Accelerated Polymer Biodegradation of Risperidone Poly(d, l-Lactide-Co-Glycolide) Microspheres

The influence of a tertiary amine, namely risperidone (pKa = 7.9) on the degradation of poly(d, l lactide-co-glycolide) (PLGA) microspheres was elucidated. Risperidone and blank microspheres were fabricated at two lactide/glycolide ratios, 65:35 and 85:15. The microspheres were characterized for drug loading by high-performance liquid chromatography, particle size by laser diffractometry, and surface morphology by scanning electron microscopy. Polymer degradation studies were carried out with drug-loaded microspheres and blank microspheres in presence of free risperidone in 0.02 M PBS containing 0.02% Tween®80 at 37°C. Molecular weight was monitored by gel permeation chromatography. Risperidone and blank microspheres had similar size distribution and were spherical with a relatively nonporous smooth surface. The presence of risperidone within the microspheres enhanced the hydrolytic degradation in both polymeric matrices with faster degradation occurring in 65:35 PLGA. The molecular weight decreased according to pseudo-first-order kinetics for all the formulations. During the degradation study, the surface morphology of drug-loaded microspheres was affected by the presence of risperidone and resulted in shriveled microspheres in which there appeared to be an intrabatch variation with the larger microspheres being less shriveled than the smaller ones. When blank microspheres were incubated in free risperidone solutions, a concentration-dependent effect on the development of surface porosity could be observed. Risperidone accelerates the hydrolytic degradation of PLGA, presumably within the microenvironment of the drug-loaded particles, and this phenomenon must be taken into consideration in designing PLGA dosage forms of tertiary amine drugs.Key words: mass loss, microencapsulation, PLGA microspheres, polymer degradation, risperidone, tertiary amine drug     

Selective delivery of rifampicin incorporated into poly(DL-lactic-co-glycolic) acid microspheres after phagocytotic uptake by alveolar macrophages, and the killing effect against intracellular Mycobacterium bovis Calmette-Guérin

Macrophages and their phagocytotic abilities play a dominant role for defense against infected organisms. However, Mycobacterium tuberculosis can survive in the phagosomes of macrophages. In this study, the effective delivery of a drug and the killing effect of tubercle bacilli within macrophages were investigated utilizing the phagocytotic uptake of rifampicin (RFP) that had been incorporated into poly(DL-lactic-co-glycolic) acid (PLGA) microspheres. The microspheres were composed of PLGA that had a monomer ratio (lactic acid/glycolic acid) of either 50/50 or 75/25. They had molecular weights from 5000 to 20,000, and diameters of 1.5, 3.5, 6.2 and 8.9 microm. The most significant factor for phagocytotic activity of macrophages was the diameter of the microspheres. By contrast, molecular weight and monomer ratio of PLGA did not influence phagocytosis. The amount of RFP delivered into cells was also investigated. RFP-PLGA microspheres composed of PLGA with a molecular weight of 20,000 and monomer ratio of 75/25 showed the highest amount of delivery (4 microg/1 x 10(6) cells). Fourteen days after infection, the survival rate of treated intracellular bacilli was 1% when compared with untreated cells. There was almost no killing effect of free RFP (4 or 15 microg/ml) on intracellular bacilli. In vivo efficacy of RFP-PLGA was also examined in rats infected with M. tuberculosis Kurono. Intratracheal administration of RFP-PLGA microspheres was shown to be superior to free RFP for killing of intracellular bacilli and preventing granuloma formation in some lobes. These results suggest that phagocytotic activity could be part of a new drug delivery system that selectively targeted macrophages.     

