Acid Polypeptide Research Articles

Heat-induced aggregation of subunits/polypeptides of soybean protein: Structural and physicochemical properties

To reveal the nature of thermal aggregation of soybean protein at subunit level, structure and physicochemical properties of αα'- and β-subunits isolated from β-conglycinin, acidic polypeptide, and basic polypeptide from glycinin, as well as β-conglycinin and glycinin, were characterized before and after heat treatment. The transmission electron microscopy (TEM) images showed that β-conglycinin, αα'-subunits and acidic polypeptide formed regular thermal aggregates, which exhibited high solubility, high ζ-potential value, and small particle size. While glycinin, β-subunit, and basic polypeptide aggregated to insoluble clusters with large particle size distribution. The results of size exclusion chromatography and non-reducing electrophoresis showed that the disulfide bond was the important force in stabilizing the protein conformation of thermal aggregates in β-conglycinin, glycinin, and their isolated subunits/polypeptides but β-subunit. The results of surface hydrophobicity and intrinsic fluorescence spectra showed that the thermal aggregations of β-subunit and basic polypeptide were mainly driven by hydrophobic interactions.

The C-terminal stretch of glycine-rich proline-rich protein (SbGPRP1) from Sorghum bicolor serves as an antimicrobial peptide by targeting the bacterial outer membrane protein.

The C-terminal stretch in SbGPRP1 (Sorghum glycine-rich proline-rich protein) acts as an antimicrobial peptide in the host innate defense mechanism. Cationic antimicrobial proteins or peptides can either bind to the bacterial membrane or target a specific protein on the bacterial membrane thus leading to membrane perturbation. The 197 amino acid polypeptide of SbGPRP1 showed disordered structure at the N-terminal end and ordered conformation at the C-terminal end. In the present study, the expression of N-SbGPRP1, C-SbGPRP1, and ∆SbGPRP1 followed by antimicrobial assays showed potential antimicrobial property of the C-terminal peptide against gram-positive bacteria Bacillus subtilis and phytopathogen Rhodococcus fascians. The SbGPRP1 protein loses its antimicrobial property when the 23 amino acid sequence (GHGGHGVFGGGYGHGGYGHGYGG) from position 136 to 158 is deleted from the protein. Thus, it can be concluded that the 23 amino acid sequence is vital for the said antimicrobial property. NPN assay, SEM analysis, and electrolyte leakage assays showed potent antimicrobial activity for C-SbGPRP1. Overexpression of the C-SbGPRP1 mutant protein in tobacco followed by infection with Rhodococcus fascians inhibited bacterial growth as shown by SEM analysis. To determine if C-SbGPRP1 might target any protein on the bacterial membrane we isolated the bacterial membrane protein from both Bacillus subtilis and Rhodococcus fascians. Bacterial membrane protein that interacted with the column-bound C-SbGPRP1 was eluted and subjected to LC-MS/MS. LC-MS/MS data analysis showed peptide hit with membrane protein YszA from Bacillus subtilis and a membrane protein from Rhodococcus fascians. Isolated bacterial membrane protein from Bacillus subtilis or Rhodococcus fascians was able to reduce the antimicrobial activity of C-SbGPRP1. Furthermore, BiFC experiments showed interactions between C-SbGPRP1 and YszA protein from Bacillus subtilis leading to the conclusion that bacterial membrane protein was targeted in such membrane perturbation leading to antimicrobial activity.

Identification and Expression Analysis of Dsx and Its Positive Transcriptional Regulation of IAG in Black Tiger Shrimp (Penaeus monodon).

