The in vitro production of an anthocyanin from callus cultures of Oxalis linearis

Callus growth and the production of anthocyanins were sustained on the salts and vitamins of Murashige and Skoog. Callus growth was stimulated at a concentration of 8–32 M -naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-d). Benzyladenine (BA) and zeatin at 8 M inhibited callus growth whereas isopentenyladenine (iP) stimulated callus growth. NAA repressed anthocyanin production with an increase in NAA from 8–32 M. Anthocyanin synthesis was promoted by an increase in 2,4-d from 0.5 to 2 M and decreased thereafter up to a concentration 32 M 2,4-d. A concentration of 8 M BA, thidiazuron and zeatin, respectively stimulated pigment production. Sucrose stimulated callus growth at 60 mM and pigment production at 120–360 mM.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - iP isopentenyladenine - TZ thidiazuron-N-phenyl-N-1,2,3-thiadiazol-5-yl-urea - Bu-HCl Butanol-2N HCl - BAW Butanol-acetic acid-water     

The role of muscle development in the transition to endothermy in nestling bank swallows,Riparia riparia

Summary We examined the transition from ectothermy to endothermy in nestling bank swallows (Riparia riparia) by measuring the peak metabolic response to cold (PMR) in groups of nestlings. Additionally aerobic capacity, as assessed by citrate synthase activity (CS), and contractile function, as assessed by myofibrillar ATPase activity (mATPase) were measured in the pectoralis and mixed leg muscles during development. During the first 65% of their growth (from 2–12 g) bank swallows do not increase their metabolic rate in response to cold (Fig. 1). Between 12 and 16 g the PMR increased from 4 to more than 10 ml O₂ (g·h)^–1. Citrate synthase activity increased throughout development, starting at 20 moles (min·g fresh mass)^–1 in both tissues and increasing to 150 and 50 moles (min·g)^–1 in the pectoralis and leg muscles, respectively (Fig. 5). The augmented aerobic capacity combined with large increases in muscle mass undoubtedly contributes to the improved thermoregulatory abilities of older nestlings. However, muscle mass and aerobic capacity increase continuously and do not show the sharp transition noted in PMR. In the leg muscle mATPase activity is constant throughout growth, but in the pectoralis muscle it undergoes an abrupt increase from 0.5 moles (min·mg myofibrillar protein)^–1 in animals weighing less than 12 g to 0.9 moles (min·mg)^–1 in nestlings weighing more than 15 g (Fig. 6). The similar pattern of development of PMR and mATPase suggests a critical role for muscle development in the transition to endothermy in this species.Abbreviations CS citrate, synthase - mATPase myofibrillar adenosine triphosphatase - PMR peak metabolic rate during cold stress - rate of oxygen consumption     

Effect of androgens on histochemical fibre type

Summary The temporal muscles of the guinea pig show a sexual differentiation reflected in their histochemical enzyme pattern. Using histochemical methods for mitochondrial (SDH, -GPDH), and glycolytic enzymes (phosphorylase, LDH) it could be shown, that in adult animals the male muscle is a white muscle with marked activity of glycolytic enzymes, the female muscle a red muscle displaying high activity of mitochondrial enzymes. This differential enzyme pattern can be converted by the application of testosterone to the female type during the postnatal development. The male sex hormone thus affects the histochemical enzyme pattern of the muscle, converting the red, female into a white, male muscle in the female guinea pig.     

Electrophoretic variation between class II molecules expressed on HLA-DRw8 homozygous typing cells reveals multiple distinct haplotypes

