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     

Identification of embryonal antigens of maize: Globulins as primary reserve proteins of the embryo

Normal and -irradiated caryopses of Zea mays L. were germinated, and the degradation of embryonal antigens (EA) was followed in the endosperms, scutella and embryonic axes of the seedlings, using double immunodiffusion, immunoelectrophoresis and quantitative immunoprecipitation. The predominant transient EA were presumed to be storage proteins related to the reserve globulins of dicotyledonous seeds. Therefore globulins were isolated from maize scutella, purified by (NH₄)₂SO₄ fractionation and isoelectric precipitation, and the molecular weights of the polypeptide units were estimated by discontinuous sodium-dodecyl-sulphate slab electrophoresis. The globulins were found to be identical with the predominant EA and amounted to about 40% of the protein nitrogen in the embryos of mature, non-germinated caryopses. The presumed reserve function of the globulins and the characteristic time course of their degradation in embryonic axes and scutella of maize seedlings are discussed in relation to the two-step pattern of mobilization of nitrogen reserves in germinating cereal caryopses.Abbreviations AG, AR and AS antisera to globulins and to root and scutellum proteins, respectively - CA common antigens - DAP days after pollination - EA embryonal antigens - ME 2-mercaptoethanol - SDS-PAGE discontinuous sodium-dodecylsulphate slab polyacrylamide-gel electrophoresis - TCA trichloroacetic acid     

Lateral diffusion in the plasma membrane of maize protoplasts with implications for cell culture

Plasma-membrane dynamics in live protoplasts from maize (Zea mays L.) roots were characterized and examined for relationships as to the ability of the protoplasts to synthesize new cell walls and develop to cells capable of division. The lateral diffusion-coefficients and mobile fractions of fluorescence-labeled plasma-membrane proteins and lipids were measured by fluorescence photobleaching recovery. Small but significant effects on the diffusion of membrane proteins were observed after treatments with oryzalin or amiprophosmethyl, microtubule-disrupting drugs that increased the mobile fraction, and after treatments with cytochalasins B or D, microfilament-disrupting drugs that decreased the diffusion coefficient. A number of parameters were tested for correlative effects on membrane dynamics and protoplast performance in culture. Protoplasts isolated with a cellulase preparation from Trichoderma viride showed faster membrane-protein diffusion and a lower frequency of development to cells capable of division than did protoplasts isolated with a cellulase preparation from T. reesei. Membrane proteins in maize A632, a line less capable of plant regeneration from callus, diffused with a smaller diffusion coefficient but a greater mobile fraction than did membrane proteins in maize A634, a line with greater regeneration capacity. The plasma membranes of A632 and A634 protoplasts also differed with regard to lateral-diffusion characteristics of phospholipid and sterol probes, although the presence of both rapidly and slowly diffusing lipid components indicated the apparent existence of lipid domains in both A632 and A634. The protoplasts of the two lines did not differ significantly, however, in either wall regeneration or frequency of development to cells capable of division.Abbreviations and symbols D lateral diffusion coefficient - FITC fluorescein-5-isothiocyanate - FPR fluorescence photobleaching recovery - LY Lucifer yellow - LY-Chol dilithium 4-amino-N-[(-(carbo(5-cholesten-3-yl)oxy)hydrazinocarbonyl)aminol]-1,8-naphthalimide-3,6-disulfonate - LY-DC_16:0PE dilithium 4-amino-N-[3-(-(dipalmitoyl-sn-glycero-3-phosphoethanol-amino)ethylsulfonyl)phenyl]-1,8-naphthalimide-3,6-disulfonate     

