Linear correlation between thermal stability and folding kinetics of lysozyme

We have studied the refolding and thermal denaturation of hen egg white lysozyme in a wide range of pH values (from 1.5 to 9.4) using stopped-flow circular dichroism (CD) and differential scanning calorimetry (DSC). A linear correlation was found between the thermal denaturation temperature (T(m)) and the logarithm of the refolding rate of the slow folding phase of hen egg white lysozyme (lnk(2)).     

Molecular basis of ATP-sensitive K+ channels in rat vascular smooth muscles

ATP-sensitive K+ (K(ATP)) channels couple metabolic changes to membrane excitability in vascular smooth muscle cells (SMCs). While the electrophysiological properties of K(ATP) channels have been examined, little is known about the molecular basis of K(ATP) complex in vascular SMCs. We identified and cloned four K(ATP) subunit genes from rat mesenteric artery, namely rvKir6.1, rvKir6.2, rvKirSUR1, and rvSUR2B. These clones showed over 99.6% amino acid sequence identity with other previously reported isoforms. The mRNA expression patterns of the K(ATP) subunits varied among rat aorta, mesenteric artery, pulmonary artery, tail artery, hepatic artery, and portal vein. Heterologous co-expression of rvKir6.1 and rvSUR2B yielded functional K(ATP) channels that were inhibited by glibenclamide, and opened by pinacidil. Our results for the first time reported the expression of four K(ATP) subunits in same vascular tissues, unmasking the diversity of native K(ATP) channels in vascular SMCs.     

Comparison of the expression of Bacillus thuringiensis full-length and N-terminally truncated vip3A gene in Escherichia coli

AIMS: Studies were performed to demonstrate the function of the putative signal peptide of Vip3A proteins in Escherichia coli. METHODS AND RESULTS: The full-length vip3A-S184 gene was isolated from a soil-isolated Bacillus thuringiensis, and the vip3AdeltaN was constructed by deleting 81 nucleotides at the 5'-terminus of vip3A-S184. Both were transformed and expressed in E. coli. About 19.2% of Vip3A-S184 proteins secreted soluble proteins and others formed inclusion bodies in the periplasmic space. In contrast, the Vip3AdeltaN was insoluble and formed inclusion bodies in the cytoplasm. Bioassay indicated that Vip3A-S184 showed different toxicity against Spodoptera exigua, Helicoverpa armigera and S. litura, but Vip3AdeltaN showed no toxicity to either of them because of the deletion of the first 27 amino acids at the N-terminus. CONCLUSIONS: The results suggest that the deleted N-terminal sequences were essential for the secretion of Vip3A-S184 protein in E. coli and might be required for toxicity. SIGNIFICANCE AND IMPACT OF THE STUDY: The function of the putative signal peptide of Vip3A protein in E. coli was investigated. These would be helpful to make clear the unknown secretion pathway of Vip3A protein in B. thuringiensis and determine the receptor-binding domain or toxic fragment of Vip3A-S184 protein.     

Additional virulence genes and sonication enhance <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated loblolly pine transformation

Additional virulence (vir) genes in Agrobacterium tumefaciens and sonication were investigated for their impact on transformation efficiency in loblolly pine (Pinus taeda L.). Mature zygotic embryos of loblolly pine were co-cultivated with disarmed A. tumefaciens strain EHA105 containing either plasmid vector pCAMBIA1301 or vector pCAMBIA1301 with an additional 15.8-kb fragment carrying extra copies of the Vir B, Vir C, and Vir G regions from the supervirulent plasmid pTOK47. pCAMBIA1301 contains hygromycin resistance and the beta-glucuronidase (GUS) reporter gene. Expression of GUS was observed after 3-6 days of co-cultivation, with peak expression at approximately 21 days. The highest numbers of GUS-expressing areas were visible up to 21 days after co-cultivation, declining rapidly thereafter. Both transient and stable transformation efficiencies increased when the explants were sonicated before co-cultivation and/or the additional virB, virC, and virG genes were included with the pCAMBIA1301 plasmid T-DNA. Use of the plasmid with additional vir genes and sonication dramatically enhanced the efficiency of Agrobacterium-mediated gene transfer not only in transient expression but also in the recovery of hygromycin-resistant lines. Stably transformed cultures and transgenic plants were produced from embryos transformed with A. tumefaciens EHA105 carrying pCAMBIA1301 or pCAMBIA1301+pTOK47 in the three families of loblolly pine. The presence of the introduced GUS and hygromycin phosphotransferase genes in the transgenic plants was confirmed by polymerase chain reaction and Southern hybridization analyses.     

