Osteopontin Deficiency Suppresses Tumor Necrosis Factor-α-Induced Apoptosis in Chondrocytes.

OBJECTIVE: Apoptosis of chondrocytes in articular cartilage has been observed in rheumatoid arthritis patients. However, molecules involved in such chondrocyte apoptosis in arthritic joints have not been fully understood. We previously observed that apoptosis of chondrocytes is enhanced in a murine arthritis model induced by injection with anti-type II collagen antibodies and lipopolysaccharide (mAbs/LPS), and osteopontin (OPN) deficiency suppresses chondrocyte apoptosis in this arthritis model in vivo. To understand how OPN deficiency renders resistance against chondrocyte apoptosis, we examined the cellular basis for this protection. DESIGN: Chondrocytes were prepared from wild-type and OPN-deficient mouse ribs, and tumor necrosis factor (TNF)-α-induced cell death was examined based on lactate dehydrogenase (LDH) release assay and TUNEL assay. RESULTS: TNF-α treatment induced LDH release in wild-type chondrocytes, while OPN deficiency suppressed such LDH release in the cultures of these cells. TNF-α-induced increase in the number of TUNEL-positive cells was observed in wild-type chondrocytes, while OPN deficiency in chondrocytes suppressed the TNF-α induction of TUNEL-positive cells. OPN deficiency suppressed TNF-α-induced increase in caspase-3 activity in chondrocytes in culture. Furthermore, OPN overexpression in chondrocytes enhanced TNF-α-induced apoptosis. CONCLUSION: These results indicated that the presence of OPN in chondrocytes is involved in the susceptibility of these cells to TNF-α-induced apoptosis.

Combinatory effects of androgen receptor deficiency and hind limb unloading on bone.

Male sex hormones play a critical role in regulation of bone metabolism. In male mice lacking androgen receptor (AR), osteopenia and high turnover state in bone remodeling have been reported. However, androgen receptor's role in disuse-induced osteopenia is not known. Therefore, we examined the effects of AR deficiency on unloading-induced bone loss. Wild type or androgen receptor deficient mice (ARKO) were subjected to hind limb unloading (HU) or normal housing (Control). The groups of mice were as follows; wild type control mice (Group WT-Cont), ARKO control mice (Group ARKO-Cont), wild type HU mice (Group WT-HU), and ARKO-HU mice (Group ARKO-HU). HU reduced cancellous bone mass in ARKO (ARKO-HU) by about 70% compared to ARKO-Cont and this reduction rate was over two-fold more than that of wild type (WT-HU) (reduction by less than 30% compared to WT-Cont). Combination of ARKO and HU (ARKO-HU) resulted in the least levels of cortical bone mass and bone mineral density among the four groups. ARKO-HU group indicated the highest levels of systemic bone resorption marker, deoxypyridinoline. Osteoclast development levels in the cultures in ARKO-HU derived bone marrow cells were the highest among the four groups. These data suggest that combination of androgen receptor deficiency and hind limb unloading results in exacerbation of disuse-induced osteopenia due to the enhanced levels of bone resorption.

Deficiency of CIZ, a nucleocytoplasmic shuttling protein, prevents unloading-induced bone loss through the enhancement of osteoblastic bone formation in vivo.

Disuse osteoporosis is a major cause to increase the risk of fractures in bed-ridden patients whose numbers are increasing in our modern society. However, the mechanisms underlying the sensing of mechanical stress in bone are largely unknown. CIZ localizes at cell adhesion plaque and transfers into nuclear compartments and activates promoters of the genes encoding enzymes, which degrade matrix proteins to link signals from the cell adhesion site to nuclear events. We examined whether this nucleocytoplasmic shuttling protein would be involved in mediation of mechanical stress signaling. Unloading based on tail suspension reduced bone volume in wild-type mice. In contrast, CIZ-deficient mice revealed suppression in such reduction of bone mass due to unloading. Histomorphometric analysis revealed that unloading suppressed the levels of osteoblastic bone formation parameters, and such suppression of bone formation parameters was blocked by CIZ-deficiency. Osteoclastic bone resorption parameters were similar regardless of CIZ-deficiency after 2-week unloading. Mineralized nodule formation in the cultures of bone marrow cells obtained from the bone of mice subjected to unloading was suppressed in wild-type mice. CIZ deficiency blocked such reduction in nodule formation induced by unloading. These data indicated that nucleocytoplasmic shuttling protein, CIZ, plays a pivotal role in the response of bone mass in unloading condition.