Doublesex (Dsx) is a polymorphic transcription factor of the DMRTs family, which is involved in male sex trait development and controls sexual dimorphism at different developmental stages in arthropods. However, the transcriptional regulation of the Dsx gene is largely unknown in decapods. In this study, we reported the cDNA sequence of PmDsx in Penaeus monodon, which encodes a 257 amino acid polypeptide. It shared many similarities with Dsx homologs and has a close relationship in the phylogeny of different species. We demonstrated that the expression of the male sex differentiation gene Dsx was predominantly expressed in the P. monodon testis, and that PmDsx dsRNA injection significantly decreased the expression of the insulin-like androgenic gland hormone (IAG) and male sex-determining gene while increasing the expression of the female sex-determining gene. We also identified a 5′-flanking region of PmIAG that had two potential cis-regulatory elements (CREs) for the PmDsx transcription. Further, the dual-luciferase reporter analysis and truncated mutagenesis revealed that PmDsx overexpression significantly promoted the transcriptional activity of the PmIAG promoter via a specific CRE. These results suggest that PmDsx is engaged in male reproductive development and positively regulates the transcription of the PmIAG by specifically binding upstream of the promoter of the PmIAG. It provides a theoretical basis for exploring the sexual regulation pathway and evolutionary dynamics of Dmrt family genes in P. monodon.

Identification and expression of a transforming growth factor beta (TGF-β) homologue in the tropical liver fluke Fasciola gigantica.

Liver flukes, Fasciola spp., are veterinary and medically important parasites infecting numerous species of economically important animals in addition to humans on a global scale. The components of transforming growth factor beta (TGF-β) signalling are widely distributed throughout the animal kingdom and are considerably conserved. Through shared common signal transduction mechanisms, crosstalk of TGF-β signalling between a host and the parasite during infection is possible. Herein, we have identified and undertaken the molecular characterisation of a putative TGF-β homologue from the tropical liver fluke F. gigantica (FgTLM). A FgTLM cDNA was 3557bp inlength, itencoded for 620 amino acid polypeptide which consisted of 494 amino acids of prodomain and 126 amino acids comprising the mature protein. FgTLM displayed characteristic structures of mammalian TGF-β ligands that were unique to the inhibin-β chain, monomer of activin. A phylogenetic analysis revealed the high degree of conservation with TGF-β molecules from trematode species. Interestingly, the sequence of amino acid in the active domain of FgTLM was completely identical to FhTLM from F. hepatica. FgTLM expressed throughout the lifecycle of F. gigantica but was highly expressed in developmental active stages. The dynamics of expression of FgTLM during the developmental stages of F. gigantica was comparable to the pattern of TGF-β expression in F. hepatica. Our findings demonstrated that FgTLM exhibits a high level of similarity to FhTLM in the context of both amino acid sequence and the life stage expression patterns. These similarities underline the possibility that the FgTLM molecule might have the same properties and functions as FhTLM in biological processes of the immature parasites and host immune evasion. Consequently, the specific biological functions of FgTLM on either parasite or relevant hosts need to be defined experimentally.

Knockdown of GFAT disrupts chitin synthesis in Hyphantria cunea larvae

Glutamine-fructose-6-phosphate transaminase (GFAT) has been reported to regulate the hexosamine biosynthetic pathway as the first rate-limiting enzyme. As a key enzyme that catalyzes the substrate of glycosylation modification, which has a wide-ranging effect on cellular functions. However, there are few studies on the relationship between GFAT and chitin metabolism in insects. In the present study, the GFAT gene from Hyphantria cunea was identified based on transcriptome and bioinformatic analysis. The role of HcGFAT in regulating development and chitin synthesis was analyzed by RNA interference (RNAi) in H. cunea larvae. The full-length HcGFAT gene (2028 bp) encodes a 676 amino acid (aa) polypeptide had typical structural features of the SIS and Gn_AT_II superfamily. Phylogenetic analyses showed that GFAT of H. cunea shares the highest homology and identity with GFAT of Ostrinia furnacalis. Expression profiles indicated that HcGFAT was expressed throughout larval, pupal and three tissues (midgut, fat body, epidermis), and highly expressed in the last instar of larvae and strongly expressed in epidermis among three tissues. Bioassay results showed that knockdown of HcGFAT repressed larval growth and development, resulting in a significant loss of larval body weight. Meanwhile, HcGFAT knockdown also significantly caused larval developmental deformity. Knockdown of HcGFAT regulated the expression of four other critical genes in the chitin synthesis pathway (HcGNA, HcPAGM, HcUAP, HcCHSA), and ultimately resulted in decreased chitin content in the epidermis. In summary, these findings indicated that GFAT plays a critical role in larval growth and development, as well as chitin synthesis in H. cunea.