Topography of the enteric nervous system in Peyer's patches of the porcine small intestine

The mechanisms of intercommunication between the immune and nervous systems are not fully understood. In the case of the intestine, the enteric nervous system is involved in the regulation of immune responses. It was therefore decided to employ immunohistochemical techniques to investigate the structural organization of the enteric nervous system in Peyer's patches of the porcine small intestine. Using antibodies against various nervous system-specific markers (protein gene product 9.5, neuron-specific enolase, neurofilament 200, S-100 protein and the glial fibrillary acidic protein), an intimate and specific structural association could be demonstrated between enteric nerves and the compartments of Peyer's patches: follicles, interfollicular regions and domes. Peyer's patches have a close topographical relationship to the two submucosal plexuses. Enteric nerves are located around the follicle in the interfollicular area — the so-called traffic area-and in the dome area, which plays an important role in the uptake and presentation of antigens.     

Occluding junctions in the epithelia of the gut-associated lymphoid tissue (GALT) of the rabbit ileum and caecum

Summary The zonulae occludentes of the dome epithelia and adjacent non-dome epithelia in four locations of the gut-associated lymphoid tissue (GALT) in the rabbit ileum and caecum (Peyer's patches, sacculus rotundus, caecal lymphoid patches, appendix) were studied in freeze-fracture replicas. In all locations the zonulae occludentes of the dome epithelium are composed of more junctional strands than in the corresponding non-dome epithelium. In the dome epithelia of Peyer's and caecal lymphoid patches the zonulae occludentes show considerable structural variation; the number of superimposed strands is 10 (range 5–18). In the dome epithelia of sacculus rotundus and appendix, in addition to zonulae occludentes, extended networks of junctional strands (fasciae occludentes) are present particularly between M-cells and enterocytes. The zonulae occludentes consist of 8 to 9 (range 5–15) superimposed strands; the fasciae occludentes extend up to a depth of 20m on the lateral membranes. The presence of the fasciae occludentes correlates with the appearance of regularly shaped clusters of lymphocytes, which are most developed in the dome epithelia of sacculus rotundus and appendix. These results suggest (1) that in contrast to the dome epithelia of Peyer's and caecal lymphoid patches those of sacculus rotundus and appendix are compartmentalized, and (2) that the mobility of lymphocytes and diffusion of antigens in the dome epithelia of sacculus rotundus and appendix is restricted.     

Validation and quantitation of an in vitro M-cell model

This study has evaluated an in vitro model of the follicle-associated epithelia that overlie Peyer's patches of the small intestine. The model shares many phenotypic characteristics of M cells in vivo. Co-cultures of the human adenocarcinoma cell line Caco-2 and freshly isolated Peyer's patch cells were established. Fluorescence microscopy and quantitative image analysis were used to validate the model against known markers of M-cell phenotype. Apical expression of alkaline phosphatase was down-regulated in co-cultures and villin was re-distributed from the apical membrane to the cytoplasm. alpha5beta1 integrin was found on the apical surfaces of the monolayers and B and T lymphocytes integrated into the monolayers. Particle transport was temperature-dependent in co-cultures, indicating that a transcytotic route was responsible. This model provides opportunities to study factors that influence M-cell development, assess putative Peyer's patch targeting in oral vaccine technologies, and study intestinal uptake in vitro.     

Immune cells associated with M cells in the follicle-associated epithelium of Peyer's patches in the rat

Summary Follicle-associated epithelium of Peyer's patches can be differentiated from nearby villous epithelium by the presence of M cells which are antigen-sampling epithelial cells, and by an increase in intraepithelial lymphocytes that are in close contact with M cells. The phenotype of the immune cells close to the M cells of the follicle-associated epithelium of rat Peyer's patches was determined by immunohistochemistry and compared with that of the intra-epithelial lymphocytes of the villous epithelium. Lymphoid T cells, predominantly of the cytotoxic/suppressor phenotype, were observed both in follicle-associated epithelium and in villous epithelium. Lymphoid B cells, mainly immunoblasts and plasma cells containing intracytoplasmic IgM, were present only in the follicle-associated epithelium, near M cells. Macrophages were also present, in contact with M cells, in follicle-associated epithelium, but not in villous epithelium. In addition, M cells bore Ia molecules on their apical membranes. These findings reinforce the concept of immune specialization of the follicle-associated epithelium, by demonstrating that this epithelium contains all the effector cells of immune responses.     

