Functional cell phenotype induction with TGF-ß1 and collagen-polyurethane scaffold for annulus fibrosus rupture repair.

Appropriate cell sources, bioactive factors and biomaterials for generation of functional and integrated annulus fibrosus (AF) tissue analogues are still an unmet need. In the present study, the AF cell markers, collagen type I, cluster of differentiation 146 (CD146), mohawk (MKX) and smooth muscle protein 22α (SM22α) were found to be suitable indicators of functional AF cell induction. In vitro 2D culture of human AF cells showed that transforming growth factor ß1 (TGF-ß1) upregulated the expression of the functional AF markers and increased cell contractility, indicating that TGF-ß1-pre-treated AF cells were an appropriate cell source for AF tissue regeneration. Furthermore, a tissue engineered construct, composed of polyurethane (PU) scaffold with a TGF-ß1-supplemented collagen type I hydrogel and human AF cells, was evaluated with in vitro 3D culture and ex vivo preclinical bioreactor-loaded organ culture models. The collagen type I hydrogel helped maintaining the AF functional phenotype. TGF-ß1 supplement within the collagen I hydrogel further promoted cell proliferation and matrix production of AF cells within in vitro 3D culture. In the ex vivo IVD organ culture model with physiologically relevant mechanical loading, TGF-ß1 supplement in the transplanted constructs induced the functional AF cell phenotype and enhanced collagen matrix synthesis. In conclusion, TGF-ß1-containing collagen-PU constructs can induce the functional cell phenotype of human AF cells in vitro and in situ. This combined cellular, biomaterial and bioactive agent therapy has a great potential for AF tissue regeneration and rupture repair.

ICTV Virus Taxonomy Profile: Nodaviridae.

The family Nodaviridae includes two genera, Alphanodavirus and Betanodavirus. The family name derives from the Japanese village of Nodamura where Nodamura virus was first isolated from Culex tritaeniorhynchus mosquitoes. Virions are non-enveloped and spherical in shape with icosahedral symmetry (T=3) and diameters ranging from 25 to 33 nm. The genome consists of two molecules of single-stranded positive-sense RNA: RNA1 and RNA2. The virion capsid consists of 180 protein subunits arranged on a T=3 surface lattice. Alphanodaviruses infect insects, whereas betanodaviruses are pathogens of fish. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Nodaviridae, which is available at www.ictv.global/report/nodaviridae.

ICTV virus taxonomy profile: Sarthroviridae.

The family Sarthroviridae includes a single genus, Macronovirus, which in turn includes a single species, Macrobrachium satellite virus 1. Members of this species, named extra small virus, are satellite viruses of Macrobrachium rosenbergii nodavirus, an unclassified virus related to members of the family Nodaviridae. Both viruses have isometric, spherical virions, infect giant freshwater prawns and together cause white tail disease, which is responsible for mass mortalities and severe economic losses in hatcheries and farms. Infection is caused by both vertical and horizontal transmission of virus. Aquatic insects act as a carrier to transmit the disease in prawns. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Sarthroviridae, which is available at www.ictv.global/report/sarthroviridae.

Transplantation of activated nucleus pulposus cells after cryopreservation: efficacy study in a canine disc degeneration model.

Transplantation of activated nucleus pulposus (NP) cells obtained by coculturing NP cells and bone marrow mesenchymal stromal cells having cell-to-cell contact has been shown to be effective in animal models and, more recently, in human clinical trials. If the NP cells can be cryopreserved, then autologous cell transplantation could be offered to patients as and when required. In a previous study, we confirmed that activated NP cells can be obtained by coculturing with mesenchymal cells after cryopreservation. However, the in vivo effects of cell transplantation therapy using activated NP cells prepared from cryopreserved cells are not known. In this in vivo canine model, we compared indicators of disc degeneration in animals that received transplanted activated normal NP cells, transplanted cryopreserved NP cells, and no cell transplantation after induction of disc degeneration. The intervertebral disc height on radiographs and T2-weighted magnetic resonance imaging were significantly higher in both cell transplantation groups compared with the degenerated disc group. Macroscopic and histological findings demonstrated attenuated disc degeneration in the two transplanted groups. Intense staining of proteoglycan and collagen type II was seen in green fluorescent protein-labelled transplanted cells, which suggested that the cells had survived and were functioning after transplantation. No significant differences were observed between the two transplanted groups. Transplanted activated cryopreserved NP cells induced a similar attenuation of intervertebral disc degeneration as that of conventionally activated NP cells. These findings suggest that the use of cryopreserved cells specific to a patient's condition has potential in transplantation therapy.

