Differential phosphorylation of multiple sites in protein 4.1 and protein 4.9 by phorbol ester-activated and cyclic AMP-dependent protein kinases

The phosphorylation of the membrane skeleton components protein 4.1 and protein 4.9 in intact erythrocytes is shown to increase in the presence of either 1 microM 12-O-tetradecanoyl phorbol 13-acetate or 2 mM dibutyryl cAMP. The phosphorylation induced by these protein kinase activators is compared by two-dimensional tryptic peptide mapping. In both proteins, the pattern of peptides phosphorylated in the presence of 12-O-tetradecanoyl phorbol 13-acetate differs from the pattern of peptides phosphorylated in the presence of dibutyryl cAMP. The relative locations of the phosphorylated sites on protein 4.1 have been determined using limited proteolysis by alpha-chymotrypsin.     

Identification of the functional site of erythrocyte protein 4.1 involved in spectrin-actin associations

Peptides produced by mild chymotryptic digestion of human erythrocyte protein 4.1 mimic the ability of intact 4.1 to promote the binding of spectrin to F-actin. This complex-promoting activity was found to reside in an 8-kDa peptide which was fully functional when dissociated from other protein 4.1-derived peptides, indicating that noncovalent complexes of multiple peptides were not essential for activity. The 8-kDa peptide was incorporated into a ternary complex with spectrin and F-actin in approximately stoichiometric amounts. Amino acid composition and two-dimensional peptide mapping show that the 8-kDa active peptide is located within the 10-kDa region of protein 4.1 which contains a cAMP-dependent phosphorylated site.     

Cytochrome P-450 from the Mesocarp of Avocado (Persea americana)

The microsomal fraction from the mesocarp of avocado (Persea americana) is one of few identified rich sources of plant cytochrome P-450. Cytochrome P-450 from this tissue has been solubilized and purified. Enzymatic assays (p-chloro-N-methylaniline demethylase) and spectroscopic observations of substrate binding suggest a low spin form of the cytochrome, resembling that in the microsomal membrane, can be recovered. However, this preparation of native protein is a mixture of nearly equal proportions of two cytochrome P-450 polypeptides that have been resolved only under denaturing conditions. Overall similarities between these polypeptides include indistinguishable amino acid compositions, similar trypsin digest patterns, and cross reactivity with the same antibody. The amino terminal sequences of both polypeptides are identical, with the exception that one of them lacks a methionine residue at the amino terminus. This sequence exhibits some similarities with the membrane targeting signal found at the amino terminus of most mammalian cytochromes P-450.     

Protein 4.1 is involved in a structural thermotropic transition of the red blood cell membrane detected by a spin-labeled stearic acid

Proteins involved in a structural transition in red blood cell membranes detected at 8 +/- 1.5 degrees C by a stearic acid spin-label have been investigated. Calcium loading of red blood cells with ionophore A23187 caused the disappearance of the 8 degrees C transition. Protein 4.1 appears to be the most susceptible protein to Ca2+ treatment. Antibodies specific for spectrin, band 3 (43K cytoplasmic domain), and protein 4.1 have been utilized as specific probes to modify membrane thermotropic properties. The 8 degrees C transition was eliminated by anti-4.1 protein antibodies but was not modified by the other antibodies. To further characterize the protein(s) involved in the transition, ghosts were subjected to sequential extraction of skeletal proteins. The extraction of band 6, spectrin, and actin did not modify the 8 degrees C transition. In contrast, high-salt extraction (1 M KCl) of spectrin-actin-depleted vesicles, a procedure that extracts proteins 2.1 and 4.1, was able to eliminate the 8 degrees C transition. Rebinding of purified protein 4.1 to the high salt extracted vesicles restored the 8 degrees C transition. These results indicate the involvement of protein 4.1 in the transition and suggest a functional membrane association of this protein. The binding of protein 4.1 to the membrane seems to contribute significantly to the thermotropic properties of red blood cells.     

