Expression pattern and raft association of NIPSNAP3 and NIPSNAP4, highly homologous proteins encoded by genes in close proximity to the ATP-binding cassette transporter A1

The highly homologous genes NIPSNAP3 and NIPSNAP4, with 87% amino acid identity, are members of the NIPSNAP family with putative roles in vesicular trafficking. NIPSNAP3 mRNA and NIPSNAP4 mRNA and protein were detected in multiple tissues and cells at varying degrees. Interestingly, NIPSNAP3 is most highly expressed in skeletal muscle, where NIPSNAP4 has a low mRNA abundance. NIPSNAP4 was found associated with membranes and partly localized in rafts. The ubiquitous expression of the highly conserved NIPSNAPs and their association with membranes further support an important cellular function of these proteins probably linked to vesicular trafficking. The NIPSNAP3 and NIPSNAP4 genes are located in close proximity to the 3' end of the ATP-binding cassette transporter A1 (ABCA1), whose mutations cause familial high-density lipoprotein deficiency syndromes. The adjacent genomic location and the finding that ABCA1 is a regulator of vesicular trafficking may indicate a functional relation of these proteins, even though NIPSNAP4 does not interact directly with ABCA1 nor is its expression altered in cells with mutated ABCA1.     

Damaging and protective cell signalling in the untargeted effects of ionizing radiation

The major adverse consequences of radiation exposures are attributed to DNA damage in irradiated cells that has not been correctly restored by metabolic repair processes. However, the dogma that genetic alterations are restricted to directly irradiated cells has been challenged by observations in which effects of ionizing radiation arise in non-irradiated cells. These, so called, untargeted effects are demonstrated in cells that are the descendants of irradiated cells either directly or via media transfer (radiation-induced genomic instability) or in cells that have communicated with irradiated cells (radiation-induced bystander effects). Radiation-induced genomic instability is characterized by a number of delayed responses including chromosomal abnormalities, gene mutations and cell death. Bystander effects include increases or decreases in damage-inducible and stress-related proteins, increases or decreases in reactive oxygen and nitrogen species, cell death or cell proliferation, cell differentiation, radioadaptation, induction of mutations and chromosome aberrations and chromosomal instability. The phenotypic expression of untargeted effects and the potential consequences of these effects in tissues reflect a balance between the type of bystander signals produced and the responses of cell populations to such signals, both of which may be significantly influenced by cell type and genotype. Thus, in addition to targeted effects of damage induced directly in cells by irradiation, a variety of untargeted effects may also make important short-term and long-term contributions to determining overall outcome after radiation exposures.     

Interactions of abscisic acid and sugar signalling in the regulation of leaf senescence

Leaf senescence can be triggered by a high availability of carbon relative to nitrogen or by external application of abscisic acid (ABA). Most Arabidopsis mutants with decreased sugar sensitivity during early plant development are either ABA insensitive (abi mutants) or ABA deficient (aba mutants). To analyse the interactions of carbon, nitrogen and ABA in the regulation of senescence, wild-type Arabidopsis thaliana (L.) Heynh. and aba and abi mutants were grown on medium with varied glucose and nitrogen supply. On medium containing glucose in combination with low, but not in combination with high nitrogen supply, senescence was accelerated and sucrose, glucose and fructose accumulated strongly. In abi mutants that are not affected in sugar responses during early development (abi1-1 and abi2-1), we observed no difference in the sugar-dependent regulation of senescence compared to wild-type plants. Similarly, senescence was not affected in the sugar-insensitive abi4-1 mutant. In contrast, the abi5-1 mutant did exhibit a delay in senescence compared to its wild type. As ABA has been reported to induce senescence and ABA deficiency results in sugar insensitivity during early development, we expected senescence to be delayed in aba mutants. However, the aba1-1 and aba2-1 mutants showed accelerated senescence compared to their wild types on glucose-containing medium. Our results show that, in contrast to sugar signalling in seedlings, ABA is not required for the sugar-dependent induction of leaf senescence. Instead, increased sensitivity to osmotic stress could have triggered early senescence in the aba mutants.Abbreviations ABA Abscisic acid - aba Abscisic acid deficient - abi Abscisic acid insensitive - F_v/F_m Maximum efficiency of photosystem II photochemistry     

<Emphasis Type="Italic">Arabidopsis</Emphasis> constitutive photomorphogenic mutant, <Emphasis Type="Italic">bls1</Emphasis>,displays altered brassinosteroid response and sugar sensitivity

We have isolated an Arabidopsis mutant impaired in light- and brassinosteroid (BR) induced responses, as well as in sugar signalling. The bls1 (brassinosteroid, light and sugar1) mutant displays short hypocotyl, expanded cotyledons, and de-repression of light-regulated genes in young seedlings, and leaf differentiation and silique formation on prolonged growth in dark. In light, the bls1 mutant is dwarf and develops a short root, compact rosette, with reduced trichome number, and exhibits delayed bolting. The activity of the BR inducible TCH4 and auxin inducible SAUR promoters, fused with GUS gene, is also altered in seedlings harbouring bls1 mutant background. In addition, the bls1 mutant is hypersensitive to metabolizable sugars. The short hypocotyl phenotype in dark, short root phenotype in light and sugar hypersensitivity could be rescued with BR application. Moreover, the bls1 mutant also showed higher expression of a BR biosynthetic pathway gene CPD, which is known to be feedback-regulated by BR. Using a genome-wide AFLP mapping strategy, the bls1 mutant has been mapped to a 1.4Mb region of chromosome 5. Since no other mutant with essentially a similar phenotype has been assigned to this region, we suggest that the bls1 mutant defines a novel locus involved in regulating endogenous BR levels, with possible ramifications in integrating light, hormone and sugar signalling.     

