The Effects of Brown Rust and Phosphate on Cold Acclimation in Winter Barley

The green leaf area of winter barley, cv. Sonja, sampled fromthe field at different times during winter was always greatestin plants grown at high soil phosphate and smallest in plantsgrown at low soil phosphate, and at each fertilizer level wasgreater in healthy plants than in plants infected by rust (Pucciniahordei). In leaves that survived the coldest period of winter,the percentage area that was damaged was increased by rust infectionwhich prevented the ameliorating effects of high soil P. Rustand low P interacted to reduce the increases in leaf area andshoot d. wt that occurred when higher temperatures prevailedin spring. Under controlled conditions in the laboratory, phosphate reducedthe injury suffered when plants not acclimated to low temperatureswere exposed to freezing conditions, but this effect was removedby rust infection. After rust infection, freezing temperatureswere damaging even to acclimated plants, particularly if grownwith low soil P. Evidence of visible symptoms, and quantitativemeasurements of electrolyte efflux from intact leaves, chlorophyllfluorescence in vivo, and ethane and ethylene evolution fromcold-acclimated plants, showed that infection raised the minimumtemperature at which tissues could survive without injury. Infectedleaves were more sensitive to low temperature post-sporulationthan presporulation. Measurements of electrolyte efflux andchlorophyll fluorescence on plants growing under cold conditionsshowed that infection inhibited the processes of acclimationto low temperatures. Winter barley, Puccinia hordei, injury, low temperature, acclimation     

Neurophysins as markers of vasopressin and oxytocin release. A study in carcinoma of the lung

Vasopressin-neurophysin (hNpI), oxytocin-neurophysin (hNpII) and blood osmolality were assayed before any treatment in basal conditions in 35 patients suffering from lung carcinoma (20 oat cell, 6 undifferentiated and 9 well-differentiated epidermoid cell carcinomas). Plasma vasopressin (antidiuretic hormone, ADH) was also assayed in 7 of the 20 patients suffering from oat cell carcinoma. We found a close correlation (r = 0.98) between plasma ADH and hNpI levels in the 7 patients. Further, hNpI was elevated in 13 out of the 20 oat cell carcinoma patients and in none of the epidermoid-cell carcinoma group; however, searching for an abnormality of ADH secretion as reflected by a detectable plasma hNpI level together with subnormal plasma osmolality revealed 2 additional positive results in the oat cell carcinoma group, and 2 out of the 6 in the undifferentiated-cell carcinoma group. hNpII was increased together with an increase in hNpI in 6 oat cell carcinoma patients; it was specifically increased without hNpI increment in 2 additional oat cell carcinoma patients and in 2 patients of the undifferentiated-cell carcinoma group (different from the 2 positive for the hNpI-osmolality ratio). hNpI and hNpII were normal in the majority of undifferentiated and all of the differentiated epidermoid-cell carcinoma group. Hence, our results show that simultaneous measurements of hNpI, hNpII, and blood osmolality could detect abnormalities in 17 out of 20 oat cell carcinoma patients, in 4 of the 9 undifferentiated-cell carcinoma patients, but in none of the differentiated epidermoid-cell carcinoma patients, suggesting that the neurophysin assay can be used for the early detection of oat cell- and possibly other neuroendocrine-derived carcinomas.     

Electrostatic forces at helix-coil boundaries in DNA

The Tm of internal loop-forming (dA.dT)N domains in pBR322 DNA has been measured over a tenfold range of [Na+]. The slopes SN = dTm/d log [Na+] are linear and decrease in magnitude with decreasing loop size N, signaling a reduction in Na+ released during the transition of these domains to the coil state. Values of SN decrease linearly with increasing N-1 in accordance with the expectation of a simple model for the occurrence of a gradient of long-range electrostatic forces at helix-coil boundaries, and extrapolate almost precisely to the value of S infinity observed for (dA.dT) infinity. These results indicate (1) less counterion is released per phosphate residue from the finite loop than from the infinite-sized loop, and (2) the difference in binding is constant for each boundary formed and independent of the size of the loop within the range examined: approximately 350 base pair (bp) greater than N greater than 71 bp. The slope of the dependence of SN on N-1 indicates the region of higher charge density at the boundary extends at least 18 A into the coil and probably 40-50 A before dropping to a value characteristic of the unperturbed coil. The free energy for excess counterion binding at boundaries can be expressed by -delta G/RT = 10.47 log[Na+] + 5.234 When the loop entropy function in a statistical mechanical algorithm for the dissociation of DNA is weighted by this quantity, calculated Tm are seen to vary by only +/- 0.09 degrees C from observed.     

Recolonisation of four small streams in central Scotland following drought conditions in 1984

Four streams in the Loch Ard Forest in central Scotland dried out almost completely during a drought in the summer of 1984. The recovery of the invertebrate populations in the streams was studied from September 1984 until March 1985 when most of the insect larvae and nymphs were almost full-grown.The appearance of very small larvae belonging to several insect orders within a month of the streams' filling up suggests that they had survived the drought as eggs, or eggs had been laid by adults soon after the drought ended.Statistical analysis showed that with the exception of 3 taxa there was no significant difference between the numbers of animals in November 1984 samples and in samples collected in March 1985.Comparison of samples from March 1984 and March 1985 showed significant difference in population size for a few species; with 2 exceptions the 1984 samples (ie before drought) were larger. In the long term the overall effect of the drought on the invertebrate communities seems to have been limited.     

