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

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

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

To study the expression of angiotensin converting enzyme 2 (ACE2) and angiotensin (Ang) 1-7 specific receptor Mas protain in renal blood vessels of metabolic syndrome ( MS) rats and its anti-oxidative effect. A total of 80 male SD rats were divided into four groups: the normal control group (NC, the same volume of normal saline), the MS group (high fat diet), the MS + Astragali Radix group (MS + HQ, 6 g x kg(-1) x d(-1) in gavage) and the MS + Valsartan group (MS + XST, 30 mg x kg(-1) x d(-1) in gavage). After four weeks of intervention, their general indexes, biochemical indexes and blood pressure were measured; plasma and renal tissue Ang II, malondialdehyde (MDA) and superoxide demutase (SOD) levels were measured with radioimmunoassay. The protein expressions of Mas receptor, AT1R, ACE and ACE2 were detected by western blot analysis. According to the result, compared with the NC group, the MS group and the MS + HQ group showed significant increases in systolic and diastolic pressures, body weight, fasting glucose, fasting insulin, triglycerides, free fatty acid and Ang II level of MS rats (P < 0.05). The MS + XST group showed notable decreases in systolic and diastolic pressures than that of the MS group. The MS group showed significant increases in the SOD activity and NO level and decrease in the MDA level after being intervened with Astragali Radix. ACE and AT1R protein expressions in renal tissues of the MS group were higher than that in the NC group, but with lower ACE2 and -Mas receptor expressions (all P < 0.05). Compared with the MS group, the MS + HQ group showed significant increase in Mas receptor expression in renal tissues, whereas the MS + XST group showed notable decrease in AT1R (all P < 0.05). In conclusion, Astragali Radix can increase the Mas receptor expressions in renal tissues, decrease ACE expression and change local Ang II, MDA, NO and SOD in kidneys, so as to protect early damages in renal tissues.
Angiotensin I ; metabolism ; Animals ; Astragalus Plant ; chemistry ; Blood Glucose ; metabolism ; Blood Pressure ; drug effects ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Kidney ; drug effects ; injuries ; metabolism ; Male ; Malondialdehyde ; metabolism ; Metabolic Syndrome ; drug therapy ; genetics ; metabolism ; physiopathology ; Peptide Fragments ; metabolism ; Peptidyl-Dipeptidase A ; genetics ; metabolism ; Proto-Oncogene Proteins ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, G-Protein-Coupled ; genetics ; metabolism ; Signal Transduction ; drug effects

Analysis of the T-cell receptor (TCR) repertoire of innate CD4+ T cells selected by major histocompatibility complex (MHC) class II-dependent thymocyte-thymocyte (T-T) interaction (T-T CD4+ T cells) is essential for predicting the characteristics of the antigens that bind to these T cells and for distinguishing T-T CD4+ T cells from other types of innate T cells. Using the TCRmini Tg mouse model, we show that the repertoire of TCRalpha chains in T-T CD4+ T cells was extremely diverse, in contrast to the repertoires previously described for other types of innate T cells. The TCRalpha chain sequences significantly overlapped between T-T CD4+ T cells and conventional CD4+ T cells in the thymus and spleen. However, the diversity of the TCRalpha repertoire of T-T CD4+ T cells seemed to be restricted compared with that of conventional CD4+ T cells. Interestingly, the frequency of the parental OT-II TCRalpha chains was significantly reduced in the process of T-T interaction. This diverse and shifted repertoire in T-T CD4+ T cells has biological relevance in terms of defense against diverse pathogens and a possible regulatory role during peripheral T-T interaction.
Amino Acid Sequence ; Animals ; Antigens, Surface/metabolism ; CD4-Positive T-Lymphocytes/cytology/*immunology/*metabolism ; Cell Communication ; Cell Differentiation/genetics/immunology ; Clonal Evolution ; Histocompatibility Antigens Class II/*immunology ; *Immunity, Innate ; Immunophenotyping ; Lymphocyte Count ; Mice ; Mice, Knockout ; Mice, Transgenic ; Peptide Fragments/chemistry ; Phenotype ; Receptors, Antigen, T-Cell/chemistry/*genetics/metabolism ; Receptors, Antigen, T-Cell, alpha-beta/chemistry/genetics ; Spleen/cytology ; Thymocytes/cytology/immunology/metabolism

