Differential expression of microRNAs may regulate pollen development in Brassica oleracea.

MicroRNAs (miRNAs) are a class of non-coding endogenous negative regulators that regulate gene expression at both the transcriptional and post-transcriptional levels. However, little is known about the expression characteristics of miRNAs during pollen development in Brassica oleracea. In this study, five known and three novel miRNAs were identified and their expression patterns were compared in the flower buds of B. oleracea using stem-loop reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR. The results revealed that the eight miRNAs were constantly expressed during pollen development but exhibited different expression patterns during the five developmental stages of the flower buds between the cytoplasmic male sterile (CMS) line and its fertile maintainer. The highest miRNA expression levels occurred at the uninucleate microspore stage in the fertile line Bo01-12B and at the bicellular pollen stage in the CMS line Bo01-12A. Potential target genes for the miRNAs were predicted and analyzed, and suggested that miRNAs are involved in the regulation of target genes related to pollen development. The results of this study further our understanding of the regulatory role of miRNAs in pollen development.

Efficacy of type a botulinum toxin injections and infrared polarized light on treating chronic migraine.

OBJECTIVE: To investigate the clinical value of the combination of ultrasound-and-hyponome-guided type A botulinum toxin injection and infrared polarized light on treating chronic migraine. PATIENTS AND METHODS: Ninety-one patients with chronic migraine were randomly divided into four groups: in the control group (group A, 22 cases in total), nimodipine was used in the treatment of chronic migraine for two months; in the infrared polarized light therapy group (group B, 22 cases in total), infrared polarized light was adopted in the treatment of chronic migraine for 50-60d; in the botulinum toxin treatment group (group C, 24 cases in total), ultrasound-and-hyponome-guided type A botulinum toxin was injected into frontal, temporal, and occipital muscles in treating chronic migraine; in the joint treatment group (group D, 23 cases in total), ultrasound-and-hyponome-guided type A botulinum toxin injection in group C and infrared polarized light in group B were both used here in the treatment of chronic migraine. Infrared polarized light therapy lasted 50-60d and the time of study lasted six months. The survey would include the conditions of patients with chronic migraine three months before treatment and at one, three and six months after treatment. Patients were asked to fill the MIDAS (migraine disability assessment questionnaire) and were graded on the evaluation scale of life quality, so that the researchers would be able to compare attack frequency, duration of attack, attack severity, the use of painkillers and their recovery from chronic migraine, and then observe their adverse reactions. RESULTS: Eleven cases dropped out during the treatment, three cases in A group, two cases in group B, four cases in group C and two cases in group D. One, three and six months after treatment, the MIDAS scores in group A, B, C and D were significantly lower than before the treatment. Hence, the differences were statistically significant (p < 0.01). The scores in quality of life rating scale were significantly higher than pre-treatment scores, so the difference was statistically significant (p < 0.01). The MIDAS scores and quality of life rating scale scores in group D were compared with those in group A, B, and C respectively, and the differences were statistically significant (p < 0.05). Two patients were recorded with dizziness, and the dizziness disappeared after two weeks with no treatments at all. Forehead lines and crow's feet of 21 patients shallowed or disappeared in varying degrees after the injection. CONCLUSIONS: The combination of ultrasound-and-hyponome-guided type A botulinum toxin injection and infrared polarized light on treating chronic migraine demonstrated a significant clinical effect.

Rhythmic auditory stimulation with visual stimuli on motor and balance function of patients with Parkinson's disease.

