Matrilin-3 is dispensable for mouse skeletal growth and development

Matrilin-3 belongs to the matrilin family of extracellular matrix (ECM) proteins and is primarily expressed in cartilage. Mutations in the gene encoding human matrilin-3 (MATN-3) lead to autosomal dominant skeletal disorders, such as multiple epiphyseal dysplasia (MED), which is characterized by short stature and early-onset osteoarthritis, and bilateral hereditary microepiphyseal dysplasia, a variant form of MED characterized by pain in the hip and knee joints. To assess the function of matrilin-3 during skeletal development, we have generated Matn-3 null mice. Homozygous mutant mice appear normal, are fertile, and show no obvious skeletal malformations. Histological and ultrastructural analyses reveal endochondral bone formation indistinguishable from that of wild-type animals. Northern blot, immunohistochemical, and biochemical analyses indicated no compensatory upregulation of any other member of the matrilin family. Altogether, our findings suggest functional redundancy among matrilins and demonstrate that the phenotypes of MED disorders are not caused by the absence of matrilin-3 in cartilage ECM.     

Phosphatidyl inositol 3-kinase-like serine/threonine protein kinases (PIKKs) are required for DNA damage-induced phosphorylation of the 32 kDa subunit of replication protein A at threonine 21

Replication protein A (RPA) is a single-stranded DNA (ssDNA) binding protein involved in various processes, including nucleotide excision repair and DNA replication. The 32 kDa subunit of RPA (RPA32) is phosphorylated in response to various DNA-damaging agents, and two protein kinases, ataxia-telangiectasia mutated (ATM) and the DNA-dependent protein kinase (DNA-PK) have been implicated in DNA damage-induced phosphorylation of RPA32. However, the relative roles of ATM and DNA-PK in the site-specific DNA damage-induced phosphorylation of RPA32 have not been reported. Here we generated a phosphospecific antibody that recognizes Thr²¹-phosphorylated RPA32. We show that both DNA-PK and ATM phosphorylate RPA32 on Thr²¹ in vitro. Ionizing radiation (IR)-induced phosphorylation of RPA32 on Thr²¹ was defective in ATM-deficient cells, while camptothecin (CPT)-induced phosphorylation of RPA32 on Thr²¹ was defective in cells lacking functional DNA-PK. Neither ATM nor DNA-PK was required for etoposide (ETOP)-induced RPA32 Thr²¹ phosphorylation. However, two inhibitors of the ATM- and Rad3-related (ATR) protein kinase activity prevented ETOP-induced Thr²¹ phosphorylation. Inhibition of DNA replication prevented both the IR- and CPT-induced phosphorylation of Thr²¹, whereas ETOP-induced Thr²¹ phosphorylation did not require active DNA replication. Thus, the regulation of RPA32 Thr²¹ phosphorylation by multiple DNA damage response protein kinases suggests that Thr²¹ phosphorylation of RPA32 is a crucial step within the DNA damage response.     

Study of cell killing and morphology on S180 by ultrasound activating hematoporphyrin derivatives

The inhibition of ascitic S180 and induced sarcoma 180 in vivo was studied with the combination of hematoporphyrin derivatives (HpD) and ultrasound (US) at the frequency of 1.1 MHz and different intensities by light microscopy observation, electronic microscopy observation, cytochemical analysis and fluorescence labeling. The present study indicated that the injury of ascitic S180 increased as time passed and the inhibitory effect was stronger in US plus HpD group than that in other groups. Our results also indicated that the changes in cell structure, cytochrome C oxidase activity, the degradation and missing of DNA were the important factors that inhibited the tumor cell growth and even induced celldeath. The phenomenon of apoptosis of tumor cells indicated that cell death andinduced apoptosis exist in the treatment of sonodynamic therapy (SDT). Our study investigated the mechanism underlying the killing effect of S180 induced by USactivating HpD by the observation of cell morphology and dynamic changes from seminal injury to succeeded injury even to death. It would provide rich referencefor the study of SDT.     

