[Differential population screening and efficacy analysis of (125)I implantation after radiofrequency ablation of primary liver cancer].

Objective: To investigate the clinical efficacy radiofrequency ablation (RFA) combined with (125)I radioactive seed implantation in treatment of hepatocellular carcinoma HCC with the tumor diameter 3-5 cm. Methods: One hundred patients with HCC diagnosed clinically or pathologically with Barcelona staging of B or C in Lishui Central Hospital from February 2012 to September 2017 were retrospectively analyzed. Of the included 100 cases, 89 were males and 11 were females with the mean age of 18-80 (57±11) years old.According to the treatment modality, the subjects were divided into control group (RFA, n=67) and combined group (RFA+(125)I, n=33). Patients in control group were only received RFA and cases in combined group received RFA plus sequenced with (125)I implantation therapy. The prognosis of progression free survival (PFS) and overall survival (OS) between the two groups were compared through the Kaplan-Meier curve and Log-rank test. Results: The median follow-up time period was 6-55 months in the last follow-up time point of Dec 30, 2017. The median PFS were 4-55 (23.0±4.7) and 1-53 (12.0±1.6) months for combined and control groups respectively with significant statistical difference (P=0.015). The median OS were 6-55 (42.0±7.9) and 2-55 (38.0±2.8) months for combined and control groups with the trend of improvement in combined group, but without statistical difference (P=0.444). Subgroup analysis further indicated that the PFS was significant improved in patients with residual tumor lesions who received the combined treatment (PFS: 18 vs 9 months, P=0.025). However, there was no statistical difference for PFS between the control and combined treatment groups for cases without residual tumor lesions after RAF treatment(P=0.685). Conclusions: PFS was obviously increased in HCC patients(tumor diameter 3-5 cm) who received(125)I implantation after radiofrequency ablation, especially for cases with residual tumor lesions.

MiR-10b suppresses the growth and metastasis of colorectal cancer cell by targeting FGF13.

OBJECTIVE: MicroRNA (miRNA) plays important roles in the progression of different cancers. In this study, we investigated the precise role of miR-10b in the growth and metastasis of colorectal cancer (CRC) cell. PATIENTS AND METHODS: The levels of miR-10b in CRC cell lines were detected using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) assay. A series of functional assays, including cell proliferation, colony formation, wound healing and transwell invasion were conducted using miR-10b transfected cells. The expressions of E-cadherin and N-cadherin were assessed by immunofluorescence staining. Xenograft model and lung metastasis model were constructed to investigate the impact of miR-10b on the growth and metastasis of CRC cell in vivo. RESULTS: We revealed that miR-10b was markedly down-regulated in CRC. The up-regulation of miR-10b suppressed the growth, colony formation, migration and invasion of CRC cell. Furthermore, the xenograft model indicated that miR-10b inhibited CRC cell growth and lung metastasis in vivo by targeting fibroblast growth factor 13 (FGF13). In addition, we demonstrated that the overexpression of FGF13 rescued the suppressive effects of miR-10b on CRC cells growth, migration and invasion. Finally, the knockdown of FGF13 was able to mimic the inhibitory effects of miR-10b on the progression of CRC cells. CONCLUSIONS: These results demonstrate that miR-10b plays an important role in growth, migration and invasion of CRC by targeting FGF13.

[Clinical value of (125)I radioactive seed implantation plus transcatheter arterial chemoembolization combined with radiofrequency ablation in treating patient with sub-capsular hepatocellular carcinoma].

