Callus formation from root protoplasts of Quercus rubra L. (red oak)

Root protoplasts of Quercus rubra L. were isolated from 12 day old seedlings with an enzyme mixture containing Cellulase R1O + Rhozyme HP150 + Macerozyme R1O, supplemented with cysteine and bovine serum albumin.Protoplasts were purified by a Ficoll density gradient centrifugation and cultured at low density in a liquid medium. The modified woody plant medium, containing 2.2 M benzyladenine + 1.8; M zeatin + 5.3 M -naphthaleneacetic acid + 2.2 M dichlorophenoxyacetic acid, allowed sustained divisions and formation of microcalluses.Protoplast — derived microcallus developed into green and compact callus when transferred to an agarose solidified medium, supplemented with casein hydrolysate and indole 3-acetic acid (devoid of 2,4 dichlorophenoxyacetic acid) and placed under low illumination.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - BA benzyladenine - BSA bovine serum albumin - 2,4-D 2,4-dichlorophenoxyacetic acid - FW fresh weight - GA₃ gibberellic acid - MES 2-(N-morpholino) ethanesulfonic acid - MS medium Murashige and Skoog medium (1962) - NAA -naphthaleneacetic acid - WPM woody plant medium (Lloyd and Mc Cown, 1981)     

Genetic analysis of resistance to whitebacked planthopper in twenty-one varieties of rice,Oryza sativa L.

Summary The inheritance of resistance to whitebacked planthopper Sogatella furcifera (Horvath) was studied in 21 rice varieties. Reactions of F₁; F₂ and F₃ progenies of the crosses of 21 resistant varieties with the susceptible variety TN 1 revealed that a single dominant gene governs resistance in Mushkan 41, Santhi, Siahnakidar 195, SM2-34, Tirisurkh 251, Zirijowaian 245, 18, 24A, 39, 76 S, 78, 180, 213 B, 267, 293, CI 6037-4, NP97, S39 JKW and Bansphul. In varieties 65 and 274 A, resistance is governed by one dominant and one recessive gene which segregate independently of each other. Tests for allelism with the Wbph 1 gene originally identified in N 22 revealed that the dominant gene present in all the test varieties is the same as Wbph 1. Further studies are required to determine the allelic relationships of the recessive gene found in varieties 65 and 274 A.     

The response of a cytokinin resistant mutant is highly specific and permits a new cytokinin bioassay

We have previously isolated six independent cytokinin-resistant mutants of Nicotiana plumbaginifolia which define three complementation groups, zeal, zea2 and zea3. We report here the characterization of the phenotypic response to cytokinin treatment of the mutant 1–64, belonging to the zeal group, and the result of the study of the specificity of this response. The phenotype of this mutant grown in the presence of cytokinin concentrations higher than 0.1 M is characterized by a hypertrophy of the cotyledons and hypocotyl which results in an increase of plantlet fresh weight. This hypertrophy is correlated to cytokinin concentration in a range between 0.01 to 10 M. The specificity of this response has been verified by using adenine and urea type cytokinins, as well as enantiomers of methylzeatin and methylbenzyladenine which differ widely in their cytokinin activities. We show that the high specificity of the hypertrophic response to cytokinins can be used as a convenient bioassay to screen the cytokinin activity of adenine or urea type molecules.Abbreviations zeatin [6-(4-hydroxy-3-methylbut-trans-2-enylamino)purine] - iP isopentenyladenine [6-(3-methylbut-2-enylamino)purine] - BA benzyladenine [6-(benzylamino)purine] - (R)-(+)-MeZea [(R)--methylzeatin] - (S)-(–)-MeZea [(S)--methylzeatin] - (R)-(+)-MeBA [(R)--methylbenzyladenine] - (S)-(–)-MeBA [(S)--methylbenzyladenine] - CPPU N-(2-chloro-4-pyridyl)-N-phenylurea - thidiazuron N-(1,2,3-thiadiazol-5-pyridyl)-N-phenylurea The authors dedicate this paper to the memory of Jean-Pierre Bourgin, Director of the Laboratoire de Biologie Cellulaire, who died suddenly on October 29, 1994.     