Calcium-dependent phospholipid-binding proteins in plants

There is evidence that Ca²⁺ can regulate vesicle-mediated secretion in plant cells, but the mechanism for this is not known. One possibility is that Ca²⁺ -dependent phospholipid-binding proteins (annexins) couple the Ca²⁺ stimulus to the exocytotic response. Using a protocol developed for the isolation of animal annexins we have identified proteins in maize (Zea mays L.) coleoptiles that have similar characteristics to annexins. The predominant polypeptide species run as a doublet of relative molecular mass (M_r) 33000–35000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE); another less-abundant protein of M_r 23000 is also present. In the presence of Ca²⁺ these proteins bind to liposomes composed of acidic phospholipids. Calcium-sensitivity of binding differs for each protein and is also influenced by the pH of the buffer used for the liposome-binding assay. Antiserum raised to the 33 to 35-kDa doublet purified on SDS-PAGE recognises the doublet in crude extracts from maize and proteins of similar M_r in Tradescantia virginiana and tobacco Nicotiana tabacum L. The antiserum also recognises p68 (Annexin VI) from chicken gizzard extracts, indicating homology between animal annexins and the maize proteins. For the maize proteins to be involved in the regulation of exocytosis, binding to phospholipids would be expected to occur at physiological levels of Ca²⁺. The characteristics of the maize annexin-like proteins are described and attention drawn to the marked effect of pH in lowering the requirement for Ca²⁺ for phospholipid binding.Abbreviations DEAE diethylaminoethyl - EGTA ethylene glycol-bis (-aminoethyiether)-N,N,N,N-tetraacetic acid - kDa kilodalton(s) - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis This work was funded by the Agricultural and Food Research Council. Our thanks also to Professor P. Lowry and Dr R. Woods, Department of Biochemistry, University of Reading for facilities and advice for antiserum production, and C. Boustead, Department of Biochemistry, University of Leeds for advice on immunoblotting and phospholipid-binding assays.     

Interactions between HMG proteins and the core sequence of DNaseI hypersensitive site 2 in the locus control region (LCR) of the human β-like globin gene cluster

HMG proteins are abundant chromosomal non-histone proteins. It has been suggested that the HMG proteins may play an important role in the structure and function of chromatin. In the present study, the binding of HMG proteins (HMG1/2 and HMG14/17) to the core DNA sequence of DNaseI hypersensitive site 2 (HS2core DNA sequence, -10681-10970 bp) in the locus control region (LCR) of the human β-like globin gene cluster has been examined by using both thein vitro nucleosome reconstitution and the gel mobility shift assays. Here we show that HMG1/2 can bind to the naked HS2core DNA sequence, however, HMG14/17 cannot. Using thein vitro nucleosome reconstitution we demonstrate that HMG14/17 can bind to the HS2core DNA sequence which is assembled into nucleosomes with the core histone octamer transferred from chicken erythrocytes. In contrast, HMG1/2 cannot bind to the nucleosomes reconstitutedin vitro with the HS2core DNA sequence. These results indicate that the binding patterns between HMG proteins and the HS2core DNA sequence which exists in different states (the naked DNA or thein vitro reconstituted nucleosomal DNA) are quite different. We speculate that HMG proteins might play a critical role in the regulation of the human β-like globin gene’s expression.     

Properties of a blue-light-absorbing photoreceptor kinase localized in the plasma membrane of the coleoptile tip region

The blue-light-sensing apical part of coleoptiles of grasses is responsible for the first positive phototropic bending reaction of this organ. The photoreceptor responsible has been shown to be localized to the plasma membrane (PM) of this tip region. An approximately 100-kDa protein moiety of this receptor is rapidly phosphorylated upon irradiation. Properties of this protein kinase reaction were studied in vitro by using PMs from the maize (Zea mays L.) coleoptile tip region: (i) The substrate for the blue-light-triggered phosphorylation of the 100-kDa protein was found to be ATP as well as GTP. However, the affinity of the involved protein kinase for the substrate GTP was lower than for ATP. (ii) Experiments were undertaken to find out whether a photoreceptor moiety acts as an autophosphorylating protein kinase or whether the photoreceptor protein, when activated by light, becomes the target of an extrinsic protein kinase. Two studied extrinsic protein kinases (50 and 55 kDa) of the coleoptile tip were found not to be involved in the lightdependent protein phosphorylation. The degree of phosphorylation of the 100-kDa protein on isolated plasma membranes upon irradiation at 0 °C was scarcely different from a reaction at 30 °C, in contrast to the background protein phosphorylations which decreased with decreasing temperature. This result points to an autophosphorylation mechanism at the receptor. (iii) In mixing experiments, solubilized membranes from maize coleoptiles were irradiated and added to unirradiated membrane proteins from pea (Pisum sativum L.) epicotyls followed by addition of [-³²P]ATP. Unirradiated proteins from pea were not phosphorylated by light-activated (autophosphorylatable) maize protein kinases. (iv) It is suggested that the blue-light-sensitive photoreceptor localized to the PM of the phototropically active tip region of coleoptiles has an autophosphorylatable kinase domain which is able to use ATP or GTP as substrate.Abbreviation PM plasma membrane The author is deeply indebted to Mrs Elke Stransky for her excellent technical assistance and performance of the experiments.     

