Effect of Gamma Irradiation on Embryogenic Avocado Cultures and Somatic Embryo Development

Embryogenic avocado cultures were exposed to ionizing irradiation in order to determine its effect on proliferation and subsequent somatic embryo development. The approximate PD₅₀ as determined by linear regression is 35 Gy 2 weeks after irradiation for Fuerte 2.11.1 and 4 weeks after irradiation for T362 2.11.1. Irradiation of embryogenic cultures did not significantly affect the number of early stage Fuerte 2.11.1 somatic embryos that developed directly from irradiated cultures; however, 10–50 Gy inhibited somatic embryo development. Irradiation of T362 2.11.1 embryogenic cultures at 25–50 Gy inhibited the number of intermediate and mature stages of somatic embryos that developed directly from irradiated cultures, and 50 Gy inhibited somatic embryo maturation. Inhibition of somatic embryo development could be partially offset by proliferation of irradiated embryogenic cultures as suspensions. Irradiation up to 10 Gy significantly increased the number of mature Fuerte 2.11.1 somatic embryos that developed from suspension cultures. Irradiation with doses up to 25 Gy stimulated development of heart stage T362 2.11.1 somatic embryos; however, mature somatic embryo development was suppressed at dosages of 10 Gy and greater.     

Molecular markers associated with seed weight in two soybean populations

Seed weight (SW) is a component of soybean, Glycine max (L.) Merr., seed yield, as well as an important trait for food-type soybeans. Two soybean populations, 120 F₄-derived lines of YoungxPI416937 (Pop1) and 111 F₂-derived lines of PI97100xCoker 237 (Pop2), were mapped with RFLP makers to identify quantitative trait loci (QTLs) conditioning SW across environments and populations. The genetic map of Pop1 consisted of 155 loci covering 973 cM, whereas Pop2 involved 153 loci and covered 1600 cM of map distance. For Pop1, the phenotypic data were collected from Plains, GA., Windblow, N.C., and Plymouth, N.C., in 1994. For Pop2, data were collected from Athens, GA., in 1994 and 1995, and Blackville, S.C., in 1995. Based on single-factor analysis of variance (ANOVA), seven and nine independent loci were associated with SW in Pop1 and Pop2, respectively. Together the loci explained 73% of the variability in SW in Pop1 and 74% in Pop2. Transgressive segregation occurred among the progeny in both populations. The marker loci associated with SW were highly consistent across environments and years. Two QTLs on linkage group (LG) F and K were located at similar genomic regions in both populations. The high consistency of QTLs across environments indicates that effective marker-assisted selection is feasible for soybean SW.     

Sucrose in storage media and cultivar affects post-storage regrowth of <Emphasis Type="Italic">in vitro</Emphasis> Hosta propagules

The goal of this research was to investigate if culturing in high sucrose (5%) liquid media during multiplication phase (stage II) would enhance endogenous sugar levels and dry matter sufficiently to allow storage of in vitro plants in sugar free media without adversely affecting post-storage recovery. Hosta tokudama Newberry Gold (NBG) and Hosta Striptease were cultured in Murashige and Skoog (MS) media containing 5% sucrose during stage II and transferred to rooting phase (stage III) in MS medium without (0%) sucrose or with 3% sucrose for 4weeks. At the end of stage III, cultures were stored, with the remaining media, at 10°C with 5molm^–2s^–1 photosynthetic photon flux (PPF) from cool white fluorescent lamps for 7 or 14weeks with or without a 2-week dark period prior to removal from storage. In both cultivars, stage III plants cultured in 3% sucrose media had higher soluble sugar levels and greater shoot and root biomass than those cultured in 0% sucrose media. Shoot and root soluble sugars decreased during storage. Shoot growth ceased during storage in both media. Root dry matter continued to increase in plants stored in 3% sucrose media but did not change in 0% sucrose media. Plants cultured in 3% sucrose media had less leaf chlorosis and less mortality after 7 or 14weeks of low temperature storage than the plantlets from sugar free media. Extending the storage period from 7 to 14weeks or introduction of 2-week dark period at the end of storage did not affect leaf chlorosis or plant mortality during acclimatization. Post-storage growth varied with the cultivar. Benefit of having sucrose in storage media was to develop a strong root system that aided the acclimatization and post-storage growth following 7 or 14week storage. Sucrose loading by culturing plants in liquid media containing 5% sucrose did not allow storage in sugar free media without adversely affecting post-storage growth in both cultivars.     