Regulators of G-protein signaling (RGS) 4, insertion into model membranes and inhibition of activity by phosphatidic acid

Regulators of G-protein signaling (RGS) proteins are critical for attenuating G protein-coupled signaling pathways. The membrane association of RGS4 has been reported to be crucial for its regulatory activity in reconstituted vesicles and physiological roles in vivo. In this study, we report that RGS4 initially binds onto the surface of anionic phospholipid vesicles and subsequently inserts into, but not through, the membrane bilayer. Phosphatidic acid, one of anionic phospholipids, could dramatically inhibit the ability of RGS4 to accelerate GTPase activity in vitro. Phosphatidic acid is an effective and potent inhibitor of RGS4 in a G alpha(i1)-[gamma-(32)P]GTP single turnover assay with an IC(50) approximately 4 microm and maximum inhibition of over 90%. Furthermore, phosphatidic acid was the only phospholipid tested that inhibited RGS4 activity in a receptor-mediated, steady-state GTP hydrolysis assay. When phosphatidic acid (10 mol %) was incorporated into m1 acetylcholine receptor-G alpha(q) vesicles, RGS4 GAP activity was markedly inhibited by more than 70% and the EC(50) of RGS4 was increased from 1.5 to 7 nm. Phosphatidic acid also induced a conformational change in the RGS domain of RGS4 measured by acrylamide-quenching experiments. Truncation of the N terminus of RGS4 (residues 1-57) resulted in the loss of both phosphatidic acid binding and lipid-mediated functional inhibition. A single point mutation in RGS4 (Lys(20) to Glu) permitted its binding to phosphatidic acid-containing vesicles but prevented lipid-induced conformational changes in the RGS domain and abolished the inhibition of its GAP activity. We speculate that the activation of phospholipase D or diacylglycerol kinase via G protein-mediated signaling cascades will increase the local concentration of phosphatidic acid, which in turn block RGS4 GAP activity in vivo. Thus, RGS4 may represent a novel effector of phosphatidic acid, and this phospholipid may function as a feedback regulator in G protein-mediated signaling pathways.     

Cell surface accumulation of a truncated transmembrane prion protein in Gerstmann-Straussler-Scheinker disease P102L

A familial prion disorder with a proline to leucine substitution at residue 102 of the prion protein (PrP(102L)) is typically associated with protease-resistant PrP fragments (PrP(Sc)) in the brain parenchyma that are infectious to recipient animals. When modeled in transgenic mice, a fatal neurodegenerative disease develops, but, unlike the human counterpart, PrP(Sc) is lacking and transmission to recipient animals is questionable. Alternate mice expressing a single copy of PrP(102L) (mouse PrP(101L)) do not develop spontaneous disease, but show dramatic susceptibility to PrP(Sc) isolates from different species. To understand these discrepant results, we studied the biogenesis of human PrP(102L) in a cell model. Here, we report that cells expressing PrP(102L) show decreased expression of the normal 18-kDa fragment on the plasma membrane. Instead, a 20-kDa fragment, probably derived from transmembrane PrP ((Ctm)PrP), accumulates on the cell surface. Because the 20-kDa fragment includes an amyloidogenic region of PrP that is disrupted in the 18-kDa form, increased surface expression of 20-kDa fragment may enhance the susceptibility of these cells to PrP(Sc) infection by providing an optimal substrate, or by amplifying the neurotoxic signal of PrP(Sc). Thus, altered susceptibility of PrP(101L) mice to exogenous PrP(Sc) may be mediated by the 20-kDa (Ctm)PrP fragment, rather than PrP(102L) per se.     

Goat Uterine DBA+ Leukocytes Differentiation and Cytokines Expression Respond Differently to Cloned versus Fertilized Embryos

