The C-terminal HDEL sequence is sufficient for retention of secretory proteins in the endoplasmic reticulum (ER) but promotes vacuolar targeting of proteins that escape the ER

Proteins are co-translationally transferred into the endo-plasmic reticulum (ER) and then either retained or transported to different intracellular compartments or to the extracellular space. Various molecular signals necessary for retention in the ER or targeting to different compartments have been identified. In particular, the HDEL and KDEL signals used for retention of proteins in yeast and animal ER have also been described at the C-terminal end of soluble ER processing enzymes in plants. The fusion of a KDEL extension to vacuolar proteins is sufficient for their retention in the ER of transgenic plant cells. However, recent results obtained using the same strategy indicate that HDEL does not contain sufficient information for full retention of phaseolin expressed in tobacco. In the present study, an HDEL C-terminal extension was fused to the vacuolar or extracellular (Δpro) forms of sporamin. The resulting SpoHDEL or ΔproHDEL, as well as Spo and Δpro, were expressed at high levels in transgenic tobacco cells ( Nicotiana tabacum cv BY2). The intracellular location of these different forms of recombinant sporamin was studied by subcellular fractionation. The results clearly indicate that addition of an HDEL extension to either Spo or Δpro induces accumulation of these sporamin forms in a compartment that co-purifies with the ER markers NADH cytochrome C reductase, binding protein (BiP) and calnexin. In addition, a significant SpoHDEL or ΔproHDEL fraction that escapes the ER retention machinery is transported to the vacuole. From these results, it may be proposed that, in addition to its function as an ER retention signal, HDEL could also act in quality control by targeting chaperones or chaperone-bound proteins that escape the ER to the plant lysosomal compartment for degradation.     

Evolution of the Mitochondrial Cytochrome Oxidase II Gene in Collembola

The sequence of the mitochondrial COII gene has been widely used to estimate phylogenetic relationships at different taxomonic levels across insects. We investigated the molecular evolution of the COII gene and its usefulness for reconstructing phylogenetic relationships within and among four collembolan families. The collembolan COII gene showed the lowest A + T content of all insects so far examined, confirming that the well-known A + T bias in insect mitochondrial genes tends to increase from the basal to apical orders. Fifty-seven percent of all nucleotide positions were variable and most of the third codon positions appeared free to vary. Values of genetic distance between congeneric species and between families were remarkably high; in some cases the latter were higher than divergence values between other orders of insects. The remarkably high divergence levels observed here provide evidence that collembolan taxa are quite old; divergence levels among collembolan families equaled or exceeded divergences among pterygote insect orders. Once the saturated third-codon positions (which violated stationarity of base frequencies) were removed, the COII sequences contained phylogenetic information, but the extent of that information was overestimated by parsimony methods relative to likelihood methods. In the phylogenetic analysis, consistent statistical support was obtained for the monophyly of all four genera examined, but relationships among genera/families were not well supported. Within the genus Orchesella, relationships were well resolved and agreed with allozyme data. Within the genus Isotomurus, although three pairs of populations were consistently identified, these appeared to have arisen in a burst of evolution from an earlier ancestor. Isotomurus italicus always appeared as basal and I. palustris appeared to harbor a cryptic species, corroborating allozyme data. Received: 12 January 1996 / Accepted: 10 August 1996     

Lisofylline prevents leak, but not neutrophil accumulation, in lungs of rats given IL-1 intratracheally

Hybertson, Brooks M., Stuart L. Bursten, Jonathan A. Leff,Young M. Lee, Eric K. Jepson, Chris R. Dewitt, John Zagorski, Hyun G. Cho, and John E. Repine. Lisofylline prevents leak, but not neutrophil accumulation, in lungs of rats given IL-1intratracheally. J. Appl. Physiol.82(1): 226-232, 1997.Interleukin-1 (IL-1) is increased in lunglavages from patients with the acute respiratory distress syndrome, andadministering IL-1 intratracheally causes neutrophil accumulation and aneutrophil-dependent oxidative leak in lungs of rats. In the presentstudy, we found that rats pretreated intraperitoneally with lisofylline[(R)-1-(5-hydroxyhexyl)-3,7-dimethylxanthine (LSF)], an inhibitor of lysophosphatidic acid acyl transferase, which reduces the production of unsaturated phosphatidic acid species,did not develop the lung leak or the related ultrastructural abnormalities that occur after intratracheal administration of IL-1.However, rats pretreated with LSF and then given IL-1 intratracheally did develop the same elevations of lung lavage cytokine-induced neutrophil chemoattractant (CINC) levels and the same increased numbersof lung lavage neutrophils as rats given IL-1 intratracheally. Lungs ofrats given IL-1 intratracheally also had increased unsaturated phosphatidic acid and free acyl (linoleate, linolenate) concentrations compared with untreated rats, and these lipid responses were prevented by pretreatment with LSF. Our results reveal that LSF decreases lungleak and lung lipid alterations without decreasing neutrophil accumulation or lung lavage CINC increases in rats given IL-1 intratracheally.