Unloading-induced bone loss was suppressed in gold-thioglucose treated mice.

Loss of mechanical stress causes bone loss. However, the mechanisms underlying the unloading-induced bone loss are largely unknown. Here, we examined the effects of gold-thioglucose (GTG) treatment, which destroys ventromedial hypothalamus (VMH), on unloading-induced bone loss. Unloading reduced bone volume in control (saline-treated) mice. Treatment with GTG-reduced bone mass and in these GTG-treated mice, unloading-induced reduction in bone mass levels was not observed. Unloading reduced the levels of bone formation rate (BFR) and mineral apposition rate (MAR). GTG treatment also reduced these parameters and under this condition, unloading did not further reduce the levels of BFR and MAR. Unloading increased the levels of osteoclast number (Oc.N/BS) and osteoclast surface (Oc.S/BS). GTG treatment did not alter the basal levels of these bone resorption parameters. In contrast to control, GTG treatment suppressed unloading-induced increase in the levels of Oc.N/BS and Oc.S/BS. Unloading reduced the levels of mRNA expression of the genes encoding osteocalcin, type I collagen and Cbfa1 in bone. In contrast, GTG treatment suppressed such unloading-induced reduction of mRNA expression. Unloading also enhanced the levels of fat mass in bone marrow and mRNA expression of the genes encoding PPARgamma2, C/EBPalpha, and C/EBPbeta in bone. In GTG-treated mice, unloading did not increase fat mass and the levels of fat-related mRNA expression. These results indicated that GTG treatment suppressed unloading-induced alteration in bone loss.

Development of tendon structure and function: regulation of collagen fibrillogenesis.

In the tendon, the development of mature mechanical properties is dependent on the assembly of a tendon-specific extracellular matrix. This matrix is synthesized by the tendon fibroblasts and composed of collagen fibrils organized as fibers, as well as fibril-associated collagenous and non-collagenous proteins. All of these components are integrated, during development and growth, to form a functional tissue. During tendon development, collagen fibrillogenesis and matrix assembly progress through multiple steps where each step is regulated independently, culminating in a structurally and functionally mature tissue. Collagen fibrillogenesis occurs in a series of extracellular compartments where fibril intermediates are assembled and mature fibrils grow through a process of post-depositional fusion of the intermediates. Linear and lateral fibril growth occurs after the immature fibril intermediates are incorporated into fibers. The processes are regulated by interactions of extracellular macromolecules with the fibrils. Interactions with quantitatively minor fibrillar collagens, fibril-associated collagens and proteoglycans influence different steps in fibrillogenesis and the extracellular microdomains provide a mechanism for the tendon fibroblasts to regulate these extracellular interactions.

Seven-hour fluorescence in situ hybridization technique for enumeration of Enterobacteriaceae in food and environmental water sample.

AIMS: A fluorescent in situ hybridization (FISH) technique using an Enterobacteriaceae-specific probe (probe D) to target 16S rRNA was improved in order to enumerate, within a single working day, Enterobacteriaceae present in food and environmental water samples. METHODS AND RESULTS: In order to minimize the time required for the FISH procedure, each step of FISH with probe D was re-evaluated using cultured Escherichia coli. Five minutes of ethanol treatment for cell fixation and hybridization were sufficient to visualize cultured E. coli, and FISH could be performed within 1 h. Because of the difficulties in detecting low levels of bacterial cells by FISH without cultivation, a FISH technique for detecting microcolonies on membrane filters was investigated to improve the bacterial detection limit. FISH with probe D following 6 h of cultivation to grow microcolonies on a 13 mm diameter membrane filter was performed, and whole Enterobacteriaceae microcolonies on the filter were then detected and enumerated by manual epifluorescence microscopic scanning at magnification of x100 in ca 5 min. The total time for FISH with probe D following cultivation (FISHFC) was reduced to within 7 h. FISHFC can be applied to enumerate cultivable Enterobacteriaceae in food (above 100 cells g-1) and environmental water samples (above 1 cell ml-1). CONCLUSIONS: Cultivable Enterobacteriaceae in food and water samples were enumerated accurately within 7 h using the FISHFC method. SIGNIFICANCE AND IMPACT OF THE STUDY: A FISHFC method capable of evaluating Enterobacteriaceae contamination in food and environmental water within a single working day was developed.

