[Evaluation of the efficacy and safety of iron therapy in patients with paroxysmal nocturnal hemoglobinuria complicated with iron deficiency anemia].

Objective: To evaluate the efficacy and safety of iron supplement in patients who have paroxysmal nocturnal hemoglobinuria (PNH) with iron deficiency. Methods: We performed analyses on the clinical data of 48 patients who accepted oral and/or intravenous iron treatment. Forty-eight consecutive PNH patients with iron deficiency who visited our hospital between November 2011 and August 2018 were enrolled in the study. Results: Total 30 patients received oral iron; 18 patients received intravenous iron supplements, including 6 who did not respond to oral iron. The median PNH clone size was 90.2% (38.5%-99.9%) in the granulocytes and 69.7% (27.6%-98.1%) in the red blood cells. The response rate was 56% (20/36) in patients who received oral iron, and the hemoglobin concentration increased 21 (10-52) g/L compared to that at baseline. Sixteen out of eighteen (89%) patients responded to intravenous iron; 6 patients who did not respond to oral iron received intravenous iron, and the hemoglobin level of 5 patients increased. Patients exhibited increased LDH levels and deepen urine after iron supplementation; however, no severe adverse events, such as thrombosis and iron-related adverse effects, were noted. Conclusion: Iron treatment is safe and effective in increasing the hemoglobin level in PNH patients with iron deficiency; those who did not respond to oral iron could benefit from intravenous iron supplement.

Melatonin mitigates cadmium and aluminium toxicity through modulation of antioxidant potential in Brassica napus L.

Melatonin has emerged as an essential molecule in plants, due to its role in defence against metal toxicity. Aluminium (Al) and cadmium (Cd) toxicity inhibit rapeseed seedling growth. In this study, we applied different doses of melatonin (50 and 100 µm) to alleviate Al (25 µm) and Cd (25 µm) stress in rapeseed seedlings. Results show that Al and Cd caused toxicity in rapeseed seedling, as evidenced by a decrease in height, biomass and antioxidant enzyme activity. Melatonin increased the expression of melatonin biosynthesis-related Brassica napus genes for caffeic acid O-methyl transferase (BnCOMT) under Al and Cd stress. The genes BnCOMT-1, BnCOMT-5 and BnCOMT-8 showed up-regulated expression, while BnCOMT-4 and BnCOMT-6 were down-regulated during incubation in water. Melatonin application increased the germination rate, shoot length, root length, fresh and dry weight of seedlings. Melatonin supplementation under Al and Cd stress increased superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, proline, chlorophyll and anthocyanin content, as well as photosynthesis rate. Both Cd and Al treatments significantly increased hydrogen peroxide and malondialdehyde levels in rapeseed seedlings, which were strictly counterbalanced by melatonin. Analysis of Cd and Al in different subcellular compartments showed that melatonin enhanced cell wall and soluble fractions, but reduced the vacuolar and organelle fractions in Al- and Cd-treated seedlings. These results suggest that melatonin-induced improvements in antioxidant potential, biomass, photosynthesis rate and successive Cd and Al sequestration play a pivotal role in plant tolerance to Al and Cd stress. This mechanism may have potential implications in safe food production.

[Identification of Two GLOBOSA-like MADS-box Genes in Tea Plant (Camellia sinensis [L.] O. Kuntze)].

Tea plant (Camellia sinensis [L.] O. Kuntze) is a woody crop of high economic importance worldwide; however, information on the molecular mechanisms underlying the regulation of flower development in this species is limited. In the present study, two GLOBOSA (GLO) -like MADS-box genes, CsGLO1 and CsGLO2, were isolated from C. sinensis 'Ziyangzhong' and were characterized to elucidate their roles in flower development. We found that CsGLOl and CsGLO2 are nuclear-localized transcription factors without transactivation ability but with a robust interaction. They have similar patterns of expression, both mainly restricted to petals and stamens. Moreover, ectopic expression of either CsGLO1 or CsGLO2 in Arabidopsis thaliana resulted in a partial conversion of sepals to petals, suggesting full GLOBOSA functional activity. Our results indicate that CsGLO1 and CsGLO2 paralogs might redundantly contribute to petal and stamen, providing the first insight into their role in tea plant flower development.