     

Evidence for Linkage of Bipolar Disorder to Chromosome 18 with a Parent-of-Origin Effect

A susceptibility gene on chromosome 18 and a parent-of-origin effect have been suggested for bipolar affective disorder (BPAD). We have studied 28 nuclear families selected for apparent unilineal transmission of the BPAD phenotype, by using 31 polymorphic markers spanning chromosome 18. Evidence for linkage was tested with affected-sib-pair and LOD score methods under two definitions of the affected phenotype. The affected-sib-pair analyses indicated excess allele sharing for markers on 18p within the region reported previously. The greatest sharing was at D18S37: 64% in bipolar and recurrent unipolar (RUP) sib pairs (P = .0006). In addition, excess sharing of the paternally, but not maternally, transmitted alleles was observed at three markers on 18q: at D18S41, 51 bipolar and RUP sib pairs were concordant for paternally transmitted alleles, and 21 pairs were discordant (P = .0004). The evidence for linkage to loci on both 18p and 18q was strongest in the 11 paternal pedigrees, i.e., those in which the father or one of the father's sibs is affected. In these pedigrees, the greatest allele sharing (81%; P = .00002) and the highest LOD score (3.51; θ = 0.0) were observed at D18S41. Our results provide further support for linkage of BPAD to chromosome 18 and the first molecular evidence for a parent-of-origin effect operating in this disorder. The number of loci involved, and their precise location, require further study.     

Prothrombin cleavage by human vascular smooth muscle cells: A potential alternative pathway to the coagulation cascade

Thrombin is a potent mitogen for human vascular smooth muscle cells (HVSMC) and its enzymatic activity is required for this function. The present study demonstrates that prothrombin is also mitogenic for HVSMC due to the generation of enzymatically active thrombin which occurs upon incubation of prothrombin with the cells. Analysis by SDS-PAGE, immunoblotting, and amino acid sequencing revealed that prothrombin incubated with HVSMC undergoes limited proteolysis. Prethrombin 1 was formed through cleavage at R¹⁵⁵-S¹⁵⁶. Cleavage at R²⁷¹-T²⁷² generated fragment 1.2 and prethrombin 2 whilst cleavage at R²⁸⁴-T²⁸⁵ yielded truncated prothrombin 2 (prethrombin 2′). However, cleavage at R³²⁰-I³²¹ which, during prothrombin activation produces two-chain α-thrombin, was not detectable. Studies on HVSMC-conditioned medium revealed that a similar pattern of prothrombin cleavage occurred by a cell-secreted factor(s). Amidolytic activity analysis indicated that 1–3% catalytically active thrombin-like activity was generated upon incubation of prothrombin with HVSMC-conditioned medium. By treating conditioned medium with various classes of proteinase inhibitors or hirudin, it was determined that prothrombin is cleaved by a cell-derived serine proteinase-like factor(s) at R²⁷¹-S²⁷² and by α-thrombin at R¹⁵⁵-S¹⁵⁶ and R²⁸⁴-T²⁸⁵. Antibodies neutralising the activity of either urokinase, tissue plasminogen activator, or factor Xa failed to alter the prothrombin cleaving activity of conditioned medium. This activity which may catalyse an alternative pathway for the generation of thrombin, was eluted from a gel filtration column as a single peak with apparent molecular mass of 30–40 kDa. © 1995 Wiley-Liss, Inc.     

Chilling-sensitive mutants of arabidopsis

Twenty-one mutants ofArabidopsis thaliana were isolated that developed chlorosis or necrosis upon incubation at low temperature (10°C to 15°C). Crosses among mutants in different phenotypic classes showed that mutants in three of four classes were found in a small number of loci. This article is reproduced fromWeeds World, vol. 1. For electronic access toWeeds World, see PMBR 12(4):302–303.     

Regulation of nitrogen-metabolizing enzymes in the commercial Mushroom Agaricus bisporus

Mycelium of Agaricus bisporus strain Horst U1 was grown in batch cultures on different concentrations of ammonium, glutamate, and glucose to test the effect of these substrates on the activities of NADP-dependent glutamate dehydrogenase (NADP-GDH, EC 1.4.1.4), NAD-dependent glutamate dehydrogenase (NAD-GDH, EC 1.4.1.2.), and glutamine synthetase (GS, EC 6.3.1.2.). When grown on ammonium, the activities of NADP-GDH and GS were repressed. NAD-GDH activity was about 10 times higher than the activities of NADP-GDH and GS. At concentrations below 8 mM ammonium, NADP-GDH and GS were slightly derepressed. When glutamate was used as the nitrogen source, activities of NADP-GDH and GS were derepressed; compared with growth on ammonium, the activities of these two enzymes were about 10 times higher. Activities of GDHs showed no variation at different glutamate concentrations. Activity of GS was slightly derepressed at low glutamate concentrations. Growth of A. bisporus on both ammonium and glutamate as nitrogen sources resulted in enzyme activities comparable to growth on ammonium alone. Activities of NADP-GDH, NAD-GDH, and GS were not influenced by the concentration of glucose in the medium. In mycelium starved for nitrogen, the activities of NADP-GDH, NAD-GDH, and GS were derepressed, while in carbon-starved mycelium the activity of GS and both GDHs was repressed.     

The cytidylyltransferase superfamily: Identification of the nucleotide-binding site and fold prediction

The crystal structure of glycerol-3-phosphate cytidylyltransferase from B. subtilis (TagD) is about to be solved. Here, we report a testable structure prediction based on the identification by sequence analysis of a superfamily of functionally diverse but structurally similar nucleotide-binding enzymes. We predict that TagD is a member of this family. The most conserved region in this superfamily resembles the ATP-binding HiGH motif of class I aminoacyI-tRNA synthetases. The predicted secondary structure of cytidylyltransferase and its homologues is compatible with the α/β topography of the class I aminoacyl-tRNA synthetases. The hypothesis of similarity of fold is strengthened by sequence-structure alignment and 3D model building using the known structure of tyrosyl tRNA synthetase as template. The proposed 3D model of TagD is plausible both structurally, with a well packed hydrophobic core, and functionally, as the most conserved residues cluster around the putative nucleotide binding site. If correct, the model would imply a very ancient evolutionary link between class I tRNA synthetases and the novel cytidylyltransferase superfamily. © 1995 Wiley-Liss, Inc.