T-cell repertoire in a strain of transgenic C57BL/6 mice with the HLA-DRA gene on the X-chromosome

We have established a strain of transgenic mice in which the HLA-DRA gene was integrated into the X-chromosome and the xenogeneic mixed isotype molecule, DRE^b, was expressed in a cell type-specific manner, although the transgenic DRA gene contained only 268 base pairs of the 5-flanking region. The DRE^b molecules expressed in the transgenic mice functioned as major histocompatibility complex (MHC) class II to select T-cell repertoire, and to stimulate mixed lymphocyte reaction. In female transgenic mice homozygous for HLA-DRA (DR-B6-F-homo) and male transgenic mice (DR-B6-M), DRE^b molecules were expressed in almost all of the MHC class II A^b-positive cells. In contrast, the expression of DRE^b molecules in female transgenic mice hemizygous for HLA-DRA (DR-B6-F-hemi) was found only in part of the A^b positive cells, and the proportion of cells expressing the DRE^b molecules varied due to random inactivation of one of the X-chromosomes. Clonal deletions of the T cells and mature thymocytes bearing Tcrb-V5 and Tcrb-V11, which are eliminated from the peripheral repertoire in mice expressing self-superantigen and MHC class II E molecules, were incomplete in DR-B6-F-hemi as compared with those in DR-B6-F-homo, and were correlated with the proportion of DRE^b-positive spleen cells. These observations suggested that the number of bone marrow-derived cells expressing DRE^b molecules was critical for clonal deletions of Tcrb-V5⁺ and Tcrb-V11⁺ T cells in the thymus.     

The use of BRM-activated killer cells in adoptive immunotherapy: A pilot study with nine advanced cancer patients

Adoptive immunotherapy using MHC-nonrestricted-lymphocytes, peripheral blood T cells and NK cells was devised. Peripheral blood mononuclear cells (3 x 107) were selected by immobilization to anti-CD3 monoclonal antibody for 4 days and cultured for 2 weeks in the presence of IL-2. Thereafter they were reactivated by 500 U/ml of IFN- and 1000 U/ml of IL-2 for 1 hour. Enhancement of NK and LAK activities was confirmed. Peripheral blood T cells proliferated in response to immobilized anti-CD3 antibody (3% to 30%). Approximately 6 x 109 BRM-activated killer (BAK) cells composed of CD56+ T cells and CD56+ NK cells, were dispensed to cancer patients via intravenous drip infusion. Nine patients were treated with BAK cells every 2 weeks or every month on an outpatient basis. During the course of adoptive immunotherapy, the crossed affinity immunoelectrophoresis (CAIE) pattern of serum immunosuppressive acidic protein (IAP) was analysed. Both the production and glycosylation pattern of IAP is changed in response to tumor enlargement and may therefore act as a marker of the disease progression. During the course of BAK therapy, the glycosylation IAP pattern of 6 patients changed from tumor (T) to normal (N). In addition, the performance status of all patients was maintained at 90–100% of the Karnofsky scale and any side effects including fever were not observed during treatments with BAK cells. Moreover, the overall quality of life (QOL) of the patients, scored at the Face scale was favorable. In addition, blood levels of activated T cells producing IFN- were assayed as an indication marker of BAK therapy. The normal range of IFN- producing T cells comprised 6.9 ± 0.9% of peripheral blood mononuclear cells (PBMC), according to a single cell FACScan analyses of PBMCs derived from normal individuals. IFN- producing T cells of Patients No. 8 and 9, who received extensive chemotherapy before initiation of BAK therapy, comprised only 0.2% and 2% of PBMC, respectively. These patients died 3 and 6 months after beginning BAK therapy. Peripheral blood T cells of Patients Nos. 1–7 proliferated in response to immobilized anti-CD3 antibody and the frequency of IFN- producing T cells in PBMC preparation of these patients were over 3% before initiation of BAK therapy. Since our data show a positive correlation between survival time and initial T cell counts, a low frequency of these cells may contraindicate BAK therapy.     