OBJECTIVE: Discuss the effect of rhythmic auditory stimulation with visual stimuli on motor and balance function in patients with Parkinson's disease (PD). PATIENTS AND METHODS: One hundred and sixteen patients with PD participated in this study. The control group used a routine drug treatment for eight weeks. The comprehensive treatment group used conventional drug treatment with sound rhythm metronome released as the rhythmical auditory stimulation, in accordance with the ground fixed ribbon rhythmic visual stimulation walking training for eight weeks. After four and eight weeks, the two groups of subjects took the walking parameters test, and used the disease Parkinson score scale to assess the damaged degree of motor function of PD patients. The Berg Balance Scale was used to evaluate the balance function of the PD patients. A six minute walk test was used to evaluate the walking motor function of the patients. RESULTS: The comparison between the groups suggests that after treatment of rhythmic auditory stimulation with visual stimulation group, the step size increased, frequency decreased, pace increased, and PD score scale part II decreased. As well, the PD score scale part III reduced, the six minute walking distance increased, and the Berg Balance Scale score increased significantly. There were significant differences compared with the control group after the treatment (p < 0.01). Comparison of time points suggests that after rhythmic auditory stimulation with visual stimulation group trained for eight weeks, the step size increased, frequency decreased, pace increased, and PD score scale part II were reduced. As well the PD score scale part III reduced, six minute walking distance increased, Berg Balance Scale increased. There were significant differences compared with the parameters of training for four weeks (p < 0.01). CONCLUSIONS: Rhythmic auditory stimulation with visual stimulation can improve motor and balance function of patients with PD.

Silymarin efficacy against influenza A virus replication.

The anti-influenza A/PR/8/34 virus activity of silymarin was evaluated in MDCK cells and investigated the effect of silymarin on synthesis of viral mRNAs. Silymarin was investigated for its antiviral activity against influenza A/PR virus using a cytopathic effect (CPE) reduction method. Silymarin exhibited anti-influenza A/PR/8/34 virus activity of 98% with no cytotoxicity at the concentration of 100 µg/ml reducing the formation of a visible CPE. Oseltamivir also did show moderate antiviral activity of about 52% against influenza A/PR/8/34 virus at the concentration of 100 µg/ml. Furthermore, the mechanism of anti-influenza virus action in the inhibition of viral mRNA synthesis was analyzed by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), and the data indicated an inhibitory effect in late viral RNA synthesis compared with oseltamivir in the presence of 100 µg/ml of silymarin. Therefore, the potential of silymarin for use in treating influenza virus infection merits greater attention.

In vitro anti-enterovirus 71 activity of gallic acid from Woodfordia fruticosa flowers.

AIMS: The anti-enterovirus 71 (EV71) activity of six Nepalese plants' extracts and gallic acid (GA) isolated from Woodfordia fruticosa Kurz (family; Lythaceae) flowers were evaluated in Vero cells. METHODS AND RESULTS: The anti-EV71 activity of tested compounds was evaluated by a cytopathic effect reduction method. Our results demonstrated that flowers' extracts of W. fruticosa exerted strong anti-EV71 activity, with a 50% inhibitory concentration (IC(50)) of 1.2 microg ml(-1) and no cytotoxicity at a concentration of 100 microg ml(-1), and the derived therapeutic index (TI) was more than 83.33. Rivabirin showed no antiviral activity against EV71. Furthermore, GA isolated from W. fruticosa flowers exhibited a higher anti-EV71 activity than the extract of W. fruticosa flowers, with an IC(50) of 0.76 microg ml(-1) and no cytotoxicity at a concentration of 100 microg ml(-1), and the derived TI was 99.57. CONCLUSIONS: This study demonstrated that flower extracts of W. fruticosa possessed anti-EV71 activity and GA isolated from these flowers showed stronger anti-EV71 activity than that the extracts. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that the GA from W. fruticosa flowers may be used as a potential antiviral agent.

Antiviral activity of raoulic acid from Raoulia australis against Picornaviruses.

RNA viruses are a major source of respiratory diseases worldwide. The lack of effective therapeutical treatment underlines the importance of research for new antiviral compounds. Raoulic acid is a principal ingredient of the plant Raoulia australis Hook. F. Antiviral assay using cytopathic effect (CPE) reduction method showed that raoulic acid possessed strong antiviral activity against human rhinovirus 2 (HRV2) with a 50% inhibition concentration (IC(50)) value of less than 0.1mug/ml, human rhinovirus 3 (HRV3) with a IC(50) value of 0.19 microg/ml, coxsackie B3 (CB3) virus with IC(50) values of 0.33 microg/ml, coxsackie B4 (CB4) virus with IC(50) values of 0.40 microg/ml, and enterovirus 71 (EV71) virus with IC(50) values of less than 0.1 microg/ml. However, the compound did not possess antiviral activity against influenza A (Flu A/PR, Flu A/WS, H1N1) and B viruses at four concentrations ranging from 0.1 to 100 microg/ml.

