In Vitro Functional Characterisation of Cytochrome P450 (CYP) 2C19 Allelic Variants <Emphasis Type="Italic">CYP2C19*23</Emphasis> and <Emphasis Type="Italic">CYP2C19*24</Emphasis>

Cytochrome P450 (CYP) 2C19 is essential for the metabolism of clinically used drugs including omeprazole, proguanil, and S-mephenytoin. This hepatic enzyme exhibits genetic polymorphism with inter-individual variability in catalytic activity. This study aimed to characterise the functional consequences of CYP2C19*23 (271 G>C, 991 A>G) and CYP2C19*24 (991 A>G, 1004 G>A) in vitro. Mutations in CYP2C19 cDNA were introduced by site-directed mutagenesis, and the CYP2C19 wild type (WT) as well as variants proteins were subsequently expressed using Escherichia coli cells. Catalytic activities of CYP2C19 WT and those of variants were determined by high performance liquid chromatography-based essay employing S-mephenytoin and omeprazole as probe substrates. Results showed that the level of S-mephenytoin 4′-hydroxylation activity of CYP2C19*23 (V _max 111.5 ± 16.0 pmol/min/mg, K _m 158.3 ± 88.0 μM) protein relative to CYP2C19 WT (V _max 101.6 + 12.4 pmol/min/mg, K _m 123.0 ± 19.2 μM) protein had no significant difference. In contrast, the K _m of CYP2C19*24 (270.1 ± 57.2 μM) increased significantly as compared to CYP2C19 WT (123.0 ± 19.2 μM) and V _max of CYP2C19*24 (23.6 ± 2.6 pmol/min/mg) protein was significantly lower than that of the WT protein (101.6 ± 12.4 pmol/min/mg). In vitro intrinsic clearance (CL_int = V _max/K _m) for CYP2C19*23 protein was 85.4 % of that of CYP2C19 WT protein. The corresponding CL_int value for CYP2C19*24 protein reduced to 11.0 % of that of WT protein. These findings suggested that catalytic activity of CYP2C19 was not affected by the corresponding amino acid substitutions in CYP2C19*23 protein; and the reverse was true for CYP2C19*24 protein. When omeprazole was employed as the substrate, K _m of CYP2C19*23 (1911 ± 244.73 μM) was at least 100 times higher than that of CYP2C19 WT (18.37 ± 1.64 μM) and V _max of CYP2C19*23 (3.87 ± 0.74 pmol/min/mg) dropped to 13.4 % of the CYP2C19 WT (28.84 ± 0.61 pmol/min/mg) level. Derived from V _max/K _m, the CL_int value of CYP2C19 WT was 785 folds of CYP2C19*23. K _m and V _max values could not be determined for CYP2C19*24 due to its low catalytic activity towards omeprazole 5′-hydroxylation. Therefore, both CYP2C19*23 and CYP2C19*24 showed marked reduced activities of metabolising omeprazole to 5-hydroxyomeprazole. Hence, carriers of CYP2C19*23 and CYP2C19*24 allele are potentially poor metabolisers of CYP2C19-mediated substrates.     

Effects of Temperature and Humidity on Laser Diffraction Measurements to Jet Nebulizer and Comparison with NGI

Laser diffraction (LD) and next generation impactor (NGI) are commonly used for the evaluation of inhaled drug formulations. In this study, the effect of temperature and humidity on the assessment of the nebulizer particle size distribution (PSD) by LD was investigated, and the consistency between NGI and LD measurements was evaluated. There was an increase in particle size with higher temperature or lower humidity. The particle population with a diameter less than 1 μm was significant at a temperature of 5°C or at relative humidity >90%; however, the same particle population became undetectable when temperature increased to 39°C or at relative humidity of 30–45%. The results of the NGI and LD measurements of aerosol generated from three types of jet nebulizers were compared. A poor correlation between the NGI and LD measurements was observed for PARI LC (2.2 μm) (R ²?=?0.893) and PARI LC (2.9 μm) (R ²?=?0.878), while a relatively good correlation (R ²?=?0.977) was observed for the largest particle size nebulizer (PARI TIA (8.6 μm)). We conclude that the ambient environment and the nebulizer have significant impacts on the performance and consistency between these instruments. These factors should be controlled in the evaluation of inhaled aerosol drug formulations when these instruments are used individually or in combination.     

