OBJECTIVETo investigate the distribution, changes and a possible role for retinal dopamine transporter (DAT) in experimental myopia in chickens.

METHODSTwo-day-old chickens were divided into four groups. Chicken eyes were fitted with lenses of -10D,-20D and translucent goggles unilaterally. Normal eyes were used as controls. After 3 wk, all chickens were given an intramuscular injection of (125)I-beta-CIT 2beta-carbomethoxy-3beta-(4-iodophenyl)tropane and sacrificed two hours post injection. Retinal pigment epithelium (RPE) and the neural retina were obtained together or RPE was dissected out from the neural retina. Radioactive DAT from each specimen was assayed by gamma-counter.

RESULTSRetinal DAT was detected in RPE specimens rather than in the neural retina in all eyes. Radioactive DAT in myopic eyes was higher, compared with control eyes.

CONCLUSIONSRetinal DAT is mainly located in the RPE and may be involved in the formation of lens induced myopia (LIM) and form deprivation myopia (FDM). These methods may provide a new approach for further studying the role of the dopamine system in experimental myopia.

Animals ; Chickens ; Dopamine Plasma Membrane Transport Proteins ; Eye ; growth & development ; Membrane Glycoproteins ; Membrane Transport Proteins ; analysis ; physiology ; Myopia ; metabolism ; Nerve Tissue Proteins ; Retina ; chemistry

Streptomyces coelicolor is the model species among streptomycetes. Until now, proteomic analyses of S. coelicolor have been conducted using two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry method, few integral membrane proteins were identified due to the hydrophobic and low-abundance nature of these proteins. In this work, 154 possible inner membrane proteins from S. coelicolor were identified using high pH-proteinase K sample preparation method and multidimensional protein identification technology, among them 44 are integral membrane proteins containing at least one transmembrane domain, most peptides and their corresponding proteins were identified experimentally for the first time.
Bacterial Proteins ; analysis ; Cell Membrane ; chemistry ; Genome, Bacterial ; genetics ; Mass Spectrometry ; methods ; Membrane Proteins ; analysis ; Proteome ; analysis ; genetics ; Streptomyces coelicolor ; chemistry ; genetics

BACKGROUNDThe diagnosis of Parkinson's disease is presently based on non-specific symptoms. However, radionuclide dopamine transporters imaging can provide specific diagnostic tool for Parkinson's disease. This study was designed to investigate the effects of imaging of dopamine transporters with 99mTc-TRODAT-1 in early diagnosis or differential diagnosis of Parkinson's disease.

METHODSNine normal monkeys were used to establish N-methyl-4-phenyl-1, 2, 3, 6-tetra-hydropyridine (MPTP) hemi-Parkinsonian animal models, and they were subjected to imaging. Twenty-nine patients with Parkinson's disease, 12 age-matched healthy volunteers, and 18 age-matched patients with Parkinson's syndrome were investigated. Single photon emission computer tomography (SPECT) was performed 3 hours after intravenous injection of 740 MBq 99mTc-TRODAT-1. Striatum specific uptake of 99mTc-TRODAT-1 was calculated according to the ratio of striatum (ST) to cerebellum (CB) in dopamine transporters uptake.

RESULTSIn normal monkeys, bilateral ratio of ST/CB was 2.34 +/- 0.41. After the injection of MPTP, uptake rate of 99mTc-TRODAT-1 at damaged region was much lower than that at the contralateral region, resulting in a significant difference in the ratio of ST/CB (right: ST/CB = 1.73 +/- 0.35; left: ST/CB = 1.90 +/- 0.30), especially in hemi-Parkinsonian model monkeys (right: ST/CB = 1.29 +/- 0.17; left: ST/CB = 1.80 +/- 0.33). The ratios of ST/CB were 1.57 +/- 0.17 and 1.61 +/- 0.14 for the right and left respectively in the healthy volunteers, 1.04 +/- 0.29 and 1.06 +/- 0.30 in the age-matched patients with Parkinson's disease, and 1.56 +/- 0.17 and 1.59 +/- 0.18 in the age-matched patients with Parkinson's disease syndrome. A significant difference was noted between group of Parkinson's disease, normal controls and Parkinson's disease syndrome.