The differential expression and effects of Drosha on metamorphosis of Japanese flounder (paralichthys olivaceus)

Drosha is critical for producing mature microRNAs (miRNAs) from their precursor molecules and small interfering RNAs (siRNAs). Although Drosha has been well characterized in most vertebrate species, identifying the Japanese flounder Drosha has remained elusive. In this study, we cloned the Japanese flounder Drosha gene, which has 4,122 nucleotides (nt), including a 5’-untranslated region (UTR) of 14 nt, a 3’-UTR of 230 nt, and a 3,879 nt open reading frame (ORF) encodes a 1,292 amino acid polypeptide. Then, we analyzed the conservation and phylogenetic evolution of Drosha in some species. Real-time quantitative PCR revealed that Drosha mRNA is highly expressed in the brain, and a lower amount of mRNA was also found in muscle than in other tissues. Drosha plays a vital role in controlling flounder development and metabolism, and its mRNA levels were relatively high at 36 dph (days post-hatching) in the period of metamorphosis and growth of the Japanese flounder. Drosha expression showed in advance with a peak at 23 dph following TH (thyroid hormone) treatment. To further investigate the role of Drosha in metamorphosis, we performed siRNA knockdown of Drosha in flounder embryonic cells (FEC) cells. The result shows that the Drosha-specific siRNA significantly down-regulated Drosha mRNA and miR-1,133,17,214,181a,181b levels, while primary miRNA (pri-miRNA) levels were not significantly different from negative control (NC). These results suggest that Drosha plays a vital role in Japanese flounder development and metamorphosis through processing to produce mature miRNAs, providing essential information for further studying of the part of the Drosha gene in the Japanese flounder development.

Functional characterization of MEKK3 in the intestinal immune response to bacterial challenges in grass carp (Ctenopharyngodon idella).

Mitogen-activated protein kinase kinase kinase 3 (MEKK3) is an evolutionarily conserved Ser/Thr protein kinase of the MEKK family that is essential for the host immune response to pathogen challenges in mammals. However, the immune function of MEKK3s in lower vertebrate species, especially in bony fish, remains largely unknown. In this study, a fish MEKK3 (designated CiMEKK3) gene was cloned and identified from grass carp (Ctenopharyngodon idella). The present CiMEKK3 cDNA encoded a 620 amino acid polypeptide containing a conserved S-TKc domain and a typical PB1 domain. Several potential immune-related transcription factor-binding sites, including activating protein 1 (AP-1), nuclear factor kappa B (NF-κB) and signal transducer and activator of downstream transcription 3 (STAT3), were observed in the 5’ upstream DNA sequence of CiMEKK3. A phylogenetic tree showed that CiMEKK3 exhibits a close evolutionary relationship with MEKK3s from Cyprinus carpio and Carassius auratus. Quantitative real-time PCR analysis revealed that CiMEKK3 transcripts were widely distributed in all selected tissues of healthy grass carp, with a relatively high levels observed in the gill, head kidney and intestine. Upon in vitro challenge with bacterial pathogens (Aeromonas hydrophila and Aeromonas veronii) and pathogen-associated molecular patterns (PAMPs) (lipopolysaccharide (LPS), peptidoglycan (PGN), L-Ala-γ-D-Glu-mDAP (Tri-DAP) and muramyl dipeptide (MDP)), the expression levels of CiMEKK3 in the intestinal cells of grass carp were shown to be significantly upregulated in a time-dependent manner. In vivo injection experiments revealed that CiMEKK3 transcripts were significantly induced by MDP challenge in the intestine; however, these effects could be inhibited by the nutritional dipeptides carnosine and Ala-Gln. Moreover, subcellular localization analysis and luciferase reporter assays indicated that CiMEKK3 could act as a cytoplasmic signal-transducing activator involved in the regulation of NF-κB and MAPK/AP-1 signaling cascades in HEK293T cells. Taken together, these findings strongly suggest that CiMEKK3 plays vital roles in the intestinal immune response to bacterial challenges, which will aid in understanding the pathogenesis of inflammatory bowel disease in bony fish.

