Ethylene and cytokinin interaction in the morphogenesis of Pelargonium × hortorum L.H. Bailey in vitro

Pelargonium × hortorum ‘Grand Prix’ which is susceptible to leaf yellowing and ‘Bergpalais’ which is not susceptible to leaf yellowing were chosen for the experiments. Ethylene production and action as well as the associated morphological response of Pelargonium shoots grown in the presence of a precursor of ethylene biosynthesis 1-aminocyclopropane-1-carboxylic acid (ACC), ethylene inhibitors: α-aminooxyacetic acid (AOA) and silver nitrate (AgNO₃) and different cytokinins: (meta-topolin) (mT) or 6-benzylaminopurine (BAP) were studied. It was found that ‘Grand Prix’ was more sensitive to ethylene than ‘Bergpalais’ and it showed the leaf yellowing in response to 0.1 mg l^?1 ACC. Moreover, it was noted that ACC added separately or together with cytokinin influenced Pelargonium morphogenesis. Depending on the concentration of ACC (0.1–2.0 mg l^?1), it either stimulated or inhibited shoot and root formation as well as the growth of shoots and leaf blades. ACC-induced leaf yellowing in ‘Grand Prix’ was effectively inhibited by mT. In contrast, BAP did not enhance shoot quality. Simultaneously, the presence of mT in the medium resulted in up to a twofold increase in the ethylene production by ‘Grand Prix’ shoots throughout the culture period compared with the shoots growing on the BAP-medium. The inhibitor of ethylene action (AgNO₃) added with cytokinin prevented the yellowing of Pelargonium shoots, but simultaneously influenced the formation of mature shoots with limited long-term multiplication potential. The shoots of P. × hortorum ‘Grand Prix’ treated with AgNO₃ and mT emitted two- and sevenfold more ethylene after 11th and 21st day of culture compared with those treated with AgNO₃ and BAP. It is suggested that mT inhibits the early senescence of Pelargonium in vitro by decreasing its sensitivity to ethylene.     

Microenvironmental influences of apoptosis in vivo and in vitro

The apoptosis program of physiological cell death elicits a range of non-phlogistic homeostatic mechanisms—“recognition, response and removal”—that regulate the microenvironments of normal and diseased tissues via multiple modalities operating over short and long distances. The molecular mechanisms mediate intercellular signaling through direct contact with neighboring cells, release of soluble factors and production of membrane-delimited fragments (apoptotic bodies, blebs and microparticles) that allow for interaction with host cells over long distances. These processes effect the selective recruitment of mononuclear phagocytes and the specific activation of both phagocytic and non-phagocytic cells. While much evidence is available concerning the mechanisms underlying the recognition and responses of phagocytes that culminate in the engulfment and removal of apoptotic cell bodies, relatively little is yet known about the non-phagocytic cellular responses to the apoptosis program. These responses regulate inflammatory and immune cell activation as well as cell fate decisions of proliferation, differentiation and death. Here, we review current knowledge of these processes, considering especially how apoptotic cells condition the microenvironments of normal and malignant tissues. We also discuss how apoptotic cells that persist in the absence of phagocytic clearance exert inhibitory effects over their viable neighbors, paying particular attention to the specific case of cell cultures and highlighting how new cell-corpse-clearance devices—Dead-Cert^® Nanoparticles—can significantly improve the efficacy of cell cultures through effective removal of non-viable cells in the absence of phagocytes in vitro.     

Impaired drug-binding capacities of in vitro and in vivo glycated albumin

Albumin, the major circulating protein in blood, can undergo increased glycation in diabetes. One of the main properties of this plasma protein is its strong affinity to bind many therapeutic drugs, including warfarin and ketoprofen. In this study, we investigated whether or not there were any significant changes related to in vitro or in vivo glycation in the structural properties and the binding of human albumin to both therapeutic drugs. Structural parameters, including redox state and ketoamine contents of in vitro and in vivo glycated purified albumins, were investigated in parallel with their affinity for warfarin and ketoprofen. High-performance liquid chromatography was used to determine the free drug concentrations and dissociation constants according to the Scatchard method. An alternative method based on fluorescence spectroscopy was also used to assess drug-binding properties. Oxidation and glycation levels were found to be enhanced in albumin purified from diabetic patients or glycated with glucose or methylglyoxal, after determination of their ketoamine, free thiol, amino group and carbonyl contents. In parallel, significant impairments in the binding affinity of in vitro and in vivo glycated albumin, as indicated by the higher dissociation constant values and confirmed by higher free drug fractions, were observed. To a lesser extent, this alteration also significantly affected diabetic albumin affinity, indicated by a lower static quenching in fluorescence spectroscopy. This work provides useful information supporting in vivo diabetic albumin could be the best model of glycation for monitoring diabetic physiopathology and should be valuable to know if glycation of albumin could contribute to variability in drugs response during diabetes.     

