Cytochemical evidence for the presence of actin in the nucleus of the voodoo lily appendix

Immunoflorescence microscopy of sections of the voodoo lily Sauromatum guttatum appendix stained with monoclonal antibodies against -smooth muscle actin and cytoplasmic actin revealed different staining intensity of different parts of the cell. The anti-cytoplasmic-actin recognized antigens present mainly in the cytoplasm, and the anti--smooth muscle-actin recognized more intensively antigens present in the nuclei. A positive staining of the nucleus was also obtained with FITC–phalloidin confirming the presence of actin in its filamenous form in the nucleus. The presence of a nuclear -smooth muscle-actin-like protein was further confirmed by confocal laser microscopy. On Western blots, the two anti-actins labelled a protein band that comigrated with standard actin at the approximate molecular weight of 43kDa. Several other proteins interacted with the two antibodies to a different degree. The monoclonal antibodies against -tubulin subunit stained only the periphery of the cytoplasm and anti-pan cytoplasmic myosin stained the cytoplasm weakly. On a Western blot, anti--tubulin subunit primarily recognized a protein band at the appropriate molecular weight of 50kDa. This is the first cytochemical evidence for the presence of -smooth muscle-actin-like protein in the plant nucleus.     

Type-VI collagen in the human iris and ciliary body

Summary The distribution of type-VI collagen in the human iris and ciliary body was investigated by means of immunohistochemical techniques and compared with that of type-IV collagen, fibronectin and laminin. As has been described for other tissues, type-VI collagen surrounds type-I and-III collagen fibers. The aggregated from of type-IV collagen (the long-spacing or curly collagen), which has already been described in the trabecular meshwork and sclera, was also observed at the ciliary muscle tips surrounding the anterior elastic tendons of this muscle. In addition, staining for type-VI collagen was seen directly adjacent to the basement membranes of the ciliary muscle cells, the iris muscles, the uveal vascular endothelia and nerves, but not adjacent to the epithelial basement membranes. The staining did not form a discrete line like the immunoreaction for type-IV collagen, but bundles of marked fibrils extended into the surrounding connective tissue. We assume that type-VI collagen similar to type-VII collagen forms part of an anchoring system for these tissues. As type-VII collagen has been described only in connection with epithelial basement membranes, both type-VI and type-VII collagens may represent anchoring fibrils, however for different tissue components.     

Submikroskopische Zytologie des Thymus von Ambystoma mexicanum

Zusammenfassung Der Thymus von Ambystoma mexicanum ist von einer Bindegewebskapsel umgeben, läßt aber histologisch keine eindeutige Differenzierung in Rinde und Mark erkennen. Er besteht aus 10–14 großen lymphoiden Zellen, zwischen denen sich zahlreiche epitheliale Retikulumzellen befinden, die durch Desmosomen miteinander verbunden sind. Mesenchymale Retikulumzellen konnten nicht beobachtet werden. Im Bereich der subkapsulären Zone sind häufig Mitosen zu erkennen. Die lymphoiden Zellen unterscheiden sich submikroskopisch nicht wesentlich von den Rindenlymphozyten des Säugerthymus, sind aber etwa doppelt so groß. Typisch für sie ist der etwa 0,5 große Nukleolus. Auch die Retikulumzellen lassen keine besonderen Strukturen im Zytoplasma erkennen, enthalten aber keine vakuolären Einschlüsse wie bei Säugern. Des öfteren treten intrazelluläre Zysten in Erscheinung, in deren Lumen zahlreiche Zilien und Mikrovilli hineinragen. Vereinzelt können Mastzellen beobachtet werden, die in ihrem Zytoplasma nahezu homogene Granula in großer Anzahl enthalten.

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Summary The thymus of Ambystoma mexicanum does not show a clear differentiation in cortical and medullary zone and consists of lymphoid cells which are 10–14 large. Between them numerous epithelial reticular cells exist being connected by desmosomes. Mesenchymal reticular cells could not be found. Mitoses can be frequently seen in the subcapsular cortical zone. Submicroscopically the lymphoid cells do not show essential differences to the cortical lymphocytes of the thymus of mammalians but they are about twice as large. A nucleolus with a diameter of about 0.5 is characteristic of the lymphoid cells. The reticular cells do not show any special structures in their cytoplasm as well but they do not contain vacuolar inclusions like mammalians. Frequently, intracellular cysts appear in the reticular cells having many cilia and microvilli. Some mast-cells may be observed containing numéros nearly homogenous granules in their cytoplasm.