Studies on the molecular markers of rhizomes of some Alpinia species.

The rhizomes of Alpinia jianganfeng are used as a traditional Chinese medicine, Jian Gan Feng, to cure rheumatism in Guangdong, China. The rhizomes of some other species of the genus Alpinia such as A. japonica, A. suishaensis and A. nanchuanensis are also used as Jian Gan Feng in Southwest China. However, the identification of the original plants of the crude drugs is difficult. The internal transcribed spacers and the 5.8S coding region of nuclear ribosomal DNA of the four species were sequenced and analyzed. The DNA markers have been determined and they can be used for the molecular identification of these medicinal plants.

Characters of nrDNA ITS region sequences of fruits of Alpinia galanga and their adulterants.

The fruits of Alpinia galanga (L.) Sw. are used as a traditional Chinese medicine; but the dry fruits of A. conchigera, A. suishaensis, A. maclurei and A. polyantha are also used as the medicine in local areas. Because dry fruits of these related plants are similar to those of Alpinia galanga (L.) Sw. in odor, morphological characters and chemical components, and even anatomical characters, it is difficult to identify the medicine. Nuclear ribosomal DNA internal transcribed spacer (ITS) regions of the five taxa were directly sequenced using an automated sequencer. Sequence analysis showed that the ITS 1 ranges from 177 to 178 base pairs (bp), and the ITS 2 from 225 to 234 bp. The size of the 5.8S coding region is 164 bp for all species. Also, the pairwise sequence divergence is higher and some molecular markers were determined. According to these molecular markers, Alpinia galanga (L.) Sw. and the related species can easily be distinguished from each other. Therefore, evidence from nrDNA ITS sequence variation can identify the medicine at the DNA level.

Authentication of Chinese crude drug, Gecko, by allele-specific diagnostic PCR.

Based on the sequences of the mitochondrial 12S rRNA gene fragment of 17 samples from Gekkonidae, Salamandridae, Agamidae and Hynobiidae, respectively, a pair of allele-specific primers was designed for differentiating the Chinese medicinal material Gecko from its adulterants by PCR. The results of amplification with the primers indicate that amplicons from the templates of Gekko gecko were clearly revealed by agarose gel electrophoresis, whereas no evident amplicons were found from other species. The primers were employed to identify crude drug samples from different sources. Among a total of 9 samples, 3 were diagnosed as genuine Gecko. This result is consistent with morphological identification and DNA sequence analyses.

[Study on relationship between the reflux laryngitis and gastroesophageal reflux disease].

OBJECTIVE: To investigate the relationship between reflux laryngitis and gastroesophageal reflux disease (GRED). METHOD: 130 Patients of refractory chronic laryngitis with upper endoscopy and ambulatory 24-hr esophageal pH-metry. The patients of gastroesophageal reflux disease were divided into two groups. Group A was treated with antacids and motile medications. Group B was control. RESULT: 45 (34.6%) gastroesophageal reflux disease were found in 130 cases. 21 cases(91.3%) were healed in group A. 3 of 22 cases (13.6%) were healed in control (P < 0.01). CONCLUSION: The gastroesophageal reflux disease was an important cause of the reflux laryngitis which symptoms and signs can be healed by antacids and motile medications.

[Study on allele-specific diagnostic PCR of the traditional Chinese medicines of the deers].

AIM: To develop a convenient and accurate method of DNA molecular marker for the identification of traditional Chinese medicines made of deers, consisting of pilose antler, penis and testis, tendon and foetus. METHODS: Based on the analysis of DNA sequence of mitochondrial Cyt b gene from original animals of both genuine crude drugs, Cervus nippon and Cervus elaphus, and adulterants, a pair of allele-specific primers named as ILu01-L and ILu01-H were designed for distinguishing geniune crude drugs of deers from their adulterants. RESULTS: The results of diagnostic PCR annealing at 64 degrees C for original animals showed that a 365 bp fragment was only amplified from DNA templates of Cervus nippon and Cervus elaphus. For the identification of medicinal materials total of 43 samples from 6 packages were tested under the same reaction conditions except for DNA templates extracted from these crude drugs. Only 9 samples mentioned above was shown to generate positive amplificon. The result indicate that of 8 samples from 1 package of pilose antler and only 1 sample of deer tendon was genuine crude drug. After that, 3 amplified fragments selected randomly were performed with sequencing analysis with the purpose of verifying the results from diagnostic PCR. Data from sequencing confirmed the reliability of diagnostic PCR identification. CONCLUSION: The diagnostic primers designed in the present study were highly specific for Cervus nippon and Cervus elaphus, and they could be used for the authentication of traditional Chinese medicines made from the deer. The quality of the crude drugs of the deer in the current market is a problem and more effective quality control for these traditional Chinese medicines is urgently needed.