CONCLUSIONThe results suggest that 99mTc-TRODAT-1 dopamine transporters SPECT has clinical application value in early diagnosis or differential diagnosis of Parkinson's disease.

Adult ; Aged ; Animals ; Dopamine Plasma Membrane Transport Proteins ; Female ; Haplorhini ; Humans ; Male ; Membrane Glycoproteins ; analysis ; Membrane Transport Proteins ; analysis ; Middle Aged ; Nerve Tissue Proteins ; analysis ; Organotechnetium Compounds ; Parkinson Disease ; diagnosis ; Radiopharmaceuticals ; Tomography, Emission-Computed, Single-Photon ; Tropanes

Humans ; In Situ Hybridization, Fluorescence ; Membrane Proteins ; analysis ; NF-kappa B ; analysis ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; Tissue Array Analysis ; methods ; Tumor Cells, Cultured

In the study presented here, identification, purification, and partial characterization of a hemin-regulated protein in Prevotella nigrescens were carried out. The results of this study confirm that the availability of hemin influences the expression of a selected membrane protein as well as the growth rate of P. nigrescens ATCC 33563. The 50 kDa cell envelope associated protein, whose expression is hemin regulated, is considered to be a putative hemin-binding protein from P. nigrescens. Disulfide bonds were not present in this protein, and N'-terminal amino acid sequence analysis revealed that this protein belongs to a new, so far undescribed protein. The 50 kDa protein was found to be rich in hydrophilic amino acids, with glycine comprising approximately 60% of the total amino acids. The study described here is the first to identify, purify, and biochemically characterize a putative hemin-binding protein from P. nigrescens. Work is in progress to further characterize the molecular structure of this protein.
Amino Acids ; Glycine ; Hemin ; Membrane Proteins ; Molecular Structure ; Prevotella nigrescens* ; Prevotella* ; Sequence Analysis, Protein

Cell Membrane ; metabolism ; Gastrointestinal Stromal Tumors ; diagnosis ; metabolism ; Humans ; Immunohistochemistry ; Proto-Oncogene Proteins c-kit ; analysis

OBJECTIVETo evaluate the biological characteristics of Campylobacter jejuni (CJ) cultured on different culture media and their expression abundance of outer membrane proteins (OMPs).

METHODSCJ was cultured on the improved Bull's medium yolk agar, improved Bull's blood agar or improved Bull's agar for 48 h. The biological characteristics of the bacteria, including the colony feature, morphology, motility, biochemistry, and results of indirect fluorescence test were observed and compared. OMP of the cultured CJ was extracted using 0.2 mol/L and glycine-hydrochloride buffered solution (pH 2.2) and identified by SDS-PAGE to compare the expression abundance of the OMPs with molecular weight of 28-31 kD.

RESULTSCJ exhibited typical biological characteristics with larger cell body and more rapid growth on improved Bull's medium yolk agar than those on improved Bull's blood agar and improved Bull's agar. The bacteria grown on improved Bull's medium yolk agar showed also greater expression abundance of the OMPs with molecule mass between 28 kD and 31 kD.

CONCLUSIONImproved Bull's medium yolk agar allows rapid growth of CJ with typical biological characteristics and enhanced expression of the OMPs with molecular weight of 28 -31 kD, and can be widely used in CJ subunit vaccine development, CJ epidemiological survey, CJ food safety examination, and CJ quarantine.

Bacterial Outer Membrane Proteins ; analysis ; metabolism ; Campylobacter jejuni ; growth & development ; metabolism ; Culture Media

OBJECTIVETo establish enzyme-linked immunosorbent assay (ELISA) for quantitative detection of Golgi protein73 (GP73) in serum.

METHODSA sandwich reaction was preformed with horseradish peroxidase labeled monoclonal antibody of GP73 as the catalytic enzyme. Several reactions liquid's concentration and reaction conditions were optimized. The method was evaluated in all aspects such as linear range, sensitivity, specificity, stability and so on.