Excitatory Sulfur-Containing Amino Acid-Induced Release of [3H]GABA from Rat Olfactory Bulb

The effect of L-cysteine sulfinic acid (CSA) and L-homocysteic acid (HCA) on the release of tritiated -amino butyric acid ([³H]GABA), from the external plexiform layer (EPL) of the rat olfactory bulb, was compared with that of glutamate. These amino acids induced release of GABA was strongly inhibited by the glutamate uptake blocker, pyrrolidine-2,4-dicarboxylate (2,4,PDC) (50 M), while it was not inhibited by the specific GABA uptake blockers nipecotic acid (0.5 mM) or NO-711 (5M). Only the HCA induced GABA release was 60% inhibited by -alanine (0.5 mM), a glial GABA uptake blocker and 78% by the NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (AP-5) (100 M). The non-NMDA receptor antagonists 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline (CNQX) up to 500 M had no effect on HCA or CSA stimulated GABA release. These results bring evidence for an excitatory role of HCA and CSA together with glutamate on GABAergic neuronal or glial elements, in the olfactory bulb. This role could be mediated through the reversal of the glutamate or/and the glial GABA transporter and through the activation of a NMDA type receptor.     

Clasmatosis: Certain qualitative and quantitative peculiarities of this phenomenon in the lymphoid organs of the rat

Summary Clasmatosis in the spleen, thymus, submaxillary lymph nodes, Peyer's patches and bone marrow has been studied in Wistar male rats by standard cytological methods, and a method of obtaining a fraction of cytoplasmic fragments (leptons) from the spleen has been elaborated. A conjectural scheme of the successive stages of this phenomenon has been drawn up; according to this scheme, the lepton detached from the clasmatocyte undergoes a series of transformations and again splits off a fragment from itself (secondary clasmatosis).Micrometric measurements of the diameters of free lymphocytes, clasmatocytes, and bound and free leptons have made it possible to establish that: 1. the size of the lepton does not depend on the size of its producer cell; 2. the largest percentage of clasmatocytes occurs among cells with a diameter of 9 (about 35%); 3. the lymphocytes of the spleen are smaller than the clasmatocytes of the same organ, and the free leptons are smaller than the bound leptons. The assumption is made that this difference is due in the former case to clasmatosis itself, and in the latter to formation of secondary leptons.Clasmatosis is traced in all stages of differentiation of lymphoid cells, which lose their pyroninophilic granularity along with their loss of cytoplasm.The different lymphoid organs have been compared according to their relative content of clasmatocytes and free leptons. The second, more distinct index increases in the following succession: thymus, submaxillary lymph nodes, spleen, Peyer's patches. The difference between the first and the last is 15.In the bone marrow of intact rats clasmatosis is practically absent.The results have been discussed in relation to the possible connection of clasmatosis with the differentiation of lymphoid cells.The authors are very grateful to N. S.Nikishova for her help in the cytological part of this work and to M. L.Kopnova for the microphotographs.     

In vitro organogenesis of elephant apple (Feronia limonia)

Elephant apple (Feronia limonia L.). was micropropagated on MS medium containing 4.4 M benzyladenine and 4.6 M kinetin using cotyledon explants taken from in vitro-grown seedlings. Adventitious buds formed on the cotyledon developed into shoots that were rooted in half-strength MS medium containing 0.57 M indoleacetic acid and 0.49 M indolebutyric acid. Plants were successfully established in soil.Abbreviations BA 6-benzyladenine - IAA 3-indoleacetic acid - IBA 3-indolebutyric acid - MS Murashige & Skoog     

PLGA Microsphere-Mediated Growth Hormone Release Hormone Expression Induces Intergenerational Growth