[rDNA its sequencing of Herba Dendrobii (Huangcao)].

AIM: To study the genetic diversity of ITS sequences of Herba Dendrobii (Huangcao) and analyze the utility of ITS sequences in molecular authentication of Herba Dendrobii (Huangcao) and phylogenetic of Dendrobium. METHODS: The ITS gene fragment was amplified using a pair of primers. The PCR products were purified and sequenced by the methods of Sanger Dideoxy. RESULTS: The DNA sequence of 228-233 bp ITS 1, 242-247 bp ITS 2 gene fragments and 5.8S rDNA were obtained from 14 samples of Dendrobium. The interspecific substitution varies from 11.79% to 31.58% at ITS 1 and 10.29% to 25.30% at ITS 2. The intraspecific substitution of D. nobile is 0.87% at ITS 1 and without difference at ITS 2. The substitution between Dendrobium and outgroup varies from 23.56% to 36.89% at ITS 1 and 26.52% to 33.31% at ITS 2. The phylogenetic tree based on ITS 1 and ITS 2 data was set up. CONCLUSION: The ITS 1 and ITS 2 gene fragments were highly conservative at intraspecific level in Dendrebium, while they were less conservative at interspecific level in D. nobile. They were least conservative between Dendrobium and outgroup. Hence, the sequence of this fragment is a good molecular marker for authentication of the Huangcao. But, further study is necessary for the phylogenetic of Dendrobium.

[Identification of Chinese crude drug snake gallbladder by DNA molecular marker].

AIM: It is difficult to identify the Chinese crude drug snake gallbladder accurately by morphological and microscopical characteristics or chemical components only. In order to solve the problem, the technique based on DNA molecular marker was introduced into the authentication of snake gallbladder. METHODS: DNA templates were extracted from the membrane or the bile of snake gallbladder, and also from the muscle of the original animal Elaphe schrenckii. About 400 bp DNA fragments of 12S rRNA gene were amplified from the templates and sequenced subsequently. RESULTS: Enough amounts of DNA templates could be extracted from a bit of membrane or bile of snake gallbladder. The sequence of amplicons from the membrane, bile and muscle of the same individual were identical completely. CONCLUSION: The technique of DNA molecular marker could be used for the authentication of snake gallbladder and bile. The results indicate that the technique could be used for the identification of crude drugs from other animal secretion. DNA sequence analysis also demonstrated that the origins of commercial snake gallbladder were complicated and more efficient quality control was necessary for supervising the crude drug in the market.

[Nuclear ribosomal RNA small subunit (18S rRNA) nucleotide sequencing and characterization of sailonggu(whole bone of Myospalax baileyi Thomas)].

OBJECTIVE: Sequencing the nuclear ribosomal RNA small subunit (18S rRNA) gene of Myospalax baileyi (Cricetidae) to develop an ultimate and definitive means for origin identification of genuine Sailonggu. METHODS: The total DNA was prepared from dried tail tissues. The nuclear 18S rRNA gene region was amplified by PCR using a consensus primer set and its nucleotide sequence was determined by PCR direct sequencing. The characteristic analysis of 18S rRNA sequences was generated using software program Genetyx-SV/R Version 10.1. RESULTS: The entire 18S rRNA gene region of M. baileyi spanded 1851 bp in length. Although multiple alignment of sequence indicates that there are only lower homology (72.04%-72.18%) comparing with its two alias Mus musculus (GenBank Accession number X00686) and Rattus norvegicus (M11188) (Muridae), their highly conservative domain is located in 1020-1509 nt. There are many variable sites from upstream of 5'-end, which could provide a novel information for molecular recognition of Sailonggu. CONCLUSION: DNA sequencing could be a useful and reliable tool in the origin identification of genuine Sailonggu.