High rate of fetal mortality in ruminant somatic cell nuclear transfer (SCNT) pregnancies is due, at least in part, to immune-mediated abortion of fetuses. In the present study, goat uterine leukocytes were isolated by Dolichos biflorus agglutinin (DBA) coated magnetic beads, and with majority being were CD56⁺CD16^- in phenotype with low levels of perforin and Granzyme B expression. The responses of the isolated cells to SCNT and in vitro fertilization (IVF) embryos conditioned mediums containing hormone steroids were compared by measuring their phenotype and cytokines expression. The results showed there was a 2-fold increase in the numbers of isolated uterine leukocytes after incubation with different conditioned mediums for 120 h. However, significantly lower percentage and absolute numbers of uterine CD56⁺CD16^- leukocytes incubated with SCNT conditioned mediums were detected as compared with those incubated with IVF conditioned mediums (P < 0.05). The group treated with progesterone (P₄) or the combination of P₄ and 17β-estradiol (E₂) were associated with significantly higher percentage and absolute numbers of CD56⁺CD16^- cells as compared with those treated with E₂ alone (P < 0.05). Furthermore, in the presence of steroids, the isolated leukocytes incubated with SCNT conditioned mediums associated with greater levels of IFN-γ secretion and expression, as well as lesser levels of VEGF, as compared with those treated with IVF conditioned mediums (P < 0.05). In conclusion, this study demonstrates that SCNT embryos have a profound effect on the phenotype expression of goat uterine DBA⁺ leukocytes, as well as the secretion and expression of IFN-γ and VEGF by these cells in vitro.     

Inverse Association of Serum Vitamin D in Relation to Carotid Intima-Media Thickness in Chinese Postmenopausal Women

Objective

This study aimed to investigate the relationship between serum vitamin D level and carotid intima-media thickness (C-IMT) in Chinese postmenopausal women.

Methods

Nine hundred and twenty six Chinese postmenopausal women without carotid artery plaque or history of cardiovascular disease were selected for analysis. Measurements of serum 25 hydroxyvitamin D₃ (25(OH)D₃) concentration and C-IMT were made by electrochemiluminescence immunoassay and B-mode ultrasound, respectively. Trend analysis was conducted according to tertiles of C-IMT.

Results

The median serum 25(OH)D₃ level was 11.03 ng/mL, with an interquartile range of 8.22–14.70. A decreasing trend of serum 25(OH)D₃ level was accompanied by increased C-IMT tertiles (P for trend = 0.001). Correlation analysis found an inverse relationship between serum 25(OH)D₃ level and C-IMT (r = –0.113, P = 0.001). After adjustment for confounding factors, multiple regression analysis showed that serum 25(OH)D₃ level independently and negatively associated with C-IMT (Standard β = –0.112, P < 0.001). Moreover, the inverse correlation of serum 25(OH)D₃ with C-IMT was also found in a subgroup of women with normal glucose tolerance, blood pressure and body mass index, and without undergoing lipid-lowering therapy (standard β = –0.140, P = 0.018).

Conclusions

Serum 25(OH)D₃ level was inversely correlated with C-IMT in Chinese postmenopausal women.     

Patterns of Circulating Fibroblast Growth Factor 21 in Subjects with and without Type 2 Diabetes

Background

Fibroblast growth factor 21 (FGF21) exerts wide-range effects on carbohydrate and lipid metabolism. However, its perturbation in type 2 diabetes mellitus (T2DM) remains elusive. Besides, previous human studies in T2DM simply investigated fasting or stimulated levels of FGF21. The current study sought to evaluate the temporal changes of circulating FGF21 in subjects with and without T2DM.

Methods

Ten patients with T2DM and 16 normal controls (NC) were recruited. Participants were categorized as obese (BMI≥25 kg/m²) or lean (BMI<25 kg/m²). Blood samples were drawn every 30 min within 7 hours (8 a.m.-3 p.m.). Serum FGF21, blood glucose, insulin, free fatty acids (FFAs) and adiponectin were measured in all subjects.

Results

The peak levels of FGF21 were observed in the fasting state (8 a.m.) both in T2DM and NC groups (267.35 ±158.72 ng/L vs. 178.93±121.37 ng/L, P = 0.096). FGF21 AUC did not differ significantly between the two groups (T2DM: 949.4±471.47 ng/L; NC: 883.13±561.40 ng/L, P = 0.770). Obese subjects had higher FGF21 levels than lean ones in patients either with or without T2DM. The pattern of FFAs closely resembled that of FGF21. Correlation analysis showed that temporal levels of FGF21 were significantly related to FFAs (r = 0.749, P = 0.002),but not blood glucose, insulin or adiponectin (all P> 0.05).

Conclusions

These findings suggest that the pattern of circulating FGF21 does not differ significantly between T2DM and NC,although T2DM patients showed a trend toward higher fasting FGF21 than healthy subjects. The pattern of circulating FFAs is significantly associated with that of FGF21.