Phloem sap proteins from Cucurbita maxima and Ricinus communis have the capacity to traffic cell to cell through plasmodesmata

In angiosperms, the functional enucleate sieve tube system of the phloem appears to be maintained by the surrounding companion cells. In this study, we tested the hypothesis that polypeptides present within the phloem sap traffic cell to cell from the companion cells, where they are synthesized, into the sieve tube via plasmodesmata. Coinjection of fluorescently labeled dextrans along with size-fractionated Cucurbita maxima phloem proteins, ranging in size from 10 to 200 kDa, as well as injection of individual fluorescently labeled phloem proteins, provided unambiguous evidence that these proteins have the capacity to interact with mesophyll plasmodesmata in cucurbit cotyledons to induce an increase in size exclusion limit and traffic cell to cell. Plasmodesmal size exclusion limit increased to greater than 20 kDa, but less than 40 kDa, irrespective of the size of the injected protein, indicating that partial protein unfolding may be a requirement for transport. A threshold concentration in the 20–100 nM range was required for cell-to-cell transport indicating that phloem proteins have a high affinity for the mesophyll plasmodesmal binding site(s). Parallel experiments with glutaredoxin and cystatin, phloem sap proteins from Ricinus communis, established that these proteins can also traffic through cucurbit mesophyll plasmodesmata. These results are discussed in terms of the requirements for regulated protein trafficking between companion cells and the sieve tube system. As the threshold value for plasmodesmal transport of phloem sap proteins falls within the same range as many plant hormones, the possibility is discussed that some of these proteins may act as long-distance signaling molecules.

Circadian-Regulated Transcription of the psbD Light-Responsive Promoter in Wheat Chloroplasts1

The level of mRNAs derived from the plastid-encoded psbD light-responsive promoter (LRP) is controlled by a circadian clock(s) in wheat (Triticum aestivum). The circadian oscillations in the psbD LRP mRNA level persisted for at least three cycles in continuous light and for one cycle in continuous dark, with maxima in subjective morning and minima in subjective early night. In vitro transcription in chloroplast extracts revealed that the circadian cycles in the psbD LRP mRNA level were dominantly attributed to the circadian-regulated transcription of the psbD LRP. The effects of various mutations introduced into the promoter region on the psbD LRP activity in vitro suggest the existence of two positive elements located between −54 and −36, which generally enhance the transcription activity, and an anomalous core promoter structure lacking the functional “−35” element, which plays a crucial role in the circadian fluctuation and light dependency of psbD LRP transcription activity.

Accumulation of a Clock-Regulated Transcript during Flower-Inductive Darkness in Pharbitis nil1

To clarify the molecular basis of the photoperiodic induction of flowering in the short-day plant Pharbitis nil cv Violet, we examined changes in the level of mRNA in cotyledons during the flower-inductive photoperiod using the technique of differential display by the polymerase chain reaction. A transcript that accumulated during the inductive dark period was identified and a cDNA corresponding to the transcript, designated PnC401 (P. nil C401), was isolated. RNA-blot hybridization verified that levels of PnC401 mRNA fluctuated with a circadian rhythm, with maxima between 12 and 16 h after the beginning of the dark period) and minima of approximately 0. This oscillation continued even during an extended dark period but was damped under continuous light. Accumulation of PnC401 mRNA was reduced by a brief exposure to red light at the 8th h of the dark period (night-break treatment) or by exposure to far-red light at the end of the light period (end-of-day far-red treatment). These results suggest that fluctuations in levels of PnC401 mRNA are regulated by phytochrome(s) and a circadian clock and that they are associated with photoperiodic events that include induction of flowering.

On the role of the weather in the deaths of R. F. Scott and his companions

Robert Falcon Scott and his companions reached the South Pole in January of 1912, only to die on their return journey at a remote site on the Ross Ice Shelf, about 170 miles from their base camp on the coast. Numerous contributing causes for their deaths have been proposed, but it has been assumed that the cold temperatures they reported encountering on the Ross Ice Shelf, near 82–80°S during their northward trek toward safety, were not unusual. The weather in the region where they perished on their unassisted trek by foot from the Pole remained undocumented for more than half a century, but it has now been monitored by multiple automated weather stations for more than a decade. The data recorded by Scott and his men from late February to March 19, 1912, display daily temperature minima that were on average 10 to 20°F below those obtained in the same region and season since routine modern observations began in 1985. Only 1 year in the available 15 years of measurements from the location where Scott and his men perished displays persistent cold temperatures at this time of year close to those reported in 1912. These remarkably cold temperatures likely contributed substantially to the exhaustion and frostbite Scott and his companions endured, and their deaths were therefore due, at least in part, to the unusual weather conditions they endured during their cold march across the Ross Ice Shelf of Antarctica.

Chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein, subsequent folding, and development of fluorescence

The present paper describes the total chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein (GFP). The molecule is made up of 238 amino acid residues in a single polypeptide chain and is nonfluorescent. To carry out the synthesis, a procedure, first described in 1981 for the synthesis of complex peptides, was used. The procedure is based on performing segment condensation reactions in solution while providing maximum protection to the segment. The effectiveness of the procedure has been demonstrated by the synthesis of various biologically active peptides and small proteins, such as human angiogenin, a 123-residue protein analogue of ribonuclease A, human midkine, a 121-residue protein, and pleiotrophin, a 136-residue protein analogue of midkine. The GFP precursor molecule was synthesized from 26 fully protected segments in solution, and the final 238-residue peptide was treated with anhydrous hydrogen fluoride to obtain the precursor molecule of GFP containing two Cys(acetamidomethyl) residues. After removal of the acetamidomethyl groups, the product was dissolved in 0.1 M Tris⋅HCl buffer (pH 8.0) in the presence of DTT. After several hours at room temperature, the solution began to emit a green fluorescence (λmax = 509 nm) under near-UV light. Both fluorescence excitation and fluorescence emission spectra were measured and were found to have the same shape and maxima as those reported for native GFP. The present results demonstrate the utility of the segment condensation procedure in synthesizing large protein molecules such as GFP. The result also provides evidence that the formation of the chromophore in GFP is not dependent on any external cofactor.

Dynamic weighting in Monte Carlo and optimization

Dynamic importance weighting is proposed as a Monte Carlo method that has the capability to sample relevant parts of the configuration space even in the presence of many steep energy minima. The method relies on an additional dynamic variable (the importance weight) to help the system overcome steep barriers. A non-Metropolis theory is developed for the construction of such weighted samplers. Algorithms based on this method are designed for simulation and global optimization tasks arising from multimodal sampling, neural network training, and the traveling salesman problem. Numerical tests on these problems confirm the effectiveness of the method.

Symplasmic Constriction and Ultrastructural Features of the Sieve Element/Companion Cell Complex in the Transport Phloem of Apoplasmically and Symplasmically Phloem-Loading Species1

The ultrastructural features of the sieve element/companion cell complexes were screened in the stem phloem of two symplasmically loading (squash, [Cucurbita maxima L.] and Lythrum salicaria L.) and two apoplasmically loading (broad bean [Vicia faba L.] and Zinnia elegans L.) species. The distinct ultrastructural differences between the companion cells in the collection phloem of symplasmically and apoplasmically phloem-loading species continue to exist in the transport phloem. Plasmodesmograms of the stem phloem showed a universal symplasmic constriction at the interface between the sieve element/companion cell complex and the phloem parenchyma cells. This contrasts with the huge variation in symplasmic continuity between companion cells and adjoining cells in the collection phloem of symplasmically and apoplasmically loading species. Further, the ultrastructure of the companion cells in the transport phloem faintly reflected the features of the companion cells in the loading zone of the transport phloem. The companion cells of squash contained numerous small vacuoles (or vesicles), and those of L. salicaria contained a limited number of vacuoles. The companion cells of broad bean and Z. elegans possessed small wall protrusions. Implications of the present findings for carbohydrate processing in intact plants are discussed.

Temperature dependence of the isotope chemistry of the heavy elements.

The temperature coefficient of equilibrium isotope fractionation in the heavy elements is shown to be larger at high temperatures than that expected from the well-studied vibrational isotope effects. The difference in the isotopic behavior of the heavy elements as compared with the light elements is due to the large nuclear isotope field shifts in the heavy elements. The field shifts introduce new mechanisms for maxima, minima, crossovers, and large mass-independent isotope effects in the isotope chemistry of the heavy elements. The generalizations are illustrated by the temperature dependence of the isotopic fractionation in the redox reaction between U(VI) and U(IV) ions.

Chronic, topical exposure to benzo[a]pyrene induces relatively high steady-state levels of DNA adducts in target tissues and alters kinetics of adduct loss.

