OBJECTIVETo identify pathogenic mutations of ANTXR2 gene in a patient with juvenile hyaline fibromatosis.

METHODSGenomic DNA was extracted from peripheral venous blood sample from the patient. All coding exons (exons 1-17) and splicing sites of the ANTXR2 gene were amplified with PCR. Potential mutations were detected with direct sequencing of the PCR products. 100 unrelated healthy subjects were used as the controls. CLUSTALX (1.81) was employed to analyze cross-species conservation of the mutant amino acid. Impact of the mutations was analyzed with software including SIFT, PolyPhen-2 and MutationTaster.

RESULTSA compound heterozygous mutation c.1074delT/c.1153G>C, was identified, among which c.1153G>C has not been reported previously and was predicted to be probably damaging. Both mutations were not found among the 100 healthy controls.

CONCLUSIONThe patient's condition may be attributed to the compound heterozygous mutations of c.1074delT and c.1153G>C of the ANTXR2 gene. Above results has facilitated molecular diagnosis for this patient.

Child, Preschool ; Female ; Heterozygote ; Humans ; Hyalinosis, Systemic ; diagnosis ; genetics ; Mutation ; Receptors, Peptide ; genetics

Animals ; Anthrax ; microbiology ; Antigens, Bacterial ; genetics ; Bacillus anthracis ; genetics ; Bacterial Toxins ; genetics ; Gene Expression Regulation, Bacterial ; Humans ; Receptors, Peptide ; genetics

OBJECTIVE: To explore the clinical characteristics and genetic basis for a pair of twins affected with hyaline fibromatosis syndrome (HFS). METHODS: Clinical data of the twins were retrospectively analyzed. High-throughput sequencing was carried out to detect potential pathogenic variants. CLUSTALX was employed to analyze cross-species conservation of the mutant amino acids. Impact of the mutations was predicted by using software including PolyPhen-2 and Mutation taster. RESULTS: The pair of twins have featured growth and intelligence retardation, and were found to carry compound heterozygous variants of the ANTXR2 gene including c.1214G>A and c.1074delT, among which c.1214G>A was unreported previously. Both variants were predicted to be pathogenic. In addition to growth and mental delay, the pair of twins also featured hyperplasia of the gum and soft tissue-like masses of the auricle. The younger brother had rupture of the auricle mass during follow-up. CONCLUSION The patients' condition can probably be attributed to the compound heterozygous variants of the ANTXR2 gene. Above finding has facilitated molecular diagnosis of the patients.
Asian Continental Ancestry Group/genetics* ; China ; Humans ; Hyalinosis, Systemic/genetics* ; Male ; Mutation ; Pedigree ; Receptors, Peptide/genetics* ; Retrospective Studies

OBJECTIVE: To investigate the effect of regulation of VIPhybrid, an unselective antagonist of vasoactive intestinal peptide receptors (VIPR), on the formation and development of form deprivation myopia (FDM) in chick and the expression of protein and mRNA of VIP on the retina and choroids of in chicks. METHODS: Seventy-two 1-day-old yellow healthy leghorn chicks were assigned into 6 groups (12 in each group). Eyes in Group I were covered on the right as a blank control group. Eyes in GroupII were those eyes having been injected with 20 microL saline into vitreous cavity and then covered as a negative control group. Eyes in GroupIII,IV and V were injected with 20 microL VIPhybrid with low (3*10(-12) mol/L), middle (3*10(-10) mol/L) and high (3*10(-8) mol/L) dosage into vitreous cavity and then covered as experimental groups. The above groups had been continuously covered for 1 week. Eyes in Group VI were uncovered and uninjected as a normal control group. Diopter was detected using retinoscopic refraction. The eyeball axis was determined using ophthalmological ultra-A. The expression of protein and mRNA of VIP on retina-choroids-sclera were investigated by SP immunohistochemistry staining and RT-PCR. RESULTS: Form deprivation for 1 week induced high myopia eyes and elongated eyeball axis in GroupI and GroupII, and there was no difference between the 2 groups (P>0.05). The diopter and eyeball axis were significantly reduced in Group III, IV, and V as compared with Group I and II (P<0.01), but the diopter was higher and the eyeball axis was longer than those of Group VI. The diopter and eyeball axis had negative correlation with the concentration gradient of VIPhybrid. The expressions of protein and mRNA of VIP in Group III, IV, and V were down-regulated as compared with those of Group I and I I(P<0.01)and also down-regulated with the increase of concentration of VIPhybrid. CONCLUSION VIPhybrid can decrease the development of FDM in chicks, which may provide a new pathway for drug therapy of myopia in human beings.
Animals ; Animals, Newborn ; Chickens ; Myopia ; etiology ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Vasoactive Intestinal Peptide ; antagonists & inhibitors ; Recombinant Fusion Proteins ; pharmacology ; Retina ; metabolism ; Vasoactive Intestinal Peptide ; biosynthesis ; genetics

