Lattices of Type I Collagen Select Invasive Variants of K-ras Oncogene-Transfected NIH3T3 with Less Cellular fibronectin

A clone of NIH3T3 transformant (H3) can yield subcutaneous tumors and experimental pulmonary metastasis in nude mice. Compared to H3 in culture, the cells after in vivo tumor growth (H3-N) acquired enhanced tumorigenicity and metastatic ability. Also, indirect immunofluorescence revealed that cellular fibronectin (c-FN) of H3-N was decreased remarkably. We have studied the interactions between H3 and extracellular matrices to elucidate these phenomena. In the present study, we observed the effect of NIH3T3, H3, and H3-N cultured in type I collagen gel. Morphologically in the collagen gel, NIH3T3 assumed an extensive elongated fiber-like shape, H3 assumed a moderately elongated shape, and H3-N assumed a round or spindle shape with short pseudopodia. Compared to conventional cultures on dishes, cell proliferation of all three types was suppressed in collagen gel, but the degree of the suppression was least in H3-N. As a result, H3-N grew fastest in collagen gel. The variants which acquired growth advantage in the subcutaneum of mice also kept it in collagen gel. H3 cells were cultured in type I collagen gel for 4 weeks, a period comparable to that of tumor formation in nude mice. The cells after this long-term culture (H3-C) acquired enhanced tumorigenicity and metastatic ability nearly equal to that of H3-N. FACS analysis revealed that the c-FN of H3-C had decreased to a value comparable to that of H3-N. This means that type I collagen gel as well as subcutaneous tissues could select variants of H3 with less c-FN through proliferation. Moreover, it is suspected that lattices of type I collagen regulate cell proliferation of fibroblast via c-FN. This revised version was published online in August 2006 with corrections to the Cover Date.     

Adjuvant Activity of 6-Amino-6-Deoxy-Muramyldipeptides and Their Acylamino Derivatives on the Induction of Delayed Hypersensitivity to Azobenzenearsonate-N-Acetyl-l-Tyrosine in Guinea Pigs

The 6-amino-6-deoxy-N-acetylmuramyldipeptides and their 6-acylamino derivatives were shown to be active as adjuvants on the induction of delayed-type hypersensitivity to azobenzenearsonate-N-acetyl-l-tyrosine in guinea pigs. However, 6-acylamino-6-deoxy-N-(acyl)muramyldipeptides were inactive as adjuvants.     

Identification and characterization of temperature-sensitive mild mutations in three Japanese patients with nonsevere forms of very-long-chain acyl-CoA dehydrogenase deficiency

Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is clinically classified into severe, intermediate, and myopathic forms. We identified mutations in three unrelated Japanese patients with VLCAD deficiency: two with the myopathic form and one with the intermediate form, all compound heterozygotes of K264E/M437V, A416T/1798delA, and P89S/IVS16-3delAA, respectively. We characterized four missense mutations, K264E, M437V, A416T, and P89S, by transisent expression analysis, using SV40-transformed fibroblasts derived from a VLCAD-null patient, as recipient cells. In transient expression of the wild-type VLCAD cDNA, VLCAD activity at 30 degrees C was higher than at 37 degrees C. Moreover, this temperature-sensitive character is more evident in all the mutant proteins tested than in wild type. Based on characterization of the five missense mutations identified in four Japanese patients, including data on one patient with the myopathic form previously reported, patients with the nonsevere forms (intermediate or myopathic forms) have missense mutations with residual activities in at least one allele. Expression analysis at 30 degrees C may be more useful for evaluating these missense mutations, compared with that at 37 degrees C.     

