Assay systems for screening food and natural substances that have anti-hyperuricemic activity: uric acid production in cultured hepatocytes and purine bodies-induced hyperuricemic model mice

Hyperuricemia is characterized by the high uric acid (UA) level in serum (or plasma) and has been considered to be an important risk factor for gout. In the present study, we have attempted to construct an assay system for UA production in vitro employing cultured AML12 hepatocytes. UA levels in balanced salt solution (BSS) in the presence of UA precursor nucleosides, adenosine, inosine, guanosine and xanthine, at 12.5, 25, and 100 µM were significantly higher than BSS alone and their effects were dose-dependent, while all the UA precursors did not significantly increase intracellular UA levels. Hence, UA levels in BSS were thereafter adopted as an index of UA production. UA production from nucleosides was significantly higher than that from nucleotides (GMP, IMP and AMP). UA production from guanosine and inosine in combination (GI mixture) as well as nucleosides increased time-dependently and almost linearly up to 2 h. Selecting GI mixture, effects of allopurinol, a widely used anti-hyperuricemic agent, and quercetin, a well-known polyphenol in onion and strawberry, on UA production were examined. Both allopurinol and quercetin dose-dependently (0.1, 0.3 and 1 μM for allopurinol and 10, 30, and 100 μM for quercetin) and significantly reduced UA production in the hepatocytes. They also significantly reduced hyperuricemia induced by intraperitoneal injection of UA precursor purine bodies to mice at a single oral dose of 10 (allopurinol) or 200 (quercetin) mg/kg body weight. This assay system for UA production in cultured hepatocytes is considered to be useful to search for novel anti-hyperuricemic compounds in foods and natural resources with possibility to have human health benefits.     

Blood purine measurements as a rapid real-time indicator of reversible brain ischaemia

To preserve the disequilibrium between ATP and ADP necessary to drive cellular metabolism, enzymatic pathways rapidly convert ADP to adenosine and the downstream purines inosine and hypoxanthine. During ischaemia, these same pathways result in the production of purines. We performed a prospective observational study to test whether purine levels in arterial blood might correlate with brain ischaemia. We made real-time perioperative measurements, via microelectrode biosensors, of the purine levels in untreated arterial blood from 18 patients undergoing regional anaesthetic carotid endarterectomy. Pre-operatively, the median purine level was 2.4 μM (95% CI 1.3–4.0 μM); during the cross-clamp phase, the purines rose to 6.7 μM (95% CI 4.7–11.5 μM) and fell back to 1.9 μM (95% CI 1.4–2.7 μM) in recovery. Three patients became unconscious during carotid clamping, necessitating insertion of a temporary carotid shunt to restore cerebral blood flow. In these, the pre-operative median purine level was 5.4 μM (range 4.7–6.1 μM), on clamping, 9.6 μM (range 9.4–16.1 μM); during shunting, purines fell to below the pre-operative level (1.4 μM, range 0.4–2.9 μM) and in recovery 1.8 μM (range 1.8–2.6 μM). Our results suggest that blood purines may be a sensitive real-time and rapidly produced indicator of brain ischaemia, even when there is no accompanying neurological obtundation.     

In vitro propagation, proscillaridin A production and antibacterial activity in Drimia robusta

Drimia robusta is a threatened traditional medicinal plant extensively used in South Africa. Rapid in vitro mass propagation of the species was developed for commercial cultivation from leaf explants using various concentrations and combinations of plant growth regulators and organic elicitors. The highest number of regenerated shoots per explant (14.6 ± 0.54) was obtained on Murashige and Skoog (MS) medium supplemented with a combination of 2.27 μM thidiazuron (TDZ), 2.22 μM benzyladenine (BA) and 20 μM glutamine. Adventitious shoots were rooted and the plantlets were successfully acclimatized (100 %) in a vermiculite-soil mixture (1:1 v/v) in the greenhouse. Proscillaridin A (PsA) content and the antibacterial activity of in vitro and ex vitro regenerated plants were evaluated in different tissues in comparison to naturally-grown plants. The highest content of PsA (19.68 μg mg^?1 DW) was recorded in roots of ex vitro plants which were grown on MS medium containing 2.27 μM TDZ, 2.22 μM BA and 100 mg l^?1 casein hydrolysate. In vitro regenerated plants grown on MS medium containing 2.27 μM TDZ, 2.22 μM BA and 50.8 μM MBZ gave high antibacterial activity (MIC of 0.156 mg ml^?1) against both Gram-positive and Gram-negative bacteria. Using this protocol the regenerated plants can be used in traditional medicine as an alternative to naturally collected plants.     