Numerous musculoskeletal disorders are caused by thickened ligament, tendon stiffness, or fibrosis of joint capsule. Relaxin, a peptide hormone, can exert collagenolytic effect on ligamentous and fibrotic tissues. We hypothesized that local injection of relaxin could be used to treat entrapment neuropathy and adhesive capsulitis. Because hormonal effect depends on the receptor of the hormone on the target cell, it is important to confirm the presence of such hormonal receptor at the target tissue before the hormone therapy is initiated. The aim of this study was to determine whether there were relaxin receptors in the ligament, tendon, and joint capsular tissues of rats and to identify the distribution of relaxin receptors in these tissues. Transverse carpal ligaments (TCLs), inguinal ligaments, anterior cruciate ligaments (ACLs), Achilles tendons, and shoulder joint capsules were obtained from male Wistar rats. Western blot analysis was used to identify relaxin receptor isoforms RXFP1 and RXFP2. The distribution of relaxin receptors was determined by immunohistochemical staining. The RXFP1 isoform was found in all tissues examined. The RXFP2 isoform was present in all tissues but the TCLs. Its expression in ACLs tissues was relatively weak compared to that in other tissues. Our results revealed that RXFP1 and RXFP2 were distributed in distinctly different patterns according to the type of tissue (vascular endothelial cells, fibroblast-like cells) they were identified.
Animals ; Blotting, Western ; *Gene Expression Regulation ; Immunohistochemistry ; Ligaments/*metabolism ; Male ; Rats ; Rats, Wistar ; Receptors, G-Protein-Coupled/*genetics/metabolism ; Receptors, Peptide/*genetics/metabolism ; Shoulder Joint/*metabolism ; Tendons/*metabolism

OBJECTIVETo investigate the expression of human adrenomedullin (ADM) and its receptor-receptor activity modifying protein 2/calcitonin receptor-like receptor (RAMP2/CRLR) mRNA in the tissues of normal adrenal medulla and pheochromocytoma.

METHODSTotal RNA was extracted from normal adrenal medulla and pheochromocytomas. The expression of ADM and RAMP2/CRLR mRNA were studied by reverse transcription-polymerase chain reaction. The ratios of ADM/GAPDH, RAMP2/ GAPDH, CRLR/GAPDH were used to evaluate the expression levels of ADM, RAMP2 and CRLR mRNA.

RESULTSExpressions of ADM and its receptor- RAMP2/CRLR mRNA were detected in normal adrenal medulla and pheochromocytoma tissues. ADM/GAPDH were 0.48+/-0.09 and 0.75+/-0.24, RAMP2/ GAPDH 0.79+/-0.12 and 1.29+/-0.30, CRLR/GAPDH 0.40+/-0.08 and 0.87+/-0.22 in normal adrenal medulla and pheochromocytomas, respectively (P < 0.05).