Objective: To analyze the clinical efficacy and safety of (125)I radioactive seed implantation in the treatment of sub-capsular hepatocellular carcinoma (sub-HCC) with sequential radiofrequency ablation and transcatheter arterial chemoembolization (TACE). Methods: The clinical data of 76 cases with advanced HCC with sub-capsular nodules including 68 males and 8 females, with an average age of (58±9) years, ranging from 33 to 78 years, enrolled in Lishui Central Hospital from January 2010 to December 2016 were collected.The average maximum diameter of tumor is (5.7±2.3) cm, ranging from 3.1 cm to 12.0 cm.The patients were divided into TACE+ RFA group and (125)I + TACE+ RFA group with 38 cases in each group.The overall survival (OS) and progression free survival(PFS) were calculated.The clinical efficiency and adverse events were evaluated. Results: The disease control rate were 84.2%(32/38) in (125)I + TACE+ RFA group and 63.2% (24/38) in TACE+ RFA group, χ(2)=4.34, P= 0.04.The median PFS were 18 months in (125)I + TACE+ RFA group and 11 months in TACE+ RFA group, χ(2)=4.84, P=0.03.The FPS cumulative rate in (125)I + TACE+ RFA group were higher than that in TACE+ RFA group at 6 months (94.7%±3.6% vs 81.3%±6.4%, Z=24.1>2.58, P=0.00), 1 year (89.2%±5.1% vs 40.7%±8.3%, Z=13.3>2.58, P=0.00) and 2 year (55.9%±8.6% vs 29.6%±8.2%, Z=7.2>2.58, P=0.00). The median OS were 42 months in (125)I + TACE+ RFA group and 30 months in TACE+ RFA group, χ(2)=4.76, P=0.029.The survival cumulative rate in (125)I+ TACE+ RFA group were higher than that in TACE+ RFA group at 1 year (92.1%±4.4% vs 83.8%±6.1%, Z=23.5>2.58, P=0.00), 2 year (75.8%±7.0% vs 59.8%±8.4%, Z=12.43>2.58, P=0.00), 3 year (59.0%±8.2% vs 41.7%±8.9%, Z=8.3>2.58, P=0.00), 5 year (34.2%±8.2% vs 18.2%±8.1%, Z=5.5>2.58, P=0.00). In addition, there was no statistical difference in liver function and complications between TACE+ RFA group and (125)I+ TACE+ RFA group. Conclusion: (125)I radioactive seed implantation plus TACE combined with RFA treatment is an effective and safe treatment for sub-capsular hepatocellular carcinoma.

Renal outcome after kidney-transplantation in Korean patients with lupus nephritis.

We investigated renal outcome of kidney-transplantation in 19 Korean recipients with biopsy-proven lupus nephritis and compared it with 18 Korean age- and gender-matched recipients without lupus nephritis who were diagnosed with end-stage renal disease caused by renal diseases other than lupus nephritis in a single centre. We reviewed histological findings of kidneys and calculated cumulative dose of immunosuppressive agents. We assessed renal flare of systemic lupus erythematosus, recurrence of lupus nephritis and graft failure as prognosis. The mean age of recipients with lupus nephritis was 43.5 years and all patients were female. Six patients had class III, 10 had class IV and three had class V. There were no meaningful differences in demographic data, renal replacement modality, cumulative doses of immunosuppressants and prognosis between recipients with and without lupus nephritis. Eight patients experienced renal flare of systemic lupus erythematosus, but there were no cases of recurrence of lupus nephritis or graft failure in recipients with lupus nephritis. Kidney-recipients with class IV lupus nephritis exhibited a lower cumulative renal flare of systemic lupus erythematosus free survival rate than those with class III lupus nephritis. In conclusion, renal outcome of kidney-transplantation in patients with lupus nephritis is similar to that in those without lupus nephritis, and class IV was associated with renal flare of systemic lupus erythematosus.

Serum Wisteria floribunda agglutinin-positive Mac-2-binding protein can reflect systemic lupus erythematosus activity.

Serum Mac-2-binding protein (M2BP) is elevated in various chronic inflammatory diseases, and evidence suggests that glycosylation of M2BP induces discrete biological effects. However, the role of serum M2BP in systemic lupus erythematosus (SLE) is still unclear. Recently, a Wisteria floribunda agglutinin-positive-M2BP (WFA+-M2BP) immunoassay has shown promise in detecting highly glycosylated M2BP. In this study, by using WFA+-M2BP immunoassay, we measured serum M2BP in 203 SLE patients and evaluated its clinical significance. Eighty patients were classified as having active SLE and 123 patients as having inactive SLE. The median serum M2BP was higher in patients with active SLE than in those with inactive SLE (2.1 vs. 0.9, p < 0.001). In multivariate linear regression analysis, serum M2BP, anti-dsDNA, C3 and erythrocyte sedimentation rate (ESR) were associated with SLEDAI-2K. Serum M2BP also strongly correlated with laboratory variables related to SLEDAI-2K, ESR and C-reactive protein. Furthermore, multivariate logistic regression analysis demonstrated that serum M2BP was useful in predicting active SLE. Finally, following immunosuppressive treatment, elevated serum M2BP significantly decreased along with improvement in disease activity. These findings suggest that serum M2BP might contribute to the inflammatory process in SLE, and measuring serum M2BP might be a useful marker to assess SLE disease activity.

Identification of dipeptidyl peptidase-IV inhibitory peptides from mare whey protein hydrolysates.

