Expression of messenger RNAs for insulin-like growth factors and their receptors in bovine fetuses at early gestation from embryos produced in vivo or in vitro

The objective of this study was to determine the effects of in vitro embryo production on physical development and levels of expression of mRNAs for insulin-like growth factor (IGF) ligands (IGF1, IGF2), their receptors (IGF1R, IGF2R), and IGF binding protein-2 (IGFBP2) in bovine fetuses during early gestation. In vivo embryos were recovered from superovulated Holstein cows. For production of embryos in vitro, Holstein oocytes were matured, fertilized, and subsequently cultured in M199 with 10% serum to 168 hpi. On Day 70 of gestation, fetuses (in vivo, n = 14; in vitro, n = 13) were recovered, serum samples collected, and physical measurements recorded. Semi-quantitative RT-PCR assays were used to determine the levels of expression of mRNAs for IGF1, IGF2, IGF1R, and IGF2R in fetal liver and skeletal muscle. Western blots were used to assess levels of IGFBP2 in fetal serum. Fetal body weight did not differ with treatment; however, production of embryos in vitro was associated with decreased crown-nose length and a tendency for increased paired kidney weight, which became significant when expressed on a per bodyweight basis. There was no effect of treatment on levels of IGFBP2 in fetal serum. Levels of IGF1 mRNA in fetal liver were decreased (P < 0.001) in the in vitro group. Levels of IGF2R mRNA in both liver and skeletal muscle were also decreased (P < 0.01) in fetuses from the in vitro group. In summary, fetuses at Day 70 of gestation from embryos produced in vitro had shortened crown-nose length and increased kidney weight on a per bodyweight basis, as well as decreased expression of mRNAs for IGF1 in liver and IGF2R in both liver and skeletal muscle, compared with fetuses from embryos produced in vivo. In conclusion, in vitro embryo culture was associated with subtle changes in fetal development as well as altered expression of both imprinted and non-imprinted genes.     

Validating the in vitro antimicrobial activity of Artemisia afra in polyherbal combinations to treat respiratory infections

Artemisia afra is one of the most widely used medicinal plants in African traditional medicine and is commonly administered in polyherbal combinations to treat respiratory infections. Focussing on plant volatiles, the aim of this study was to provide scientific evidence for the antimicrobial activity of A. afra (principle plant) in combination with essential oils from three medicinal aromatic plants; Agathosma betulina, Eucalyptus globulus and Osmitopsis asteriscoides. In vitro minimum inhibitory concentration (MIC) assays were undertaken on four pathogens (Enterococcus faecalis ATCC 29212, Moraxella catarrhalis ATCC 23246, Klebsiella pneumoniae NCTC 9633 and Cryptococcus neoformans ATCC 90112) to determine antimicrobial efficacy of the oils and their combinations. The fractional inhibitory concentration (FIC) and isobolograms were used to interpret pharmacodynamic interactions such as synergy, antagonism or additive profiles. The antimicrobial activity of the individual oils mostly displayed moderate activity. Predominantly, additive interactions were noted. The most prominent synergistic interaction (FIC value of 0.5) was observed when A. afra was combined with O. asteriscoides in the 8:2 ratio (eight parts A. afra with two parts O. asteriscoides) against C. neoformans. No antagonistic interactions were evident.     

In vitro and in vivo interaction of AtRma2 E3 ubiquitin ligase and auxin binding protein 1

E3 ubiquitin (Ub) ligases play diverse roles in cellular regulation in eukaryotes. Three homologous AtRmas (AtRma1, AtRma2, and AtRma3) were recently identified as ER-localized Arabidopsis homologs of human RING membrane-anchor E3 Ub ligase. Here, auxin binding protein 1 (ABP1), one of the auxin receptors in Arabidopsis, was identified as a potential substrate of AtRma2 through a yeast two-hybrid assay. An in vitro pull-down assay confirmed the interaction of full-length AtRma2 with ABP1. AtRma2 was transiently expressed in tobacco (Nicotiana benthamiana) plants through an Agrobacterium-mediated infiltration method and bound ABP1 in vivo. In vitro ubiquitination assays revealed that bacterially-expressed AtRma2 ubiquitinated ABP1. ABP1 was poly-ubiquitinated in tobacco cells and its stability was significantly increased in the presence of MG132, a 26S proteasome inhibitor. This suggests that ABP1 is controlled by the Ub/26S proteasome system. Therefore, AtRma2 is likely involved in the cellular regulation of ABP1 expression levels.     

