Growth and Oleanolic Acid Accumulation of Polyscias fruticosa Cell Suspension Cultures.

BACKGROUND: Oleanolic acid is an oleanane triterpene found in many plant species all over the world. This compound is also a major saponin in leaves of Polyscias fruticosa and possesses several promising pharmacological activities, such as hepatoprotective effects, and antiinflammatory, antioxidant, or anticancer activities. OBJECTIVE: The objective of the present work is to establish cell suspension culture of P. fruticosa, investigate the influence of several factors such as plant growth regulators and carbon source on cell growth, and determine their oleanolic acid content. METHODS: Cell culture was established by using 2 g fresh weight of 30 day old friable callus derived from in vitro stem segment in 50 mL of liquid medium with a shaking speed of 220 rpm. The culture was then incubated at 25±2ºC with a shaking speed of 120 rpm in the period of 12 h daylight at a light intensity of about 6.75 µmol/m2/s. Cell growth was measured by fresh and dry biomass at 16 h day. Oleanolic acid content was determined using HPLC analysis. RESULTS & DISCUSSION: The study results showed that MS medium containing 2% sucrose as a carbon source, supplemented with 1 mg/L 6-benzylaminopurine and 0.5 mg/L 2,4-dichlorophenoxyacetic acid was the most appropriate growth medium. Cell biomass and oleanolic acid content reached the highest values of 0.43 g dry weight/flask and 25.4 mg/g dry weight, respectively. CONCLUSION: These results indicated the potential production of oleanolic acid, a compound with high pharmacological value, from P. fruticosa cell culture.

Expression of Escherichia coli Heat-Labile Enterotoxin B Subunit in Centella (Centella asiatica (L.) Urban) via Biolistic Transformation.

BACKGROUND: Heat-Labile enterotoxin B subunit (LTB) produced by Escherichia coli, a non-toxic protein subunit with potential biological properties, is a powerful mucosal and parenteral adjuvant which can induce a strong immune response against co-administered antigens. OBJECTIVE: In the present study, LTB protein, encoded by the optimized ltb (also known synthetic ltb, s-ltb) gene in centella plant (Centella asiatica) for use as an antigen, has been discussed. METHODS: The s-ltb gene was cloned into a plant expression vector, pMYO51, adjacent to the CaMV 35S promoter and was then introduced into centella plant by biolistic transformation. PCR amplification was conducted to determine the presence of s-ltb gene in the transgenic centella plant. The expression of s-ltb gene was analyzed by immunoblotting and quantified by ELISA. In vitro activity of LTB protein was determined by GM1-ELISA. RESULTS: PCR amplification has found seven transgenic centella individuals. However, only five of them produced LTB protein. ELISA analysis showed that the highest amount of LTB protein detected in transgenic centella leaves was about 0.8% of the total soluble protein. GM1-ELISA assay indicated that plant LTB protein bound specifically to GM1-ganglioside, suggesting that the LTB subunits formed active pentamers. CONCLUSION: The s-ltb gene that was successfully transformed into centella plants by the biolistic method has produced a relatively high amount of plant LTB protein in the pentameric quaternary structure that has GM1-ganglioside binding affinity, a receptor on the intestinal epithelial membrane.

Aza-capped cyclodextrins for intra-cavity metal complexation.

Aza-capped, methylated cyclodextrins (CDs) were obtained in high yields by reacting the soft nitrogen nucleophile 2-nitrobenzenesulfonamide with either A,B-dimesylated CDs in basic media or their diol analogues under Mitsunobu reaction conditions followed by deprotection with thiophenol. A methyl pyridine substituent was grafted on the N atom of these secondary amines. When built on an α-CD scaffold, the resulting tertiary amine no longer undergoes nitrogen inversion at room temperature and behaves as a confining ligand, opening the way to intra-cavity metal complexation and promoting the formation of supramolecular helices.

An action spectrum for ultraviolet radiation-induced immunosuppression in humans.

BACKGROUND: The immune-suppressive effects of sunlight play a central role in skin carcinogenesis. Ultraviolet (UV) B radiation is highly immunosuppressive even at suberythemal doses, and longwave UVA is now also recognized to cause immunosuppression in humans. The relative contributions of UVA and UVB to immunosuppression by incidental daily sun exposure are, however, unclear. OBJECTIVES: We previously determined wavelength dependencies for immunosuppression by UVB and UVA wavebands in humans. We now aimed to calculate relative and solar immune-suppressive effectiveness across the UVB and UVA spectra. METHODS: We used the nickel model of recall contact hypersensitivity to determine UV immunosuppression dose responses and minimum immune suppression doses (MISDs) at 11 narrowbands from 289 to 392 nm. The relative immune-suppressive effectiveness of each narrowband was then determined as 1/MISD vs. wavelength. This curve was multiplied by the solar spectrum to show the relative immune-suppressive effectiveness of each waveband in sunlight. RESULTS: We found peaks of immune-suppressive effectiveness in the UVB waveband at 300 nm and in the UVA at 370 nm. Because of the far greater amount of longwave UVA in sunlight, the relative solar immune-suppressive effectiveness of UVA was threefold higher than that of UVB at doses equivalent to sun exposure from normal daily activities. CONCLUSIONS: Longwave UVA, which abuts the visible light spectrum and is less effectively filtered by sunscreens than UVB, is likely to be the largest contributor to immunosuppression resulting from incidental daily sun exposure.

