[Intein-Mediated Protein trans-Splicing of the Recombinant Streptavidin on Magnetosomes].

When expressing streptavidin recombinant polypeptide on magnetosomes (called bacterial magnetic nanoparticles, or BMPs), the presence of endogenous bacterial biotin might be detrimental. In the study, the streptavidin monomer fragment (S1-116) was fused with the intein N-terminal (termed precursor S1-116-IN), and S1-116-IN was expressed in E. coli (BL21). Meanwhile, the SA117-160 fragment was fused with the C-terminal intein, and then this chimeric polypeptide was expressed on magnetosomes by fusion with magnetosome membrance protein MamF. In the in vitro protein splicing system, the purified engineered magnetosomes (BMP-SA117-160-IC) and the S1-116-IN precursor were mixed. Intein-mediated trans-splicing reaction was induced to produce the functional magnetic beads BMP-SA. Our results indicate that intein-mediated protein trans-splicing may lead to efficient synthesis of the recombinant streptavidin on the magnetosomes, showing its promising potential to produce other functional magnetic nanoparticles.

Extraction of proteins and preliminary characterization of physicochemical properties in Toona sinensis fruit.

We investigated the extraction of Toona sinensis fruit proteins and preliminarily characterized their physicochemical properties. The results showed that optimal extraction occurred under conditions of pH 10.5, a duration of 40 min, a liquid-to-solid ratio of 25:1, and a temperature of 40°C by an orthogonal design using T. sinensis fruit protein as the index and single factor. The total nitrogen content was 13.8 g/100 g and included 17 different amino acids. The glutamate level was highest at 35.37%, followed by arginine at 15.31%. The isoelectric point of T. sinensis fruit protein was between 6.8 and 10.0 with a typical absorption peak by infrared chromatography. Three protein bands were analyzed using SDS-polyacrylamide gel electrophoresis, with relative molecular weights of 55, 51, and 22 kDa. This study provides a theoretical basis for the comprehensive utilization of T. sinensis fruit by further investigating the biological activity of its proteins.

Mur (MNS 10) screening with a novel loop-mediated isothermal amplification assay in Zhongshan, China.

BACKGROUND: The distribution of the Mur blood group antigen is 5-7% in the south of China, and a much higher prevalence is observed in some areas of the region. Anti-Mur can cause hemolytic disease of the newborn and severe transfusion reactions. OBJECTIVES: Genetic testing is more ideal than conventional serological tests because antibodies for detection are usually not available. METHODS: In this study, a novel loop-mediated isothermal amplification (LAMP) assay for the detection of Mur blood group antigen was established. RESULTS: Fifteen of 275 (5·5%) samples were confirmed by LAMP as Mur antigen positive. All the Mur antigen-positive samples were GP.Mur subtype which was confirmed with sequencing. CONCLUSION: The LAMP method has identical results with conventional serology method but more suitable for large-scale screening.

The incidence of thyroid abnormalities in adults irradiated for lymphoma.

The aim of the study was to investigate the incidence of thyroid abnormalities in neck irradiated lymphoma patients. Of the 298 patients who had irradiation to the neck for lymphoma between 1966-1988, 174 were found to be alive and free of disease. These patients were invited to participate in the study. From the 174, 93 were able to participate (group 1). Two control groups were recruited; both were sex and aged matched. One group (group 2) consisted of lymphoma patients who were treated with chemotherapy (n=39) or irradiation to areas other than the neck (n=16). The other group (group 3) consisting of healthy volunteers (n=35) recruited from hospital staff and minor surgery attendees, had never had lymphoma or radiotherapy. All participants were required to complete a past medical history and thyroid symptom questionnaire, had blood taken for assays of thyroid stimulating hormone (TSH), thyroglobulin antibodies, thyroid peroxidase antibodies, sodium iodide symporter antibodies and TSH receptor antibodies and underwent ultrasound and clinical examination of the neck. A significant percentage of patients who had been irradiated in the neck had abnormalities on ultrasound, compared to groups 2 and 3 (77% vs 42% vs 24%). Abnormal TSH levels were found to be significantly more common in neck irradiated patients compared to the other groups (50% vs 9% vs 5%). There is a clear difference between neck irradiated patients and control groups. The importance of screening irradiated patients for thyroid abnormalities is re-emphasised.

Use of human menopausal gonadotropins in the treatment of endometrial defects associated with recurrent miscarriage: preliminary report.

OBJECTIVE: To test the hypothesis that controlled ovarian stimulation by gonadotropins, which enhances estrogen priming, is of beneficial value in the treatment of endometrial defects associated with recurrent miscarriage. DESIGN: A retrospective, observational, nonrandomized study. SETTING: A regional recurrent miscarriage clinic in a teaching hospital. PATIENT(S): Twenty-one subjects with otherwise unexplained recurrent miscarriage who had retarded endometrial development in the mid-luteal phase. Endometrial biopsies were timed by the luteinizing hormone surge. INTERVENTION(S): Controlled ovarian stimulation using human menopausal gonadotropins and repeat endometrial biopsy in the treatment cycle in 13 subjects. MAIN OUTCOME MEASURE(S): Histological dating of endometrial biopsy in treatment cycles and miscarriage rate in treatment and nontreatment cycles. RESULT(S): Eleven (85%) of the 13 biopsies in the treatment cycle were found to be normal. The miscarriage rate in the treatment group, 2 of 13, was significantly lower than that in the nontreatment group (7/12) (chi2 5.0, P<.05). CONCLUSION(S): In this small series, preliminary experience suggests that controlled ovarian stimulation by human menopausal gonadotropins in the follicular phase is an effective treatment for luteal phase defect associated with recurrent pregnancy loss. There is now a case for a prospective, controlled study to confirm the value of such a treatment.