Effect of reverse photoperiod on in vitro regeneration and piperine production in Piper nigrum L.

In this study, a novel approach for in vitro regeneration of Piper nigrum L. has been applied in order to increase healthy biomass, phytochemicals and piperine production via reverse photoperiod (16hD/8hL). Leaf portions of the seed-derived plants were placed on an MS-medium fortified with different PGRs. Under 16hD/8hL, thidiazuron (TDZ; 4.0 mg L⁻¹) and BA (1.5 mg L⁻¹) was found to be the most effective (< 90%) in callus induction. Two concentrations (1.5, 2.0 mg L⁻¹) of the IBA produced > 80% shoots from callus cultures. Healthy shoots were transferred to rooting medium and higher percentage of rooting (< 90%) was observed on IBA (1.5 mg L⁻¹). These in vitro tissues were subjected to amino acid analysis, spectrophotometry, and HPLC. ARG, SER, THR, and TYR were the most abundant components out of 17 amino acids. Higher amino acid production was observed under normal photoperiod (16hL/8hD) than under reverse photoperiod (16hD/8hL). The highest total phenolic content (TPC; 9.91 mg/g-DW) and flavonoid content (7.38 mg/g-DW) were observed in callus cultures incubated under 16hL/8hD than other tissues incubated under 16hD/8hL photoperiod. Higher DPPH and PoMo activities were observed in tissues incubated under 16hL/8hD photoperiod, while ABTS and Fe²⁺ chelating activities were found higher in tissues incubated under reverse photoperiod. Significant quantities of piperine content were observed in all tissues except callus cultures. These results suggest that reverse photoperiod is a promising approach for callus induction, phytochemicals and piperine production for commercial applications.     

Amelioration of intracerebroventricular streptozotocin induced cognitive impairment by Evolvulus alsinoides in rats: In vitro and in vivo evidence

Evolvulus alsinoides, also known as Shankpushapi, is a commonly used traditional medicine for enhancing memory. We evaluated the in vitro free radical scavenging and enzymes [acetylcholinesterase, butyrylcholinestrase, glycogen synthase kinase-3-β (GSK-3-β), rho kinase (ROCK II), prolyl endopeptidase (PEP), catechol-O-methyl transferase (COMT) and lipoxygenase (LOX)] inhibitory activities of aqueous and hydro-alcoholic extracts of E. alsinoides. Hydro-alcoholic extract of E. alsinoides demonstrated more free radical scavenging activity as compared to aqueous extract. Hydro-alcoholic extract also showed higher cholinesterase, GSK-3-β, ROCK II, PEP, COMT and LOX enzyme inhibitory activities as compared to aqueous extract. Phytochemical analysis revealed more flavanoids in hydro-alcoholic extract as compared to aqueous extract but no significant difference in phenolic content of the two extracts was observed. Based on in vitro data, hydro-alcoholic extract (100, 300 and 500 mg/kg, p.o.) was selected for in vivo study in intracerebroventricularly injected streptozotocin (STZ) induced cognitive impairment in male Wistar rats. Elevated plus maze, passive avoidance and Morris water maze were used for assessment of cognitive function on 14th, 21st and 28th day after STZ injection. Oxidative stress parameters (malondialdehyde, reduced glutathione, nitric oxide levels and superoxide dismutase activity), cholinergic dysfunction and rho kinase (ROCK II) expression were studied in cerebral cortex and hippocampus of rat brain at the end of the study. Hydro-alcoholic extract of E. alsinoides dose dependently prevented STZ induced cognitive impairment by reducing the oxidative stress, improving cholinergic function and preventing the increase in rho kinase expression. The results suggest an anti-Alzheimer potential of hydro-alcoholic extract of E. alsinoides.     

In vitro activity of pacifastin-like inhibitors in relation to their structural characteristics

Information on the structural characteristics and inhibitory activity of the pacifastin family is restricted to a handful of locust pacifastin-related inhibitors. In this report the optimization of a bacterial recombinant expression system is described, resulting in the high yield production of pacifastin-like inhibitors of the desert locust. Subsequently, the relative inhibitory activity of these peptides towards mammalian, locust and caterpillar digestive peptidases has been compared. In general, the enzyme specificity of locust pacifastin-like inhibitors towards trypsin- or chymotrypsin-like peptidases corresponds to the nature of the P1-residue at the reactive site. In addition, other structural characteristics, including specific core interactions, have been reported to result in a different affinity of pacifastin members towards digestive trypsin-like enzymes from mammals and arthropods. One remarkable observation in this study is a specifically designed pacifastin-like peptidase inhibitor, which, unlike other inhibitors of the same family, does not display this specificity and selectivity towards digestive enzymes from different animals.     

