Patterns of protein synthesis in salivary glands ofDrosophila melanogaster during larval and prepupal development

Summary Patterns of protein synthesis in the salivary glands ofDrosophila melanogaster have been studied throughout late larval and prepupal development by pulse labelling the tissues with³⁵S-methionine. Specific changes to the pattern of proteins synthesized during development are found and the significance of these changes is discussed in view of the known changes in gene (puffing) activity which occur at the same times. We review the problem of salivary gland function in prepupalDrosophila.     

In vitro propagation of Asparagus maritimus – A rare Mediterranean salt-resistant species

Asparagus maritimus L. Miller is a rare species growing of the Mediterranean region and is morphologically similar to A. officinalis. In order to establish an efficient in vitro propagation protocol, explants were excised from spear segments and cultured on Murashige and Skoog (1962) medium containing 3% sucrose and various concentrations of growth regulators. The best shoot initiation (3–4 per explant) was achieved on a medium containing 0.88 M N⁶-benzyladenine (BA), 0.93 M kinetin, 1.07 M -naphthaleneacetic acid (NAA) and 3.90 M ancymidol. Shoot initiation could also be achieved without ancymidol but the shoots were thinner and longer. A very high shoot multiplication rate was achieved on media supplemented with 3% sucrose, 1.07 M NAA, 0.93 M kinetin, 0.44 M BA and various concentrations of ancymidol. The lowest concentration of ancymidol (0.39 M) significantly promoted the highest shoot multiplication rate (11.9 shoots/crown). For root formation, media were supplemented with 6% sucrose, 1.07 M NAA and various concentrations of ancymidol. Rooting frequency increased with higher ancymidol concentration up to 5.07 M (82.0% rooting). The number of ex vitro shoots formed was strongly correlated (r=0.66) with the length of roots formed in vitro, which was the highest at a 1.95 M ancymidol.     

Inhibitory effect of GA3 on the development of thidiazuron-induced somatic embryogenesis in geranium (Pelargonium xhortorum Bailey) hypocotyl cultures

Somatic embryogenesis in geranium (Pelargonium xhortorum Bailey cv Scarlet Orbit Improved) can be achieved by incubating hypocotyl explants on MS medium supplemented with thidiazuron (TDZ; 10 M for 3 days followed by subculture on medium devoid of any plant growth regulators. The presence of gibberellins (GAs) during both the induction and expression phases of embryogenesis was significantly detrimental to somatic embryo formation on the hypocotyl explants. The addition of the GA-synthesis inhibitors paclobutrazol, uniconazole or ancymidol during the period of growth and differentiation of somatic embryos increased the number of somatic embryos formed on each explant. However, paclobutrazol added during the period of induction had no significant influence on somatic embryo formation. Results suggest that both exogenously supplied as well as endogenous GAs play a role, albeit a negative one, on somatic embryogenesis of geranium.Abbreviations MS Murashige and Skoog (1962) medium - MSO basal medium devoid of any plant growth regulator - TDZ N-phenyl-N1,2,3-thidiazol-5-ylurea (thidiazuron)     

Purine glucosylating activity in cell suspension cultures ofSolanum tuberosum L.

Cell suspension cultures ofSolanum tuberosum L. cv. Adretta were established from leaf-derived calluses. In the search for purine glucosylating activity, the metabolism of 6-benzylaminopurine was studied. The main metabolite of BA was isolated and identified as 6-benzylaminopurine 7--d-glucopyranoside indicating the occurrence of purine glucosylating activity.Abbreviations BA 6-Benzylaminopurine - [3G]BA BA 3--d-glucopyranoside - [7G]BA BA 7--d-glucopyranoside - [9G]BA BA 9--d-glucopyranoside - RA Radioactivity - R _T Retention Time     

Structures of Oligosaccharides Derived from <Emphasis Type="Italic">Cladosiphon okamuranus</Emphasis> Fucoidan by Digestion with Marine Bacterial Enzymes