Solvent assistance in regiospecific disulfide formation in dimethylsulfoxide

This paper describes a convenient oxidative refolding method that exploits the dual property of dimethylsulfoxide (DMSO) as an oxidant and a solvent 'chaperon' in assisting disulfide formation in peptides. Synthetic peptides with preferred secondary structures were used as models. For -sheet peptides, an active fragment of fibroblast growth factor containing the tetrapeptide Arg-Ser-Arg-Arg at a reverse turn was used. A disulfide scan consisting of 16 analogs is designed in which different pairs of amino acids on the antiparallel -sheets flanking the active determinants are replaced with cysteine. DMSO in aqueous buffer at pH 6 or 8 was found to minimize aggregation due to -sheet formation in all 16 -stranded peptides and provided monocyclic disulfide peptides within 7 h. In contrast, air oxidation required a significantly longer duration for completion under similar conditions, and only 8 of 16 peptides formed intramolecular disulfides. Rate accelerations at pH 8 were found in exo-disulfide formation involving peptides with amino terminal cysteine, irrespective of oxidation conditions. The exo-disulfide effect in accelerating disulfide formation may be useful for regiospecific disulfide formation. For -helix peptides, the two-disulfide endothelin (ET) was used as a model. DMSO in combination with trifluoroethanol (TFE) was found to favor the desired bicyclic 1,4-disulfide bridged ET (1,4-ET) over the incorrectly folded 1,3-ET. Under aqueous conditions at pH 5–11, 1,4-ET to 1,3-ET was formed in the ratio of approximately 3:1, while the use of DMSO and TFE increased the ratio to 11:1. This solvent combination may stabilize an -helical stretch found in ET and contribute to enhanced selectivity. Thus, our results show that DMSO in disulfide formation in an aqueous or helix-promoting solution may serve as an oxidant and a 'chaperon' solvent system to provide regiospecificity for oxidative disulfide formation.     

The ability of acidic pH,growth inhibitors,and glucose to increase the proton motive force and energy spilling of amino acid-fermenting <Emphasis Type="Italic">Clostridium sporogenes</Emphasis> MD1 cultures

Clostridium sporogenes MD1 grew rapidly with peptides and amino acids as an energy source at pH 6.7. However, the proton motive force (p) was only –25 mV, and protonophores did not inhibit growth. When extracellular pH was decreased with HCl, the chemical gradient of protons (ZpH) and the electrical membrane potential () increased. The p was –125 mV at pH 4.7, even though growth was not observed. At pH 6.7, glucose addition did not cause an increase in growth rate, but increased to –70 mV. Protein synthesis inhibitors also significantly increased . Non-growing, arginine-energized cells had a of –80 mV at pH 6.7 or pH 4.7, but was not detected if the F₁F₀ ATPase was inhibited. Arginine-energized cells initiated growth if other amino acids were added at pH 6.7, and and ATP declined. At pH 4.7, ATP production remained high. However, growth could not be initiated, and neither nor the intracellular ATP concentration declined. Based on these results, it appears that C. sporogenes MD1 does not need a large p to grow, and p appears to serve as a mechanism of ATP dissipation or energy spilling.Mandatory disclaimer: Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, and exclusion of others that may be suitable.     

Temperature coefficients of amide proton NMR resonance frequencies in trifluoroethanol: A monitor of intramolecular hydrogen bonds in helical peptides?

Summary 2D ¹H NMR spectroscopy of two -helical peptides which differ in their amphipathicity has been used to investigate the relationships between amide-proton chemical shifts, amide-proton exchange rates, temperature, and trifluoroethanol (TFE) concentration. In 50% TFE, in which the peptides are maximally helical, the amide-proton chemical shift and temperature coefficient patterns are very similar to each other in each peptide. Temperature coefficients from –10 to –6 ppb/K, usually indicative of the lack of intramolecular hydrogen bonds, were observed even for hydrophobic amino acids in the center of the -helices. However, slow hydrogen isotope exchange for residues from 4 to 16 in both 18-mer helices indicates intact intramolecular hydrogen bonds over most of the length of these peptides. Based on these anomalous observations, we suggest that the pattern of amide-proton shifts in -helices in H₂O/TFE solvents is dominated by bifurcated intermolecular hydrogen-bond formation between the backbone carbonyl groups and TFE. The amide-proton chemical shift changes with increasing temperature may be interpreted by a disruption of intermolecular hydrogen bonds between carbonyl groups and the TFE in TFE/water rather than by the length of intramolecular hydrogen bonds in -helices. Supplementary Material is available upon request, comprising seven pages with listings of experimental details and the NMR shift data for the two peptides.     