Adoptions expérimentales de larves entre des fourmis de genres différents (II):Myrmica laevinodis Nylander etAnergates atratulus Schenck

Résumé Nous avons fait élever des larves d'Anergates atratulus par des ouvrières deMyrmica laevinodis à 22°C. Pour y parvenir, il n'est pas utile de faire hivernerensemble les larves d'Anergates et les ouvrières deMyrmica. La présence de larves autochtones n'empêche pas lesMyrmica d'élever des larves d'Anergates. Dans toutes les expériences lesMyrmica ont été soumises au fridavant de recevoir des larves d'Anergates. Aucune reine deMyrmica n'a été utilisée dans ces expériences.Sur les 64 larves d'Anergates que nous avons utilisées, 38 se sont transformées en imagos. C'est au début de l'adoption et au moment des métamorphoses que périrent la plupart des 26Anergates perdus. Les femelles vécurent en général 2 ou 3 jours et cherchèrent très tôt à quitter le nid natal. Les mâles vécurent 2 à 3 semaines.

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Summary Larvae ofAnergates atratulus were experimentally reared by workers ofMyrmica laevinodis, at 22°C. An overwintering of both larvae ofAnergates and workers ofMyrmica is not necessary for the success of that experiment. The presence of larvae ofMyrmica does not keep theMyrmica from rearing larvae ofAnergates. The workers ofMyrmica have been cooled, in all the experiments, before receiving larvae ofAnergates. No queen ofMyrmica have been used in that experiments.38 of the 64 larvae ofAnergates used became imagos. Most of the 26 lostAnergates died at the beginning of the adoption and during the metamorphosis. The females lived generally 2 or 3 days and tried, very early, to leave their native nest. The males lived 2 or 3 weeks.

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Anergates atratulus Myrmica laevinodis, 22 . bmecme Anergates Myrmica. Myrmica Anergates. Myrmica Anergates. Myrmica . 64 Anergates , 38 . 26 Anergates 2 3 . 2 3 .

     

Modification of the properties of a translocation in the German cockroach by selection

Summary A stock of Blattella germanica bearing the interchange T(3; 12)/3;12 was subjected to close inbreeding with selection for random disjunction at metaphase I. After 3–4 generations of selection, interchange quadrivalent chiasma frequency decreased, variability in free bivalent chiasma frequency increased sharply, and individuals with either random or directed disjunction were present in the stock. Random disjunction was modified from a ratio of 2112 (adj.-1; alt.-1; adj.-2; alt.-2) to a ratio of 1111. After 7–8 generations of selection, chiasma frequency appeared to stabilize at lower than normal levels and variability decreased for both quadrivalents and free bivalents. Directed disjunction was modified from a ratio of 2114 to 1112, and no individuals with the original high level of directed disjunction were detected. Chains-of-four tended to orient randomly, especially in individuals where the ring quadrivalents showed directed disjunction. Relaxation of inbreeding, but not selection, produced an increase in chiasma frequency and variability in both free bivalents and quadrivalents, but the modified ratios for both random and directed disjunction were retained. These results are discussed with respect to inbreeding effects and genetic control of chiasma frequency and metaphase I disjunction in interchange quadrivalents.     

Activation of immunoglobulin control elements in trasgenic mice

To assess the role interleukins and mitogens play in regulating immunoglobulin (Ig) gene expression via the Ig enhancer and promoter, transgenic mice carrying two different Ig gene regulatory regions were generated. One, EkCAT, contains the Ig heavy chain enhancer (E) and the light chain promoter driving the chloramphenicol acetyltransferase (CAT) gene. In the other, EkCAT, CAT is under the control of the promoter alone. E and relative activity were assessed by CAT assay. In EkCAT mice, low CAT expression was consistently found in spleen, bone marrow, mesenteric lymph node, and thymus but not in brain, lung, or kidney. In EkCAT mice, CAT expression was detectable just above background in lymphoid tissues, suggesting a basic level of tissue specificity in the absence of the enhancer. Whole spleen cell cultures prepared from the mice were treated with lymphokines and mitogens. Lipopolysaccharide (LPS), concanavilin A (Con A), interleukin 6 (IL-6), and interferon- (IFN-) increased CAT expression to varying extents in cells derived from EkCAT mice but not in spleen cells prepared from EkCAT mice. Thus, the presence of E, in addition to the promoter, is essential for the stimulation of CAT expression mediated by these factors. B cells from EkCAT mice were separated by density into populations of small and large cells. In untreated small B cells, no CAT expression was detected and only addition of LPS resulted in an increase in CAT expression. In large B cells, CAT was expressed at a low level without addition of exogenous factors. Incubation with LPS, IL-6, Con A and IFN- caused CAT expression to increase several-fold. This transgenic system provides a means to identify exogenous factors that activate Ig enhancers and promoters.This work has been submitted in partial fulfillment of the requirements for the doctoral degree from the George Washington University.     