A fucoidan-utilizing marine bacterium, Fucophilus fucoidanolyticus, was cultivated in medium containing fucoidan from Cladosiphon okamuranus. The C. okamuranus fucoidan was digested into oligosaccharides with the intracellular enzymes of F. fucoidanolyticus, and their structures were determined by nuclear magnetic resonance analyses. Some of their structures are represented by one general structural formula, (-3L-Fucp1-3L-Fucp(4-O-sulfate)1-3L-Fucp(4-O-sulfate)1-3(D-GlcpUA1-2)L-Fucp1)_m-3L-Fucp1-3L-Fucp(4-O-sulfate)1-3L-Fucp(4-O-sulfate) 1-3L-Fucp (m = 0, 1, 2, or 3). We concluded that all oligosaccharides obtained were derived from a sulfated-fucose-containing polysaccharide of C. okamuranus, which has a repeating unit of (-3L-Fucp1-3L-Fucp(4-O-sulfate)1-3L-Fucp(4-O-sulfate)1-3(D-GlcpUA1-2)L-Fucp1-).     

Experiments on the water budget of densely and sparsely needled spruces [Picea abies (L.) Karst.] in a declining stand

Summary The osmotic potentials of needles were compared from numerous trees that had been classified according to needle loss along an altitude profile. With the increasing degree of damage of the trees, the maximum and minimum values deviated more strongly above and below the common mean of all samples. The level of water content of the needles unequivocally reflected the vitality of the trees. Experiments covering a whole vegetation period were performed on a tree pair selected from a natural stand. They were designed to demonstrate differences in water balance between the healthy and damaged state of the trees. For the damaged tree, measurements of the water potentials of single needles showed a greater reduction of potential during the course of the day compared to the healthy tree. Recovery in the evenings was slower and often incomplete. The osmotic potentials of damaged and healthy shoots measured in individual needles also differed both predawn and especially after transpiration stress. Depending on weather and soil desiccation, the differences were more or less pronounced. In the damaged tree, the rises in potential after saturation of the twigs fell far short of the healthy tree. Reduction of water potential, osmotic potential and relative water content under comparable stress conditions suggest a reduced tolerance of drought by damaged trees. In the context of earlier experiments this result was ascribed to a lack of stomatal control, and long-term pollution effects were thus explained as a specific disturbance of hydroregulation.     

Analog of vertebrate anionic sites in blood-brain interface of larval Drosophila

The blood-brain barrier ensures brain function in vertebrates and in some invertebrates by maintaining ionic integrity of the extraneuronal bathing fluid. Recent studies have demonstrated that anionic sites on the luminal surface of vascular endothelial cells collaborate with tight junctions to effect this barrier in vertebrates. We characterize these two analogous barrier factors for the first time on Drosophila larva by an electron-dense tracer and cationic gold labeling. Ionic lanthanum entered into but not through the extracellular channels between perineurial cells. Tracer is ultimately excluded from neurons in the ventral ganglion mainly by an extensive series of (pleated sheet) septate junctions between perineurial cells. Continuous junctions, a variant of the septate junction, were not as efficient as the pleated sheet variety in blocking tracer. An anionic domain now is demonstrated in Drosophila central nervous system through the use of cationic colloidal gold in LR White embedment. Anionic domains are specifically stationed in the neural lamella and not noted in the other cell levels of the blood-brain interface. It is proposed that in the central nervous system of the Drosophila larva the array of septate junctions between perineurial cells is the physical barrier, while the anionic domains in neural lamella are a charge-selective barrier for cations. All of these results are discussed relative to analogous characteristics of the vertebrate blood-brain barrier.     

Conformational energy analysis of the leucine repeat regions of C/EBP, GCN4, and the proteins of the myc, jun, and fos oncogenes

It has been recently proposed that certain DNA binding proteins (including C/EBP, GCN4 and themyc, jun, andfos oncogene proteins) share a common structural motif based on helix-promoting regions containing heptad repeat sequences of leucines. It has been suggested that this structure is critical to the biological activity of these proteins, since it facilitates the formation of functional dimers held together by interdigitating leucine side-chains along the hydrophobic interfaces between long -helical regions of the polypeptide chains in a configuration termed the leucine zipper. In this paper, conformational energy analysis is used to determine the preferred three-dimensional structures of the leucine repeat regions of these proteins. The results indicate that, in all cases, the global minimum energy conformation for these regions is an amphipathic -helix with the leucine side-chains arrayed on one side in such a way to favor leucine zipper dimerization. Furthermore, amino acid substitutions in these regions (such as Pro for Leu), that are known to inhibit dimer formation and prevent DNA binding, are found to produce significant conformational changes that disrupt the amphipathic helical structure. Thus, these results provide support for the proposed leucine zipper configuration as a critical structural feature of this class of DNA binding proteins.     