Authentication of an animal crude drug, Zaocys, by diagnostic PCR.

A pair of diagnostic primers for distinguishing the Chinese crude drug Zaocys (Zaocys dhumandes) from its substitutes was designed based on the sequence data of the original animal of the drug and substitutes. Total DNAs were extracted from genuine crude drug and 5 of its substitutes, as well as from 12 species of original animal of the snake crude drug. Diagnostic PCRs were performed using the primers with these total DNAs as a template, annealing at 60-65 degrees C. Positive amplifications were obtained from all DNA templates of Zaocys, whereas negative amplifications were obtained from that of others. The results indicate that Zaocys samples could be definitely distinguished from its substitutes by diagnostic PCR, and no incorrect discrimination was found under the same reaction conditions. The advantages of the method in the authentication of crude drugs are also discussed in the present paper.

[A study of low temperature catalyst for Sabatier reaction].

Objective. To develop a low temperature catalyst for the Sabatier CO2 reduction of the atmospheric regeneration system and lower the start-up temperature of the Sabatier reaction. Method. A low temperature catalyst was designed from the considerations of the active composition, the choice of the carrier, the production method and condition of the catalyst. Then the performance of the newly developed low temperature catalyst was tested. Result. A new low temperature catalyst for the Sabatier reaction using Ru as the active composition and using r-Al2O3 as the carrier was developed. The start-up temperature was lower than 110 degrees C and the start-up time was 8 min; The conversion efficiency of the lean component (H2 or CO2) was over 95 percent when the temperature of the reactor was from 200 degrees C to 300 degrees C; The reaction product water was nearly colorless, transparent and neutral. Conclusion. The test results showed that the goals of the design are achieved and it is worthwhile to make further studies on the low temperature catalyst.

[Application of RAPD in the taxonomy of the genus Fritillaria].

OBJECTIVE: To provide the molecular data for the right application of the Chinese medicine "Beimu". METHOD: Using the technology of RAPD, we studied the relationship among 12 samples of Beimu. RESULT: The total genomic DNA of all the samples are about 21 Kb in size. Among 20 primers used, the five primers can reatedly generate a certain specified amplified band type, 27 bands were recored from all amplified products and 25 polymorphic fragments were found in it. The size of amplified fragments is between 450 bp and 1904 bp. CONCLUSION: The similarity within species is higher than those between species. The relationship of Fritillaria anhuiensis and F. puqiensis is the farest, while F. thunbergii and F. puqiensis is the closest.

[Clinical study on effect of shenqi fuzheng injection combined with chemotherapy in treating gastric cancer].

OBJECTIVE: To assess the clinical effect of Shenqi Fuzheng Injection (SQFZI) combined with chemotherapy in treating gastric cancer. METHODS: One hundred and twenty patients were randomly divided into 3 groups, the combined therapy group (Group A), the chemotherapy group (Group B) and the SQFZI group (Group C) and the effect on remission and stabilization of patients were observed. RESULTS: The remission rate and stabilizing rate of Group A were 16.1% and 87.1%, those of Group B were 13.5% and 64.9%, the difference between the two groups was significant, P < 0.05. The symptom and living quality improving rate of Group A were 75.8% and 43.5% respectively, those of Group C were 61.9% and 57.1% and of Group B were 35.1% and 29.7% respectively. SQFZI showed good protective effect on hemopoietic system, 4.8% patients in Group A with WBC count lower than 4 x 10(9)/L, while the percentage reached 21.6% in Group B, the difference between the two groups was significant, P < 0.05. Moreover, SQFZI could raise activity of NK cell, macrophage and T-lymphocyte subgroups, without any injury on heart, liver and kidney function or other adverse reaction. CONCLUSION: SQFZI combined with chemotherapy has certain effect of remission and stabilization on gastric cancer, the clinical effect is significant to patients with Qi-Deficiency, and is reliable and safe.

