Regulation Fe65 localization to the nucleus by SGK1 phosphorylation of its Ser566 residue

Fe65 is characterized as an adaptor precursor (APP) through its PID2 element, as well as with the other members of the APP protein family. With the serum- and glucocorticoid-induced kinase 1 (SGK1) substrate specificity information, we found that the putative site of phosphorylation in Fe65 by SGK1 is present on its Ser(566) residue in (560)CRVRFLSFLA(569)(X60469). Thus, we demonstrated that Fe65 and the fluorescein-labeled Fe65 peptide FITC-(560)CRVRFLSFLA(569) are phosphorylated in vitro by SGK1. Phosphorylation of the Ser(566) residue was also demonstrated using a Ser566 phospho-specific antibody. The phospho Fe65 was found mainly in the nucleus, while Fe65 S556A mutant was localized primarily to the cytoplasm. Therefore, these data suggest that SGK1 phosphorylates the Ser(566) residue of Fe65 and that this phosphorylation promotes the migration of Fe65 to the nucleus of the cell.     

Mucin secretion by the human colon cell line LS174T is regulated by bile salts

We recently reported that bile salts play a role in the regulation of mucin secretion by cultured dog gallbladder epithelial cells. In this study we have examined whether bile salts also influence mucin secretion by the human epithelial colon cell line LS174T. Solutions of bile salts were applied to monolayers of LS174T cells. Mucin secretion was quantified by measuring the secretion of [3H]GlcNAc labeled glycoproteins. Both unconjugated bile salts as well as taurine conjugated bile salts stimulated mucin secretion by the colon cells in a dose-dependent fashion. Hydrophobic bile salts were more potent stimulators than hydrophilic bile salts. Free (unconjugated) bile salts were more stimulatory compared with their taurine conjugated counterparts. Stimulation of mucin secretion by LS174T cells was found to occur at much lower bile salt concentrations than in the experiments with the dog gallbladder epithelial cells. The protein kinase C activators PMA and PDB had no stimulatory effect on mucin secretion. We conclude that mucin secretion by the human colon epithelial cell line LS174T is regulated by bile salts. We suggest that regulation of mucin secretion by bile salts might be a common mechanism, by which different epithelia protect themselves against the detergent action of bile salts, to which they are exposed throughout the gastrointestinal tract.      

Enzymatic characterization of a maltogenic amylase from Lactobacillus gasseri ATCC 33323 expressed in Escherichia coli

A gene corresponding to a maltogenic amylase (MAase) in Lactobacillus gasseri ATCC 33323 (lgma) was cloned and expressed in Escherichia coli. The recombinant LGMA was efficiently purified 24.3-fold by one-step Ni-NTA affinity chromatography. The final yield and specific activity of the purified recombinant LGMA were 68% and 58.7 U/mg, respectively. The purified enzyme exhibited optimal activity for beta-CD hydrolysis at 55 degrees C and pH 5. The relative hydrolytic activities of LGMA to beta-CD, soluble starch or pullulan was 8:1:1.9. The activity of LGMA was strongly inhibited by most metal ions, especially Zn(2+), Fe(2+), Co(2+) and by EDTA. LGMA possessed some unusual properties distinguishable from typical MAases, such as being in a tetrameric form, having hydrolyzing activity towards the alpha-(1,6)-glycosidic linkage and being inhibited by acarbose.     

Tritaenia Maegdefrau et Rudolf, Mesozoic 'Sciadopitys-like' leaves in mass accumulations

The Late Jurassic to Early Cretaceous genus Tritaenia Maegdefrau et Rudolf 1969 is problematic because of: (1) missing authentic material of its type species, T. linkii (Roemer 1839) Maegdefrau et Rudolf; and (2) Watson and Harrison's (1998) synonymization of T. linkii with Pseudotorellia heterophylla Watson. This paper: (1) rectifies the status of T. linkii on the basis of newly recovered specimens carrying the original author's authentication; and (2) gives the basis for rejecting Watson and Harrison's claim that T. linkii and T. crassa (Seward) Bose et Manum 1991 represent linear leaves of the heterophyllous taxon Pseudotorellia heterophylla. The three species of Tritaenia known to date (T. crassa, T. linkii, T. scotica) are reviewed, and the genus is compared with other Mesozoic so-called 'Sciadopitys-like' hypostomatic leaves with a median stomatal zone, many of which occur in mass accumulations such as T. linkii. Deciduousness is indicated for T. linkii and T. crassa by their occurrence in mass accumulations and the possession of well-developed abscission scars. Known mass accumulations of fossil foliage are reviewed and their implications for palaeoenvironmental interpretations discussed.     

