Advances in physiological activities and synthesis of β-nicotinamide mononucleotide / 生物工程学报

Nicotinamide mononucleotide (NMN) is one of the key precursors of coenzyme Ⅰ (NAD+). NMN exists widely in a variety of organisms, and β isomer is its active form. Studies have shown that β-NMN plays a key role in a variety of physiological and metabolic processes. As a potential active substance in anti-aging and improving degenerative and metabolic diseases, the application value of β-NMN has been deeply explored, and it is imminent to achieve large-scale production. Biosynthesis has become the preferred method to synthesize β-NMN because of its high stereoselectivity, mild reaction conditions, and fewer by-products. This paper reviews the physiological activity, chemical synthesis as well as biosynthesis of β-NMN, highlighting the metabolic pathways involved in biosynthesis. This review aims to explore the potential of improving the production strategy of β-NMN by using synthetic biology and provide a theoretical basis for the research of metabolic pathways as well as efficient production of β-NMN.

Systematic identification of chemical forms of key terpene synthase in Cinnamomum camphora / 中国中药杂志

Cinnamomum camphora is an important economic tree species in China. According to the type and content of main components in the volatile oil of leaf, C. camphora were divided into five chemotypes, including borneol-type, camphor-type, linalool-type, cineole-type, and nerolidol-type. Terpene synthase(TPS) is the key enzyme for the formation of these compounds. Although several key enzyme genes have been identified, the biosynthetic pathway of(+)-borneol, which has the most economic value, has not been reported. In this study, nine terpenoid synthase genes CcTPS1-CcTPS9 were cloned through transcriptome analysis of four chemical-type leaves. After the recombinant protein was induced by Escherichia coli, geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) were used as substrates for enzymatic reaction, respectively. Both CcTPS1 and CcTPS9 could catalyze GPP to produce bornyl pyrophosphate, which could be hydrolyzed by phosphohydrolase to obtain(+)-borneol, and the product of(+)-borneol accounted for 0.4% and 89.3%, respectively. Both CcTPS3 and CcTPS6 could catalyze GPP to generate a single product linalool, and CcTPS6 could also react with FPP to generate nerolidol. CcTPS8 reacted with GPP to produce 1,8-cineol(30.71%). Nine terpene synthases produced 9 monoterpene and 6 sesquiterpenes. The study has identified the key enzyme genes responsible for borneol biosynthesis in C. camphora for the first time, laying a foundation for further elucidating the molecular mechanism of chemical type formation and cultivating new varieties of borneol with high yield by using bioengineering technology.

Consensus to 99mTechnetium-Pyrophosphate Scintigraphy in the Diagnosis of Transthyretin-related Cardiac Amyloidosis / 罕见病研究

Transthyretin-related amyloid cardiomyopathy (ATTR-CM) is a disease caused by the depo-sition of insoluble amyloid fibers formed by the misfolding of transthyretin precursor protein in the intercellular space of cardiomyocytes. This lesion may lead to myocardial dysfunction, cogestive heart failure, and death.When diagnosed earlier, the patient can be treated with drugs as soon as possible to intervene in the progress of the disease, so as to effectively improve the patient's prognosis.99mtechnetium-pyrophosphate (99Tcm-PYP)single-photon emission computed tomography (SPECT) has been widely used in the imaging examination of cardiac amyloidosis (CA) in recent years. While achieving early non-invasive diagnosis, accurate pathological classification can be obtained through Perugini visual score analysis, semi-quantitative analysis of heart to contralateral lung (H/CL) ratio, and SPECT image analysis. This article presents the application, methods, and the precautions of 99Tcm-PYPSPECT in the diagnosis of ATTR-CM, aiming to provide clinical reference for the application of this technology.