Gas exchange,carbon balance and stomatal traits in wild and cultivated rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) genotypes

Carbon balancing within the plant species is an important feature for climatic adaptability. Photosynthesis and respiration traits are directly linked with carbon balance. These features were studied in 20 wild rice accessions Oryza spp., and cultivars. Wide variation was observed within the wild rice accessions for photosynthetic oxygen evolution or photosynthetic rate (A), dark (R _d), and light induced respiration (LIR) rates, as well as stomatal density and number. The mean rate of A varied from 10.49 μmol O₂ m^?2 s^?1 in cultivated species and 13.09 μmol O₂ m^?2 s^?1 in wild spp., The mean R _d is 2.09 μmol O₂ m^?2 s^?1 and 2.31 μmol O₂ m^?2 s^?1 in cultivated and wild spp., respectively. Light induced Respiration (LIR) was found to be almost twice in wild rice spp., (16.75 μmol O₂ m^?2 s^?1) compared to cultivated Oryza spp., Among the various parameters, this study reveals LIR and A as the key factors for positive carbon balance. Stomatal contribution towards carbon balance appears to be more dependent on abaxial surface where several number of stomata are situated. Correlation analysis indicates that R _d and LIR increase with the increase in A. In this study, O. nivara (CR 100100, CR 100097), O. rufipogon (IR 103404) and O. glumaepatula (IR104387) were identified as potential donors which could be used in rice breeding program. Co-ordination between gas exchange and patchiness in stomatal behaviour appears to be important for carbon balance and environmental adaptation of wild rice accessions, therefore, survival under harsh environment.     

The relationship between adiposopathy and glucose-insulin homeostasis is not affected by moderate-intensity aerobic training in healthy women with obesity

The contribution of adiposopathy to glucose-insulin homeostasis remains unclear. This longitudinal study examined the potential relationship between the adiponectin/leptin ratio (A/L, a marker of adiposopathy) and insulin resistance (IR: homeostasis model assessment (HOMA)), insulin sensitivity (IS: Matsuda), and insulin response to an oral glucose tolerance test before and after a 16-week walking program, in 29 physically inactive pre- and postmenopausal women with obesity (BMI, 29–35 kg/m²; age, 47–54 years). Anthropometry, body composition, VO₂max, and fasting lipid-lipoprotein and inflammatory profiles were assessed. A/L was unchanged after training (p =?0.15), despite decreased leptin levels (p <?0.05). While the Matsuda index tended to increase (p =?0.07), HOMA decreased (p <?0.05) and fasting insulin was reduced (p <?0.01) but insulin area under the curve (AUC) remained unchanged (p =?0.18) after training. Body fatness and VO₂max were improved (p <?0.05) while triacylglycerols increased and HDL-CHOL levels decreased after training (p <?0.05). At baseline, A/L was positively associated with VO₂max, HDL-CHOL levels, and Matsuda (0.37?< ρ <?0.56; p <?0.05) but negatively with body fatness, HOMA, insulin AUC, IL-6, and hs-CRP levels (??0.41?< ρ <???0.66; p <?0.05). After training, associations with fitness, HOMA, and inflammation were lost. Multiple regression analysis revealed A/L as an independent predictor of IR and IS, before training (partial R² =?0.10 and 0.22), although A/L did not predict the insulin AUC pre- or post-intervention. A significant correlation was found between training-induced changes to A/L and IS (r =?0.38; p <?0.05) but not with IR or insulin AUC. Although changes in the A/L ratio could not explain improvements to glucose-insulin homeostasis indices following training, a relationship with insulin sensitivity was revealed in healthy women with obesity.     

Impact of elevated CO<Subscript>2</Subscript> in <Emphasis Type="Italic">Casuarina equisetifolia</Emphasis> rooted stem cuttings inoculated with <Emphasis Type="Italic">Frankia</Emphasis>

