of protease-specific B-cell responses. Sera from mice infected with Staphylococcus aureus wild-type strain 8325-4 and two of its isogenic mutants devoid of protease expression were analyzed for the occurrence of polyclonal B-cell activation and the presence of specific antibodies against a set of exoproteases and superantigens. Furthermore, the functional properties of anti-V8-protease antibodies were analyzed in vitro. Polyclonal activation was manifested by increased levels of total serum IgG and IgM in all infected animals and by antibodies to staphylococcal toxins and aureolysin whether these antigens were present in the inoculate or not. Importantly, Staphylococcus aureus mutant lacking the V8-protease did not trigger a response against this enzyme. In contrast, strains expressing the V8-protease elicited V8-protease antibodies, proving the antigen-specific nature of this response. In vitro tests revealed that these antibodies had the capacity to inhibit the V8 protease activity in a dose-dependent manner. It was concluded that exposure to Staphylococcus aureus, in addition to a massive polyclonal B-cell response, gives rise to production of exoprotease-specific antibodies displaying functional properties.     

Simplified screening for the detection of soluble fusion constructs expressed in <Emphasis Type="Italic">E. coli</Emphasis> using a modular set of vectors

Background  

The solubility of recombinant proteins expressed in bacteria is often disappointingly low. Several strategies have been developed to improve the yield and one of the most common strategies is the fusion of the target protein with a suitable partner. Despite several reports on the successful use of each of these carriers to increase the solubility of some recombinant proteins, none of them was always successful and a combinatorial approach seems more efficient to identify the optimal combination for a specific protein. Therefore, the efficiency of an expression system critically depends on the speed in the identification of the optimal combination for the suitable fusion candidate in a screening process. This paper describes a set of expression vectors (pETM) designed for rapid subcloning, expression and subsequent purification using immobilized metal affinity chromatography (IMAC).     

A regulatory role for p38 delta MAPK in keratinocyte differentiation. Evidence for p38 delta-ERK1/2 complex formation

p38 MAPK isoforms are important in the regulation of a variety of cellular processes. Among the four described p38 isoforms, p38 alpha, beta, and delta are expressed in keratinocytes (Dashti, S. R., Efimova, T., and Eckert, R. L. (2001) J. Biol. Chem. 276, 8059-8063). However, very little is known about how individual p38 isoforms regulate keratinocyte function. In the present study, we use okadaic acid (OA) as a tool to study the role of p38 MAPKs as regulators of keratinocyte differentiation. We demonstrate that OA activates p38 delta but not other p38 isoforms. p38 delta activation is increased as early as 0.5 h after OA addition, and activity is maximal at 8 and 24 h. ERK1 and ERK2 activity are reduced on an identical time course. We show that p38 delta forms a complex with ERK1/2, and overexpression of p38 delta inhibits ERK1/2 activity without reducing ERK1/2 level. Thus, p38 delta may directly suppress ERK1/2 activity. Additional studies show that p38 delta is expressed in the epidermis, suggesting a role for p38 delta in regulating differentiation. To evaluate its function, we show that increased p38 delta activity is associated with increased levels of AP1 and CAATT enhancer binding protein factors, increased binding of these factors to the involucrin (hINV) promoter, and increased expression. Moreover, these responses are maintained in the presence of SB203580, an agent that inhibits p38 alpha and beta, further suggesting a central role for the p38 delta isoform. Dominant-negative p38 also inhibits these responses. These unique observations suggest that p38 delta is the major p38 isoform driving suprabasal hINV gene expression and that p38 delta directly regulates ERK1/2 activity via formation of a p38 delta-ERK1/2 complex.     