Association of a single nucleotide variant in the human tumour necrosis factor alpha promoter region with decreased bone mineral density.

BACKGROUND: Tumour necrosis factor alpha (TNFalpha) has come to be regarded as a potential osteoporotic factor, because it has stimulatory effects on cells of the osteoclast lineage and has been implicated in the pathogenesis of bone loss associated with oestrogen deficiency. We recently described genetic linkage between the TNFalpha locus and human osteoporosis by sib-pair analysis. However, the molecular mechanism by which this locus regulates bone mineral density (BMD) remains unknown. AIM: We investigated whether the observed linkage reflects a sequence variation which might affect expression of the TNFalpha gene or alter the function of TNFalpha protein. SUBJECTS AND METHODS: We examined three single-nucleotide polymorphisms (SNPs) of the TNFalpha gene in a group of 390 postmenopausal Japanese women living in northern Japan. Minor-allele frequencies for the three SNPs (-1031C, -863A and -857T) in this population were 0.16, 0.13 and 0.20, respectively. RESULTS: Among the three SNPs examined, we observed a significant correlation only between the presence of a T allele at nt -1031 and decreased BMD, by analysis of variance. Among the three genotypic groups at nt -1031, mean BMD values were significantly higher in the T-negative genotype (C/C homozygotes; mean SD = 0.342 +/- 0.052 g cm(-2)), compared with T-positive genotypes (T/T homozygotes, 0.309 +/- 0.062 g cm(-2); p = 0.0253 and T/C heterozygotes, 0.305 +/- 0.062 g cm(-2); p = 0.0164). CONCLUSIONS: Given the lines of evidence from different genetic studies, we suggest that TNFalpha may play a role in pathogenesis of osteoporosis.

Three TNFalpha single nucleotide polymorphisms in the Japanese population.

BACKGROUND: Tumour necrosis factor-alpha (TNFalpha) is an essential regulator of immune responses and is implicated to relate to several types of disease susceptibilities. Population information on polymorphisms is essential for the study of genetic diseases. AIM: To obtain accurate information about single nucleotide polymorphisms (SNPs) in the TNFalpha gene in the Japanese population. SUBJECTS AND METHODS: The entire TNFalpha gene was screened for SNPs by directly sequencing 48 chromosomes derived from 24 unrelated Japanese individuals. Allele frequencies of each polymorphism were determined and compared with those previously reported in other populations. RESULTS: Three SNPs, -308G/A at nt -308, IVS1 + 125G/A at nt 492 and IVS3 + 104G/A at nt 1359 were observed, of which one (IVS3 + 104G/A at nt 1359) was novel. In addition, allele frequencies of -308G/A were remarkably different from those presented in the NCBI dbSNP, indicating a significant ethnic difference. CONCLUSIONS: The polymorphisms and allele frequencies obtained in this study will be useful for genetic studies of common diseases such as osteoporosis and rheumatoid arthritis in the Japanese population.

Oligonucleotide probe for detecting Enterobacteriaceae by in situ hybridization.

AIMS: To develop oligonucleotide probes for visualizing bacteria belonging to Enterobacteriaceae. METHODS AND RESULTS: 24-mer oligonucleotide probe (probe D) was designed by comparison of 16S rDNA sequences of 35 species of Enterobacteriaceae, eight species of Vibrionaceae and six species of Pasteurellaceae. The sequence of the probe corresponding to the complementary sequence of a position 1251-1274 of Escherichia coli 16S rRNA was found to be a highly conserved region of 16S rDNA sequence in Enterobacteriaceae different from that of Vibrionaceae and Pasteurellaceae. The fluorescent dye-labelled probe was tested for the specificity by in situ hybridization and epifluorescence microscopy. Seventy-six out of 78 strains belonging to Enterobacteriaceae were visualized in an optimal hybridization condition. No bacterial strains belonging to Vibrionaceae (31 strains) and Gram-positive bacteria (three strains) were visualized. CONCLUSIONS: In situ hybridization using probe D allows the detection of bacterial cells belonging to Enterobacteriaceae without false positive reaction. SIGNIFICANCE AND IMPACT OF THE STUDY: In situ hybridization techniques using the probe D are potential tools for detecting Enterobacteriaceae in food and water samples.

