PARP9-DTX3L ubiquitin ligase targets host histone H2BJ and viral 3C protease to enhance interferon signaling and control viral infection.

Enhancing the response to interferon could offer an immunological advantage to the host. In support of this concept, we used a modified form of the transcription factor STAT1 to achieve hyper-responsiveness to interferon without toxicity and markedly improve antiviral function in transgenic mice and transduced human cells. We found that the improvement depended on expression of a PARP9-DTX3L complex with distinct domains for interaction with STAT1 and for activity as an E3 ubiquitin ligase that acted on host histone H2BJ to promote interferon-stimulated gene expression and on viral 3C proteases to degrade these proteases via the immunoproteasome. Thus, PARP9-DTX3L acted on host and pathogen to achieve a double layer of immunity within a safe reserve in the interferon signaling pathway.

Comparative transcriptome analysis provides clues to molecular mechanisms underlying blue-green eggshell color in the Jinding duck (Anas platyrhynchos).

BACKGROUND: In birds, blue-green eggshell color (BGEC) is caused by biliverdin, a bile pigment derived from the degradation of heme and secreted in the eggshell by the shell gland. Functionally, BGEC might promote the paternal investment of males in the nest and eggs. However, little is known about its formation mechanisms. Jinding ducks (Anas platyrhynchos) are an ideal breed for research into the mechanisms, in which major birds lay BGEC eggs with minor individuals laying white eggs. Using this breed, this study aimed to provide insight into the mechanisms via comparative transcriptome analysis. RESULTS: Blue-shelled ducks (BSD) and white-shelled ducks (WSD) were selected from two populations, forming 4 groups (3 ducks/group): BSD1 and WSD1 from population 1 and BSD2 and WSD2 from population 2. Twelve libraries from shell glands were sequenced using the Illumina RNA-seq platform, generating an average of 41 million clean reads per library, of which 55.9% were mapped to the duck reference genome and assembled into 31,542 transcripts. Expression levels of 11,698 genes were successfully compared between all pairs of 4 groups. Of these, 464 candidate genes were differentially expressed between cross-phenotype groups, but not for between same-phenotype groups. Gene Ontology (GO) annotation showed that 390 candidate genes were annotated with 2234 GO terms. No candidate genes were directly involved in biosynthesis or transport of biliverdin. However, the integral components of membrane, metal ion transport, cholesterol biosynthesis, signal transduction, skeletal system development, and chemotaxis were significantly (P < 0.05) overrepresented by candidate genes. CONCLUSIONS: This study identified 464 candidate genes associated with duck BGEC, providing valuable information for a better understanding of the mechanisms underlying this trait. Given the involvement of membrane cholesterol contents, ions and ATP levels in modulating the transport activity of bile pigment transporters, the data suggest a potential association between duck BGEC and the transport activity of the related transporters.

An EAV-HP insertion in 5' Flanking region of SLCO1B3 causes blue eggshell in the chicken.

The genetic determination of eggshell coloration has not been determined in birds. Here we report that the blue eggshell is caused by an EAV-HP insertion that promotes the expression of SLCO1B3 gene in the uterus (shell gland) of the oviduct in chicken. In this study, the genetic map location of the blue eggshell gene was refined by linkage analysis in an F(2) chicken population, and four candidate genes within the refined interval were subsequently tested for their expression levels in the shell gland of the uterus from blue-shelled and non-blue-shelled hens. SLCO1B3 gene was found to be the only one expressed in the uterus of blue-shelled hens but not in that of non-blue-shelled hens. Results from a pyrosequencing analysis showed that only the allele of SLCO1B3 from blue-shelled chickens was expressed in the uterus of heterozygous hens (O*LC/O*N). SLCO1B3 gene belongs to the organic anion transporting polypeptide (OATP) family; and the OATPs, functioning as membrane transporters, have been reported for the transportation of amphipathic organic compounds, including bile salt in mammals. We subsequently resequenced the whole genomic region of SLCO1B3 and discovered an EAV-HP insertion in the 5' flanking region of SLCO1B3. The EAV-HP insertion was found closely associated with blue eggshell phenotype following complete Mendelian segregation. In situ hybridization also demonstrated that the blue eggshell is associated with ectopic expression of SLCO1B3 in shell glands of uterus. Our finding strongly suggests that the EAV-HP insertion is the causative mutation for the blue eggshell phenotype. The insertion was also found in another Chinese blue-shelled breed and an American blue-shelled breed. In addition, we found that the insertion site in the blue-shelled chickens from Araucana is different from that in Chinese breeds, which implied independent integration events in the blue-shelled chickens from the two continents, providing a parallel evolutionary example at the molecular level.