Regeneration of Elaeagnus angustifolia from leaf segments of in vitro-derived shoots

Shoot regeneration was achieved from in vitro-produced leaves of Elaeagnus angustifolia L. Half-leaf explants from the terminal part of the shoot produced more shoots than explants from the basal part of the in vitro-derived shoots on agar-solidified WPM medium supplemented with 1 M benzyladenine (BA). In liquid medium of the same formulation, compact shoots that did not elongate were formed on the explants. Leaf cross-section explants (1 mm thick) produced shoots both on solid and liquid medium with 1 M BA, whereas again compact shoots were formed with 10 M BA. Further shoot development on these explants was promoted by their transfer to fresh solid medium containing 1 M BA and 1 M gibberellic acid (GA₃).Abbreviations BA benzyladenine - GA₃ gibberellic acid - WPM woody plant medium     

Comparison of the carbohydrate moieties of recombinant soluble Fcε receptor (sFcε RII/sCD23) expressed inSaccharomyces cerevisiae and Chinese hamster ovary cells. Different O-glycosylation sites are used by yeast and mammalian cells

Recombinant human soluble low affinity receptor for the Fc portion of IgE (sFcRII/sCD23) was produced inSaccharomyces cerevisiae or Chinese hamster ovary cells and subjected to carbohydrate analysis. Applied methods included analytical SDS-PAGE, reversed phase HPLC, methylation analysis and sequential degradation with exoglycosidases. The results revealed that sFcRII derived from Chinese hamster ovary cells is glycosylated exclusively at Ser-147, containing mainly the trisaccharide Sia(2–3)Gal(1–3)GalNAc, whereas the yeast derived glycoprotein was glycosylated at Ser-167 and contained only -mannosyl residues. It is shown here for the first time that different amino acids of a given protein can be O-glycosylated when expressed in yeast or Chinese hamster ovary cells.     

A structural model for elongation factor 1 (EF-1) and phosphorylation by protein kinase CKII

EF-1a binds aminoacyl-tRNA to the ribosome with the hydrolysis of GTP; the complex facilitates the exchange of GDP for GTP to initiate another round of elongation. To examine the subunit structure of EF-1 and phosphorylation by protein kinase CKII, recombinant , , and subunits from rabbit were expressed in E. coli and the subunits were reconstituted into partial and complete complexes and analyzed by gel filtration. To determine the availability of the and subunits for phosphorylation by CKII, the subunits and the reconstituted complexes were examined as substrates for CKII. Formation of the nucleotide exchange complex increased the rate of phosphorylation of the subunit and reduced the Km, while addition of to or the complex inhibited phosphorylation by CKII. However, a had little effect on phosphorylation of . Thus, the and subunits in EF-1 were differentially phosphorylated by CKII, in that phosphorylation of was altered by association with other subunits, while the site on was always available for phosphorylation by CKII. From the availability of the subunits for phosphorylation by CKII and the composition of the reconstituted partial and complete complexes, a model for the subunit structure of EF-1 consisting of (22)2 is proposed and discussed.     

Transformation of Atriplex gmelini plants from callus lines using Agrobacterium tumefaciens

Atriplex gmelini plants were regenerated via organogensis from hypocotyl explants. Callus lines were induced from the hypocotyl explants on Linsmaier and Skoog (LS) medium supplemented with 1 M benzyladenine and 5 M -naphthaleneacetic acid in the dark. Shoots were regenerated from the callus lines on LS medium supplemented with 20 M thidiazuron and 0.1 M -naphthaleneacetic acid under a high-intensity light condition (450 mol m^–2 s^–1). The regenerated shoots were rooted on LS medium without growth regulators to obtain fully developed plants. We succeeded in transforming Atriplex gmelini from callus lines using Agrobacterium tumefaciens.     