     

Effect of 17β-estradiol on cells stained for FSHβ and/or LHβ in the dog pituitary gland

Summary The effect of long-term treatment (52 weeks) with high doses of 17-estradiol (1.28 mg/kg/week intramuscularly) on gonadotrophs was studied in the pituitary gland of the beagle bitch. For immunochemical staining the immunoperoxidase technique and antisera to the specific beta () subunits of FSH and LH were employed. For control purposes antisera to the following hormones were also used: bovine TSH, canine GH, canine PRL and porcine ACTH¹. In the pars distalis and pars tuberalis of control bitches, in addition to the cells which react solely with antisera to either LH or FSH, most cells were reactive to both antisera. The cells stained for FSH were less numerous than those shown to contain LH. TSH, PRL, GH and ACTH/MSH were localized in distinctly different cell types in the pars distalis of all control animals. In the treated bitches, almost complete regression of cells classically identified as gonadotrophs and stained for LH was observed. On the other hand, using the antiserum to FSH, selective immunochemical staining was localized in cells fitting the morphological characteristics of TSH cells. All these cells were also stained for TSH. However, a few cells were also shown to react solely with the antiserum to TSH. These cells, which seem to contain both TSH and FSH, were further clearly differentiated from PRL, GH and ACTH/MSH cells on the basis of their cytological features, intraglandular distribution and by immunochemical double staining. These observations support the concept that the one cell-one hormone theory may not necessarily apply to the glycoprotein hormones of the dog pituitary gland.Abbreviations of Hormones cited in this Paper ACTH Adrenocorticotropin - FSH Follicle Stimulating Hormone - GH Growth Hormone - LH Luteinizing Hormone - MSH Melanocyte Stimulating Hormone - PRL Prolactin - TSH Thyrotropin The authors are grateful to Mrs. K. Oertel for carrying out the experimental work on animals, to Mrs. B. Schilk and Miss U. Tüshaus for their excellent technical assistance, and to Dr. P. Günzel for his advice and encouragement     

A study of the HLA-D region in patients with classic Kaposi's sarcoma

The HLA-D region in nine Sardinian patients with classic Kaposi's sarcoma was studied with two restriction enzymes, Eco RI and Eco RV, and two cDNA probes, DR and DQ. A total of 41 polymorphic restriction fragments were identified. One, an 11.5 kb Eco RV DQ fragment, was present in three of the patients but in none of the controls; a second, an 8.0 kb Eco RV DR fragment, was present in six patients and all the controls. No single fragment was identified which was significantly over or under-represented in either group.     

Der Anteil akustischer Komponenten an der Ausl?sung der Balz m?nnlicher Zebrafinken (Taeniopygia guttata castanotis)

Zusammenfassung Im Gegensatz zu optischen Komponenten des Auslösers Weibchen ist die Balz männlicher Zebrafinken durch reinen akustischen Kontakt mit dem nicht auslösbar. Akustischer Kontakt erhöht aber den Gesamterregungspegel des und führt im Vergleich zur Darbietung rein optischer Attrappen zu einer Intensivierung der Balz.

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Influence of acoustic stimuli on courtship behaviour of male Zebra Finches

Summary Courtship behaviour of male zebra finches is elicited by the presence of stuffed dummies, but not by acoustic contact with a female. Acoustic contact, however, enhances the arousal of the male and leads to a higher frequency of courtship behaviour if compared with experiments, where solely optic components of the releaser female are offered.

     

The β-1,3-glucan-binding protein from the crayfish Pacifastacus leniusculus,when reacted with a β-1,3-glucan,induces spreading and degranulation of crayfish granular cells

Summary A -1,3-glucan-binding protein (GBP) was purified from crayfish plasma, and incubated with laminarin (L), a -1,3-glucan. The GBP reacted with laminarin (GBP-L) induced strong spreading and partial degranulation of isolated and separated crayfish granular haemocytes. However, neither the GBP nor laminarin alone induced any changes in the crayfish granular cells. When monolayers of granular haemocytes were incubated with 20 g of GBP-L, more than 82% of the haemocytes were affected. The activity of GBP-L on granular cells was dose-dependent and a plateau was reached at 10 g of GBP-L. The degranulation of crayfish haemocytes induced by GBP-L seemed to occur by a regulated exocytosis, since it was strongly inhibited by specific blockers of this process such as SITS or calmidazolium. Monospecific anti-GBP antibodies also totally blocked the effect of GBP-L on crayfish granular cells. Indirect immunofluoresence staining demonstrated that the GBP-L could bind to the surface of granular cells, whereas GBP did not bind or bound very weakly to the haemocyte surface.     

