Expression of pH-sensitive green fluorescent protein in Arabidopsis thaliana

This is the first report on using green fluorescent protein (GFP) as a pH reporter in plants. Proton fluxes and pH regulation play important roles in plant cellular activity and therefore, it would be extremely helpful to have a plant gene reporter system for rapid, non‐invasive visualization of intracellular pH changes. In order to develop such a system, we constructed three vectors for transient and stable transformation of plant cells with a pH‐sensitive derivative of green fluorescent protein. Using these vectors, transgenic Arabidopsis thaliana and tobacco plants were produced. Here the application of pH‐sensitive GFP technology in plants is described and, for the first time, the visualization of pH gradients between different developmental compartments in intact whole‐root tissues of A. thaliana is reported. The utility of pH‐sensitive GFP in revealing rapid, environmentally induced changes in cytoplasmic pH in roots is also demonstrated.     

Separation of peptides by reverse-phase high-performance liquid chromatography using propyl- and cyanopropylsilyl supports

The separation of peptides and proteins by reverse-phase high-performance liquid chromatography with cyanopropylsilyl and large-pore propylsilyl supports, together with aqueous trifluoroacetic acid/acetonitrile gradients, was studied. Operating parameters (trifluoroacetic acid concentration, flow rate, and gradient slope) were evaluated using different enzymatic digests of horse cytochrome c and bovine serum albumin. Peptides ranging in size from five amino acids to 68 kDa could be separated on the propylsilyl column in a single chromatographic run. The cyanopropylsilyl column is suitable as a supplement to the use of the large-pore column for medium size (5-20 amino acids) peptides. The chromatographic supports and conditions presented here offer a simple, sensitive, and rapid separation system for a wide size range of peptides and proteins. They extend the versatility of separation methodology for these molecules.     

Factors determining the reconstructive denaturation of proteins in sodium dodecyl sulfate solutions. Further circular dichroism studies on structural reorganization of seven proteins

The factors determining the onset and extent of reconstructive denaturation of proteins were considered by comparing circular dichroism (CD) data of seven proteins and previously published findings. The effects of sodium dodecyl sulfate (SDS) on the conformation of the following proteins were tested: lysozyme, the mitogens fromPhytolacca americana (fractions Pa2 and Pa4), lectin fromWistaria floribunda, ovine lutropin, a Bence Jones protein, and histone H2B. While the helix content of lysozyme was raised by SDS slightly, in the Bence Jones protein andW. floribunda lectin it increased from near zero to about 25–30%. In histone H2B the helix content was raised by SDS even to about 48%. However, no clear indication of helix formation could be observed in the mitogens and lutropin, even at low pH or 2.0–2.5. The tertiary structure of the proteins was perturbed by SDS. It was concluded that the reorganization of secondary structure of the proteins was favored by the following factors: (1) presence of helicogenic amino acid sequences in the protein, (2) availability of positively charged sites of the basic amino acids for interactions with the dodecyl ion, (3) absence of a large surplus of negatively charged sites on the surface of protein, and (4) absence of extensive disulfide cross-linking within the macromolecule. Both hydrophobic and electrostatic interactions occur in reconstructive denaturation, and the newly formed helices are stabilized by hydrophobic shielding by the alkyl chains of the alkyl sulfate.     

Simulation of protein misfolding using a lattice model

The structure of the tightly bound complex of the globular myosin head with F-actin is the key to understanding important details of the mechanism of how the actin-myosin motor functions. The current notion on this complex is based on the docking of known atomic structures of constituent proteins into low-resolution electron-density maps. The atomic structure of the complex was refined by the molecular mechanics method, which consists in minimizing the energy of molecular interaction and which makes it possible to optimize not only the relative position of protein backbones as rigid bodies, but also the position of side chains on the protein interface. The structure calculated using ICM-Pro software, on the one hand, is close to the model obtained using electron microscopy; on the other hand, it ensures the best calculated interaction energy and accounts for the results of mutagenesis experiments. On the basis of the structure obtained, we can suggest the molecular mechanisms underlying the actin-activated release of ATP hydrolysis products from myosin and the decrease in the affinity of myosin for actin upon binding of nucleotides.     

The reactivity of neonatal rabbits to the mammary pheromone as a probe for viability

Newborn rabbits depend on a daily nursing interaction with the mother to gain milk and to survive. During this interaction, they localise and seize the nipples displaying a typical behaviour triggered by maternal odour cues. The mammary pheromone constitutes such a signal in domestic rabbits: it elicits sucking-related movements in more than 90% of the pups. However, some newborns remain unresponsive to the presentation of the pheromone, even pups apparently healthy and highly motivated to suck. The main goal of the present study was therefore to explore the link between the unresponsiveness of rabbit pups to the mammary pheromone and their growth and survival in breeding conditions. To that end, 293 newborns from 30 litters were tested for their head searching-oral grasping responses to the mammary pheromone on days 1 and 3, and their milk intake and mortality were followed up from days 1 to 21. It was hypothesised that unresponsive newborns would have subsequent difficulties in finding nipples, sucking and surviving. Early weight and success in milk intake were further considered as mediating factors in growth and viability. The results showed that pups that were unresponsive to the mammary pheromone on day 1 were less successful in gaining milk and had a higher rate of mortality than the responsive pups. However, this impact was modulated by the weight of pups: it appeared only in the lightest newborns. Moreover, this impact vanished on day 3. On the other hand, the pup weight and sucking success on days 1 to 3 strongly influenced viability and growth during the period extending from days 1 to 21. Taken together, the results show that the day-1 responsiveness of rabbit pups to the mammary pheromone can be considered as an indicator of individual viability in pups having a small weight (<48 g on day 1). The predictive validity of the pups’ pheromonal reactivity seems however time-limited as it works only during the first, but crucial, postnatal days.     