Structural analysis of water-soluble polysaccharides in the fruiting body of Dictyophora indusiata and their in vivo antioxidant activities

The water-soluble Dictyophora indusiata polysaccharides (DIP) were extracted from the fruiting body of D. indusiata. The structural features of purified DIPs I and II were investigated. The results indicated that DIP I was composed of glucose (Glc) and mannose (Man) with molecular weight of 2100 kDa, while DIP II comprised of xylose (Xyl), galactose (Gal), glucose (Glc) and Man with molecular weight of 18.16 kDa. The glycosidic linkage of DIP I was composed of →1)-Glc-(6→: →1)-Man-(3,6→ with the ratio of 5.6:1.0, while DIP II was composed of →1)-Glc-(6→: →1)-Man-(3,6→: →1)-Xyl-(5→: →1)-Gal-(3→: →1)-Gal-(6→: with the ratio of 4.9: 15.5: 7.8: 1.0: 5.7. DIP significantly (P < 0.05) decreased the malondialdehyde (MDA), lipofuscin levels and increased the superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activities of mice. The strong in vivo antioxidant activity indicated DIP had great potential as functional food.     

Validation of internal control genes for quantitative gene expression studies in chickpea (Cicer arietinum L.)

The real-time polymerase chain reaction (PCR) data requires normalization with an internal control gene expressed at constant levels under all the experimental conditions being analyzed for accurate and reliable gene expression results. In this study, the expression of 12 candidate internal control genes, including ACT1, EF1α, GAPDH, IF4a, TUB6, UBC, UBQ5, UBQ10, 18SrRNA, 25SrRNA, GRX and HSP90, in a diverse set of 18 tissue samples representing different organs/developmental stages and stress conditions in chickpea (Cicer arietinum L.) has been validated. Their expression levels vary considerably in various tissue samples analyzed. The expression levels of EF1α and HSP90 are most constant across various organs/developmental stages analyzed. Similarly, the expression levels of IF4a and GAPDH are most constant across various stress conditions. A set of two most stable genes is found sufficient for accurate and reliable normalization of real-time PCR data in the given set of tissue samples of chickpea. The genes with most constant expression identified in this study should be useful for normalization of gene expression data in a wide variety of tissue samples in chickpea.     

Tris(3-hydroxypropyl)phosphine is superior to dithiothreitol for in vitro assessment of vitamin K 2,3-epoxide reductase activity

Use of the reductant dithiothreitol (DTT) as a substrate for measuring vitamin K 2,3-epoxide reductase (VKOR) activity in vitro has been reported to be problematic because it enables side reactions involving the vitamin K₁ 2,3-epoxide (K₁>O) substrate. Here we characterize specific problems when using DTT and show that tris(3-hydroxypropyl)phosphine (THPP) is a reliable alternative to DTT for in vitro assessment of VKOR enzymatic activity. In addition, the pH buffering compound imidazole was found to be problematic in enhancing DTT-dependent non-enzymatic side reactions. Using THPP and phosphate-based pH buffering, we measured apparent Michaelis–Menten constants of 1.20 μM for K₁>O and 260 μM for the active neutral form of THPP. The K_m value for K₁>O is in agreement with the value that we previously obtained using DTT (1.24 μM). Using THPP, we successfully eliminated non-enzymatic production of 3-hydroxyvitamin K₁ and its previously reported base-catalyzed conversion to K₁, both of which were shown to occur when DTT and imidazole are used as the reductant and pH buffer, respectively, in the in vitro VKOR assay. Accordingly, substitution of THPP for DTT in the in vitro VKOR assay will ensure more accurate enzymatic measurements and assessment of warfarin and other 4-hydroxycoumarin inhibition constants.     