CONCLUSIONADM exerts a possible autocrine or paracrine effect in the adrenal. ADM may be involved in the pathogenesis of pheochromocytoma.

Adrenal Gland Neoplasms ; metabolism ; Adrenal Medulla ; metabolism ; Adrenomedullin ; Adult ; Calcitonin Gene-Related Peptide ; biosynthesis ; genetics ; Female ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; Male ; Membrane Proteins ; biosynthesis ; genetics ; Middle Aged ; Peptides ; genetics ; metabolism ; Pheochromocytoma ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Receptor Activity-Modifying Protein 2 ; Receptor Activity-Modifying Proteins ; Receptors, Adrenomedullin ; Receptors, Calcitonin ; biosynthesis ; genetics ; Receptors, Peptide ; metabolism

OBJECTIVETo screen human monoclonal Fab fragments against HIV-1 gp120 peptide binding chemokine receptor.

METHODSA synthesized polypeptide containing 23 amino acid residues of the gp120 antigen epitope binding chemokine receptor was coated as the solid-phase antigen. After biopanning from the HIV-1 phage Fab antibody library, the acquired positive clones were tested and sequenced.

RESULTSOne clone of human phage Fab monoclonal antibody against HIV-1 gp120 polypeptide was acquired. It has high affinity, specificity and inhibition rate and it belongs to IgG I subclass and kappa type. Its Vh H and V kappa were derived from Vh III and V kappa III.

CONCLUSIONSThe human phage Fab fragment against HIV-1 gp120 antigen site binding chemokine receptor was acquired.

Amino Acid Sequence ; Antibodies, Monoclonal ; genetics ; Antibodies, Viral ; genetics ; Bacteriophages ; genetics ; Cloning, Molecular ; HIV Envelope Protein gp120 ; Humans ; Immunoglobulin Fab Fragments ; genetics ; In Vitro Techniques ; Molecular Sequence Data ; Peptide Library ; Receptors, Chemokine ; metabolism ; Receptors, HIV ; immunology

Through phage display, we tried to find out whether the N-terminal fragment of glucogan-like peptide 1 receptor (nGLP-1R) still had binding activity to Exendin-4 after missing one or two gene segments. By error-prone PCR, We constructed a randomly mutated phage display peptide library with different length of the N-terminal (21-145 residues) extracellular domain of glucogan-like peptide 1 receptor (GLP-1R) from rat lung. A mutant named EP16 without binding activity was found by ELISA. Through sequence alignment we found that EP16 missed the first 20 and last 10 amino acids and the 52nd tryptophan was mutated to arginine. In order to determine why Ep16 did not show its binding ability to Exendin-4, a wild type EP16 without the first 20 and last 10 amino acids and nGLP-1R(W52R) was constructed in which the 52nd tryptophan was mutated to arginine. The contrastive analysis showed that the substitution of W52R led to a markedly reduced binding ability of EP16. The mutation of the conserved W52 could change the biologic activity of the protein. The lack of the first 20 and last 10 amino acids had no effect on its biologic activity. Therefore, the mutation of a single amino acid residue of the key sequence could change the biologic activity of the nGLP-1R.
Amino Acid Sequence ; Amino Acid Substitution ; Animals ; Binding Sites ; Glucagon-Like Peptide-1 Receptor ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Mutation ; Peptide Fragments ; chemistry ; genetics ; metabolism ; Peptides ; metabolism ; Protein Binding ; Rats ; Receptors, Glucagon ; chemistry ; genetics ; metabolism ; Venoms ; metabolism

OBJECTIVETo establish Caco2 cell line with stable expression of glucagon like peptide-2 receptor( GLP-2R) , in order to establish an in vitro model for the study of protective mechanism of GLP-2 of the intestinal tract.