Hypoxanthine Effect on Equilibrative and Concentrative Adenosine Transport in Human Lymphocytes. Implications in the Phatogenesis of Lesch-Nyhan Syndrome

We postulated that increased levels of hypoxanthine, a main characteristic of hypoxanthine phosphoribosyltransferase (HPRT) deficiency, may influence adenosine function which could be related to some of the neurological features of the Lesch-Nyhan syndrome. We have examined the effect of hypoxanthine on different adenosine transporters in peripheral blood lymphocytes from control subjects. Increased hypoxanthine concentrations (25 μM) significantly decreased adenosine transport. The equilibrative adenosine transporters (79.6% of the adenosine transport), both NBTI sensitive and NBTI insensitive, were affected significantly. In contrast, the concentrative adenosine transporters were not influenced by hypoxanthine. These results supports the hypothesis that increased hypoxanthine levels influence equilibrative (predominantly NBTI-insensitive type) adenosine transporters.     

In vitro shoot growth, direct organogenesis and somatic embryogenesis promoted by silver nitrate in Coffea dewevrei

Direct differentiation of shoot buds in Coffea dewevrei was evident from the seedling shoots with collar region and also from collar region end of hypocotyl segments in presence of 40 μM AgNO₃, 8.88 μM of BA and 2.85 μM of IAA. Apart from this, shoot end of hypocotyl explants mainly supported yellow friable callus or somatic embryos. Subsequent transfer to the same medium induced secondary somatic embryogenesis. The collar region of the hypocotyl explants not only showed direct organogenesis by producing 1–3 shoots per explant and also able to produce globular somatic embryos and embryogenic yellow friable callus. Similarly direct somatic embryogenesis along with yellow friable embryogenic callus formation on 1/2 strength MS medium comprising 1.47 μM IAA, 2.22 μM BA and 40 μM AgNO₃ was noticed from cut portion of in vitro leaf and stalk of regenerated plants. The microshoots rooted well upon subculturing onto the same medium in 6 weeks and showed 60 % survival in green house and resumed growth upon hardening.     

In vitro organogenesis from internode derived callus cultures of Capsicum annuum L.

An efficient protocol of shoot organogenesis and plant regeneration from internode derived callus has been developed for Capsicum annuum. Optimal callus was developed from internodal segments on Murashige and Skoog (MS) medium supplemented with 10 μM 2,4-dichlorophenoxy acetic acid (2,4-D) and 2.0 μM 6-benzyladenine (BA). Shoot differentiation was achieved from the surface of callus when transferred on shoot induction medium containing BA and thidiazuron (TDZ) alone or in combination. The highest number of de novo adventitious shoots (25.4?±?1.42) and shoot length (4.6?±?0.37 cm) was recorded on MS medium supplemented with 5.0 μM BA and 2.5 μM TDZ. The individual elongated shoots were rooted well on MS medium supplemented with 1.0 μM Indole-3-butyric acid (IBA). The in vitro raised plantlets with properly developed shoot and roots were acclimatized successfully and grew well in the greenhouse. All the regenerated plants appeared normal with respect to morphology and growth characteristics with 85% survival rate.     

