Reaction specificities of the ε-ionone-forming lycopene cyclase from rice (Oryza sativa) elucidated in vitro

Lycopene cyclases responsible for the formation of ε-ionone rings (LCYe) mark a plant-specific bifurcation of carotenogenesis. We investigated purified rice LCYe (OsLCYe) in a liposome-based biphasic assay system. OsLCYe depends on reduced flavin cofactors stabilizing a transient state formed during the non-redox cyclization reaction. In contrast to OsLCYb, OsLCYe produces predominantly monocyclic products and monocyclic carotene intermediates are not suitable substrates. Determination of the OsLCYe reaction specificities and the combined use of OsLCYb allow the characterization of the reaction sequence leading to heterocyclic carotenoids. It was also found that 5-cis-lycopene, which was thought to be decisive for ε-cyclization, was not involved in the reaction, with OsLCYe acting as an exclusion filter for this naturally occurring isomer.     

Long-term in vitro generation of amoebocytes from the Indian horseshoe crab Tachypleus gigas (Müller)

Amoebocyte is the single type of cell circulating in the horseshoe crab hemolymph, which plays a major role in the defense system of the animal. Granules present in these cells are sensitive to nanogram quantities of bacterial endotoxins, which form the basis of the Limulus amoebocyte lysate (LAL) test. Normally, amoebocytes for the production of the LAL are collected by cardiac puncture; hence, development of the in vitro culture system for amoebocytes will reduce the variability of the lysate and help to conserve the 400 million-yr-old living fossil. In the present investigation we have attempted organ culture of gill flaps that have been shown to be the source of amoebocytes. The gill flaps were cultured at 28 degrees C on a rocker platform in a modified L-15 medium supplemented with 10% v/v horseshoe crab serum. This led to the release of amoebocytes outside the gill flaps for a period of 6-8 wk with a more or less steady number of amoebocytes during the weekly harvest. No significant difference was seen in the yield of amoebocytes from male and female horseshoe crabs. Confocal laser microscopy studies revealed significant difference in the size of amoebocytes released in vitro as compared with those obtained in vivo. Thus, we have optimized the culture conditions for the long-term generation of amoebocytes in vitro from the Indian horseshoe crab Tachypleus gigas by reducing the incidence of contamination, simulating in vivo conditions for the organ culture of gill flaps, and improvising the nutritional status using the modified L-15 medium, providing the desired osmolarity and pH.     

Assembly of microfibrils in vivo and in vitro from (1»3)-β-d-glucan synthesized by protoplasts of Saccharomyces cerevisiae

Polymer chains of (13)--d-glucan were dissolved with 1 M NaOH at 4° C from native microfibrillar protoplast nets. The chains associated into microfibrils during NaOH neutralization or dialysis. In contrast to the native microfibrils which are of uniform width individually (10 to 20 nm) and arranged in flat bundles, the microfibrils formed in vitro showed no band formation and consisted of fibrous spindle-shaped subunits of variable width or loose elementary fibrils about 1.7 nm wide. X-ray diagrams of native nets indicated a fairly high crystallinity and were different for wet and dry specimens. They corresponded to those of paramylon. Precipitated glucans produced diagrams different from the former and revealing a lower crystallinity especially with the dry samples.The X-ray pattern, combined with other data, allowed the precipitated microfibrils to be identified as aggregates of molecular strands composed each of three intertwined helical glucan chains. Since these triple helical chains are about 1.7 nm wide the elementary fibrils of this width can represent only single triple-helical strands. These helices have 7 glucose residues per turn and therefore a low symmetry which explains the poor crystallizing properties. The 7 membered helix represents a basic difference with the well crystallized native glucan which is built of highly symmetrical triple helices with 6 glucose residues per turn. Since 6₁ helical conformation is not formed in vitro at normal temperatures its generation in vivo must be due to the action of synthesizing enzymes at the protoplast membrane. The intertwining of these helices and crystallization of the strands are determined by their symmetry and physical properties of the chains. This characterizes the native microfibrils as products of self-assembly of enzymegenerated 6₁ helices.     