The association of very low-density lipoprotein receptor (VLDLR) haplotypes with egg production indicates VLDLR is a candidate gene for modulating egg production.

The very low-density lipoprotein receptor (VLDLR) transports egg yolk precursors into oocytes. However, our knowledge of the distribution patterns of VLDLR variants among breeds and their relationship to egg production is still incomplete. In this study, eight single nucleotide polymorphisms (SNPs) that account for 87% of all VLDLR variants were genotyped in Nick Chick (NC, n=91), Lohmann Brown (LohB, n=50) and Lueyang (LY, n=381) chickens, the latter being an Chinese indigenous breed. Egg production by NC and LY chickens was recorded from 17 to 50 weeks. Only four similar haplotypes were found in NC and LohB, of which two accounted for 100% of all NC haplotypes and 92.5% of LohB haplotypes. In contrast, there was considerable haplotypic diversity in LY. Comparison of egg production in LY showed that hens with NC-like haplotypes had a significantly higher production (p < 0.05) than those without the haplotypes. However, VLDLR expression was not significantly different between the haplotypes. These findings indicate a divergence in the distribution of VLDLR haplotypes between selected and non-selected breeds and suggest that the near fixation of VLDLR variants in NC and LohB is compatible with signature of selection. These data also support VLDLR as a candidate gene for modulating egg production.

Refine localizations of functional variants affecting eggshell color of Lueyang black-boned chicken in the SLCO1B3.

Table eggs with color-uniformity shell are visually attractive for consumers. Lueyang black-boned chicken (LBC) lays colorful eggs, which is undesirable for sale of table eggs, but provides a segregating population for mapping functional variants affecting eggshell color. SLCO1B3 was identified as the causative gene for blue eggs in the Dongxiang and Araucana chickens. The aim of this study is to map functional variants associated with chicken eggshell color in the SLCO1B3. Eggshell color of LBC (n = 383) was measured using the L*a*b color space. SLCO1B3 was resequencing using a subset (n = 30) of 383 samples. Linkage disequilibrium among 139 SNP was analyzed. Association of 16 SNP in the SLCO1B3 and 8 in CPOX, ALAS1, and ABCG2 genes with L*a*b were tested by a polygenic model (LMM) and a polygenic/oligogenic mixed model (BSLMM). Chromatin state annotations were retrieved from the UCSC database. Effect of SLCO1B3 variants distributed in mapping and upstream 1.6-kb regions on promoter activities were analyzed using dual-luciferase reporter assay. One hundred and thirty-nine variants maintained low linkage disequilibrium with 80% of r2 less than 0.226. Fifteen SLCO1B3 variants were significantly associated with a*, of which 1B3_SNP108 was showed the strongest association and the largest effect on a*. In the BSLMM, 1B3_SNP108 alone appeared in the Markov chain Monte Carlo as major variants in 100% of posterior inclusion probability. None of variants in CPOX, ALAS1, and ABCG2 were significantly associated with color indexes except that 2 ALAS1 variants were associated with L*. 1B3_SNP108 distributes in the Intron4 where 6 active enhancers and 1 ATAC island were enriched. However, 1B3_SNP108-containing constructs showed negligible activities in the reporter assay. No significant differences of activities between haplotypes were found for five 5'-deleted promoter constructs. The data recognizes 1B3_SNP108 as a valuable marker for breeding of eggshell color. Functional variants are localized in the region adjacent to the 1B3_SNP108 due to low linkage disequilibrium in the LBC. Our findings extend the role of SLCO1B3 from a causative gene for blue eggs to a major regulator driving continuous variation of LBC eggshell color.