Two-dimensional (2D) gel electrophoresis of immunoprecipitated HLA-DR antigens from eight homozygous typing cells (HTC) expressing the HLA-DRw8 specificity revealed a clustering of polymorphic chain patterns into distinct electrophoretic variants. The variant patterns correlate with three discrete HLA-D clusters that are defined in the mixed leukocyte culture reaction (MLR) using DRw8-positive HTC. These HLA-D clusters have been provisionally designated Dw8.1, detected primarily in Caucasoids, Dw8.2, detected primarily in American Indians, and Dw8.3, detected predominantly in Orientals. All three HLA-Dw8.1 cell lines express a single DR-locus product as defined by immunoprecipitation with a DR-specific monoclonal antibody, P4.1. This DR chain is identical among the Dw8.1 cell lines and different from the DR chains of the Dw8.2 and Dw8.3 cell lines. Two separate Dw8.2 HTC express a shared DR chain that is slightly more basic than the 8.1 DR molecule; interestingly, one of these lines also expresses an additional DR-like chain not found in the other cells. Thus, the two lines defining the Dw8.2 cluster share one distinct class 11 molecule, but differ in another and therefore are not biochemically HLA-identical. Cells from the Dw8.3 cluster are likewise distinct from all other Dw8 clusters. One additional DRw8-positive HTC has been analyzed and found to be distinct from the Dw8.1, 8.2 and 8.3 clusters by both MLR and 2D gels. lmmunoprecipitates using monoclonal antibody 1B5 [anti-DR and anti-DQ(DS)] identify additional polymorphic class II variants among the cell lines tested. These data indicate that HLA-DRw8 is a public serologic specificity present on class II molecules expressed on multiple distinct haplotypes. These haplotypes differ from each other in expression of polymorphic class II molecules encoded by at least two HLA loci. They also differ in HLA-D, even though they all type as HLA-DRw8 homozygous. In Dw8.2, variation in expressed chains is not reflected in variation in HLA-D, indicating that MLR, as well as serologic typing, does not detect the full degree of allelic polymorphism within HLA.     

Detection and partial characterization of two antigenically distinct β-amylases in developing kernels of wheat

A -amylase (EC 3.2.1.2) was identified in the outer pericarp (P) of developing seeds of wheat (Triticum aestivum L.) and compared with the well known -amylase which is synthesized during seed development in the starchy endosperm (E). The enzyme P already exists in the tissues before anthesis and vanishes at the time when E starts to accumulate. The isoelectric-focusing patterns of P and E are very similar. The relative molecular weight (M_r) of P is slightly higher than that of E (66 and 64.5 kDa, respectively). Both P and E exhibit common epitopes in addition to epitopes specific for each of them. The two enzymes were identified in small amounts in the green tissues of the developing seeds (inner pericarp and testa). No antigenic difference was detected between P and the -amylases of roots and leaves.Abbreviations P pericarp -amylase - E endosperm -amylase - IS1 anti--amylase immune serum - IS2 anti- and anti- amylase immune serum - IS3 anti- amylase immune serum - IEF isoelectric focusing - IgG immunoglobulin G The authors thank Dr. P. Ziegler (Universität Bayreuth, FRG) for stimulating discussion and for useful suggestions during the writing of the text. The authors thank Miss C. Mayer for her skillful technical assistance.     

Meiotic studies ofElymus nutans andE. jacquemontii (Poaceae,Triticeae) and their hybrids withPseudoroegneria spicata and seventeenElymus species

Hybridizations ofElymus nutans andE. jacquemontii were carried out with one species ofPseudoroegneria (S genome), and 20Elymus species, each containing either of the SH, SY, SYH, or SYW genomes. Chromosome configurations were analysed at metaphase I of the two target taxa and their interspecific hybrids. It is concluded that (i)E. nutans is an allohexaploid containing the SYH genomes, andE. jacquemontii is an allotetraploid having the SY genomes; (ii) the genomic affinity is associated with the geographic distance between the species studied; (iii) minor genomic structural rearrangements have occurred within the hexaploid taxon ofE. nutans.     