Incontinentia pigmenti case series: clinical spectrum of incontinentia pigmenti in 53 female patients and their relatives.

A retrospective case series of 53 female patients with incontinentia pigmenti (IP) including 28 secondary cases (female relatives of probands) was reviewed and compared with other series in an attempt to estimate more accurately the true disease burden of patients with IP. We found that, while the frequency of the first three cutaneous stages of IP was comparable with previous studies, none of the secondary cases manifested any serious neurological complications but all displayed stage IV pale anhidrotic reticulate lines on their posterior calves. This important clinical feature of IP in secondary cases has previously been under-represented in studies that often involved only paediatric probands. Hence, mildly affected cases of IP are often undiagnosed and under-represented in case series to date, possibly leading to inappropriately high estimates of neurological and eye involvement. With the availability of genetic testing, it is now feasible to confirm the variability of the phenotype and the risk of complications in IP.

Sudden permanent hearing loss following anterior inferior cerebellar artery infarction.

Anterior inferior cerebellar artery infarction with the only sequel being a permanent unilateral hearing loss is described. The damage was confirmed by magnetic resonance imaging. Hearing loss of vascular cause may be more common and permanent than realised, and missed if the other neurological deficits have resolved.

Autoradiographic evidence that intrastriatal administration of adenosine A(1) receptor antisense oligodeoxynucleotide decreases adenosine A(1) receptors in the rat striatum and cortex.

In this study, the effect of a phosphorothioated A(1) adenosine receptor antisense oligodeoxynucleotide on A(1) receptor density and mRNA in the striatum and cortex of rats was determined. Receptor autoradiography and in situ hybridization revealed a reduction in striatal and cortical A(1) receptor density and cortical A(1) receptor mRNA, respectively, in antisense-treated brains but not in those treated with a mismatch oligonucleotide. There was no change in A(2) receptor binding. These data imply that the corticostriatal pathway synthesizes A(1) receptors and transports them to its terminals.

Kappa-opioid receptor antisense oligonucleotide injected into rat hippocampus causes hypertension.

Bi-hippocampal microinjection treatment (1 microg per side, twice a day for 5 days) with an antisense phosphorothioate oligodeoxynucleotide antisense oligodeoxynucleotide to the rat kappa-opioid receptor, caused hypertension in normotensive Wistar Kyoto (WKY) rats and increased the blood pressure of spontaneously hypertensive rats (SHR). Systolic blood pressure in WKY rats increased from 121+/-4 to 153+/-6 mm Hg, and in SHR systolic blood pressure increased from 153+/-4 to 183+/-5 mm Hg. Similar results were observed with mean blood pressure, however, there were no changes in heart rate. No significant responses were seen with either vehicle or missense injections. Radioligand binding studies indicated that there was a significant decrease in apparent kappa-opioid receptor density due to antisense oligodeoxynucleotide treatment. The results are in accord with our earlier suggestions that the kappa-opioid system in the hippocampus may have a role in the neural control of blood pressure.

Intrastriatal adenosine A1 receptor antisense oligodeoxynucleotide blocks ethanol-induced motor incoordination.

Intrastriatal administration of a 21-mer phosphorothioate antisense oligodeoxynucleotide targeting the adenosine A1 receptor blocked ethanol-induced motor incoordination in the rat and reduced striatal adenosine A1 receptor content, as judged by specific binding of the A1-specific ligand 8-cyclopentyl-1,3-dipropylxanthine (Bmax = 0.350 +/- 0.07, Kd = 1.87 +/- 0.50 nM). No effect upon striatal adenosine A2 receptor content was observed (Bmax = 0.415 +/- 0.04, Kd = 13.13 +/- 1.25 nM) with the A2-specific ligand 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine. A mismatched control oligodeoxynucleotide of identical G-C base composition and general sequence structure was without effect on adenosine A1 receptor (Bmax = 0.666 +/- 0.11, Kd = 1.32 +/- 0.27 nM) or adenosine A2 receptor content (Bmax = 0.501 +/- 0.08; Kd = 14.65 +/- 1.82 nM) or ethanol-induced motor incoordination. These results confirm an important role of the striatal adenosine A1 receptor in mediating certain motor-related physiological effects of ethanol.

Characterization of the effects of a thymine glycol residue on the structure, dynamics, and stability of duplex DNA by NMR.

A duplex DNA containing a single thymine glycol (5,6-dihydroxy-5,6-dihydrothymidine) has been studied by NMR and other methods. Oxidative stress, ionizing radiation, and other causes can induce the oxidation of thymine to thymine glycol. The presence of thymine glycol is known to have significant biological consequences, and there are repair enzymes for thymine glycol in a wide range of organisms. These studies have been carried out on the DNA duplex of d(C1G2C3A4G5Tg6C7A8G9C10C11) paired with d(G22C21G20T19C18A17G16T15C14G13G12), with Tg indicating thymine glycol. The presence of thymine glycol lowers the thermal stability of duplex DNA. The NMR results indicate that thymine glycol induces a large, localized structural change in duplex DNA with the thymine glycol base being extrahelical as well as the opposing base on the complementary strand. This structural information is consistent with the biological consequences of thymine glycol in DNA.