Delivery of small interfering RNA with a synthetic collagen poly(Pro‐Hyp‐Gly) for gene silencing in vitro and in vivo

Silencing gene expression by small interfering RNAs (siRNAs) has become a powerful tool for the genetic analysis of many animals. However, the rapid degradation of siRNA and the limited duration of its action in vivo have called for an efficient delivery technology. Here, we describe that siRNA complexed with a synthetic collagen poly(Pro‐Hyp‐Gly) (SYCOL) is resistant to nucleases and is efficiently transferred into cells in vitro and in vivo, thereby allowing long‐term gene silencing in vivo. We found that the SYCOL‐mediated local application of siRNA targeting myostatin, coding a negative regulator of skeletal muscle growth, in mouse skeletal muscles, caused a marked increase in the muscle mass within a few weeks after application. Furthermore, in vivo administration of an anti‐luciferase siRNA/SYCOL complex partially reduced luciferase expression in xenografted tumors in vivo. These results indicate a SYCOL‐based non‐viral delivery method could be a reliable simple approach to knockdown gene expression by RNAi in vivo as well as in vitro.     

Methyladeninylcobamide functions as the cofactor of methionine synthase in a Cyanobacterium, Spirulina platensis NIES-39

To clarify the physiological function of pseudovitamin B₁₂ (or adeninylcobamide; AdeCba) in Spirulina platensis NIES-39, cobalamin-dependent methionine synthase (MS) was characterized. We cloned the full-length Spirulina MS. The clone contained an open reading frame encoding a protein of 1183 amino acids with a molecular mass of 132 kDa. Deduced amino acid sequences of the Spirulina MS contained critical residues identical to cobalamin-, zinc-, S-adenosylmethionine-, and homocysteine-binding motifs. The recombinant Spirulina enzyme showed higher affinity for methyladeninylcobamide than methylcobalamin as a cofactor. These results indicate that Spirulina cells can utilize AdeCba synthesized as the cofactor for MS.     

A novel glucosylation reaction on anthocyanins catalyzed by acyl-glucose-dependent glucosyltransferase in the petals of carnation and delphinium

Glucosylation of anthocyanin in carnations (Dianthus caryophyllus) and delphiniums (Delphinium grandiflorum) involves novel sugar donors, aromatic acyl-glucoses, in a reaction catalyzed by the enzymes acyl-glucose-dependent anthocyanin 5(7)-O-glucosyltransferase (AA5GT and AA7GT). The AA5GT enzyme was purified from carnation petals, and cDNAs encoding carnation Dc AA5GT and the delphinium homolog Dg AA7GT were isolated. Recombinant Dc AA5GT and Dg AA7GT proteins showed AA5GT and AA7GT activities in vitro. Although expression of Dc AA5GT in developing carnation petals was highest at early stages, AA5GT activity and anthocyanin accumulation continued to increase during later stages. Neither Dc AA5GT expression nor AA5GT activity was observed in the petals of mutant carnations; these petals accumulated anthocyanin lacking the glucosyl moiety at the 5 position. Transient expression of Dc AA5GT in petal cells of mutant carnations is expected to result in the transfer of a glucose moiety to the 5 position of anthocyanin. The amino acid sequences of Dc AA5GT and Dg AA7GT showed high similarity to glycoside hydrolase family 1 proteins, which typically act as β-glycosidases. A phylogenetic analysis of the amino acid sequences suggested that other plant species are likely to have similar acyl-glucose-dependent glucosyltransferases.     