To improve animal growth, growth hormone-releasing hormone (GHRH) expression vectors that maintain constant GHRH expression can be directly injected into muscles. To deliver the GHRH expression vectors, biodegradable microspheres have been used as a sustained release system. Although administering GHRH through microspheres is a common practice, the intergenerational effects of this delivery system are unknown. To investigate the intergenerational effects of polylactic-co-glycolic acid (PLGA) encapsulated plasmid-mediated GHRH supplements, pCMV-Rep-GHRH microspheres were injected into pregnant mice. Growth and expression of GHRH were measured in the offspring. RT-PCR and immunohistochemistry reveal GHRH expression 3–21 days post-injection. The proportion of GH-positive cells in the GHRH treated offspring was 48.2% higher than in the control group (P < 0.01). The GHRH treated offspring were 6.15% (P < 0.05) larger than the control offspring. At day 49 post-injection, IGF-I serum levels were significantly higher in the treatment group than in the control group. This study confirms that intramuscular expression of GHRH mediated by PLGA microspheres significantly enhances intergenerational growth.     

Comparison between (RS)-nipecotic acid and GABA transport in cultured astrocytes: Coupling with two sodium ions

The sodium ion dependency of the uptake of (RS)-nipecotic acid into astrocytes in primary cultures has been studied by performing kinetic analysis at different sodium ion concentrations (16–151 mM).V _max of the saturable component of the astroglial (RS-nipecotic acid uptake is clearly affected by the sodium ion concentration whereasK _m surprisingly remains unaffected. At high (RS)-nipecotic acid concentrations (50 M), uptake rates as a function of the sodium ion concentration were clearly sigmoid. This sigmoid shape was not obvious at lower concentrations of (RS)-nipecotic acid. The calculated Hill coefficients corresponding to all (RS)-nipecotic acid concentrations studied were approximately two. From these results it is concluded that (RS)-nipecotic acid uptake into astrocytes in primary cultures, like astroglical GABA uptake, requires the binding of at least two sodium ions per (RS)-nipecotic acid molecule transported.     

Characteristics of Cl−-dependentl-[35S]cysteic acid transport into rat brain synaptic membrane vesicles

Uptake ofl-[³⁵S]cysteic acid (L-CA) in rat synaptic membrane vesicles was investigated. Preincubation with either 10 mMl-glutamic acid (L-Glu), 25 mM L-CA, 10 mMdl-homocysteic acid, or 25 mMdl-2-amino-4-phosphonobutyrate on membrane vesicles enhanced L-[³⁵S]CA and L-[³H]Glu uptake. Na⁺ (5 mM) and omission of Cl^– from the assay medium decreased L-[³⁵S]CA uptake into both 10 mM L-Glu-loaded and non-loaded membrane vesicles. The anion transport blockers, 4-acetamide-4-isothiocyano-2,2-disulfonic acid stibene (SITS) and 4,4-diisothiocyano-2,2-disulfonic acid stilbene (DIDS), inhibited L-[³⁵S]CA uptake in a dose-dependent manner. The maximal uptake rate for L-[³⁵S]CA was decreased by 50 M SITS, while the apparent Km value of L-CA was not changed. SITS increased the EC₅₀ value of Cl^– for L-[³⁵S]CA uptake from 5 mM to 10 mM with reduction of the maximal effect. These results suggested that L-[³⁵S]CA uptake into synaptic membrane vesicles was mediated by a SITS-sensitive hetero-exchange transport with non-labeled substrates.Abbreviations SITS 4-Acetamide-4-isothiocyano-2,2-disulfonic acid stilbene - DIDS 4,4-Diisothiocyano-2,2-disulfonic acid stilbene - CA Cysteic acid - APB 2-Amino-4-phosphonobutyrate - CSA Cysteine sulfinic acid - EGTA Ethyleneglycol bis(aminoethylether) tetraacetate - GABA -Aminobutyric acid     