Grouper interferon-induced protein 35, a CP-interacting protein, inhibits fish nodavirus replication via positively regulating host interferon and inflammatory immune response

Interferon (IFN)-induced protein 35 (IFI35, also known as IFP35), a member of IFN induced genes (ISGs), participates in virus infection, cancer progression and the chronic inflammatory diseases. However, its roles during fish nodavirus infection still remained largely unknown. In the present study, a homolog of IFI35 from orange spotted grouper (Epinephelus coioides) (EcIFI35) was cloned and characterized. The open reading frame of EcIFI35 was composed of 1,128 bp, and encoded a 375 amino acid polypeptide, which contained two conserved N-myc-interactor (Nmi)/IFP35 domains (NIDs). Homology analysis indicated that EcIFI35 shared 95.73% and 31.96% identity with homologs of giant grouper (E. lanceolatus) and human (Homo sapiens), respectively. The transcription of EcIFI35 was significantly up-regulated in grouper spleen (GS) cells after challenged with red-spotted grouper nervous necrosis virus (RGNNV), polyinosinic:polycytidylic acid [poly(I:C)] or lipopolysaccharide (LPS). The subcellular localization analysis showed that EcIFI35 encoded a cytoplasmic protein. The ectopic expression of EcIFI35 inhibited RGNNV replication by reducing viral genes transcription and protein synthesis. Co-immunoprecipitation (Co-IP) assay demonstrated that EcIFI35 interacted with RGNNV coat protein (CP), and partly co-localized with CP. EcIFI35 overexpression promoted the expression of IFN-related molecules and pro-inflammatory factors, including IFN regulatory factor 7 (IRF7), mitochondrial antiviral signaling protein (MAVS) and myxovirus resistance gene I (MxI), nuclear factor κB (NF-κB), interleukin 6 (IL-6) and IL-8. Together, our results revealed that EcIFI35 interacted with CP and inhibited fish nodavirus replication through positively regulated host innate immune response.

Identification and subcellular localization analysis of CCTα in microsporidian Nosema bombycis.

CCT is a chaperonin which is widely present in eukaryotic cells and mainly involves in the folding and assembly of cytoskeletal proteins β-tubulin and actin. The alpha subunit of CCT(CCTα) plays a pivotal role in the folding and assembly of cytoskeletal protein(s) as an individuals or complexes. In this study, we report cloning, characterization and expression of the CCTα of Nosema bombycis (NbCCTα) for the first time. The NbCCTα gene contains a complete ORF of 1629bp in length that encodes a 542-amino acid polypeptide. The NbCCTα is 59.662kDa molecular weight in size with an isoelectric point (pI) of 5.81, no signal peptide or transmembrane domain. The IFA results showed that the NbCCTα was co-localized with actin and β-tubulin in the cytoplasm, nucleus, nuclear membrane and plasma membrane of N. bombycis in the process of proliferation. qPCR analysis showed that the relative expression level of NbCCTα increased from 24h to 96h post-infection (hp.i) of N. bombycis, and reached the highest at 96hp.i. The relative expression level of NbCCTα gene after RNAi was restrained at a low level from 48hp.i to 96hp.i. Knockdown of NbCCTα gene down-regulated the expression of Nbβ-tubulin and Nbactin genes. These results imply that NbCCTα may play an important role in the lifecycle of N. bombycis.