Application value of delay-phase 99Tc m-PYP scintigraphy and SPECT imaging for diagnosis of transthyretin-related cardiac amyloidosis / 中华核医学与分子影像杂志

Objective:To investigate the application of different imaging methods of 99Tc m-pyrophosphate (PYP) in the diagnosis and pathological classification of cardiac amyloidosis (CA). Methods:A total of 31 patients (22 males, 9 females, age 21-81(57.2±13.4) years) with suspected CA who underwent 99Tc m-PYP dual-phase scintigraphy (early-phase: 1 h, delay-phase: 2-3 h) and SPECT/CT (1 h) between December 2018 and December 2019 in Peking Union Medical College Hospital were retrospectively included. Taking clinical diagnosis as the standard, the results of visual score (≥2, positive) and semi-quantitative values (heart to contralateral lung (H/CL)≥1.5, positive) of 99Tc m-PYP uptake in dual-phase scintigraphy and SPECT/CT imaging were analyzed. One-way analysis of variance and Bonferroni test were used to analyze the data. Results:Among 31 patients with suspected CA, 15 were clinically diagnosed as CA (5 patients with transthyretin-related CA (ATTR-CA) and 10 patients with light chain CA (AL-CA)) and 16 were diagnosed as non-CA. All 5 patients with ATTR-CA had positive dual-phase scintigraphy and SPECT/CT imaging results. Three out of 10 patients with AL-CA had positive early-phase scintigraphy whereas negative delay-phase scintigraphy and SPECT/CT imaging results. Sixteen patients who were clinically diagnosed as non-CA had negative dual-phase scintigraphy and SPECT/CT imaging results. The sensitivity (5/5), specificity (10/10), positive predictive value (5/5), negative predictive value (10/10) and accuracy (15/15) of delay-phase scintigraphy and SPECT/CT imaging were the same. Among 31 patients, 16 patients carried transthyretin-related (TTR) gene mutation, and 4 of them who clinically diagnosed as variant ATTR (ATTRv) had positive image findings while 12 of them who not clinically diagnosed as CA had negative image findings. There were significant differences in H/CL between ATTR-CA group and AL-CA group in early-phase (2.11±0.24 vs 1.31±0.07) and delay-phase (2.02±0.19 vs 1.30±0.05; F values: 75.41 and 87.15, Bonferroni test, both P<0.01). Conclusions:99Tc m-PYP delay-phase scintigraphy and SPECT/CT have high diagnostic efficiencies in ATTR-CA, helping to determine the pathological classification of CA; while early-phase scintigraphy has false positive results. Moreover, 99Tc m-PYP imaging is helpful to detect CA in patients with TTR gene mutation.

Effect of isopentenyl pyrophosphate translocation on the biosynthesis of triptolide / 生物工程学报

Triptolide has wide clinical applications due to its anti-inflammatory, anti-tumor and immunosuppressive activities. In this study, we investigated the effect of blocking isopentenyl pyrophosphate (IPP) translocation on the biosynthesis of triptolide by exogenously adding D,L-glyceraldehyde (DLG) to the suspension cells of Ttripterygium wilfordii at different stages (7 d, 14 d). Subsequently, the cell viability, biomass accumulation, triptolide contents, as well as the profiles of the key enzyme genes involved in the upstream pathway of triptolide biosynthesis, were analyzed. The results showed that IPP translocation is involved in the biosynthesis of triptolide. IPP is mainly translocated from the plastid (containing the MEP pathway) to the cytoplasm (containing the MVA pathway) in the early stage of the culture, but reversed in the late stage. Blocking the translocation of IPP affected the expression of key enzyme genes involved in the upstream pathway of triptolide, which in turn affected the accumulation of triptolide. Understanding the characteristics and mechanism of IPP translocation provides a theoretical basis for further promoting triptolide biosynthesis through synthetic biology.

Research Advances of Human Homologue of Mouse Progressive Ankylosis Protein and Bone and Joint Diseases / 中国医学科学院学报

The human homologue of mouse progressive ankylosis protein(ANKH)is an inorganic pyrophosphate transport regulator,which regulates tissue mineralization by controlling the level of inorganic pyrophosphate.It plays an important role in the pathogenesis and development of bone and joint diseases,such as ankylosing spondylitis,craniometaphyseal dysplasia,and articular cartilage calcification.This review summarizes the progress of research on ANKH and the above-mentioned diseases.