On the relationship between speech- and nonspeech-evoked auditory brainstem responses.

Auditory brainstem response (ABR) reflects activation of the neural generators along the ascending auditory pathway when a sound is heard. In this study, we explored the relationship between brainstem encoding of click and speech signals in normal-learning children and in those with language-based learning problems. To that end, ABR was recorded from both types of stimuli. We found that the normal pattern of correlation between click- and speech-evoked ABRs was disrupted when speech-evoked ABRs were delayed. Thus, delayed responses to speech were not indicative of clinically abnormal responses to clicks. We conclude that these two responses reflect largely separate neural processes and that only processes involved in encoding complex signals such as speech are impaired in children with learning problems.

Enhanced level of n-3 fatty acid in membrane phospholipids induces lipid peroxidation in rats fed dietary docosahexaenoic acid oil.

The effect of dietary docosahexaenoic acid (DHA, 22:6n-3) oil with different lipid types on lipid peroxidation was studied in rats. Each group of male Sprague-Dawley rats was pair fed 15% (w/w) of either DHA-triglycerides (DHA-TG), DHA-ethyl esters (DHA-EE) or DHA-phospholipids (DHA-PL) for up to 3 weeks. The palm oil (supplemented with 20% soybean oil) diet without DHA was fed as the control. Dietary DHA oils lowered plasma triglyceride concentrations in rats fed DHA-TG (by 30%), DHA-EE (by 45%) and DHA-PL (by 27%), compared to control. The incorporation of dietary DHA into plasma and liver phospholipids was more pronounced in the DHA-TG and DHA-EE group than in the DHA-PL group. However, DHA oil intake negatively influenced lipid peroxidation in both plasma and liver. Phospholipid peroxidation in plasma and liver was significantly higher than control in rats fed DHA-TG or DHA-EE, but not DHA-PL. These results are consistent with increased thiobarbituric acid reactive substances (TBARS) and decreased alpha-tocopherol levels in plasma and liver. In addition, liver microsomes from rats of each group were exposed to a mixture of chelated iron (Fe(3+)/ADP) and NADPH to determine the rate of peroxidative damage. During NADPH-dependent peroxidation of microsomes, the accumulation of phospholipid hydroperoxides, as well as TBARS, were elevated and alpha-tocopherol levels were significantly exhausted in DHA-TG and DHA-EE groups. During microsomal lipid peroxidation, there was a greater loss of n-3 fatty acids (mainly DHA) than of n-6 fatty acids, including arachidonic acid (20:4n-6). These results indicate that polyunsaturation of n-3 fatty acids is the most important target for lipid peroxidation. This suggests that the ingestion of large amounts of DHA oil enhances lipid peroxidation in the target membranes where greater amounts of n-3 fatty acids are incorporated, thereby increasing the peroxidizability and possibly accelerating the atherosclerotic process.

Dynamic readjustment of parental methylation patterns of the 5'-flank of the mouse H19 gene during in vitro organogenesis.

Gametic marks are stably propagated in order to manifest parent of origin-specific expression patterns of imprinted genes in the developing conceptus. Although the character of the imprint has not yet been fully elucidated, there is compelling evidence that it involves a methylation mark. This is exemplified by a region upstream of the H19 gene, which is not only methylated in a parent of origin-specific manner, but also regulates the silencing of the maternal Igf2 and paternal H19 alleles, respectively. We show here that the parental-specific methylation patterns within the differentially methylated domain (DMD) are perturbed in the soma during in vitro organogenesis. Under these conditions, the paternal DMD allele becomes partially demethylated, whereas the maternal DMD allele gains methylation. Despite these effects, there were no changes in allelic Igf2 or H19 expression patterns in the embryo. Finally, we show that although TSA derepresses the paternal H19 allele in ectoplacental cone when in vitro developed, there is no discernible effect on the methylation status of the paternally inherited 5'-flank in comparison to control samples. Collectively, this data demonstrates that the parental mark is sensitive to a subset of environmental cues and that a certain degree of plasticity of the gametic mark is tolerated without affecting the manifestation of the imprinted state.

