Biosynthesis of raw starch degrading β-cyclodextrin glycosyltransferase by immobilized cells of <i>Bacillus licheniformis</i> using potato wastewater

The study was sought to enhance the synthesis of thermal stable β-cyclodextrin glycosyltransferase (β-CGTase) using potato wastewater as a low-cost medium and assess the degree to which it is efficient for industrial production of β-cyclodextrin (β-CD) from raw potato starch. Thermophilic bacteria producing β-CGTase was isolated from Saudi Arabia and the promising strain was identified as Bacillus licheniformis using phylogenetic analysis of the 16S rRNA gene. Alginate-encapsulated cultures exhibited twice-fold of β-CGTase production more than free cells. Scanning electron microscopy (SEM) of polymeric capsules indicated the potential for a longer shelf-life, which promotes the restoration of activity in bacterial cells across semi-continuous fermentation of β-CGTase production for 252 h. The optimal conditions for β-CGTase synthesis using potato wastewater medium were at 36 h, pH of 8.0, and 50°C with 0.4% potato starch and 0.6% yeast extract as carbon and nitrogen sources, respectively. The purified enzyme showed a specific activity of 63.90 U/mg with a molecular weight of ∼84.6 kDa as determined by SDS-PAGE analysis. The high enzyme activity was observed up to 60°C, and complete stability was achieved at 75°C. High levels of activity and stability were shown at pH 8.0, and the pH range from 7.0–10.0, respectively. The enzyme has an appreciable affinity for raw potato starch with a Km of 5.7 × 10⁻⁶ M and a Vmax of 87.71 µmoL/mL/min. β-CD production was effective against 25 U/g of raw potato starch. The outcomes demonstrated its feasibility to develop a fermentation process by integrating the cost-effective production of β-CGTase having distinctive properties for β-CD production with ecofriendly utilization of potato wastewater.     

Apoptogenic effect of the lipophilic <i>o</i>-naphthoquinone CG 10-248 on rat hepatocytes: light and electron microscopy studies

CG 10-248 (3,4-dihydro-2,2-dimethyl-9-chloro-2H-naphtho[1,2b]pyran- 5,6-dione; CG-NQ), a β-lapachone analogue, modified the ultrastructure of rat hepatocytes, as demonstrated by light and electron microscopy. After 4 h incubation with 100 µM CG-NQ, the following effects were observed: (a) nuclear chromatin condensation; (b) chromatin fragmentation; (c) displacement of mitochondria, concentrated around the nucleus; (d) disruption or expansion of mitochondrial outer or inner membranes, respectively; (e) displacement and alteration of endoplasmic reticulum (rough and smooth); (f) decrease of microvilli; (g) blebbing of plasma membrane and production of apoptotic bodies formed by folding of plasma membrane fragments around mitochondria or peroxysomes; and (h) production of hydrogen peroxide. Expression of such effects varied according to hepatocyte samples and taken together strongly support an apoptotic action of CG-NQ dependent on “reactive oxygen species”.     

Antibacterial activity of lactose-binding lectins from <i>Bufo arenarum</i> skin

Amphibians respond to microbial infection through cellular and humoral defense mechanisms such as antimicrobial protein secretion. Most humoral defense proteins are synthetized in the skin. In this study we isolated two β-galactoside-binding lectins with molecular weights of 50 and 56 KDa from the skin of Bufo arenarum. These lectins have significant hemagglutination activity against trypsinized rabbit erythrocytes, which was inhibited by galactose-containing saccharides. They are water-soluble and independent of the presence of calcium. The antimicrobial analysis for each lectin was performed. At µmolar concentration lectins show strong bacteriostatic activity against Gram negative bacteria (Escherichia coli K12 4100 and wild strains of Escherichia coli and Proteus morganii) and Gram positive bacteria (Enterococcus faecalis). The antibacterial activity of these lectins may provide an effective defense against invading microbes in the amphibian Bufo arenarum.     

