Nipah virus glycoprotein: production in baculovirus and application in diagnosis

A method for serological diagnosis of Nipah virus (NiV) is described. DNA encoding truncated G protein of NiV was cloned into the pFastBac HT vector, and the fusion protein to His-tag was expressed in insect cells by recombinant baculovirus. The resulting His-G recombinant fusion protein was purified by affinity chromatography and used as the coating antigen for serological testing by indirect enzyme-linked immunosorbant assay (ELISA). When tested against a panel of swine serum samples, the recombinant G protein-based ELISA successfully discriminated all 40 samples previously determined to be serum neutralizing test (SNT) positive from 11 SNT negatives samples. The data show that the recombinant G protein exhibits the antigenic epitopes and conformation necessary for specific antigen-antibody recognition. The main advantage of the recombinant G protein-based NiV ELISA compared to an ELISA using whole virus antigen is the use of a single antigenic protein instead of inactivated whole virus which is required to be prepared under high risk and cost. This test is suitable for routine diagnosis of NiV and also for epidemiological surveys as it allows highly reliable testing of a large number of sera rapidly.     

A simple method for computer quantification of stage REM eye movement potentials

We describe a simple method for computer quantification of eye movement (EM) potentials during REM sleep. This method can be applied by investigators using either period-amplitude (PA) or Fast Fourier Transform (FFT) spectral EEG analysis without special hardware or computer programming. It provides good correlations with visual ratings of EM in baseline sleep and after administration of GABAergic hypnotics. We present baseline data for both PA and FFT measures for 16 normal subjects, studied for 5 consecutive nights. Both visually rated and computer-measured EM density (EMD) showed high night-to-night correlations across baseline and drug nights and the computer measures detected the EMD suppression that is produced by GABAergic drugs. Measurement of EM in addition to stage REM provides biologically significant information and application of this simple computer method, which does not require pattern recognition algorithms or special hardware, could provide reliable data that can be compared across laboratories.     

Frataxin expression rescues mitochondrial dysfunctions in FRDA cells

Friedreich's ataxia (FRDA) is the result of mutations in the nuclear-encoded frataxin gene, which is expressed in mitochondria. Several lines of evidence have suggested that frataxin is involved in mitochondrial iron homeostasis. We have transfected the frataxin gene into lymphoblasts of FRDA compound heterozygotes (FRDA-CH) with deficient frataxin expression to produce FRDA-CH-t cells in which message and protein are rescued to near-physiological levels. FRDA-CH cells were more sensitive to oxidative stress by challenge with free iron, hydrogen peroxide and the combination, consistent with a Fenton chemical mechanism of pathophysiology, and this sensitivity was rescued to control levels in FRDA-CH-t cells. Iron challenge caused increased mitochondrial iron levels in FRDA-CH cells, and a decreased mitochondrial membrane potential (MMP), both of which were rescued in FRDA-CH-t cells. The rescue of the low MMP, and high mitochondrial iron concentration by frataxin overexpression suggests that these cellular phenotypes are relevant to the central pathophysiological process in FRDA which is aggravated by exposure to free iron. However, even at physiological iron concentrations, FRDA-CH cells had decreased MMP as well as lower activities of aconitase and ICDH (two enzymes supporting MMP), and twice the level of filtrable mitochondrial iron (but no increase in total mitochondrial iron), and the observed phenotypes were either fully or partially rescued in FRDA-CH-t cells. Free iron is known to be toxic. The observation that frataxin deficiency (either directly or indirectly) causes an increase in filtrable mitochondrial iron provides a new hypothesis for the mechanism of cell death in this disease, and could be a target for therapy.     

高师贫困生自我效能感与主观幸福感的特点及关系研究

目的分析高师贫困生自我效能感、主观幸福感的特点及关系，为心理健康教育提供依据。方法采用一般自我效能感量表、生活满意度量表、快乐感量表，对218名贫困生和216名非贫困生进行测查。结果①高师贫困生生活满意度、快乐感显著低于非贫困生（t=4．811，P〈0．001；t=2．504，P〈0．05），自我效能感差异不显著。②高师贫困生自我效能感存在性别差异（t=2．117，P〈0．05），男生的自我效能感显著高于女生；生活满意度年级差异显著（F=3．547，P〈0．05），大一学生的生活满意度显著高于大二和大三学生，大二和大三之间的差异不显著。③高师贫困生自我效能感与生活满意度、快乐感呈显著正相关（r=0．349，P〈0．01；r=0．436，P〈0．01）。自我效能感水平越高，幸福感越强。结论高师贫困生主观幸福感相对较低，自我效能感与主观幸福感显著相关，通过提升自我效能感水平可以增强主观幸福感。     