[Effect of hyperbaric oxygen on cerebral microcirculation and tissue cells in animals with cerebral ischemic injury].

Changes of cerebral microcirculation and tissue cells after Hyperbaric Oxygen (HBO) exposure were observed in 136 gerbils with cerebral ischemia by observation of meningeal microcirculation pathological study in cerebral tissues and determination of Na, K-ATPase. It is indicated that HBO may be helpful in improving microcirculatory dynamics and other microcirculatory functions, and enhancing cerebral tissue cell activity and cell function, as well as increasing oxygen content. It is suggested that HBO (250 approximately 300kPa) may play a role in protecting vessel endothelial cells and nerve cells.

[Molecular genetic marker identification of traditional Chinese drug hippocampus].

Ancient DNA technique was used to extract DNA from 5 species of Chinese traditional drugs Hippocampus. The 12S rRNA gene fragment and cytochrome b gene fragment were amplified from DNA extract using Polymerase Chain Reaction (PCR) method. Restriction Fragment Length Polymorphism (RFLP) analysis and DNA sequence analysis were performed. The RFLP analysis method can identify 2 species of Hippocampus. The molecular genetic markers produced by DNA sequence method can identify all 5 species of Hippocampus. This method will be valuable for the identification of other animal drugs.

[Molecular identification of the Chinese drug turtle shells].

This paper reports a new method to identify the Chinese drug turtle shells using PCR product direct sequencing method. DNA was extracted from tissues of the Chinese three-keeled pond turtle Chinemys reevesii and 20 other species of turtles occurring in China and Southeast Asian countries. One hundred and ten base pairs of mitochondrial 12S rRNA gene fragment were amplified from the extract using PCR technique and obtained sequences. These sequences were used to construct 12S rRNA gene fragment sequence database for the 21 turtle species. Comparison of these sequences indicated that the sequence from the Chinese three-keeled pond turtle is different from that of all the other 20 turtle species. The sequence divergence is 3.7-15.7%. DNA was extracted from 0.1-0.5 g of shell from 19 turtle shells provided by the Jiangsu Institute for Drug Control and 12S rRNA gene fragment was amplified and sequenced. Comparison of the sequences from the 19 turtle shells and 12S rRNA gene fragment sequence database indicated that only 3 samples are shells of the Chinese three-keeled pond turtle specified in the Phamacopoeia of the People's Republic of China and the others are substitutes. The technique used in the present paper was found to be effective and reliable for the identification of turtle shells.

[Sequencing of Cyt b gene fragments and PCR identification of "jinqian baihuashe" (Bungarus parvus) and its adulterants].

DNAs extracted from both "Jinqian Baihuashe" (Bungarus parvus) and its adulterants and original animals of the crude snake drugs were used as templates for Cyt b gene fragment amplification. The sequence data of the fragments showed that the differences of the sequence between Bungarus parvus and its adulterants were far greater than that between intraspecific variations of Bungarus parvus. Therefore, the Cyt b gene fragment was a good molecular genetic marker for the authentication of Bungarus parvus. On the basis of the sequence data, a pair of specialized primers, BuL-1 and BuH-1 was designed for the PCR identification of Bungarus parvus. The effectiveness of the primers were examined at a series of anneal temperatures. The results showed that Bungarus parvus samples could be absolutely distinguished when the anneal temperatures were 60 degrees C-65 degrees C, whereas no incorrect or missing discrimination was found at these temperatures. The results also showed that the powder of Bungarus parvus which was mixed with powders of three other crude snake drugs may be detected by the PCR identification. This indicates that PCR identification may be a new method for examining the compositions of Chinese patent medicine.

[A preliminary study on the identification of crude snake drugs by molecular genetic markers].

DNA was amplified from 11 samples of crude snake drugs by 2 random primers. "Wushaoshe" (Zaocys dhumnades) and its substitutes, Elaphe taeniurus, E. rufodorsata, E. carinata and Sinonatrix annularis, and "Jinqian Baihyashe" (Bungarus multicinctus) and its adulterants, Dinodon rufozonatum and Sinonatrix annularis, can be distinguished by electrophoresis pattern of the amplified products. The results showed that the molecular genetic marker technique can be used as a new method for the identification of crude snake drugs.