RESULTSThe linear range was 25-500 ng/ml. The detection limit was 18.5 ng/ml. Inter-assay and intra-assay RSD were both less than 10%. The recoveries of three different spiked concentration samples were 95.3%, 92.6% and 103.7%. After stored at 4 degrees C and 37 degrees C for 3, 5, 7 days, the analysis showed correlation coefficient higher than 0.98 and RSD lower than 10%.

CONCLUSIONEstablished ELISA for quantity determination of serum GP73 has high accuracy, sensitivity and repeatability.

Antibodies, Monoclonal ; analysis ; Enzyme-Linked Immunosorbent Assay ; methods ; Humans ; Membrane Proteins ; blood ; Sensitivity and Specificity

OBJECTIVE: To determine the frequencies of deafness gene mutations among patients with non-syndromic hearing loss (NSHL) from northern Jiangsu province. METHODS: A total of 117 patients with NSHL were enrolled. The coding region of GJB2 gene, IVS7-2A>G and 2168A>G mutations of SLC26A4 gene, and 1555A>G and 1494C>T mutations of mitochondrial DNA 12S rRNA were subjected to Sanger sequencing. Patients in whom no mutation was detected were further tested by targeted gene capture and high-throughput sequencing. RESULTS: Among the 117 patients, 86 (73.50%) were found to carry mutations. GJB2 gene mutations were found in 61 patients (52.14%), including 22 (18.80%) with homozygous mutations and 39 (33.33%) with heterozygous mutations. SLC26A4 gene mutations were found in 19 patients (16.24%), including 4 (3.42%) with homozygous mutations and 15 with heterozygous mutations (14.53%). Mitochondrial 12S rRNA gene mutation was found in 6 patients (5.13%). Targeted gene capture and high-throughput sequencing of 8 patients identified 4 further cases, including 1 with RDX gene 129_130del and 76_79del compound heterozygous mutations, 1 with OTOF gene 1274G>C homozygous mutation, 1 with SLC26A4 gene 919-2A>G and IVS16-6G>A compound heterozygous mutation, and 1 with SLC26A4 gene 919-2A>G and A1673T compound heterozygous mutation. CONCLUSION The frequency of mutation among patients with NSHL from north Jiangsu was 73.50%, and GJB2 gene was most commonly mutated.
China ; Connexins ; DNA Mutational Analysis ; DNA, Mitochondrial ; Hearing Loss ; genetics ; Humans ; Membrane Proteins ; Mutation ; Sulfate Transporters

OBJECTIVETo investigate the clinical features and proline-rich transmembrane protein 2 (PRRT2) gene mutation in patients with paroxysmal kinesigenic dyskinesia (PKD).

METHODClinical information was collected at Peking University First Hospital from January 2004 to July 2014. In total, 10 patients with PKD were recruited, and all were males. Among them, four patients were the probands from four PKD families and the other six patients were sporadic cases. Clinical information was analyzed. Peripheral blood samples for DNA study were collected from PKD patients and their family members. Genomic DNA was extracted using standard procedures. Mutation analysis of PRRT2 was performed by Sanger sequencing after PCR.

RESULTOf the 10 patients, the median age of dyskinesias onset was 10 years, ranging from 4 to 13 years. The description of their attacks were abnormal involuntary movements provoked by sudden movements, without loss of consciousness. Five patients exhibited dystonia, two patients exhibited choreoathetosis, and three patients had mixed (dystonia and choreoathetosis) dyskinesias. The duration of the attacks lasted for 3 to 30 seconds. The frequency ranged from once per month to twenty times per day. PRRT2 mutations, c. 649_650insC (p. R217PfsX8), were found in all the four PKD families. Mutation c. 649_650insC was also detected in two of the six sporadic PKD cases, inheriting from their asymptomatic mother.

CONCLUSIONThe onset age of PKD could be in the early childhood. The clinical features of the familial cases and sporadic cases showed no difference. The attacks manifested as dystonia, choreathetosis, or mixed. PRR2 mutations could be identified in familial or sporadic cases with PKD. Mutation c. 649_650insC is the hotspot mutation of PRRT2 gene.

Adolescent ; Child ; Child, Preschool ; DNA Mutational Analysis ; Dystonia ; genetics ; Humans ; Male ; Membrane Proteins ; genetics ; Mutation ; Nerve Tissue Proteins ; genetics