A new method of subcutaneous placement of free jejunal flaps to reconstruct a diversionary conduit for swallowing in complicated pharyngoesophageal injury

Choking is a serious problem in pharyngoesophageal reconstruction, which may occur following tumor ablation of the pharynx or following corrosive injury involving the epiglottis and other parts of the upper airway. To prevent choking and the risk of severe pulmonary complications, patients have to give up oral intake and assume feeding via jejunostomy for the rest of their lives. After reconstruction of the esophagus, eight patients experienced frequent choking and aspiration. With a free jejunal flap, the inlet for food could be separated from the route of the upper airway by a diversion technique. The jejunum segment was transferred microsurgically to reconstruct the cervical esophagus, with its inlet at the buccogingival sulcus. There were no surgical complications related to either the free jejunal flap transfer or the donor site. Postoperatively, patients require re-education of their pattern of swallowing, but after the rehabilitation period all patients reported a satisfactory oral intake through the reconstructed esophagus to the abdomen without choking. There were no episodes of aspiration following reconstruction. With this new method to create a separate food pathway, patients can resume oral intake safely without choking and without permanent jejunostomy. This technique offers a useful solution for patients who suffer from recurrent choking and aspiration following injury or ablation of the pharynx.     

Extracellular ATP inhibits root gravitropism at concentrations that inhibit polar auxin transport

Raising the level of extracellular ATP to mM concentrations similar to those found inside cells can block gravitropism of Arabidopsis roots. When plants are grown in Murashige and Skoog medium supplied with 1 mM ATP, their roots grow horizontally instead of growing straight down. Medium with 2 mM ATP induces root curling, and 3 mM ATP stimulates lateral root growth. When plants are transferred to medium containing exogenous ATP, the gravity response is reduced or in some cases completely blocked by ATP. Equivalent concentrations of ADP or inorganic phosphate have slight but usually statistically insignificant effects, suggesting the specificity of ATP in these responses. The ATP effects may be attributable to the disturbance of auxin distribution in roots by exogenously applied ATP, because extracellular ATP can alter the pattern of auxin-induced gene expression in DR5-beta-glucuronidase transgenic plants and increase the response sensitivity of plant roots to exogenously added auxin. The presence of extracellular ATP also decreases basipetal auxin transport in a dose-dependent fashion in both maize (Zea mays) and Arabidopsis roots and increases the retention of [(3)H]indole-3-acetic acid in root tips of maize. Taken together, these results suggest that the inhibitory effects of extracellular ATP on auxin distribution may happen at the level of auxin export. The potential role of the trans-plasma membrane ATP gradient in auxin export and plant root gravitropism is discussed.     

Structural basis for phosphodependent substrate selection and orientation by the SCFCdc4 ubiquitin ligase

Cell cycle progression depends on precise elimination of cyclins and cyclin-dependent kinase (CDK) inhibitors by the ubiquitin system. Elimination of the CDK inhibitor Sic1 by the SCFCdc4 ubiquitin ligase at the onset of S phase requires phosphorylation of Sic1 on at least six of its nine Cdc4-phosphodegron (CPD) sites. A 2.7 A X-ray crystal structure of a Skp1-Cdc4 complex bound to a high-affinity CPD phosphopeptide from human cyclin E reveals a core CPD motif, Leu-Leu-pThr-Pro, bound to an eight-bladed WD40 propeller domain in Cdc4. The low affinity of each CPD motif in Sic1 reflects structural discordance with one or more elements of the Cdc4 binding site. Reengineering of Cdc4 to reduce selection against Sic1 sequences allows ubiquitination of lower phosphorylated forms of Sic1. These features account for the observed phosphorylation threshold in Sic1 recognition and suggest an equilibrium binding mode between a single receptor site in Cdc4 and multiple low-affinity CPD sites in Sic1.     

Heregulin is sufficient for the promotion of tumorigenicity and metastasis of breast cancer cells in vivo

Resistance of breast carcinomas to hormonal therapy is a clinical obstacle for the treatment of breast cancer. The molecular mechanisms and the factors involved in the progression of tumors from an estrogen (E2)-dependent to an E2-independent phenotype are not entirely understood. Heregulin (HRG) is a pleiotropic growth factor that binds to the erbB family of receptors, which are correlated with breast cancer progression and an aggressive phenotype in the breast carcinomas overexpressing the receptors. Previous studies in transgenic mice have shown that HRG is sufficient to induce mammary gland transformation and proliferation in the presence of hormonal stimulation. However, these studies did not address the important issue of the E2 independence that is part of the progression of breast cancer. In this study, we investigated the role of HRG in E2 independence. We were able to determine that HRG up-regulation was sufficient for the development of mammary tumors in the absence of E2 stimulation, a situation that mimics the progression of the human disease. We demonstrated that in ovariectomized nude mice, HRG induced E2 independence and antiestrogen resistance and promoted metastasis and preneoplastic transformation of the adjacent mouse mammary tissue. We show that one of the mechanisms by which HRG achieves the aggressive phenotype may be mediated via an increase in activated mitogen-activated protein kinase, an increase in a matrix-degrading enzyme, MMP-9, and the overexpression of vascular endothelial growth factors. The up-regulation of these genes occurred in the absence of any additional stimulation, in an autocrine manner. Our data provide new insights into the mechanisms of breast cancer progression in vivo, and reinforce the important role that HRG plays in this process.