Short communication: Difructose anhydride III promotes calcium absorption from the duodenum in cattle.

Difructose anhydride (DFA) III promotes the intestinal absorption of calcium via a paracellular pathway in rats. In dairy cows, DFA III reaches the duodenum without being degraded by ruminal bacteria and hence could be used to control hypocalcemia. The aims of the present study were to investigate the percentage of DFA III that appears in the duodenum of cows and to determine the effect of DFA III on calcium absorption from duodenal fluid. The first experiment was performed in 3 ruminally and duodenally cannulated dry Holstein cows in a 3 × 3 Latin square design. Each experimental period lasted 7 d. On the first day, the cows were ruminally fed one of the following treatments: 0 (DFA0), 50 (DFA50), or 100 (DFA100) g/d of DFA III, using cobalt-EDTA as a liquid phase marker. Difructose anhydride III was detected in duodenal fluid 1 h after feeding, and its concentration peaked 4 h after feeding, in a dose-dependent manner. The percentages of DFA III that appeared in the duodenum after the DFA50 and DFA100 treatments were 69.1 ± 7.0% and 67.9 ± 5.6%, respectively. The second experiment used the everted duodenal sacs of cattle (n = 7 in each group). Sacs were incubated in artificial mucosal fluid containing 1 mM DFA III or no DFA III (control) for 60 min with 100% O2 in a water bath at 37 °C. After incubation, the calcium concentration of the artificial serosal fluid in the everted sacs was measured. Calcium absorption was higher in the DFA III-treated group than in the control group (803 ± 161 and 456 ± 74 nmol/cm of sac, respectively). The above results demonstrate that approximately 70% of administered DFA III reached the duodenum of cows intact. Moreover, similar to its effects on calcium absorption in rats, DFA III promoted calcium absorption via a paracellular pathway in the duodenum of cows.

Peroxisome assembly factor-2, a putative ATPase cloned by functional complementation on a peroxisome-deficient mammalian cell mutant.

Rat peroxisome assembly factor-2 (PAF-2) cDNA was isolated by functional complementation of peroxisome deficiency of a mutant CHO cell line, ZP92, using transient transfection assay. This cDNA encodes a 978-amino acid protein with two putative ATP-binding sites. PAF-2 is a member of a putative ATPase family, including two yeast gene products essential for peroxisome assembly. A stable transformant of ZP92 with the cDNA was morphologically and biochemically restored for peroxisome biogenesis. Fibroblasts derived from patients deficient in peroxisome biogenesis (complementation group C) were also complemented with PAF-2 cDNA, indicating that PAF-2 is a strong candidate for the pathogenic gene of group C peroxisome deficiency.

Short communication: effect of difructose anhydride III on serum immunoglobulin G concentration in newborn calves.

Difructose anhydride (DFA) III is an indigestible disaccharide that promotes paracellular absorption of calcium, magnesium, and other minerals in the intestine by acting on epithelial tight junctions. This study aimed to elucidate the effect of DFA III on serum IgG concentration. One hundred and twenty Holstein and Holstein/Japanese Black crossbred calves were randomly divided into 4 groups of 30 to receive untreated colostrum (DFA0) or colostrum containing 3, 6, or 18 g of DFA III (DFA3, DFA6, or DFA18, respectively). At 24 h after birth, both serum IgG (ranging from 16.4 to 21.2 mg/mL) and apparent efficiency of absorption (26.0 to 37.2%) showed increases with the amount of DFA III intake. By multiple regression analysis, the standardized partial regression coefficient for DFA III was 0.25, the second highest following that for the colostrum IgG concentration (0.80), indicating a positive effect of DFA III on serum IgG. A positive linear regression was found between colostrum IgG and serum IgG concentrations at 24h of age. These results indicate that IgG absorption occurred as a nonsaturable process, which might be characteristic of gradient-dependent paracellular transport. Thus, it was concluded that DFA III improves not only minerals but IgG absorption in calves.