Variation in protein levels obtained from human blood cells and biofluids for platelet, peripheral blood mononuclear cell, plasma, urine and saliva proteomics

Blood cells and biofluid proteomics are emerging as a valuable tool to assess effects of interventions on health and disease. This study is aimed to assess the amount and variability of proteins from platelets, peripheral blood mononuclear cells (PBMC), plasma, urine and saliva from ten healthy volunteers for proteomics analysis, and whether protein yield is affected by prolonged fasting. Volunteers provided blood, saliva and morning urine samples once a week for 4 weeks after an overnight fast. Volunteers were fasted for a further 24 h after the fourth sampling before providing their final samples. Each 10 mL whole blood provided 400–1,500 μg protein from platelets, and 100–600 μg from PBMC. 30 μL plasma depleted of albumin and IgG provided 350–650 μg protein. A sample of morning urine provided 0.9–8.6 mg protein/dL, and a sample of saliva provided 70–950 μg protein/mL. None of these yields were influenced by the degree of fasting (overnight or 36 h). In conclusion, in contrast to the yields from plasma, platelets and PBMC, the protein yields of urine and saliva samples were highly variable within and between subjects. Certain disease conditions may cause higher or lower PBMC counts and thus protein yields, or increased urinary protein levels.     

The control of hyperhomocysteinemia through thiol exchange mechanisms by mesna

In hyperhomocysteinemic patients, after reaction with homocysteine-albumin mixed disulfides (HSS-ALB), mesna (MSH) forms the mixed disulfide with Hcy (HSSM) which can be removed by renal clearance, thus reducing the plasma concentration of total homocysteine (tHcy). In order to assess the HSS-ALB dethiolation via thiol exchange reactions, the distribution of redox species of cysteine, cysteinylglycine, homocysteine and glutathione was investigated in the plasma of healthy subjects: (i) in vitro, after addition of 35 μM reduced homocysteine (HSH) to plasma for 72 h, followed by MSH addition (at the concentration range 10–600 μM) for 25 min; (ii) in vivo, after oral treatment with methionine (methionine, 200 mg/kg body weight, observation time 2–6 h). In both experiments the distribution of redox species, but not the total amount of each thiol, was modified by thiol exchange reactions of albumin and cystine, with changes thermodynamically related to the pKa values of thiols in the corresponding mixed disulfides. MSH provoked a dose–response reversal of the redox state of aged plasma, and the thiol action was confirmed by in vivo experiments. Since it was observed that the dimesna production could be detrimental for the in vivo optimization of HSSM formation, we assume that the best plasma tHcy lowering can be obtained at MSH doses producing the minimum dimesna concentration in each individual.     

In vitro plantlet regeneration and Agrobacterium tumefaciens-mediated genetic transformation of Indian Kino tree (Pterocarpus marsupium Roxb.)

The objective of the present study was to develop a protocol for in vitro plantlet regeneration and Agrobacterium tumefaciens-mediated genetic transformation using immature cotyledon explants of Indian Kino tree (Pterocarpus marsupium Roxb.). Immature cotyledon explants excised from 9-day-old axenic seedlings produced optimal callus on Murashige and Skoog (MS) medium supplemented with 1.07 μM α-naphthalene acetic acid (NAA), after 2 weeks of culture. When the above said callus was incubated on MS + 8.90 μM 6-benzylaminopurine (BAP) + 1.07 μM NAA, a regeneration frequency of 60.41 % with shoot number and length 12.2 ± 0.85 and 1.4 ± 0.13, respectively, was observed. For further shoot multiplication and elongation, these cultures were transferred onto MS + 4.40 μM BAP. Elongated shoots dipped in 19.60 μM indole-3-butyric acid (IBA) for 24 h and then cultured on ½MS + 2.85 μM IBA, 75 % shoots developed roots and 95 % of plantlets survived in field condition. Organogenic callus was co-cultivated with the A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301with ß-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes and grown on MS + 8.90 μM BAP + 1.07 μM NAA (RM) + 200 μM acetosyringone for 2 days and then transferred to MS + 8.90 μM BAP + 1.07 μM NAA + 20 mg/l hygromycin + 250 mg/l cefotaxime (SIM) and 4.40 μM BAP + 15 mg/l hygromycin + 200 mg/l cefotaxime (SEM). The putatively transformed shoots were subsequently rooted on ½MS + 2.85 μM IBA + 20 mg/l hygromycin (SRM), after pulse treatment for 24 h with 19.60 μM IBA. Successful gene transfer into putatively transformed plantlets was confirmed by histochemical GUS assay, PCR and RT-PCR analysis. Southern blot analysis of regenerated plantlets confirmed the integration of hpt gene in transgenic plantlets. In the present study, a rate of 20.92 % transformation frequency was achieved and the genetic transformation protocol presented here may pave way for genetic manipulation of this multipurpose legume tree.     