Proliferation and germination of somatic embryos from embryogenic suspension cultures in <Emphasis Type="Italic">Phœnix dactylifera</Emphasis>

The present study demonstrates a procedure for the rapid development of a high number of somatic embryos from embryogenic suspension culture. This method might be efficient for mass propagation of Phnix dactylifera L. Embryogenic callus placed in liquid medium with 10^–5M ABA yielded an average 72 embryos per 100ml of culture medium within 2months, while those placed on solid medium yielded an average of 33, 20 and 16 embryos per 100ml of culture medium respectively for 10^–7, 10^–6 and 10^–5 M ABA after 4months. The combination of 2,4-DIchlorophenoxyacetic acid (2,4-D) (4.5×10^–7M), glutamine (6.7×10^–4M), and ABA (10^–5M) (L8 liquid medium) showed a beneficial effect on somatic embryos production compared to 2,4-D and glutamine alone, while this combination significantly (p<0.05) increased the accumulation of storage proteins (144 and 138mgg^–1 DW respectively for Jihel and Bousthami noir cultivars) in somatic embryos. The somatic embryos which underwent maturation on medium containing only 4.5×10^–7M 2,4-D and 10^–5M ABA (L6 liquid medium) accumulated more sugars (292 and 265mgg^–1 DW respectively for Jihel and Bousthami noir) than those matured on any other liquid medium. Histological studies revealed that somatic embryos (developed in L6 and L8 liquid media) accumulated less reserve compounds (proteins and sugars) than zygotic embryos. The addition of activated charcoal (0.25 and 0.5gl^–1) and phytagel^® (2.5gl^–1) to the germination medium may be useful for enhancing the germination of Phnix dactyliferasomatic embryos.     

Susceptibility of clinical isolates of yeasts to anti-fungal agents

The antimicrobial activity of amphotericin B, 5-fluorocytosine, nystatin, clotrimazole and miconazole were compared in vitro against 244 strains of yeasts that had been isolated from clinical specimens. The yeasts used in this study included 20 species of Candida, Cryptococcus, Saccharomyces Geotrichum, Rhodotorula, Torulopsis and Trichosporon. The majority of the strains (78%) had an MIC of 0.5 g/ml for amphotericin B, 81% an MIC of 1 g/ml for 5-fluorocytosine, 99% 8 g/ml for nystatin, 91%, 8.0 g/ml for clotrimazole and 98% had an MIC of 4.0 for miconazole. Of the anti-fungal agents tested, 5-fluorocytosine and nystatin were found to have the greatest antifungal activity.     

Myosatellite cells associated with different muscle fibre types in the Atlantic hagfish (Myxine glutinosa,L.)

Summary The incidence of myosatellite cells associated with white and red muscle fibres of the parietal muscle and red fibres of the craniovelar muscle was estimated by quantitative electron microscopy in the Atlantic hagfish (Myxine glutinosa, L.). Myosatellite cell nuclei constitute 3, 11 and 23 % of the total number of nuclei inside the basal lamina of the three types of muscle fibres, respectively. However, the total number of nuclei is highest in white fibres, most of the nuclei belonging to striated muscle cells. Myosatellite cell profiles in transverse sections constitute 23, 41 and 61 % of the number of muscle fibre profiles of the three types, respectively. The intervals between adjacent myosatellite cells are 135 m in white fibres, 55 m in red parietal fibres, and only 25 m in craniovelar fibres. Since craniovelar fibres are also comparatively thin, myosatellite cells constitute a significant fraction of the volume inside the basal lamina in these fibres. The myosatellite cells are 30–50 m long and up to 5 m thick. Some myosatellite cells possess few organelles, whereas others appear to contain many free ribosomes, granular endoplasmic reticulum, prominent Golgi apparatus and lysosome-like bodies.This investigation was supported by the Norwegian Research Council for Science and the Humanities (NAVF grant No. C20.30–37). The authors are indebted to Jorunn Line Vaaland and Berit Branil for technical assistance, and to Dr. Finn Walvig, Biological Station, University of Oslo, Drøbak, for supplying the hagfish     

Genetics of Ia-like alloantigens in chickens and linkage withB major histocompatibility complex

Two sets of backcross matings were performed to test for linkage between genes coding for the Ia-like antigens (Ia) and the B erythrocyte antigens (Ea-B) of the chicken. Evidence is presented which indicates that the la antigens are determined by a single codominant locus and that theEa-B and Ia loci are on the same chromosome. Failure to detect a single recombinant between theEa-B and Ia loci out of 208 progeny suggests close linkage of the two genes with a map distance of up to about 2 centimorgans. The Ia genes are thus included in theB major histocompatibility complex of the chicken.     