Purification and characterization of ten new rice NaCl-soluble proteins: identification of four protein-synthesis inhibitors and two immunoglobulin-binding proteins

Ten new proteins from rice (Oryza saliva L. cv. Bahia) including four protein-synthesis inhibitors and two immunoglobulin E (IgE)-binding proteins have been isolated and characterized. These proteins as well as one previously known component, -globulin, were purified from a 0.5 M NaCl extract of rice endosperm by a new, apparently non-denaturing, isolation procedure developed for rice proteins. The method is based on extractions of this complex protein mixture with a diluted volatile salt solution and an aqueous solution of ethanol. This preliminary step results in an improvement in the separation of these proteins, thus facilitating their subsequent purification by reversed-phased high-performance liquid chromatography. These new proteins have similar relative molecular masses (M_rs) from 11000 to 17000. The purity of the proteins was analyzed by micro two-dimensional gel electrophoresis. Four of these components were found to be in-vitro protein-synthesis inhibitors in a cell-free system from rat brain. The NH₂-terminal amino-acid sequences of these four inhibitors were determined from 12 to 26 cycles after direct blotting of the separated proteins from electrophoresis gels. Three of these proteins with M_rs between 16000 and 17000 showed a high degree of homology ranging from 57% to 75% but seem to be unrelated to the fourth inhibitor. In addition, the -globulin and one of the new low-molecular-weight proteins of M_r 12500 seemed to show allergenic properties since they bound IgE antibodies from the sera of hypersensitive patients. Boths proteins have blocked NH₂-terminal amino acids.Abbreviations HMW high molecular weight - IgE immunoglobulin E - LMW low molecular weight - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - RP-HPLC reversed-phase high-performance liquid chromatography - SDS sodium dodecyl sulphate We thank F. Soriano and F. Colillia for technical assistance, and Shirley McGrath for secretarial work. We also appreciate the cheerful assistance of the members of Instituto Nacional de Semillas, specially Mr. L. Solaices, who provided samples of rice. This work was supported by a grant from Comision Asesora de Investigación Ciéntifica y Técnica.     

ent-Kaurene biosynthesis in a cell-free system from wheat (Triticum aestivum L.) seedlings and the localisation of ent-kaurene synthetase in plastids of three species