In vitro regeneration in chile pepper (Capsicum annuum L.) from ‘half-seed explants’

An in vitro regeneration protocol has been developed from half-seed explants of a mild (cv. New Mexico-6) and a pungent (cv. Rajpur Hirapur) chile pepper (Capsicum annuum L). Imbibed seeds were cut into two parts such that one portion contained the cotyledons and a part of the hypocotyl (part A) while the other part had the proximal part of the hypocotyl and the radicle (part B). These explants were cultured on MS medium with or without cytokinins (KIN, BA, ZEA, 2iP). Cytokinins dramatically increased both the percentage of explants forming buds as well as the number of buds per explant, and also hastened the rate of bud production. The relative efficacy of cytokinins in inducing the formation of leafy buds varied in the two cultivars. However, the best response was observed with ZEA in both cultivars. The highest percentage of bud formation was recorded after presoaking part B explants for 72 hours. The elongation growth of leafy buds was severely inhibited in the continuous presence of high concentrations of cytokinins, and frequently the buds became quite thick, ill-defined and vitreous. Within 3–5 weeks of transfer to Magenta boxes containing vermiculite and soil (1:3), 70–85% of the rooted hypocotyls developed 1–2 elongated shoots. Following transfer to pots, these plantlets grew into normal plants.Abbreviations BA benzylamino purine - IAA indole-3-acetic acid - 2iP 6--dimethyl (allyl) amino purine - KIN kinetin - MS Murashige and Skoog medium - NAA napthaleneacetic acid - ZEA zeatin     

Alloantigenic variant selection in vitro from a mouse lymphoma — Sarcoma hybrid

TwoH-2 ^a +/H-2 ^s — alloantigenic loss variants have been obtained from anH-2 ^a +/H-2 ^s + mouse lymphoma/sarcoma hybrid, YACIR/MSWBS, by in vitro selection, with repeated exposure toanti-H-2 ^s serum and complement. Loss of the MSWBS derivedH-2 ^s complex was associated with the loss of the morphologically distinguishable, translocated MSWBS derived chromosomes 17. The morphologically normal YACIR-derived chromosomes 17 were maintained.     

IL-1ra delivered from poly(lactic-co-glycolic acid) microspheres attenuates IL-1β-mediated degradation of nucleus pulposus in vitro

Introduction

Inflammation plays a key role in the progression of intervertebral disc degeneration, a condition strongly implicated as a cause of lower back pain. The objective of this study was to investigate the therapeutic potential of poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with interleukin-1 receptor antagonist (IL-1ra) for sustained attenuation of interleukin-1 beta (IL-1β) mediated degradative changes in the nucleus pulposus (NP), using an in vitro model.

Methods

IL-1ra was encapsulated in PLGA microspheres and release kinetics were determined over 35 days. NP agarose constructs were cultured to functional maturity and treated with combinations of IL-1β and media conditioned with IL-1ra released from microspheres at intervals for up to 20 days. Construct mechanical properties, glycosaminoglycan content, nitrite production and mRNA expression of catabolic mediators were compared to properties for untreated constructs using unpaired Student''s t-tests.

Results

IL-1ra release kinetics were characterized by an initial burst release reducing to a linear release over the first 10 days. IL-1ra released from microspheres attenuated the degradative effects of IL-1β as defined by mechanical properties, glycosaminoglycans (GAG) content, nitric oxide production and mRNA expression of inflammatory mediators for 7 days, and continued to limit functional degradation for up to 20 days.

Conclusions

In this study, we successfully demonstrated that IL-1ra microspheres can attenuate the degradative effects of IL-1β on the NP for extended periods. This therapeutic strategy may be appropriate for treating early-stage, cytokine-mediated disc degeneration. Ongoing studies are focusing on testing IL-1ra microspheres in an in vivo model of disc degeneration, as a prelude to clinical translation.     

Induction of methionine-sulfoxide reductases protects neurons from amyloid β-protein insults in vitro and in vivo

Self-assembly of amyloid β-protein (Aβ) into toxic oligomers and fibrillar polymers is believed to cause Alzheimer's disease (AD). In the AD brain, a high percentage of Aβ contains Met-sulfoxide at position 35, though the role this modification plays in AD is not clear. Oxidation of Met(35) to sulfoxide has been reported to decrease the extent of Aβ assembly and neurotoxicity, whereas surprisingly, oxidation of Met(35) to sulfone yields a toxicity similar to that of unoxidized Aβ. We hypothesized that the lower toxicity of Aβ-sulfoxide might result not only from structural alteration of the C-terminal region but also from activation of methionine-sulfoxide reductase (Msr), an important component of the cellular antioxidant system. Supporting this hypothesis, we found that the low toxicity of Aβ-sulfoxide correlated with induction of Msr activity. In agreement with these observations, in MsrA(-/-) mice the difference in toxicity between native Aβ and Aβ-sulfoxide was essentially eliminated. Subsequently, we found that treatment with N-acetyl-Met-sulfoxide could induce Msr activity and protect neuronal cells from Aβ toxicity. In addition, we measured Msr activity in a double-transgenic mouse model of AD and found that it was increased significantly relative to that of nontransgenic mice. Immunization with a novel Met-sulfoxide-rich antigen for 6 months led to antibody production, decreased Msr activity, and lowered hippocampal plaque burden. The data suggest an important neuroprotective role for the Msr system in the AD brain, which may lead to development of new therapeutic approaches for AD.     