Carcinogen-DNA adduct measurements may become useful biomarkers of effective dose and/or early effect. However, validation of this biomarker is required at several levels to ensure that human exposure and response are accurately reflected. Important in this regard is an understanding of the relative biomarker levels in target and nontarget organs and the response of the biomarker under the chronic, low-dose conditions to which humans are exposed. We studied the differences between single and chronic topical application of benzo[a]pyrene (BAP) on the accumulation and removal of BAP-DNA adducts in skin, lung, and liver. Animals were treated with BAP at 10, 25, or 50 nMol topically once or twice per week for as long as 15 weeks. Animals were sacrificed either at 24, 48, or 72 hr after the last dose at 1 and 30 treatments, and after 24 hr for all other treatment groups. Adduct levels increased with increasing dose, but the slope of the dose-response was different in each organ. At low doses, accumulation was linear in skin and lung, but at high doses the adduct levels in the lung increased dramatically at the same time when the levels in the skin reached apparent steady state. In the liver adduct, levels were lower than in target tissues and apparent steady-state adduct levels were reached rapidly, the maxima being independent of dose, suggesting that activating metabolism was saturated in this organ. Removal of adducts from skin, the target organ, was more rapid following single treatment than with chronic exposure. This finding is consistent with earlier data, indicating that some areas of the genome are more resistant to repair. Thus, repeated exposure and repair cycles would be more likely to cause an increase in the proportion of carcinogen-DNA adducts in repair-resistant areas of the genome. These findings indicate that single-dose experiments may underestimate the potential for carcinogenicity for compounds that follow this pattern.

Foldons, protein structural modules, and exons.

Foldons, which are kinetically competent, quasi-independently folding units of a protein, may be defined using energy landscape analysis. Foldons can be identified by maxima in a scan of the ratio of a contiguous segment's energetic stability gap to the energy variance of that segment's molten globule states, reflecting the requirement of minimal frustration. The predicted foldons are compared with the exons and structural modules for 16 of the 30 proteins studied. Statistical analysis indicates a strong correlation between the energetically determined foldons and Go's geometrically defined structural modules, but there are marked sequence-dependent effects. There is only a weak correlation of foldons to exons. For gammaII-crystallin, myoglobin, barnase, alpha-lactalbumin, and cytochrome c the foldons and some noncontiguous clusters of foldons compare well with intermediates observed in experiment.

Betidamino acids: versatile and constrained scaffolds for drug discovery.

Betidamino acids (a contraction of "beta" position and "amide") are N'-monoacylated (optionally, N'-monoacylated and N-mono- or N,N'-dialkylated) aminoglycine derivatives in which each N'acyl/alkyl group may mimic naturally occurring amino acid side chains or introduce novel functionalities. Betidamino acids are most conveniently generated on solid supports used for the synthesis of peptides by selective acylation of one of the two amino functions of orthogonally protected aminoglycine(s) to generate the side chain either prior to or after the elongation of the main chain. We have used unresolved Nalpha-tert-butyloxycarbonyl-N'alpha-fluorenylmethoxycarbonyl++ + aminoglycine, and Nalpha-(Nalpha-methyl)-tert-butyloxycarbonyl-N'alpha-fluo renylmethoxycarbonyl aminoglycine as the templates for the introduction of betidamino acids in Acyline [Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(Ac)-D4Aph(A c)-Leu-Ilys-Pro-DAla-NH2, where 2Nal is 2-naphthylalanine, 4Cpa is 4-chlorophenylalanine, 3Pal is 3-pyridylalanine, Aph is 4-aminophenylalanine, and Ilys is Nepsilon-isopropyllysine], a potent gonadotropin-releasing hormone antagonist, in order to test biocompatibility of these derivatives. Diasteremneric peptides could be separated in most cases by reverse-phase HPLC. Biological results indicated small differences in relative potencies (<5-fold) between the D and L nonalkylated betidamino acid-containing Acyline derivatives. Importantly, most betide diastereomers were equipotent with Acyline. In an attempt to correlate structure and observed potency, Ramachandran-type plots were calculated for a series of betidamino acids and their methylated homologs. According to these calculations, betidamino acids have access to a more limited and distinct number of conformational states (including those associated with alpha-helices, beta-sheets, or turn structures), with deeper minima than those observed for natural amino acids.

The old problems of glass and the glass transition, and the many new twists.