In vitro morphogenesis in Selaginella microphylla (Kunth.) Spring

Selaginella, an extant genus of primitive vascular plants, has survived over 400 million years of evolution. In vitro morphogenesis in Selaginella microphylla is considered for the first time to establish a well-documented aseptic culture on half- strength Murashige and Skoog’s basal medium with 2ip (4.92–49.21 μM), or Kn (4.65–46.47 μM) or GA₃ (2.89–28.90 μM) for shoot multiplication, and with different concentrations of IBA (4.9–49 μm) to initiate root cultures. GA₃ was instrumental for shoot multiplication as well as induction of reproductive structures in each and every leaf axil. On the other hand, it is observed that IBA alone in S. microphylla can act as signal molecules for induction of enormous numbers of root masses from a few existing roots. An interesting pattern of re-differentiation has also been observed where apical portions of large numbers of roots were converted to green shoot apical meristems. Further differentiation produced tiny green shoots. Distinct bipolarity was noted in shoots when they were isolated from root masses and appeared as embryo-like structures. Chromosome analysis from in vitro sporophytic plants revealed 2n = 16 chromosomes, indicating chromosomal stability. The interesting in vitro pattern of morphogenesis obtained in S. microphylla may provide new insights into totipotency of plants.     

In vitro propagation of <Emphasis Type="Italic">Psoralea corylifolia</Emphasis> L. by somatic embryogenesis in cell suspension culture

An effective protocol was developed for in vitro propagation of Psoralea corylifolia via somatic embryogenesis in cell suspension culture. Embryogenic callus was obtained on Murashige and Skoog (MS) medium supplemented with 6 μM naphthaleneacetic acid (NAA) and 30 μM glutamine from transverse TCLs from 10-day-old hypocotyl explants with a 96.4% frequency. Embryogenic callus produced a higher number of somatic embryos (123.7 ± 1.24 per gram fresh weight callus) on MS medium containing 30 g l^?1 sucrose, 1 μM NAA, 4 μM benzyladenine (BA), 15 μM glutamine and 2 μM abscisic acid (ABA) after 4 weeks of culture. Somatic embryos successfully germinated (97.6%) on ½ MS medium containing 20 g l^?1 sucrose, 8 g l^?1 agar and supplemented with 2 μM BA, 1 μM ABA and 2 μM gibberellic acid (GA₃) within 2 weeks of culture. Somatic embryos developed into normal plants, which hardened with 100% efficiency in soil in a growth chamber. Plants were successfully transferred to greenhouse and subsequently established in the field. Plant survival percentage in the field differed with seasonal variations. Average psoralen content of 12.9 μg g^?1 DW was measured in different stages of somatic embryo development by high-performance liquid chromatography (HPLC). This protocol will be helpful for efficient propagation of elite clones on a mass scale, conservation efforts of this species and for secondary metabolites production studies.     

