OsAGAP, an ARF-GAP from rice, regulates root development mediated by auxin in Arabidopsis

Arf (ADP-ribosylation factor) proteins, which mediate vesicular transport, have little or no intrinsic GTPase activity. They rely on the action of GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) for their function. In the present study the OsAGAP gene in rice, which encoded a protein with predicted structure similar to ArfGAP, was identified. The purified OsAGAP-GST fusion protein was able to stimulate the GTPase activity of rice Arf. Furthermore, OsAGAP can rescue the defect of vesicular transport in the yeast gcs1 delta glo3 delta double-mutant cells. Transgenic Arabidopsis with OsAGAP constitutively expression showed reduced apical dominance, shorter primary roots, increasing number of longer adventitious roots. Many of the phenotypes can be phenocopied by treatment of exogenous indoleacetic acid level (IAA) in wild-type plants. Determination of whole-plant IAA level showed that there is a sharp increase of free IAA in OsAGAP transgenic Arabidopsis seedlings. In addition, removal of the 4-day-old shoot apex could inhibit the adventitious root formation in the transgenic seedlings. These results suggest OsAGAP, an ARF-GAP of rice, maybe involved in the mediation of plant root development by regulating auxin level.     

A trifluoromethyl substituted organoimido derivative of the hexametalate cluster: synthesis, crystal structure and bioactivity of [Mo6O17(NAr)2]2- (Ar=o-CF3C6H4)

The hexamolybdate cluster has been functionalized with fluorine-containing aromatic amine by the DCC (N,N'-dicyclohexylcarbodiimide) dehydrating protocol and a novel bifunctionalized arylimido derivative of hexamolybdate bearing the electron-withdrawing trifluoromethyl group, (Bu(4)N)(2)[Mo(6)O(17)(NAr)(2)] (Ar=o-CF(3)C(6)H(4)), has been prepared. Complete assignments were achieved for the title compound by elemental analysis, IR, (1)H NMR, UV/visible and single-crystal X-ray diffraction analyses. The preliminary agricultural biological activity test indicated that it has some herbicidal and insecticidal activities.     

The molecular origins of specificity in the assembly of a multienzyme complex

The pyruvate dehydrogenase (PDH) multienzyme complex is central to oxidative metabolism. We present the first crystal structure of a complex between pyruvate decarboxylase (E1) and the peripheral subunit binding domain (PSBD) of the dihydrolipoyl acetyltransferase (E2). The interface is dominated by a "charge zipper" of networked salt bridges. Remarkably, the PSBD uses essentially the same zipper to alternately recognize the dihydrolipoyl dehydrogenase (E3) component of the PDH assembly. The PSBD achieves this dual recognition largely through the addition of a network of interfacial water molecules unique to the E1-PSBD complex. These structural comparisons illuminate our observations that the formation of this water-rich E1-E2 interface is largely enthalpy driven, whereas that of the E3-PSBD complex (from which water is excluded) is entropy driven. Interfacial water molecules thus diversify surface complementarity and contribute to avidity, enthalpically. Additionally, the E1-PSBD structure provides insight into the organization and active site coupling within the approximately 9 MDa PDH complex.     

Arabidopsis membrane steroid binding protein 1 is involved in inhibition of cell elongation

A putative Membrane Steroid Binding Protein (designated MSBP1) was identified and functionally characterized as a negative regulator of cell elongation in Arabidopsis thaliana. The MSBP1 gene encodes a 220-amino acid protein that can bind to progesterone, 5-dihydrotestosterone, 24-epi-brassinolide (24-eBL), and stigmasterol with different affinities in vitro. Transgenic plants overexpressing MSBP1 showed short hypocotyl phenotype and increased steroid binding capacity in membrane fractions, whereas antisense MSBP1 transgenic plants showed long hypocotyl phenotypes and reduced steroid binding capacity, indicating that MSBP1 negatively regulates hypocotyl elongation. The reduced cell elongation of MSBP1-overexpressing plants was correlated with altered expression of genes involved in cell elongation, such as expansins and extensins, indicating that enhanced MSBP1 affected a regulatory pathway for cell elongation. Suppression or overexpression of MSBP1 resulted in enhanced or reduced sensitivities, respectively, to exogenous progesterone and 24-eBL, suggesting a negative role of MSBP1 in steroid signaling. Expression of MSBP1 in hypocotyls is suppressed by darkness and activated by light, suggesting that MSBP1, as a negative regulator of cell elongation, plays a role in plant photomorphogenesis. This study demonstrates the functional roles of a steroid binding protein in growth regulation in higher plants.     

Synthesis of a lipid conjugate of SO3Le(a) and its enhancement on liposomal binding to activated platelets

3'-O-Sulfated Le(a) (SO3Le(a)) is one of the most potent natural oligosaccharide ligands of selectins. The specific interactions between SO3Le(a) and E-/P-selectins are critical in the inflammation process. This paper described an efficient synthesis of a lipid conjugate of SO3Le(a) and its combination with phospholipid and cholesterol to form SO3Le(a)-coated liposomes by the freeze-thaw and extrusion method. The size (D = 78 nm) and stability of the resultant glycoliposomes were comparable to that of liposomes without the glycoconjugate. It was further observed that the incorporation of SO3Le(a) into liposomes could significantly enhance their adhesion to activated platelets as a result of the specific binding between SO3Le(a) on the glycoliposome and the P-selectin on activated platelets. The glycoliposome constructs may be useful for antiinflammation or for targeted delivery of drugs to endothelial cells that express E- and P-selectins.     