Rats that consume caffeine show decreased brain protein synthesis

The effect of caffeine on protein synthesis in brain and liver was studied. When caffeine was added to a post-mitochondrial supernatant from rat brain protein synthesis was inhibited, i.e. 1 mM caffeine about 20%. The effect on protein synthesis of two weeks administration of large doses of caffeine in the drinking fluid of rats was also measured. Caffeine decreased protein synthesis in rat brain by about 32% and 20% compared with ad libitum and pair-fed controls. Protein synthesis was calculated taking into account the levels of free leucine determined by HPLC: 0.10 mol/g brain of ad libitum; 0.11 for pair-fed and 0.07 for caffeine. The pattern of proteins synthesized was not significantly altered by caffeine as shown by gel-electrophoresis and fluorography. There was no effect on protein synthesis of liver. The possible significance of these results is briefly discussed.     

Studies on fish scale formation and resorption

Summary Electron microscopic investigation of scales of the goldfish Carassius auratus revealed that the lamellae of fibrillary plates contain sheet-like structures composed of vertically oriented collagen fibers embedded in an organic matrix. The fibers (TC fibers) are smaller in diameter (35–45 nm) than those of the lamellae and the matrix is stained intensely with lead citrate.The sheet-like structures as well as the lamellae are formed by fibroblasts located beneath the lamellae. The orientation of the collagen fibers of the sheets and the lamellae seems to be controlled by the orientation of the ridges and invaginations of the surface of the fibroblasts.The fibrillary plate of C. auratus was found to be partially calcified. Calcification was initiated by the deposition of needle-like or flaky crystals of hydroxyapatite in the organic matrix of the sheet-like structure and proceeded into the TC fibers and the matrix region of the lamellae. The potassium pyroantimonate-osmium tetroxide method showed a heavy concentration of calcium in the osteoblasts, fibroblasts, and in the matrix regions of the fibrillary plate. Calcium-containing precipitates were also present in the hole zone of the collagen fibers in the lamellae, but the significance of this location in calcification remains to be elucidated.Contribution No. 285, Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina, Columbia, South Carolina, 29208, USA     

Prokaryotic endosymbionts in the chloroplast stroma of the dinoflagellateWoloszynskia pascheri

Summary Chloroplasts of the freshwater dinoflagellate,Woloszynskia pascheri, were found to contain small, double membrane-bound bodies that appear to be modified bacteria existing in this organelle as endosymbionts. These chloroplast endosymbionts (CESs) contain thin filaments, which observations on thin-sectioned and Feulgen-stained material indicate to be strands of naked DNA. They also possess putative prokaryotic-sized ribosomes. The outer of the two membranes that surround a CES may be expanded to form cisternae or tubules, which frequently connect with the outer membrane of adjacent CESs. Considering their appearance in relation to free-living bacteria, and their apparently benign presence in the dinoflagellate host, it is suggested that the CESs have been involved in a symbiotic relationship withW. pascheri for some time.     

Induction of cellulose- and xylan-degrading enzyme complex in the yeast Trichosporon cutaneum

The specificity of induction of cellulose- and xylan-degrading enzymes was investigated on the yeast strain Trichosporon cutaneum CCY 30-5-4 using series of compounds structurally related to cellulose and xylan, including monosaccharides, glycosides, glucooligosaccharides and xylooligosaccharides. Determination of activities of secreted cellulase and -xylanase, intracellular, cell wall bound and extracellular -glucosidase and -xylosidase revealed that: (1) The synthesis of xylan-degrading enzymes is induced in the cell only by xylosaccharides, 1,3--xylobiose, 1,2--xylobiose, 1,4--xylosyl-L-arabinose, 1,4--xylobiose and thioxylobiose being the best inducers. The xylan-degrading enzymes show different pattern of development in time and discrete cellular localization, i.e. intracellular -xylosidase precedes extracellular -xylanase. (2) A true cellulase is not inducible by glucosaccharides and cellulose. Negligible constitutive cellulase activity was detected which was about two orders lower than an induced cellulase in the typical cellulolytic fungus Trichoderma reesei QM 9414. (3) The best inducer of intracellular -glucosidase splitting cellobiose was thiocellobiose in a wide range of concentration (0.1–10 mM), whereas xylosaccharides at high concentrations induced -xylosidase of xylobiose type and a non-specific aryl -D-glucosidase.The results were confirmed by growing cells on cellulose and xylan. T. cutaneum was found to be a xylan-voracious yeast, unable to grow on cellulose.     