Identification and subcellular localization analysis of membrane protein Ycf 1 in the microsporidian Nosema bombycis.

Microsporidia are obligate intracellular parasites that can infect a wide range of vertebrates and invertebrates including humans and insects, such as silkworm and bees. The microsporidium Nosema bombycis can cause pebrine in Bombyx mori, which is the most destructive disease in the sericulture industry. Although membrane proteins are involved in a wide range of cellular functions and part of many important metabolic pathways, there are rare reports about the membrane proteins of microsporidia up to now. We screened a putative membrane protein Ycf 1 from the midgut transcriptome of the N. bombycis-infected silkworm. Gene cloning and bioinformatics analysis showed that the Ycf 1 gene contains a complete open reading frame (ORF) of 969 bp in length encoding a 322 amino acid polypeptide that has one signal peptide and one transmembrane domain. Indirect immunofluorescence results showed that Ycf 1 protein is distributed on the plasma membrane. Expression pattern analysis showed that the Ycf 1 gene expressed in all developmental stages of N. bombycis. Knockdown of the Ycf 1 gene by RNAi effectively inhibited the proliferation of N. bombycis. These results indicated that Ycf 1 is a membrane protein and plays an important role in the life cycle of N. bombycis.

Nuclear Factor-κB Plays a Positive Role in TNFα Expression in Golden Pompano, Trachinotus ovatus (Linnaeus 1758)

Tumour necrosis factor-α (TNFα) is a multifarious mediator of lymphoid tissue growth and antimicrobial defence mechanisms, and it acts as a pro-inflammatory regulator. The function of TNFα in parasite infection and the underlying mechanism through which nuclear factor-κB (NF-κB) regulates TNFα remain largely unclear in teleosts. In the present study, TNFα (ToTNFα) from golden pompano (Trachinotus ovatus) was identified, and its sequence features and expression levels were determined. The genomic DNA sequence is composed of 1,130 bp, consists of four exons and three introns, and encodes 341 amino acid polypeptides. The putative protein sequence shares 34.7%–61.9% identity with fish TNFα and possesses a TNF family signature and two conserved cysteine residues. Moreover, the expressions of ToNF-κB and ToTNFα are constitutively expressed in all examined tissues, with higher levels observed in the immune relevant tissues. Both ToNF-κB and ToTNFα transcription was increased in the local infection sites (skin and gill) and system immune tissues (liver, spleen and head kidney) after Cryptocaryon irritans stimulation. In addition, to investigate whether ToNF-κB is a regulator of ToTNFα, promoter analysis was performed. The region from -970 to +79 bp is known as the core promoter by different truncated mutants of ToTNFα. Subsequently, the activity of the ToTNFα-p2 promoter was dramatically reduced after targeted mutation of the M6-binding site. Additionally, an electrophoretic mobile shift assay (EMSA) verified that ToNF-κB interacted with the M6-binding site in the ToTNFα promoter region to control the expression of ToTNFα. In conclusion, the present study provides the positive regulation of TNFα transcription by NF-κB and contributes to a better understanding of the transcriptional mechanism of TNFα in fish.