Two regulatory elements in the 5' region of the mouse mitochondrial malate dehydrogenase gene.

We previously reported that the promoter region of the mouse mitochondrial malate dehydrogenase gene is located within the 160 base-pairs (bp) region upstream from the initiation codon, named P1, and that the -160/-131 bp region contains sequence(s) essential for the promoter activity of mitochondrial malate dehydrogenase gene. To define more precisely the regulatory mechanism of this gene, a 3' deletion analysis was performed using constructs with a constant 3' boundary at -130 bp. We obtained evidence of an additional positive regulatory element located between -393/-249 bp, named P2, the function of which can be separated from that of the P1. DNase I footprinting and gel-sift mobility experiments showed that nuclear protein binding to the P2 was not inhibited in the presence of the P1 sequences.

Prevalence of antibodies to hepatitis C virus in relation to surrogate markers in a blood donor population of Singapore.

Prevalence of antibodies to HCV is studied among a blood donor population in Singapore and its relationship to surrogate markers was examined. Sequential serum samples from 4,091 blood donors were tested for the presence of anti-HCV using the second generation immunoassay (Abbott). 275 random serum samples were tested for anti-HBc and ALT. All the samples positive for anti-HCV were also tested for anti-HBc and ALT. Only 22 of the 4,091 donor samples (0.54%) were repeatedly reactive for anti-HCV. Of the 275 random samples tested, 43 samples (15.6%) were positive for anti-HBc and 24 (8.7%) had ALT levels more than 45 IU/l. None of these 67 samples were positive for anti-HCV. Only 3 of the 22 anti-HCV positive samples (13.6%) were positive for anti-HBc and only 6 samples (27.2%) had ALT level more than 45 IU/l. The prevalence of anti-HCV among the donors is only 0.54% which is much lower than the prevalence of HBV. An important finding is that about 60% of the donors positive for anti-HCV had no detectable surrogate markers. Exclusion of blood donors positive for anti-HBc, if implemented in an area where the prevalence of HBV infection is relatively high will result in the loss of blood donors estimated to be 15.6% and the use of raised ALT will result in a further loss of 6.1% of the blood donors.

A novel Fmoc-based anchorage for the synthesis of protected peptides on solid phase.

A novel bifunctional compound, 9-(hydroxymethyl)-2-fluoreneacetic acid, was synthesized, coupled to benzhydrylamine-resin, and evaluated for its application to the solid phase synthesis of protected peptide fragments. Anchor-bond cleavage was achieved with 15% piperidine/DMF. A protected heptapeptide, Boc-Val-Val-Ser(Bzl)-His(Tos)-Phe-Asn-Lys-(Z)-OH, corresponding to the sequence (1-7) of rat-transforming growth factor-alpha, was synthesized using this new support with an overall yield of 46%.

Regulatory regions of the mitochondrial and cytosolic isoenzyme genes participating in the malate-aspartate shuttle.

The malate-aspartate shuttle, consisting of mitochondrial and cytosolic aspartate aminotransferase and mitochondrial and cytosolic malate dehydrogenase, is a major pathway for the transport of reducing equivalents from cytosol to mitochondria in mammals. To elucidate molecular mechanisms regulating metabolic coordination between the mitochondria and the cytosol, we analyzed the 5'-flanking regulatory regions of the complete set of mouse isoenzyme genes playing a pivotal role in the shuttle. Deletion analysis and an in vivo transfection assay, using NIH3T3 cells, revealed that all the promoter regions are located within the 300-base pair regions upstream from the initiation codon. Subsequently, DNase I footprinting analyses using NIH3T3 cell nuclear extracts led to identification of several protein binding sites within these promoter regions. A synthetic oligomer containing the consensus binding site sequence for CTF/NFI, a transcription factor for RNA polymerase II, competed for the binding of proteins to the promoter regions of cytosolic aspartate aminotransferase and mitochondrial and cytosolic malate dehydrogenase genes, but not for that of the mitochondrial aspartate amino-transferase gene. On the other hand, a synthetic oligomer containing the consensus binding site sequence for Sp1, which activates transcription from promoters containing properly positioned GC boxes, competed for protein(s) binding to the promoter region of the mitochondrial aspartate aminotransferase gene.

Molecular structures and evolution of mouse isozyme genes functioning in the malate-aspartate shuttle.

To examine molecular mechanisms of transcription of mammalian isozyme genes functioning in the malate-aspartate shuttle and to observe structural and evolutionary relationships, we investigated gene organizations of cAspAT and mAspAT, and cMDH and mMDH, and isolated and characterized cDNAs and genomic DNAs for these isozymes in mice. The deduced amino acid sequences of mouse cAspAT and mAspAT showed about 47%, and those of mouse cMDH and mMDH, about 23% overall homology. Surprisingly, the homology between the mouse cMDH and thermophilic bacterial MDH, as well as the homology between the mouse mMDH and E. coli MDH, markedly exceeds the intraspecies sequence homology between mMDH and cMDH from mice. The first duplication of a common ancestral MDH gene should thus have occurred long before the emergence of the eukaryotic cells, and subsequently, the mammalian mMDH and E. coli MDH genes have evolved from one of the duplicates. The mammalian cMDH and Thermus flavus MDH genes have no doubt evolved from one of the other duplicates. Moreover, structural organizations of the two-pairs of isozyme genes indicated that introns antedate the divergence of these mitochondrial and cytosolic isozyme genes. The 5' ends of all four isozyme genes lacked the TATA and CAAT boxes characteristic of eukaryotic promoters but did contain G + C-rich sequences and multiple transcription-initiation sites. We found several highly conserved regions in the 5' flanking sequences between mAspAT and cAspAT, between mMDH and mAspAT, and between cMDH and cAspAT genes.