Specific antibodies to the N-termini of the interpeptide bridges of peptidoglycan

The synthetic peptides Gly₅--Ahx and l-Ala₃--Ahx, with structural similarity to the interpeptide bridge peptides of staphylococci or micrococci, respectively, were covalently linked to human serum albumin via their carboxylgroups. Antisera to these synthetic peptidyl-protein antigens contained fairly high amounts of antibodies with specificity to the N-terminal parts of the peptide chains attached to the carrier proteins. Antisera to (Gly₅--Ahx)₂₀-albumin gave, without exception, strong precipitin reactions in latex-agglutination with staphylococcal peptidoglycans. The antisera completely failed, however, in any reaction with peptidoglycans of micrococci or other bacteria which did not have these oligo-glycine peptides typical for staphylococci. On the contrary, antisera to (l-Ala₃--Ahx)₂₂-albumin strongly precipitated micrococcal peptidoglycans with oligo-l-alanine interpeptide bridges (e.g. Micrococcus varians, Micrococcus reseus), but showed no significant reaction with peptidoglycans of staphylococci or other bacteria lacking oligo-l-alanine interpeptide bridges.Abbreviations Use Ac acetyl- - -Ahx -amino caproic acid - ATCC American Type Culture Collection, Rockville, Md., U.S.A. - CCM Czechoslovak Collection of Microorganisms, Brno, CSSR - DSM Deutsche Sammlung für Mikroorganismen, München, FRG - IMRU Institute of Microbiology, Rutgers University, N.J., U.S.A. - Kiel Bundesanstalt für Milchforschung, Kiel, FRG - NPS o-nitrophenylsulphenyl- - -OMe methyl ester - -OSu succinimide ester - Z- benzyloxycarbonyl     

Identification and characterization of gibberellin-insensitive mutants selected from among dwarf mutants of rice

In rice, many dwarf mutants have been isolated and characterized. We have investigated the relationship between dwarfism and the gibberellin (GA)-mediated control of physiological processes. Twenty-three rice cultivars and mutants (9 normal, 3 semi-dwarf, 11 dwarf) were analyzed in terms of two GA-mediated processes, namely, elongation of shoots and production of -amylase activity in the endosperm. As a result, we identified four different groups (groups N, T, D and E). Two-dimensional plotting of the extent of induction of -amylase in the endosperm versus the extent of enhancement of shoot elongation upon treatment with exogenous gibberellic acid (GA₃) provided a useful method for the rapid allocation of large numbers of dwarf mutants of rice to the various groups. Members of group N (normal type), which included all normal cultivars and semi-dwarf mutants, showed a slight increase in elongation of shoots and a remarkable increase in production of -amylase with the application of GA₃ during germination. All of the dwarf mutants were classified as being members of the other three groups. Members of group T (Tan-ginbozu type), including three dwarf mutants, were highly responsive to exogenous GA₃ in terms of elongation of shoots and production of -amylase, with associated lower levels of endogenous GA. In contrast, members of the other three groups, including group N, had normal levels of endogenous GAs. Members of group D (Daikoku type) were only slightly responsive to exogenous GA₃, an indication that they are GA-insensitive mutants. Members of group E (Ebisu type) had responses to GA₃ similar to those of group N, not only in terms of elongation of shoots but also in terms of -amylase production, an indication that they are dwarf mutants that can be considered as neither GA-deficient nor GA-insensitive mutants. We also examined a GA-insensitive mutant selected from among 19 near-isogenic dwarf lines of Shiokari, and we concluded that the d-1 gene is associated with the phenotype of GA-insensitive dwarf mutants.     

Does Formate Reduce α-Ketoglutarate and Ammonia to Glutamate?

The reported reduction of -ketoglutarate and ammonia by formate is much slower than described (Morowitz et al., 1995). The formate reduction if any is small under these conditions. Glutamate is produced from a reduction by a second molecule of -ketoglutarate involving an oxidative decarboxylation.     