The role of macrophages in the tissues regeneration stimulated by the biomaterials

Allogenic grafted tissues are subjected to biodegradation and replaced by the regenerate. To minimize the immune response and improve the rebuilding of tissues there was developed a technology to treat tissues with a cells elimination and dosed out extraction of proteoglycanes (Alloplant^®). With aim to clarify the role of macrophages in the tissues regeneration resulting implantation the biomaterials 112 rats were injected the allogenic and xenogenic (rabbits) pulverized biomaterials in the form of suspension. Injections were performed subcutaneously into the animals back by the base of the tail. The control group (14 rats) were injected a physiologic saline. Animals were killed by ether inhalation on day 2, 4, 7, 14, 30, 90 and 180 and tissue sections were studied by light and electron microscopy. The study showed the key role of the macrophages in resorption of the allogenic biomaterial and formation of the newly-formed tissue. Implantation of the biomaterial induced activity a great number of the mature macrophages, which completely lysed and resorbed the biomaterial particles. Expression TNF was significantly higher whereas expression TGF-1 was significantly lower. With xenogenic biomaterial implantation there were less macrophages, their activity was restricted. Macrophages containing large vacuoles with an active endo- and exocytosis were revealed in the allogenic biomaterial implantation and were named matrix-forming macrophages. We may suppose that these macrophages synthesize (or re-synthesize) proteoglycan component of the newly-formed collagen fibers. There was put forward a hypothesis about the two component mechanism of the collagen fibers formation.     

Membrane specialisation of the extra-junctional sarcolemma of normal and denervated insect muscle

Summary Corrugated areas of sarcolemma were observed in serial transverse sections. Complexes of intramembranous particles that appear after freeze-fracture replication were concluded to represent the same specialisation. These specialisations, up to 14 m x 2 m, are orientated parallel to the long axis of the muscle. Intramembranous particles are concentrated along the peaks of the corrugations, and are associated with the P-face. Corresponding pits are found in the E-face. Fourteen and thirty days after sectioning the excitatory motor-nerve supply to the muscle, corrugated areas 0.5–1 m x 0.5–1 m are found. Occurring singly or in groups, their orientation with respect to the long axis of the muscle is more variable than those of control muscles. Thin sections reveal no complementary areas on adjacent fibres or intracellular submembrane attachments or specialisations. A structural role is therefore unlikely. Mitochondria are frequently found in close association with these specialisations. Their possible role as receptors or transmembrane transport systems is discussed.This work was supported by an SRC Project grant to IRD     

GroE-mediated folding of bacterial luciferases in vivo

In this study we present evidence indicating that GroE chaperonins mediate de novo protein folding of heterodimeric and monomeric luciferases under heat shock or sub-heat shock conditions in vivo. The effects of additional groESL and groEL genes on the bioluminescence of Escherichia coli cells expressing different bacterial luciferase genes at various temperatures were directly studied in cells growing in liquid culture. Data indicate that at 42° C GroESL chaperonins are required for the folding of the subunit polypeptide of the heterodimeric luciferase from the mesophilic bacterium Vibrio harveyi MAV (B392). In contrast, the small number of amino acid substitutions present in the luciferase subunit polypeptide from the thermotolerant V. harveyi CTP5 suppresses this requirement for GroE chaperonins, and greatly reduces interaction between the subunit polypeptide and GroEL chaperonin. In addition, GroESL are required for the de novo folding at 37° C of a MAV luciferase fusion polypeptide that is functional as a monomer. No such requirement for luciferase activity is observed at that temperature with a fusion of the CTP5 and subunit polypeptides, although GroE chaperonins can still mediate folding of the CTP5 fusion luciferase. Bacterial luciferases provide a unique system for direct observation of the effects of GroE chaperonins on protein folding and enzyme assembly in living cells. Furthermore, they offer a sensitive and simple assay system for the identification of polypeptide domains required for GroEL protein binding.     