Comparison of HMOX1 expression and enzyme activity in blue-shelled chickens and brown-shelled chickens.

Blue egg coloring is attributed to biliverdin derived from the oxidative degradation of heme through catalysis by heme oxygenase (HO). The pigment is secreted into the eggshell by the shell gland. There is uncertainty as to whether the pigment is synthesized in the shell gland or in other tissues. To investigate the site of pigment biosynthesis, the expression of heme oxygenase (decycling) 1 (HMOX1), a gene encoding HO, and HO activity in liver and spleen were compared between blue-shelled chickens (n = 12) and brown-shelled chickens (n = 12). There were no significant differences in HMOX1 expression and HO activity in these tissues between the two groups. Since the liver and spleen, two important sites outside the shell gland where heme is degraded into biliverdin, CO and Fe(2+), did not differ in HO expression and activity we conclude that the pigment is most likely synthesized in the shell gland.

A new molecular mechanism supports that blue-greenish egg color evolved independently across chicken breeds.

Chicken blue-greenish coloration (BGC) was known as a classic Mendel trait caused by a retrovirus (EAV-HP) insertion in the SLCO1B3 gene. Lueyang black-boned chicken (LBC) BGC is light and varies continuously, implying that LBC BGC may be controlled by a new molecular mechanism. The aim of this study was to provide an insight into the molecular basis of LBC BGC. The EAV-HP was detected in the BGC (n = 105) and non-BGC LBC (n = 474) using a PCR-based method. The association of SLCO1B3 expression in shell glands and sequence variants in a 1.6-kb region upstream from the transcription start site of SLCO1B3 with eggshell color and biliverdin (pigment for BGC) concentration was studied. Promoter activities of haplotypes in the 1.6-kb region were analyzed by luciferase reporter assay. This study did not found the EAV-HP in BGC and Non-BGC LBC, but detected a strong positive correlation between levels of SLCO1B3 expression in shell glands and biliverdin concentrations. A total of 31 SNP were found in the 1.6-kb region. Twenty-two of 31 SNP formed 42 types of haplotypes in the re-sequenced samples (n = 94). Haplotype 4 was present in higher frequency in the BGC (52%) than Non-BGC (3%). Haplotype 13 was significantly associated with Non-BGC (Non-BGC vs. BGC = 26% vs. 6%). In line with the above associations, Haplotype 4 showed higher (P < 0.05) levels of SLCO1B3 expression in shell glands, biliverdin concentration, and promoter activity than Haplotype 13. This study confirms that LBC BGC is not caused by the EAV-HP, but remains to be associated with the change of SLCO1B3 expression. Haplotype 4 accounts to some extents for the molecular basis of LBC BGC. The new molecular mechanism supports LBC BGC independently evolved.

Effect of 3-Mercaptopropyltriethoxysilane Modified Illite on the Reinforcement of SBR.

To achieve the sustainable development of the rubber industry, the substitute of carbon black, the most widely used but non-renewable filler produced from petroleum, has been considered one of the most effective ways. The naturally occurring illite with higher aspect ratio can be easily obtained in large amounts at lower cost and with lower energy consumption. Therefore, the expansion of its application in advanced materials is of great significance. To explore their potential use as an additive for reinforcing rubber, styrene butadiene rubber (SBR) composites with illites of different size with and without 3-mercaptopropyltriethoxysilane (KH580) modification were studied. It was found that the modification of illite by KH580 increases the K-illite/SBR interaction, and thus improves the dispersion of K-illite in the SBR matrix. The better dispersion of smaller size K-illite with stronger interfacial interaction improves the mechanical properties of SBR remarkably, by an increment of about nine times the tensile strength and more than ten times the modulus. These results demonstrate, except for the evident effect of particle size, the great importance of filler-rubber interaction on the performance of SBR composites. This may be of great significance for the potential wide use of the abundant naturally occurring illite as substitute filler for the rubber industry.

Distributed contrastive learning for medical image segmentation.