Partial androgen insensitivity with phenotypic variation caused by androgen receptor mutations that disrupt activation function 2 and the NH(2)- and carboxyl-terminal interaction

Partial androgen insensitivity with sex phenotype variation in two unrelated families was associated with missense mutations in the androgen receptor (AR) gene that disrupted the AR NH(2)-terminal/carboxy terminal interaction. Each mutation caused a single amino acid change within the region of the ligand-binding domain that forms activation function 2 (AF2). In one family, the mutation I737T was in alpha helix 4 and in the other F725L was between helices 3 and 4. Neither mutation altered androgen binding as determined by assays of mutant AR in the patient's cultured genital skin fibroblasts or of recombinant mutant receptors transfected into COS cells. In transient cotransfection assays in CV1 cells, transactivation with the AR mutants at low concentrations of DHT was reduced several fold compared with wild-type AR but increased at higher concentrations. Defects in NH(2)-terminal/carboxy terminal interactions were identified in mammalian two hybrid assays. In similar assays, there was reduced binding of the p160 coactivators TIF2/SRC2 and SRC1 to the mutant AR ligand binding domains (LBD). In the family with AR I737T, sex phenotype varied from severely defective masculinization in the proband to a maternal great uncle whose only manifestation of AIS was severe gynecomastia. He was fertile and passed the mutation to two daughters. The proband of the F725L family was also incompletely masculinized but was raised as a male while his half-sibling by a different father was affected more severely and reared as a female. These studies indicate that the function of an AR AF2 mutant in male development can vary greatly depending on the genetic background.     

Drosophila mutants with progressive atrophy in dorsal longitudinal muscles

We have characterized six chemically induced mutations of the Drosophila dlm-defective (d l m d) gene. The mutants are flightless, but they have an otherwise normal appearance. By electron microscopic examination, a focal atrophy was found in their dorsal longitudinal muscle (DLM) fibers, but no abnormalities in nerve conduction or synaptic transmission were detected by electrophysiological tests. The nerve-evoked muscle spike also seemed to be normal, except that the resting potential of DLM in mutant flies was lower and their membrane excitability was higher than those in the wild type flies. The possible causes of the DLM degeneration in this strain are discussed.     

Characterization of knitted polymeric scaffolds for potential use in ligament tissue engineering

Different scaffolds have been designed for ligament tissue engineering. Knitted scaffolds of poly-L-lactic acid (PLLA) yarns and co-polymeric yarns of PLLA and poly(glycolic acid) (PLGA) were characterized in the current study. The knitted scaffolds were immersed in medium for 20 weeks, before mass loss, molecular weight, pH value change in medium were tested; changes in mechanical properties were evaluated at different time points. Results showed that the knitted scaffolds had 44% porosity. There was no significant pH value change during degradation, while there was obvious mass loss at initial 4 week, as well as smooth molecular weight drop of PLLA. PLGA degraded more quickly, while PLLA kept its integrity for at least 20 weeks. Young's modulus increased while tensile strength and strain at break decreased with degradation time; however, all of them could maintain the basic requirements for ACL reconstruction. It showed that the knitted polymeric structures could serve as potential scaffolds for tissue-engineered ligaments.     

Identification of two mutations in a compound heterozygous child with dihydrolipoamide dehydrogenase deficiency

An infant girl with elevated blood lactate, pyruvate, and plasma branched-chain amino acids was diagnosed with dihydrolipoamide dehydrogenase (E3; dihydrolipoamide: NAD+ oxidoreductase, EC 1.8.1.4) deficiency. Activities of the pyruvate dehydrogenase complex and E3 from patient were 26 and 2% of controls in blood lymphocytes, and 11 and 14% in cultured skin fibroblasts, respectively. Western blot analysis demonstrated that the amount of E3 protein in fibroblasts from the patient and her father was about half of controls, while Northern blot analysis showed normal amounts of E3 RNA. DNA sequencing of cloned full-length E3 cDNAs from the patient revealed two mutations in separate alleles. One is a single base insertion of an extra adenine in the last codon of the leader peptide sequence (TAC-->TAAC) leading to a nonsense mutation which results in the premature termination of the precursor E3 polypeptide (Y35X). The other is a missense mutation due to substitution of guanine for adenine, causing an Arg-->Gly substitution at amino acid 460 of the mature protein (R460G) which triggers the loss of E3 activity probably by structural change in the E3 dimer. DNA sequencing of E3 cDNAs from the parents demonstrated that the nonsense mutation was inherited from the father and the missense mutation was inherited from the mother.