Milk-clotting protease production by <Emphasis Type="Italic">Nocardiopsis</Emphasis> sp. in an inexpensive medium

The milk-clotting and proteolytic activities of extracellular enzyme preparations from Nocardiopsis sp. were investigated under different culture conditions. A soybean flour medium was used, with concentrations of soybean flour and of glucose varying from 0.25 to 1% w/v and from 0 to 1% w/v, respectively. Growth was monitored with 2ml samples withdrawn from the culture medium at 8-h intervals, for determination of total protein, proteolytic activity, milk-clotting activity and sugar reduction. The best milk-clotting protease production, with a specific activity of 24.49U/mg at 40h, was obtained in the glucose-free medium containing soybean flour 1% w/v.     

Characterization of solute/proton cotransport in plasma membrane vesicles from Ricinus cotyledons,and a comparison with other tissues

Plasma membrane vesicles, purified by aqueous two-phase partitioning, were used to investigate the presence of sugar and amino acid carriers in cotyledons and roots of Ricinus communis L. and in roots of red beet (Beta vulgaris L.). Artificial pH and electrical gradients were generated across the plasma membrane, and [¹⁴C]acetate and [¹⁴C]tetraphenylphosphonium were used to demonstrate the presence of an internal alkaline pH gradient and an internal negative membrane potential, respectively. In Ricinus cotyledons, uptake of sucrose was more strongly inhibited than that of glutamine by p-chloromercuribenzenesulphonic acid, phlorizin and phenylglyoxal. The sucrose transport system showed a high degree of substrate specificity with only the presence of maltose and phenyl--glucoside significantly affecting sucrose uptake; in contrast, the glutamine transport system was inhibited by a number of other amino acids. pH+gD-driven glutamine uptake showed saturation kinetics with a K _m of 0.35 mol · m^–3. Sucrose and glutamine -driven uptake was pH dependent with an optimum in the acidic range (pH 6.25) and a decrease at higher pH values. Vesicles obtained from cotyledons and roots of Ricinus showed different transport properties. In the cotyledons, gDH+gD-driven transport for both sucrose and glutamine were observed at similar levels; however, in the root tissue, pH--driven glutamine transport was the dominant uptake process. Uptake rates for glucose and fructose were low in the cotyledons whereas, in the roots, glucose and sucrose transport were slightly higher than that of fructose. In vesicles from red beet tissue there was a different uptake profile, with evidence of proton-coupled cotransport systems for sucrose and glucose, but lower uptake of glutamine and fructose. The results are discussed in relation to the reported different pathways for loading and unloading of solutes in these tissues.Abbreviations CCCP carbonyl cyanide-m-chlorophyenyl hydrazone - DEPC diethyl pyrocarbonate - NEM N-ethylmaleimide - PCMBS p-chloromercuribenzenesulfonic acid - TPP tetraphenylphosphonium ion - gDH⁺ proton electrochemical potential gradient - membrane potential We would like to thank the SERC(UK) and the Royal Society for financial support.     

Voltammetric determination of dissolved iron and its speciation in freshwater

Analytical methods were developed to determine the concentration of total dissolved iron and its chemical speciation in freshwater using cathodic stripping voltammetry (CSV) with 1-nitroso-2-naphthol (NN) at pH 8.1. The concentrations of total dissolved iron in river water that iron concentration was certified and in natural water samples from Lake Kasumigaura were determined successfully. The natural iron ligand concentration and the conditional stability constant were determined by ligand competition between NN and the natural ligands present in the sample. In the water samples from Lake Kasumigaura, the concentrations of total dissolved iron and natural ligand were 47.8 ± 4.4nM and 80.0 ± 19.6nM and the conditional stability constant (K_FeL) was 10^25.9±0.4M^–1 (n = 3). The value of K_FeL was greater than any reported K_FeL for seawater. More than 99.9% of the dissolved iron existed as organic species due to the very high value of the conditional stability constant. The inorganic iron concentration calculated from these results was 10^–13.4M, indicating that the inorganic iron level in Lake Kasumigaura was similar to that in the open ocean and therefore that iron can be a limiting factor for algal growth in Lake Kasumigaura. This is the first report of the complexation of iron(III) and inorganic iron levels in lake water determined by CSV.     