Supervised deep learning needs a large amount of labeled data to achieve high performance. However, in medical imaging analysis, each site may only have a limited amount of data and labels, which makes learning ineffective. Federated learning (FL) can learn a shared model from decentralized data. But traditional FL requires fully-labeled data for training, which is very expensive to obtain. Self-supervised contrastive learning (CL) can learn from unlabeled data for pre-training, followed by fine-tuning with limited annotations. However, when adopting CL in FL, the limited data diversity on each site makes federated contrastive learning (FCL) ineffective. In this work, we propose two federated self-supervised learning frameworks for volumetric medical image segmentation with limited annotations. The first one features high accuracy and fits high-performance servers with high-speed connections. The second one features lower communication costs, suitable for mobile devices. In the first framework, features are exchanged during FCL to provide diverse contrastive data to each site for effective local CL while keeping raw data private. Global structural matching aligns local and remote features for a unified feature space among different sites. In the second framework, to reduce the communication cost for feature exchanging, we propose an optimized method FCLOpt that does not rely on negative samples. To reduce the communications of model download, we propose the predictive target network update (PTNU) that predicts the parameters of the target network. Based on PTNU, we propose the distance prediction (DP) to remove most of the uploads of the target network. Experiments on a cardiac MRI dataset show the proposed two frameworks substantially improve the segmentation and generalization performance compared with state-of-the-art techniques.

Identification of the Core Promoter and Variants Regulating Chicken CCKAR Expression.

Decreased expression of chicken cholecystokinin A receptor (CCKAR) attenuates satiety, which contributes to increased food intake and growth for modern broilers. The study aims to define the core promoter of CCKAR, and to identify variants associated with expression activity. A 21 kb region around the CCKAR was re-sequenced to detect sequence variants. A series of 5'-deleted promoter plasmids were constructed to define the core promoter of CCKAR. The effects of sequence variants located in promoter (PSNP) and conserved (CSNP) regions on promoter activity were analyzed by comparing luciferase activity between haplotypes. A total of 182 variants were found in the 21 kb region. There were no large structural variants around CCKAR. pNL-328/+183, the one with the shortest insertion, showed the highest activity among the six promoter constructs, implying that the key cis elements regulating CCKAR expression are mainly distributed 328 bp upstream. We detected significant activity differences between high- and low-growth associated haplotypes in four of the six promoter constructs. The high-growth haplotypes of constructs pNL-1646/+183, pNL-799/+183 and pNL-528/+183 showed lower activities than the low-growth haplotypes, which is consistent with decreased expression of CCKAR in high-growth chickens. Lower expression of the high-growth allele was also detected for the CSNP5-containing construct. The data suggest that the core promoter of CCKAR is located the 328 bp region upstream from the transcription start site. Lower expression activities shown by the high-growth haplotypes in the reporter assay suggest that CSNP5 and variants located between 328 bp and 1646 bp upstream form a promising molecular basis for decreased expression of CCKAR and increased growth in chickens.

Circ_0003159 upregulates LIFR expression through competitively binding to miR-221-3p/miR-222-3p to block gastric cancer development.

Gastric cancer (GC) remains a major cause of cancer-related deaths. Increasing studies suggest that cancer development is accompanied by the deregulation of circular RNAs. We investigated the function of circ_0003159 in GC. The expression levels of circ_0003159, miR-221-3p/miR-222-3p and leukemia inhibitory factor receptor (LIFR) mRNA were measured by real-time quantitative polymerase chain reaction. Cell colony formation ability was assessed by colony formation assay, and cell viability was assessed by cell counting kit-8 assay. Cell apoptosis was assessed by flow cytometry assay and caspase3 activity. Cell migration and invasion were assessed by transwell assay. Glycolysis energy metabolism was assessed by 5'-triphosphate production, glucose uptake and lactate production. The protein levels of related marker proteins and LIFR were detected by western blot. The relationship between circ_0003159 and miR-221-3p/miR-222-3p, or LIFR and miR-221-3p/miR-222-3p was obtained from bioinformatics tools and verified by dual-luciferase reporter assay. A cancer tumorogenicity xenograft experiment in nude mice was conducted to determine the role of circ_0003159 in tumor growth by AGS cells. Our results showed that circ_0003159 expression was decreased in GC tissues and cells. Circ_0003159 overexpression sequestered GC cell viability, migration, invasion and glycolysis and induced cell apoptosis. MiR-221-3p and miR-222-3p were targets of circ_0003159, and the inhibition of miR-221-3p and miR-222-3p also blocked GC cell viability, migration, invasion and glycolysis and promoted cell apoptosis. LIFR was a common target of miR-221-3p and miR-222-3p. Interestingly, LIFR knockdown reversed the effects of circ_0003159 overexpression on GC cell behaviors. Circ_0003159 increased the expression level of LIFR by targeting miR-221-3p and miR-222-3p. The tumorigenicity assay showed that circ_0003159 overexpression inhibited tumor growth in vivo. In conclusion, circ_0003159 inhibited GC development in vitro and in vivo by enriching the level of LIFR via direct binding to miR-221-3p/miR-222-3p.