Failure of androgenesis in Miscanthus × giganteus in vitro culture of cytologically unbalanced microspores

Miscanthus × giganteus is a popular energy crop, which due to its hybrid origin is only vegetatively reproduced. Asexual embryogenesis in anther and microspore culture leading to double haploids production could allow to regain the ability for sexual reproduction and to increase the biodiversity of the species. Therefore, the goal of this paper was to investigate the requirements of androgenesis in Miscanthus. The standard protocols used for monocotyledonous plants were applied with many modifications regarding the developmental stage of the explants at the time of culture initiation, stress treatment applied to panicles and isolated anthers as well as various chemical and physical parameters of in vitro culture conditions. Our results indicated that the induction of androgenesis in M. × giganteus is possible. However, the very low efficiency of the process and the lack of regeneration ability of the androgenic structures presently prevent the use of this technique.     

The control of hepatic glycogen metabolism in an in vitro model of sepsis

Culturing hepatocytes with a combination of LPS, TNF-α, IL-1β and IFN-γ resulted in an inhibition of glucose output from glycogen and prevented the repletion of glycogen in freshly cultured cells. The reduced glycogen mobilisation correlated with the lower cell glycogen content and reduced rate of glycogen synthesis from [U-¹⁴C]glucose rather than alterations in either total phosphorylase or phosphorylase a activity. There was no change in the percentage of glycogen exported as glucose nor the production of lactate plus pyruvate indicating that redistribution of the Gluc-6-P cannot explain the failure of the liver to export glucose. Although changes in glycogen mobilisation correlated with NO production, inhibition of NO synthase by inclusion of L-NMMA in the culture medium failed to prevent the inhibition of either glycogen accumulation or mobilisation by the proinflammatory cytokines, precluding the involvement of NO in this response. LPS plus cytokine treatment had no effect on total glycogen synthase activity although the activity ratio was lowered, indicative of increased phosphorylation. The inhibition of glycogen synthesis correlated with a fall in the intracellular concentrations of Gluc-6-P and UDP-glucose and in the absence of measured changes in kinase activity, it is suggested that the fall in Gluc-6-P reduces both substrate supply and glycogen synthase phosphatase activity. The fall in Gluc-6-P coincided with a reduction in total glucokinase and hexokinase activity within the cells, but no significant change in either the translocation of glucokinase or glucose-6-phosphatase activity. This demonstrates direct cytokine effects on glycogen metabolism independent of changes in glucoregulatory hormones.     

In vitro morphogenesis patterns from shoot apices of sugar cane are determined by light and type of growth regulator

The regeneration patterns of shoot apices derived from in vitro plants of four varieties of sugar cane in response to different growth regulators and light were evaluated. The cellular origin of the regeneration processes was also investigated. Explants cultivated on medium supplemented with NAA and incubated under light showed direct bud regeneration from cells of external layers of the ground parenchyma of the stem. Explants cultivated in the dark on medium supplemented with low concentrations of picloram (PIC) or 2,4D (4.0 and 4.5 μM, respectively) showed callus formation derived from the ground parenchyma of stem and development of preembryogenic masses derived from bundle sheath cells facing the phloem tissue of immature leaves. Somatic embryos at further developmental stages were visible following transfer to medium devoid of growth regulators and incubation under light. When incubated under light since the begining of the experiment, explants cultivated in the presence of higher PIC or 2,4D concentrations (40 and 22.6 μM, respectively) first displayed direct organogenesis from external layers of the ground parenchyma of the stem, followed by the development of organogenic calluses. Preembryogenic masses were also observed from bundle sheath cells of immature leaves. However, in contrast to the cultures pre-incubated in darkness for 30 days, the subsequent stages of embryo development were not detected. The regeneration efficiency of calluses induced by 2,4D and PIC was generally increased following desiccation in laminar flow or incubation on medium solidified with phytagel.     