Different modes of sodium-D-glucose cotransporter-mediated D-glucose uptake regulation in Caco-2 cells

We recently reported that a considerable amount of the sodium-D-glucose cotransporter SGLT1 present in Caco-2 cells, a model for human enterocytes, is located in intracellular compartments attached to microtubules (Kipp H, Khoursandi S, Scharlau D, and Kinne RKH. Am J Physiol Cell Physiol 285: C737–C749, 2003). A similar distribution pattern was also observed in enterocytes in thin sections from human jejunum, highlighting the validity of the Caco-2 cell model. Fluorescent surface labeling of live Caco-2 cells revealed that the intracellular compartments containing SGLT1 were accessible by endocytosis. To elucidate the role of endosomal SGLT1 in the regulation of sodium-dependent D-glucose uptake into enterocytes, we compared SGLT1-mediated D-glucose uptake into Caco-2 cells with the subcellular distribution of SGLT1 after challenging the cells with different stimuli. Incubation (90 min) of Caco-2 cells with mastoparan (50 µM), a drug that enhances apical endocytosis, shifted a large amount of SGLT1 from the apical membrane to intracellular sites and significantly reduced sodium-dependent -[¹⁴C]methyl-D-glucose uptake (–60%). We also investigated the effect of altered extracellular D-glucose levels. Cells preincubated (1 h) with D-glucose-free medium exhibited significantly higher sodium-dependent -[¹⁴C]methyl-D-glucose uptake (+45%) than did cells preincubated with high D-glucose medium (100 mM, 1 h). Interestingly, regulation of SGLT1-mediated D-glucose uptake into Caco-2 cells by extracellular D-glucose levels occurred without redistribution of cellular SGLT1. These data suggest that, pharmacologically, D-glucose uptake can be regulated by a shift of SGLT1 between the plasma membrane and the endosomal pool; however, regulation by the physiological substrate D-glucose can be explained only by an alternative mechanism. endosomes; enterocytes     

The fine structure of the polian vesicles of holothurians

Summary The Polian vesicles are tubules of variable length consisting of four layers: an external peritoneal epithelium, a connective tissue layer, a muscular layer, and an inner epithelium. The two simple epithelia produce a polysaccharide in their Golgi complexes. Their cellular junctions consist of an extensive zonula adhaerens, and an apical septate desmosome. Their surfaces possess microvilli and cilia. A basement membrane is lacking. The muscular layer is composed of paramyosin fibers. The connective tissue layer contains abundant collagen fibers that have a period of 640 Å and are embedded in an acid mucopolysaccharide matrix. Amoebocytes containing waste products and an acid mucopolysaccharide enter the Polian vesicles from the coelom, pass through them, and empty into the lumen along with material secreted by the inner epithelium. While establishing close contact with cells of the muscular layer and inner epithelium the amoebocytes seem to transfer part of their contents to these cells.Polian vesicles appear to be a very primitive excretory organ.Research performed under C.N.R. contract.     

A protein delivery system: biodegradable alginate-chitosan-poly(lactic-co-glycolic acid) composite microspheres

In the present study we developed alginate-chitosan-poly(lactic-co-glycolic acid) (PLGA) composite microspheres to elevate protein entrapment efficiency and decrease its burst release. Bovine serum albumin (BSA), which used as the model protein, was entrapped into the alginate microcapsules by a modified emulsification method in an isopropyl alcohol-washed way. The rapid drug releases were sustained by chitosan coating. To obtain the desired release properties, the alginate-chitosan microcapsules were further incorporated in the PLGA to form the composite microspheres. The average diameter of the composite microcapsules was 31+/-9microm and the encapsulation efficiency was 81-87%, while that of conventional PLGA microspheres was just 61-65%. Furthermore, the burst releases at 1h of BSA entrapped in composite microspheres which containing PLGA (50:50) and PLGA (70:30) decreased to 24% and 8% in PBS, and further decreased to 5% and 2% in saline. On the contrary, the burst releases of conventional PLGA microspheres were 48% and 52% in PBS, respectively. Moreover, the release profiles could be manipulated by regulating the ratios of poly(lactic acid) to poly(glycolic acid) in the composite microspheres.     