Different photoreceptors within the same retina express unique combinations of potassium channels

Single electrode current and voltage clamp recordings in Calliphora, and whole-cell voltage clamp recordings in Drosophila were used to characterise the voltage-gated K channels in both major classes of photoreceptors, R7/8 (long visual fibres, LVFs) and R1-6 (short visual fibres, SVFs). R7/8 were identified by their unique spectral properties, ca. 3–4 fold higher input resistances and 3–4 fold lower cell capacitance. In Calliphora SVFs possess both fast and slow activating delayed rectifier potassium conductances. Drosophila SVFs possess a slowly inactivating delayed rectifier (I_Ks), a very rapidly inactivating A channel encoded by the Shaker gene (I_A), and, in a minority of cells, a third K conductance with intermediate kinetics (I_Kf). In both specs the LVFs lack the slowest component, but exhibit the faster K conductance(s) with properties indistinguishable from those in the SVFs. These findings add to established evidence demonstrating the significant role played by potassium channels in tuning the photoreceptor membrane. The results also suggest that R1-6 photoreceptors and R7/8 form inputs to visual subsystems tuned to different temporal frequencies.Abbreviations LVF long visual fibre - SVF short visual fibre - R1-6 retinular cells 1 to 6 inclusive - R7/8 retinular cell 7 and 8 - I _A rapidly inactivating A type potassium conductance; channel coded by Shaker gene - I _Kf rapidly activating, slowly inactivating delayed rectifier-like potassium conductance - I _Ks slowly activating, slowly inactivating delayed rectifier-like potassium conductance - I _KDs slowly activating delayed rectifier potassium conductance - I _KDf rapidly activating delayed rectifier potassium conductance     

Expression of secondary alcohol dehydrogenase in methanogenic bacteria and purification of the F420-specific enzyme from Methanogenium thermophilum strain TCI

In four species of methanogens able to grow with secondary alcohols as hydrogen donors the expression and properties of secondary alcohol dehydrogenase (sec-ADH) were investigated. Cells grown with 2-propanol and CO₂ immediately started to oxidize secondary alcohols to ketones if transferred to new media. In the presence of H₂, such cells reduced ketones or aldehydes to alcohols. In the absence of H₂, aldehydes were dismutated (without growth) to primary alcohols and fatty acids. None of these reactions was catalyzed by cells grown with only H₂ and CO₂ at non-limiting concentration. This indicated an induction or derepression of sec-ADH by its substrate. Apparently, sec-ADH in all strains enabled not only the reduction of ketones or aldehydes, but also the dismutation of the latter. Sec-ADH was also expressed if strains were grown on H₂ and CO₂ in the presence of non-oxidizable, tertiary alcohols. Methanogenium thermophilum expressed sec-ADH even without added alcohol when H₂ became limiting. From this species, an F₄₂₀-specific sec-ADH was purified; the final gel filtration chromatography yielded a single protein peak that coincided with the activity. The enrichment was 12-fold, the activity recovery 26%. SDS polyacrylamide gel electrophoresis indicated that the enzyme was a homodimer with an apparent M _r of 79,000. At the pH optimum around 4.2, the specific activity for oxidation of 2-propanol (130 mM) and reduction of acetone (20 mM) was 176 and 110 mol/ min·mg, respectively (40°C). The apparent K _m for 2-propanol and acetone (with 15 M F₄₂₀) was 2.5 and 0.25 mM, respectively. Aldehydes also were reduced.Non-standard abbreviations ADH alcohol dehydrogenase - Bis-Tris bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane - F₄₂₀ N-(N-L-lactyl--L-glutamyl)-L-glutamic acid phosphodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin-5-phosphate - Mb. Methanobacterium - Mg. Methanogenium - Ms. Methanospirillum - OD₅₇₈ optical density at 578 nm - SDS sodium dodecyl sulfate     