Selective dimerization of cysteines in glycopeptides and phosphopeptides

Summary To study the influence of phosphorylation and oxidation on the repeat domains of human Tau protein, we faced the challenge to selectively dimerize two cysteine-containing peptides in the presence of a nearby phosphate group. To this end, we were able to demonstrate the utility of a selective dimerization approach by forming disulfide bonds in unprotected phosphopeptides and extended the methodology to unprotected glycopeptides. Activation of one cysteine of a peptide chain with 2,2-dithiodipyridine and coupling this thiopyridyl-peptide to another peptide chain, containing an unprotected cysteine residue, yielded the mixed dimers in high purities and reasonable yields. Phosphate or sugar side chains on either peptide component remained unaffected during the activation and dimerization processes. While for mixed dimers the activated peptides were isolated by chromatography, homodimers were obtained by a simple one-pot reaction after 1 h. We demonstrate that cysteines can be dimerized in unprotected phosphopeptides and glycopeptides, without any side reactions affecting these posttranslational modifications.Abbreviations DCM dichloromethane - DMF N,N-dimethylformamide - DTP 2,2-dithiodipyridine - Fmoc 9-fluorenylmethyloxycarbonyl - HPLC high-performance liquid chromatography - MALDI matrix-assisted laser desorption/ionization - MS mass spectrometry - NFT neurofibrillary tangles - PHF paired helical filaments - PKC protein kinase C - RP reversed phase - human Tau protein - TFA trifluoroacetic acid Parts of this paper were presented at the 24th European Peptide Symposium in Edinburgh, Scotland, U.K., September 8–13, 1996.     

Feeding Ecology of Black-headed Uacaris (Cacajao melanocephalus melanocephalus) in Pico da Neblina National Park,Brazil

From June 1994 to October 1995, I conducted a study of the feeding ecology of one group of black-headed uacaris (Cacajao melanocephalus melanocephalus) in Pico da Neblina National Park, Brazil, near the Venezuelan border (01°10N to 00°26S, 65°03W to 66°52W). Of a total of 429 feeding records, 89% corresponded to fruits from which black-headed uacaris ate ripe and unripe seeds, mesocarps and arils. Seeds were the single most important food item year-around, corresponding to 81% of the feeding records for fruits. Black-headed uacaris used 120 different plant species in 32 families as food. The 3 most important plant species in their diet, Micrandra spruceana, Eperua leucantha, and Hevea braziliensis (all trees), accounted for 38% of the feeding records for fruits. These species produced fruits that were protected by very hard green husks, were barochorous, did not have fleshy mesocarps, and were slow to mature, thus being available as unripe fruits for long periods. The second and third most important species in the diet of black uacaris, Eperua leucantha and Hevea braziliensis, were dominant in the forest: approximately 30% of trees 10 cm dbh sampled in a 2-ha botanical transect at the study site. I discuss the formation of large social groups of black-headed uacaris in Pico da Neblina National Park in light of the peculiar floristic composition of the study site.     

Rooting apple cultivars in vitro: Interactions among light,temperature, phloroglucinol and auxin

Delicious apple (Malus domestica Borkh.) and several of its strains, which have been difficult to root in vitro, were successfully propagated with rooting percentages up to 100%. The combination of treatments used to achieve this result included placing the shoots on rooting medium in the dark at 30°C for the first week of the rooting stage, then moving them to a regime of 16 hr light-8 hr dark at 25°C. The rooting medium contained half strength Murashige and Skoog salts plus 1.2 M thiamine HCl, 0.56 mM myo-inositol, 1 mM phloroglucinol (PG), 1.4 M indolebutyric acid (IBA), 1.3 M gibberellic acid (GA₃), 87.6 mM sucrose, and 7 g l^–1 Difco Bacto agar. Dark treatment applied during the proliferation stage (etiolation) was less effective than one applied at the beginning of the rooting stage. The optimum length of dark treatment during rooting was 4 to 7 days. Increasing the temperature from 25°C to 30°C improved rooting of Delicious, Royal Red Delicious, and Vermont Spur Delicious in the absence of PG but generally had less effect in the presence of PG. Further increase in temperature to 35°C stimulated rooting of Royal Red Delicious but reduced rooting of Vermont Spur Delicious. Transfer of the cuttings to auxin-free medium after 1 week had no effect on percentage rooting and increased the number of roots per cutting for only 1 of 4 cultivars tested and then only in the presence of PG. In general PG stimulated rooting of Delicious and its strains, but had no effect on Golden Delicious.     