In this paper I review the ways in which the glassy state is obtained both in nature and in materials science and highlight a "new twist"--the recent recognition of polymorphism within the glassy state. The formation of glass by continuous cooling (viscous slowdown) is then examined, the strong/fragile liquids classification is reviewed, and a new twist-the possibility that the slowdown is a result of an avoided critical point-is noted. The three canonical characteristics of relaxing liquids are correlated through the fragility. As a further new twist, the conversion of strong liquids to fragile liquids by pressure-induced coordination number increases is demonstrated. It is then shown that, for comparable systems, it is possible to have the same conversion accomplished via a first-order transition within the liquid state during quenching. This occurs in the systems in which "polyamorphism" (polymorphism in the glassy state) is observed, and the whole phenomenology is accounted for by Poole's bond-modified van der Waals model. The sudden loss of some liquid degrees of freedom through such weak first-order transitions is then related to the polyamorphic transition between native and denatured hydrated proteins, since the latter are also glass-forming systems--water-plasticized, hydrogen bond-cross-linked chain polymers (and single molecule glass formers). The circle is closed with a final new twist by noting that a short time scale phenomenon much studied by protein physicists-namely, the onset of a sharp change in d/dT ( is the Debye-Waller factor)--is general for glass-forming liquids, including computer-simulated strong and fragile ionic liquids, and is closely correlated with the experimental glass transition temperature. The latter thus originates in strong anharmonicity in certain components of the vibrational density of states, which permits the system to access the multiple minima of its configuration space. The connection between the anharmonicity in these modes, vibrational localization, the Kauzmann temperature, and the fragility of the liquid is proposed as the key problem in glass science.

The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: molecular cloning and functional expression.

The biosynthesis of gibberellins (GAs) after GA12-aldehyde involves a series of oxidative steps that lead to the formation of bioactive GAs. Previously, a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14.11.-] was isolated by immunoscreening a cDNA library from liquid endosperm of pumpkin (Cucurbita maxima L.) with antibodies against partially purified GA 20-oxidase. Here, we report isolation of a genomic clone for GA 20-oxidase from a genomic library of the long-day species Arabidopsis thaliana Heynh., strain Columbia, by using the pumpkin cDNA clone as a heterologous probe. This genomic clone contains a GA 20-oxidase gene that consists of three exons and two introns. The three exons are 1131-bp long and encode 377 amino acid residues. A cDNA clone corresponding to the putative GA 20-oxidase genomic sequence was constructed with the reverse transcription-PCR method, and the identity of the cDNA clone was confirmed by analyzing the capability of the fusion protein expressed in Escherichia coli to convert GA53 to GA44 and GA19 to GA20. The Arabidopsis GA 20-oxidase shares 55% identity and > 80% similarity with the pumpkin GA 20-oxidase at the derived amino acid level. Both GA 20-oxidases share high homology with other 2-oxoglutarate-dependent dioxygenases (2-ODDs), but the highest homology was found between the two GA 20-oxidases. Mapping results indicated tight linkage between the cloned GA 20-oxidase and the GA5 locus of Arabidopsis. The ga5 semidwarf mutant contains a G-->A point mutation that inserts a translational stop codon in the protein-coding sequence, thus confirming that the GA5 locus encodes GA 20-oxidase. Expression of the GA5 gene in Ara-bidopsis leaves was enhanced after plants were transferred from short to long days; it was reduced by GA4 treatment, suggesting end-product repression in the GA biosynthetic pathway.

Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis.

Fluorescent dye-labeled DNA primers have been developed that exploit fluorescence energy transfer (ET) to optimize the absorption and emission properties of the label. These primers carry a fluorescein derivative at the 5' end as a common donor and other fluorescein and rhodamine derivatives attached to a modified thymidine residue within the primer sequence as acceptors. Adjustment of the donor-acceptor spacing through the placement of the modified thymidine in the primer sequence allowed generation of four primers, all having strong absorption at a common excitation wavelength (488 nm) and fluorescence emission maxima of 525, 555, 580, and 605 nm. The ET efficiency of these primers ranges from 65% to 97%, and they exhibit similar electrophoretic mobilities by gel electrophoresis. With argon-ion laser excitation, the fluorescence of the ET primers and of the DNA sequencing fragments generated with ET primers is 2- to 6-fold greater than that of the corresponding primers or fragments labeled with single dyes. The higher fluorescence intensity of the ET primers allows DNA sequencing with one-fourth of the DNA template typically required when using T7 DNA polymerase. With single-stranded M13mp18 DNA as the template, a typical sequencing reaction with ET primers on a commercial sequencer provided DNA sequences with 99.8% accuracy in the first 500 bases. ET primers should be generally useful in the development of other multiplex DNA sequencing and analysis methods.