The effect of avermectins on feeding,salivary fluid secretion,and fecundity in some ixodid ticks

We tested the effects of the potent acaricides, avermectin B₁a (AVM) and 22,23-dihydroavermectin B₁ (ivermectin; IVM) when injected directly into partially fed and fully engorged female ticks. When injected into small ticks (Amblyomma hebraeum Koch), neither drug (up to 100 g/kg b.w.) inhibited subsequent engorgement nor affected oviposition latency, weight of total egg mass laid nor viability of laid eggs. At higher concentrations (1000 and 5000 g/kg b.w.), AVM and IVM were markedly toxic. When injected into engorged ticks, both drugs increased oviposition latency, and reduced fecundity at about 75–100 g/kg b.w. Vitellogenesis, as assessed by a spectrophotometric assay of the ovaries, was not inhibited. Also at 50–100 g/kg b.w., AVM and IVM caused paralysis of the abdominal dorso-ventral muscles and the leg muscles. Both drugs, at 7 days post-injection, proved detrimental to salivary gland function in both small and large ticks, but had little effect on salivary gland weight. At concentrations which did not inhibit oviposition (20–50 g/kg b.w.) many of the eggs dried out even though they were kept at high RH. We then demonstrated inAmblyomma americanum, Dermacentor andersoni andD. albipictus that removal of egg wax (by extraction with hexane) induced a marked increase in water permeability. IVM neither increased water permeability ofD. andersoni eggs nor diminished the amount of egg wax deposited on the surface of the eggs, when injected posteriorly through the alloscutum. However, injection of IVM, dimethylsulphoxide (vehicle for IVM) or distilled water through the articulation between the capitulum and scutum (anterior injection), did markedly reduce the wax coating and increased egg permeability. We suggest that anterior injection damages Gené's organ and thus causes the latter effects.     

The carrot,not the stick: appetitive rather than aversive gustatory stimuli support associative olfactory learning in individually assayed <Emphasis Type="Italic">Drosophila</Emphasis> larvae

The ability to learn is universal among animals; we investigate associative learning between odors and tastants in larval Drosophila melanogaster. As biologically important gustatory stimuli, like sugars, salts, or bitter substances have many behavioral functions, we investigate not only their reinforcing function, but also their response-modulating and response-releasing function. Concerning the response-releasing function, larvae are attracted by fructose and repelled by sodium chloride and quinine; also, fructose increases, but salt and quinine suppress feeding. However, none of these stimuli has a nonassociative, modulatory effect on olfactory choice behavior. Finally, only fructose but neither salt nor quinine has a reinforcing effect in associative olfactory learning. This implies that the response-releasing, response-modulating and reinforcing functions of these tastants are dissociated on the behavioral level. These results open the door to analyze how this dissociation is brought about on the cellular and molecular level; this should be facilitated by the cellular simplicity and genetic accessibility of the Drosophila larva.     

Regeneration of Cucumis sativus var. sativus and C. sativus var. hardwickii,C. melo,and C. metuliferus from explants through somatic embryogenesis and organogenesis

Regeneration in six inbred lines or F₁ hybrids of Cucumis sativus was achieved on Murashige & Skoog's medium containing various concentrations of 2,4-D/BA, NAA/BA, NAA/Z or NAA/K. The range of regeneration frequency for cotyledon, leaf and petiole explants was 0–38, 0–75 and 14–96%, respectively, after 6–8 weeks in culture. Only one subculture of calli to growth regulator-free medium was required for regeneration. Preincubation of explants in the dark for 2–3 weeks was essential to achieve optimal regeneration. Highest frequency of plantlet formation occurred with petiole explants incubated on NAA/BA (5.0/2.5 M), NAA/Z (5.0/5.0 M) or 2,4-D/BA (5.0/5.0 M). Approximately 80% of these plantlets survived after transplanting to greenhouse soil, and they flowered and set fruit. The F₁ hybrid, Endeavor, gave the highest regeneration frequency of 91% on 2,4-D/BA at 5.0/5.0 M. Formation of somatic embryos was observed on 2,4-D/BA, while organogenesis and embryogenesis both were evident on NAA/BA and NAA/Z. Cotyledonary explants yielded the lowest frequency (ca. 7%) of plantlet formation in this study. Plantlets of C. sativus var. hardwickii and an F₁ hybrid of C. sativus x C. s. var hardwickii were regenerated on NAA/Z and NAA/K at frequencies of 15–65%, predominantly by the formation of somatic embryos. Shoots were obtained from cotyledon and leaf explants of C. metuliferus on IAA/BA (7.5/5.0 M) and from leaf and petiole explants of C. melo on NAA/BA (5.0/2.5 M), but plantlets were recovered only in C. melo.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxy-acetic acid - IAA indoleacetic acid - K kinetin - MS Murashige & Skoog's medium - NAA naphthaleneacetic acid - Z zeatindihydroside     