Efficient plant regeneration from petiole explants of West Indian gherkin (Cucumis anguria L.) via indirect organogenesis

An efficient protocol for in vitro organogenesis was achieved from callus-derived immature and mature petiole explants of West Indian gherkin (Cucumis anguria L.). Calluses were induced from immature petiole explants excised on 7-day-old in vitro seedlings and mature petiole explants of 40-day-old in vivo plants. The maximum frequency of immature petiole explants (98.0 %) and mature petiole (91.5 %) produced green, compact organogenic callus in Murashige and Skoog (MS) medium with Gamborg (B5) vitamins containing 30 g l^?1 sucrose, 8.0 g l^?1 agar and 4.0 μM naphthalene acetic acid (NAA) with 2.0 μM benzyl amino purine (BAP) after two successive subculture at 11 days interval. Adventitious shoots were produced from the organogenic callus when it was transferred to MSB₅ medium supplemented with 3.0 μM TDZ, 1.0 μM NAA and 0.05 mM L-glutamine with shoot induction frequency of immature petiole 45 shoots and mature petiole 40 shoots per explant. The shoots were excised from callus and elongated in MSB₅ medium fortified with 3.0 μM gibberellic acid (GA₃). Then elongated shoots were rooted in half strength MSB₅ medium supplemented with 3.0 μM indole 3-butyric acid (IBA). Histological analyses of the regeneration process confirmed the indirect organogenesis pattern. Plantlets with well-developed shoot and root systems were successfully acclimatized (95 %) in winter season and exhibited normal morphology and growth characteristics. The survival percentage differed with seasonal variations.     

Characterization of colchicine binding with normal and glycated albumin: In vitro and molecular docking analysis

The transport of more than 90% of the drugs viz. anticoagulants, analgesics, and general anesthetics in the blood takes place by albumin. Hence, albumin is the prime protein needs to be investigated to find out the nature of drug binding. Serum albumin molecules are prone to glycation at elevated blood glucose levels as observed in diabetics. In this piece of work, glycation of bovine serum albumin (BSA) was carried out with glyceraldehyde and characterized by molecular docking and fluorometry techniques. Glycation of BSA showed 25% loss of free amino groups and decreased protein fluorescence (60%) with blue shift of 6 nm. The present study was also designed to evaluate the binding of colchicine (an anti-inflammatory drug) to native and glycated BSA and its ability to displace 8-analino-1-nephthalene sulfonic acid (ANS), from the BSA–ANS complex. Binding of ANS to BSA showed strong binding (K_a = 4.4 μM) with native conformation in comparison to glycated state (K_a = 8.4 μM). On the other hand, colchicine was able to quench the fluorescence of native BSA better than glycated BSA and also showed weaker affinity (K_a = 23 μM) for glycated albumin compared with native state (K_a = 16 μM). Molecular docking study showed that both glyceraldehyde and colchicine bind to common residues located near Sudlow’s site I that explain the lower binding of colchicine in the glycated BSA. Based on our results, we believe that reduced drugs-binding affinity to glycated albumin may lead to drugs accumulation and precipitation in diabetic patients.     

In vitro organogenesis from leaf and transverse thin cell layer derived callus cultures of Talinum triangulare (Jacq.) Willd.

Talinum triangulare is an important medicinal herb used traditionally in the treatment of various diseases. The present study was intended to develop a rapid and efficient protocol for indirect organogenesis from leaf discs and transverse thin cell layer (tTCL) of internodal explants of T. triangulare. Best callusing response (100 %) was observed with tTCL explants on Murashige and Skoog (MS) medium supplemented with 4.44 μM 6-benzyl amino purine and 5.37 μM α-naphthalene acetic acid (NAA). High frequency shoot regeneration (96.67 %) was obtained from tTCL derived calli on MS medium supplemented with a combination of 0.45 μM thidiazuron and 0.27 μM NAA, by producing 9.20 ± 0.35 shoots with a shoot length of 2.74 ± 0.03 cm. In vitro rooting of the microshoots was recorded on half-strength MS medium containing 2.46 μM indole-3-butyric acid by eliciting 15.20 ± 0.27 roots with a length of 4.25 ± 0.11 cm. The rooted shoots were acclimatized on garden soil, sand and coco pith (1:1:3 v/v) planting substrate. The plantlets were successfully established under field conditions with 100 % survival rate. The hardened plants exhibited homogeneity and no observable morphological variations were detected among the regenerants and the mother plants of T. triangulare.     