Impact of different levels of elevated CO ₂ on the activity of Frankia (Nitrogen-fixing actinomycete) in Casuarina equisetifolia rooted stem cuttings has been studied to understand the relationship between C. equisetifolia, Frankia and CO₂. The stem cuttings of C. equietifolia were collected and treated with 2000 ppm of Indole Butyric Acid (IBA) for rooting. Thus vegetative propagated rooted stem cuttings of C. equisetifolia were inoculated with Frankia and placed in the Open top chambers (OTC) with elevated CO₂ facilities. These planting stocks were maintained in the OTC for 12 months under different levels of elevated CO₂ (ambient control, 600 ppm, 900 ppm). After 12 months, the nodule numbers, bio mass, growth, and photosynthesis of C. equisetifolia rooted stem cuttings inoculated with Frankia were improved under 600 ppm of CO₂. The rooted stem cuttings of C. equisetifolia inoculated with Frankia showed a higher number of nodules under 900 ppm of CO₂ and cuttings without Frankia inoculation exhibited poor growth. Tissue Nitrogen (N) content was also higher under 900 ppm of CO₂ than ambient control and 600 ppm levels. The photosynthetic rate was higher (17.8 μ mol CO₂ m^?2 s^?1) in 900 ppm of CO₂ than in 600 ppm (13.2 μ mol CO₂ m^?2 s^?1) and ambient control (8.3 μ mol CO₂ m^?2 s^?1). This study showed that Frankia can improve growth, N fixation and photosynthesis of C. equietifolia rooted stem cuttings under extreme elevated CO₂ level conditions (900 ppm).     

Milk and Dairy Products Intake Is Associated with Low Levels of Lead (Pb) in Workers highly Exposed to the Metal

Lead (Pb) is a toxic metal, frequently associated with occupational exposure, due to its widespread use in industry and several studies have shown high Pb levels in workers occupationally exposed to the metal. The aim of this study was to evaluate the influence of milk and dairy products (MDP) on Pb levels in blood (B-Pb), plasma (P-Pb), and urine (U-Pb), in workers from automotive battery industries in Brazil. The study included 237 male workers; information concerning diet and lifestyle were gathered through a questionnaire, and B-Pb, P-Pb, and U-Pb were determined by ICP-MS. Mean B-Pb, P-Pb, and U-Pb were 21 ± 12, 0.62 ± 0.73 μg/dL, and 39 ± 47 μg/g creatinine, respectively. Forty three percent of participants declared consuming ≤3 portions/week of MDP (classified as low-MDP intake), while 57% of individuals had >3portions/week of MDP (high-MDP intake). B-Pb and P-Pb were correlated with working time (r _s  = 0.21; r _s  = 0.20; p < 0.010). Multivariable linear regressions showed a significant influence of MDP intake on B-Pb (β = ?0.10; p = 0.012) and P-Pb (β = ?0.16; p < 0.010), while no significance was seen on U-Pb. Our results suggest that MDP consumption may modulate Pb levels in individuals highly exposed to the metal; these findings may be due to the Pb-Ca interactions, since the adverse effects of Pb are partially based on its interference with Ca metabolism and proper Ca supplementation may help to reduce the adverse health effects induced by Pb exposure.     

The aromatic cytokinin <Emphasis Type="Italic">meta-</Emphasis>topolin promotes in vitro propagation,shoot quality and micrografting in <Emphasis Type="Italic">Corylus colurna</Emphasis> L.

The role of 4.1 or 8.2 μM meta-topolin (mT) on shoot multiplication, rooting and ex vitro acclimatization of micropropagated Corylus colurna L., a promising non-suckering rootstock for hazelnut (Corylus avellana L.), was examined in comparison to N⁶-benzyladenine (BA), the most used cytokinin in tissue culture of Corylus spp. The influence of 8.2 μM mT and BA on photosynthetic pigments content and antioxidant enzymes activity, catalase (CAT) and guaiacol peroxidase (POD), in regenerated shoots, and on the preparation of the rootstock for micrografting was also evaluated. The highest shoot multiplication was recorded on medium containing 8.2 μM mT and an overall positive effect of mT on growth and quality of micropropagated shoots was found. The highest chlorophyll a content (1.236 mg g^?1 fresh weight, FW) and chlorophyll a/b ratio (2.48), and the lowest total carotenoids content (0.292 mg g^?1 FW) and CAT activity (25.8 μmol min^?1 mg^?1 protein) were detected after 8.2 μM mT application, while no significant differences were found in chlorophyll b content and POD activity between the two cytokinins. The best rhizogenesis response (98% for 4.1 μM and 100% for 8.2 μM mT) and ex vitro acclimatization competence (higher than 78%) were exhibited from shoots multiplied on mT. Furthermore, the multiplication of rootstock on mT allowed obtaining the highest (70%) response of successful micrografting. The present findings provide the first evidence of the successful applicability of mT in C. colurna tissue culture and development of micrografted plantlets.     