Characterization of TRIM16, a member of the fish-specific finTRIM family, in olive flounder Paralichthys olivaceus

Tripartite motif-containing (TRIM) proteins are conserved throughout the metazoan kingdom, and the TRIM subset finTRIM is highly diversified in fish. We isolated TRIM16 cDNA, a member of the finTRIM family, from the olive flounder Paralichthys olivaceus (PoTRIM16). PoTRIM16 contained a 1,725-bp coding sequence encoding a 574-amino acid polypeptide, which in turn contained a really interesting new gene (RING) finger domain, B-box-type zinc finger (B-BOX), nuclease SbcCD subunit C (SbcC), structural maintenance of chromosome (SMC prok B), and stonustoxin (SNTX) subunit alpha (SPRY-PRY-SNTX). Multiple alignment of related sequences revealed that PoTRIM16 showed 86.63–97.40% identity with fish orthologues, and a phylogenetic tree was constructed of vertebrates. PoTRIM16 mRNA was detected in all tissues examined; levels were highest in the eye and ovary. PoTRIM16 mRNA expression was investigated during early development. Under VHSV infection, PoTRIM16 mRNA was downregulated in the liver of P. olivaceus. This is the first study to characterize fish-specific finTRIM in P. olivaceus, which may play a role in the immune response against virus infection.

MiR-137 modulates coelomocytes autophagy by targeting Atg13 in the sea cucumber Apostichopus japonicus

MicroRNAs (miRNAs), as important regulators of host immune responses, play an crucial position in the interaction between host and pathogen by inhibiting the target gene's transcriptional and post-transcriptional expression. A well-validated tumor suppressor, Previously, miR-137 was found to be variably expressed in the sick sea cucumber Apostichopus japonicus specimens by high-throughput sequencing. To further investigate the mechanism of miR-137 regulation of SUS, we identified Atg13 from sea cucumber by dual luciferase reporter assay and RACE (designated as AjAtg13) and was able to serve as a target gene for miR-137. The full-length cDNA of AjAtg13 is a 2197 bp fragment containing an ORF (open reading frame) of 1149 bp and encodes a total of 382 amino acid polypeptides with a predicted molecular weight of 41.7 kDa. Further expression profiling analysis showed increased mRNA levels of AjAtg13 and reduced expression levels of miR-137 in LPS-stimulated sea cucumber coelomocytes, hinting that miR-137 may negatively regulate AjAtg13. MiR-137 targets AjAtg13 through binding to the 3′UTR region by dual-luciferase reporter gene analysis. MiR-137 overexpression in coelomocytes repressed the expression of autophagy related genes, such as AjAtg13, AjLC3, at the same time, it significantly inhibited autophagy and reduced the ability to clear Vibrio splendidus. Conversely, inhibition of miR-137 significantly upregulated the expression of AjAtg13, promoted autophagy and increased clearance of V. splendidus. Subsequent interference with AjAtg13 also significantly inhibits autophagy. In summary, our results suggested that miR-137 could promote coelomocytes autophagy to restrict bacterial invasion by aiming at AjAtg13 in pathogen-stimulated sea cucumbers.

Comprehensive Analysis of Role of Cyclin-Dependent Kinases Family Members in Colorectal Cancer.

BackgroundCyclin-dependent kinases (CDKs) are cell cycle regulators, and abnormal activation can accelerate tumor cell proliferation. However, The relation between CDKs dysregulation to colorectal cancer incidence and progression have not been examined in detail. Methods:Differences in CDKs expression between colorectal cancer and normal tissues, associations between expression and clinical prognosis, incidence and frequencies of CDKs gene mutations, and the influences of CDKs on tumor infiltration by immune cells were examined by analyses of Oncomine, Gene Expression Profiling Interactive Analysis, Kaplan-Meier plotter, cBioPortal, GeneMANIA, and TIMER databases.ResultsColorectal cancer tissues showed enhanced expression levels of CDKs 1/2/4/5/6/8/12/13/19 but reduced CDK3 expression. CDK7 was highly expressed in some colorectal cancer tissues but downregulated in others. Expression levels of CDK1/3/4/7/8/10/11b/13/18/19/20 were correlated with clinical stage, and CDK 5/10/12/16 expression levels predicted prognosis and survival. Differential CDKs expression correlated with cell cycle progression, amino acid polypeptide modifications, and activation of other protein kinases. Expression levels of all CDKs except CDK16 were correlated with infiltration of CD4+T, CD8+T, B and Tregs cells.ConclusionsCDK 1 and 4 could be used as diagnostic biomarkers for CRC. CDK 5/10/12/16 can be utilized as prognostic biomarkers.