Soybean seedling emergence at high temperatures

Bragg and Cobb soybean [Glycine max (L.) Merr.] seeds were germinated in sand at temperatures ranging from 25 to 40°C. Emergence decreased with increasing temperature above 37°C, with virtually no emergence at 40°C. Emergence of 12 other cultivars at 38°C ranged from 25 to 95%. Foster and Coker 338 were more sensitive to high temperature than the other cultivars.     

Enzyme-linked immunosorbent assays for the measurement of blood group A and B glycosyltransferase activities

ELISA assays have been developed for (1–3)N-acetylgalactosaminyltransferase (blood group A transferase) and (1–3)galactosyltransferase (blood group B transferase) activities. In these assays, microtitre plates coated with the bovine serum albumin conjugate of a synthetic Fuc1–2Gal-R acceptor substrate are incubated with the appropriate nucleotide donor (UDP-GalNAc or UDP-Gal) and human serum as the enzyme source. The resulting trisaccharide products Fuc1–2(GalNAc1–3)Gal-R-BSA or Fuc1–2(Gal1–3)Gal-R-BSA are detected and quantified with monoclonal antibodies selected not to cross-react with the substrate structure. With less than a microliter of human serum, product formation is proportional to enzyme concentration and to time of incubation of up to 90 min.     

Structural modifications of oxidized insulin B-chain induced by glycerol addition

H-NMR studies of the bovine insulin S-sulfonatedB-chain are reported in H₂O/D₂O (9/1) and inglycerol-d₅ (5 M) using two-dimensional NMRspectroscopy. The first results show that the oxidizedinsulin B-chain secondary structure differs from thatof native insulin by a loss of the -helixbetween the two disulfide bridges and that theglycerol favours the structuring of the peptide.     

Modification of length, hydrophobic properties and electric charge of Bacillus subtilis alpha-amylase signal peptide and their different effects on the production of secretory proteins in B. subtilis and Escherichia coli cells

Summary Bacillus subtilis -amylase signal peptide, which consists of 33 amino acids, is functional in Escherichia coli cells.Lysine, glutamic acid, leucine, leucyl-leucine, or leucyl-leucyl-leucine was inserted between positions 28 and 29 of the -amylase signal peptide using site directed mutagenesis. DNAs encoding the wild-type and modified signal peptides were then fused in-frame to DNAs encoding the mature regions of the -lactamase of pBR322 and a thermostable -amylase. The secretion of -lactamase in E. coli cells was more inhibited by the modified signal peptides than that in B. subtilis cells, although the degree of inhibition varied and the inhibitory effect of each signal peptide was found to be similar in the two strains. In contrast, the difference in the inhibitory effect of each modified signal peptide was no longer detected in the case of the production of thermostable -amylase, except for the insertion of glutamic acid. Nearly 50% of thermostable -amylase in the precursor form was accumulated in the intracellular fraction of E. coli cells containing the DNAs for the modified signal peptides. The insertion of glutamic acid inhibited the secretion of the two enzymes in both B. subtilis and E. coli cells.     

Are we building enough bridges between paleolimnology and aquatic ecology?

Opening keynote lecture to the Aquatic Ecology and Palaeolimnological Interpretation session at the Vth International Symposium on Palaeolimnology, held at Ambleside, U.K., Sept. 1, 1989.     

Template-directed oligomerization of 3-isoadenosine 5′-phosphate

Summary Template-directed oligomerization of an activated derivative of 3-isoadenosine 5-phosphate (piA) on polyuridylic acid [poly(U)] was studied. The reaction of ImpiA is more efficient than the corresponding reaction of ImpA, and produces 3–5-linked oligomers while the reaction of ImpA gives only 2–5-linked oligomers. The base pairing between piA and poly(U) in this system is probably of the Hoogsteen type (involving the 6-amino group and N7 of 3-isoadenosine) rather than of the Watson-Crick type.     

A 500-MHz1H-NMR study on theN-linked carbohydrate chain of bromelain

The 500-MHz¹H-NMR characteristics of theN-linked carbohydrate chain Man1-6[Xyl1-2]Man1-4GlcNAc1-4[Fuc1-3]GlcNAc1-NAsn of the proteolytic enzyme bromelain (EC 3.4.22.4) from pineapple stem were determined for the oligosaccharide-alditol and the glycopeptide, obtained by hydrazinolysis and Pronase digestion, respectively. The¹H-NMR structural-reporter-groups of the (1–3)-linked fucose residue form unique sets of data for the alditol as well as for the glycopeptide.