Testing the cost of reproduction with the rotifer <Emphasis Type="Italic">Brachionus</Emphasis><Emphasis Type="Italic">patulus</Emphasis> at different pH levels

In the present work, we tested the hypothesis that the cost of reproduction was evident under stressful conditions with the rotifer Brachionus patulus at different pH levels (5–10 at 1 unit intervals). We used sublethal pH levels (pH 5, 9, and 10) to simulate stressful conditions. We analyzed the correlations between age-specific fecundity (m ₁, m ₂, m ₃, …) versus future survival (l _x + 1, l _x + 2, l _x + 3, … for the entire lifespan) (survival costs) and future expectation of reproduction (\( V_{ 1}^{*} , \, V_{ 2}^{*} , \, V_{ 3}^{*} , \ldots \) for the entire lifespan) (reproductive costs), using the data obtained from life table demographic studies of B. patulus under stressful and favorable (pH 6, 7, and 8) pH levels. The results showed that significant negative correlations were observed between age-specific fecundity and future survival and future expectation of reproduction at all tested pH levels, indicating that costs of reproduction exist in the rotifer B. patulus under stressful and favorable pH conditions. However, the percentage of statistically significant negative correlations from total correlations of survival and reproductive costs differed greatly, depending on the tested pH conditions. The percentage of significant negative correlation of reproductive costs is significantly higher under stressful pH conditions (pH 5, 9, and 10) than favorable pH conditions (pH 6, 7, and 8). For survival costs, the same trends are also observed, suggesting that the costs of reproduction were more obvious under stressful pH than favorable pH.     

Evaluation of Mineral Concentrations in Maternal Serum Before and After Birth and in Newborn Cord Blood Postpartum—Preliminary Study

The mineral levels in maternal serum change during pregnancy and may be correlated with those of newborn cord blood. The aim of this study was to evaluate the concentrations of calcium (Ca), magnesium (Mg), zinc (Zn), iron (Fe), and copper (Cu) in maternal blood before and after delivery and in umbilical cord vein and artery serum. The study was carried out in 64 Caucasian pregnant women who delivered in a district hospital in Greater Poland region, aged 28.1 ± 5.4 years, with a mean gestational age of 39.2 ± 1.3 weeks. Blood samples were taken from women 2–8 h before delivery and immediately after childbirth. The umbilical cord artery and vein blood of newborns was obtained immediately after childbirth. The levels of minerals in serum were determined by flame atomic absorption spectrometry. A significant drop in the concentrations of Mg (17.71 ± 1.51 vs 17.07 ± 1.61 μg/ml; p < 0.007), Fe (1.08 ± 0.46 vs 0.82 ± 0.35 μg/ml; p < 0.0004), and Zn (0.63 ± 0.17 vs 0.46 ± 0.16; p < 0.0001) in maternal serum was found after delivery. Moreover, higher levels of Ca, Fe, and Zn and lower levels of Cu were observed in the umbilical vein (Ca: 102.80 ± 7.80 μg/ml; p < 0.0001, Fe: 1.96 ± 0.43 μg/ml; p < 0.0001, Zn: 0.65 ± 0.16 μg/ml; p < 0.0001, Cu: 0.36 ± 0.09 μg/ml; p < 0.0001) and in the umbilical artery cord blood (Ca: 98.07 ± 8.18 μg/ml; p < 0.0001, Fe: 1.63 ± 0.30 μg/ml; p < 0.0001, Zn: 0.65 ± 0.15 μg/ml; p < 0.0001, and Cu: 0.36 ± 0.10 μg/ml; p < 0.0001) compared to the maternal serum (Ca: 85.05 ± 10.76 μg/ml, Fe: 0.82 ± 0.35 μg/ml, Zn: 0.46 ± 0.16 μg/ml, and Cu: 1.90 ± 0.35 μg/ml). Fe levels in the cord artery serum negatively correlated with blood loss during delivery (R = ?0.48; p = 0.01), while the Ca concentration in the maternal serum after birth decreased with the age of the women (R = ?0.25; p = 0.03). In conclusion, it seems that the process of birth alters the mineral levels in pregnant women’s blood. Moreover, it was found that blood loss and the age of the mother are associated with mineral concentrations in the maternal serum and cord artery blood.     