Growth of Flavobacterium psychrophilum in fish serum correlates with pathogenicity.

Flavobacterium psychrophilum isolates, obtained from ayu, Plecoglossus altivelis, three species of salmonids and two species of cyprinids in Japan, were used in this study. Bacteria were inoculated to serum prepared from ayu or red spotted masu trout (RSMT), Oncorhynchus masou ishikawae, and incubated at 18 °C for 24 h. All isolates (n = 19) from ayu grew well with a 9- to 116-fold increase of CFU in ayu serum, while CFU decreased markedly in RSMT serum. In contrast, isolates (n = 17) from fish species other than ayu exhibited no growth in ayu serum, but some isolates from salmonids survived or grew (1.2-23.5 fold increase of CFU) in RSMT serum. The isolates that could not survive or grow in ayu and RSMT sera grew well in both heat-inactivated sera of ayu and RSMT. Experimental infection by intraperitoneal injection showed that ayu isolates examined were all pathogenic to ayu but not to RSMT, while none of the isolates from salmonids and cyprinids were pathogenic to ayu but some showed pathogenicity to RSMT. These results indicate that the in vitro growth ability of F. psychrophilum isolates in fish serum correlates well with their pathogenicity to fish, particularly in ayu.

Genotyping of Edwardsiella ictaluri isolates in Japan using amplified-fragment length polymorphism analysis.

AIM: The major objective of the present study was to clarify genetic relationship of isolates of Edwardsiella ictaluri in Japan, which was first found from ayu Plecoglossus altivelis in Japanese rivers in 2007. METHODS AND RESULTS: Ten isolates of Edw. ictaluri in 2007-2008 from ayu and the 1 isolate from bagrid catfish Pelteobagrus nudiceps in Japan were subjected to amplified-fragment length polymorphism (AFLP) analysis. The strains isolated from catfish in United States (ATCC strains) or Indonesia were used as reference strains. The AFLP profiles were all the same among the isolates from Japan, while the polymorphic DNA bands were observed among the strains from United States or Indonesia. The isolates from Japan and Indonesia constituted a genogroup different from the ATCC strains on a dendrogram constructed from the AFLP profiles. CONCLUSION: No DNA polymorphisms were found among Japanese Edw. ictaluri isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: A single clonality of the Edw. ictaluri isolates in Japan suggests the single source of the organism, and the infection in ayu is in the early stage of epidemics.

Protection conferred against viral nervous necrosis by simultaneous inoculation of aquabirnavirus and inactivated betanodavirus in the sevenband grouper, Epinephelus septemfasciatus (Thunberg).

An aquabirnavirus (ABV) and a formalin-inactivated betanodavirus [redspotted grouper nervous necrosis virus (RGNNV)] were investigated for their potential to prevent RGNNV-induced viral nervous necrosis (VNN) in the sevenband grouper, Epinephelus septemfasciatus (Thunberg). Three groups of fish were injected intramuscularly with ABV, intraperitoneally with inactivated RGNNV (iRGNNV) or with both ABV and iRGNNV. At 3, 7, 14, 21 and 28 days post-injection (p.i.), fish were challenged by intramuscular injection of RGNNV. Control fish, which received neither ABV nor iRGNNV, showed high mortalities in all RGNNV challenges. Fish that received only ABV exhibited relative percent survival (RPS) of >60 against RGNNV challenges at 3, 7, 14 and 21 days p.i., but not at 28 days p.i., while fish that received only iRGNNV showed significantly higher protection against RGNNV challenges only at 21 and 28 days p.i. In contrast, fish that received both ABV and iRGNNV showed 60 or higher RPS against all RGNNV challenges. Fish inoculated with iRGNNV with or without ABV exhibited similar high titres of neutralizing antibodies to RGNNV at 14, 21 and 28 days p.i. These results indicate that combined inoculation with iRGNNV and ABV conferred both rapid non-specific and delayed specific protection against VNN.

