Cytoplasmic localization of PML particles in laminopathies

There is growing evidence that laminopathies, diseases associated with mutations in the LMNA gene, are caused by a combination of mechanical and gene regulatory distortions. Strikingly, there is a large variability in disease symptoms between individual patients carrying an identical LMNA mutation. This is why classical genetic screens for mutations appear to have limited predictive value for disease development. Recently, the widespread occurrence of repetitive nuclear ruptures has been described in fibroblast cultures from various laminopathy patients. Since this phenomenon was strongly correlated with disease severity, the identification of biomarkers that report on these rupture events could have diagnostic relevance. One such candidate marker is the PML nuclear body, a structure that is normally confined to the nuclear interior, but leaks out of the nucleus upon nuclear rupture. Here, we show that a variety of laminopathies shows the presence of these cytoplasmic PML particles (PML CPs), and that the amount of these protein aggregates increases with severity of the disease. In addition, between clinically healthy individuals, carrying LMNA mutations, significant differences can be found. Therefore, we postulate that detection of PML CPs in patient fibroblasts could become a valuable marker for diagnosis of disease development.     

Protoplast Fusion of Lactobacillus casei

Lactobacillus casei ATCC 7469 was successfully converted to protoplasts by treatment with endo-7V-acetyl muramidase in sucrose phosphate buffer. For full hydrolysis of cell walls, a high concentration of sucrose and a cold shock were necessary. Mg²⁺ ions enhanced the stability of protoplasting cells. The cell wall regeneration of protoplasts was more effective on gelatin-induced regeneration medium than with the soft overlay method. The optimal concentration of gelatin was 2.5%. The frequency of regeneration was found to be about 6% for the protoplast prepared by enzyme treatment for 20 min. The mutants having streptomycin resistance and rifampicin resistance, as selection markers for the detection of fusion, were isolated by UV irradiation and NTG treatment. These mutants were stable for at least several transfers. Protoplast fusion was carried out using PEG (50% solution of polyethyleneglycol, M.W. 6,000). The frequency of protoplast fusion was found to be about 10^-5.     

Effects of deer bone extract on the expression of pro-inflammatory cytokine and cartilage-related genes in monosodium iodoacetate-induced osteoarthritic rats

Deer bone extract has the potential to relieve the discomfort or the articular cartilaginous damage associated with osteoarthritic (OA) and may be useful as a natural supplement for OA treatment without serious side effects. We analyzed the expression of pro-inflammatory cytokine and cartilage-related genes in monosodium iodoacetate-induced OA rats. Increases in the levels of serum pro-inflammatory cytokines, such as interleukin-1β, interleukin-6, and tumor necrosis factor-α were significantly inhibited by the administration of deer bone extract (p?<?0.05). Decreases in the expression of collagen type II (COL2) and tissue inhibitors of metalloproteinases (TIMPs) mRNAs in the cartilage were significantly inhibited by deer bone extract treatment (p?<?0.05). The deer bone extract significantly suppressed the expression of matrix metalloproteinases (MMPs) mRNAs in the cartilage. The deer bone extract induced the up-regulation of COL2 and TIMP mRNAs and the down-regulation of MMP mRNAs by suppressing the expression of pro-inflammatory cytokine mRNAs.     

Involvement of hydrogen sulfide and homocysteine transsulfuration pathway in the progression of kidney fibrosis after ureteral obstruction

Hydrogen sulfide (H₂S) produced by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) in the transsulfuration pathway of homocysteine plays a number of pathophysiological roles. Hyperhomocysteinemia is involved in kidney fibrosis. However, the role of H₂S in kidney fibrosis remains to be defined. Here, we investigated the role of H₂S and its acting mechanism in unilateral ureteral obstruction (UO)-induced kidney fibrosis in mice. UO decreased expressions of CBS and CSE in the kidney with decrease of H₂S concentration. Treatment with sodium hydrogen sulfide (NaHS, a H₂S producer) during UO reduced UO-induced oxidative stress with preservations of catalase, copper-zinc superoxide dismutase (CuZnSOD), and manganese superoxide dismutase (MnSOD) expression, and glutathione level. In addition, NaHS mitigated decreases of CBS and CSE expressions, and H₂S concentration in the kidney. NaHS treatment attenuated UO-induced increases in levels of TGF-β1, activated Smad3, and activated NF-κB. This study provided the first evidence of involvement of the transsulfuration pathway and H₂S in UO-induced kidney fibrosis, suggesting that H₂S and its transsulfuration pathway may be a potential target for development of therapeutics for fibrosis-related diseases.     

Sequence polymorphisms in ribosomal RNA genes and variations in chromosomal loci of Oenothera odorata and O. laciniata

Numerous studies on Oenothera species have been investigated for the physiological and ecological characteristics. However, no such an information based on molecular cytogenetic has yet been introduced, in turn, is very essential for identifying sequence polymorphisms of rRNA genes with their loci on mitotic phases for further biological researches. In this study, sequence variations of rRNA genes in Oenothera odorata and O. laciniata were examined to identify informative factors as unique or repeat sequences in intra- and inter-specific variations. Intra-specific variation revealed that the sequences of the rRNA genes including the spacer regions were highly conserved revealing only a few variations. From the inter-specific variation, spacer regions of species were completely different as (1) non-homologous sequences in NTS and (2) different type repeat sequences in ITS 1, 2 and 5.8S rRNA, whereas the remaining coding regions were highly conserved. FISH was carried out on mitotic phases using the 5S rDNA of the analyzed sequences. From the interphase and metaphase chromosomes of the examined species, two loci of 5S rDNA in O. odorata and four loci in O. laciniata were confirmed on the telomeric region of the short arm. Due to the small size and unclear centromere of the chromosomes, karyotype could not be completed. However, we confirmed that the chromosomes are organized by meta- and acrocentric chromosomes and the chromosomes with identified loci were assumed to be paired by the location of loci at the telomeric region on the short arm with relative lengths.     

Cloning and characterization of Tc1 family-derived PPTN related transposons from ridged-eye flounder (Pleuronichthys cornutus) and inshore hagfish (Eptatretus burgeri)

The Tc1 transposable element is the most widespread family among animal transposon and these elements consist of an inverted repeat (IR) sequence flanking a transposase gene that belongs to Class II type transposon, which is highly conserved in the genome of the nematode C. elegans. In order to characterize Tc1-like transposable elements from several fishes, PPTN (Tc1-like transposon was isolated from Pleuronectes platessa, marine flatfish species) IR primer-specific amplified elements were cloned from the genomic DNA of several fishes. Transposable elements were found in ridged-eye flounder (Pleuronichthys cornutus) and inshore hagfish (Eptatretus burgeri) and named as PCTN and EBTN, respectively. Amino acid sequence alignment and phylogenetic analysis confirmed that the PPTN-like transposons belonged to the Tc1 superfamily of transposons, but they comprised a unique clade of Tc1-like transposons. The IR-PCR analysis using MMTS-IR and PPTN-IR specific primers from Paralichthys olivaceus (Paralichthyidae), Paraplagusia japonica (Cynoglossidae), P. yokohamae (Pleuronectidae) and Pagurus cornutus (Pleuronectidae) (within the same order, Pleuronectiformes but different families) exhibited mutually exclusive distribution of Tc1 family-derived PPTN and MMTS-like transposons in these fish genomes. These results indicate that Tc1 family-derived PPTN and MMTS related Tc1-like transposable elements have uniquely evolved in piscine genome, and can be used as phylogenetic markers for the distribution of subfamilies of Tc1-like transposon and the involvement of horizontal and vertical transmission in the evolution of fish genome.