A cell-free system capable of converting [¹⁴C]geranylgeranyl diphosphate to ent-[¹⁴C]kaurene and to an unidentified acid-hydrolysable compound was obtained from the basal portions of 5-d-old shoots of wheat seedlings (Triticum aestivum L.). By means of marker enzyme activities, the synthesis of ent-kaurene and the unknown compound could be quantitatively assigned to a plastid fraction obtained by Percoll-gradient centrifugation of the homogenate. The enzyme activities were located within the plastids, probably in the stroma, because they withstood trypsin treatment of the intact plastids, and the plastids had to be broken to release the activity, which was then obtained in soluble form. Plastid membranes had no activity. Plastid stroma preparations obtained from pea (Pisum sativum L.) shoot tips and pumpkin (Cucurbita maxima L.) endosperm also yielded ent-kaurene synthetase activity, but did not form the unknown compound. The exact nature of the active plastids was not ascertained, but the use of methods for proplastid isolation was essential for full activity, and the active tissues are all known to contain high proportions of proplastids, developing chloroplasts or leucoplasts. We therefore believe that ent-kaurene synthesis may be limited to these categories. Mature chloroplasts from the wheat leaves did not contain ent-kaurene synthetase activity and did not yield the unknown component. Incorporation of [¹⁴C]geranylgeranyl diphosphate into ent-[¹⁴C]kaurene and the unknown component was assayed by high-performance liquid chromatography with on-line radiocounting. ent-[¹⁴C]Kaurene was identified by Kovats retention index and full mass spectra obtained by combined gas chromatography-mass spectrometry. The unknown component was first believed to be copalyl diphosphate, because it yielded a compound on acid hydrolysis, which migrated like copalol on high-performance liquid chromatography and gave a mass spectrum very similar to that of authentic copalol. However, differences in the mass spectrum and in retention time on capillary gas chromatography excluded identity with copalol. Furthermore, the unhydrolysed compound was not converted to ent-kaurene by a cell-free system from C. maxima endosperm as copalyl diphosphate would have been.Abbreviations ADH alcohol dehydrogenase - AMO 1618 2isopropyl-4-(trimethylammoniumchloride)-5-methylphenyl piperi-dine-1-carboxylate - BSA bovine serum albumin - DTT dithioth-reitol - GA_n gibberellin A_n - GAPDH NADP⁺-glyceraldehyde 3-phosphate dehydrogenase - GC-MS combined gas chromatography-mass spectrometry - GGPP all trans-isomer of geranyl-geranyl diphosphate - KS ent-kaurene synthetase - MDH malate dehydrogenase - MAA mevalonate activating activity - SOR shikimate oxidoreductase We thank Mrs. Gudrun Bodtke and Mrs. Dorothee Dasbach for able technical assistance, Prof. L.N. Mander (Australian National University, Canberra, Australia) for ent-[²H₂]kaurene and Dr. Yuji Kamiya (RIKEN, Saitama, Japan) for geranylgeraniol and copalol. The work was supported by the Deutsche Forschungsgemeinschaft.     

Effect of cyproterone acetate,d-norgestrel and progesterone on cells of the pars distalis of the adenohypophysis in the beagle bitch

Summary The effects of short-term (8 weeks) treatment with different doses of cyproterone acetate (CPA), d-norgestrel (d-N) and progesterone on cells of the pars distalis, as revealed by the immunoperoxidase technique, were studied in cycle-synchronized beagle bitches (first anoestrus). Pituitary glands from non-treated primiparous beagle bitches at the 6th and 9th week of pregnancy were also included. For immunochemical staining specific antisera to the following hormones were used: canine GH, canine PRL, porcine ACTH, bovine TSH, bovine LH and human FSH. Morphological features of high secretory activity in GH cells were evident even after the human oral contraceptive doses of CPA and d-N, and after a dose as low as 0.1 mg/kg/day subcutaneously (s.c.) of progesterone. In contrast, PRL cells did not show any significant treatment-related effects except in those animals which received the highest dose of d-N (0.5 mg/kg/day per os). In this group, as well as in all pregnant bitches, hyperplasia and hypertrophy of PRL cells were found. In the animals treated with the highest doses of CPA (4.0 mg/kg/day per os) and progesterone (42.5 mg/kg/day s.c.) as well as in pregnant bitches, ACTH/MSH and TSH cells showed marked atrophy and regressive changes. Similar morphological signs of depressed secretory activity were also observed in the cells shown to contain FSH and/or LH as a result of treatment with the highest dose of progesterone and at the 9th week of pregnancy. These structural responses indicate that quantitative and/or qualitative differences may exist between progesterone, the synthetic progesterone derivative CPA and the nortestosterone type progestagen d-N with regard to their effect on pituitary hormone secretion in the beagle bitch.Abbreviations ACTH Adrenocorticotropin - FSH Follicle Stimulating Hormone - GH Growth Hormone - LH Luteinizing Hormone - MSH Melanocyte Stimulating Hormone - PRL Prolactin - TSH Thyrotropin - CRH Corticotropin Releasing Hormone - TRH Thyrotropin Releasing Hormone The authors are grateful to Dr. Christel Schöbel and Mrs. P. Kurth 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     