Iliopectineal Bursitis Associated With Calcium Pyrophosphate Crystal Deposition Disease: A Case Report / 日本農村医学会雑誌

A 93-year-old man with a history of calcium pyrophosphate deposition disease (CPPD) in the right shoulder and wrist had severe pain in his right lower limb in the absence of trauma. He was transported by ambulance to our hospital. He was febrile (38℃) and swelling of the inguinal region was noted on physical examination. Laboratory examination showed elevated C-reactive protein (19.1 mg/dL, normal range ＜ 0.3 mg/dL) and white blood cell count (9600/μl, normal range ＜ 9000/μL). X-ray showed calcifications near the hip bilaterally, computed tomography showed distension of the iliopectineal bursa, and magnetic resonance imaging showed a cystic lesion of the iliopectineal bursa. Aspiration was performed under ultrasound guidance. Fluid analysis under an optical microscope revealed calcium pyrophosphate crystals. Bacterial examination was negative. Iliopectineal bursitis associated with CPPD was diagnosed, and he was treated conservatively with NSAIDs. To our knowledge, only 5 cases of iliopectineal bursitis associated with CPPD have been reported in Japan, and here we present the details of this rare case.

Phosphoribosyl-pyrophosphate synthetase 2 (PRPS2) depletion regulates spermatogenic cell apoptosis and is correlated with hypospermatogenesis / 亚洲男科学杂志(英文版)

Phosphoribosyl-pyrophosphate synthetase 2 (PRPS2) is a rate-limiting enzyme and plays an important role in purine and pyrimidine nucleotide synthesis. Recent studies report that PRPS2 is involved in male infertility. However, the role of PRPS2 in hypospermatogenesis is unknown. In this study, the relationship of PRPS2 with hypospermatogenesis and spermatogenic cell apoptosis was investigated. The results showed that PRPS2 depletion increased the number of apoptotic spermatogenic cells in vitro. PRPS2 was downregulated in a mouse model of hypospermatogenesis. When PRPS2 expression was knocked down in mouse testes, hypospermatogenesis and accelerated apoptosis of spermatogenic cells were noted. E2F transcription factor 1 (E2F1) was confirmed as the target gene of PRPS2 and played a key role in cell apoptosis by regulating the P53/Bcl-xl/Bcl-2/Caspase 6/Caspase 9 apoptosis pathway. Therefore, these data indicate that PRPS2 depletion contributes to the apoptosis of spermatogenic cells and is associated with hypospermatogenesis, which may be helpful for the diagnosis of male infertility.

Effect of Strong Light Stress on Growth, Physiological and Biochemical and Gene Expression of Key Enzymes in Atractylodes lancea / 中国实验方剂学杂志

Objective::To explore the effect of strong light stress on the growth, physiological and biochemical and key enzyme gene expression of the Atractylodes lancea, in order to provide the scientific basis for the standardized cultivation of the A. lancea. Method::The two-year-old A. lancea seedlings were taken as experimental materials. Poplar forest (light transmittance between 18.26%-36.04%) was taken as control group(ck). Different density shading networks were used to simulate different degrees of high light stress (51.10%, 80.73%, 100%) in late July. The growth state of A. lancea was observed. On the 0th, 5th, 10th, 15th, 20th days, the physiological and biochemical indexes of malondialdehyde (MDA) content, cell membrane permeability, proline (Pro) content, antioxidant enzyme activity and chlorophyll content in the leaves of A. lancea were measured. The relative expression levels of 3-hydroxy-3-methylglutarate monoacyl coenzyme A reductase (3-hydroxy-3-methylglutaryl coenzyme A, HMGR) and farnesyl pyrophosphate synthase gene (farnesyl pyrophosphate synthase, FPPS) in leaves of A. lancea under intense light stress were determined by real-time fluorescence quantitative PCR(Real-time PCR). Result::After strong light stress, the color of the leaves of A. lancea changed from dark green to light green and yellowish green, and the burn of leaves became more and more serious. The contents of MDA, conductivity and Pro showed an upward trend with the increase of transmittance. Peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) tended to increase first and then decrease. The chlorophyll content decreased with the increase of light transmittance. The relative expression of HMGR in leaves of A. lancea decreased with the increase of light transmittance, while FPPS increased first and then decreased. Conclusion::The results showed that A. lanceaa could alleviate the inhibition of strong light stress by increasing the activity of antioxidant enzymes and regulating the content of osmotic pressure under certain strong light stress. Excessively strong intensity light stress leads to disequilibrium of metabolic mechanism of A. lancea, and seriously inhibits the plant growth.