Type IIB procollagen NH(2)-propeptide induces death of tumor cells via interaction with integrins alpha(V)beta(3) and alpha(V)beta(5).

Cartilage is resistant to tumor invasion. In the present study, we found that the NH(2)-propeptide of the cartilage-characteristic collagen, type IIB, PIIBNP, is capable of killing tumor cells. The NH(2)-propeptide is liberated into the extracellular matrix prior to deposition of the collagen fibrils. This peptide adheres to and kills cells from chondrosarcoma and cervical and breast cancer cell lines via the integrins alpha(v)beta(5) and alpha(v)beta(3). Adhesion is abrogated by blocking with anti alpha(v)beta(5) and alpha(v)beta(3) antibodies. When alpha(v) is suppressed by small intefering RNA, adhesion and cell killing are blocked. Normal chondrocytes from developing cartilage do not express alpha(v)beta(3) and alpha(v)beta(5) integrins and are thus protected from cell death. Morphological, DNA, and biochemical evidence indicates that the cell death is not by apoptosis but probably by necrosis. In an assay for invasion, PIIBNP reduced the number of cells crossing the membrane. In vivo, in a tumor model for breast cancer, PIIBNP was consistently able to reduce the size of the tumor.

Research Note: Fine mapping of sequence variants associated with body weight of Lueyang black-boned chicken in the CCKAR gene.

Cholecystokinin A receptor (CCKAR) is a key receptor mediating satiety. Previous studies found that decreased expression of CCKAR attenuated satiety, and thus contributed to the high-growth of broiler chickens. The objective of this study is to map sequence variants associated with the growth of chickens in the CCKAR. The CCKAR and upstream 1.4 kb genomic sequences were resequenced to find out all sequence variants using 35 Lueyang black-boned chickens (LBC). Haplotypes were reconstructed using the PHASE program. Linkage disequilibrium between variants was analyzed using the Haploview software. Associations of 33 tag SNPs that captured 89% of all variants with body weight of LBC (n = 675) at 16 (BW16), 20 (BW20) weeks of age and the onset (BWOEP) of egg production were tested using linear mixed models. A total of 126 SNPs were found and formed 41 haplotypes in 35 resequenced samples. Average length of haplotype blocks is 129 bp, indicating that LBC maintains low linkage disequilibrium at the CCKAR locus. Eleven of 33 tag SNPs were significantly associated with BW16, but not with BW20 and BWOEP. These significantly associated variants were most (8/11) distributed in a 2 kb region (chr4:73206169-73208244) around the Exon3. They together with 33 captured variants potentially disrupted binding sites of 471 transcription factors. Twelve variants can disrupt appetite (FOXO1) or lipid metabolism-related TF (AR and C/EBP) motifs. This study recognized chr4:73206169-73208244 as a key region harboring functional variants affecting the growth of chickens.

Study on the Use of CTAB-Treated Illite as an Alternative Filler for Natural Rubber.