Stability of the maize chromosomal high-mobility-group proteins,HMGa and HMGb,in vivo

Chromosomal non-histone high-mobility-group (HMG) proteins represent essential components of eukaryotic chromatin and have also been isolated from a variety of plants. In maize, studies on structure and function of the two larger of the four major HMG proteins have recently been performed and are now extended by analysis of theirin vivo stability using pulse-chase experiments in a cell suspension culture. The half-life of the analyzed HMGa and HMGb proteins was found to be 65 h or more than 78 h, respectively.     

Localization of gonadotrophic hormones in the dog pituitary gland

Summary Using the immunoperoxidase technique and antisera to the specific beta () subunits of FSH and LH¹, selective immunochemical staining was localized mostly in the same cell type in the pars distalis and pars tuberalis of the dog pituitary gland. However, some cells were consistently shown to react solely with antisera to either LH or FSH. The cells stained for FSH were at least 1.5 times less numerous than those shown to contain LH. In the pars distalis of adult male dogs the immunoreactive gonadotrophs varied greatly in their relative proportion and were mostly shown to be much less numerous than in bitches in the anestrus phase of the sexual cycle. These cells were found to be positive to aldehyde fuchsin, alcian blue, periodic acid-Schiff (PAS) and aniline blue. The performic acid-alcian blue (pH 0.2)-PAS-orange G procedure stained the FSH/LH cells blue or turquoise, demonstrating TSH cells (blue-purple), ACTH/MSH cells (red-purple) and PRL cells (orange-red). The FSH/LH cells were further differentiated from other functional cell types of the pars distalis on the basis of their typical cytological features, intraglandular distribution and by immunochemical double staining. These observations support the concept that the one cell-one hormone theory may not apply to gonadotrophic hormones, although some cells seem to be the source of either FSH or LH.Abbreviations for Pituitary 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 Dr. H. Wiemann for the statistical evaluation and to Mrs. B. Schilk and Miss U. Tüshaus for their excellent technical assistanceRecipient of a Research Scholarship from the Arabic Republic of Egypt     

Localization of thyrotropin (TSH) in the dog pituitary gland

Summary Using the immunoperoxidase technique and antisera to the specific beta () subunits of bovine and rat TSH¹, selective immunocytochemical staining was localized in a specific cell population in the pars distalis of the dog pituitary gland. These TSH cells were found to be positive to aldehyde fuchsin, alcian blue, periodic acid-Schiff (PAS) and aniline blue. With the performic acidalcian blue (pH 0.2) -PAS-orange G procedure these cells stained blue-purple, demonstrating FSH/LH cells (blue or turquoise), ACTH/MSH cells (redpurple) and PRL cells (orange-red). The TSH cells were further differentiated from other functional cell types of the pars distalis on the basis of their typical cytological features, intraglandular distribution and by immunocytochemical double staining. In the pars distalis of adult male dogs the TSH cells were mostly shown to be smaller in size and less numerous than in bitches in the anestrous phase of the sexual cycle. Moreover, cytological alterations in the immunoreactive thyrotrophs in the pituitary of male and female dogs generally paralleled the spontaneous changes in thyroid function associated with thyroid atrophy and/or pituitary insufficiency, and thyroid hyperplasia or goiter. In conclusion, because of their specificity and high potency, the antisera to the -subunits of bovine and rat TSH represent an effective tool for the selective immunocytochemical localization of TSH in the dog pituitary. This allows the study of the morphology and function of TSH cells under different physiological, pathological and experimental conditions.Abbreviations for 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 - TRH TSH Releasing Hormone - CG Chorionic Gonadotropin The authors are grateful to Mrs. B. Schilk and Miss U. Tüshaus for their excellent technical assistance     