Trisomics in diploid alfalfa

Eight triploids were produced by pollinating male sterile alfalfa tetraploids with diploid lines of closely related species involving Medicago sativa, M. falcata and M. coerulea. Seeds were produced on all but one of the triploids by crossing them with diploid and tetraploid lines. Primary trisomic plants were obtained from the crosses with diploid lines and studies on their fertility and trisomic transmission are reported. A brief review of the cytogenetic evidence indicates that the closely related species involved in these trisomics appear to be forms of a single polymorphic species and that cultivated tetraploid alfalfa behaves essentially as an autotetraploid. Thus, it is proposed that linkage groups established with these diploid trisomics will also represent the linkage groups of cultivated alfalfa.Contribution No. 190 from the Ottawa Research Station.     

Establishment by the rat lymph node method of epitope-defined monoclonal antibodies recognizing the six different α chains of human type IV collagen

A group of rat monoclonal antibodies recognizing the six different chains of human type IV collagen have been established by our novel method. The method is designated the rat lymph node method in which enlarged medial iliac lymph nodes of a rat injected with an antigen emulsion via hind footpads are used as a source of B cells for cell fusion to produce hybridomas. The immunogens used were synthetic peptides having non-consensus amino acid sequences near the carboxyl termini of type IV collagen chains. Hybridomas were screened both by ELISA with synthetic peptides and by indirect immunofluorescence with cryostat sections of human kidneys. Because the epitopes of all antibodies were determined by multipin-peptide scanning, they were confirmed to be isoform-specific. They are useful for identification of chains of type IV collagen at the protein level in normal and abnormal conditions. The combined use of synthetic peptides as immunogens, the rat lymph node method as making monoclonal antibodies, and the multipin-peptide scanning as epitope mapping is found to be a strong tool for identification of peptides and proteins whose amino acid sequences are known or have been deduced.     

Glycoconjugates of the Rat Ciliary Body Epithelium: A Lectin Histochemical and Protein Blotting Study

The present study sought to identify and partially characterize the glycoconjugates specific to the double-layered ciliary body epithelium of the rat eye by lectin histochemistry and lectin blottings. Hydrated paraffin sections of Carnoy-fixed Sprague-Dawley rat eyes were stained with a panel of 21 different biotinylated lectins, followed by streptavidin-peroxidase and the glucose oxidase-diaminobenzidine-nickel staining procedure. The results of lectin histochemistry revealed that the inner epithelial layer was rich in GlcNAc(1,4)GlcNAc, -Gal, Gal(1,3)GalNAc, GalNAc(1,3)GalNAc/Gal, GalNAc(1,6)Gal, Fuc(1,2)Gal(1,4)GlcNAc and Gal(1,4)GlcNAc(1,2)Man(1,6) sugar residues as shown by its positive reactivities with S-WGA, PWA, DSA, GS-I-B4, PNA, DBA, SBA, WFA, UEA-I, LTA and PHA-E. The reactivities of GS-I-B4, PNA, DBA and SBA were restricted to the inner layer at the tips of the ciliary processes. On the other hand, the outer epithelial layer was stained evenly by DSA and Jacalin, and partly by MAA, showing that this epithelial layer was rich in GlcNAc(1,4)GlcNAc, Gal(1,3)GalNAc and NeuAc(2,3)Gal disaccharides. These lectin binding patterns of the ciliary body epithelium suggest a topographical and functional difference in this double cell-layered epithelium. Their possible roles in the secretion of aqueous humour and production of ciliary zonule are discussed. Some identified lectin markers specific to these two cell layers may be useful for further experimental studies. Glycoproteins extracted from the dissected ciliary body were separated by SDS-PAGE electrophoresis and analyzed by protein blottings with 8 different lectins. The results showed that at least 10 major membrane-bound glycoproteins, with molecular weights ranging from 30 to 150kD, rich in -GlcNAc, -Gal, /-GalNAc and NeuAc(2,6)Gal residues, were present in the microsomal fraction.     

Locations and central projections of neurons associated with the retrocerebral neuroendocrine complex of the cockroach Periplaneta americana (L.)