Fillers are indispensable for rubber composites. Carbon black as an efficient reinforcing filler is most widely used in the rubber industry. However, the utilization of nonrenewable feedstock, energy consumption, and footprint for making carbon black lead to the seeking of alternative substitutes for carbon black, which is of great significance. Here in this work, the possibility of illite, a most common mineral in sedimentary rocks, as an alternative filler for natural rubber (NR) is determined. It is found that pristine illite slows the curing rate and decreases the cross-linking density of NR, which results in the inferior performance of NR. This is associated with the weak filler-rubber interaction, which is a vital factor in deciding the performance of rubber composites. Therefore, illite has been modified using hexadecyl trimethyl ammonium bromide (CTAB), a commonly used cation surfactant, for improving the filler-rubber interaction. The thus obtained C-illite is confirmed to be efficient for (i) enhancing the illite-NR interaction, (ii) improving the dispersion of illite in the NR matrix, and (iii) accelerating the curing process of NR with increased cross-linking density. All of these lead to significantly improved mechanical properties and wear resistance of the C-illite/NR composites, e.g., a 71.88% increase of the modulus at 300% strain compared to the pure NR and a 23.79% reduction of the DIN abrasion volume compared to the NR filled with 40 phr pristine illite. This illustrates the high possibility of CTAB-modified illite with an optimal particle size as a promising alternative filler of carbon black for reinforcing rubbers.

Inducible expression of Wnt7b promotes bone formation in aged mice and enhances fracture healing.

There remain unmet clinical needs for safe and effective bone anabolic therapies to treat aging-related osteoporosis and to improve fracture healing in cases of nonunion or delayed union. Wnt signaling has emerged as a promising target pathway for developing novel bone anabolic drugs. Although neutralizing antibodies against the Wnt antagonist sclerostin have been tested, Wnt ligands themselves have not been fully explored as a potential therapy. Previous work has demonstrated Wnt7b as an endogenous ligand upregulated during osteoblast differentiation, and that Wnt7b overexpression potently stimulates bone accrual in the mouse. The earlier studies however did not address whether Wnt7b could promote bone formation when specifically applied to aged or fractured bones. Here we have developed a doxycycline-inducible strategy where Wnt7b is temporally induced in the bones of aged mice or during fracture healing. We report that forced expression of Wnt7b for 1 month starting at 15 months of age greatly stimulated trabecular and endosteal bone formation, resulting in a marked increase in bone mass. We further tested the effect of Wnt7b on bone healing in a murine closed femur fracture model. Induced expression of Wnt7b at the onset of fracture did not affect the initial cartilage formation but promoted mineralization of the subsequent bone callus. Thus, targeted delivery of Wnt7b to aged bones or fracture sites may be explored as a potential therapy.

Phototoxic effect of UVR on wild type, ebony and yellow mutants of Drosophila melanogaster: life span, fertility, courtship and biochemical aspects.

Melanin plays an important role in protecting organisms from ultraviolet radiation (UVR). Therefore, it is possible that differently colored strains can show different sensitivities to UVR. In the present work, life span, fertility and courtship behavior of wild type (w), ebony (e) and yellow (y) strains of Drosophila melanogaster were studied to evaluate their sensitivity to ultraviolet (UV). Because a range of phototoxic effects of UVR are mediated through generation of free radicals, levels of free radicals, lipid peroxide (malondialdehyde, MDA) and superoxide dismutase (SOD) activity of three strains were examined to indicate their antioxidant defending ability and oxidative status. It was shown that w always had the highest lifespan and fertility not only in the control but also in UV-exposed groups. Moreover, lifespan and fertility of e were significantly higher (P<0.0001) than those of y in the UV-exposed groups, but not for the control. On the other hand, UV exposure had an adverse effect on courtship of flies. Stronger electron paramagnetic resonance (EPR) signals could be detected in w, e and y exposed to 5 min UV. And there were more significant changes of EPR signals in y than in w and e. UVR had no significant (P=0.1782) effect on the SOD activities. After pooling data from the control and UV-exposed groups, we found that w had a significantly (P<0.05) higher level of SOD activity, but e and y were nearly at the same levels (P>0.05). MDA levels were increased in the UV dose-dependent manner (P=0.0495). In conclusion, our results suggested that UVR can decrease life span and fertility of flies and do harm to courtship, which may be due to oxidative damage to flies tissues (e.g. central nervous system) induced by free radicals. w had the highest tolerance to UVR, which may be ascribed to its advantage of survival under the natural condition and at high level of SOD activity. Then differences of pigment between e and y in absorbing UV, shielding against UV and scavenging free radicals produced by UVR should be responsible for their different sensitivity to UVR.