Hevein: an antifungal protein from rubber-tree (Hevea brasiliensis) latex

Several chitin-binding proteins were isolated from the bottom fraction of Hevea brasiliensis (Müll.) Arg. latex. One of these chitin-binding proteins is hevein, a small monomeric protein which strongly resembles the lectin from stinging nettle (Urtica dioica L.). Like the latter, hevein showed strong antifungal activity against several fungi in vitro. The possible involvement of this protein in the defense against invasion by potentially pathogenic fungi is discussed.Abbreviations FPLC fast protein liquid chromatography - M_r apparent molecular mass - SDS-PAGE Sodium dodecyl sulp-hatepolyacrylamide gel electrophoresis - UDA Urtica dioica agglutinin - WGA Wheat-germ agglutinin This work is supported in part by NIH grant and grants of the National Fund for Scientific Research (Belgium): W.J.P. is senior research associate, and W.F.B. senior research assistant of this fund. J.V.P. receives a fellowship of the Belgian Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw.     

Resolution of two molecular forms of sucrose-phosphate synthase from maize,soybean and spinach leaves

Two forms of sucrose-phosphate synthase (EC 2.4.1.14) were resolved from leaves of three species, maize (Zea mays L. cv. Pioneer 3184), soybean (Glycine max (L.) Merr., cv. Ransom) and spinach (Spinacia oleracea L. cv. Resistoflay) by hydroxyapatite Ultrogel chromatography, using a 75-mM (designated peak 1) and 250-mM (peak 2) K-phosphate discontinuous-gradient elution. Rechromatography of the two forms showed that they were not readily interconvertible. The distribution of activity between the two forms differed among species and changed during purification of the enzyme. Recovery of peak-1 activity was specifically lowered when maize leaf extracts were prepared in the absence of magnesium, indicating that the two forms may differ in stability. In addition, the forms of the enzyme from maize differed in the extent of glucose-6-phosphate activation. These results provide evidence for the existence of multiple forms of sucrose-phosphate synthase in leaves of different species and that the forms differ in regulatory properties.Abbreviations Fru6P fructose 6-phosphate - Glc6P glucose 6-phosphate - HAU hydroxyapatite Ultrogel - Pi inorganic phosphate - SPS sucrose-phosphate synthase - UDP uridine 5-diphosphate - UDPG uridinediphosphate glucose Cooperative investigations of the United States Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh. Paper No. 10511 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh. Supported in part by USDA Competitive Research Grant No. 85-CRCR-1-1568     

Immunolocalization of pheromone-binding protein and general odorant-binding protein in olfactory sensilla of the silk moths Antheraea and Bombyx

The distribution of odorant-binding proteins among olfactory sensilla of three moth species was studied by immuno-electron microscopy. Two polyclonal antisera were used in a post-embedding labelling protocol on sections of cryo-substituted antennae. The first was directed against the pheromone-binding protein (PBP) of Antheraea polyphemus, the second against the general odorant-binding protein (GOBP) of the same species. Immunoblots showed that these antisera were highly specific; both antisera did, however, cross-react with related proteins in the related species A. pernyi, and in the bombycid moth B. mori. PBP and GOBP were localized only in olfactory sensilla trichodea and sensilla basiconica, the principal site being the sensillum lymph surrounding the sensory dendrites. In the males of all three species, the pheromone-sensitive long sensilla trichodea exclusively contained PBP. the majority of the sensilla basiconica in both sexes in these species contained GOBP; these sensilla are known to respond to plant and other general odours. Some sensilla were not labelled by either antiserum; presumably, these held an odorantbinding protein of a different subfamily. Never were PBP and GOBP co-localized in the same sensillum. Two observations deserve special attention: (1) PBP was also found in a few sensilla in females, and (2) in B. mori, where the long sensilla trichodea have a different functional specificity in males (pheromone) and females (plant odours), the expression of the odorant-binding protein (males: PBP; females: GOBP) is similarly different. The distinct and complex distribution pattern of odorant-binding proteins supports the notion that these proteins participate in stimulus recognition.Dedicated to Professor Ya.A. Vinnikov on the occasion of his 85. birthdayThis work was partly supported by DFG grant ste 501/3-1.     