Oral Ferric Pyrophosphate Formulation Utilization Surveillance Study To Assess Clinical Impact On Hemoglobin Levels: Maxiim-Hemoglobin Study

After hemorrhage, anemia is the most common cause of maternal mortality and leading cause of maternal morbidity in India. Theprevalence rates of anemia in pregnancy in India is estimated to be >50%. Iron deficiency anemia (IDA) is the most commontype of anemia in pregnancy in India, which I can be as high as 80–90%.Aims and Objectives: The present survey was initiated in pursuit of analyzing the effectiveness and safety of oral ferricpyrophosphate (FPP) formulation given once to twice daily for treatment and prophylaxis of IDA in pregnancy.Materials and Methods: This was a questionnaire-based retrospective survey. Each gynecologist was given this survey bookletcontaining questionnaire. Clinical response was assessed by measuring rise in mean hemoglobin (Hb) levels at baseline, week4, and week 8, after giving oral FPP formulation for 8 weeks.Results: A total of 60 gynecologists participated and completed the survey, which involved 1073 pregnant subjects and patientssuffering from IDA (864 patients, i.e., 80%). Mean Hb level at baseline was found to be 8.98 g/dl, 10.03 at week 4, and 10.99 atweek 8. Thus, rise of Hb from baseline to week 8 was found to be 2.01 g/dl. Adverse events were reported in only 10 patients(<0.09%), none requiring discontinuation of therapy. 98% of the participants agreed good acceptability of oral FPP formulation.Conclusion: Findings of the present survey suggests that oral FPP formulation therapy can serve as potent choice of therapyfor IDA in pregnancy, both therapeutically and prophylactically

Prominent Inflammatory Features of Monocytes/Macrophages in Acute Calcium Pyrophosphate Crystal Arthritis: a Comparison with Acute Gouty Arthritis

Calcium pyrophosphate (CPP) crystals can present as acute inflammatory arthritis which is known as an acute CPP crystal arthritis. Although monocytes/macrophages have been shown to play a role in the initiation of crystal-mediated inflammatory responses, differences in their phenotypes between acute CPP crystal arthritis and acute gouty arthritis have not yet been investigated. We examined the immunological characteristics of synovial monocytes/macrophages in patients with acute CPP crystal and acute gouty arthritis. CD14⁺CD3⁻CD19⁻CD56⁻ cell frequencies in synovial fluid mononuclear cells (SFMCs) were measured. Expression of pro- and anti-inflammatory cytokines and markers was determined. The SFMCs were dominated by a population of monocytes/macrophages in acute CPP crystal arthritis similar to that in acute gout. Synovial monocytes/macrophages showed the phenotypes of infiltrated monocytes as shown by expression of CD88, C-C chemokine receptor type 2, myeloid-related protein (MRP)8 and MRP14 but not proto-oncogene tyrosine-protein kinase MER. Comparatively, the CD14⁺ cells from patients with acute CPP crystal arthritis had similar high levels of IL-1β and TNF-α production but significantly lower expression of IL-10 and M2 marker (CD163). The monocytes/macrophages had the capacity to produce IL-8 in response to CPP crystals. Proinflammatory features were more dominant in monocytes/macrophages during acute CPP crystal arthritis than those during acute gouty arthritis.

Effect of Mangiferin on mRNA Expressions of Hepatic PRPS, PRPPAT and Cerebral HGPRT in Hyperuricemic Mice Induced by Potassium Oxonate / 中国实验方剂学杂志

Objective:To investigate the effect of mangiferin on the mRNA expression of phosphoribosylpyrohoosphate synthetase (PRPS), phosphate ribose pyrophosphate amide transferase (PRPPAT) in liver and hypoxanthine-guanine phosphate transfer enzyme (HGPRT) in brain of hyperuricemic mice induced by potassium oxonate. Method:Hyperuricemic mice were induced through intraperitoneal injection with uricase inhibitor potassium oxonate. The serum uric acid level was determined by the phosphotungstic acid method. The mRNA expression levels of PRPS and PRPPAT in liver as well as HGPRT in brain of hyperuricemic mice were measured by reverse transcriptase polymerase chain reaction (RT-PCR). Result:An intraperitoneal injection with potassium oxonate caused a marked increase in serum uric acid level, compared with normal control group (P-1 was able to significantly reduce serum uric acid levels, compared with hyperuricemic control group (PPConclusion:The hyporuricemic effect of mangiferin might not be related with PRPS, PRPPAT and HGPRT in therapeutic dose.