Purification and characterization of flavone-specific β-glucuronidase from callus cultures of Scutellaria baicalensis Georgi

-Glucuronidase from callus cultures of Scutellaria baicalensis Georgi was purified to apparent homogeneity by fractionated ammonium-sulfate precipitation and chromatography on diethylaminoethyl-cellulose, hydroxylapatite and baicalin-conjugated Sepharose 6B. A 650-fold purification was obtained by this purification system. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified protein migrated as a single band with a molecular mass of 55 kDa. We determined that the native enzyme has a molecular mass of 230 kDa using gel-filtration chromatography. These results suggested that the enzyme exists as a homotetramer composed of four identical 55-kDa subunits. The enzyme showed a broad pH optimum between 7.0 and 8.0. The K _m values were 9 M, 10 M, 30 M and 40 M for luteolin 3 -O--d-glucuronide, baicalin, wogonin 7-O--d-glucoronide and oroxlin 7-O--d-glucuronide, respectively. The enzyme was most active with flavone 7-O--d-glucuronides.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - pI isoelectric point - R _t retention time     

Lethal genes on the sex chromosomes concealed in a population of the mosquito Aedes aegypti L.

A population has been examined in which an overall parity between the sexes hides considerable between-family variation in sex ratio. A proportion of families show highly distorted sex ratios, with either an excess of females or an excess of males. Distorted sex ratios are invariably associated with mortality in the immature stages at a level appropriate to the action of recessive lethal genes. It has been shown that 26% of M-bearing (Y) chromosomes and at least 24% of m-bearing (X) chromosomes carry a recessive lethal gene.Two such genes have been investigated. l kills males and, in a cross between two heterozygotes, gives rise to a sex ratio close to 2:1 (excess families). k kills females and, in a cross between two heterozygotes, gives rise to a sex ratio close to 1:2 (excess families). Selection for excess or excess did not increase the level of sex ratio distortion.No crossing over occurs between k and the M/m locus whereas l shows 5–10% recombination with M/m. A test for allelism confirmed that l and k are not allelic. The penetrance of k is complete whereas l shows somewhat less than full penetrance. The penetrance of l has been improved by selection.The high frequency of lethals remained in the population during the two year period of study. There was evidence for heterosis preserving this frequency, the heterozygotes living longer and producing more progeny. However lethals were no longer to be found after four further years of laboratory culture.     

The human genes for the α and γ subunits of the mast cell receptor for immunoglobulin E are located on human chromosome band 1823

The receptor with high affinity for immunoglobulin E (FcERI) is a key molecule in triggering the allergic reaction. It is tetrameric complex of one subunit, one subunit, and two disulfide-linked subunits. This receptor is present exclusively on mast cells and basophils. Molecules identical to the subunit of FcRI also form cell surface complex with other Fc receptors such as mouse FcRIIa in macrophages and most probably with human FcRIII (CD16) in natural killer (NK) cells. Here we show by in situ hybridization that the human genes for the (FCER1A) and subunits (FCER1 G) of FcERI and the gene for FcRIII (FCGR3, CD16) are located on human chromosome band 1823.     

Fromβ-lactams toα- andβ-amino acid derived peptides

Summary The potential of-lactams as intermediates for the access to- and-amino acid-derived peptides is shortly reviewed, with major focus on the technologies developed in our group. The two general strategies lie, on one side, in the oxidative ring expansion of 3-hydroxy-lactams toN-carboxy-amino acid anhydrides or Leuch's anhydrides and subsequent coupling with-amino acid esters and, on the other side, in the nucleophilic ring opening ofN-Boc--lactams. Both approaches have been successfully applied to the synthesis of,-diamino acid,-amino--hydroxy acid, polyhydroxylated-amino acid,,-disubstituted-amino acid,-amino acid,-amino--hydroxy acid and,-disubstituted-amino acid derived peptides. Because of the mild reaction conditions needed for the above transformations and the highly stereoselective procedures employed for the construction of the starting-lactam ring, the whole process allows the production of optically pure final products.     