A Potential Role of the Translation Elongation Factor eef1a1 in Gonadal High-Temperature Perception in Chinese Tongue Sole (Cynoglossus semilaevis).

Simple SummaryThe eukaryotic elongation factor 1 alpha (eef1a) gene is vital for protein translation by delivering aminoacylated tRNAs to the A/P site of the ribosome via the GTP-dependent reaction. Here, the Chinese tongue sole (Cynoglossus semilaevis) eef1a1 gene was identified, and its potential role in gonadal high-temperature perception was assessed. The full-length sequence of eef1a1 cDNA was 1809 base pair (bp) encoding a putative protein of 461 amino acids. The expression levels of eef1a1 in the ovary were significantly higher than that in the testis from 6 mpf to 3 ypf. Under high-temperature induction during sex differentiation, eef1a1 was significantly down-regulated in males, while the difference was not detected in females. Furthermore, the rapid response of eef1a1 to environmental high temperature was assessed in vitro. Our findings suggest that C. semilaevis eef1a1 might be essential for the molecular response regulatory network of external temperature affecting internal sex differentiation.The eukaryotic translation elongation factor 1 alpha (eef1a) gene has a well-defined role in protein synthesis. However, its role in external temperature perception and internal sex differentiation and development is still unclear. In this study, eef1a1 was identified and functionally analyzed in Chinese tongue sole (Cynoglossus semilaevis). The eef1a1 cDNA, 1809 bp in length, had a 1386 bp open reading frame (ORF) that encoded a 461 amino acid polypeptide containing one EF-1_alpha domain. eef1a1 expression levels were investigated across different tissues and during gonadal development. In the gonad, eef1a1 showed a sexually dimorphic expression pattern with a statistically higher expression level in the ovary than in the testis from 6 months postfertilization to 3 years postfertilization. Under high temperature (28 °C) treatment during C. semilaevis sex differentiation (from 30 days postfertilization to 3 months postfertilization), eef1a1 was statistically down-regulated in males, while the difference was not detected in females. In addition, the dual-luciferase assay exhibited that eef1a1 can respond to high temperature rapidly. Based on these results, C. semilaevis eef1a1 might have a dual role in the perception of external temperature changes and sex differentiation regulation.

Mechanism and efficacy of parathyroid hormone in osteoporosis

Background: Parathyroid hormone (PTH) and teriparatide, which is a PTH analog, are special options of osteoanabolic therapies for osteoporosis treatment. Their efficacy in improving the bone mineral density (BMD) and reducing fracture risk is considered to have a long-awaited justification.Current Concepts: PTH is an 84-amino acid polypeptide that plays a key role in the calcium homeostasis of the human body. It yields anabolic effects on the bone by intermittent injections. During the first 6 months of PTH treatment, bone formation increases rapidly and bone resorption rises gradually. PTH is believed to be maximally anabolic during the anabolic window. In the phase III trial of teriparatide, significant reductions in vertebral and non-vertebral fractures were identified. Teriparatide has been studied in drug-induced osteoporosis, postmenopausal women with osteoporosis, and men with osteoporosis. The data derived from clinical trials with teriparatide showed an increase of the BMD in the treatment group compared to that of the placebo. Selective advantages on fracture healing have been shown in some studies; however, consensus on the relationship between teriparatide and bone healing is unclear.Discussion and Conclusion: Teriparatide has been approved for osteoporosis treatment in various situations, including postmenopausal women, men with osteoporosis, and cases with glucocorticoid-induced osteoporosis. Additionally, it can be useful in high risk for fracture groups, if fractures persist despite bone resorption inhibitor therapy, and if bone resorption inhibitor administration is difficult and contraindicated.