Plant regeneration from different explant types of Bituminaria bituminosa and furanocoumarin content along plant regeneration stages

The first protocol for in vitro plant regeneration from different explants of Bituminaria bituminosa, a pasture and medicinal species, has been established. Three explant types (petiole, leaflet and petiole-leaflet attachment “PLA”) cultured on media with different combinations of benzylaminopurine (BA; 5.0, 10.0 or 20.0 μM) and naphthalene acetic acid (NAA) or indole acetic acid (IAA; 0.5 or 5.0 μM) were tested for calli induction, and with 5 μM BA + 0.5 μM NAA or IAA for shoot development. The average number of shoots (≥5 mm) per callus depended on the explant type and the calli induction medium. The highest average number of shoots per callus was achieved by culturing leaflet and PLA explants on 5 μM IAA + 10 μM BA for calli induction and on 0.5 μM IAA + 5 μM BA for shoot development, and by culturing petiole explants on 0.5 μM NAA + 10 μM BA followed by a second culture on 0.5 μM NAA + 5 μM BA. The highest frequency of shoot rooting was achieved with 10.0 μM NAA and 1.0 μM gibberellic acid (GA₃). Rooted plants were acclimatised in a culture chamber, reaching 96 % survival. Acclimatised plants were transferred to a greenhouse and finally to the field, reaching 100 % survival. The furanocoumarin (FC) accumulation was evaluated in organogenic calli, in vitro shoots, ex vitro plants in the greenhouse and in ex vitro plants in the field (after 1 and 4 months of acclimatisation). The content of FCs depended on the plant material evaluated, being higher in ex vitro plants in the field (up to 9,824 μg g^?1 DW total FC) and lowest in organogenic calli (up to 50 μg g^?1 DW total FC). This effect may be due to cell organization, longer exposure to environmental factors and the developmental stage.     

Novel 3-fluoro-6-methoxyquinoline derivatives as inhibitors of bacterial DNA gyrase and topoisomerase IV

Novel (non-fluoroquinolone) inhibitors of bacterial type II topoisomerases (NBTIs) are an emerging class of antibacterial agents. We report an optimized series of cyclobutylaryl-substituted NBTIs. Compound 14 demonstrated excellent activity both in vitro (S. aureus MIC₉₀ = 0.125 μg/mL) and in vivo (systemic and tissue infections). Enhanced inhibition of Topoisomerase IV correlated with improved activity in S. aureus strains with mutations conferring resistance to NBTIs. Compound 14 also displayed an improved hERG IC₅₀ of 85.9 μM and a favorable profile in the anesthetized guinea pig model.     

Insight into the Anti-Inflammatory Mechanism of Action of Atrial Natriuretic Peptide,a Heart Derived Peptide Hormone: Involvement of COX-2, MMPs,and NF-kB Pathways

We sought to determine the in vivo anti-inflammatory activity of atrial natriuretic peptide (ANP) using 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced acute and chronic skin inflammatory mice model. ANP treatment (2 μg/kg body weight/day/i.p. for acute and 0.5 μg/kg body weight/day/i.p. for chronic inflammatory study) was started after 30 min of TPA application. The standard drug, aspirin (ASP) (20 μg/kg body weight/day/i.p.; 10 μg/kg body weight/day/i.p., respectively) was used as a positive control for the both acute and chronic study. TPA alone treated mice exhibited a marked increase in the ear length (7 ± 0.08 vs. 13 ± 0.7 mm, p < 0.001) as well as in ear weight (80 ± 1.3 vs. 130 ± 1.5 mg, p < 0.001) as compared with control mice. Upon treatment with ANP, the increased ear length and weight were reverted back to near normal level. Similarly, ANP treatment markedly suppressed the TPA-induced chronic skin inflammatory lesion (5.7 ± 0.2 vs. 0.95 ± 0.05, p < 0.001) as compared with TPA-induced mouse skin. TPA-induced alterations in the levels of serum C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), total white blood cell (TWBC), serum tumor necrosis factor-α (TNF-α), natriuretic peptide receptor-A (NPR-A), cyclooxygenase-2 (COX-2), matrix metalloproteinase-2/-9 (MMP-2/-9) and nuclear factor kappa B (NF-κB) (p < 0.01, respectively) were reverted back to near normal levels. The results of the present study clearly show the in vivo anti-inflammatory activity of ANP, which is comparable with that of a standard drug, ASP. Our results suggest that ANP elicits its anti-inflammatory activity by down-regulating the expressions of NPR-A, COX-2, MMPs and NF-κB.     