Distribution of endogenous gibberellins in dwarf and giant off-types banana (Musa AAA,cv.‘Grand nain’) plants from in vitro propagation

GA₃ and GA₂₀ were quantified in leaf extracts from true-to-type and somaclonal variants (dwarf and giant) of Musa AAA cv. Grand nain by GC-MS-SIM after purification on reverse- and normal-phase HPLC and detection by ELISA with GA₃ antibodies and by a dwarf rice bioassay. GA₃ concentration in dwarf plants was 811 ng g^–1 dry weight. For normal and giant plants, the endogeneous GA₃ levels were respectively 3.6 and 4.6 times higher. The GA₂₀ concentration in the giant plant was 68 ng g^–1 of dry weight. This concentration was, respectively, 4.6 and 7.3 times higher than those of normal and dwarf plants. These results suggest that the somaclonal variations affecting banana plant height are associated with modifications in GA metabolism.Abbreviations HPLC High Performance Liquid Chromatography - GC-MS Gas Chromatography-Mass Spectrometry - SIM Selected Ion Monitoring - GA Gibberellin - BSA Bovine Serum Albumin - PB Phosphate Buffer     

Olfactory epithelium progenitors: insights from transgenic mice and in vitro biology

The rodent olfactory epithelium (OE) is capable of prolonged neurogenesis, beginning at E10 in the embryo and continuing throughout adulthood. Significant progress has been made over the last 10 years in revealing the signals that drive induction, differentiation and survival of its Olfactory Receptor Neurons (ORNs). Our understanding of the identity of specific progenitors or precursors that respond to these signals is, however, less well developed, and the search is still on for the elusive, definitive multipotent neuro-glial OE "Stem cell". Here, we review several lines of evidence that support the existence of a heterogeneous population of neural and glial progenitors in the olfactory mucosa, and highlight the differences in the identity and activity of progenitors found in the embryonic and adult OE. In particular, we show how recent advances in mouse transgenesis, and in the development of in vitro assays of progenitor activity, have helped to demonstrate the existence of multiple classes of olfactory mucosa-based progenitors.     

Propagation of Musa textilis Neé plants from apical meristem slices in vitro

Cultures were propagated from apical meristem slices of Musa textilis plants. They were cultured in vitro in light on either MS medium containing BAP (10 mg/l), but without edamin or MS mineral salts supplemented with 100 mg/l each of inositol, tyrosine, ascorbic acid; 150 mg/l citric acid; 2 mg/l cysteine; 0.4 mg/l thiamine HCl; 3% sucrose and 0.5–0.8% agar. Shoot initials were induced using media containing 5–10 mg/l BAP. Further promotion of shoot induction was achieved when BAP (1–3 mg/l) was supplemented with either NAA (1 mg/l) or adenine sulphate (80–160 mg/l). Shoot initials were multiplied on media containing 3–5 mg/l BAP, 0.1 mg/l IBA and 160–200 mg/l adenine sulphate. Plantlets generated roots on media without adenine sulphate but containing 1–1.5% sucrose and either NAA (0.1–1 mg/l) or IBA (2–10 mg/l). Plantlets were transferred into pots in the greenhouse 7 days after rooting.     

Effects of heavy metal (Pb) on arbuscular mycorrhizal fungi in vitro

The responses of Ri-TDNA-transformed roots and arbuscular mycorrhizal fungi established on Ri-TDNA-transformed roots to lead-amended media was investigated in vitro. At exposure to increasing concentrations of lead (2–10 mg/l[ppm]), three Ri-TDNA-transformed root clones viz., Swa, Swb and Swc, exhibited profuse growth. At exposure to increasing concentrations of lead (0.1–5 ppm), the dual cultures of Ri-TDNA-transformed roots and arbuscular mycorrhizal fungi., Glomus lamellosum/Swa, Glomus intraradices/Swb and Glomus proliferum/Swc, exhibited tolerance to 5 ppm of lead. When subjected to one physiological stress, either exposure to Pb or inoculation with AM fungi, Ri-TDNA-transformed root clones exuded more phenols in the growth medium than retained in the roots. When subjected to dual physiological stress, mycorrhizal Ri-TDNA-transformed roots growing on Pb-enriched medium, the total phenol content increased in the roots and exudation into the medium decreased.     