In vitro culture of Aloe Barbadensis Mill.: Micropropagation from vegetative meristems

A method for rapid and highly effective plant micropropagation from vegetative meristems was established for Aloe barbadensis Mill. Plant micropropagation was achieved culturing apices on medium containing 1.1 M 2,4-dichlorophenoxyacetic acid and 2.3 M kinetin for 15–30 days. High morphogenetic ability was maintained by transferring explants (after 60 days) on media containing 0.11 M 2,4-dichlorophenoxyacetic acid and 2.2 M 6-benzylaminopurine.     

The arrangement of F-actin and microtubules during germination of Mucor rouxii sporangiospores

The distribution of F-actin microfilaments and microtubules was analyzed in germinating sporangiospores of Mucor rouxii by labeling with rhodamine-tagged phalloidin and by immunofluorescence microscopy. The transition from isodiametrical to apical growth was accompanied by a switch from uniform distribution of F-actin patches to a polarized accumulation of F-actin material at the germ tube tips. Immunoblotting of cell-free extracts of M. rouxii with a monoclonal anti-porcine -tubulin antibody (TU-01) disclosed two discrete bands of -tubulin suggesting the existence of two -tubulin genes in this fungus. Immunofluorescence microscopy of germinating cells stained with the same antibody revealed an elaborate network of cytoplasmic microtubules that persisted during the entire germination process and extended into the apex of the germ tube. Although their precise roles remain undetermined, the observed arrangement of cytoskeletal elements during germination is consistent with their presumed involvement in cell wall morphogenesis: the long axial microtubules serving as long-distance conveyors of wall-building vesicles to the apical region while the concentrated F-actin patches mark the participation of microfilaments in the zone of intense vesicle exocytosis at the hyphal apex.Abbreviations DAPI 4,6-diamidino-2-phenylindole - DTT dithiothreitol - EGTA Ethylene glycol-bis (beta-aminoethyl ether) - N,N,N,N tetraacetic acid - F-actin Filamentous actin - MES 2-(N0morpholino)-ethanesulfonic acid - PIPES Piperazine-N,N-bis-2-ethanesulfonic acid - PMSF Phenyl-methylsulphonyl fluoride - TBS Tris-buffered saline     

Analysis of the physiological effects of the antibiotic streptozotocin on Escherichia coli K 12 and other sensitive bacteria

The antibiotic streptozotocin under a variety of growth conditions rapidly and irreversibly inactivates the capacity to divide or to form colonies of a series of sensitive bacteria, containing the phosphoenolpyruvate-dependent sugar-phosphotransferase system. Cells can be sensitized towards the drug by pregrowth in N-acetyl-glucosamine and can be protected by adding this amino-glucoside to the medium. Starvation for energy, especially for phosphoenolpyruvate, or prevention of the induction of a transport system involved in streptozotocin uptake will protect the cells, while a block in protein synthesis does not. The killed cells neither lyse, nor are they transformed into spheroplasts. At first, the capacity of such dead cells to respire, to swim actively or to keep the cytoplasmic membrane impermeable for small molecules remains intact. Their capacity for over-all RNA and protein synthesis, and for carbohydrate and amino acid uptake by facilitated diffusion or active transport is not affected. However, they loose rapidly their ability to take up carbohydrates by the phospheonolpyruvate dependent process of group translocation or to synthesize inducible enzymes, e.g. the enzyme -galactosidase. These inhibitory effects apparently are caused by the accumulation of phosphorylated, toxic derivates of the antibiotic and eventually lead to a pronounced bacteriostasis. Killing of the cells seems to be caused by a direct effect of the strongly mutagenic drug on replicating DNA.Non-Standard Abbreviations IPTG isopropyl--D-thiogalactopyranoside - MIC minimal inhibitory concentration - NAG N-acetyl-D-glucosamine - oNP ortho-nitrophenolat - oNPG o-nitrophenyl--D-galactopyranoside - PEP phosphoenolpyruvate - PTS phosphoenolpyruvate dependent sugar: phosphotransferase system - Stz streptozotocin - TCA trichloroacetic acid     