Micropropagation of <i>Ilex dumosa</i> (Aquifoliaceae) from nodal segments in a tissue culture system

Micropropagation of Ilex dumosa var. dumosa R. (“yerba señorita”) from nodal segments containing one axillary bud was investigated. Shoot regeneration from explants of six-year-old plants was readily achieved in ¹/₄ strength Murashige and Skoog medium (¹/₄ MS) plus 30 gr·L^-1 sucrose and supplemented with 4.4 µM BA. Further multiplication and elongation of the regenerated shoots were obtained by subculture in a fresh medium of similar composition with 1.5 gr·L^-1 sucrose. Rooting induction from shoots were achieved in two steps: 1) 7 days in ¹/₄ MS (30 gr·L^-1 sucrose, 0.25 % Phytagel^®) with 7.3 µM IBA and 2) 21 days in the same medium without IBA and 20 µM of cadaverine added. Regenerated plants were successfully transferred to soil. This micropropagation schedule can be implemented in breeding programs of Ilex dumosa.     

Organogenesis and plant regeneration of <i>Arachis villosa</i> Benth. (Leguminosae) through leaf culture

With the aim of developing an efficient plant regeneration protocol, leaflet explants of three accessions of Arachis villosa Benth. (S2866, S2867 and L97) were cultured on basic Murashige and Skoog medium supplemented with different combinations of plant growth regulators: α-naphthalenacetic acid, indole-3-butyric acid, 6-benzylaminopurine, kinetin and thidiazuron. The accession L97 was the only one able to differentiate buds through indirect organogenesis. The most suitable combination for bud regeneration was the basic medium added with 13.62 μM thidiazuron and 4.44 μM 6-benzylaminopurine. These results show the important role of the genotype in morphogenetic responses and the organogenetic effect of thidiazuron in Arachis villosa accession L97. A thidiazuron lacking media (only 0.54 μM α-naphthalenacetic acid, 13.95 μM kinetin and 13.32 μM 6-benzylaminopurine were added) promoted the elongation of the regenerated buds. Adventitious rooting was achieved 90 days after the isolated shoots were transferred to a rooting medium containing 0.54 μM α-naphthalenacetic acid.     

Edematogenic and myotoxic activities of the duvernoy’s gland secretion of <i>Philodryas olfersii</i> from the north-east region of Argentina

Philodryas olfersii is found in South America, from Amazonas to Patagonia. It is important to characterize the venom of P. olfersii, who inhabits the North-East region of Argentina, since snake venoms are known to exhibit considerable variability in composition and biological activities. In this work, mice weighing 18-20 g (n = 4 for each experimental group) were used. For the edematogenic activity mice were injected s.c. in the right foot pad with 50 µl of solutions containing different amounts of venom, whereas the left foot pad was injected with 50 µl of PBS. Two hours after injection mice were killed by cervical dislocation and both feet were cut off and weighed individually. For the myotoxic activity mice were injected i.m. with 100 µl of solutions containing 40 µg of venom. Blood samples were extracted after 1, 3, 6, 8, 10, 12, 14, 16 and 24 h of venom injection to determinate serum CPK activity and mice were sacrificed at the same time intervals to obtain the inoculated gastrocnemius muscle. They were fixed with Bouin solution and stained with Hematoxylin-Eosin. Results showed that P. olfersii venom exhibits a high edematogenic activity (MED = 0.31 µg) and a moderate myotoxic activity. Myonecrosis reached its highest level after 12 h of venom injection as shown by plasmatic CPK levels (5,401 ± 330 IU/l) and microscopic assay. It demonstrates the potential toxicity of the venom of P. olfersii, who inhabits the North-East region of Argentina. It also reinforces the original warning concerning the potential danger of bites by colubrids.     

