The pharmacophore of the human C5a anaphylatoxin.

We have determined which amino acids contribute to the pharmacophore of human C5a, a potent inflammatory mediator. A systematic mutational analysis of this 74-amino acid protein was performed and the effects on the potency of receptor binding and of C5a-induced intracellular calcium ion mobilization were measured. This analysis included the construction of hybrids between C5a and the homologous but unreactive C3a protein and site-directed mutagenesis. Ten noncontiguous amino acids from the structurally well-defined 4-helix core domain (amino acids 1-63) and the C-terminal arginine-containing tripeptide were found to contribute to the pharmacophore of human C5a. The 10 mostly charged amino acids from the core domain generally made small incremental contributions toward binding affinity, some of which were independent. Substitutions of the C-terminal amino acid Arg 74 produced the largest single effect. We also found the connection between these 2 important regions to be unconstrained.     

The 5′ untranslated region of Arabidopsis thaliana calmodulin cDNA is an independent cDNA containing an open reading frame

A cDNA clone, pAUK1, with an open reading frame (ORF) coding for a hypothetical 164-amino-acid protein was isolated from an Arabidopsis thaliana (L.) Heynh cDNA library. The clone was attached, tail to tail, to the 3′ end of A. thaliana hexokinase cDNA. An almost identical sequence had been previously described as the 5′ untranslated region (5′ UTR) of A. thaliana calmodulin cDNA (ACaM-2). Sequence comparison with three additional A. thaliana truncated cDNA clones which appear in a database (GenBank) supports the conclusion that pAUKl is identical to the 5′ UTR of ACaM-2 and that the 5′ UTR of ACaM-2 is an independent cDNA artificially linked to A. thaliana calmodulin cDNA.     

光信号转导因子HY5调控拟南芥分枝的功能研究

光是影响植物分枝的重要外在环境因素,但光信号因子HY5（ELONGATED HYPOCOTYL5）是否调控植物分枝目前尚不清楚。创制了HY5转基因超表达植株,并获得商业化T-DNA插入突变体纯合植株。通过比较野生型（WT）、超表达植株（HY5-OE）、突变体（hy5-215）的分枝数目发现,与野生型相比,超表达植株分枝数目显著增加,而突变体分枝数目则显著减少。进一步比较这些遗传材料的拟南芥植株分枝的负调控关键因子BRC1（BRANCHED1）转录本水平差异,发现与野生型相比,超表达植株中BRC1转录本显著下调、突变体中显著上调。研究结果表明,HY5通过抑制拟南芥分枝关键负调控因子BRC1的转录水平,进而促进拟南芥的分枝。研究结果为阐明HY5调控分枝的生物学功能提供了一定的理论依据。     

Subunit-specific modulation of [(3)H]MK-801 binding to NMDA receptors mediated by dopamine receptor ligands in rodent brain

Dopamine D1 receptor (D1R) ligands may directly interact with the NMDA receptor (NMDAR), but detailed knowledge about this effect is lacking. Here we identify D1R ligands that directly modulate NMDARs and examine the contributions of NR2A and NR2B subunits to these interactions. Binding of the open channel blocker [(3)H]MK-801 in membrane preparations from rat- and mouse brain was used as a biochemical measure of the functional state of the NMDAR channel. We show that both D1R agonist A-68930 and dopamine receptor D2 antagonist haloperidol can decrease [(3)H]MK-801 binding with increased potency in membranes from the NR2A(-/-) mice (i.e. in membranes containing NR2B only), as compared to the inhibition obtained in wild-type membranes. Further, a wide range of D1R agonists such as A-68930, SKF-83959, SKF-83822, SKF-38393 and dihydrexidine were able to decrease [(3)H]MK-801 binding, all showing half maximal inhibitory concentrations ~20 μM, and with significant effects occurring at or above 1 μM. With membranes from D1R(-/-) mice, we demonstrate that these effects occurred through a D1R-independent mechanism. Our results demonstrate that dopamine receptor ligands can selectively influence NR2B containing NMDARs, and we characterize direct inhibitory NMDAR effects by different D1R ligands.     

Once phosphorylated, tyrosines in carboxyl terminus of protein-tyrosine kinase Syk interact with signaling proteins, including TULA-2, a negative regulator of mast cell degranulation