Summary Retrograde diffusion and precipitation of Co²⁺ reveals in the ipsilateral pars lateralis (PL) and contralateral pars intercerebralis (PI) of the brain neurons that enter the corpus cardiacum (CC), and, possibly, the corpus allatum (CA) on each side. The PL group consists of 29.6±8.4 somata that fill. Of these, 5.6±0.6 exceed 25 m in diameter, 14.3±2.7 range from 15–25 m, and 9.6±7.6 are smaller than 15 m. After CoCl₂ was applied to the right CC-CA of two males, 239 and 265 somata in the left PI stained. Except for 16 ranging from 30–45 m and chiefly located anteriorly, a majority of these somata measured 10–25 m.The only somata revealed by staining whole brains with the performic acid-resorcin fuchsin method are neurosecretory cells 10–20 m in diameter located within the PI. In starved adult males there are 92.4±8.1 on the right, and 93.2±6.9 on the left. The largest somata in the PL group contain numerous granules that stain with paraldehyde fuchsin. These somata also fill with Co²⁺, and belong to neurosecretory cells that extend into the CC-CA.The cerebral distribution of branches from the PL group, and the relationship of these to the corpora pedunculata, central body, and arborizations from the PI decussation are described.     

Presence of β-endorphin-like immunoreactivity in the anterior pituitary gland of rat and man and evidence for the differential localization with ACTH

Summary Two antisera, Y-10 and Y-18 were raised in rabbits against synthetic human -endorphin conjugated to bovine serum albumin and keyhole limpet haemocyanin respectively. Antiserum Y-10 has been shown by radioimmunoassay to be highly specific for human -endorphin with minimal or no cross-reactivity against other pituitary peptides whilst antiserum Y-18 crossreacted on an equimolar basis against -endorphin and -lipotropin. When used in the immunohistochemical procedure, both antisera specifically stained the corticotrophs in human anterior pituitary tissue. A similar effect was observed when antiserum Y-18 was applied to rat anterior pituitary tissue in the immunohistochemical procedure. Y-10 antiserum, on the other hand, stained not only rat corticotrophs but also somatotrophs. The somatotrophin staining could not be attributed to the enkephalins reported to be present in these cells.The non-specific -endorphin antiserum Y-18 was used to stain anterior pituitaries from dehydrated and adrenalectomized rats as well as rats of the Brattleboro strain. In tissues from the three experimental animals, cells that stained positively for -endorphin did not give a positive immunoreaction for ACTH and vice versa in some other sections. It is concluded that under the physiological conditions, formalin fixation of the tissue causes the proopiocortin molecule to be trapped in a conformation such that either ACTH or -endorphin-like determinants are available for reacting with the appropriate antiserum.This work was financed by the Medical Research Council of New Zealand, NIH Research Program Project Grant HD-12303 and by U.S.P.H.S. Grant NS-16304 from NIH. We thank Drs Guillemin, Bloom and Ling for samples of -endorphin and -endorphin antisera     

A method for long-term micropropagation of Phaseolus coccineus L.

A method for long-term plant regeneration of Phaseolus coccineus L, is described. Shoot-tips and cotyledonary nodes cultured on a Murashige and Skoog medium supplemented with N⁶-benzylaminopurine, 10 M, and -naphthaleneacetic acid, 1M, formed multiple bud-shoots. These shoots were transferred to medium containing BAP 1 M, NAA 0.1 M, and gibberellic acid 3 M to promote shoot growth and further shoot multiplication. Rooting was achieved in medium with 11 M indole-3-acetic acid. Rooted plants grew to maturity and were fertile. Cultures have maintained their ability to regenerate plants for more than two years. A sample of 30 regenerated plants (R₀) was tested for chromosome number, all of them being diploid; seven isozymatic systems were electrophpretically analyzed in 82 R₀ regenerated plants. No differences were observed in their electrophoretic patterns in comparison with those shown by seedlings. Histological studies revealed the origin of buds from calluses via organogenesis.Abbreviations BAP N⁶-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic acid - ADH alcohol dehydrogenase - GOT glutamic-oxaloacetic transaminase - MDH malate dehydrogenase - 6PGD 6-phosphogluconate dehydrogenase - PGI Phosphoglucose isomerase - PGM phosphoglucose mutase - SK shikimate dehydrogenase