Solution conformations of proline rings in proteins studied by NMR spectroscopy

Summary Three different conformations of proline rings in a protein in solution, Up, Down and Twist, have been distinguished, and stereospecific assignments of the pyrrolidine -, - and -hydrogens have been made on the basis of ¹H-¹H vicinal coupling constant patterns and intraresidue NOEs. For all three conformations, interhydrogen distances in the pairs -³, ³-³, ²-², ²-², and ³-³ (2.3 Å) are shorter than those in the pairs -², ²-³, ³-², ²-³, and ³-² (2.7–3.0 Å), resulting in stronger NOESY cross peaks. For the Up conformation, the ³-² and ²-³ spin-spin coupling constants are small (<3 Hz), and weak cross peaks are obtained in a short-mixing-time (10 ms) TOCSY spectrum; all other vicinal coupling constants are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. For the Down form, the -², ²-³, and ³-² vicinal coupling constants are small, leading to weak TOCSY cross peaks; all other couplings again are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. In the case of a Twist conformation, dynamically averaged coupling constants are anticipated. The procedure has been applied to bovine pancreatic trypsin inhibitor and Cucurbita maxima trypsin inhibitor-V, and ring conformations of all prolines in the two proteins have been determined.     

Molecular cloning of the β-cyclodextrin synthetase gene from an alkalophilic Bacillus and its expression in Escherichia coli and Bacillus subtilis

Summary The gene for -CGTase from an alkalophilic bacterium, Bacillus sp. #1011, was cloned in an Escherichia coli phage D69 and recloned in an E. coli plasmid pBR322 and a B. subtilis plasmid pUB110. An E. coli recombinant plasmid pTUE202 and a B. subtilis plasmid pTUB703 were selected from ten plasmids, because the transformants by each of the two plasmids produced the highest amount of extracellular -CGTase in each strain. The plasmids were stably maintained and expressed in each bacterial strain. A common DNA region of approximately 2.5 kb was defined in the ten plasmids, and the enzymatic activity was lost when a part of the common region was deleted. The major product of hydrolysis from starch by the -CGTases of E. coli [pTUB202] and B. subtilis [pTUB703] was -CD as in the case of the enzyme of the parental Bacillus sp. #1011.Abbreviations -CGTase -cyclodextrin synthetase - -CD -cyclodextrin - -CD -cyclodextrin - -CD -cyclodextrin - [] designates plasmid-carrier state     

Comparative residual toxicities of pesticides to the predator Agistemus industani (Acari: Stigmaeidae) on citrus in Florida

Residual toxicities of registered and selected experimental pesticides used on citrus against Agistemus industani Gonzalez (Acari: Stigmaeidae) were compared. Pesticides considered highly toxic to A. industani were: abamectin 0.15 EC at 731ml/ha+FC 435-66 petroleum oil at 46.8l/ha, pyridaben 75WP at 469g/ha, ethion 4EC at 7.01l/ha+FC 435-66 petroleum oil at 46.8l/ha, propargite 6.55 EC at 3.51l/ha, chlorfenapyr 2SC at 1.46l/ha applied alone or in combination with FC 435-66 petroleum oil at 46.8l/ha, sulphur 80DF at 16.81kg/ha, dicofol 4EC at 7.01l/ha, fenbutatin oxide 50WP at 2.24kg/ha, benomyl 50WP at 2.24kg/ha, benomyl 50WP at 1.68kg/ha+ferbam 76 GF at 5.60kg/ha, ferbam 76GF at 11.21kg/ha, neem oil 90EC at 46.8l/ha, and copper hydroxide DF (40% metallic copper) at 4.48kg metallic copper/ha+FC 435-66 petroleum oil at 46.8l/ha. Pesticides that were moderately to slightly toxic included: copper sulphate 98% at 4.48kg metallic copper/ha+FC 435-66 petroleum oil at 46.8l/ha, fenbuconazole 2F at 280ml/ha+FC 435-66 petroleum oil at 46.8l/ha, FC 435-66 petroleum oil applied alone at 46.8l/ha or 23.4l/ha, and diflubenzuron 25WP at 1.40kg/ha. Pesticides that were non-toxic included: fenbuconazole 2F at 585ml/ha, malathion 57EC at 5.85l/ha, FC 435-66 petroleum oil at 46.8l/ha, carbaryl 80S at 3.36kg/ha, chlorpyrifos 4EC at 4.68l/ha, and formetanate 92SP at 1.12kg/ha. Understanding the toxic effects of field weathered pesticides against key predacious mite species is important for effective IPM. The results of this study provide a comparison of direct and indirect toxic effects of various pesticides to A. industani under field conditions.     