In vitro anticancer properties of selected <Emphasis Type="Italic">Eucalyptus</Emphasis> species

In spite of the recent advancements in oncology, the overall survival rate for pancreatic cancer has not improved over the last five decades. Eucalypts have been linked with cytotoxic and anticancer properties in various studies; however, there is very little scientific evidence that supports the direct role of eucalypts in the treatment of pancreatic cancer. This study assessed the anticancer properties of aqueous and ethanolic extracts of four Eucalyptus species using an MTT assay. The most promising extracts were further evaluated using a CCK-8 assay. Apoptotic studies were performed using a caspase 3/7 assay in MIA PaCa-2 cells. The aqueous extract of Eucalyptus microcorys leaf and the ethanolic extract of Eucalyptus microcorys fruit inhibited the growth of glioblastoma, neuroblastoma, lung and pancreatic cancer cells by more than 80% at 100 μg/mL. The E. microcorys and Eucalyptus saligna extracts showed lower GI₅₀ values than the ethanolic Eucalyptus robusta extract in MIA PaCa-2 cells. Aqueous E. microcorys leaf and fruit extracts at 100 μg/mL exerted significantly higher cell growth inhibition in MIA PaCa-2 cells than other extracts (p < 0.05). Statistically similar IC₅₀ values (p > 0.05) were observed in aqueous E. microcorys leaf (86.05 ± 4.75 μg/mL) and fruit (64.66 ± 15.97 μg/mL) and ethanolic E. microcorys leaf (79.30 ± 29.45 μg/mL) extracts in MIA PaCa-2 cells using the CCK-8 assay. Caspase 3/7-mediated apoptosis and morphological changes of cells were also witnessed in MIA PaCa-2 cells after 24 h of treatment with the extracts. This study highlighted the significance of E. microcorys as an important source of phytochemicals with efficacy against pancreatic cancer cells. Further studies are warranted to purify and structurally identify individual compounds and elucidate their mechanisms of action for the development of more potent and specific chemotherapeutic agents for pancreatic cancer.     

Comparative Pharmaceutical Evaluation of Candesartan and Candesartan Cilexetil: Physicochemical Properties, <Emphasis Type="Italic">In Vitro</Emphasis> Dissolution and <Emphasis Type="Italic">Ex Vivo In Vivo</Emphasis> Studies

The aim of the present work is to answer the question is it possible to replace the ester prodrug candesartan cilexetil (CC) by its active metabolite candesartan (C) to bypass the in vivo variable effect of esterase enzymes. A comparative physicochemical evaluation was conducted through solubility, dissolution, and stability studies; additionally, ex vivo permeation and in vivo studies were assessed. C demonstrated higher solubility over CC at alkaline pH. Moreover, dissolution testing using the pharmacopeial method showed better release profile of C even in the absence of surfactant in the testing medium. Both drugs demonstrated a slight degradation in acidic pH after short-term stability. Instead, shifting to alkaline pH of 6.5 and 7.4 showed superiority of C solution stability compared to CC solution. The ex vivo permeation results demonstrated that the parent compound C has a significant (P < 0.05) enhanced permeation compared to its prodrug from CC, that agreed with in vivo results in which C suspension reached significantly (P < 0.05) higher C _max of 1.39 ± 0.59 μg/mL at T _max of 0.66 ± 0.11 h, while CC suspension reached C _max of 0.47 ± 0.22 μg/mL at T _max of 2.00 ± 0.27 h, a lag period of 40 min is needed prior to detection of any absorbed CC in plasma. Those findings are not in agreement with the previously reported rationale on the prodrug formation owing to the poor permeability of the parent compound, suggesting the possibility of marketing the parent drug candesartan for clinical use similarly to azilsartan and its prodrug.     

In Vitro Antimicrobial Activity and Downregulation of Virulence Gene Expression on <Emphasis Type="Italic">Helicobacter pylori</Emphasis> by Reuterin

