A Comparison of the Effect of Salt on Polypeptides and Translatable mRNAs in Roots of a Salt-Tolerant and a Salt-Sensitive Cultivar of Barley

The effect of salt stress on polypeptide and mRNA levels in roots of two barley (Hordeum vulgare L.) cultivars differing in salt tolerance (cv CM 72, tolerant; cv Prato, sensitive) was analyzed using two-dimensional polyacrylamide gel electrophoresis. Preliminary experiments indicated that germination of Prato was inhibited significantly in the presence of NaCl, but growth of the surviving Prato seedlings was not substantially different from that of CM 72. Fluorographs of two-dimensional gels containing in vivo labeled polypeptides or in vitro translation products were computer analyzed to identify and quantitate changes that resulted when plants were grown in the presence of 200 millimolar NaCl for 6 days. The patterns of in vivo labeled polypeptides and in vitro products of CM 72 and Prato were qualitatively the same. Salt caused quantitative changes in numerous polypeptides and translatable mRNAs, but, overall, the changes were relatively small. Salt did not induce the synthesis of unique polypeptides or translatable mRNAs and did not cause any to disappear. Because of the similarities of the two cultivars with respect to growth and polypeptide patterns and the slight changes in polypeptide and translation product levels caused by salt, specific polypeptides or translatable mRNAs that are related to salt tolerance in barley could not be identified.     

Germin-Like Polypeptides Increase in Barley Roots during Salt Stress

The 26 kilodalton, isoelectric point 6.3 and 6.5 (Gs1 and Gs2) polypeptides that increase in barley (Hordeum vulgare L.) roots during salt stress were isolated and identified. Both Gs1 and Gs2 had high sequence similarity to germin, a protein that increases significantly in germinating wheat seeds. Like germin, Gs1 and Gs2 were resistant to proteases and were glycosylated. Immunoblots were probed with antibodies to Gs1 and Gs2 to determine the distribution of these polypeptides among organs and cell-free fractions. Gs1 and Gs2 were present in roots and coleoptiles, but absent from leaves. In roots, Gs1 and Gs2 were present in the mature region, but not the tip. Gs1 and Gs2 increased in roots, but decreased in coleoptiles in response to salt stress. Gs1 and Gs2 were distributed among the soluble, microsomal, and cell wall fractions of roots, but the majority of Gs1 and Gs2 was present in the soluble fraction. Although Gs1 and Gs2 were heat stable, their synthesis was not affected by abscisic acid treatment. Gs2 accumulated during abscisic acid treatment, whereas Gs1 did not. However, a 25.5 kilodalton, isoelectric point 6.1 polypeptide that was immunologically related to Gs1 did accumulate with abscisic acid treatment.     

Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Aggregation-dependent turnover of flagellar adhesion molecules in chlamydomonas gametes

Previous studies on flagellar adhesion in chlamydomonas (Snell, W. and S. Roseman. 1979. J. Biol. Chem. 254:10820-10829.) have shown that as gametes adhere to flagella isolated from gametes of the opposite mating type, the adhsiveness of the added flagella but not of the gametes is lost. The studies reported here show that the addition of protein synthesis inhibitors (cycloheximide [CH] or anisomycin) to the medium of such cell- flagella mixtures causes the cells to lose their adhesiveness. This loss, however, occurs only after the cells have interacted with 4-8 flagella/cell and does not occur if the cells are kept in CH (7 h) without aggregating. The availability of an impotent (imp) mating type plus (MT(+)) mutant (provided by U.W. Goodenough), which adheres but is unable to undergo the fusion that normally follows adhesion, made it possible to determine whether a similar loss of adhesiveness occurs in mixtures of matting type minus (mt(-)) and imp mt(+) gametes. In the absence of inhibitor, mt(-) and imp mt(+) gametes adhered to each other (without fusing) for several hours; however, in the presence of CH or anisomycin, the gametes began to de-adhere 35 min after mixing, and, by 90 min, 100 percent of the cells were single again. This effect was reversible, and the rapid turnover of cells were single again. This effect was reversible, and the rapid turnover of molecules involved in adhesion occurred only during adhesion inasmuch as gametes pretreated for 4 h with CH were able to aggregate in CH for the same length of time as nonpretreated cells aggregated in CH. By the addition of CH at various times after the mt(-) and imp mt(+) gametes were mixed, measurements were made of the “pool size” of the molecules involved in adhesion. The pool reached a minimum after 25 min of aggregation, rapidly increased for the next 25 min, and then leveled off at the premixing level. These results suggest that flagellar adhesion in chlamydomonas causes modification of surface molecules (receptors, ligands), which brings about their inactivation and stimulates their replacement.     