Comparison between pyrophosphate method and infrared spectrophotometry for determination of silicon dioxide content in dust / 中华劳动卫生职业病杂志

Objective@#To discuss the difference between pyrophosphoric acid method and infrared spectrophotometry for the determination of silica content in dust.@*Methods@#The content of silica in the laboratory comparison samples organized by CDC Occupational Health Institute in China in 2018, and purchased quality control samples were determined by pyrophosphate method. Meanwhile, the samples were qualitatively and quantitatively analyzed by infrared spectrophotometry, and the results obtained by the two methods were compared.@*Results@#Four samples (062C1、062C2、GDOHZKTG012-1、GDOHZKTG012-2) were detected by pyrophosphate method and infrared spectrophotometry. The results of pyrophosphate method were 55.49%, 5.24%, 4.90% and 54.72%, respectively. The results of infrared spectrophotometry were 0.91%, 1.87%, 1.29% and 1.16% respectively.@*Conclusion@#The content of silica in dust determined by pyrophosphate method is higher than that by infrared spectrophotometry.

Cloning and expressive characteristics of GPS gene and its correlation with saponins content in Eleutherococcus senticosus / 中草药

Objective To clone cDNA sequence of geranyl pyrophosphate synthase (GPS) gene from Eleutherococcus senticosus and analyze genetic characteristics, gene expression level in different organs and the correlation between GPS gene expression and saponins content. Methods RNA was extracted from E. senticosus and reverse transcribed into cDNA. Gene specific primers were designed according to the unigene (c37362.graph_c0) of GPS from transcriptome sequencing data. The full length of the GPS gene cDNA was amplified by PCR. The expression level of GPS gene in different organs was analyzed by qRT-PCR. The content of E. senticosus saponins was detected by spectrophotometry method. Results GPS gene cDNA was cloned from E. senticosus and encodes 419 amino acids with full length of 1 260 bp. GPS protein located in mitochondria does not have transmembrane region. The GPS gene was expressed in each organ and had the highest expression in blade, which is 5.26 times in root. The relative expression of GPS gene and content of saponin showed the same trend and significant positive correlation (r = 0.851, P < 0.05). Conclusion The whole length of cDNA sequence of GPS gene is cloned for the first time, and there is a positive correlation between the expression level of GPS gene and the saponin content of E. senticosus.

Effect of endophytic fungi on expression of key enzyme genes in polysaccharide and alkaloid synthesis from Dendrobium officinale / 中草药

Objective: To analyze the effect of endophytic fungi GXRz2, GXRz3 and GXRz10 on content of poly saccharide and alkaloid, and the expression of key enzyme genes UGPase, HMGR, and FPS in Dendrobium officinale. Methods: The endophytic fungi liquid were added to D. officinale seedlings. Polysaccharide and alkaloid content were measured by spectrophotometry method. With 18S rRNA as internal control gene, the expression of key enzyme genes was detected by real-time quantitative PCR method. Results: It was found that the content of polysaccharide in D. officinale was higher, mainly concentrated in stem, but the lowest in root. And the content of alkaloid in D. officinale was lower, mainly accumulated in leaf, but the lowest in root. In addition, the three endophytic fungi strains could promote accumulation of polysaccharide and alkaloid in D. officinale to a certain extent. The expression of UGPase, HMGR and FPS genes in D. officinale induced by different strains was detected by real-time quantitative PCR. The results showed that endophytic fungi GXRz3 and GXRz10 could significantly increase the expression of UGPase, HMGR and FPS genes in D. officinale. In the polysaccharide synthesis pathway, the UGPase gene had the highest relative expression in the stem, followed by the leaf, and the least in the root. In the alkaloid synthesis pathway, HMGR gene had the highest relative expression in the stem, followed by the leaf, and the least in the root. However, FPS gene had the highest relative expression in the leaf, followed by the stem, and the least in the root. Conclusion: Endophytic fungi may affect the synthesis of polysaccharide by regulating the expression of UGPase. Considering the accumulation of polysaccharide, it is speculated that UGPase may be a key enzyme in the polysaccharide synthesis pathway of D. officinale. Endophytic fungi may affect the synthesis of alkaloid by regulating the expression of HMGR and FPS. Considering the accumulation of alkaloid, it is speculated that FPS may be a key enzyme in the alkaloid synthesis pathway of D. officinale.