Cloning of a sesquiterpene cyclase and its functional expression by domain swapping strategy.

Sesquiterpene cyclase, the first committed step enzyme from the general isoprenoid building block farnesyl pyrophosphate (FPP) for the synthesis of phytoalexin capsidiol, was isolated from the UV-C treated leaves of Capsicum annuum. This sesquiterpene cyclase, termed as CASC2 showing 77% amino acid identity with the previously cloned sesquiterpene cyclase CASC1, was composed of 560 amino acids with a calculated molecular mass of 64,907. The mRNA expression pattern of CASC2 was very similar to that of CASC1 during the time course of UV-C irradiated leaves of pepper on RNA blot analysis by using each specific probe. The heterologous expression in Escherichia coli using the CASC2 full length failed; however the chimeric construct of CASC2 in which the amino terminal 164 amino acid substituted by the equivalent portion of either CASC1 or tobacco sesquiterpene cyclase was capable of expressing the functional sesquiterpene cyclase activities. The radio-labeled enzymatic products catalyzed by the partially purified chimeric CASC2 were comigrated with authentic radio-labeled sesquiterpene on thin layer chromatography.

Cloning, developmental expression, and immunohistochemistry of cyclooxygenase 2 in the endometrium during embryo implantation and gestation in the mink (Mustela vison).

Cyclooxygenase (COX) is the first rate-limiting enzyme in the biosynthesis of PGs. There are two isoforms, COX-1, a constitutive enzyme and COX-2, the induced form, products of two different genes. In this study, we report COX-2 complementary DNA cloning, uterine expression, and immunohistochemical localization in the mink uterus during postimplantation gestation. The open reading frame of mink COX-2 contains 1812 nucleotides encoding 604 amino acids. The homologies are 86%, 83%, 83%, 83%, 85%, and 84% in nucleotides and 86%, 87%, 87%, 85%, 86%, and 88% in amino acids with human, mouse, rat, guinea pig, sheep, and rabbit, respectively. All domains associated with biological activity are conserved in the mink. Northern analysis revealed a transcript of 4.2 kb for COX-2 in mink uterus and adrenal. Semiquantitative RT-PCR showed that COX-2 messenger RNA is not present during diapause. The abundance of COX-2 messenger RNA reached its maxima (P < 0.05) on days 3-5 of postimplantation, gradually decreased through day 9, and was not present thereafter. By immunohistochemistry, COX-2 was present in uterine epithelium, stroma, and necks of endometrial glands at sites of implantation. COX-2 expression appears to be induced in the endometrium by the embryo and may play a role in implantation and placentation in the mink.

Oxidative stability of docosahexaenoic acid-containing oils in the form of phospholipids, triacylglycerols, and ethyl esters.

The peroxidative stability of docosahexaenoic acid (DHA)-containing oils (DHA at 10.7 mol% of the total fatty acids), in the form of phospholipids (PL), triacylglycerols (TG), and ethyl esters (EE) with the same constituent fatty acids, was investigated in the dark at 25 degrees C in a bulk phase, and compared with that of control palm oil (supplemented with 20% soybean oil). The oxygen absorption of the DHA-containing oil was significantly lower in the form of PL than in the form of TG and EE during a 10-week oxidation, and the oxygen absorption of PL was almost equivalent to that of the control oil. A gas chromatographic analysis showed that 90% of initial DHA was retained in the form of PL after the 10-week oxidation, while TG and EE respectively more rapidly decayed with the loss of 97% and 64% of DHA. Tocopherol in the form of TG and EE had also completely decayed after the oxidation, while 37% of the initial tocopherol remained in the form of PL. The peroxide and carbonyl values of TG and EE showed large increases after the oxidation, but no such increase was observed for PL. These results show that DHA-containing oil in the form of PL was more resistant to the oxidative degradation of DHA than that in the form of TG and EE in a bulk phase.