Fine structure and development of proteoplasts in primary leaves of mung bean

Summary The developing primary leaves of mung bean seedlings contain plastids, called proteoplasts, which are modified for protein storage. The proteoplast has a large protein inclusion with a granular matrix that is bound by a single membrane. Proteoplasts of this type are located in a layer of tissue one cell thick between the palisade and spongy mesophyll cells. The cell layer containing proteoplasts (P layer) differentiates within a few days after seed imbibition. Proteoplast precursors are distinguished by the development of membrane-bound protein sacs within the stroma. The protein sacs coalesce to form a spherical protein body. The P layer is short lived in primary leaves of seedlings grown in light and degeneration of these cells begins soon after proteoplast differentiation. As the cell layer degenerates, proteoplast contents become very electron dense. Within two days, the P layer breaks down and disappears as adjacent cells enlarge and differentiate. In contrast, this specialized cell layer remains intact, with little change in proteoplast fine structure, over a corresponding period in etiolated seedlings.     

Extraction of wheat endosperm proteins for proteome analysis

Total protein extracts of wheat endosperm are widely used for the analysis of the highly abundant gliadins and glutenins. In this review, the most popular total endosperm extraction methods are compared for their effectiveness in proteome coverage. A drawback of total endosperm extracts is that the enormous dynamic range of protein abundance limits the detection, quantification, and identification of low abundance proteins. Protein fractionation is invaluable for improving proteome coverage, because it reduces sample complexity while enriching for specific classes of less abundant proteins. A wide array of techniques is available for isolating protein subpopulations. Sequential extraction is a method particularly suited for subfractionation of wheat endosperm proteins, because it takes advantage of the specific solubility properties of the different classes of endosperm proteins. This method effectively separates the highly abundant gliadins and glutenins from the much less abundant albumins and globulins. Subcellular fractionation of tissue homogenates is a classical technique for isolating membranes and organelles for functional analysis. This approach is suitable for defining the biochemical processes associated with amyloplasts, specialized organelles in the endosperm that function in the synthesis and storage of starch. Subproteome fractionation, when combined with 2-DE and protein identification, provides a powerful approach for defining endosperm protein composition and providing new insights into cellular functions.     

Effect of paracetamol (acetaminophen) and ibuprofen on body temperature in acute ischemic stroke PISA, a phase II double-blind, randomized, placebo-controlled trial [ISRCTN98608690]

Background

Body temperature is a strong predictor of outcome in acute stroke. In a previous randomized trial we observed that treatment with high-dose acetaminophen (paracetamol) led to a reduction of body temperature in patients with acute ischemic stroke, even when they had no fever. The purpose of the present trial was to study whether this effect of acetaminophen could be reproduced, and whether ibuprofen would have a similar, or even stronger effect.

Methods

Seventy-five patients with acute ischemic stroke confined to the anterior circulation were randomized to treatment with either 1000 mg acetaminophen, 400 mg ibuprofen, or placebo, given 6 times daily during 5 days. Treatment was started within 24 hours from the onset of symptoms. Body temperatures were measured at 2-hour intervals during the first 24 hours, and at 6-hour intervals thereafter.

Results

No difference in body temperature at 24 hours was observed between the three treatment groups. However, treatment with high-dose acetaminophen resulted in a 0.3°C larger reduction in body temperature from baseline than placebo treatment (95% CI: 0.0 to 0.6 °C). Acetaminophen had no significant effect on body temperature during the subsequent four days compared to placebo, and ibuprofen had no statistically significant effect on body temperature during the entire study period.

Conclusions

Treatment with a daily dose of 6000 mg acetaminophen results in a small, but potentially worthwhile decrease in body temperature after acute ischemic stroke, even in normothermic and subfebrile patients. Further large randomized clinical trials are needed to study whether early reduction of body temperature leads to improved outcome.     

Evolutionary distances for protein-coding sequences: modeling site- specific residue frequencies

Estimation of evolutionary distances from coding sequences must take into account protein-level selection to avoid relative underestimation of longer evolutionary distances. Current modeling of selection via site-to-site rate heterogeneity generally neglects another aspect of selection, namely position-specific amino acid frequencies. These frequencies determine the maximum dissimilarity expected for highly diverged but functionally and structurally conserved sequences, and hence are crucial for estimating long distances. We introduce a codon- level model of coding sequence evolution in which position-specific amino acid frequencies are free parameters. In our implementation, these are estimated from an alignment using methods described previously. We use simulations to demonstrate the importance and feasibility of modeling such behavior; our model produces linear distance estimates over a wide range of distances, while several alternative models underestimate long distances relative to short distances. Site-to-site differences in rates, as well as synonymous/nonsynonymous and first/second/third-codon-position differences, arise as a natural consequence of the site-to-site differences in amino acid frequencies.