Inhibition of dipeptidyl peptidase-IV (DPP-IV) activity is a promising strategy for treatment of type 2 diabetes. In the current study, DPP-IV inhibitory peptides were identified from mare whey protein hydrolysates obtained by papain. The results showed that all the mare whey protein hydrolysates obtained at various hydrolysis durations possessed more potent DPP-IV inhibitory activity compared with intact whey protein. The 4-h hydrolysates showed the greatest DPP-IV inhibitory activity with half-maximal inhibitory concentration of 0.18 mg/mL. The 2 novel peptides from 4-h hydrolysate fractions separated by successive chromatographic steps were characterized by liquid chromatography-electrospray ionization tandem mass spectrometry. The novel peptides Asn-Leu-Glu-Ile-Ile-Leu-Arg and Thr-Gln-Met-Val-Asp-Glu-Glu-Ile-Met-Glu-Lys-Phe-Arg, which corresponded to ß-lactoglobulin 1 f(71-77) and ß-lactoglobulin 1 f(143-155), demonstrated DPP-IV inhibitory activity with half-maximal inhibitory concentrations of 86.34 and 69.84 µM, respectively. The DPP-IV inhibitory activity of the 2 peptides was retained or even improved after simulated gastrointestinal digestion in vitro. Our findings indicate that mare whey protein-derived peptides may possess potential as functional food ingredients in the management of type 2 diabetes.

[Association between ultrasound screening frequency and mortality in patients with hepatocellular carcinoma].

Objective: To discuss the association between ultrasound screening frequency and total mortality in patients with HCC before diagnosing HCC, and explore the optimal ultrasound screening frequency for HCC high-risk groups. Methods: Retrospectively collected clinical data of 615 cases of liver cirrhosis who developed to HCC from January 1, 2010 to December 31, 2015. Before diagnosing HCC, all patients were divided into five groups according to ultrasound screening frequency: 0-6, 7-12, 13-24, 25-36 months and not screened within 3 years (never screened). The chance to receive curative therapy, 5-year cumulative mortalities and independent factors of mortality in patients with HCC were analyzed. Results: Chances to receive curative therapy among the 0-6, 7-12, 13-24, 25-36 months and never screened groups were 38.2%, 27.2%, 25.4%, 23.8% and 19.7%, respectively (P<0.05). The 5-year overall mortality rates were 76.4%, 77.7%, 79.3%, 82.5% and 84.6%, respectively. Compared with 0-6 months, the adjusted OR of mortality for the other groups were 1.112, 1.235, 1.305 and 1.451, respectively (all P<0.05). Multivariate analysis showed that ultrasound screening frequency, curative treatment and Child-Pugh (class A/B) were the factors to affect long-term survival in patients with HCC (all P<0.05). Conclusion: For HCC high-risk groups, optimal ultrasound screening frequency is within 6 months, and high-frequency ultrasound screening can increase the chance of receiving curative treatment, reduce total mortality, and improve overall survival.

[Efficacy analysis of radiofrequency ablation combined with 125I seed for multiple nodular hepatocellular carcinoma].

OBJECTIVE: To investigate the clinical efficacy of radiofrequency ablation(RFA)combined with (125)I seed in patients with multiple nodular hepatocellular carcinoma(HCC). METHODS: The clinical data of 47 patients with multiple nodular HCC confirmed clinically or pathologically in the Fifth Affiliated Hospital of Wenzhou Medical University between January 2009 and September 2014 were retrospectively analyzed. All patients were divided into two groups depending on treatment: RFA group(n=29); RFA combined with (125)I seed group(n=18). Survival time was estimated with the Kaplan-Meier analysis. The survival curve was compared by Log-rank test. RESULTS: The clinical data and tumor situation of patients between two groups did not show significant differences. The follow-up time ranged from 2 to 55 months. At the end of the study, the median progression-free survival of two groups were 18 months and 11 months, and the differences were statistically significant(P=0.036). The overall survival rates in RFA combined with (125)I seed group at 1, 2, and 3 years (94.4%, 78.0%, 66.8%, respectively) were higher than those in RFA group (88.5%, 68.6%, 57.1%, respectively). However, the differences between the two groups were not statistically significant(P=0.554). CONCLUSIONS: For multiple nodular HCC, RFA combined with (125)I seed can prolong progression-free survival, have an advantage in local control rate, and is better than single RFA at short-term curative effect.However, its long-term outcomes should be further explored by a large-sample, multi-center and randomized trial.