Characterization of Spetex‐1, a new component of satellite fibrils associated with outer dense fibers in the middle piece of rodent sperm flagella

Spetex‐1, which has been isolated by differential display as a haploid spermatid‐specific gene, encodes a protein with two coiled‐coil motifs located in the middle piece of flagella in rodent spermatozoa. The middle piece of flagella is composed of axoneme and peri‐axonemal elements including outer dense fibers (ODFs) and satellite fibrils. Pre‐embedding immunoelectron microscopy clearly demonstrated that Spetex‐1 is located at satellite fibrils associated with ODFs in the middle piece of flagella of rat spermatozoa. Extraction of Spetex‐1 from spermatozoa by SDS or urea required dithiothreitol, suggesting crosslinking by disulfide bond is involved in the assembly of satellite fibrils containing Spetex‐1. We identified putative Spetex‐1 orthologs in many animal species, and both cysteine residues and coiled‐coil motifs were well conserved in mammalian orthologs of Spetex‐1. When Spetex‐1 was co‐transfected into COS‐7 cells with myc‐tagged Tektin4, another filamentous protein associated with ODFs, the two molecules were co‐localized in various sizes of aggregates in the cells. These data suggested that Spetex‐1, a new component of satellite fibrils, might be involved in the structural stability of the sperm flagellar middle piece and functions in co‐operation with Tektin4. Mol. Reprod. Dev. 77: 363–372, 2010. © 2010 Wiley‐Liss, Inc.     

Expression of connexins 36, 43, and 45 during postnatal development of the mouse retina

Gap junction channels formed by connexins (Cx) may play essential roles in some processes that occur during retinal development, such as apoptosis and calcium wave spread. The present study was undertaken to determine the distribution pattern of Cx36, Cx43, and Cx45 by immunofluorescence, as well as their gene expression levels by quantitative PCR during postnatal development of the mouse retina. Our results showed an increased expression of neuronal Cx36 from P1 until P10, when this Cx reached adult levels, and it was mainly distributed in the outer and inner plexiform layers. In turn, Cx43 was almost absent in retinal progenitor cells at P1, it became more prominent in glial cell processes about P10, and did not change until adulthood. Double-labeling studies in situ and in vitro with antivimentin, a Müller cell marker, confirmed that Cx43 was expressed by these cells. In addition, quantitative PCR showed that Cx43 and vimentin shared very similar temporal expression patterns. Finally, in contrast to Cx36 and Cx43, Cx45 mRNA was strongly down-regulated during development. In early postnatal days, Cx45 was seen ubiquitously distributed throughout the retina in cells undergoing proliferation and differentiation, as well in differentiated neurons. In adult retina, this protein had a more restricted distribution both in neurons and glial cells, as confirmed in situ and in vitro. In conclusion, we observed a distinct temporal expression pattern for Cx36, Cx43, and Cx45, which is probably related to particular roles in retinal function and maintenance of homeostasis during development of the mouse retina.     

The expression and possible roles of chemokine CXCL11 and its receptor CXCR3 in the human endometrium

IFN-gamma secreted by a human embryo and trophoblast cells during implantation is suggested to play an important role in implantation and pregnancy. In the present study, we explored expression and possible functions of CXCL11, a CXC chemokine strongly induced by IFN-gamma, and its receptor CXCR3 in the human endometrium. Secreted CXCL11 protein was not detected in cultured endometrial stromal cells (ESC) but was detected in cultured endometrial epithelial cells (EEC). IFN-gamma stimulated the protein levels of CXCL11 in a dose-dependent manner in EEC and ESC. CXCL11 secreted from EEC with 100 ng/ml IFN-gamma was 220-fold of the control, and 100-fold as compared with that secreted from ESC with the same dose of IFN-gamma. CXCR3 was expressed in EEC, ESC, and trophoblast cells. Addition of IFN-gamma to EEC increased the chemotactic activity of its culture medium to trophoblast cells and T cells, and the effect was suppressed by immunoneutralization with Abs of three CXCR3 ligands, including anti-CXCL11 Ab. CXCL11 significantly increased BrdU incorporation of ESC, which was inhibited by a p42/44 MAPK pathway inhibitor PD98059. In contrast, CXCL11 significantly decreased BrdU incorporation and increased the release of lactate dehydrogenase and the positive staining of annexin V in EEC. These findings suggest that IFN-gamma promotes implantation by stimulating EEC to produce CXCL11, which induces migration of trophoblast cells and T cells, proliferation of ESC, and apoptosis of EEC.