Functional characterisation and comparative analysis of two C-type lectins with different key motifs from mud crab

C-type lectins are pattern recognition receptors (PRRs) involved in innate immunity. They possess one or more carbohydrate recognition domains (CRDs) belonging to Ca2+-dependent carbohydrate binding proteins. Herein, two C-type lectins were identified from the mud crab Scylla paramamosain. The open reading frame (ORF) of SpCTL-E encodes 160 amino acids including a CRD and a signal peptide, while the SpCTL-F ORF encodes a 244 amino acid polypeptide with a signal peptide and a CRD, and the key motifs for carbohydrate binding specificity are Asp-Pro-Asn (DPN) and Gln-Pro-Asp (QPD), respectively. DPN is a newly discovered motif, while QPD is typical of C-type lectins. The binding specificity toward different pathogen-associated molecular patterns (PAMPs) was analysed, and both bound three PAMPs: lipopolysaccharide (LPS), glucan (GLU) and peptidoglycan (PGN). SpCTL-F bound more strongly than SpCTL-E. SpCTL-E and SpCTL-F bound to five and seven microbial species, respectively, both agglutinated Gram-negative bacteria and fungi, and both promoted the phagocytosis activity of hemocytes toward Escherichia coli, and both enhanced the encapsulation activity of hemocytes towards Ni-NTA beads. SpCTL-E inhibited the growth of Micrococcus luteus and E. coli, while SpCTL-F also inhibited Vibrio parahaemolyticus. Thus, SpCTL-E and SpCTL-F are important PRRs with a broad nonself-recognition spectrum and play a key role in pathogen elimination.

Identification, expression and subcellular localization of Orc1 in the microsporidian Nosema bombycis

As a typical species of microsporidium, Nosema bombycis is the pathogen causing the pébrine disease of silkworm. Rapid proliferation of N. bombycis in host cells requires replication of genetic material. As eukaryotic origin recognition protein, origin recognition complex (ORC) plays an important role in regulating DNA replication, and Orc1 is a key subunit of the origin recognition complex. In this study, we identified the Orc1 in the microsporidian N. bombycis (NbOrc1) for the first time. The NbOrc1 gene contains a complete ORF of 987 bp in length that encodes a 328 amino acid polypeptide. Indirect immunofluorescence results showed that NbOrc1 were colocalized with Nbactin and NbSAS-6 in the nuclei of N. bombycis. Subsequently, we further identified the interaction between the NbOrc1 and Nbactin by CO-IP and Western blot. These results imply that Orc1 may be involved in the proliferation of the microsporidian N. bombycis through interacting with actin.

Conjugation in the eukaryotic single-celled organism Euplotes aediculatus (Protozoa, Ciliophora): A focus on nuclear divisions, morphogenesis and pheromones

Ciliates are unique in having nuclear dimorphism (i.e., possessing both a germline micronucleus and a somatic macronucleus in one cell), and undergo dramatic changes during their sexual process (known as conjugation). Here we investigate the nuclear events and morphogenetic processes during conjugation in the freshwater species Euplotes aediculatus. In addition, three conjugation-related pheromone genes were sequenced and analyzed. The results indicate that: (i) the 65-hour-long conjugation process includes five nuclear divisions, i.e., three prezygotic divisions (mitosis, meiosis I and II) and two postzygotic divisions; (ii) the ciliature reorganizes twice during conjugation with the frontoventral-transverse cirral anlagen regenerating during both morphogenetic events and breaking apart in a 3:3:3:2:2 pattern; and (iii) each of three pheromone genes contains a 444 bp coding region and encodes 147 amino acid polypeptides. These results suggest a broadly shared morphogenetic pattern during conjugation in Euplotes species and provide new information for studies on the evolutionary history of pheromone genes.

Metabolic Bone Disease of Prematurity.

Metabolic Bone Disease of Prematurity.