Transport of sodium and chloride across earthworm skin in vitro

We present a new invertebrate model for the study of epithelial sodium transport in tight epithelia, the earthworm integument. Dissected segments of earthworm integument were mounted in modified Ussing chambers and perfused with either pond water (PW) or earthworm ringer solution (ERS) on the apical side. In order to investigate ion transport under near-in vivo physiological conditions, measurements were performed under current-clamp conditions by monitoring the transepithelial potential (V _T), as well as the transepithelial resistance (R _T). These were recorded continuously and the virtual short circuit current (I _SC) was calculated. The integument has a high transepithelial resistance (R _T=9,037±502 Ω cm² for PW, n=24, and 11,055±1,320 Ω cm² for ERS, n=32). V _T was −3.7±2.2 mV for PW (n=24) and −1.5±1.0 mV for ERS (n=32), and I _SC was −0.57±0.30 μA/cm² for PW (n=24) and −0.44±0.24 μA/cm² for ERS (n=32). Only under PW, but not under ERS conditions, was there a pronounced inhibition of I _SC by low doses of amiloride or its analogues phenamil and benzamil. The resistance of the paracellular pathway was found to be very high. The terrestrial oligochaete Lumbricus seems especially adapted to the environmental conditions because it has an ultra-tight integument and a very fast up- and down-regulation of apical Na⁺ channels.     

Assessment of polyembryony in lemon: rescue and in vitro culture of immature embryos

The 27 lemon cultivars analysed could be considered slightly or moderately polyembryonic, with 25 to 43% of seeds being polyembryonic and from 1.3 to 1.6 embryos per seed. On this basis, it is necessary to rescue zygotic embryos at an immature stage. Rescue and in vitro embryo development have been studied in two Citrus limon polyembryonic cultivars. Sucrose (50 and 70 g/l) was combined with Murashige and Skoog and Gamborg’s B5 media and tested for optimal growth response. An important effect of genotype was observed: embryos from cultivar ‘Eureka’ had greater survival, germination percentage, and radical development. While the sucrose concentration in the medium did not have an effect on germination, the medium affected the embryo survival and root development of the seedlings, Gamborg’s B5 medium giving the best results. The ability to form plants in vitro was affected by an increase of embryo developmental stage. The germination and seedling height were greater with embryos of seeds collected 135–150 days after anthesis.     

Analysis of different cell death processes of prepubertal rat oocytes in vitro

The processes of cell death were studied in vitro in populations of oocytes isolated from prepubertal rats. In order to identify apoptosis, the externalized phosphatidylserine was recognized with Annexin-V coupled to FITC and the fragmentation of DNA was demonstrated by means of electrophoresis. Oocytes were tested for autophagy by means of the incorporation of monodansylcadaverine and monitoring Lc3-I/Lc3-II by western blot. The expression of mRNA marker genes of autophagy and of apoptosis was studied by means of RT–PCR in pure populations of oocytes. Some oocytes expressed at least one of the following markers: caspase-3, lamp1 and Lc3. Some oocytes were positive to Annexin-V or to monodansylcadaverine. However, most of them were simultaneously positive to both markers. The relative frequency of oocytes simultaneously positive to markers of apoptosis and autophagy did not change in the different ages studied. The transformation of Lc3-I in Lc3-II was present in all populations of oocytes studied. The mRNAs for caspase-3, lamp1 and Lc3 were present in all populations of oocytes analyzed. Our results demonstrate that oocytes of rats from new born to prepubertal age are eliminated by means of three different cell death processes: apoptosis, autophagy and a mixed event in which both routes to cell death participate in the same cell.     

Phosphorylation of Recombinant Tristetraprolin In vitro

Tristetraprolin/zinc finger protein 36 (TTP/ZFP36) binds and destabilizes some proinflammatory cytokine mRNAs. TTP-deficient mice develop a profound inflammatory syndrome due to excessive production of proinflammatory cytokines. TTP gene expression is induced by various factors including insulin, cinnamon, and green tea extracts. Previous studies have shown that TTP is highly phosphorylated in vivo and multiple phosphorylation sites are identified in human TTP. This study evaluated the potential protein kinases that could phosphorylate recombinant TTP in vitro. Motif scanning suggested that TTP was a potential substrate for various kinases. SDS-PAGE showed that in vitro phosphorylation of TTP with p42 and p38 MAP kinases resulted in visible electrophoretic mobility shift of TTP to higher molecular masses. Autoradiography showed that TTP was phosphorylated in vitro by GSK3b, PKA, PKB, PKC, but not Cdc2, in addition to p42, p38, and JNK. These results demonstrate that TTP is a substrate for a number of protein kinases in vitro.     