ABC transporters do not contribute to extracellular translocation of hyaluronan in human breast cancer in vitro

Extracellular translocation of the polysaccharide, hyaluronan (HA) has been thought to be mediated via its transmembrane synthetic enzyme, hyaluronan synthase (HAS) but recent studies have indicated that the ATP-Binding-Cassette (ABC) transporter, MRP5 contributes to this process. Liberated and cell-associated HA contributes to breast cancer initiation and progression, and therefore the inhibition of ABC transporters and consequently HA transport could provide therapeutic benefit in the treatment of breast cancer. Quantitation of ABC transporter genes, MRP1-5, BCRP and MDR1 were determined in six breast cancer cell lines selected for their differential HA synthetic rates. Low endogenous expression of transporters was detected but no significant correlation existed between ABC transporter and HAS gene expression or HA production. A dose titration of up to ten times the IC₅₀ of ten small molecule ABC transporter inhibitors did not significantly inhibit HA export in four breast cancer cell lines. Unlike the changes observed after inhibition of HA synthesis by the characterised inhibitor 4-MU, inhibition of ABC transporters did not alter the cell morphology, HA glycocalyx or the intracellular quantity or localisation of HA. Collectively these data indicate that ABC transporters do not contribute to the extracellular transport of HA in breast cancer, supporting a role for the hyaluronan synthase in translocation.     

The in vitro and in vivo apoptotic effects of Mahonia oiwakensis on human lung cancer cells

Both the root and stem bark of Mahonia species were popular folk medicines. The plant has several proven biological activities including anti-bacterial, anti-fungal, and anti-inflammatory effects. However, Mahonia has not been studied for its anticancer effects. In the present study, we made extracts from Mahonia oiwakensis (MOE), a selected species in Taiwan, and investigated their effects on various human lung cells. We found that MOE-induced apoptotic death in human A549 non-small-cell lung carcinoma (NSCLC) cells in a dose- and time-dependent manner. Treatment with the extracts also caused an increase in the sub-G1 fraction of cells, chromosome condensation, and DNA fragmentation. The mitochondrial-mediated pathway was implicated in this MOE-induced apoptosis as evidenced by the activation of the caspase cascade, cleavage of poly (ADP-ribose) polymerase (PARP), disruption of mitochondrial membrane potential, and release of cytochrome C. A higher ratio of Bax/Bcl-2 proteins and cleavage of Bid were also observed in MOE-induced cell apoptosis. In A549 tumor-xenografted nude mice, MOE also retarded in vivo proliferation (P < 0.05) and induced apoptosis in tumor cells, as shown by a decrease in Ki-67-positive staining (P < 0.05) and increased transferase-mediated dUTP nick-end labeling (TUNEL)-positive staining (P < 0.05). In conclusion, MOE inhibits the growth of human lung cancer cells in vitro and in vivo, suggesting that it may have therapeutic potential against human lung cancer.     

Methylerythritol phosphate pathway to isoprenoids: Kinetic modeling and in silico enzyme inhibitions in Plasmodium falciparum

The methylerythritol phosphate (MEP) pathway of Plasmodium falciparum (P. falciparum) has become an attractive target for anti-malarial drug discovery. This study describes a kinetic model of this pathway, its use in validating 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) as drug target from the systemic perspective, and additional target identification, using metabolic control analysis and in silico inhibition studies. In addition to DXR, 1-deoxy-d-xylulose 5-phosphate synthase (DXS) can be targeted because it is the first enzyme of the pathway and has the highest flux control coefficient followed by that of DXR. In silico inhibition of both enzymes caused large decrement in the pathway flux. An added advantage of targeting DXS is its influence on vitamin B1 and B6 biosynthesis. Two more potential targets, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase, were also identified. Their inhibition caused large accumulation of their substrates causing instability of the system.     

The induction of trehalose and glycerol in Saccharomyces cerevisiae in response to various stresses

Trehalose and glycerol have been implicated as potential stress protectants that accumulate in yeasts during various stress conditions. We investigated the levels of glycerol and trehalose and the expression profiles of genes involved in their metabolism to determine their involvement in the response of Saccharomyces cerevisiae XQ1 to thermal, sorbitol and ethanol stresses. The results showed that the genes involved in the synthesis and degradation of trehalose and glycerol were stress induced, and that trehalose and glycerol were synthesized simultaneously during the initial stages (a sensitive response period) of diverse stress treatments. Trehalose accumulated markedly under heat treatment, but not under sorbitol or ethanol stress, whereas glycerol accumulated strikingly under sorbitol stress conditions. Interestingly, extracellular trehalose seemed to be involved in protecting cells from damage under unfavorable conditions. Moreover, our results suggest that the stress-activated futile ATP cycles of trehalose and glycerol turnover are of general importance during cellular stress adaptation.     