Neutralizing antibody levels for protection against betanodavirus infection in sevenband grouper, Epinephelus septemfasciatus (Thunberg), immunized with an inactivated virus vaccine.

Abstract An inactivated betanodavirus, red-spotted grouper nervous necrosis virus (RGNNV), is a vaccine candidate for viral nervous necrosis (VNN). The present study was conducted to examine inoculation doses of the vaccine and neutralizing antibody titre levels to protect fish against VNN. Young sevenband grouper, Epinephelus septemfasciatus, averaging 25.4 g, were immunized at 25 degrees C water temperature by a single intraperitoneal injection of formalin-inactivated RGNNV. Fish immunized at vaccine doses of 10(8.5), 10(8.0), 10(7.5), 10(7.0) and 10(6.5) TCID(50) per fish produced antibodies at mean titres of 1:907, 1:511, 1:259, 1:197 and 1:96, respectively, at 20 days post-immunization (p.i.). Neutralizing antibodies were not detected in any control fish (titre <1:80). When fish were challenged with RGNNV (10(5.0) and 10(4.0) TCID(50)/fish) at 20 days p.i., cumulative mortalities of the fish groups immunized with 10(8.5), 10(8.0), 10(7.5) and 10(7.0) TCID(50) per fish were significantly lower than those of the control group, and the relative percent survival values were higher than 60% in fish groups immunized with 10(7.5) TCID(50) per fish or higher doses. However, no significant differences were found in mortality between the group immunized with 10(6.5) TCID(50) per fish and the control group. From these results, it was deduced that the minimum effective inoculation dose of the vaccine is 10(7.0) TCID(50) per fish and the minimum mean neutralizing antibody titre giving significant protection is approximately 1:200. This antibody titre level is a possible measure of vaccine efficacy against VNN in sevenband grouper, instead of a virus challenge test.

Immune responses of Asian sea bass, Lates calcarifer Bloch, against an inactivated betanodavirus vaccine.

Asian sea bass, Lates calcarifer (Bloch), exhibited strong immune responses against a single injection of the formalin-inactivated red-spotted grouper nervous necrosis virus (RGNNV), a betanodavirus originally isolated in Japan. Fish produced neutralizing antibodies at high titre levels from days 10 (mean titre 1:480) to 116 (1:1280), with the highest titre at day 60 post-vaccination (1:4480). When fish were challenged with the homologous RGNNV at day 54 post-vaccination, there were no mortalities in both the vaccinated and unvaccinated control fish. However, a rapid clearance of the virus was observed in the brains and kidneys of vaccinated fish, followed by a significant increase in neutralizing-antibody titres. Furthermore, the vaccine-induced antibodies potently neutralized Philippine betanodavirus isolates (RGNNV) in a cross-neutralization assay. The present results indicate the potential of the formalin-inactivated RGNNV vaccine against viral nervous necrosis (VNN) of Asian seabass.

Genetic heterogeneity of betanodaviruses in juvenile production trials of Pacific bluefin tuna, Thunnus orientalis (Temminck & Schlegel).

Pacific bluefin tuna, Thunnus orientalis (Temminck & Schlegel), is one of the most important commercially exploited fish species in the world, and juvenile production techniques have been developed for its culture and stock enhancement in Japan. However, recent juvenile production has often failed because of the occurrence of viral nervous necrosis caused by betanodaviruses. In this study, we examined the genetic variability of betanodaviruses detected in the diseased juveniles to understand the transmission of the disease in a tuna hatchery. A total of 94 nucleotide sequences of betanodavirus (partial sequence of the coat protein gene, RNA2) were obtained from fish samples by reverse-transcriptase polymerase chain reaction amplification and 13 haplotypes were recognized among the sequences. The haplotype distributions in the viral populations from the diseased juveniles were related to the broodstocks from which the juveniles originated, suggesting that vertical transmission had occurred in the hatchery. The statistical parsimony network of viral haplotypes suggests that the nucleotide substitutions among the samples were accumulated in a recent population growth.