Silencing ECHS1 attenuates the proliferation and induces the autophagy of hepatocellular carcinoma via impairing cell metabolism and activating AMPK.

Hepatocellular carcinoma (HCC) is among the most common cancers in the world with a low survival rate. Our previous study showed Short chain enoyl-CoA hydratase (ECHS1) could bind to HBsAg (HBs) and that ECHS1's localization in mitochondria induced HepG2 cell apoptosis. However, the role of the ECHS1 in energy metabolism and autophagy during hepatocellular carcinoma development remains undefined. We aimed to determine what ECHS1 does to energy metabolism and its effects on HCC progression. We performed CCK-8, EdU assays in hepatocellular carcinoma cell lines (HepG2 and HuH7) with stable ECHS1 knock-down. ATP and NADP+/NADPH levels were measured using an colorimetric assay. Our data demonstrated that ECHS1 silencing inhibited cell proliferation and induced autophagy. ECHS1 knockdown did not increase fatty acid synthesis, but decreased cellular ATP. This resulted in AMP-activated protein kinase (AMPK) activation and induced HCC cell autophagy. Our results showed that silencing ECHS1 to attenuate proliferation and induce autophagy may make it a novel cancer therapy target.

Transforming growth factor-ß gene silencing using adenovirus expressing TGF-ß1 or TGF-ß2 shRNA.

Tumor cells secrete a variety of cytokines to outgrow and evade host immune surveillance. In this context, transforming growth factor-ß1 (TGF-ß1) is an extremely interesting cytokine because it has biphasic effects in cancer cells and normal cells. TGF-ß1 acts as a growth inhibitor in normal cells, whereas it promotes tumor growth and progression in tumor cells. Overexpression of TGF-ß1 in tumor cells also provides additional oncogenic activities by circumventing the host immune surveillance. Therefore, this study ultimately aimed to test the hypothesis that suppression of TGF-ß1 in tumor cells by RNA interference can have antitumorigenic effects. However, we demonstrated here that the interrelation between TGF-ß isotypes should be carefully considered for the antitumor effect in addition to the selection of target sequences with highest efficacy. The target sequences were proven to be highly specific and effective for suppressing both TGF-ß1 mRNA and protein expression in cells after infection with an adenovirus expressing TGF-ß1 short hairpin RNA (shRNA). A single base pair change in the shRNA sequence completely abrogated the suppressive effect on TGF-ß1. Surprisingly, the suppression of TGF-ß1 induced TGF-ß3 upregulation, and the suppression of TGF-ß2 induced another unexpected downregulation of both TGF-ß1 and TGF-ß3. Taken together, this information may prove useful when considering the design for a novel cancer immunogene therapy.

c-Cbl shRNA-expressing adenovirus sensitizes TRAIL-induced apoptosis in prostate cancer DU-145 through increases of DR4/5.

We previously demonstrated that the downregulation of Casitas B-lineage lymphoma (c-Cbl) can sensitize tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in two different ways. One way is to block the rapid degradation of TRAIL receptors, which can sustain TRAIL-induced apoptosis for a long time. Here, we designed a replication-defective adenovirus expressing the short hairpin RNA (shRNA) against c-Cbl to test the possibility of developing a cancer gene therapy that can act as a sensitizer of TRAIL. As expected from the results of our previous study that used a stable cell line with downregulated c-Cbl, infection with the c-Cbl shRNA-expressing adenovirus led to an increase in the death receptor 4 (DR4) and DR5 levels, which is known to be a cause for the increase of TRAIL-induced apoptosis. In conclusion, we demonstrated that c-Cbl shRNA-expressing adenovirus is able to sensitize TRAIL-induced apoptosis in vivo as well as in vitro.

Nucleotide-dependent conformational changes in a protease-associated ATPase HsIU.

BACKGROUND: The bacterial heat shock locus ATPase HslU is an AAA(+) protein that has structures known in many nucleotide-free and -bound states. Nucleotide is required for the formation of the biologically active HslU hexameric assembly. The hexameric HslU ATPase binds the dodecameric HslV peptidase and forms an ATP-dependent HslVU protease. RESULTS: We have characterized four distinct HslU conformational states, going sequentially from open to closed: the empty, SO(4), ATP, and ADP states. The nucleotide binds at a cleft formed by an alpha/beta domain and an alpha-helical domain in HslU. The four HslU states differ by a rotation of the alpha-helical domain. This classification leads to a correction of nucleotide identity in one structure and reveals the ATP hydrolysis-dependent structural changes in the HslVU complex, including a ring rotation and a conformational change of the HslU C terminus. This leads to an amended protein unfolding-coupled translocation mechanism. CONCLUSIONS: The observed nucleotide-dependent conformational changes in HslU and their governing principles provide a framework for the mechanistic understanding of other AAA(+) proteins.