Amplification of plasmid copy number by thymidine kinase expression in Saccharomyces cerevisiae

Summary A 2 m circle-based chimaeric plasmid containing the yeast LEU2 and the Herpes Simplex Virus type 1 thymidine kinase (HSV-1 TK) genes was constructed. Transformants grown under selective conditions for the LEU2 gene harboured the plasmid at about 15 copies per cell whilst selection for the HSV-1 TK gene led to an increase to about 100 copies per cell. Furthermore, the plasmid copy number could be controlled by the stringency of selection for the TK gene, and the increase in TK gene dosage was reflected in an increase in intracellular thymidine kinase activity. The mitotic stability of the plasmid in high-copy and low-copy number cells was determined. High-copy number cells showed a greater mitotic stability. The relationship of TK expression to plasmid copy number may be useful for the isolation of plasmid copy number mutants in yeast and the control of heterologous gene expression.     

A fluorometric assay of ceramide glycanase with 4-methylumbelliferyl β-d-lactoside derivatives

4-Methylumbelliferyl 6-O-benzyl--d-lactoside (6Bn-MU-Lac) and some related compounds were synthesizedvia different selective reactions including phase-transfer glycosylation. Their suitability as substrates for a fluorometric assay of ceramide glycanase (CGase) was evaluated. Among others, the 6Bn-MU-Lac, which is resistant to exogalactosidase, was found to be a suitable substrate for routine assay of the CGase activity. For American leech CGase, theK _m value is 0.232 mM at pH 5. Abbreviations: CGase, ceramide glycanase; Gal, galactose; Glc, Glucose; Lac, lactose; MU, 4-methylumbelliferone; MU-Lac, 4-methylumbelliferyl -d-lactoside; bBn-Lac, 6-O-benzyl-lactose; 6Bn-MU-Lac, 4-methylumbelliferyl 6-Obenzyl--d-lactoside; 46Bd-MU-Lac, 4-methylumbelliferyl 4,6-O-benzylidene--d-lactoside; MU-Cel, 4-methylumbellifery -d-cellobioside; 46Bd-MU-Cel, 4-methylumbelliferyl 4,6-O-benzylidene--d-cellobioside; TLC, thin layer chromatography;¹H-NMR, proton nuclear magnetic resonance; GSL, glycosphingolipids; CSA, 10-camphorsulfonic acid. See Scheme 1 for chemical structures.     

Corticosteroid-binding globulin synthesis and distribution in rat white adipose tissue

The lactone isolated from Fusarium termed L659,699 is a potent specific inhibitor of the enzyme 3hydroxi3methylglutaril coenzyme A (HMG-CoA) synthase. In cultures of smooth muscle cells (SMC) isolated from aortic-arch of control (CSMC) and 5% of cholesterol diet (Ch-SMC) treated chicks, the incorporation of (¹⁴C)acetate to lipids (cholesterol, triacylglycerides and cholesterol ester) were greater in ChSMC cultures than in CSMC and the presence of 0.05 M L659,699 for 2 h in the incubation medium decrease the synthesis of cholesterol however the triacylglycerides synthesis increase. The effect of inhibitor is stronger in young cultures (3–4 steps) than in the older ones (11–12 steps). In young CSMC and ChSMC cultures the inhibition of cholesterol and triacylglycerides synthesis by L659,699 was reversal.