Constitutive precoupling to G(i) and increased agonist potency in the alpha(2B)-adrenoceptor

The human alpha(2B)-adrenoceptor (alpha(2B)-AR) was mutated by substituting the D(3.49) aspartate in position 109 with an alanine (alpha(2B)-D109A) in the conserved DRY sequence at the cytoplasmic face of TM3. We studied the effects of the mutation on agonist binding and on receptor activation in CHO cells, including possible inverse agonism monitored by measuring intracellular Ca(2+) concentrations ([Ca(2+)](i)). The mutated receptor had increased binding affinity for agonists, especially dexmedetomidine (3.8-fold). The increased affinity was abolished by pretreatment of the cells with pertussis toxin. The mutation produced constitutive receptor activity evidenced as increased basal [Ca(2+)](i) and increased potency and efficacy of agonists to elicit Ca(2+) responses. The imidazoline derivative RX821002 functioned as an inverse agonist only through the alpha(2B)-D109A, reducing [Ca(2+)](i). The results thus indicate that this mutation causes constitutive receptor-G(i)-protein precoupling, and that the D(3.49) aspartate residue of the DRY motif is involved in controlling coupled and uncoupled conformations of alpha(2B)-AR.     

New and old roles of the double-stranded RNA-binding domain

RNA-binding proteins can strongly regulate and influence the cellular function and fate of an RNA molecule. Of the many described nucleic acid-binding domains, the double-stranded RNA-binding domain (dsRBD) is a highly specialized example found in a wide variety of proteins with diverse cellular functions. Mostly present in multiple copies and highly homologous to one another, the individual functional specificity of dsRBDs is now becoming apparent. Here we review recent evidence showing that single dsRBDs within individual proteins are capable of distinct in vivo functions. Not only does this enable dsRBD-containing proteins to increase their functional diversity but it also reveals novel and unexpected roles that dsRBDs can perform.     

Genetically engineered intracellular single-chain antibodies in gene therapy

The delineation of the molecular basis of cancer allows for the possibility of specific intervention at the molecular level for therapeutic purposes. To a large extent, the genetic lesions associated with malignant transformation and progression are being identified. Thus, not only in the context of inherited genetic diseases, but also for many acquired disorders, characteristic aberrancies of patterns of gene expression may be precisely defined. It is therefore clear that elucidation of the genetic basis of inherited and acquired diseases has rendered gene therapy both a novel and rational approach for these disorders. To this end, three main strategies have been developed: mutation compensation, molecular chemotherapy, and genetic immunopotentiation. Mutation compensation relies on strategies to ablate activated oncogenes at the level of DNA (triplex), messenger RNA (antisense or ribozyme), or protein (intracellular single-chain antibodies), and augment tumor suppressor gene expression. This article will review in detail practical procedures to generate a single-chain intracellular antibody (scFv). We will emphasize in this article the different steps in our protocol that we have employed to develop scFvs to a variety of target proteins.     

KCl activates phospholipase D at two different concentration ranges: distinguishing between hyperosmotic stress and membrane depolarization

Hyperosmotic stress induces the rapid formation of phosphatidic acid (PA) in Chlamydomonas moewusii via the activation of two signalling pathways: phospholipase D (PLD) and phospholipase C (PLC), the latter in combination with diacylglycerol kinase (DGK) (Munnik et al., 2000). A concomitant increase in cell Ca(2+) becomes manifest as deflagellation. When KCl was used as osmoticum we found that two concentration ranges activated deflagellation: one between 50 and 100 mm and another above 200 mm. Deflagellation in low KCl concentrations was complete within 30 sec whereas in high concentrations it took 5 min. PLC was not activated, as it was by high KCl concentrations that cause hyperosmotic stress. Moreover PLD was activated more strongly by low than by high KCl concentrations. Potassium was the most potent monovalent cation based on the induction of deflagellation and the formation of PA and PBut. During treatment, the external medium acidified, indicating an increase in H(+)-ATPase activity in order to re-establish the membrane potential. Activation of PLD and deflagellation at low KCl concentrations were abrogated by treatment with La(3+), Gd(3+) and EGTA, indicating the dependency on extracellular Ca(2+). This suggests that low concentrations of KCl depolarize the plasma membrane, resulting in the activation of H(+)-ATPases and opening voltage-dependent Ca(2+) +/- channels, observed as deflagellation and an increase in PLD activity.