Intracellular degradation of two structurally different polyhydroxyalkanoic acids accumulated in Pseudomonas putida and Pseudomonas citronellolis from mixtures of octanoic acid and 5-phenylvaleric acid.

From a set of mixed carbon sources, 5-phenylvaleric acid (PV) and octanoic acid (OA), polyhydroxyalkanoic acid (PHA) was separately accumulated in the two pseudomonads Pseudomonas putida BM01 and Pseudomonas citronellolis (ATCC 13674) to investigate any structural difference between the two PHA accumulated under a similar culture condition using one-step culture technique. The resulting polymers were isolated by chloroform solvent extraction and characterized by fractional precipitation and differential scanning calorimetry. The solvent fractionation analysis showed that the PHA synthesized by P. putida was separated into two fractions, 3-hydroxy-5-phenylvalerate (3HPV))-rich PHA fraction in the precipitate phase and 3-hydroxyoctanoate (3HO)-rich PHA fraction in the solution phase whereas the PHA produced by P. citronellolis exhibited a rather little compositional separation into the two phases. According to the thermal analysis, the P. putida PHA exhibited two glass transitions indicative of the PHA not being homogeneous whereas the P. citronellolis PHA exhibited only one glass transition. It was found that the structural heterogeneity of the P. putida PHA was caused by a significant difference in the assimilation rate between PV and OA. The structural heterogeneity present in the P. putida PHA was also confirmed by a first order degradation kinetics analysis of the PHA in the cells. The two different first-order degradation rate constants (k(1)), 0.087 and 0.015/h for 3HO- and 3HPV-unit, respectively, were observed in a polymer system over the first 20 h of degradation. In the later degradation period, the disappearance rate of 3HO-unit was calculated to be 0.020 h. The k(1) value of 0.083/h, almost the same as for the 3HO-unit in the P. putida PHA, was obtained for the P(3HO) accumulated in P. putida BM01 grown on OA as the only carbon source. In addition, the k(1) value of 0.015/h for the 3HPV-unit in the P. putida PHA, was also close to 0.019/h for the P(3HPV) homopolymer accumulated in P. putida BM01 grown on PV plus butyric acid. On the contrary, the k(1) values for the P. citronellolis PHA were determined to be 0.035 and 0.029/h for 3HO- and 3HPV-unit, respectively, thus these two relatively close values implying a random copolymer nature of the P. citronellolis PHA. In addition, the faster degradation of P(3HO) than P(3HPV) by the intracellular P. putida PHA depolymerase indicates that the enzyme is more specific against the aliphatic PHA than the aromatic PHA.

Physiological and biochemical characteristics of poly gamma-glutamate synthetase complex of Bacillus subtilis.

An enzymatic system for poly gamma-glutamate (PGA) synthesis in Bacillus subtilis, the PgsBCA system, was investigated. The gene-disruption experiment showed that the enzymatic system was the sole machinery of PGA synthesis in B. subtilis. We succeeded in achieving the enzymatic synthesis of elongated PGAs with the cell membrane of the Escherichia coli clone producing PgsBCA in the presence of ATP and D-glutamate. The enzyme preparation solubilized from the membrane with 8 mM Chaps catalyzed ADP-forming ATP hydrolysis only in the presence of glutamate; the D-enantiomer was the best cosubstrate, followed by the L-enantiomer. Each component of the system, PgsB, PgsC, and PgsA, was translated in vitro and the glutamate-dependent ATPase reaction was kinetically analyzed. The PGA synthetase complex, PgsBCA, was suggested to be an atypical amide ligase.

Efficient gene transfer of VSV-G pseudotyped retroviral vector to human brain tumor.