Agrobacterium tumefaciens mediated gene transfer in peanut (Arachis hypogaea L.)

Transgenic peanut plants were produced using Agrobacterium mediated gene transfer. Primary leaf explants of peanut were co-cultivated with Agrobacterium tumefaciens LBA 4404 harbouring the binary plasmid pBI 121 (conferring -glucuronidase activity and resistance to kanamycin) and cultured on regeneration medium supplemented with kanamycin to select putatively transformed shoots. They were rooted and plants were transferred to soil. Stable integration and expression of the transgenes were confirmed by NPT II assay, Southern blot hybridization and GUS assay.Abbreviations BA 6-benzyladenine - GUS -glucuronidase - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - NOS nopaline synthase - NPT II neomycin phosphotransferase II - SDS Lauryl sulfate     

Subcellular and regional localization ofγ-glutamyl transpeptidase in sheep brain

Studies of the subcellular distribution of-glutamyl transpeptidase from sheep brain by discontinuous sucrose density gradient centrifugation showed that 40% of the transpeptidase activity associated with the mitochondrial-synaptosomal fraction was localized with the synaptosomal-enriched fraction. The microsomal fraction was found to have the highest specific activity when-glutamylp-nitroanalide was used as substrate. This activity, however, represented only 5% of the total-glutamyl transpeptidase activity. Approximately 90% of the total enzyme activity was apparently associated with the fraction containing cell debris and membrane fragments.The 160,000g supernatant fluid (soluble supernatant fraction) represented the least total activity, with only 1.2% recovery; however, this fraction contained two apparent forms of the enzyme. One form had a highK _mand the other a lowK _m for the substrate,-glutamylp-nitroanilide.It was observed that the enzyme-glutamyl transpeptidase was not evenly distributed in all areas of brain when the homogenate was used as the enzyme source. The brain region with the highest enzyme activity was the thalamus, which was able to form 1.10 molp-nitroanaline/min/g wet brain tissue. The cortex was found to have the lowest activity. The 40,000g supernatant fluid from each region, however, exhibited only slight distribution differences.     

A new type of putative non-visual photoreceptors in the optic lobe of beetles

A putative photoreceptor organ is described in the carabid beetle, Pachymorpha sexguttata. The elongated structure, about 20–40 m wide and more than 300 m long, is situated within the optic lobe at the fronto-dorsal rim of the lamina. It lies, deep in the head capsule, in front of the compound eyes and beneath window-like thinnings of the cuticle. The organ is composed of two types of cells: (1) clear sheath cells and (2) well-organized inner receptor cells that appear in a horseshoe-like or circular array in cross-section. Common histological features of all inner cells include a distal trunk ending in microvilli that form a rhabdom-like structure, an axon at the proximal end of the cell, lamellar and multivesicular bodies within the trunk, and clusters of small mitochondria. The organ has no shielding pigment. It is connected by thin axons to a circumscribed neuropil that parallels the organ, and thence via a fiber tract to the medulla accessoria, a possible site of the circadian pacemaker in insects. Immunoreactivity to anti-per ^s , an antibody recognizing the Drosophila period (per) protein that plays a central role in the function of the circadian pacemaker in fruit flies, is demonstratable in thin efferent terminals within the organ, in the associated neuropil and in its fiber connection to the medulla. A second receptor organ displaying the same fine structure lies near the second optic chiasm. This set of putative photoreceptors also occurs in the tenebrionid beetle, Zophobas morio, and its pupa. The possible function of these receptor organs is discussed with respect to former chronobiological data and some recently described types of extraretinal photoreceptors in arthropods.