Nutrient-induced signal transduction through the protein kinase A pathway and its role in the control of metabolism, stress resistance, and growth in yeast

Yeast cells growing in the presence of glucose or a related rapidly-fermented sugar differ strongly in a variety of physiological properties compared to cells growing in the absence of glucose. Part of these differences appear to be caused by the protein kinase A (PKA) and related signal transduction pathways. Addition of glucose to cells previously deprived of glucose triggers cAMP accumulation, which is apparently mediated by the Gpr1-Gpa2 G-protein coupled receptor system. However, the resulting effect on PKA-controlled properties is only transient when there is no complete growth medium present. When an essential nutrient is lacking, the cells arrest in the stationary phase G0. At the same time they acquire all characteristics of cells with low PKA activity, even if there is ample glucose present. When the essential nutrient is added again, a similar PKA-dependent protein phosphorylation cascade is triggered as observed after addition of glucose to glucose-deprived cells, but which is not cAMP-mediated. Because the pathway involved requires a fermentable carbon source and a complete growth medium, at least for its sustained activation, it has been called “fermentable growth medium (FGM)-induced pathway.”     

Technology platform development for targeted plasma metabolites in human heart failure

Background

Heart failure is a multifactorial disease associated with staggeringly high morbidity and motility. Recently, alterations of multiple metabolites have been implicated in heart failure; however, the lack of an effective technology platform to assess these metabolites has limited our understanding on how they contribute to this disease phenotype. We have successfully developed a new workflow combining specific sample preparation with tandem mass spectrometry that enables us to extract most of the targeted metabolites. 19 metabolites were chosen ascribing to their biological relevance to heart failure, including extracellular matrix remodeling, inflammation, insulin resistance, renal dysfunction, and cardioprotection against ischemic injury.

Results

In this report, we systematically engineered, optimized and refined a protocol applicable to human plasma samples; this study contributes to the methodology development with respect to deproteinization, incubation, reconstitution, and detection with mass spectrometry. The deproteinization step was optimized with 20% methanol/ethanol at a plasma:solvent ratio of 1:3. Subsequently, an incubation step was implemented which remarkably enhanced the metabolite signals and the number of metabolite peaks detected by mass spectrometry in both positive and negative modes. With respect to the step of reconstitution, 0.1% formic acid was designated as the reconstitution solvent vs. 6.5 mM ammonium bicarbonate, based on the comparable number of metabolite peaks detected in both solvents, and yet the signal detected in the former was higher. By adapting this finalized protocol, we were able to retrieve 13 out of 19 targeted metabolites from human plasma.

Conclusions

We have successfully devised a simple albeit effective workflow for the targeted plasma metabolites relevant to human heart failure. This will be employed in tandem with high throughput liquid chromatography mass spectrometry platform to validate and characterize these potential metabolic biomarkers for diagnostic and therapeutic development of heart failure patients.     

Enhancing thermostability of maltogenic amylase from Bacillus thermoalkalophilus ET2 by DNA shuffling

DNA shuffling was used to improve the thermostability of maltogenic amylase from Bacillus thermoalkalophilus ET2. Two highly thermostable mutants, III-1 and III-2, were generated after three rounds of shuffling and recombination of mutations. Their optimal reaction temperatures were all 80 degrees C, which was 10 degrees C higher than that of the wild-type. The mutant enzyme III-1 carried seven mutations: N147D, F195L, N263S, D311G, A344V, F397S, and N508D. The half-life of III-1 was about 20 times greater than that of the wild-type at 78 degrees C. The mutant enzyme III-2 carried M375T in addition to the mutations in III-1, which was responsible for the decrease in specific activity. The half-life of III-2 was 568 min while that of the wild-type was < 1 min at 80 degrees C. The melting temperatures of III-1 and III-2, as determined by differential scanning calorimetry, increased by 6.1 degrees C and 11.4 degrees C, respectively. Hydrogen bonding, hydrophobic interaction, electrostatic interaction, proper packing, and deamidation were predicted as the mechanisms for the enhancement of thermostability in the enzymes with the mutations.     

长春市儿童医院1998～2001年婴幼儿杯状病毒腹泻流行病学研究

人类杯状病毒(human calicivirus,HuCV)是引起儿童和成人非菌性胃肠炎的主要病原之一.为了掌握HuCV在我国的流行情况,1998年7月至2001年6月,从长春市儿童医院2343例5岁以下腹泻患儿中共收集粪便标本1264份,其中1056份来自2135例住院患儿.对轮状病毒检测为阴性的588份标本,经多价酶免疫试验(EIA)和两组引物反转录-聚合酶链反应(RT-PCR)检测HuCV,202份为阳性,其中住院患儿标本178份,HuCV检出率为16.9%.HuCV腹泻以2岁以下儿童为主(占96%),流行高峰季节为11月至次年3月.选择17株HuCV进行分子鉴定,15株属GⅡ-4群,1株属GⅡ-3群,另1株属GⅠ-2群,表明GⅡ-4群HuCV是我国流行的优势株.根据HuCV住院患儿的监测资料初步估计,HuCV腹泻住院率约为0.5‰～2.4‰.讨论了长春地区HuCV的流行趋势和疾病负担.以上结果为我国HuCV腹泻的预防和控制提供了科学依据.