In vitro micropropagation of Arctostaphylos uva-ursi (L.) Sprengel.: Comparison between two methodologies

In vitro micropropagation of Arctostaphylos uva-ursi was performed to increase the number of ground cover species able to serve as substitute for members of the Rosaceae susceptible to fire blight. Explants (node segments) excised from plants growing in the greenhouse were established in vitro on a medium containing 10 M -naphthaleneacetic acid (NAA) and activated charcoal (2 g I^-1). Using in vitro grown shoots, two propagation procedures were used:- Culture of nodal fragments with 50 M NAA resulted in the growth of 6 to 7 nodes every 4 weeks, yielding 1 700 almost rootable shoots after 4 subcultures;- Development of axillary shoots obtained with media containing 25 M benzyladenine (BA) and 20 M indoleacetic acid (IAA) yielded almost 500 rootable shoots after 4 subcultures. The rate of propagation decreased after the 3^rd subculture.Percentage of in vitro rooted shoots reached 98% with diluted micronutrients and 10 M NAA but 31% of the plants died during acclimatization.Abbreviations BA benzyladenine - BM basal medium - HID high intensity discharge - IAA indoleacetic acid - IBA indolebutyric acid - NAA -naphthaleneacetic acid - PAR photosynthetic active radiation - 2iP 2-isopentenyladenine     

Ion content and aperture in “isolated” guard cells ofCommelina communis L.

Summary Isolated guard cells ofCommelina communis L., in epidermal strips in which all cells other than guard cells have been killed by treatment at low pH, will open to a degree dependent on the K (Rb)/Cl(Br) concentration in the bathing medium. Estimates of the changes with aperture of the ion concentrations in the guard cells were made by measurement of⁸⁶Rb uptake from RbCl, of⁸²Br uptake from K⁸²Br, and of potassium activity with a potassium-sensitive microelectrode. The osmotic effects of such changes were compared with the previous estimates of the osmotic changes required to change the aperture. The results suggest that a substantial fraction of the osmotic pressure of isolated guard cells is contributed by solutes other than KCl (or other potassium salts), and that, even in stomata opened by incubation on KCl solutions, a substantial fraction of the increase in osmotic pressure associated with opening is contributed by solutes other than KCl.     

DNA markers tightly linked to a gall midge resistance gene (Gm2) are potentially useful for marker-aided selection in rice breeding

We have developed a polymerase chain reaction (PCR)-based assay that could effectively reduce the time period required to screen and select for Gall Midgeresistant rice lines under field conditions. The primers for the assay were designed on the basis of sequence information of two phenotype specific random amplified polymorphic DNA fragments which were found to be tightly linked to Gall Midge biotype-1 resistance gene (Gm2). The two RAPD fragments, F8₁₇₀₀ in the susceptible parent ARC6650 and F10₆₀₀ in the resistant parent Phalguna, were identified after screening 5450 loci using 520 random primers on genomic DNAs of ARC6650 and Phalguna. These primers, when used in a multiplexed PCR, amplified specifically a 1.7-kb and 0.6-kb fragment in the susceptible and resistant parents, respectively. When this assay was performed on genomic DNAs of 44 recombinant inbred lines derived from ARC6650 x Phalguna and 5 lines derived from other crosses where one of the parents was Phalguna, ARC6650 or their derivatives, the primers amplified a 1.7-kb fragment in all of the susceptible lines or a 0.6-kb fragment in all of the resistant ones. These markers can be of potential use in the marker-aided selection of Gall Midge biotype-1 resistant phenotypes. As screening for resistance can now be conducted independent of the availability of insects, the breeding of resistant varieties can be hastened.     