When mouse bone marrow-derived macrophages were stimulated with serum amyloid A (SAA), which is a major acute-phase protein, there was strong inhibition of osteoclast formation induced by the receptor activator of nuclear factor kappaB ligand. SAA not only markedly blocked the expression of several osteoclast-associated genes (TNF receptor-associated factor 6 and osteoclast-associated receptor) but also strongly induced the expression of negative regulators (MafB and interferon regulatory factor 8). Moreover, SAA decreased c-fms expression on the cell surface via shedding of the c-fms extracellular domain. SAA also restrained the fusion of osteoclast precursors by blocking intracellular ATP release. This inhibitory response of SAA is not mediated by the well-known SAA receptors (formyl peptide receptor 2, Toll-like receptor 2 (TLR2) or TLR4). These findings provide insight into a novel inhibitory role of SAA in osteoclastogenesis and suggest that SAA is an important endogenous modulator that regulates bone homeostasis.
Adenosine Triphosphate/metabolism ; Animals ; Cell Differentiation ; Cell Line ; Gene Expression Regulation, Developmental ; Humans ; Macrophages/*cytology/metabolism ; Mice ; Osteoclasts/*cytology/metabolism ; RANK Ligand/*metabolism ; Receptor, Macrophage Colony-Stimulating Factor/genetics ; Receptors, Formyl Peptide/metabolism ; Serum Amyloid A Protein/*metabolism ; Toll-Like Receptor 2/metabolism ; Toll-Like Receptor 4/metabolism

OBJECTIVETo investigate the effects of α3 neuronal nicotinic acetylcholine receptor (nAChR) on apoptosis and p38 signal transduction pathway in SH-SY5Y cells and to assess the roles of α3 nAChR in the pathogenesis of Alzheimer's disease (AD).

METHODSThe levels of α3 nAChR mRNA and protein were measured by real-time PCR and Western blot, respectively, in SH-SY5Y cells transfected with α3 nAChR siRNA. The mRNA level of bcl-2 and bax was measured by the real-time PCR. The siRNA transfected SH-SY5Y cells and control were then treated with 10 µmol/L Aβ25-35 for another 48 h, and the change in apoptotic rate and the levels of p-p38 and p38 were measured by flow cytometry and Western blot. Subsequently these SH-SY5Y cells were exposed to a blocker of p38 protein, and the apoptotic rate was measured again.

RESULTSCompared to the controls, the expression of α3 nAChR at mRNA and protein levels in the SH-SY5Y cells transfected with α3 nAChR siRNA decreased by 95% and 86%, respectively; the mRNA levels of bax increased 2.11 times and that for bcl-2 decreased 0.53 times. The apoptotic rate was unaffected (3.40% ± 0.20%); but it increased after Aβ25-35 treatment (24.52% ± 1.59%); the level of p-p38 protein also increased by 178% in the α3 nAChR inhibited cells treated with Aβ25-35. Compared to controls, the Aβ25-35-treated SH-SY5Y cells and the Aβ25-35-treated and siRNA-transfected cells both showed a reduction in apoptosis after treatment with p38 blocker, especially in the former.

CONCLUSIONThe siRNA silencing of α3 nAChR mRNA may enhance the effect of Aβ25-35 on the cell apoptosis by increasing the levels of p38 protein and bax mRNA and decreasing the level of bcl-2 mRNA, which may play a role in the pathogenesis of AD.

Alzheimer Disease ; etiology ; Amyloid beta-Peptides ; metabolism ; Apoptosis ; Cell Line, Tumor ; Gene Silencing ; Humans ; Neuroblastoma ; metabolism ; pathology ; Peptide Fragments ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Receptors, Nicotinic ; genetics ; metabolism ; Signal Transduction ; Transfection ; bcl-2-Associated X Protein ; genetics ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo investigate the expression of C-type natriuretic peptides (CNP) and natriuretic peptide receptor-B (NPR-B) receptor in diabetic rats renal cortex, and the regulation by Tongluo Recipe (TLR).

METHODSSixty male SD rats were divided into 3 groups: the normal control group, diabetic model group and diabetic TLR group. Each group was further divided into two subgroups of ten in each, according to 4-week or 12-week observation period. Streptozotocin (STZ)-induced diabetic rats were treated with TLR (1.0 g·kg(-1)·d(-1)) for 4 and 12 weeks, respectively. (1) The essential information was collected for comparing renal mass, serum creatinine and 24 h urine albumen on each group was calculated. (2) CNP mRNA and NPR-B mRNA were detected by realtime-polymerase chain reaction (PCR) on rats renal cortex. (3) Concentration of CNP on renal cortex or serum were analyzed by enzyme-linked immunosorbent assay (ELISA). (4) Pathological evaluation and NPR-B immunostaining for renal tissue were also performed.