METHODSThe GLP-2R/pcDNA3. 1 ( + ) plasmid was verified by restriction endonuclease and sequencing , and then it was transfected into Caco2 cells with lipofectamine. After G418 selection, the clones with stable expression of GLP-2R were obtained by limited dilution cloning and expanding. The mRNA and protein expression of GLP-2R in normal human intestine, Caco2 cells, HER293, VE cells, as well as in transfected Caco2 cells were determined with RT-PCR and Western blot.

RESULTSThe sequence of GLP-2R/pcDNA 3. 1 plasmid was correct. No expression of GLP-2R mRNA and protein was found in HER293 and VE cells, but weak expression were found in Caco2 cells, and strong expression was found in normal human intestines. The expression of GLP-2R mRNA and protein expression in Caco2/GLP-2R ( + ) cells were obviously increased after transfection.

CONCLUSIONGLP-2R has special distribution. The expression of GLP-2R is weak in normal Caco2 cells. The establishment of Caco2/GLP-2R ( + ) cellular model is beneficial for the further research of the mechanism of action of GLP-2.

Caco-2 Cells ; Cellular Structures ; metabolism ; Cloning, Molecular ; Gene Expression ; Genetic Vectors ; Glucagon-Like Peptide 2 ; genetics ; metabolism ; Glucagon-Like Peptide-2 Receptor ; Humans ; Receptors, Glucagon ; genetics ; metabolism ; Transfection

In this study, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the changes in mRNAs levels of preproadrenomedullin (ppADM) gene encoding adrenomedullin (ADM) and the essential receptor components of ADM, calcitonin receptor-like receptor (CRLR), and the receptor activity modifying protein 2 and 3 (RAMP2 and RAMP3) in the medulla oblongata, hypothalamus, midbrain, pituitary gland and adrenal gland of the stress-induced hypertensive rats. It was shown that chronic foot-shock and noise stress for 15 consecutive days induced a significant increase in systolic blood pressure (SBP) and unique changes in ppADM and its receptor components mRNAs in all areas studied. As compared with the control group, the level of ppADM mRNA, normalized against a glyceraldehydes-3-phosphate dehydrogenase (GAPDH) control, was up-regulated in the hypothalamus-pituitary-adrenal (HPA) axis, but down-regulated in the medulla oblongata and midbrain (P<0.01 and P<0.05, respectively). The relative amount of CRLR mRNA was higher in the hypothalamus than that in other areas. The level of CRLR mRNA expression was significantly increased in the medulla oblongata of the stress group (P<0.01), but decreased in the midbrain (P<0.01) as well as hypothalamus(P<0.05), as compared with that of the control group. Chronic stress for 15 consecutive days produced an increase in the level of RAMP2 mRNA expression in the medulla oblongata (P<0.01) and a decrease in the adrenal gland (P<0.01), as compared with the control. No significant stress-related changes in RAMP2 mRNA were observed in the midbrain, hypothalamus and pituitary gland. The amount of RAMP3 mRNA was relatively higher in the midbrain and hypothalamus than that in the medulla oblongata, adrenal gland and adrenal gland. Stress-induced hypertensive rats exhibited an increased RAMP3 mRNA expression in the hypothalamus and pituitary gland (P<0.01 and P<0.05, respectively) and a decrease in the adrenal gland and midbrain (P<0.05). No significant stress-related change in RAMP3 mRAN was observed in the medulla oblongata. Taken together, our results indicate that the significant changes in ppADM and its receptor components mRNAs expression in the HPA axis and autonomic centers may be related to the development of the stress-induced hypertension. Nevertheless, the pathophysiological significance of brain-derived ADM and its receptors in stress and blood pressure regulation and their roles in stress-induced hypertension still await further investigation.
Adrenomedullin ; Animals ; Brain Stem ; metabolism ; Hypertension ; etiology ; metabolism ; Hypothalamo-Hypophyseal System ; metabolism ; Male ; Peptides ; genetics ; metabolism ; Pituitary-Adrenal System ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Receptors, Adrenomedullin ; Receptors, Peptide ; biosynthesis ; genetics ; Stress, Physiological