italic> of the Whole nif Genes of Klebsiella oxytoca,a Nitrogen Fixer in the Rhizosphere of Rice

We cloned in E. coli the whole 17 nif genes (nifQ-J) of Klebsiella oxytoca NG13 using pBR322 as a vector, and constructed a recombinant plasmid, pNOW25 (nif⁺, Ap^r, 42.6 kb). A non nif DNA fragment was deleted from the plasmid with XhoI, and a smaller plasmid, pNOK31 (nif+, Ap^r, 31.1 kb), was reconstructed.

We constructed the restriction map of the cloned nif genes. The map was the same as that of the K. pneumoniae M5a1 nif genes as to the EcoRI, HindIII, BamHI and XhoI sites, but differed considerably in the PstI, SalI and BglII sites.

E. coli KO60 containing pNOW25 or pNOK31 can grow on a N-free medium. The acetylene reduction activities of KO60 (pNOW25) and KO60 (pNOK31) were 280 nmol and 390 nmol/48 hr per 7 ml of N-free liquid medium, whereas the activity of K. oxytoca NG13 was 3800 nmol. Thus, the expressed activity of the nif system of K. oxytoca is rather low in E. coli even if the nif genes are cloned on a multicopy plasmid.     

Enhancement of exercise endurance capacity by fermented deer antler in BALB/c mice

To investigate the activity of fermented deer antler on exercise endurance capacity, we evaluated endurance capacity in five-week-old male BALB/c mice by administering the fermented deer antler extract (FA) or the non-fermented deer antler extract (NFA) and then subjected the mice to exercise in the form of swimming. The mice administered 500?mg/kg/day of FA showed a significant increase in swimming time compared with mice administered placebo (16.55?min vs. 21.64?min, P?<?0.05). Serum lactate dehydrogenase (LDH), the marker of the liver and muscle damage, was significantly lower in FA groups. However, NFA groups did not show significantly different swimming time or serum LDH from that of the control group. Moreover, the FA-500 group had significantly higher hepatic superoxide dismutase (SOD) activity after forced swimming than the control and NFA groups (P?<?0.05). These findings suggest that fermentation may increase the exercise endurance capacity of the deer antler.     

Effect of sorghum ethyl-acetate extract on benign prostatic hyperplasia induced by testosterone in Sprague–Dawley rats

ABSTRACT

Benign prostatic hyperplasia (BPH) is commonly observed in men > 50 years worldwide. Phytotherapy is one of the many treatment options. Sorghum (Sorghum bicolor L.) contains various health-improving phytochemicals with antioxidant and inhibitory activities on cell proliferation, both in vitro and in vivo. To confirm the effects of Donganme sorghum ethyl-acetate extract (DSEE) on BPH, we induced BPH in Spragye–Dawley rats using exogenous testosterone. We measured prostate weight, examined prostrates histopathologically, and analyzed mRNAs associated with male hormones and proteins associated with cell proliferation in the prostate. DSEE inhibited weight gain of the prostate; decreased mRNA expressions of androgen receptor and 5α-reductase II; and improved histopathological symptoms, the protein-expressed ratio of Bax/Bcl-2, and the oxidative status of BPH induced by testosterone in SD rats. Therefore, DSEE may have potential as a preventive or therapeutic agent against BPH.     

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.     

Gene cloning and functional analysis of yellow green leaf3 (ygl3) gene during the whole-plant growth stage in rice

Chlorophyll is an important photosynthetic pigment in the process of photosynthesis in plants and photosynthetic bacteria. Genes involved in chlorophyll biosynthesis in Arabidopsis and photosynthetic bacteria have been well documented. In rice, however, these genes have not been fully annotated. In this paper, a yellow-green leaf gene, yellow green leaf3 (ygl3) was cloned and analyzed. ygl3 encodes magnesium chelation ChlD (D) subunit, a key enzyme for chlorophyll synthesis, resulting in a yellow-green leaf phenotype in all growth stages in rice. Expression content of ygl3 is highest in the leaf blades, followed by the leaf sheaths, while there is virtually no expression of the gene in the stems and seeds. The sub-cellular structure and protein content of the photosynthetic system of the ygl3 mutant were revealed by transmission electron microscopy, BN-PAGE, and western blotting. The results show that the mutation of the ygl3 gene indirectly leads to a decrease in the protein content of the photosynthetic system and severely obstructs the formation of granum thylakoids.     

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.     

Dichloroacetate induces protective autophagy in LoVo cells: involvement of cathepsin D/thioredoxin-like protein 1 and Akt-mTOR-mediated signaling

Dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase (PDK), and recently it has been shown as a promising nontoxic antineoplastic agent. In this study, we demonstrated that DCA could induce autophagy in LoVo cells, which were confirmed by the formation of autophagosomes, appearance of punctate patterns of LC3 immunoreactivity and activation of autophagy associated proteins. Moreover, autophagy inhibition by 3-methyladenine (3-MA) or Atg7 siRNA treatment can significantly enhance DCA-induced apoptosis. To determine the underlying mechanism of DCA-induced autophagy, target identification using drug affinity responsive target stability (DARTS) coupled with ESI-Q-TOF MS/MS analysis were utilized to profile differentially expressed proteins between control and DCA-treated LoVo cells. As a result, Cathepsin D (CTSD) and thioredoxin-like protein 1 (TXNL1) were identified with significant alterations compared with control. Further study indicated that DCA treatment significantly promoted abnormal reactive oxygen species (ROS) production. On the other hand, DCA-triggered autophagy could be attenuated by N-acetyl cysteine (NAC), a ROS inhibitor. Finally, we demonstrated that the Akt-mTOR signaling pathway, a major negative regulator of autophagy, was suppressed by DCA treatment. To our knowledge, it was the first study to show that DCA induced protective autophagy in LoVo cells, and the potential mechanisms were involved in ROS imbalance and Akt-mTOR signaling pathway suppression.     

Impairment in Task-Specific Modulation of Muscle Coordination Correlates with the Severity of Hand Impairment following Stroke

Significant functional impairment of the hand is commonly observed in stroke survivors. Our previous studies suggested that the inability to modulate muscle coordination patterns according to task requirements may be substantial after stroke, but these limitations have not been examined directly. In this study, we aimed to characterize post-stroke impairment in the ability to modulate muscle coordination patterns across tasks and its correlation with hand impairment. Fourteen stroke survivors, divided into a group with severe hand impairment (8 subjects) and a group with moderate hand impairment (6 subjects) according to their clinical functionality score, participated in the experiment. Another four neurologically intact subjects participated in the experiment to serve as a point of comparison. Activation patterns of nine hand and wrist muscles were recorded using surface electromyography while the subjects performed six isometric tasks. Patterns of covariation in muscle activations across tasks, i.e., muscle modules, were extracted from the muscle activation data. Our results showed that the degree of reduction in the inter-task separation of the multi-muscle activation patterns was indicative of the clinical functionality score of the subjects (mean value = 26.2 for severely impaired subjects, 38.1 for moderately impaired subjects). The values for moderately impaired subjects were much closer to those of the impaired subjects (mean value = 46.1). The number of muscle modules extracted from the muscle activation patterns of a subject across six tasks, which represents the degree of motor complexity, was found to be correlated with the clinical functionality score (R = 0.68). Greater impairment was also associated with a change in the muscle module patterns themselves, with greater muscle coactivation. A substantial reduction in the degrees-of-freedom of the multi-muscle coordination post-stroke was apparent, and the extent of the reduction, assessed by the stated metrics, was strongly associated with the level of clinical impairment.