The type II collagen N-propeptide, PIIBNP, inhibits cell survival and bone resorption of osteoclasts via integrin-mediated signaling.

OBJECTIVE: Type IIB procollagen is characteristic of cartilage, comprising 50% of the extracellular matrix. The NH(2)-propeptide of type IIB collagen, PIIBNP, can kill tumor cells via binding to integrins α(V)ß(3) and α(V)ß(5). As osteoclasts rely on α(V)ß(3) integrins for function in bone erosion, we sought to determine whether PIIBNP could inhibit osteoclast function. METHODS: We undertook in vitro and in vivo experiments to evaluate both osteoblast and osteoclast functions in the presence of recombinant PIIBNP. Adhesion of osteoclasts to PIIBNP was analyzed by staining of attached cells with crystal violet. PIIBNP-induced cell death was evaluated by counting Trypan Blue stained cells. The mechanism of cell death was evaluated by DNA fragmentation, TUNEL staining and western blotting to detect cleaved caspases. To determine the role of α(V)ß(3) integrin, osteoclasts were pretreated with α(V) or ß(3) integrin specific siRNA before the treatment with PIIBNP. To explore PIIBNP function in vivo, a lipopolysaccharide-induced mouse calvaria lysis model was employed. RESULTS: Osteoclasts adhered to PIIBNP via an RGD-mediated mechanism. When osteoclasts were plated on extracellular matrix proteins, PIIBNP induced apoptosis of osteoclasts via caspase 3/8 activation. Osteoblasts and macrophages were not killed. Reduction of α(V) or ß(3) integrin levels on osteoclasts by siRNA reduced cell death in a dose-dependent manner. In vivo, PIIBNP could inhibit bone resorption. CONCLUSION: We conclude that PIIBNP can inhibit osteoclast survival and bone resorption via signal transduction through the α(V)ß(3) integrins. Because of this property and the cell specificity, we propose that PIIBNP may play a role in vivo in protecting cartilage from osteoclast invasion and also could be a new therapeutic strategy for decreasing bone loss.

Group VIA phospholipase A2 forms a signaling complex with the calcium/calmodulin-dependent protein kinase IIbeta expressed in pancreatic islet beta-cells.

Insulin-secreting pancreatic islet beta-cells express a Group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)beta) that contains a calmodulin binding site and protein interaction domains. We identified Ca(2+)/calmodulin-dependent protein kinase IIbeta (CaMKIIbeta) as a potential iPLA(2)beta-interacting protein by yeast two-hybrid screening of a cDNA library using iPLA(2)beta cDNA as bait. Cloning CaMKIIbeta cDNA from a rat islet library revealed that one dominant CaMKIIbeta isoform mRNA is expressed by adult islets and is not observed in brain or neonatal islets and that there is high conservation of the isoform expressed by rat and human beta-cells. Binary two-hybrid assays using DNA encoding this isoform as bait and iPLA(2)beta DNA as prey confirmed interaction of the enzymes, as did assays with CaMKIIbeta as prey and iPLA(2)beta bait. His-tagged CaMKIIbeta immobilized on metal affinity matrices bound iPLA(2)beta, and this did not require exogenous calmodulin and was not prevented by a calmodulin antagonist or the Ca(2+) chelator EGTA. Activities of both enzymes increased upon their association, and iPLA(2)beta reaction products reduced CaMKIIbeta activity. Both the iPLA(2)beta inhibitor bromoenol lactone and the CaMKIIbeta inhibitor KN93 reduced arachidonate release from INS-1 insulinoma cells, and both inhibit insulin secretion. CaMKIIbeta and iPLA(2)beta can be coimmunoprecipitated from INS-1 cells, and forskolin, which amplifies glucose-induced insulin secretion, increases the abundance of the immunoprecipitatable complex. These findings suggest that iPLA(2)beta and CaMKIIbeta form a signaling complex in beta-cells, consistent with reports that both enzymes participate in insulin secretion and that their expression is coinduced upon differentiation of pancreatic progenitor to endocrine progenitor cells.