Multiple genes, including a member of the AAA family, are essential for degradation of unassembled subunit 2 of cytochrome c oxidase in yeast mitochondria.

Cytochrome c oxidase consists of three mitochondrion- and several nucleus-encoded subunits. We previously found that in a mutant of Saccharomyces cerevisiae lacking nucleus-encoded subunit 4 of this enzyme (CoxIV), subunits 2 and 3 (CoxII and CoxIII), both encoded by the mitochondrial DNA, were unstable and rapidly degraded in mitochondria, presumably because the subunits cannot assemble normally. To analyze the molecular machinery involved in this proteolytic pathway, we obtained four mutants defective in the degradation of unassembled CoxII (osd mutants) by screening CoxIV-deficient cells for the accumulation of CoxII. All of the mutants were recessive and were classified into three different complementation groups. Tetrad analyses revealed that the phenotype of each mutant was caused by a single nuclear mutation. These results suggest strongly that at least three nuclear genes (the OSD genes) are required for this degradation system. Interestingly, degradation of CoxIII was not affected in the mutants, implying that the two subunits are degraded by distinct pathways. We also cloned the OSD1 gene by complementation of the temperature sensitivity of osd1-1 mutants with a COXIV+ genetic background on a nonfermentable glycerol medium. We found it to encode a member of a family (the AAA family) of putative ATPases, which proved to be identical to recently described YME1 and YTA11. Immunological analyses revealed that Osd1 protein is localized to the mitochondrial inner membrane. Disruption of the predicted ATP-binding cassette by site-directed mutagenesis eliminated biological activities, thereby underscoring the importance of ATP for function.

Crystal structure of N-carbamyl-D-amino acid amidohydrolase with a novel catalytic framework common to amidohydrolases.

BACKGROUND: N-carbamyl-D-amino acid amidohydrolase (DCase) catalyzes the hydrolysis of N-carbamyl-D-amino acids to the corresponding D-amino acids, which are useful intermediates in the preparation of beta-lactam antibiotics. To understand the catalytic mechanism of N-carbamyl-D-amino acid hydrolysis, the substrate specificity and thermostability of the enzyme, we have determined the structure of DCase from Agrobacterium sp. strain KNK712. RESULTS: The crystal structure of DCase has been determined to 1.7 A resolution. The enzyme forms a homotetramer and each monomer consists of a variant of the alpha + beta fold. The topology of the enzyme comprises a sandwich of parallel beta sheets surrounded by two layers of alpha helices, this topology has not been observed in other amidohydrolases such as the N-terminal nucleophile (Ntn) hydrolases. CONCLUSIONS: The catalytic center could be identified and consists of Glu46, Lys126 and Cys171. Cys171 was found to be the catalytic nucleophile, and its nucleophilic character appeared to be increased through general-base activation by Glu46. DCase shows only weak sequence similarity with a family of amidohydrolases, including beta-alanine synthase, aliphatic amidases and nitrilases, but might share highly conserved residues in a novel framework, which could provide a possible explanation for the catalytic mechanism for this family of enzymes.

Scintigraphic detection of overexpressed c-erbB-2 protooncogene products by a class-switched murine anti-c-erbB-2 protein monoclonal antibody.