In vitro propagation of <Emphasis Type="Italic">Cymbidium goeringii</Emphasis> Reichenbach fil. through direct adventitious shoot regeneration

The influence of 2,4-dichlorophenoxyacetic acid (2,4-D), benzyladenine (BA), and thidiazuron (TDZ) on direct rhizome induction and shoot formation from rhizome explants of Cymbidium goeringii was explored. Rhizome segments obtained from in vitro seed cultures of C. goeringii were placed on Murashige and Skoog (MS) medium incorporated with 5, 10, 20, or 40 µM 2,4-D and 1, 2, 4, or 8 µM BA or TDZ alone or in combination with 20 µM 2,4-D. The explants developed only rhizomes on MS medium with or without 2,4-D. The highest percent of rhizome formation (100%) was obtained on MS medium incorporated with 20 μM of 2,4-D. The morphology and number of rhizomes varied with the level of 2,4-D in the medium. Direct adventitious shoot formation was achieved on medium incorporated with BA or TDZ. The adventitious shoots produced per explant significantly increased with the supplementation of 2,4-D to cytokinin-containing medium. The highest mean of 21.8 ± 1.8 shoot buds per rhizome segment was obtained in medium fortified with 20 μM 2,4-D and 2 μM TDZ. The greatest percent of root induction (100%) and the mean of 5.3 ± 1.1 roots per shoot were achieved on ½ MS medium incorporated with 2 μM of α-naphthaleneacetic acid. About 97% of the in vitro-produced plantlets acclimatized in the greenhouse. An efficient in vitro propagation protocol was thus developed for C. goeringii using rhizome explants.     

Clonidine Suppresses the Induction of Long-Term Potentiation by Inhibiting HCN Channels at the Schaffer Collateral–CA1 Synapse in Anesthetized Adult Rats

Activation of alpha2-adrenoceptors inhibits long-term potentiation and long-term depression in many brain regions. However, effectiveness and mechanism of alpha2-adrenoceptors for synaptic plasticity at the Schaffer collateral–CA1 synapses in rat in vivo is unclear. In the present study, we investigated the effects of alpha2-adrenoceptors agonist clonidine on high-frequency stimulation (HFS)-induced long-term potentiation (LTP) and paired-pulse facilitation (PPF) at the Schaffer collateral–CA1 synapse of rat hippocampus in vivo. Clonidine (0.05, 0.1 mg/kg, ip) inhibited synaptic plasticity in a dose-dependent manner, accompanying with the decreasing of aortic pressure and heart rate (HR) in anesthetized rats. Clonidine (1.25, 2.5 μg/kg, icv, 10 min before HFS) also dose-dependently inhibited synaptic plasticity, which had no remarkable effect on HR and aortic pressure. But, 20 min after HFS, administration of clonidine (2.5 μg/kg) had no effect on LTP. The inhibitory effect of clonidine (2.5 μg/kg) on LTP was completely reversed by yohimbine (18 μg/kg, icv) and ZD7288 (5 μg/kg, icv). Moreover, the inhibition was accompanied by a significant increase of the normalized PPF ratio. Furthermore, clonidine at 1 and 10 μM significantly decreased glutamate (Glu) content in the culture supernatants of hippocampal neurons, and yohimbine at 1 and 10 μM had no effect on Glu release, while it could reverse the inhibition of clonidine (1 and 10 μM) on Glu release. In conclusion, clonidine can suppress the induction of LTP at the Schaffer collateral–CA1 synapse, and the possible mechanism is that activation of presynaptic alpha2-adrenoceptors reduces the Glu release by inhibiting HCN channels.