Linkage disequilibrium and haplotype analysis among four novel single-nucleotide polymorphisms in the human leukemia inhibitory factor (LIF) gene.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine implicated in various pathological conditions, such as rheumatoid arthritis and osteoporosis. Despite the possible importance of LIF as a therapeutic target, little is known about the bioregulation of the human LIF gene. We here sequenced the entire structure of the LIF gene of 48 alleles in the Japanese population. These experiments identified four single-nucleotide polymorphisms (SNPs) and determined their allelic frequencies from a 48-allele sequence in the Japanese population. All four SNPs found in the LIFgene were located within exon 3, that is, a C/T at nucleotide (nt) position 3951, a C/G at nt position 4376, an A/C at nt position 4442, and a G/A at nt position 5961 (nucleotide numbering starts from the ATG start codon). Based on the genotypic data, we constructed four major haplotypes in the tested population. Two-way comparisons of SNPs revealed complete linkage disequilibrium between SNPs at positions 3951, 4376, and 4442. These results may prove to be useful as genetic markers for population-based disease-association studies in osteoporosis.

Cloning, sequence analysis and expression of Pseudoalteromonas elyakovii IAM 14594 gene (alyPEEC) encoding the extracellular alginate lyase.

A gene (alyPEEC) encoding an alginate lyase of Pseudoalteromonas elyakovii IAM 14594 was cloned using the plasmid vector pUC118 and expressed in Escherichia coli. Sequencing of a 3.0kb fragment revealed a 1,197bp open reading frame encoding 398 amino acid residues. The calculated molecular mass and isoelectric point of the alyPEEC gene product are 43.2 kDa and pI 5.29. A region G(165) to V(194) in the AlyPEEC internal sequence is identical to the N-terminal amino acid sequence of the previously purified extracellular alginate lyase of P. elyakovii, and the calculated molecular mass (25.4 kDa) and isoelectric point (pI 4.78) of the region resembled those of the purified enzyme. Expression of enzymically-active alginate lyase from alyPEEC required growth of recombinant E. coli in LB broth containing 50% (v/v) artificial seawater (ASW). Alginate lyase activity with broad substrate specificity was detected in both 42 and 30 kDa products. Subcloning of the region G(165) to N(398) of AlyPEEC corresponding to the 30 kDa protein confirmed that this region of the alyPEEC gene encoded the active site of the enzyme. A region A(32) to G(164) corresponding to about 13 kDa of the N-terminal region of AlyPEEC showed about 30% identity to a putative chitin binding domain of Streptomyces chitinases, but did not exhibit any catalytic activity.

Thirteen single-nucleotide polymorphisms in the human osteopontin gene identified by sequencing of the entire gene in Japanese individuals.

Osteopontin (OPN) is one of the major noncollagenous bone matrix proteins produced by osteoblasts and osteoclasts. We systematically surveyed the entire structure of the OPN gene for single-nucleotide polymorphisms (SNPs) by directly sequencing 48 alleles derived from 24 unrelated Japanese individuals. We identified 13 SNPs in the OPN gene. Ten polymorphisms were identified in introns 1, 3, and 5; 2 in the coding region of exons 6 and 7; and 1 in the 3' untranslated region of exon 7. Allele frequencies for some of the polymorphisms were significantly different from those reported in the United States National Center for Biotechnology Information (NCBI) dbSNP database. These polymorphisms will be useful in genetic studies to evaluate the role of OPN proteins in bone metabolism.

Linkage disequilibrium and haplotype analysis among ten single-nucleotide polymorphisms of interleukin 11 identified by sequencing of the gene.