Class-switched monoclonal antibody SV2-61r recognized the extracellular domain of c-erbB-2 protooncogene products separate from the epidermal growth factor receptor. We studied the potential of SV2-61r for evaluating the amplification of c-erbB-2 protooncogene on cancer cells, which has been reported to have prognostic value in adenocarcinoma patients. Radiolabeled SV2-61r specifically bound to various adenocarcinoma cells in addition to c-erbB-2-transfected NIH-3T3 cells (A4) with the affinity constant of 4.4 x 10(8) M-1. SV2-61r injected i.v. localized well to A4 cells xenografted in nude mice. Tumor uptake and localization index of radioiodinated SV2-61r were lower than those of 111In-labeled SV2-61r, probably due to the internalization and dehalogenation of formed antibody-antigen complexes. Biodistribution and specificity of targeting were assessed by comparison among three cells, A4, lung cancer SBC-3 (c-erbB-2 weakly positive) and B-lymphoblastoid Manca cells (c-erbB-2 negative). Tumor:blood ratios, obtained 48 h after injection, were 5.63, 1.45, and 0.68, respectively, indicating the potential of 111In-labeled SV2-61r for evaluating the amplification of c-erbB-2 protooncogene on cancer cells. Because of its close relationship with carcinogenesis and the uniform expression, c-erbB-2 protooncogene products seem to be the optimal target of imaging and therapy of adenocarcinoma patients.

The reaction of tryptophan with cystine during acid hydrolysis of proteins. Formation of tryptathionine as a transient intermediate in a model system.

Under the conditions commonly used to hydrolyze proteins with 6 M HCl, tryptophan reacted with cystine to give a transient intermediate, which was isolated and identified as 2-(2-amino-2-carboxyethylthio)tryptophan (tryptathionine) by NMR studies, etc. Studies on the formation and degradation of the above compound showed that beta-3-oxoindolylalanine and cysteine, which were previously reported to be the main degradation products of tryptophan and cystine, respectively, are formed by the hydrolysis of this intermediate during the course of the reaction.

Beta-3-Oxindolylalanine: The main intermediate in tryptophan degradation occurring in acid hydrolysis of protein.

Under hydrolytic conditions using 6 M HCl, tryptophan reacted separately with dithiodiglycolic acid and cystine to give beta-3-oxindolylalanine (beta-[3-(2-indolinone)]alanine) as the main product. A compound, which eluted in the amino acid analyzer at the same position as beta-3-oxindolylalanine, was found in the acid hydrolyzate of lysozyme. The identicalness of these two compounds was established by comparison of their ultraviolet absorption spectra, elution positions on ion exchange chromatograms, etc. The "acid degradation product of tryptophan", which is known to be produced upon acid hydrolysis of tryptophan-containing proteins, must also be the same compound.

Proteolysis of canine apolipoprotein by acid proteases in canine liver lysosomes.

Canine liver lysosomes were purified by sucrose discontinuous density gradient centrifugation and then ruptured by sonication to obtain the soluble fraction. This soluble lysosomal fraction, which contained a 25-fold increase in acid phosphatase activity per mg of total protein when compared with the original homogenate, was incubated with a subfraction (1.110 less than d less than 1.210 g/cm3, HDL3) of canine high density lipoproteins (HDL) at pH 3.8. HDL3 proteolysis by lysosomal proteases, measured as the release of peptides and amino acids by the ninhydrin reaction, followed hyperbolic curves with straight lines (r = 0.99) obtained on Lineweaver-Burk plots. Km calculated from the Lineweaver-Burk plot was 635 mug of HDL3 protein per 0.5 ml of incubation mixture. Optimum HDL3 proteolysis was observed from pH 3.8 to 4.5. Incubation with the other subcellular organelle fractions did not result in HDL3 proteolysis. To evaluate the effects of enzyme inhibitors, iodoacetate, p-chloromercuribenzoate (both specific for the endopeptidase, cathepsin B (EC 3.4.22.1)) and pepstatin (specific for the endopeptidase, cathepsin D (EC 3.4.23.5) were tested. Iodoacetate and p-chloromercuribenzoate inhibited HDL3 proteolysis 100% and bovine serum albumin proteolysis 65%. Pepstatin inhibited HDL3 proteolysis 45% and bovine serum albumin proteolysis 70%. The in vitro data presented support the hypothesis that hepatic lysosomes play an important role in HDL3 catabolism in the dog. Furthermore, results obtained from enzyme inhibition studies suggest that a specific lysosomal endopeptidase, cathepsin B, may play the key role in HDL3 proteolysis.