Do western boxelder bugs sunbathe for sanitation? Inferences from in vitro experiments

When western boxelder bugs, Boisea rubrolineata (Barber) (Hemiptera: Rhopalidae), form aggregations in warm sunlight, they release from their posterior dorsal abdominal gland an odorous blend of monoterpenes with heretofore unknown biological function. In laboratory analyses and experiments, we show that bugs in warm sunlight, but not in shade, exude and spread copious amounts of monoterpenes onto their cuticle. These monoterpenes do not serve as a pheromone, but rather as a means of sanitation. They inhibit germination of conidia of the fungal pathogen Beauveria bassiana (Bals.‐Criv.) Vuill. (Hypocreales) as well as halt the growth of germinated spores. This prophylactic defense against pathogens appears adaptive for phytophagous insects, like B. rubrolineata, that are prone to infections by microbes thriving on leaf surfaces and in the insects' overwintering microhabitat.     

Trace Fossils from the Brioverian (Ediacaran–Fortunian) in Brittany (NW France)

The Ediacaran–Fortunian ichnofauna from Central Brittany (NW France) is revised for the first time since the pioneering work by Lebesconte at the end of the 19th century. The study is based on fossils from the type-localities of the historical Brioverian taxa Montfortia (traces from Montfort-sur-Meu) and Neantia (wrinkle surfaces from Néant-sur-Yvel), and on two new outcrops from Saint-Gonlay. The ichnofossil assemblage includes Helminthoidichnites tenuis, Helminthopsis tenuis, Gordia marina, Palaeophycus tubularis, ?Neonereites uniserialis, and ?Spirodesmos archimedeus. Locally, the grazing traces are associated with wrinkle surfaces considered as microbially induced sedimentary structures (MISS). The sedimentological characteristics of these deposits correspond to a shelf marine environment. Both U-Pb datings on detrital zircon and ichnostratigraphic criteria tend toward an Ediacaran age of the fossiliferous deposits, but we cannot exclude the possibility of a Fortunian age at the present state of knowledge.     

Cytotoxic functions and susceptibility to apoptosis of human CD56(bright) NK cells differentiated in vitro from CD34? hematopoietic progenitors

Cytotoxic functions and susceptibility to apoptosis are crucial aspects of NK cells suitable to counter cancer after infusion in oncologic patients. To test the feasibility and the usefulness of infusing in vitro generated NK cells, these two features were investigated in NK cells developed in vitro from CD34? hematopoietic progenitors. Purified CD34? cells were cultured for 15-30 days with FLT-3 ligand (FLT3-L) and IL-15 with or without IL-21. To induce terminal differentiation, NK cells were cultured for further 15 days with IL-15, IL-21, or their combination. A CD56(dim) /CD16? NK subset, expressing high level of perforin, granzymes, and LFA-1, appeared early in cultures with FLT3-L, IL-15, and IL-21, but it quickly died, indicating its predisposition to apoptosis. On the contrary, CD56(bright) NK cells generated after 30 days of culture with FLT3-L plus IL-15 did not show a considerable apoptosis, nevertheless only a subset of these cells expressed granzyme-B, perforin, LFA-1, and CD94-CD159a heterodimer, indicating a functional immaturity. Interestingly, further 15 days of culture with IL-21 plus IL-15 did not induce the generation of CD56(dim) cells from the CD56(bright) subset and actually inhibited IL-15-induced maturation/activation of this latter subset. In fact, IL-15 alone upregulated granzyme-B, TRAIL, Fas ligand, CD94-CD159a, LFA-1, CD16, KIRs, and TRAIL-R2 on CD56(bright) NK cells. Our results suggest that during differentiation CD56(bright) NK cells, similarly to mature activated NK cells, become highly cytotoxic and are relatively resistant to apoptosis induced by TNF family members.     

Synthetic biotinylated tetra β(1→5) galactofuranoside for in vitro aspergillosis diagnosis

The synthesis of a tetra ??(1→5) galactofuranoside was achieved using a thioglycoside donor with a methyl tert-butyl phenyl thio leaving group. This tetrasaccharide was conjugated to biotin and validated as antigen with the monoclonal antibody used for clinical detection of Aspergillus fumigatus galactomannan on streptavidin-coated microplates. Then we have shown its ability to detect antibodies associated with A. fumigatus induced disease by using sera from patients with Allergic broncho-pulmonary aspergillosis (ABPA) and correlated the results of antibody detection with those gained with a commercially available diagnostic test.     