Competition between transferrin and the serum ligands citrate and phosphate for the binding of aluminum.

A key issue regarding the speciation of Al(3+) in serum is how well the ligands citric acid and phosphate can compete with the iron transport protein serum transferrin for the aluminum. Previous studies have attempted to measure binding constants for each ligand separately, but experimental problems make it very difficult to obtain stability constants with the accuracy required to make a meaningful comparison between these ligands. In this study, effective binding constants for Al-citrate and Al-phosphate at pH 7.4 have been determined using difference UV spectroscopy to monitor the direct competition between these ligands and transferrin. The analysis of this competition equilibrium also includes the binding of citrate and phosphate as anions to apotransferrin. The effective binding constants are 10(11.59) for the 1:1 Al-citrate complexes and 10(14.90) for the 1:2 Al-citrate complexes. The effective binding constant for the 1:2 Al-phosphate complex is 10(12.02). No 1:1 Al-phosphate complex was detected. Speciation calculations based on these effective binding constants indicate that, at serum concentrations of citrate and phosphate, citrate will be the primary low-molecular-mass ligand for aluminum. Formal stability constants for the Al-citrate system have also been determined by potentiometric methods. This equilibrium system is quite complex, and information from both electrospray mass spectrometry and difference UV experiments has been used to select the best model for fitting the potentiometric data. The mass spectra contain peaks that have been assigned to complexes having aluminum:citrate stoichiometries of 1:1, 1:2, 2:2, 2:3, and 3:3. The difference UV results were used to determine the stability constant for Al(H(-1)cta)-, which was then used in the least-squares fitting of the potentiometric data to determine stability constants for Al(Hcta)+, Al(cta), Al(cta)2(3-), Al(H(-1)cta)(cta)(4-), Al2(H(-1)cta)2(2-), and Al3(H(-1)cta)3(OH)(4-).

Kinetics of metal ion exchange between citric acid and serum transferrin.

The exchange of Fe(3+), Tb(3+), In(3+), Ga(3+), and Al(3+) between the C-terminal metal-binding site of the serum iron transport protein transferrin and the low-molecular-mass serum chelating agent citrate has been studied at pH 7.4 and 25 degrees C. The removal of Ga(3+), In(3+), and Al(3+) follows simple saturation kinetics with respect to the citrate concentration. In contrast, removal of both Fe(3+) and Tb(3+) shows a combination of saturation and first-order kinetic behavior with respect to the citrate concentration. The saturation component is consistent with a mechanism for metal release in which access to the bound metal is controlled by a rate-limiting conformational change in the protein. The first-order kinetic pathway is very rapid for Tb(3+), and this is attributed to a direct attack of the citrate on the Tb(3+) ion within the closed protein conformation. It is suggested that this pathway is more readily available for Tb(3+) because of the larger coordination number for this cation and the presence of an aquated coordination site in the Tb(3+)-CO(3)-Tf ternary complex. There is relatively little variation in the k(max) values for the saturation pathway for Tb(3+), Ga(3+), Al(3+), and In(3+), but the k(max) value for Fe(3+) is significantly smaller. It is suggested that protein interactions across the interdomain cleft of transferrin largely control the release of the first group of metal ions, while the breaking of stronger metal-protein bonds slows the rate of iron release. The rates of metal binding to apotransferrin are clearly controlled in large part by the hydrolytic tendencies of the free metal ions. For the more amphoteric metal ions Al(3+) and Ga(3+), there is rapid protein binding, and the addition of citrate actually retards this reaction. In contrast, the nonamphoteric In(3+) ion binds very slowly in the absence of citrate, presumably due to the rapid formation of polymeric In-hydroxo complexes upon addition of the unchelated metal ion to the pH 7.4 protein solution. The addition of citrate to the reaction accelerates the binding of In(3+) to apoTf, presumably by forming soluble, mononuclear In-citrate complexes.