Synergistic combination of colistin with imipenem,amikacine or ciprofloxacin against <i>Acinetobacter baumannii</i> and <i>Pseudomonas aeruginosa</i> carbapenem-resistant isolated in Annaba hospital Algeria

Objective: The aim of this study is to detect in vitro the synergetic activity of colistin in combination with imipenem, amikacin or ciprofloxacin, at sub-inhibitory concentrations, against carbapenems-resistant (CR) Acinetobacter baumannii and Pseudomonas aeruginosa strains isolated from various wards in Annaba teaching hospital in eastern Algeria.
Materials and Methods: The minimal inhibitory concentrations (MIC) were determined by broth macrodilution (BMD). Carbapenemase encoding genes were screened using polymerase chain reaction (PCR). The activity of colistin in combination with second antibiotic was evaluated by the Checkerboard Technique.
Results: 39 CR P. aeruginosa and 21 CR A. baumanni strains where collected. The MIC values ranging from (0.25 to 4 µg/ml) to colistin, ≥16 µg/ml for imipenem, ≥4 µg/ml to amikacin and ≥8 µg/ml ciprofloxacin. The PCR reveals the presence of the genes bla_OXA23 (n = 12), blaOXA24 (n = 6), blaNDM1 (n = 3) in A. baumannii and blaVIM2 (n = 12) in P. aeruginosa. The combination of colistin with imipenem showed synergistic effect on 57.14% and 46.15% of A. baumannii and P. aeruginosa isolates, respectively. For colistin and amikacin, the synergistic effect is detected in 28.6% of A. baumannii and 30.8% of P. aeruginosa. While colistin and ciprofloxacin showed synergy on 14.29% and 15.38% of A. baumannii and P. aeruginosa isolates, respectively.
Conclusion: CR A. baumannii and P. aeruginosa remain the most prevalent infection agents in patients from high-risk wards at Annaba Hospital. Colistin associated with imipenem or with amikacin at sub-inhibitory concentrations gives very encouraging results allowing better management of infections caused by this type of bacteria.     

Effect of the Lipophilic <i>o</i>-Naphthoquinone CG 10-248 on Rat Liver Mitochondria Structure and Function

CG 10-248 (3,4-dihydro-2,2 dimethyl-9-chloro-2H-naphtho[1,2b]pyran-5,6-dione), a ß-lapachone analogue, modified the ultrastructure of rat liver mitochondria in vitro, in the absence of added oxidizable substrates. The condensed mitochondrial state was replaced by the orthodox or swollen state to a significant degree. The number of modified mitochondria depended on incubation time and quinone concentration, in the 25-100 µM range. Under the same experimental conditions, mitochondrial respiration was uncoupled as indicated by the increase in the rate of succinate oxidation by controlled mitochondria in metabolic state “4” (not in state “3”), and by the activation of latent F₀ F₁ -ATP synthase. Taking into account structural similarities, the results reported here may be valid for other o-naphthoquinones, such as ß-lapachone.     

Role of foliar spray of plant growth regulators in improving photosynthetic pigments and metabolites in <i>Plantago ovata</i> (Psyllium) under salt stress–A field appraisal

Salinity is one of the major abiotic factors that limit the growth and productivity of plants. Foliar application of plant growth regulators (PGRs) may help plants ameliorate the negative impacts of salinity. Thus, a field experiment was conducted at the Botanical Garden University of Balochistan, Quetta, to explore the potential role of PGRs, i.e., moringa leaf extract (MLE; 10%), proline (PRO; 1 µM), salicylic acid (SA; 250 µM), and thiourea (TU; 10 mM) in ameliorating the impacts of salinity (120 mM) on Plantago ovata, an important medicinal plant. Salinity hampered plant photosynthetic pigments and metabolites but elevated oxidative parameters. However, foliar application of PGRs enhanced photosynthetic pigments, including Chl b (21.11%), carotenoids (57.87%) except Chl a, activated the defense mechanisms by restoring and enhancing the metabolites, i.e., soluble sugars (49.68%), soluble phenolics (33.34%), and proline (31.47%), significantly under salinity stress. Furthermore, foliar supplementation of PGRs under salt stress led to a decrease of about 43.02% and 43.27% in hydrogen peroxide and malondialdehyde content, respectively. Thus, PGRs can be recommended for improved photosynthetic efficiency and metabolite content that can help to get better yield under salt stress, with the best and most effective treatments being those of PRO and MLE to predominately ameliorate the harsh impacts of salinity.     