Helicobacter pylori is an infectious agent commonly associated with gastrointestinal diseases. The use of probiotics to treat this infection has been documented, however, their potential antimicrobial metabolites have not yet been investigated. In the present study, the effect of reuterin produced by Lactobacillus reuteri on H. pylori growth and virulence gene expression was evaluated. It was observed that reuterin caused significant (P < 0.05) H. pylori growth inhibition at concentrations from 0.08 to 20.48 mM, with minimal inhibitory concentrations (MICs) of 20.48 mM for H. pylori ATCC700824 and 10.24 mM for H. pylori ATCC43504. In a reuterin bacterial killing assay, it was observed that half of the MIC value for H. pylori (ATCC700824) significantly (P < 0.01) reduced colony numbers from 5.65 ± 0.35 to 3.78 ± 0.35 Log₁₀ CFU/mL after 12 h of treatment and then increased them to 5.25 ± 0.23 Log₁₀ CFU/mL at 24 h; at its MIC value (20.48 mM), reuterin abrogated (P < 0.01) H. pylori (ATCC700824) growth after 20 h of culture. In addition, reuterin significantly (P < 0.01) reduced H. pylori (ATCC 43504) colony numbers from 5.65 ± 0.35 to 4.1 ± 0.12 Log10 CFU/mL from 12 to 24 h of treatment and abrogated its growth at its MIC value (10.24 mM), after 20 h of treatment. Reuterin did not alter normal human gastric Hs738.St/Int cell viability at the concentrations tested for H. pylori strains. Furthermore, 10 μM reuterin was shown to significantly (P < 0.01) reduce mRNA relative expression levels of H. pylori virulence genes vacA and flaA at 3 h post-treatment, whose effect was higher at 6 h post-treatment, as measured by RT-qPCR. The observed direct antimicrobial effect and the downregulation of expression of virulence genes on H. pylori by reuterin may contribute to the understanding of the mechanisms of action of probiotics against H. pylori.     

<Emphasis Type="Italic">In Vitro</Emphasis>-<Emphasis Type="Italic">In Vivo</Emphasis> Evaluation of Novel Co-spray Dried Rifampicin Phospholipid Lipospheres for Oral Delivery

The objective of this study comprises of developing novel co-spray dried rifampicin phospholipid lipospheres (SDRPL) to investigate its influence on rifampicin solubility and oral bioavailability. Solid-state techniques were employed to characterize the liposphere formulation. SDRPL solubility was determined in distilled water. BACTEC 460TB System was employed to evaluate SDRPL antimycobacterial activity. The oral bioavailability of the lipospheres was evaluated in Sprague Dawley rats. Lipospheres exhibited amorphous, smooth spherical morphology with a significant increase (p?<?0.001) in solubility of SDRPL (2:1), 350.9?±?23 versus 105.1?±?12 μg/ml and SDRPL (1:1) 306.4?±?20 versus 105.1?±?12 μg/ml in comparison to rifampicin (RMP). SDRPL exhibited enhanced activity against Mycobacterium tuberculosis, H37Rv strain, with over twofolds less minimum inhibitory concentration (MIC) than the free drug. Lipospheres exhibited higher peak plasma concentration (109.92?±?25 versus 54.31?±?18 μg/ml), faster T _max (two versus four hours), and enhanced area under the curve (AUC_0–∞) (406.92?±?18 versus 147.72?±?15 μg h/L) in comparison to pure RMP. Thus, SDRPL represents a promising carrier system exhibiting enhanced antimycobacterial activity and oral bioavailability of rifampicin.     

Interspecific variation in extracellular polysaccharide content and colony formation of <Emphasis Type="Italic">Microcystis</Emphasis> spp. cultured under different light intensities and temperatures

Single cells of five different Microcystis species (M. ichthyoblabe, M. viridis, M. flos-aquae, M. wesenbergii, and M. aeruginosa) were batch-cultured at different temperatures and light intensities: (a) 25 °C and 50 μmol photons m^?2 s^?1 (control culture); (b) 25 °C and 10 μmol photons m^?2 s^?1; and (c) 15 °C and 50 μmol photons m^?2 s^?1. The extracellular polysaccharide content was significantly higher in treatments b and c than in the control treatment. All Microcystis species existed as single cells under the control treatment but formed colonies in treatments b and c. All of the colonies were irregular with indistinct margins. M. ichthyoblabe, M. viridis, M. flos-aquae, and M. wesenbergii formed colonies with similar morphologies and their cells were loosely aggregated. In contrast, M. aeruginosa formed denser colonies with no distinct holes. The colony morphologies differed from the classic morphology of M. ichthyoblabe field-grown colonies but resembled that of small colonies found in Lake Taihu (Yangtze Delta Plain, China) during early spring. This indicates that field- and laboratory-grown colonies are governed by similar formation processes. We suggest that in laboratory and field environments, M. ichthyoblabe (or M. flos-aquae) colonies are representative of small colonies formed from single Microcystis cells, whereas the morphology of older colonies evolves to resemble M. wesenbergii and M. aeruginosa colonies.     