Decellularized ureter for tissue-engineered small-caliber vascular graft

Previous attempts to create small-caliber vascular prostheses have been limited. The aim of this study was to generate tissue-engineered small-diameter vascular grafts using decellularized ureters (DUs). Canine ureters were decellularized using one of four different chemical agents [Triton-X 100 (Tx), deoxycholate (DCA), trypsin, or sodium dodecyl sulfate (SDS)] and the histology, residual DNA contents, and immunogenicity of the resulting DUs were compared. The mechanical properties of the DUs were evaluated in terms of water permeability, burst strength, tensile strength, and compliance. Cultured canine endothelial cells (ECs) and myofibroblasts were seeded onto DUs and evaluated histologically. Canine carotid arteries were replaced with the EC-seeded DUs (n = 4). As controls, nonseeded DUs (n = 5) and PTFE prostheses (n = 4) were also used to replace carotid arteries. The degree of decellularization and the maintenance of the matrix were best in the Tx-treated DUs. Tx-treated and DCA-treated DUs had lower remnant DNA contents and immunogenicity than the others. The burst strength of the DUs was more than 500 mmHg and the maximum tensile strength of the DUs was not different to that of native ureters. DU compliance was similar to that of native carotid artery. The cell seeding test resulted in monolayered ECs and multilayered alpha-smooth muscle actin-positive cells on the DUs. The animal implantation model showed that the EC-seeded DUs were patent for at least 6 months after the operation, whereas the nonseeded DUs and PTFE grafts become occluded within a week. These results suggest that tissue-engineered DUs may be a potential alternative conduit for bypass surgery.     

A Comparative Analysis of the Murine Thymic Microenvironment in Normal,Autoimmune, and Immunodeficiency States

It is widely accepted that the thymic microenvironment regulates normal thymopoiesis through a highly coordinated and complex series of cellular and cytokine interactions. A direct corollary of this is that abnormalities within the microenvironment could be of etiologic significance in T-cell-based diseases. Our laboratory has developed a large panel of monoclonal antibodies (mAbs) that react specifically with epithelial or nonepithelial markers in the thymus. We have taken advantage of these reagents to characterize the thymic microenvironment of several genetic strains of mice, including BALB/cJ, C57BL/6J, NZB/BlnJ, SM/J, NOD/Ltz, NOD/Ltz-scid/sz, C57BL/6J-Hcph m^e/Hcph m^e, and ALY/NscJcl-aly/aly mice, and littermate control animals. We report herein that control mice, including strains of several backgrounds, have a very consistent phenotypic profile with this panel of monoclonal antibodies, including reactivity with thymic epithelial cells in the cortex, the medulla and the corticomedullary junction, and the extracellular matrix. In contrast, the disease-prone strains studied have unique, abnormal staining of thymic cortex and medulla at both the structural and cellular levels. These phenotypic data suggest that abnormalities in interactions between developing thymocytes and stromal cells characterize disease-prone mice.     

Use of an Enrichment Broth Cultivation-PCR Combination Assay for Rapid Diagnosis of Swine Erysipelas

We have previously described the creation by Tn916 mutagenesis of avirulent transposition mutants from a highly virulent strain of Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas. In this study, we cloned a 2.2-kb DNA fragment which flanked the Tn916 insertion in an avirulent mutant (strain 33H6) and evaluated the possibility that this region could be used for the specific detection of E. rhusiopathiae. According to the sequences of this region, oligonucleotide primers were designed to amplify a 937-bp fragment of the E. rhusiopathiae chromosome by PCR. The specificity of the PCR was investigated by analyzing 64 strains of Erysipelothrix species and 27 strains of other genera different from Erysipelothrix. A 937-bp DNA fragment could be amplified from all E. rhusiopathiae strains tested, and no amplification was observed by using DNAs from the other species tested. To make a rapid and definite diagnosis of swine erysipelas in slaughterhouses, we developed an enrichment broth cultivation-PCR combination assay, which used a commercially available DNA extraction kit, to identify E. rhusiopathiae in the specimens from swine with arthritis. After samples were enriched in selective broth culture, detection of E. rhusiopathiae was tested by either conventional methods or the PCR. Of 102 samples tested, 15 samples were positive by conventional methods and 12 of the 15 samples were positive by the PCR. The detection limit of the PCR was 10³ CFU per reaction mixture for the PCR-positive samples. These results indicate that this PCR technique could be used as a first-line screening technique for the specific detection of E. rhusiopathiae in specimens.     