MicroRNA-382 inhibits the proliferation of mouse spermatogonia by targeting <i>Kmt5a</i>

Spermatogenesis is a highly efficient and intricate process in the testis by which mature spermatozoa are produced daily to maintain lifelong male fertility. Essential to this process are spermatogonia capable of both proliferation and differentiation. Nevertheless, the underlying mechanisms for spermatogonial proliferation and differentiation remain poorly understood. MicroRNAs (miRNAs) are a category of non-coding small RNAs with regulatory functions by binding to the 3’ untranslated region (UTR) of the target mRNA. Previous studies have demonstrated that miRNAs are capable of modulating cell proliferation, differentiation and apoptosis, but the roles of individual miRNAs in spermatogonial fate determination remain largely elusive. Here, by using a mouse spermatogonial cell line (GC-1), we investigated the role for miRNA-382 in spermatogonial proliferation. We found that pre-miRNA-382 was expressed in spermatogonia. The luciferase reporter assay demonstrated Kmt5a but not Top1 as a target gene of miRNA-382. Overexpression of miRNA-382 by transfecting a miRNA mimic downregulated Kmt5a at both RNA and protein levels, and further reduced the proliferation and viability of spermatogonia. Knockdown of Kmt5a by RNA interference (RNAi) resulted in a uniform phenotype in spermatogonia. We therefore conclude that miRNA-382 inhibits the proliferation of mouse spermatogonia by targeting Kmt5a. Our finding extends the knowledge about the regulatory roles of miRNAs in spermatogonia and lays the groundwork for diagnosis and treatment of male infertility.     

AKT regulates IL-1β-induced proliferation and activation of hepatic stellate cells

Background: Activated hepatic stellate cells (HSCs) are closely involved in the initiation, perpetuation, and resolution of liver fibrosis. Pro-inflammatory cytokine levels are positively correlated with the transition from liver injury to fibrogenesis and contribute to HSC pathophysiology in liver fibrosis. Methods: In this study, we investigated the effect of the pro-inflammatory cytokine interleukin (IL)-1β on the proliferation and signaling pathways involved in fibrogenesis in LX-2 cells, an HSC cell line, using western blotting and cell proliferation assays. Results: IL-1β increased the proliferation rate and α-smooth muscle actin (SMA) expression of LX-2 cells in a dose-dependent manner. Within 1 h after IL-1β treatment, c-Jun N-terminal kinase (JNK), p38, and nuclear factor-κB (NF-κB) signaling was activated in LX-2 cells. Subsequently, protein kinase B (AKT) phosphorylation and an increase in α- SMA expression were observed in LX-2 cells. Each inhibitor of JNK, p38, or NF-κB decreased cell proliferation, AKT phosphorylation, and α-SMA expression in IL-1β-treated LX-2 cells. Conclusion: These results indicate that JNK, p38, and NF-κB signals converge at AKT phosphorylation, leading to LX-2 activation by IL-1β. Therefore, the AKT signaling pathway can be used as a target for alleviating liver fibrosis by the inflammatory cytokine IL-1β.     

Inhibition of focal adhesion kinase by antisense oligonucleotides enhances the sensitivity of breast cancer cells to camptothecins

This study shows a strong association between cell attachment to substratum and activation of β1-integrin-signaling with resistance to the camptothecin derivative topotecan (TPT) in breast cancer cells. We propose a mechanistic-driven approach to sensitize the cells to camptothecins. ZR-75-1 anchoragedependent breast cancer cell line, its derivative 9D3S suspension cells (9D3S-S), and 9D3S cells attached to fibronectin-coated plates (9D3S-A) were treated with TPT (1 µM) or CPT-11 (40 µM) for 48 h. Programmed cell death (PCD), as shown by poly(ADP-ribose) polymerase (PARP), pro-caspase-3 and pro-caspase-9 cleavage, was observed in 9D3S-S cells but not in ZR-75-1 or 9D3S-A cells. Because p125 focal adhesion kinase (FAK) is a transducer in the β1-integrin signaling pathway, it is essential to cell adhesion and it is overexpressed in metastatic breast cancer, we hypothesized that attenuation of FAK might enhance the sensitivity of breast cancer cells to camptothecins. Moreover, inhibition of FAK gene expression by a phosphorothioated antisense oligodeoxynucleotide targeting the portion of the gene encoding amino acids 262-268, increased the sensitivity of ZR-75-1, MDA-MB-231 and MCF7 breast cancer cells to treatment with TPT or CPT-11.     