Protein malnutrition during fetal programming induces fatty liver in adult male offspring rats

We evaluated the effects of protein malnutrition on liver morphology and physiology in rats subjected to different malnutrition schemes. Pregnant rats were fed with a control diet or a low protein diet (LPD). Male offspring rats received a LPD during gestation, lactation, and until they were 60 days old (MM group), a late LPD that began after weaning (CM), or a LPD administrated only during the gestation-lactation period followed by a control diet (MC). On day 60, blood was collected and the liver was dissected out. We found a decrease in MM rats’ total body (p < 0.001) and liver (p < 0.05) weight. These and CM rats showed obvious liver dysfunction reflected by the increase in serum glutamic pyruvic transaminase (SGOT) (MM p < 0.001) and serum glutamic pyruvic transaminase (SGPT) (MM and CM p < 0.001) enzymes, and liver content of cholesterol (MM and CM p < 0.001) and triglycerides (MM p < 0.01; CM p < 0.001), in addition to what we saw by histology. Liver dysfunction was also shown by the increase in gamma glutamyl transferase (GGT) (MM, MC, and CM p < 0.001) and GST-pi1 (MM and CM p < 0.001, MC p < 0.05) expression levels. MC rats showed the lowest increment in GST-pi1 expression (MC vs. MM; p < 0.001, MC vs. CM; p < 0.01). ROS production (MM, CM, and MC: p < 0.001), lipid peroxidation (MM, CM, and MC p < 0.001), content of carbonyl groups in liver proteins (MM and CM p < 0.001, MC p < 0.01), and total antioxidant capacity (MM, CM, and MC p < 0.001) were increased in the liver of all groups of malnourished animals. However, MM rats showed the highest increment. We found higher TNF-α (MM and CM p < 0.001), and IL-6 (MM and CM p < 0.001) serum levels and TGF-β liver content (MM p < 0.01; CM p < 0.05), in MM and CM groups, while MC rats reverted the values to normal levels. Pro-survival signaling pathways mediated by tyrosine or serine/threonine kinases (pAKT) (MM and CM p < 0.001; MC p < 0.01) and extrasellular signal-regulated kinase (pERKs) (MM p < 0.01; CM p < 0.05) appeared to be activated in the liver of all groups of malnourished rats, suggesting the presence of cells resistant to apoptosis which would become cancerous. In conclusion, a LPD induced liver damage whose magnitude was related to the developmental stage at which malnutrition occurs and to its length.     

Genetic population structure and low genetic diversity in the over-exploited sea cucumber <Emphasis Type="Italic">Holothuria edulis</Emphasis> Lesson, 1830 (Echinodermata: Holothuroidea) in Okinawa Island

Understanding genetic connectivity is fundamental for ecosystem-based management of marine resources. Here we investigate the metapopulation structure of the edible sea cucumber Holothuria edulis Lesson, 1830 across Okinawa Island, Japan. This species is of economic and ecological importance and is distributed from the Red Sea to Hawai‘i. We examined sequence variation in fragments of mitochondrial cytochrome oxidase subunit I (COI) and 16S ribosomal RNA (16S), and nuclear histone (H3) at six locations across Okinawa Island. We found higher haplotype diversity for mtDNA (COI: Hd = 0.69 and 16S: Hd = 0.67) and higher heterozygosity of nDNA (H3: H _E = 0.39) in populations from the west coast of Okinawa compared to individuals from populations on the east coast (COI: Hd = 0.40; 16S: Hd = 0.21; H3: H _E = 0.14). Overall population structure was significant (AMOVA results for COI: Φ _ST = 0.49, P < 0.0001; 16S: Φ _ST = 0.34, P < 0.0001; H3: Φ _ST = 0.12, P < 0.0001). One population in the east, Uruma, showed elevated pairwise Φ _ST values in comparisons with all other sites and a marked reduction of genetic diversity (COI: Hd = 0.25 and 16S: Hd = 0.24), possibly as a consequence of a shift to a more dominant asexual reproduction mode. Recent reports have indicated that coastal development in this area influences many marine organisms, and ecosystem degradation in this location could cause the observed decrease of genetic diversity and isolation of H. edulis in Uruma. Our study should provide valuable data to help with the urgently needed management of sea cucumber populations in Okinawa, and indicates particular attention needs to be paid to vulnerable locations.     