1H, 13C and 15N resonance assignment of phage T4 endoribonuclease RegB

RegB is involved in the control of the phage T4 life cycle. It inactivates the phage early mRNAs when their translation is no more required. We determined its structure and identified residues involved in substrate binding. For this, all backbone and 90% of side-chain resonance frequencies were assigned.     

Identification of the coding region for the putidaredoxin reductase gene from the plasmid of Pseudomonas putida

The first 12 NH₂-terminal amino acids of the Pseudomonas putida putidaredoxin reductase were shown to be Met-Asn-Ala-Asn-Asp-Asn-Val-Val-Ile-Val-Gly-Thr. Comparison of these data with the DNA sequence of the BamHI-HindIII 197-base fragment derived from the PstI 2.2-kb fragment obtained from the P. putida plasmid showed that the putidaredoxin reductase gene was downstream from the cytochrome P-450 gene and the intergenic region had the 24-nucleotide sequence TAAACACATGGGAGTGCGTGCTAA. The Shine-Dalgarno sequence GGAG was detected in this region. The initiating triplet for the reductase gene was GTG, which normally codes for valine, but in the initiating codon position codes for methionine. From the amino acid sequence and X-ray data comparisons with other flavoproteins, what appears to be the AMP binding region of the FAD can be recognized in the NH₂-terminal portion of the reductase involving residues 5–35.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Chemical modification of the hydroxylase of soluble methane monooxygenase gives one form of the protein with significantly increased thermostability and another that functions well in organic solvents

Proteolysis of the hydroxylase component of soluble methane monooxygenase (MMO) with trypsin yielded a protein which retained 50% activity in a standard MMO assay. In an H₂O₂-driven assay, in which H₂O₂ replaced two of the protein components, NADH and O₂ used in the standard assay, the proteolysed hydroxylase retained full activity for ethane, propane and propene, but had a 2–3 fold increase with methane as substrate. Several crosslinking reagents have been tested for their ability to stabilise the proteolysed form of the hydroxylase. Using polyoxyethylene bis(imidazolyl carbonyl) (M_r 3350) as the crosslinking agent, increased thermostability of the hydroxylase was observed. Activated methoxypolyethylene glycol (M_r 5000) was used to modify the hydroxylase which was now soluble in organic solvents as well as water and could be activated by H₂O₂. The glycol-modified hydroxylase functioned well in organic solvents in the catalysis of propene oxidation.     

A Temporally Explicit Production Efficiency Model for Fuel Load Allocation in Southern Africa

Abstract We present a regional fuel load model (1 km² spatial resolution) applied in the southern African savanna region. The model is based on a patch-scale production efficiency model (PEM) scaled up to the regional level using empirical relationships between patch-scale behavior and multi-source remote sensing data (spatio-temporal variability of vegetation and climatic variables). The model requires the spatial distribution of woody vegetation cover, which is used to determine separate respiration rates for tree and grass. Net primary production, grass and tree leaf death, and herbivory are also taken into account in this mechanistic modeling approach. The fuel load model has been calibrated and validated from independent measurements taken from savanna vegetation in Africa southward from the equator. A sensitivity analysis on the effect of climate variables (incoming radiation, air temperature, and precipitation) has been conducted to demonstrate the strong role that water availability has in determining productivity and subsequent fuel load over the southern African region. The model performance has been tested in four different areas representative of a regional increasing rainfall gradient—Etosha National Park, Namibia, Mongu and Kasama, Zambia, as well as in Kruger National Park, South Africa. Within each area, we analyze model output from three different magnitudes of canopy coverage (<5, 30, and 50%). We find that fuel load ranges predicted by the model are globally in agreement with field measurements for the same year. High rainfall sustains green herbaceous production late in the dry season and delays tree leaf litter production. Effect of water on production varies across the rainfall gradient with delayed start of green material production in more arid regions.     

Melanocortin‐1 receptor structure and functional regulation

The melanogenic actions of the melanocortins are mediated by the melanocortin‐1 receptor (MC1R). MC1R is a member of the G‐protein‐coupled receptors (GPCR) superfamily expressed in cutaneous and hair follicle melanocytes. Activation of MC1R by adrenocorticotrophin or α‐melanocyte stimulating hormone is positively coupled to the cAMP signaling pathway and leads to a stimulation of melanogenesis and a switch from the synthesis of pheomelanins to the production of eumelanic pigments. The functional behavior of the MC1R agrees with emerging concepts in GPCR signaling including dimerization, coupling to more than one signaling pathway and a high agonist‐independent constitutive activity accounting for inverse agonism phenomena. In addition, MC1R displays unique properties such as an unusually high number of natural variants often associated with clearly visible phenotypes and the occurrence of endogenous peptide antagonists. Therefore MC1R is an ideal model to study GPCR function. Here we review our current knowledge of MC1R structure and function, with emphasis on information gathered from the analysis of natural variants. We also discuss recent data on the regulation of MC1R function by paracrine and endocrine factors and by external stimuli such as ultraviolet light.