Interleukin (IL11) is a member of the interleukin 6 (IL6)-related cytokine subfamily, which stimulates T cell-dependent development of immunoglobulin-producing B cells. IL11 is also an important paracrine regulator of bone metabolism that induces formation of osteoclasts. In the work reported here, we sequenced the entire IL11 structural gene of 48 alleles in a Japanese test population. These experiments identified ten single-nucleotide polymorphisms (SNPs) and determined their allelic frequencies. One polymorphism was identified upstream of exon 1, one in exon 3, four in intron 4 and four in the 3' untranslated region (3'UTR) of exon 5. Based on the genotype data, we constructed six haplotypes in the tested population. Two-way comparisons of SNPs revealed two combinations in complete linkage disequilibrium, one with SNPs at nucleotide positions 2753, 3644, 5154, and 5568, and another with SNPs at positions 3686, 5141, and 5734. These results will be useful in disease-association studies where a contribution of the human IL11 gene has been suspected, especially in disorders affecting immune response and bone metabolism.

[Genetic approaches for rheumatoid arthritis associated osteopenia].

Rheumatoid Arthritis (RA) associated osteopenia is a resultant of multifactorial secondary osteoporosis, including disuse-atrophy, glucocorticoid-induced osteopenia, and RA specific activation of osteolysis. In addition, primary osteoporosis, i.e. postmenopausal and senile osteoporosis may overlap with them. Pursuits for the genetic factors for the causes of RA associated osteopenia could be clarified both from the characterization of RA associated factors and the osteoporosis associated factors. Recent progress in systematic genome-wide analysis for the association studies with multiple gene polymorphisms may improve the understandings of genetic contribution of these factors for the pathogenesis of this symptom.

Differential expression of lumican and fibromodulin regulate collagen fibrillogenesis in developing mouse tendons.

Collagen fibrillogenesis is finely regulated during development of tissue-specific extracellular matrices. The role(s) of a leucine-rich repeat protein subfamily in the regulation of fibrillogenesis during tendon development were defined. Lumican-, fibromodulin-, and double-deficient mice demonstrated disruptions in fibrillogenesis. With development, the amount of lumican decreases to barely detectable levels while fibromodulin increases significantly, and these changing patterns may regulate this process. Electron microscopic analysis demonstrated structural abnormalities in the fibrils and alterations in the progression through different assembly steps. In lumican-deficient tendons, alterations were observed early and the mature tendon was nearly normal. Fibromodulin-deficient tendons were comparable with the lumican-null in early developmental periods and acquired a severe phenotype by maturation. The double-deficient mice had a phenotype that was additive early and comparable with the fibromodulin-deficient mice at maturation. Therefore, lumican and fibromodulin both influence initial assembly of intermediates and the entry into fibril growth, while fibromodulin facilitates the progression through growth steps leading to mature fibrils. The observed increased ratio of fibromodulin to lumican and a competition for the same binding site could mediate these transitions. These studies indicate that lumican and fibromodulin have different developmental stage and leucine-rich repeat protein specific functions in the regulation of fibrillogenesis.

Assignment of Alteromonas elyakovii KMM 162T and five strains isolated from spot-wounded fronds of Laminaria japonica to Pseudoalteromonas elyakovii comb. nov. and the extended description of the species.

A marine bacterium, Alteromonas elyakovii KMM 162T, which was described recently, and five strains isolated from spot-wounded fronds of Laminaria japonica have been subjected to phylogenetic analysis, and geno- and phenotypic characterization. The phenotypic features of Pseudoalteromonas elyakovii strains were closely related to that of Pseudoalteromonas espejiana IAM 12640T, but utilization of three carbon compounds (D-mannose, L-tyrosine and trehalose) distinguished both species. The G+C content of Pseudoalteromonas elyakovii was between 38.5 and 38.9 mol%. Pseudoalteromonas elyakovii KMM 162T and the five Laminaria isolates constitute a single species different from any other Alteromonas and Pseudoalteromonas species as revealed by DNA-DNA hybridization data, especially Pseudoalteromonas distincta KMM 638T (52.4%), Pseudoalteromonas citrea KMM 216 (49.5%), Pseudoalteromonas carrageenovora NCIMB 302T (46.9%) and Pseudoalteromonas espejiana IAM 12640T (29.9%). All the data indicated that Alteromonas elyakovii KMM 162T should be reclassified as Pseudoalteromonas elyakovii and five strains isolated from Laminaria japonica have to be included in the species. Pseudoalteromonas elyakovii comb. nov. (type strain, KMM 162T = ATCC 700519T) is proposed and a set of phenotypic features which differentiate the Pseudoalteromonas species is described.