A retroviral vector constructed from the murine leukemia virus (MLV) can only express transgenes in cells undergoing mitosis, indicating its suitability as a delivery vehicle for cancer gene therapy. However, the transduction efficiency (TE) of retroviruses embedding endogenous envelope proteins in human cancer cells was found to be unsatisfactory. Recently, several research groups have demonstrated the feasibility of a retroviral vector pseudotyped with a vesicular stomatitis virus G (VSV-G) protein. In this study, the potential of VSV-G pseudotyped MLV-based retrovirus was examined as a delivery vehicle in a variety of human cancer cells including brain tumor cells in vitro and in vivo. The transduction efficiency of the 293T/G/GP/LacZ retrovirus in cell culture was superior in most cancer cells, particularly in brain tumor cells, compared with that of other retroviruses, such as PA317- or PG13-derived. The relative growth rate and phosphatidylserine expression level on the plasma membrane of target cells mainly influenced the transduction efficiency of VSV-G pseudotyped retrovirus, which suggested that both the relative growth rate and phosphatidylserine expression level were major determinants of TE. Furthermore, 293T/G/GP/LacZ could efficiently transduce human cancer cells regardless of the presence of chemical additives, whereas in other retroviruses, cationic chemical additives such as polybrene or liposomes were essential during virus infection. Finally, an average of 10% gene expression was routinely obtained exclusively in the tumor mass when 293T/G/GP/LacZ concentrated by simple ultracentrifugation was directly administrated to pre-established brain tumors in animal models (U251-N nu/nu mice or C6 Wistar rats). All told, the present study suggests that the VSV-G pseudotyped retrovirus is a suitable vector for brain tumor gene therapy.

Crystal structures of the HslVU peptidase-ATPase complex reveal an ATP-dependent proteolysis mechanism.

BACKGROUND: The bacterial heat shock locus HslU ATPase and HslV peptidase together form an ATP-dependent HslVU protease. Bacterial HslVU is a homolog of the eukaryotic 26S proteasome. Crystallographic studies of HslVU should provide an understanding of ATP-dependent protein unfolding, translocation, and proteolysis by this and other ATP-dependent proteases. RESULTS: We present a 3.0 A resolution crystal structure of HslVU with an HslU hexamer bound at one end of an HslV dodecamer. The structure shows that the central pores of the ATPase and peptidase are next to each other and aligned. The central pore of HslU consists of a GYVG motif, which is conserved among protease-associated ATPases. The binding of one HslU hexamer to one end of an HslV dodecamer in the 3.0 A resolution structure opens both HslV central pores and induces asymmetric changes in HslV. CONCLUSIONS: Analysis of nucleotide binding induced conformational changes in the current and previous HslU structures suggests a protein unfolding-coupled translocation mechanism. In this mechanism, unfolded polypeptides are threaded through the aligned pores of the ATPase and peptidase and translocated into the peptidase central chamber.

Detection of mitochondrial DNA mutations in pancreatic cancer offers a "mass"-ive advantage over detection of nuclear DNA mutations.

We sequenced the complete 16.5-kb mitochondrial genome (mtDNA) in 15 pancreatic cancer cell lines and xenografts. Homoplasmic mtDNA somatic mutations and novel variants were identified in nearly all samples. Southern blot analysis and direct sequencing of mutation sites showed that the intracellular mass of mtDNA was greatly (6-8-fold) increased in pancreatic cancer cells in relation to corresponding normal cells; this property accounted for and greatly facilitated the identification of these mutations among the dense desmoplastic host reaction characteristic of primary pancreatic cancers. Structural characteristics and mathematical modeling of the evolution of mtDNA mutations suggested that many of the mutations identified might represent a random evolution of homoplasmic variants, rather than necessarily being a product of selective pressures. Complete sequencing of the nuclear MnSOD gene, which protects cells from the mitogenic and toxic effects of oxygen radicals, did not reveal any mutations. Nevertheless, the nearly ubiquitous prevalence and high copy number of mtDNA mutations suggest that they be considered of promising clinical utility in diagnostic applications.

[Genetic transformation of hairy roots in Trichosanthes kirilowii Maxim. by Ti and Ri plasmids].

OBJECTIVE: To establish a hairy root culture system by double transformation for Trichosanthes kirilowii. METHOD: 1. Crown galls were induced by direct infection of sterile seedlings of T. kirilonii with Agrobacterium tumefaciens C58, and then the hairy roots were obtained from the regenerated plants by infection with A. rhzogenes 15834; 2. Transformation of Ti and Ri plasmids was inspected by high-pressure-paper electrophoresis; 3. The protein contents in the tissues of T. kirilowii were inspected by spectrophotometer and SDS-PAGE. RESULT: A hairy root culture system has been established successfully by double transformation with Ti and Ri plasmids in T. kirilowii. CONCLUSION: Compared with the ordinary hairy roots, the double transformed hairy roots grow faster but retain similar protein contents.