The simplest method for in vitro β-cell production from human adult stem cells

Diabetes mellitus is a challenging autoimmune disease. Biomedical researchers are currently exploring efficient and effective ways to solve this challenge. The potential of stem cell therapies for treating diabetes represents one of the major focuses of current research on diabetes treatment. Here, we have attempted to differentiate adult stem cells from umbilical cord blood-derived mesenchymal cells (UCB-MSC), Wharton's jelly-derived mesenchymal stem cells (WJ-MSC) and amniotic epithelial stem cells (AE-SC) into insulin-producing cells. The serum-free protocol developed in this study resulted in the differentiation of cells into definitive endoderm, pancreatic foregut, pancreatic endoderm and, finally, pancreatic endocrine cells, which expressed the marker genes SOX17, PDX1, NGN3, NKX6.1, INS, GCG, and PPY, respectively. Detection of the expression of the gap junction-related gene connexin-36 (CX36) using RT-PCR provided conclusive evidence for insulin-producing cell differentiation. In addition to this RT-PCR result, insulin and C-peptide protein were detected by immunohistochemistry and ELISA. Glucose stimulation test results showed that significantly greater amounts of C-peptide and insulin were released from differentiated cells than from undifferentiated cells. In conclusion, the methods investigated in this study can be considered an effective and efficient means of obtaining insulin-producing cells from adult stem cells within a week.     

In vitro studies on calcium absorption from the gastrointestinal tract in␣small ruminants

Unidirectional flux rates of Ca²⁺ across gastrointestinal tissues from sheep and goats were measured in vitro by applying the Ussing-chamber technique. Except for the sheep duodenum, mucosal to serosal Ca²⁺ flux rates (J _ms) exceeded respective flux rates in the opposite direction (J _sm) in both species and in all segments of the intestinal tract. This resulted in net Ca²⁺ flux rates␣(J _net = J _ms − J _sm) ranging between −2 and 9 nmol · cm⁻² · h⁻¹ in sheep and between 10 and 15 nmol cm⁻² · h⁻¹ in goats. In sheep, only J _net in jejunum, and in goats, J _netin duodenum and jejunum were significantly different from zero. Using sheep rumen wall epithelia, significant J _net of Ca²⁺ of around 5 nmol · cm⁻² · h⁻¹ could be detected. Since the experiments were carried out in the absence of an electrochemical gradient, significant net Ca²⁺ absorption clearly indicates the presence of active mechanisms for Ca²⁺ transport. Dietary Ca depletion caused increased calcitriol plasma concentrations and induced significant stimulations of net Ca²⁺ absorption in goat rumen. J _net of Ca²⁺ across goat rumen epithelia was significantly reduced by 1 mmol · l ⁻¹ verapamil in the mucosal buffer solution. In conclusion, there is clear evidence for the rumen as a main site for active Ca²⁺ absorption in small ruminants. Stimulation of active Ca²⁺ absorption by increased plasma calcitriol levels and inhibition by mucosal verapamil suggest mechanistic and regulatory similarities to active Ca²⁺ transport as described for the upper small intestines of monogastric species. Accepted: 31 July 1996     

A review on the “in vitro” culture of freshwater mussels (Unionoida)

Many Unionoida are considered to be extinct, endangered, or of special concern. These bivalves have complex life cycle stages that limit successful culture. In nature, the larvae (glochidia) of these bivalves must successfully parasitize a host (mainly fish) in order to metamorphose into juveniles. The two artificial methods used to obtain juvenile freshwater mussels in laboratory are either by induced attachment to host fish or by in vitro culture of glochidia. This article is focused on the in vitro method that represents a novel and alternative process to fish infestation, offering the ability to obtain larger numbers of juveniles without the need for host fishes and reducing the overall costs of propagation. In vitro culture requires a medium which fulfills the nutritional needs of each glochidia species and avoids microbial contamination. Recently, this methodology has presented excellent results with survival and transformation rates up to 94% using host fish plasma. High efficiencies on growth, and survival rates (84%) of juvenile freshwater bivalve Hyriopsis myersiana (Lea, 1856) up to 120 days were obtained when reared in adequate recirculating aquacultural systems using a very specific diet. More research is still needed to demonstrate successful propagation, mainly concerning the media nutritional composition to increase glochidia transformation and juvenile quality.