Activation of the high affinity IgE-binding receptor (FcεRI) results in the tyrosine phosphorylation of two conserved tyrosines located close to the COOH terminus of the protein-tyrosine kinase Syk. Synthetic peptides representing the last 10 amino acids of the tail of Syk with these two tyrosines either nonphosphorylated or phosphorylated were used to precipitate proteins from mast cell lysates. Proteins specifically precipitated by the phosphorylated peptide were identified by mass spectrometry. These included the adaptor proteins SLP-76, Nck-1, Grb2, and Grb2-related adaptor downstream of Shc (GADS) and the protein phosphatases SHIP-1 and TULA-2 (also known as UBASH3B or STS-1). The presence of these in the precipitates was further confirmed by immunoblotting. Using the peptides as probes in far Western blots showed direct binding of the phosphorylated peptide to Nck-1 and SHIP-1. Immunoprecipitations suggested that there were complexes of these proteins associated with Syk especially after receptor activation; in these complexes are Nck, SHIP-1, SLP-76, Grb2, and TULA-2 (UBASH3B or STS-1). The decreased expression of TULA-2 by treatment of mast cells with siRNA increased the FcεRI-induced tyrosine phosphorylation of the activation loop tyrosines of Syk and the phosphorylation of phospholipase C-γ2. There was parallel enhancement of the receptor-induced degranulation and activation of nuclear factor for T cells or nuclear factor κB, indicating that TULA-2, like SHIP-1, functions as a negative regulator of FcεRI signaling in mast cells. Therefore, once phosphorylated, the terminal tyrosines of Syk bind complexes of proteins that are positive and negative regulators of signaling in mast cells.     

Role of changing environmental parameters in leaf gas exchange of <Emphasis Type="Italic">Arbutus unedo</Emphasis> L. assessed by field and laboratory measurements

In the frame of the foreseen climate global changes we analysed the physiological responses of Arbutus unedo L. to the variations of carbon dioxide concentration, leaf temperature, and irradiance by measurements of leaf gas exchange and leaf water potential performed both in field and in the laboratory. Stomatal conductance was not affected by increase of leaf temperature. The growth conditions of potted plants likely made stomata more sensitive to the variation of external parameters than naturally growing plants. The interaction between high CO₂ concentration and temperature involved important down-regulation mechanisms in the metabolic pathway of the carbon fixation. From an ecological point of view, the ability of A. unedo to adapt to the field stress makes it highly competitive in the Mediterranean plant community.     

Glutamate levels control HT22 murine hippocampal cell death by regulating biphasic patterns of Erk1/2 activation: role of metabolic glutamate receptor 5

Extracellular glutamate concentration is a critical determinant of neuronal cell fate. We recently demonstrated that HT22 murine hippocampal cell viability was reduced by exposure to high concentrations of glutamate, whereas low concentrations promoted cell survival. Extracellular signal-regulated kinase (Erk)1/2 activation by glutamate is important for both glutamate-induced cell death and survival. In this study, we investigated the role of glutamate-induced or hydrogen peroxide (H₂O₂)-induced Erk1/2 activation in HT22 cell fate determination. Glutamate and H₂O₂ treatment similarly induced early (<1 h) Erk1/2 phosphorylation regardless of concentration. On the other hand, persistent Erk1/2 phosphorylation (16–24 h) was observed only in the presence of excess glutamate. Only the latter contributed to glutamate-induced cell death, which involved metabolic glutamate receptor 5. Our findings suggest that glutamate concentration modulates two distinct phases of Erk1/2 activation, which can explain the glutamate concentration-dependent determination of HT22 cell fate.     

Unusual evolutionary conservation of 5S rRNA pseudogenes inAspergillus nidulans: Similarity of the DNA sequence associated with the pseudogenes with the mouse immunoglobulin switch region

Summary AllAspergillus nidulans 5S rRNA pseudogenes known so far are the result of integration of an approx. 0.2-kbp-long DNA sequence into the 5S rRNA genes. This sequence, called block C, is present in at least five copies in theA. nidulans genome and seems to be associated either with 5S rRNA genes or pseudogenes. In contrast to the 78% sequence conservation of the C-block in pseudogenes, the truncated 5 halves of the pseudogenes are very highly conserved (96.9–100%). We postulate that the 5S rRNA pseudogenes are still a subject of concerted evolution. The C-block sequence shows similarity to the switch region of the mouse heavy chain immunoglobulin gene. A characteristic motif GGGTGAG is repeated several times in both sequences; the sequence conservation is 63%.     

Purification and characterization of a dicyclohexylcarbodiimide-sensitive adenosine triphosphatase complex from membranes of Escherichia coli.

The energy transducing adenosine 5′-triphosphatase (ATPase) complex was extracted with deoxycholate from $\text{Escherichia coli}$ membranes and purified 20–25 fold. The detergent-solubilized ATPase complex was inhibited more than 80% by dicyclohexylcarbodiimide (DCCD). Its sedimentation velocity coefficient was 14.7s in the presence of deoxycholate. Phospholipid stimulated its hydrolytic activity and maximized DCCD sensitivity. These parameters clearly differentiate the ATPase complex from the DCCD-insensitive, soluble ATPase prepared by extraction with EDTA at low ionic strength. The purified ATPase complex showed twelve discrete bands on lauryl sulfate gel electrophoresis. Five of these components co-electrophresed with subunits of soluble ATPase. Of the seven additional components, primarily two were precipitated with antibody to soluble ATPase. The protein which specifically reacts with DCCD co-migrated with one of these subunits.