Analysis on the molecular species and concentration of circulating ADAMTS13 in Blood.

ADAMTS13 is the metalloprotease responsible for the proteolytic degradation of von Willebrand factor (VWF). A severe deficiency of this VWF-cleaving protease activity causes thrombotic thrombocytopenic purpura. This protease, comprising 1,427 amino acid residues, is composed of multiple domains, i.e., a preproregion, a metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 motif (Tsp1), a cysteine-rich domain, a spacer domain, seven Tsp1 repeats, and two CUB domains. We prepared one polyclonal and seven monoclonal antibodies recognizing distinct epitopes spanning the entire ADAMTS13 molecule. Of these antibodies, two of the monoclonal ones, which recognize the disintegrin-like and cysteine-rich/spacer domains, respectively, abolished the hydrolytic activity of ADAMTS13 toward both a synthetic substrate, FRETS-VWF73, and the natural substrate, VWF. In addition, these antibodies blocked the binding of ADAMTS13 to VWF. These results revealed that the region between the disintegrin-like and cysteine-rich/spacer domains interacts with VWF. Employing these established polyclonal and monoclonal antibodies, we examined the molecular species of ADAMTS13 circulating in the blood by immunoprecipitation followed by Western blot analysis, and estimated the plasma concentration of ADAMTS13 by enzyme-linked immunosorbent assay. These studies indicated that the major fraction of ADAMTS13 in blood plasma consisted of the full-length form. The concentration of ADAMTS13 in normal plasma was approximately 0.5-1 microg/ml.

N-myc downstream-regulated gene 1 expression in injured sciatic nerves.

N-myc downstream-regulated gene 1 (NDRG1)/RTP/Drg1/Cap43/rit42/TDD5/Ndr1 is expressed ubiquitously and has been proposed to play a role in growth arrest and cell differentiation. A recent study showed that mutation of this gene is responsible for hereditary motor and sensory neuropathy-Lom. However, the role of this gene in the peripheral nervous system is not fully understood. In our study, rabbit polyclonal antibodies were raised against this gene product and were used to examine changes in its expression over the time course of Wallerian degeneration and ensuing regeneration after crush injury of mouse sciatic nerves. Fluorescent immunohistochemistry showed that NDRG1 was expressed over the intact nerve fibers. Double labeling with a Schwann cell (SC) marker, S-100 protein (S-100), revealed that NDRG1 was localized in the cytoplasm of S-100-positive Schwann cells (SCs). NDRG1 expression was maintained in the early stage of myelin degradation but was then markedly depleted at the end stage of myelin degradation when frequent occurrence of BrdU-labeled SCs was observed (at 7-9 days). The depletion of NDRG1 at this time point was also confirmed by Western blotting analysis. NDRG1 expression finally recovered at the stage of remyelination, with immunoreactivity stronger than that in intact nerves. These findings suggest that NDRG1 may play an important role in the terminal differentiation of SCs during nerve regeneration.

Preparation of recombinant alpha-thrombin: high-level expression of recombinant human prethrombin-2 and its activation by recombinant ecarin.

We have established a large-scale manufacturing system to produce recombinant human alpha-thrombin. In this system, a high yield of alpha-thrombin is prepared from prethrombin-2 activated by recombinant ecarin. We produced human prethrombin-2 using mouse myeloma cells and an expression plasmid carrying the chicken beta-actin promoter and mutant dihydrofolate reductase gene for gene amplification. To increase prethrombin-2 expression further, we performed fed-batch cultivation with the addition of vegetable peptone in 50 liters of suspension culture. After five feedings of vegetable peptone, the expression level of the recombinant prethrombin-2 reached 200 micro g/ml. Subsequently, the recombinant prethrombin-2 could be activated to alpha-thrombin by recombinant ecarin expressed in a similar manner. Finally, recombinant alpha-thrombin was purified to homogeneity by affinity chromatography using a benzamidine-Sepharose gel. The yield from prethrombin-2 in culture medium was approximately 70%. The activity of the purified recombinant alpha-thrombin, including hydrolysis of a chromogenic substrate, release of fibrinopeptide A, and activation of protein C, was indistinguishable from that of plasma-derived alpha-thrombin. Our system is suitable for the large-scale production of recombinant alpha-thrombin, which can be used in place of clinically available alpha-thrombin derived from human or bovine plasma.

Cellular distribution of NDRG1 protein in the rat kidney and brain during normal postnatal development.

N-myc downregulated gene 1 (NDRG1) is a 43-kD protein whose mRNA is induced by DNA damage, hypoxia, or prolonged elevation of intracellular calcium. Although NDRG1 is also upregulated during cell differentiation, there are few studies on NDRG1 expression during postnatal development. Here we investigated the expression and cellular distribution of NDRG1 protein in rat kidney and brain during postnatal development. Immunohistochemical analysis revealed that the cellular localization of NDRG1 protein in the kidney changed from the proximal convoluted tubules to the collecting ducts between postnatal days 10 and 20. In the brain, a change in cellular expression was also found from the hippocampal pyramidal neurons to the astrocytes in the gray matter during the same postnatal period. These alterations in the cellular distribution of NDRG1 were associated with shifts in the molecular assembly on Western blots. Under non-reduced conditions, the main NDRG1 band was found only around 215 kD in both kidney and brain during the early postnatal stage. After postnatal day 10, the immunoreactive bands shifted to 43 kD in the kidney and 129 kD in the brain. These changes in the cellular distribution and state of assembly may correlate with the functional maturation of both organs.

Downregulation of Cap43 gene by von Hippel-Lindau tumor suppressor protein in human renal cancer cells.

We previously identified 9 genes (i.e., thymosin beta4, secreted protein acidic and rich in cysteine, Cap43, ceruloplasmin, serum amyloid A, heat shock protein 90, LOT1, osteopontin and casein kinase Igamma) that are more highly expressed in cancerous regions than in noncancerous regions in human renal cancers. In our study, we considered the possibility that the von Hippel-Lindau (VHL) tumor suppressor gene might be able to affect the expression of these 9 genes in renal cancer cells. We first established 2 VHL-positive cell lines, 786/VHL-1 and 786/VHL-2, after the introduction of wild-type VHL into VHL-negative renal cancer 786-O cells. Of these 9 genes, expression of the Cap43 gene was specifically downregulated by VHL. Expression of Cap43 was also much lower in 4 other VHL-positive renal cancer cell lines than in VHL-negative 786-O cells. Cap43 promoter assays with several deletion or mutation constructs demonstrated that the Sp1 site in the element from -286 base pairs (bp) to -62 bp was partly responsible for VHL-induced suppression of the Cap43 gene. Immunostaining analysis with human specimens of renal cancers demonstrated that the Cap43 protein was expressed in most cancer cells and macrophages. We also observed a marked and specific increase of Cap43 mRNA levels in response to hypoxia or nickel in all VHL-positive cell lines. Cellular expression of Cap43 mRNA in response to hypoxia or nickel thus is closely associated with VHL gene expression in renal cancer cells. Although the function of the Cap43 protein remains unclear, the expression of Cap43 protein could be a molecular marker closely associated with VHL in renal cancer.