LncRNA <i>ZFAS1</i> regulates cardiomyocyte differentiation of human embryonic stem cells

Background: Cardiomyocytes derived from human embryonic stem cells (hESCs) are regulated by complex and stringent gene networks during differentiation. Long non-coding RNAs (lncRNAs) exert critical epigenetic regulatory functions in multiple differentiation processes. However, the involvement of lncRNAs in the differentiation of hESCs into cardiomyocytes has not yet been fully elucidated. Here, we identified the key roles of ZFAS1 (lncRNA zinc finger antisense 1) in the differentiation of cardiomyocytes from hESCs. Methods: A model of cardiomyocyte differentiation from stem cells was established using the monolayer differentiation method, and the number of beating hESCs-derived cardiomyocytes was calculated. Gene expression was analyzed by quantitative real-time PCR (qRT-PCR). Immunofluorescence assays were performed to assess the expression of cardiac troponin T (cTnT) and α-actinin protein in cardiomyocytes. Results: qRT-PCR showed that ZFAS1 expression in the mesoderm was significantly higher than that in embryonic stem cells, cardiac progenitor cells, and cardiomyocytes. Knockdown of ZFAS1 inhibited cardiomyocyte differentiation from hESCs, which was characterized by reduced expression of the cardiac-specific markers cTnT, α-actinin, myosin heavy chain 6 (MYH6), and myosin heavy chain 7 (MYH7). In contrast, ZFAS1 overexpression remarkably increased the percentage of spontaneously beating cardiomyocytes. In terms of the mechanism, we found that ZFAS1 is an antisense lncRNA at the 5′ end of the protein-coding gene ZNFX1. Knockdown of ZFAS1 could increase the mRNA expression level of ZNFX1. Furthermore, qRT-PCR demonstrated that the silencing of ZNFX1 led to an increase in cardiac-specific markers that predicted the promotion of cardiomyocyte differentiation. Conclusion: Altogether, these data suggest that lncRNA-ZFAS1 is required for cardiac differentiation by functionally inhibiting the expression of ZNFX1, which may provide a reference for the treatment of heart disease to a certain extent.     

Cryopreservation of <i>Cyrtopodium hatschbachii</i> Pabst (Orchidaceae) immature seeds by encapsulation-dehydration

The aim of the present study was to investigate the efficiency of the encapsulation-dehydration technique for cryopreservation of Cyrtopodium hastchbachii Pabst seeds. Immature seeds of this species were cryopreserved by an encapsulation-dehydration technique. Seeds of five immature pods, 120 days after pollination, were encapsulated in 3% calcium alginate matrix and pretreated in liquid medium supplemented with 0.08 M sucrose (24 h), 0.15 M sucrose (24 h), 0.25 M sucrose (48 h), 0.5 M sucrose (24 h) and 0.75 M sucrose (24 h) in shaker at 60 rpm. Alginate beads were dehydrated 5 h in silicagel and immersed in liquid nitrogen for 12 h. Cryopreserved beads were thawed at 30°C for 1 min, rehydrated using the same liquid mediums [0.75 M sucrose (24 h), 0.5 M sucrose (24 h), 0.25 M sucrose (48 h) and 0.15 M sucrose (24 h)] and cultivated in half strength Murashige & Skoog medium (1962) with the addition of 2 g/L activated charcoal. Sixty four percent of seeds survived and developed into acclimatized plants after being cryopreserved. In this work, the encapsulation-dehydration technique was employed for first time in Cyrtopodium hatschbachii.     