Variability of serum levels of tumor necrosis factor-alpha, interleukin 6, and soluble interleukin 6 receptor over 2 years in young women

Serum levels of tumor necrosis factor-alpha (TNF-alpha), interleukin 6 (IL-6), and soluble interleukin 6 receptor (sIL-6R) have been studied as risk factors of cardiovascular disease in longitudinal studies. However, it is unknown about their long-term intra-individual variations and whether single measurements of these cytokines and receptor are reliable biomarkers in epidemiological studies. In this study, serum levels of TNF-alpha, IL-6, and sIL-6R were assayed by ELISAs in 36 young, healthy women from whom three blood samples were collected at 12-month intervals over 2 years, and the intraclass correlation coefficients (ICC) were estimated. The ICC of 0.73 (95% CI=0.49-0.79) for TNF-alpha was comparable to those of other commonly used biomarkers, justifying its use in epidemiological studies. The ICC of 0.48 (95% CI=0.25-0.58) for IL-6 was not optimal. However, IL-6 has been demonstrated as a consistent risk factor for cardiovascular disease, suggesting it could still be a useful biomarker if its disease association is substantial. The ICC of 0.36 (95% CI=0.10-0.47) for sIL-6R was relatively low, and multiple samples would need to be collected in prospective studies for this receptor.     

Identification, expression pattern, and subcellular location of human RIP isoforms

XRIP alpha was identified as an adapter protein involved in RAP nuclear import. Several homologs were reported in mammal EST analysis, but the expression pattern and genomic organization of hRIP isoforms were not clarified yet. We isolated nine isoforms of hRIP from a premade human fetal brain library. hRIP alpha is the longest isoform with 219 residues, containing a N-terminal arginine-rich basic region, followed by an acidic region and two C-terminal Zn finger-like structures. hRIP beta deletes one Zn-finger-like structure. Three hRIP alpha isoforms and four hRIP sigma isoforms express truncated proteins due to frame shift. hRIP gamma isoforms lost the C-terminal Zn-finger-like structure. hRIP delta isoforms only contain the N-terminal arginine-rich basic region and the core sequence of the acidic region. The genomic organization of hRIP was identified by bioinformatic analysis. hRIP, containing seven exons, is located at human chromosome 17p13. hRIP was expressed in all 16 detected human tissues with a similar pattern. All EGFP-hRIP fusion proteins were located at the nucleus in the HEK293 cell. The two-polar molecular structure of hRIP might be involved in the basic cell function, and plays a role in the alternative nuclear ingress.     

Preparation and characterization of fusion protein truncated Pseudomonas Exotoxin A (PE38KDEL) in Escherichia coli

Pseudomonas Exotoxin A (PE) and truncated PE have been used to prepare immunotoxin with monoclonal antibodies. Truncated Pseudomonas Exotoxin A (PE38KDEL) was expressed with the pET-32a(+) vector in Escherichia coli under control of a T7 promoter. The recombinant protein was purified by His-Ni(2+) metal affinity chromatography and gel filtration. The biological activity of PE38KDEL was evaluated by the inhibition assay of protein synthesis in rabbit reticulocyte lysate system, and the cytotoxicity was tested in Hut 102 and hepatocellular cell lines by the MTS assay. PE38KDEL can significantly inhibit luciferase synthesis in cell-free protein synthesis assay and was slightly cytotoxic in the Hut 102 and hepatocellular cell lines. The results suggest that PE38KDEL would be useful for the preparation of more potent immunotoxins.     

Delta12-prostaglandin D2 is a potent and selective CRTH2 receptor agonist and causes activation of human eosinophils and Th2 lymphocytes

Prostaglandin D2 (PGD2) is a lipid mediator produced by mast cells, macrophages and Th2 lymphocytes and has been detected in high concentrations in the airways of asthmatic patients. There are two receptors for PGD2, namely the D prostanoid (DP) receptor and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). The proinflammatory effects of PGD2 leading to recruitment of eosinophils and Th2 lymphocytes into inflamed tissues is thought to be predominantly due to action on CRTH2. Several PGD2 metabolites have been described as potent and selective agonists for CRTH2. In this study we have characterized the activity of delta12-PGD2, a product of PGD2 isomerization by albumin. Delta12-PGD2 induced calcium mobilization in CHO cells expressing human CRTH2 receptor, with efficacy and potency similar to those of PGD2. These effects were blocked by the TP/CRTH2 antagonist ramatroban. delta12-PGD2 bound to CRTH2 receptor with a pKi of 7.63, and a 55-fold selectivity for CRTH2 compared to DP. In Th2 lymphocytes, delta12-PGD2 induced calcium mobilization with high potency and an efficacy similar to that of PGD2. delta12-PGD2 also caused activation of eosinophils as measured by shape change. Taken together, these results show that delta12-PGD2 is a potent and selective agonist for CRTH2 receptor and can cause activation of eosinophils and Th2 lymphocytes. These data also confirm the selective effect of other PGD2 metabolites on CRTH2 and illustrate how the metabolism of PGD2 may influence the pattern of leukocyte infiltration at sites of allergic inflammation.