Quantification of arabinogalactan proteins during in vitro morphogenesis induced by β-d-glucosyl Yariv reagent in Centaurium erythraea root culture

Arabinogalactan proteins (AGPs) are a family of highly glycosylated cell surface proteins located at the plasma membrane and plant cell wall. AGPs play important roles in plant growth and development. Yariv phenylglycoside (βGlcY), synthetic red-brown dye that specifically binds and precipitates AGPs, has been used for detection and quantification of AGPs in plant tissue. Graded concentrations of βGlcY (0–75 μM) were used to investigate the effect of this synthetic dye on induction of in vitro morphogenesis in Centaurium erythraea root culture on two nutrient media: ½MS and ½MS + IBA 1.0 μM. Regeneration of C. erythraea shoots on root explants was stimulated on both media supplemented with 25 μM βGlcY after 8 weeks in culture. Quantification of AGPs in different tissues of C. erythraea was determinate with single radial diffusion method. This work emphasizes clear effect of βGlcY on induction of morphogenesis in vitro in C. erythraea root culture.     

Modulations of rabbit erythrocyte ATPase activities induced by in vitro and in vivo exposure to ethanol

Alcohol intake is associated with numerous degenerative disorders, and the detrimental effects of alcohol may be due to its influence on plasma membrane and cellular transport systems. The aim of the present study was to compare in vitro and in vivo effects of ethanol on rabbit erythrocyte ATPase activities and correlate them with ethanol-induced oxidative stress. Age-matched male rabbits were given 5% ethanol in 2% sucrose solution, for 6 weeks ad libitum; control animals were given tap water. Daily intake of ethanol was 5 g/kg body weight; this experimental regimen resulted in an average serum ethanol concentration of 16.77 ± 2.00 mM/l. After this period, blood was collected, serum ethanol concentration was determined and erythrocyte membranes were prepared according to the method of Post et al. Activities of Na⁺/K⁺- and Mg²⁺-ATPases were determined. Thiobarbituric acid-reactive substance (TBARS) assay was used to detect levels of lipid peroxidation, a major indicator of oxidative stress. In vitro ethanol inhibits both Na⁺/K⁺-ATPase and Mg²⁺-ATPase, but Na⁺/K⁺-ATPase is more sensitive to the ethanol-induced inhibition. Increasing concentration of ethanol increased TBARS production, but significant difference was attained only at 5 and 12.5 mM of ethanol. Chronic ethanol consumption induced significant increase in Na⁺/K⁺- and Mg²⁺-ATPase activity, and TBARS production. Our results suggest that increased ATPase activity induced by chronic ethanol consumption is due to oxidative, induced modification of membrane phospholipids and proteins, which are responsible for inhibition of ATPase activity. Increased production of TBARS induced by in vitro exposure to ethanol is not the only factor that influences ATPases activity. Further research is needed to elucidate this relationship.     

Construction of two recombination yeast two-hybrid vectors by in vitro recombination

Recombination-based restrictionless, ligation-independent cloning has been proven to be advantageous over restriction digestion and ligation cloning. To utilize the recombination cloning and previously constructed two-hybrid cDNA libraries, a new Gateway yeast two-hybrid bait vector, pEZY202, and a new prey vector, pEZY45, were constructed. The two-hybrid vectors were generated by in vitro recombination using a protocol that can be easily adapted for the conversion of other existing vectors. The new vectors were used to assay the interaction between the WW domain of PQBP1 (PQBPww) and the WW domain binding protein WBP11. Both PQBPww and WBP11 were cloned into a Gateway donor vector by in vitro recombination. They were then subcloned into pEZY45 and pEZY202, respectively, by in vitro recombination. The binding between PQBPww and WBP11 was reported in a two-hybrid experiment using the new vectors. The results of testing the new vectors in combination with the original vectors indicated that the new bait vector could be used to screen cDNA libraries that are constructed using the original prey vectors.     