Glutamate receptors of the A5 region modulate cardiovascular responses evoked from the dorsomedial hypothalamic nucleus and perifornical area

To assess the possible function of glutamate in the interaction between the dorsomedial hypothalamic nucleus-perifornical area (DMH-PeF) and the A5 pontine region (A5), cardiovascular and respiratory changes were studied in response to electrical stimulation of the DMH-PeF (1 ms pulses, 30–50 μA given at 100 Hz for 5 s) before and after the microinjection of kynurenic acid (non-specific glutamate receptor antagonist; 50 nl, 5 nmol), MK-801 (NMDA receptor antagonist; 50 nl, 50 nmol), CNQX (non-NMDA receptor antagonist; 50 nl, 50 nmol) or MCPG (metabotropic glutamate receptor antagonist; 50 nl, 5 nmol) within the A5 region. DMH-PeF electrical stimulation elicited a pressor (p <?0.001) and tachycardic response (p <?0.001) which was accompanied by an inspiratory facilitation characterised by an increase in respiratory rate (p <?0.001) due to a decrease in expiratory time (p <?0.01). Kynurenic acid within the A5 region decreased the tachycardia (p <?0.001) and the intensity of the blood pressure response (p <?0.001) to DMH-PeF stimulation. After the microinjection of MK-801 and CNQX into the A5 region, the magnitude of the tachycardia and the pressor response were decreased (p <?0.05 and p <?0.01; p <?0.001 and p <?0.05, respectively). After MCPG microinjection into the A5 region, a decrease in the tachycardia (p <?0.001) with no changes in the pressor response was observed during DMH-PeF stimulation. The respiratory response elicited by DMH-PeF stimulation was not changed after the microinjection of kynurenic acid, MK-801, CNQX or MCPG within the A5 region. These results suggest that A5 region glutamate receptors play a role in the cardiovascular response elicited from the DMH-PeF. The possible mechanisms involved in these interactions are discussed.     

Textural and Rheological Properties of Soy Protein Isolate Tofu-Type Emulsion Gels: Influence of Soybean Variety and Coagulant Type

The contribution of soybean variety and coagulant type to the textural and rheological properties of soy protein isolate (SPI) tofu-type emulsion gels was studied. SPIs from eight soybean varieties were subjected to amino acid and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and results showed that the 11S fraction proteins (r?=?0.833, p?<?0.05) and the ratio of 11S to 7S (r =?0.920, p <?0.01) were positively correlated with the hardness of CaSO₄-induced emulsion gels and glucono-δ-lactone (GDL)-induced gels, with the correlation coefficients of 0.827 (p <?0.05) and 0.893 (p <?0.01), respectively. In the case of microbial transglutaminase (MTGase), strong relations between the content of glutamate (r =?0.886, p?<?0.01) and lysine (r =?0.810, p <?0.05) and gel hardness were found. Rheological data demonstrated that CaSO₄-induced emulsion gel was stiffer with high rigidity but gel induced by MTGase performed better elasticity. The findings of this study are of great importance to further understand the gelation mechanisms of different coagulants and provide useful information for the development of SPI-based filled tofu.     

Nitrogen-induced variations in leaf gas exchange of spring triticale under field conditions

Nitrogen (N) is the key factor limiting photosynthetic processes and crop yield. Little is known about the response of leaf gas exchange of spring triticale (Triticosecale Wittm.) to N supply. The effect of N fertilizers on different gas exchange variables, i.e., photosynthetic rate (A), transpiration rate (E), stomatal conductance (g _s), instantaneous water use efficiency (WUE) and maximum quantum yield of photosystem II (PSII) (F _v/F _m), chlorophyll index (SPAD, soil–plant analysis development), and the relationship of these variables with yield were studied in spring triticale grown under field conditions. Six treatments of N—0, 90, 180, 90 + 30, 90 + 30 + 30 kg ha^?1 (applied as ammonium nitrate, AN) and one treatment of N 90 + 30 + 30 kg ha^?1 (applied as urea ammonium nitrate solution, UAN) were compared. The analysis of variance showed that throughout the triticale growing season, N fertilization had significant effects on A, WUE, g _s and SPAD. On average, N fertilizer application increased A values by 14–70%. E and F _v/F _m values were not influenced by N fertilization levels. The effect of growth stage and year on gas exchange variables and F _v/F _m and SPAD was found to be significant. At different growth stages, A values varied and maximum ones were reached at BBCH 31–33 (decimal code system of growth stages) and BBCH 59. With aging, values of A decreased independently of N fertilization level. The gas exchange variables were equally affected by both fertilizer forms. The interplay among grain yield, leaf gas exchange variables, F _v/F _m and SPAD of spring triticale was estimated. The statistical analysis showed that grain yield positively and significantly correlated with A and SPAD values throughout the growing season.