The mitochondrial genome of the Russian wheat aphid Diuraphis noxia: Large repetitive sequences between trnE and trnF in aphids

To characterize aphid mitochondrial genome (mitogenome) features, we sequenced the complete mitogenome of the Russian wheat aphid, Diuraphis noxia. The 15,784-bp mitogenome with a high A + T content (84.76%) and strong C skew (− 0.26) was arranged in the same gene order as that of the ancestral insect. Unlike typical insect mitogenomes, D. noxia possessed a large tandem repeat region (644 bp) located between trnE and trnF. Sequencing partial mitogenome of the cotton aphid (Aphis gossypii) further confirmed the presence of the large repeat region in aphids, but with different repeat length and copy number. Another motif (58 bp) tandemly repeated 2.3 times in the control region of D. noxia. All repeat units in D. noxia could be folded into stem-loop secondary structures, which could further promote an increase in copy numbers. Characterization of the D. noxia mitogenome revealed distinct mitogenome architectures, thus advancing our understanding of insect mitogenomic diversities and evolution.     

Preparation of low-molecular-weight and high-sulfate-content chitosans under microwave radiation and their potential antioxidant activity in vitro

In the present paper microwave radiation has been used to introduce N-sulfo and O-sulfo groups into chitosan with a high degree of substitution and low-molecular weight. The sulfation of chitosan was performed in microwave ovens. It was found that microwave heating is a convenient way to obtain a wide range of products of different degrees of substitution and molecular weight only by changing reaction time or/and radiation power. Moreover, microwave radiation accelerated the degradation of sulfated chitosan, and the molecular weight of sulfated chitosan was considerably lower than that obtained by traditional heating. There are no differences in the chemical structure of sulfated chitosan obtained by microwave and by conventional technology. FTIR and 13C NMR spectral analyses demonstrated that a significantly shorter time is required to obtain a satisfactory degree of substitution and molecular weight by microwave radiation than by conventional technology. In this present paper, we also determined antioxidant activity of low-molecular-weight and high-sulfate-content chitosans (LCTS). The results showed LCTS could scavenge superoxide and hydroxyl radical. Its IC50 is 0.025 and 1.32 mg/mL, respectively. It is a potential antioxidant in vitro.     

Heme-heme and heme-ligand interactions in the di-heme oxygen-reducing site of cytochrome bd from Escherichia coli revealed by nanosecond absorption spectroscopy

Cytochrome bd is a terminal quinol:O₂ oxidoreductase of respiratory chains of many bacteria. It contains three hemes, b₅₅₈, b₅₉₅, and d. The role of heme b₅₉₅ remains obscure. A CO photolysis/recombination study of the membranes of Escherichia coli containing either wild type cytochrome bd or inactive E445A mutant was performed using nanosecond absorption spectroscopy. We compared photoinduced changes of heme d-CO complex in one-electron-reduced, two-electron-reduced, and fully reduced states of cytochromes bd. The line shape of spectra of photodissociation of one-electron-reduced and two-electron-reduced enzymes is strikingly different from that of the fully reduced enzyme. The difference demonstrates that in the fully reduced enzyme photolysis of CO from heme d perturbs ferrous heme b₅₉₅ causing loss of an absorption band centered at 435 nm, thus supporting interactions between heme b₅₉₅ and heme d in the di-heme oxygen-reducing site, in agreement with previous works. Photolyzed CO recombines with the fully reduced enzyme monoexponentially with τ ∼ 12 μs, whereas recombination of CO with one-electron-reduced cytochrome bd shows three kinetic phases, with τ ∼ 14 ns, 14 μs, and 280 μs. The spectra of the absorption changes associated with these components are different in line shape. The 14 ns phase, absent in the fully reduced enzyme, reflects geminate recombination of CO with part of heme d. The 14-μs component reflects bimolecular recombination of CO with heme d and electron backflow from heme d to hemes b in ∼ 4% of the enzyme population. The final, 280-μs component, reflects return of the electron from hemes b to heme d and bimolecular recombination of CO in that population. The fact that even in the two-electron-reduced enzyme, a nanosecond geminate recombination is observed, suggests that namely the redox state of heme b₅₉₅, and not that of heme b₅₅₈, controls the pathway(s) by which CO migrates between heme d and the medium.