The Role of Calcium Pyrophosphate Dihydrate Deposition in the Postoperative Outcome of Lumbar Spinal Stenosis Patients

STUDY DESIGN: Retrospective study.PURPOSE: This study aimed to investigate the association of surgical intervention with clinical and quality of life (QoL) outcomes in patients who underwent posterior spinal surgery for lumbar spinal stenosis (LSS) with spinal calcium pyrophosphate dihydrate deposition (SCPPD) versus that in those who underwent the surgery for LSS without SCPPD.OVERVIEW OF LITERATURE: Calcium pyrophosphate (CPP)-associated arthritis is one of the most common types of arthritis. The clinical outcomes are well studied in CPP-associated arthritis of the appendicular joints. However, few studies have investigated SCPPD.METHODS: A single-institution database was reviewed. LSS patients were categorized as those who did and did not have SCPPD, based on histologic identification. Clinical presentations and postoperative results were analyzed. Disability and QoL were assessed using the Oswestry Disability Index (ODI) and the 36-item Short-Form Health Survey.RESULTS: Thirty-four patients were enrolled, with 18 patients being allocated to the SCPPD group and 16 being allocated to the non- SCPPD group. Preoperative and postoperative pain scores were not significantly different between the groups (p=0.33 and p=0.48, respectively). The average preoperative ODI score in the SCPPD group was slightly higher than that in the non-SCPPD group (57 vs. 51, p=0.33); however, the postoperative ODI score was significantly lower (15 vs. 43, p=0.01). The postoperative physical function, vitality, and mental health of the SCPPD patients were also significantly improved (p=0.03, p=0.022, and p=0.022, respectively).CONCLUSIONS: Surgical intervention resulted in good clinical outcomes in SCPPD patients. As per our findings, total removal of CPPinvolved tissue is unnecessary. As such, surgery should be performed as indicated according to clinical presentation without considering the presence of CPPD.

Palatability of micro-encapsulated iron pyrophosphate (Ferfer®)

Background:Iron deficiency is the leading cause of anemia globally and affects 20% of world adult population. The general practice for replacement of iron reserves is oral supplementation. However, the success of this strategy solely depends on drug adherence and compliance. Where many other factors contribute to compliance, the role of tasteof the medicine can also not be neglected. The aim of this study is to evaluate the palatability of micro-encapsulated iron pyrophosphate (Ferfer) and compare its taste with previously taken other oral forms of iron supplements.Methods:The study was conducted with 231 female participants of age more than 18 years. Participants had taken one sachet of micro-encapsulated iron pyrophosphate (Ferfer) for the purpose of taste. Questionnaire based on their previous experience of iron supplements and palatability of Ferfer was asked by participants based on 0-10 mmvisual analogue scale and 5 point Likert scale evaluating responses at 0 minute, 5 minute, and at 10 minute. Data was analyzed by using SPSS version 23.Results:The women previously taking some others oral form of iron supplementation scored their supplements, a mean taste score is 2.92±2.44 on the VAS. However, micro-encapsulated iron pyrophosphate (Ferfer) received a mean score of 7.66±1.32 immediately after taking it and a score of 7.96±1.37 after 5 minutes on the 0-10 mmVAS. 81.4% patients will recommend their doctors to prescribe Ferferto them in future. Conclusions: Enhanced palatability of micro-encapsulated iron pyrophosphate sachets have resulted in increased compliance to the supplementation regimen among the participants.