RESULTS(1) CNP and NPR-B mRNA levels were detected in each treated or untreated group, with slight elevated in untreated diabetes rats administrated with STZ after 4-week and CNP mRNA level remarkable elevated at 39.21 times higher than normal control group after 12 weeks, but NPR-B mRNA level showed a remarkably down-regulation at 98.07% after 12 weeks. CNP mRNA of TLR-treated group was also elevated after 12-week treatment, but less than untreated group. (2) Concentrations of CNP in renal cortex were obviously increased in treated or untreated diabetes rats, within these groups the treatment of TLR was found more significantly on prompting CNP concentration. Comparing to normal group, serum concentrations of CNP were also increased in treated or untreated diabetic groups, but there was no difference between these diabetic groups. (3) Renal lesions like glomerular volume increased are observed mostly in the relative early stage after 4 weeks. Although TLR treated group had no significant difference in their glomerular volume, the degrees of injury of glomerulus were ameliorated, as well as the NPR-B immunostaining enhanced in glomerulus. Weakly positive immunostaining of NPR-B are observed in glomerulus of normal control, and negative in glomerulus of untreated diabetes rats administrated with STZ after 12 weeks, whereas TLR-treatment groups showed a little enhancement.

CONCLUSIONCNP and NPR-B showed different characteristic on renal cortex at different pathological period in diabetes rats, and TLR regulated their expression.

Animals ; Body Weight ; drug effects ; Diabetes Mellitus, Experimental ; complications ; drug therapy ; genetics ; pathology ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Gene Expression Regulation ; drug effects ; Hematuria ; complications ; genetics ; pathology ; Immunohistochemistry ; Kidney ; drug effects ; metabolism ; pathology ; Kidney Cortex ; drug effects ; metabolism ; pathology ; Kidney Glomerulus ; drug effects ; metabolism ; pathology ; Male ; Natriuretic Peptide, C-Type ; genetics ; metabolism ; Organ Size ; drug effects ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Atrial Natriuretic Factor ; genetics ; metabolism ; Staining and Labeling ; Streptozocin

To rapidly select potent anti-VSTM1-v2 scFv (single-chain antibody fragment) by construction and screening of a humanized scFv library in which a murine VH-CDR3 library was grafted onto a human scFv framework. A murine VH-CDR3 library was amplified from anti-VSTM1-v2 murine cDNA and grafted on human scFv (VH3-VK1) framework. Anti-VSTM1-v2 scFv templates were selected and enriched through ribosome display, TA-cloned into expression vector, and transformed into BL21 (DE3) for soluble expression of target scFv. A total of 1000 clones were randomly picked. Positive ones were first identified using colony PCR, indirect ELISA, Western blotting and then verified with sequencing and dose response ELISA. At last an anti-VSTM1-v2 humanized scFv with good binding affinity (EC50 = 21.35 nmol x L(-1)) was selected from the humanized library of 10(12) members generated in this study. This scFv antibody might have potential applications. This study provides a new approach for rapid screening of humanized antibodies.
Animals ; Complementarity Determining Regions ; genetics ; immunology ; Cytokines ; immunology ; Humans ; Immunoglobulin Fragments ; genetics ; immunology ; Immunoglobulin Heavy Chains ; genetics ; immunology ; Mice ; Peptide Library ; Protein Binding ; Receptors, Immunologic ; immunology ; Single-Chain Antibodies ; genetics ; immunology ; isolation & purification

In this study, we examined the therapeutic effects of an immune-stimulating peptide, WKYMVm, in ulcerative colitis. The administration of WKYMVm to dextran sodium sulfate (DSS)-treated mice reversed decreases in body weight, bleeding score and stool score in addition to reversing DSS-induced mucosa destruction and shortened colon. The WKYMVm-induced therapeutic effect against ulcerative colitis was strongly inhibited by a formyl peptide receptor (FPR) 2 antagonist, WRWWWW, indicating the crucial role of FPR2 in this effect. Mechanistically, WKYMVm effectively decreases intestinal permeability by stimulating colon epithelial cell proliferation. WKYMVm also strongly decreases interleukin-23 and transforming growth factor-beta production in the colon of DSS-treated mice. We suggest that the potent immune-modulating peptide WKYMVm and its receptor FPR2 may be useful in the development of efficient therapeutic agents against chronic intestinal inflammatory diseases.
Adjuvants, Immunologic/pharmacology/*therapeutic use ; Animals ; Caco-2 Cells ; Cell Proliferation ; Colitis, Ulcerative/*drug therapy/metabolism ; Colon/pathology ; Humans ; Interleukin-23/genetics/metabolism ; Intestinal Mucosa/drug effects/metabolism/pathology ; Mice ; Mice, Inbred C57BL ; Oligopeptides/pharmacology/*therapeutic use ; Permeability ; Receptors, Formyl Peptide/antagonists & inhibitors ; Transforming Growth Factor beta/genetics/metabolism