Reef-coral proteins as visual,non-destructive reporters for plant transformation

Recently, five novel fluorescent proteins have been isolated from non-bioluminescent species of reef-coral organisms and have been made available through ClonTech. They are AmCyan, AsRed, DsRed, ZsGreen and ZsYellow. These proteins are valuable as reporters for transformation because they do not require a substrate or external co-factor to emit fluorescence and can be tested in vivo without destruction of the tissue under study. We have evaluated them in a large range of plants, both monocots and dicots, and our results indicate that they are valuable reporting tools for transformation in a wide variety of crops. We report here their successful expression in wheat, maize, barley, rice, banana, onion, soybean, cotton, tobacco, potato and tomato. Transient expression could be observed as early as 24 h after DNA delivery in some cases, allowing for very clear visualization of individually transformed cells. Stable transgenic events were generated, using mannose, kanamycin or hygromycin selection. Transgenic plants were phenotypically normal, showing a wide range of fluorescence levels, and were fertile. Expression of AmCyan, ZsGreen and AsRed was visible in maize T1 seeds, allowing visual segregation to more than 99% accuracy. The excitation and emission wavelengths of some of these proteins are significantly different; the difference is enough for the simultaneous visualization of cells transformed with more than one of the fluorescent proteins. These proteins will become useful tools for transformation optimization and other studies. The wide variety of plants successfully tested demonstrates that these proteins will potentially find broad use in plant biology.Abbreviations BMS Black Mexican sweet maize - CMP Cestrum virus promoter - GUS -Glucuronidase - LUC Luciferase - nos Nopaline synthetase - pmi Phosphomannose isomerase - PCR Polymerase chain reactionCommunicated by M.C. Jordan     

Purification and characterization of antifreeze proteins from larvae of the beetle Dendroides canadensis

Summary Four antifreeze proteins (AFPs) were purified from larvae of the beetle Dendroides canadensis. The AFPs are similar in amino acid compositions, having high contents of hydrophilic amino acids (45–55 mol%) and cysteine (16 mol% Cys). Approximately half of the Cys residues form disulfide bridges, and both the disulfide bridges and free sulfhydryls are essential for activity. The N-terminals of the AFPs are blocked. The pH optimum of the AFPs is 7.8, but major loss of activity occurred only at very high pH (12.0). The detergents SDS and Triton X-100 did not inactivate the AFPs. Circular dichroism spectra indicate the presence of both and secondary structures in the AFPs, in addition to a large random structure component.Abbreviations AFP antifreeze protein - CD circular dichroism - DTT dithiothreitol - HPLC high pressure liquid chromatography - PAGE polyacrylamide gel electrophoresis - PAS periodic acid Schiff - SDS sodium dodecyl sulfate - TFA trifluoroacetic acid     

Gibberellin biosynthesis from gibberellin A12-aldehyde in a cell-free system from germinating barley (Hordeum vulgare L., cv. Himalaya) embryos

Gibberellin (GA) metabolism from GA₁₂-aldehyde was studied in cell-free systems from 2-d-old germinating embryos of barley. [¹⁴C]- or [17-²H₂]Gibberellins were used as substrates and all products were identified by combined gas chromatography-mass spectrometry. Stepwise analysis demonstrated the conversion of GA₁₂-aldehyde via the 13-deoxy pathway to GA₅₁ and via the 13-hydroxylation pathway to GA₂₉, GA₁ and GA₈. In addition, GA₃ was formed from GA₂₀ via GA₅. We conclude that the embryo is capable of producing gibberellins that can induce -amylase production in the aleurone layer. There was no evidence for 12- or 18-hydroxylation and GA₄ was neither synthesised nor metabolised by the system. All metabolically obtained GAs, with the exception of GA₃, were also found as endogenous components of the cell-free system in spite of ammonium-sulfate precipitation and desalting steps.Abbreviations GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography We thank Mrs. G. Bodtke and Mrs. B. Schattenberg for preparing the barley embryos and the Deutsche Forschungsgemeinschaft for supporting this work.