Estimation of Interaction Between Human Keratinocytes and Xenogenic Collagen in vitro

This paper describes the interaction observed between human keratinocytes and xenogenic collagen in vitro modified by HCl. Human keratinocytes were cultivated for 3–10 days, on modified and control support. Their growth, morphology and interaction with support were analyzed. It was found that on both control and experimental (modified) collagen cells proliferated in a similar way. Within 3–10 days, the culture became multilayered and mature and differentiation of cells was visible. Using electron microscope elements of basal membrane interacting with support were seen. On modified support processes of cells penetrating the support are occasionally seen. By use of the immunofluorescent, cytochemical techniques was found the presence of: BP-180 (antigen), β₄ integrin, laminin 5 and collagen IV, VII, VIIc. On the modified support the above listed elements appeared between 3 and 7 days of culture, whereas on the control between 7th and 10th days. On 10th day of culture, the presence of elements of basal membranes became less evident. Results give some hope for using xenogenic, modified collagen as support of keratinocytes culture in process of human skin engineering.     

In vitro and in vivo antioxidant activity of exopolysaccharide fractions from Bifidobacterium animalis RH

The aim of the study was to purify the exopolysaccharides (EPS) produced from Bifidobacterium animalis RH, which was isolated from the feces of Bama centenarians in Guangxi of China, and evaluate their antioxidant activities in vitro and in vivo. 2 fractions, a neutral EPS fraction (EPSa) and an acidic EPS fraction (EPSb), were obtained and compared for antioxidative activity. In vitro, they both showed remarkable inhibition effect on lipid peroxidation and strong DPPH radical scavenging activity, hydroxyl radical scavenging activity, superoxide radical scavenging activity, in which the last two were measured by the electron spin resonance (ESR). In vivo, EPSa and EPSb were orally administrated for 30 days in a d-galactose induced aged mice model. As results, they both could significantly increase the activities of SOD, CAT and total antioxidant capacity (TAOC) in serums and glutathione GST in livers. They also could inhibit significantly the formation of MDA in serums and livers, and reduce the activity of MAO and lipofuscin accumulation in mice brain. Moreover, EPSb exhibited much higher antioxidant activities than EPSa in vitro and in vivo. The results suggested that EPS fractions of Bifidobacterium animalis RH had direct and potent antioxidant activities.     

Light cytokinin interactions in shoot formation in epicotyl cuttings of Troyer citrange cultured in vitro

Ilumination did not affect the pathway of shoot regeneration at the cut edges of epicotyl explants of Troyer citrange (Moreira-Dias et al. 2000, 2001), but signigficantly affected the number of developed shoots and the response to exogenous cytokinins. Shoot regeneration at the apical end occurred through a direct organogenic pathway without callus formation. For explants incubated in the light, this regeneration did not require cytokinin addendum, but the number of shoots formed was significantly increased by benzyl adenine, but not by zeatin or kinetin. Incubation in the dark almost suppressed shoot formation at the apical end. The addition of benzyl adenine or kinetin, but not of zeatin, restored shoot formation in the dark to the value obtained in the light. At the basal end of the explants shoot regeneration occurred through an indirect organogenic pathway after the formation of a primary callus. In explants incubated in the light, callus formation and shoot growth was supported by a low (0.5–1 mg l⁻¹) benzyl adenine concentration and by zeatin. Kinetin did not support callus growth. Shoot formation was higher in the presence of benzyl adenine (0.5–1 mg l⁻¹) than of zeatin, but was inhibited by a high (5 mg l⁻¹) benzyl adenine concentration. Incubation in the dark increased callus growth and shoot formation at the basal cut as compared to explants incubated in the light. The three cytokinins tested supported callus growth and shoot formation in the dark, zeatin being the most effective and kinetin the least. In terms of number of shoots developed, the optimum cytokinin addendum depended on the pathway of organogenesis and the conditions of incubation. The maximum number of shoots developed at the apical end was obtained when the incubation was performed in the light in the presence of benzyl adenine. At the basal end, the optimal conditions were incubation in the dark in the presence of zeatin. It was not always possible to define an optimal cytokinin concentration as the curve concentration/response varied from experiment to experiment, which seemed unrelated to the endogenous cytokinin concentration in the explants.