Brief Communication: Role of Nuclear Background and in vivo Environment in Variable Segregation Behavior of the Aging-Dependent T414G Mutation at Critical Control Site for Human Fibroblast mtDNA Replication

Previous work had shown a large accumulation (up to 50% of mtDNA) of a noninherited T414G transversion at a critical control site for mtDNA replication in skin fibroblasts from the majority of human subjects above 65 years old, and its absence in younger individuals. In the present studies, long-term in vitro culture of several fibroblasts populations carrying the heteroplasmic T414G mutation revealed an outgrowth of the mutant cells by wild-type cells. This observation supported the previous conclusion that the mutation accumulation is an in vivo phenomenon, while, at the same time, indicating intrinsic physiological differences between mutant and wild-type cells. Furthermore, subcloning experiments revealed a striking mosaic distribution of the mutation in the original fibroblasts populations, as shown by its presence, in heteroplasmic or homoplasmic form, in a fraction (18–32%) of the fibroblasts, and its absence in the others. In other investigations, transfer of mitochondria from mutation-carrying fibroblasts into mtDNA-less 143B.TK^–0 206 cells revealed the persistence of the mosaic distribution of the mutation, however, with a near-complete shift to homoplasmy. The generality of the latter phenomenon would exclude a founder effect by one or few mitochondria in the transformation experiments, and would rather point to the important role of the nuclear background in the in vitro behavior of the T414G mutation. The stability of the homoplasmic mutation in ⁰ cell transformants provides a powerful tool for analyzing its biochemical effects.     

Evidence of Delayed Mesangial Transport of Human IgA in Glomeruli of ddY Mice Pretreated with Sheep Anti-type IV Collagen Serum

In order to investigate whether mesangial transport by glomeruli is delayed in ddY mice pretreated with sheep anti type IV collagen serum, the mice were administered an overload of human IgA myeloma serum. Non pretreated ddY mice used as controls and both experimental and control BALB/c mice were also processed in a similar manner. The intensities of mesangial deposition of human IgA were examined periodically and were found to correlate well with deposition of mouse IgA. Both mouse and human IgAs showed a gradual increase for up to 8 experimental weeks. In the control young ddY mice, however, the overloaded mesangial human IgA quickly disappeared, presenting no appreciable mesangial deposition of autologous IgA. In sharp contrast, both the experimental and control BALB/c mice showed an initially prolonged and rather heavy mesangial deposition of human IgA, followed by a gradual decrease and somewhat light mesangial deposition of autologous mouse IgA. These results obtained using experimental ddY mice appear to confirm the possibility that non immunological local trapping, due to retardation of mesangial transport function, causes mesangial deposition of autologous mouse IgA in this particular strain. Acta Pathol Jpn 39: 289 295, 1989.     

Arpp,a new homolog of carp,is preferentially expressed in type 1 skeletal muscle fibers and is markedly induced by denervation

In this study, we isolated and characterized a murine counterpart of the human Arpp (hArpp) gene. Sequence analysis revealed that the murine Arpp (mArpp) gene is almost identical to the Ankrd2 gene, which has recently been isolated as a mouse gene induced in stretched skeletal muscle. The mArpp gene encodes a protein of 332 amino acids that contains four well-conserved ankyrin-repeat domains in the central portion of the protein. The amino acid sequence of mArpp protein (mArpp) is highly homologous to that of mouse cardiac-restricted ankyrin-repeat protein (Carp), which is proposed to be a putative genetic marker for cardiac hypertrophy. Immunohistochemical analysis revealed that mArpp is preferentially expressed in type 1 skeletal muscle fibers, and that mArpp is localized in both the nucleus and the sarcomeric I-band of muscle fibers, suggesting that Arpp may function as a nuclear and sarcomeric protein. Furthermore, mArpp was also expressed in neurons of the cerebellum and cerebrum, the islets of Langerhans in the pancreas, and the esophageal epithelium, suggesting that mArpp may play a functional physiologic role in brain, pancreas, and esophagus as well as in type 1 muscle fibers. Interestingly, although mArpp was localized in both nucleus and cytoplasm in neurons, its localization was restricted to nucleus in pancreas and esophagus, suggesting that intracellular localization of mArpp is regulated in a tissue-specific manner. Furthermore, we found that mArpp- and Carp-expression in skeletal muscle were markedly up-regulated after denervation. Although the elevated expression level of Carp was kept only for two weeks after denervation, that of Arpp was kept at least for 4 weeks, suggesting that mArpp and Carp may play distinct functional roles in denervated skeletal muscle.