Measurement of active-site homology between potato and rabbit muscle alpha-glucan phosphorylases through use of a linear free energy relationship

The Michaelis-Menten parameters (Vmax and Km) for turnover of an extensive series of deoxy and deoxyfluoro derivatives of alpha-D-glucopyranosyl phosphate by the alpha-glucan phosphorylase from potato tuber have been determined. Very large rate reductions are observed as a consequence of each substitution, primarily due to losses in specific binding interactions, most likely hydrogen bonding, at the enzymic transition state. Comparison of the Vmax/Km values so determined with those measured for rabbit muscle alpha-glucan phosphorylase [Street et al. (1989) Biochemistry 28, 1581] reveals an astonishingly similar specificity, especially in light of the phylogenetic separation of their host organisms. This indicates that very similar hydrogen-bonding interactions between the enzyme and the substrate must be present at the transition states for the two enzymic reactions; therefore, they have very similar active sites. Quantitation of this similarity is achieved by plotting the logarithm of the Vmax/Km value for each substrate analogue with the potato enzyme against the same parameter for the muscle enzyme, yielding straight lines (p = 0.998 and 0.999) of slope 1.0 and 1.2 for the deoxy and deoxyfluoro substrates, respectively. Since the correlation coefficient of such plots is a direct measure of the similarity of the two transition-state complexes, thus of the enzyme active sites, it can be used as a measure of active-site homology between the two enzymes. The extremely high homology observed in this case is consistent with the observed sequence homology at the active site.     

Inactivation of pyruvate decarboxylase by 3-hydroxypyruvate.

Pyruvate decarboxylase from Zymomonas mobilis is inhibited by 3-hydroxypyruvate, which can also act as a poor substrate. While catalysing the decarboxylation of this alternative substrate, the enzyme undergoes a progressive but partial inactivation over several hours. The extent of inactivation depends upon the pH and upon the concentration of 3-hydroxypyruvate. After partial inactivation and removal of unchanged 3-hydroxypyruvate, enzymic activity recovers slowly. We suggest that inactivation results from accumulation of enzyme-bound glycollaldehyde, which is relatively stable, possibly because it is dehydrated to form an acetyl group.     

Phylogenetic analysis and secondary structure of the Bacillus subtilis bacteriophage RNA required for DNA packaging

An unusual RNA molecule encoded by the Bacillus subtilis bacteriophage phi 29 is a structural component of the viral prohead and is required for the ATP-dependent packaging of DNA. Here we report a model of secondary structure for this prohead RNA developed from a phylogenetic analysis of the primary sequences of prohead RNAs of related phages. Twenty-nine phages related to phi 29 were found to produce prohead RNAs. These RNAs were analyzed by their ability to replace phi 29 RNA in in vitro phage assembly, by Northern blot hybridization with a probe complementary to phi 29 RNA, and by partial and complete sequence analyses. These analyses revealed four quite different sequences ranging in length from 161 to 174 residues. The secondary structure deduced from these sequences, in agreement with earlier observations, indicated that prohead RNA is organized into two domains. The larger 5'-domain (Domain I) is composed of 113-117 residues and contains four helices. Three of these helices appear to be organized into a central stem that is interrupted by two unpaired loops and the fourth helix and loop. The smaller 3'-domain (Domain II) is composed of 40-44 residues and consists of two helices. Domains I and II are separated by 8-13 unpaired residues. Nuclease cleavage occurs readily in this single-stranded joining region, and this cleavage allows the subsequent separation of the two RNA domains. The separated Domain I is fully active in DNA packaging in vitro. The functional significance and biological role of Domain II are unknown. The phylogenetic secondary structure model provides a basis for further analysis of the role of this RNA in bacteriophage morphogenesis.     

Solid-phase synthesis of peptides with C-terminal asparagine or glutamine. An effective, mild procedure based on N alpha-fluorenylmethyloxycarbonyl (Fmoc) protection and side-chain anchoring to a tris(alkoxy)benzylamide (PAL) handle

Attempts to anchor Fmoc-asparagine or glutamine as p-alkoxybenzyl esters for solid-phase peptide synthesis are fraught with difficulties. A convenient and effective method to prepare peptides with C-terminal asparagine or glutamine involves quantitative attachment of N alpha-Fmoc-C alpha-tert.-butyl aspartate or glutamate via the free omega-carboxyl groups to a tris(alkoxy)benzylamino (PAL) support. Chain elongation proceeds normally by standard Fmoc chemistry, and treatment with acid, e.g., CF3COOH--CH2Cl2, 90 min at 25 degrees, releases the desired peptides in greater than 95% yields without side reactions at the C-terminus. Feasibility of the approach has been demonstrated by the syntheses of the C-terminal octapeptide from human proinsulin, H-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-OH, and the serum thymic factor pGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn-OH.     

Purification and partial characterization of an iron regulated outer membrane protein of B. fragilis under non-denaturing conditions

The CHAPS-PAGE gelsystem we applied gave a good separation of the proteins of Bacteroides fragilis under non-denaturing conditions. We succeeded with preparative CHAPS-PAGE in purifying an iron regulated outer membrane protein (a 44 kDa polypeptide on SDS-PAGE) of B. fragilis. This integral membrane protein proved to be a lipopolysaccharide binding protein with an isoelectric point of approximately pH 5.5. This method of purifying membrane proteins could be an important step in research into the function of membrane proteins.