Unusual reactivity of copper(I) complexes of functionalised calix[4]arenes

Two functionalised calix[4]arenes, 5,11,17,23-tetra-tert-butyl-25,27-bis(2-pyridylmethoxy)calix[4]arene (L¹) and 5,11,17,23-tetra-tert-butyl-25,27-bis(2-pyridylmethoxy)-26,28-dibutoxycalix[4]arene (L³), were prepared and characterised. The copper(I) complexes of both calix[4]arenes were synthesised and their reactivities were analysed and compared. The presence of the metal induced a radical in the case of L¹ whereas no such radical was observed in the metal complex of ligand L³.     

N-Terminal domain linkage modulates the folding properties of protein S epidermal growth factor-like modules

Protein S interacts with activated protein C to play a crucial role in blood anticoagulation, and protein S deficiency is associated with increased risk of thrombosis. Despite the large volume of functional data available for this protein, no atomic resolution structure data have yet been reported. This is due at least in part to difficulties encountered when trying to produce fragments dissected from the intact protein; however, a few successful strategies have been described. In this research we have expressed a number of constructs containing protein S epidermal growth factor-like (EGF) domains 1 and 2 in Escherichia coli and Pichia pastoris. None of the proteins produced was stably folded as assayed by solution nuclear magnetic resonance spectroscopy. We therefore constructed a series of non-native protein S EGF concatemers to investigate the role of pairwise domain linkage in domain folding. Our results demonstrate that N-terminal domain linkage can either positively or negatively impact on the refolding of an adjacent domain. Furthermore, analysis of the NMR data for EGF3-4 reveals the expected interdomain NOEs that are characteristic of an extended arrangement of calcium-binding EGF domains and a similar average [(1)H]-(15)N heteronuclear NOE value for each of the two domains. These results provide the first data in support of protein S EGF3-4 adopting the same extended domain orientation as observed for the functionally distinct proteins fibrillin-1 and the low-density lipoprotein receptor. The results also have important implications for future studies, particularly when a dissection approach is used, of tandem EGF domains from protein S and other proteins.     

Impact of staphylococcal protease expression on the outcome of infectious arthritis

The exoproteases of Staphylococcus aureus have been proposed as virulence factors during S. aureus infections. To investigate this, we used the wild-type S. aureus strain 8325-4 and its mutants devoid of aureolysin, serine protease, and cysteine protease, respectively, in a well-established model of septic arthritis in mice. The inactivation of the exoprotease genes did not affect the frequency or the severity of joint disease. We conclude that in the model of haematogenously spread staphylococcal arthritis, the bacterial proteases studied do not act as virulence factors.     

S100 protein subcellular localization during epidermal differentiation and psoriasis.

S100 proteins are calcium-activated signaling proteins that interact with target proteins to modulate biological processes. Our present studies compare the level of expression, and cellular localization of S100A7, S100A8, S100A9, S100A10, and S100A11 in normal and psoriatic epidermis. S100A7 and S100A11 are present in the basal and spinous layers in normal epidermis. These proteins appear in the nucleus and cytoplasm in basal cells but are associated with the plasma membrane in spinous cells. S100A10 is present in basal and spinous cells, in the cytoplasm, and is associated with the plasma membrane. S100A8 and S100A9 are absent or are expressed at minimal levels in normal epidermis. In involved psoriatic tissue, S100A10 and S100A11 levels remain unchanged, whereas, S100A7, S100A8, and S100A9 are markedly overexpressed. The pattern of expression and subcellular localization of S100A7 is similar in normal and psoriatic tissue. S100A8 and S100A9 are strongly expressed in the basal and spinous layers in psoriasis-involved tissue. In addition, we demonstrate that S100A7, S100A10, and S100A11 are incorporated into detergent and reducing agent-resistant multimers, suggesting that they are in vivo transglutaminase substrates. S100A8 and S100A9 did not form these larger complexes. These results indicate that S100 proteins localize to the plasma membrane in differentiated keratinocytes, suggesting a role in regulating calcium-dependent, membrane-associated events. These studies also indicate, as reported previously, that S100A7, S100A8, and S100A9 expression is markedly altered in psoriasis, suggesting a role for these proteins in disease pathogenesis.