A C-terminal fragment of fibulin-7 interacts with endothelial cells and inhibits their tube formation in culture.

We have previously demonstrated that fibulin-7 (Fbln7) is expressed in teeth by pre-odontoblast and odontoblast cells, localized in the basement membrane and dentin matrices, and is an adhesion molecule for dental mesenchyme cells and odontoblasts. Fbln7 is also expressed in blood vessels by endothelial cells. In this report, we show that a recombinant C-terminal Fbln7 fragment (Fbln7-C) bound to Human Umbilical Vein Endothelial Cells (HUVECs) but did not promote cell spreading and actin stress fiber formation. Fbln7-C binding to HUVECs induced integrin clustering at cell adhesion sites with other focal adhesion molecules, and sustained activation of FAK, p130Cas, and Rac1. In addition, RhoA activation was inhibited, thereby preventing HUVEC spreading. As endothelial cell spreading is an important step for angiogenesis, we examined the effect of Fbln7-C on angiogenesis using in vitro assays for endothelial cell tube formation and vessel sprouting from aortic rings. We found that Fbln7-C inhibited the HUVEC tube formation and the vessel sprouting in aortic ring assays. Our findings suggest potential anti-angiogenic activity of the Fbln7 C-terminal region.

Inhibition of brain tumor growth by intravenous poly (ß-L-malic acid) nanobioconjugate with pH-dependent drug release [corrected].

Effective treatment of brain neurological disorders such as Alzheimer's disease, multiple sclerosis, or tumors should be possible with drug delivery through blood-brain barrier (BBB) or blood-brain tumor barrier (BTB) and targeting specific types of brain cells with drug release into the cell cytoplasm. A polymeric nanobioconjugate drug based on biodegradable, nontoxic, and nonimmunogenic polymalic acid as a universal delivery nanoplatform was used for design and synthesis of nanomedicine drug for i.v. treatment of brain tumors. The polymeric drug passes through the BTB and tumor cell membrane using tandem monoclonal antibodies targeting the BTB and tumor cells. The next step for polymeric drug action was inhibition of tumor angiogenesis by specifically blocking the synthesis of a tumor neovascular trimer protein, laminin-411, by attached antisense oligonucleotides (AONs). The AONs were released into the target cell cytoplasm via pH-activated trileucine, an endosomal escape moiety. Drug delivery to the brain tumor and the release mechanism were both studied for this nanobiopolymer. Introduction of a trileucine endosome escape unit resulted in significantly increased AON delivery to tumor cells, inhibition of laminin-411 synthesis in vitro and in vivo, specific accumulation in brain tumors, and suppression of intracranial glioma growth compared with pH-independent leucine ester. The availability of a systemically active polymeric drug delivery system that passes through the BTB, targets tumor cells, and inhibits glioma growth gives hope for a successful strategy of glioma treatment. This delivery system with drug release into the brain-specific cell type could be useful for treatment of various brain pathologies.

Regional differences in the expression of laminin isoforms during mouse neural tube development.

Many significant human birth defects originate around the time of neural tube closure or early during post-closure nervous system development. For example, failure of the neural tube to close generates anencephaly and spina bifida, faulty cell cycle progression is implicated in primary microcephaly, while defective migration of neuroblasts can lead to neuronal migration disorders such as lissencephaly. At the stage of neural tube closure, basement membranes are becoming organised around the neuroepithelium, and beneath the adjacent non-neural surface ectoderm. While there is circumstantial evidence to implicate basement membrane dynamics in neural tube and surface ectodermal development, we have an incomplete understanding of the molecular composition of basement membranes at this stage. In the present study, we examined the developing basement membranes of the mouse embryo at mid-gestation (embryonic day 9.5), with particular reference to laminin composition. We performed in situ hybridization to detect the mRNAs of all eleven individual laminin chains, and immunohistochemistry to identify which laminin chains are present in the basement membranes. From this information, we inferred the likely laminin variants and their tissues of origin: that is, whether a given basement membrane laminin is contributed by epithelium, mesenchyme, or both. Our findings reveal major differences in basement composition along the body axis, with the rostral neural tube (at mandibular arch and heart levels) exhibiting many distinct laminin variants, while the lumbar level where the neural tube is just closing shows a much simpler laminin profile. Moreover, there appears to be a marked difference in the extent to which the mesenchyme contributes laminin variants to the basement membrane, with potential contribution of several laminins rostrally, but no contribution caudally. This information paves the way towards a mechanistic analysis of basement membrane laminin function during early neural tube development in mammals.

Identification and molecular characterization of tryptophanase encoded by tnaA in Porphyromonas gingivalis.

Indole produced via the beta-elimination reaction of l-tryptophan by pyridoxal 5'-phosphate-dependent tryptophanase (EC 4.1.99.1) has recently been shown to be an extracellular and intercellular signalling molecule in bacteria, and controls bacterial biofilm formation and virulence factors. In the present study, we determined the molecular basis of indole production in the periodontopathogenic bacterium Porphyromonas gingivalis. A database search showed that the amino acid sequence deduced from pg1401 of P. gingivalis W83 is 45 % identical with that from tnaA of Escherichia coli K-12, which encodes tryptophanase. Replacement of the pg1401 gene in the chromosomal DNA with the chloramphenicol-resistance gene abolished indole production. The production of indole was restored by the introduction of pg1401, demonstrating that the gene is functionally equivalent to tnaA. However, RT-PCR and RNA ligase-mediated rapid amplification of cDNA ends analyses showed that, unlike E. coli tnaA, pg1401 is expressed alone in P. gingivalis and that the nucleotide sequence of the transcription start site is different, suggesting that the expression of P. gingivalis tnaA is controlled by a unique mechanism. Purified recombinant P. gingivalis tryptophanase exhibited the Michaelis-Menten kinetics values K(m)=0.20+/-0.01 mM and k(cat)=1.37+/-0.06 s(-1) in potassium phosphate buffer, but in sodium phosphate buffer, the enzyme showed lower activity. However, the cation in the buffer, K(+) or Na(+), did not appear to affect the quaternary structure of the enzyme or the binding of pyridoxal 5'-phosphate to the enzyme. The enzyme also degraded S-ethyl-l-cysteine and S-methyl-l-cysteine, but not l-alanine, l-serine or l-cysteine.

Ammonia removal from livestock wastewater by ammonia-assimilating microorganisms immobilized in polyvinyl alcohol.

We isolated ammonia-assimilating microorganisms from the livestock manure treatment systems and evaluated their ammonia-assimilating ability. Many isolates utilized ammonia at high rates when they were purely cultivated in a nitrogen-limited medium to which sterilized lagoon extract had been added. Some isolates that were immobilized in polyvinyl alcohol (PVA) utilized ammonia present in the media containing viable lagoon microorganisms. Staining with 4',6'-diamidino-2-phenylindole (DAPI) indicated that the immobilized high ammonia-assimilating isolates grew dominantly within the PVA beads. High ammonia-assimilating isolates in the mixed culture containing viable lagoon microorganisms were identified as Pseudomonas spp. and member of Rhizobiaceae species by partial sequencing of the 16S ribosomal DNA.