Capsaicin exerts anti-benign prostatic hyperplasia effects via inhibiting androgen receptor signaling pathway

Background: Benign prostatic hyperplasia (BPH) is a common condition in middle-aged and elderly men. Enlargement of the prostate causes lower urinary tract symptoms. Capsaicin is a phytochemical extracted from chili peppers and exerts many pharmacological actions, such as anti-tumor and anti-inflammatory effects. Methods: Our study investigated the effect of capsaicin in vitro and in a mouse model in vivo. A prostatic stromal myofibroblast cell line (WPMY-1) was co-incubated with testosterone (1 µM) and different concentrations of capsaicin (10–100 µM) for 24 and 48 h. Capsaicin (10–100 µM) significantly inhibited testosterone-treated WPMY-1 cell growth at 48 h by MTT assay. The testosterone propionate (7.5 mg/kg)-induced BPH mouse model was used to examine the anti-proliferative effect of capsaicin. Treatment with capsaicin (10 mg/kg) for 14 days significantly attenuated prostatic hyperplasia. Finasteride was used as a positive control. Results: Capsaicin significantly decreased prostate weight and prostate index (prostate/body weight ratio) in BPH mice. The expression of 5α-reductase type II, androgen receptor (AR) and prostate specific antigen (PSA) protein expression and PSA serum were all significantly reduced in capsaicin-treated BPH mice. In addition, capsaicin also activated transient receptor potential vanilloid 1 mediated apoptosis and autophagy in BPH mice. Conclusion: These results demonstrate multiple positive effects of capsaicin in controlling prostate growth and suggest its therapeutic potential in the treatment of BPH.     

<i>Agrobacterium rhizogenes</i> vs auxinic induction for <i>in vitro</i> rhizogenesis of <i>Prosopis chilensis</i> and <i>Nothofagus alpina</i>

The induction and improvement of in vitro rhizogenesis of microshoots of Prosopis chilensis (Mol.) Stuntz and Nothofagus alpina (Poep. et Endl. Oerst.) were compared using Agrobacterium rhizogenes (Ar) versus indole-3-butyric acid (IBA) in the culture media. Microshoots of P. chilensis (1-2 cm length), coming from in vitro grown seedlings, were cultivated in a modified Broadleaved Tree Medium (BTMm) containing half salt concentration of macronutrients and 0.05 mg.L^-1 benzilaminopurine (BAP). After 30 days, microshoots with 2-4 leaves were selected and cultured in BTMm-agar in presence or abscense of Ar and in combination with IBA. For N. alpina, the apical shoots with the first 2 true leaves, from 5 weeks old seedlings, were cultured in the abovementioned medium, but with 0.15 mg.L^-1 of BAP. After 2 months, microshoots with 2-3 leaves were selected and cultured in BTMm-agar, supplemented with 5 mg.L^-1 IBA or in liquid BTMm on perlite and, in the presence or absence of A. rhizogenes (Ar) and in combination with 3 mg.L^-1 IBA. Rooting in P. chilensis reached 100.0% when Ar infection was produced in the presence of IBA, increasing both, the number and dry weight of roots. In N. alpina, 90.0% of rooting efficiency was obtained when Ar infection was produced in liquid culture and in the absence of auxin.     

N-烷基酰胺类化合物电喷雾质谱裂解机制的研究

为阐明维吾尔族药阿纳其根中N-烷基酰胺类成分的质谱裂解机制,采用电喷雾离子源(ESI)正离子模式进行多级碎片解析,对阿纳其根中分离得到5种N-烷基酰胺类成分:十-2E,4E-二烯酸-异丁基胺,十一-2E,4E-二烯-8,10二炔酸-苯乙胺,十-2E,4E-二烯酸-4-羟基苯乙胺,十二-2E,4E-二烯酸-4-羟基苯乙胺和十四-2E,4E-二烯酸-4-羟基苯乙胺,应用密度泛函(DFT)法比较不同裂解方式的优化结构,并分析其可能的质谱裂解机制。结果表明,在正离子模式下,5种N-烷基酰胺类成分均易形成[M+H]+准分子离子,并进一步发生N-位的α裂解,丢失酰胺结构中胺部分、羰基以及水等而形成碎片离子。该类化合物的裂解规律可为N-烷基酰胺类化合物的结构鉴定和定量分析奠定理论基础。