Effects of exogenous linoleic acid on fatty acid composition,receptor-mediated cAMP formation,and transport functions in rat astrocytes in primary culture

We have examined the effects of culturing neonatal rat-brain astrocytes in medium containing delipidated serum, with or without added linoleic acid (LA, 18:26), on membrane fatty-acid composition and functions. After 18–21 days in culture, polyunsaturated fatty acids (PUFA) constituted24 mol% of the total fatty acids in the astrocytes grown in delipidated media (controls); these proportions were increased by 35–40% to33 mol% when the cells were supplemented with 35M LA. Notable differences in the PUFA profiles of the cells cultured with or without added LA included: (a) higher proportions of 6 PUFA in the LA-supplemented astrocytes (25%, relative to10% in controls) that were accompanied by an increase in the ratio of 6/3 PUFA (from <2 in controls to 5), and (b) higher proportions of 20:39 and 22:39 in the control astrocytes (>5%) relative to the LA-supplemented cells (1%). The major metabolites in the 6 PUFA-enriched cells were arachidonic (20:46), adrenic (22:46) and docosapentaenoic (22:56) acids (15, 5 & 3 mol%, respectively). Enrichment of the astrocytes in 6 PUFA did not alter basal levels of cAMP, nor did it affect the amounts of cAMP formed in response to forskolin, isoproterenol, adenosine or histamine. However, dopamine-dependent increases in cAMP formation in the presence of the phosphodiesterase inhibitor, Ro 20-1724, were reduced by 25% relative to those in controls. LA supplementation modified uptake of [³H]adenosine into the astrocytes; values for K_t for a high affinity transport were increased relative to controls, and maximum capacity of a lower affinity process was reduced. Uptake of [³H]glutamate was not altered in the 6 PUFA-enriched astrocytes. This study demonstrated that cultured astrocytes take up exogenous linoleic acid and incorporate its metabolites into, phospholipid, and that the resulting changes in membrans PUFA composition modify only specific cell functional properties.Abbreviations PUFA polyunsaturated fatty acid(s) - EFA essential fatty acid(s) - LA linoleic acid - AA arachidonic acid - DHA docosahexaenoic acid - BSA bovine serum albumin - DMEM Dulbecco's modified Eagle's medium - TBARS thiobarbituric-acid-reactive substances - NECA 5-N-ethylcarboxamidoadenosine Special issue dedicated to Dr. Leon S. Wolfe.     

Regeneration of plantlets through organogenesis in the Himalayan yellow poppy,Meconopsis paniculata

Plantlet formation through organogenesis in callus cultures of Himalayan yellow poppy,Meconopsis paniculata D.Don (Prain), a threatened taxon of ornamental value, is described. Hypocotyl segments from 3-month-old laboratory-raised seedlings produced callus on agar-solidified Murashige and Skoog medium (MS) containing 10 M -naphthaleneacetic acid and 1 M kinetin. Shoots differentiated best from callus on MS containing 10 M indolebutyric acid (IBA) and 1 M 6-benzyladenine. The regenerated shoots rooted best on MS medium containing 10 M IBA. From seed germination to differentiation of plantlets through the two-step organogenesis process required 28–29 weeks.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - FAA formalin-acetic acid-alcohol - BA 6-benzyladenine - IAA indole-acetic acid - IBA indolebutyric acid - GA₃ gibberellic acid - NAA -naphthaleneacetic acid - RH relative humidity