The adaptive significance of cultural behavior: Comments and reply

Summary Fundamentally, theoretically, there is only one process underlying genetic and cultural evolution: natural selection. Organism fitness-enhancement (adaptive significance) is one of its practical mechanisms; group formation and maintenance is another, often but not always through fitness-enhancement; and need-fulfillment is still another. If Durham can accept that formulation, and switch from organism-thinking to instruction-thinking (Cloak, 1975: 178), he will free himself from two handicaps: First, he can forget his worries about reductionism and determinism (1976a: 100, 101). Under this general theory of natural selection, cultural evolutionis biological evolution, continued by other (nongenetic) means. Second, he will spare himself the appearance of anthropomorphism, mentalism, and wishy-washiness attendant on his discussion of kinds of significance, other than adaptive significance, of cultural behaviors (1976a: 102–106, 115).     

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     

Average phase difference theory and 1∶1 phase entrainment in interlimb coordination

The dynamics of coupled biological oscillators can be modeled by averaging the effects of coupling over each oscillatory cycle so that the coupling depends on the phase difference between the two oscillators and not on their specific states. Average phase difference theory claims that mode locking phenomena can be predicted by the average effects of the coupling influences. As a starting point for both empirical and theoretical investigations, Rand et al. (1988) have proposed d/dt= — K sin ), with phase-locked solutions =arcsin( /K), where is the difference between the uncoupled frequencies and K is the coupling strength. Phase-locking was evaluated in three experiments using an interlimb coordination paradigm in which a person oscillates hand-held pendulums. was controlled through length differences in the left and right pendulums. The coupled frequency _c was varied by a metronome, and scaled to the eigenfrequency _v of the coupled system K was assumed to vary inversely with _c. The results indicate that: (1) and K contribute multiplicatively to (2) =0 or = regardless of K when =0; (3) 0 or regardless of when K is large (relative to ); (4) results (1) to (3) hold identically for both in phase and antiphase coordination. The results also indicate that the relevant frequency is _c/_v rather than _c. Discussion high-lighted the significance of confirming =arcsin(/K) for more general treatments of phase-locking, such as circle map dynamics, and for the 11 phase-entrainment which characterizes biological movement systems.     

Improvement in the quality of seed storage protein by transformation of Brassica napus with an antisense gene for cruciferin

The levels of certain essential amino acids, in particular cysteine, lysine and methionine, in the seed storage protein of a commercial spring variety of rape, Brassica napus, have been increased by the introduction of an antisense gene for cruciferin, which is the most abundant storage protein in rapeseed. The antisense construct contained part of the cruA gene in an inverted orientation, and the gene was driven by the 5 flanking region of the gene for napin such that antisense RNA was expressed in a seed-specific manner. The construct was introduced by Agrobacterium-mediated gene transfer. In self-pollinated seeds (T1 seeds) of transgenic plants there was a reduction in the levels of the 11 and 2/32/3 subunits of cruciferin, whereas the level of the 44 subunit was unchanged. The total protein and lipid contents of transgenic seeds did not differ significantly from that of normal seeds. Seeds with reduced amounts of cruciferin accumulated higher amounts of napin than non-transformed seeds, but the level of oleosin was unaffected. Amino-acid analysis of the seed storage protein revealed that T1 seeds with reduced amounts of cruciferin contained higher relative levels of three essential amino acids, namely, lysine, methionine and cysteine, with increases of 10%, 8% and 32% over the respective levels in non-transgenic seeds (B. napus cv Westar).     

Effect of culture media on lymphokine-activated killer effector phenotype and lytic capacity

Summary We compared the ability of murine lymphokine-activated killer (LAK) cells grown in either a serum-supplemented standard medium (MEM plus fetal calf serum) or a serum-free medium (AIM-V) to lyse a range of tumour targets. LAK cells grown in either of the media killed a cultured murine tumour line (YAC-1 lymphoma) well and spared syngeneic self cells (concanavalin-A-stimulated splenocytes). However, a striking difference was noted in the ability of LAK cells grown in MEM plus fetal calf serum (as opposed to AIM-V) to kill modified self cells (trinitrophenol-modified concanavalin A blasts); LAK cells grown in the former always killed modified self cells better than those grown in the latter. This pattern held under a broad range of experimental manipulations and was found to be related to a relative increase in CD3-bearing LAK cells grown in the standard medium. These data suggest that the two media cannot be used interchangeably. This conclusion may have clinical implications for the use of LAK cells, as animal studies have been done using LAK cells generated in serum-containing medium and clinical studies have used LAK cells generated in serum-free medium.