Cloning and Sequence Analysis of Vibrio halioticoli Genes Encoding Three Types of Polyguluronate Lyase.

The alginate lyase-coding genes of Vibrio halioticoli IAM 14596(T), which was isolated from the gut of the abalone Haliotis discus hannai, were cloned using plasmid vector pUC 18, and expressed in Escherichia coli. Three alginate lyase-positive clones, pVHB, pVHC, and pVHE, were obtained, and all clones expressed the enzyme activity specific for polyguluronate. Three genes, alyVG1, alyVG2, and alyVG3, encoding polyguluronate lyase were sequenced: alyVG1 from pVHB was composed of a 1056-bp open reading frame (ORF) encoding 352 amino acid residues; alyVG2 gene from pVHC was composed of a 993-bp ORF encoding 331 amino acid residues; and alyVG3 gene from pVHE was composed of a 705-bp ORF encoding 235 amino acid residues. Comparison of nucleotide and deduced amino acid sequences among AlyVG1, AlyVG2, and AlyVG3 revealed low homologies. The identity value between AlyVG1 and AlyVG2 was 18.7%, and that between AlyVG2 and AlyVG3 was 17.0%. A higher identity value (26.0%) was observed between AlyVG1 and AlyVG3. Sequence comparison among known polyguluronate lyases including AlyVG1, AlyVG2, and AlyVG3 also did not reveal an identical region in these sequences. However, AlyVG1 showed the highest identity value (36.2%) and the highest similarity (73.3%) to AlyA from Klebsiella pneumoniae. A consensus region comprising nine amino acid (YFKAGXYXQ) in the carboxy-terminal region previously reported by Mallisard and colleagues was observed only in AlyVG1 and AlyVG2.

In Situ Polymerase Chain Reaction Visualization of Vibrio halioticoli Using Alginate Lyase Gene AlyVG2.

A prokaryotic in situ polymerase chain reaction (PI-PCR) technique was applied to visualize Vibrio halioticoli cells using alginate lyase gene alyVG2 as a target gene. Prior to PI-PCR, a primer set, VG2-OS3, for specific amplification of an approximately 1.0-kb fragment from V. halioticoli genomic DNA was developed with amplified fragments from V. pelagius and V. fischeri DNAs as reference strains. One-stage PI-PCR using the primer set, digoxigenin-labeled dUTP, and indirect alkaline-phosphatase-linked fluorescence detection technique (HNPP/Fast Red TR as a substrate) failed to differentiate V. halioticoli IAM14596(T) cells from ATCC25916(T) cells of the closely related species V. pelagius. However, two-stage PI-PCR adding the extension and digoxigenin-labeling step of the amplified fragment into the first amplification stage allowed us to differentiate V. halioticoli cells from V. pelagius cells.

Notchplasty in anterior cruciate ligament reconstruction: an experimental animal study.

The purpose of this study was to evaluate the effects of a notchplasty on the biomechanical and histological properties of the anterior cruciate ligament (ACL) and changes in patellar articular cartilage. We used an in situ freeze-thaw model for the ACL reconstruction performed with or without notchplasty in 36 Japanese white rabbits. The cross-sectional area of the regenerated ACLs without a notchplasty (6.4 +/- 0.4 mm(2)) was statistically smaller than that of the ACLs with a notchplasty (7.0 +/- 0.3 mm(2)), and the cross-sectional area of the normal ACLs (5.4 +/- 0.5 mm(2)) was statistically smaller than that of both other types. However, the mechanical strength of the ACL with the notchplasty was identical to that of the ACL without a notchplasty. Although the notchplasty areas were covered with fibrous scar tissue, caliper measurement and histological examination showed no obvious osteochondral reconstitution in the notchplasty sites of any of the specimens. Regarding the deleterious effect of notchplasty on patellar articular cartilage, the extent of the slight degenerative changes was about the same in the group with a 1-mm notchplasty and in the control group.