Cloning and prokaryotic expression of CDS region from GGPPS gene of Panax notoginseng / 中草药

Objective: In order to study the function of geranylgeranyl pyrophosphate synthase (GGPPS) gene, the CDS nucleotide sequence of GGPS was cloned from Panax notoginseng, and its prokaryotic expression was performed. Methods: The primers were designed according to the reported GGPPS gene sequence in Genbank, and the coding sequence was obtained by RT-PCR. The prokaryotic expression vector was constructed and transformed into Escherichia coli BL21 for the expression under the induction of isopropyl β-D-1-thiogalactopyranoside (IPTG). Results: The CDS of GGPS gene had a full length of 1 032 bp coding for 343 amino acids. Results of SDS-PAGE showed that a 29 000—44 000 protein was achieved and the recombinant protein was mainly in the form of insoluble inclusion body. Conclusion: The CDS nucleotide sequence of GGPPS gene was successfully cloned, and the stable prokaryotic expression was established. This study will provide a foundation for the further functional researches of GGPPS gene in P. notoginseng.

Prokaryotic expression, function verification and immunoassay activity of farnesyl pyrophosphate synthase of Alisma orientale (Sam.) Juzep / 药学学报

Farnesyl pyrophosphate synthase of Alisma orientale (Sam.) Juzep. (AoFPPS) is considered as one of the important rate-limiting enzymes in the biosynthetic pathway of protostane triterpenes. In order to investigate the expression and function of AoFPPS, the gene (accession No. HQ724508) was cloned into a bacterial expression vector pCzn1, then the combined plasmid pCzn1-AoFPPS was transformed into Escherichia coli BL21, and a fusion protein was obtained after induction. The fusion protein was purified by Ni resin, and the function was verified through in vitro enzymatic reaction. High performance liquid chromatography (HPLC) analysis revealed that the products were able to catalyze the synthesis of farnesyl pyrophosphate (FPP). High purity recombinant protein was used to immunize New Zealand rabbits to generate a polyclonal antibody. The titer of the antibody was determined by enzyme linked immunosorbent assay (ELISA), and Western blot results demonstrated that the antibody could specifically recognize the AoFPPS protein in A. orientale (Sam.) Juzep. So,the method of rapid immunoassay to detect AoFPPS was established. This study lays the foundation for further study of the AoFPPS gene expression, regulation and mechanism of action in A. orientale (Sam.) Juzep., and it also provides a scientific basis on improving the quality of Alismatis Rhizoma using the plant genetic engineering.

Age-Related Prevalence of Periodontoid Calcification and Its Associations with Acute Cervical Pain

STUDY DESIGN: Prospective study. PURPOSE: To assess the prevalence of periodontoid calcification and its associations with acute cervical pain. OVERVIEW OF LITERATURE: Calcium pyrophosphate dihydrate (CPPD) deposition disease is a common rheumatological disorder that occurs especially in elderly patients. Although CPPD crystals induce acute arthritis, these crystals are not usually symptomatic. Calcification surrounding the odontoid process (periodontoid calcification) has been reported to induce inflammation, resulting in acute neck pain. This disease is called crowned dens syndrome. Whether calcification induces inflammation or whether the crystals are symptomatic remains unclear. METHODS: The prevalence of periodontoid calcification at the atlas transverse ligament was examined by computed tomography of the upper cervical spine in patients suspected of brain disease but no cervical pain (control group, n=296), patients with pseudogout of the peripheral joints but no cervical pain (arthritis group, n=41), and patients with acute neck pain (neck pain group, n=22). Next, the correlation between the prevalence of periodontoid calcification and symptoms was analyzed. RESULTS: In the control group, 40 patients (13.5%) showed periodontoid calcification with no significant difference in the prevalence with gender. The prevalence of calcification increased significantly with age (p=0.002). In the arthritis group, 26 patients (63.4%) reported periodontoid calcification. In the neck pain group, 14 patients (63.6%) reported periodontoid calcification. Multiple logistic regression analysis by age and group revealed that higher age, inclusion in the arthritis group, and inclusion in the neck pain group significantly affected the prevalence of calcification. CONCLUSIONS: Our results cumulatively suggest that periodontoid calcification is an aging-related reaction and that calcification per se does not always cause neck pain. Periodontoid calcification was observed more frequently in patients with pseudogout of the peripheral joints and in those with acute neck pain than in asymptomatic control patients.