Evidence for multiple catalytic sites of human acrosin from kinetic evaluation of fructose-induced acrosin inhibition

Acrosin (acrosomal proteinase; EC 3.4.21.10) is a sperm-specific serine proteinase implicated in sperm penetration of the mammalian oocyte. Previously, we had shown that human acrosin, unlike human trypsin (EC 3.4.21.4), was inhibited by beta-D-fructose and related carbohydrates. The present study was undertaken to more fully elucidate the mechanism of action of fructose as an acrosin inhibitor, and to further differentiate the kinetic properties of acrosin from those of trypsin. Fructose produced a complex pattern of inhibition. At relatively low concentrations (10-60 mM), fructose acted as a competitive inhibitor with an apparent inhibition constant of 13 mM. In contrast, at high concentrations (80-320 mM), fructose behaved as a noncompetitive inhibitor, with an apparent inhibition constant of 205 mM. A Hill plot of enzyme activity as a function of fructose concentration suggested only a single binding site for fructose (slope = -0.90). The pattern of inhibition is not consistent with an enzyme containing only a single catalytic site, based either upon steady-state or rapid equilibrium assumptions; however, good agreement between observed and simulated data were obtained based upon the assumption of two catalytic sites with equal or similar binding and catalytic constants. The data suggested that fructose interacts with a single binding site (Ki = 8 mM) which alters both catalytic sites to produce an enzyme species having a higher apparent Michaelis constant and lower kcat as compared to the uninhibited enzyme. Fructose had no effect upon the rate of acrosin inactivation by either diisopropylfluorophosphate or tosyl-lysine-chloromethylketone, suggesting that neither substrate binding nor acylation were altered by this agent. The above data indicate substantial differences between the catalytic properties of human acrosin and those of trypsin.     

Tropical surface temperature response to vegetation cover changes and the role of drylands

Vegetation cover creates competing effects on land surface temperature: it typically cools through enhancing energy dissipation and warms via decreasing surface albedo. Global vegetation has been previously found to overall net cool land surfaces with cooling contributions from temperate and tropical vegetation and warming contributions from boreal vegetation. Recent studies suggest that dryland vegetation across the tropics strongly contributes to this global net cooling feedback. However, observation-based vegetation-temperature interaction studies have been limited in the tropics, especially in their widespread drylands. Theoretical considerations also call into question the ability of dryland vegetation to strongly cool the surface under low water availability. Here, we use satellite observations to investigate how tropical vegetation cover influences the surface energy balance. We find that while increased vegetation cover would impart net cooling feedbacks across the tropics, net vegetal cooling effects are subdued in drylands. Using observations, we determine that dryland plants have less ability to cool the surface due to their cooling pathways being reduced by aridity, overall less efficient dissipation of turbulent energy, and their tendency to strongly increase solar radiation absorption. As a result, while proportional greening across the tropics would create an overall biophysical cooling feedback, dryland tropical vegetation reduces the overall tropical surface cooling magnitude by at least 14%, instead of enhancing cooling as suggested by previous global studies.     

Kinetics of in vivo inhibition of tissue cathepsin D by pepstatin A

1. We have investigated the kinetics of inhibition of cathepsin D in heart, liver and skeletal muscle of CD-1 mice following administration of 25, 50, 100 and 200 mg/kg i.p. of pepstatin A, a specific inhibitor of this protease. 2. In the liver, a significant inhibition of cathepsin D occurred up to at least 15 days, whereas, in heart and skeletal muscle, this inhibition lasted for a much shorter period of time. 3. These results show that the recovery of enzyme activity to normal values is dose-dependent and that, at the same dose level, marked differences occur in the recovery of enzyme activity in these organ tissues, the liver being the most sensitive one.     

Model vs. playback experiments: The impact of sensory mode on predator-specific escape responses in saki monkeys

Although experimentally simulating predator presence helps improve sample sizes in studies of free-ranging animals, few studies have examined whether auditory playbacks and visual models produce similar results. Additionally, it is unclear if anti-predator strategies are specific to predator hunting styles in understudied Neotropical pitheciid primates, limiting what we can generalize about this phenomenon across this taxonomic order. We conducted predator simulation experiments to assess whether wild Rylands' bald-faced saki monkeys (Pithecia rylandsi) recognize predators based solely on acoustic cues, exhibit predator-specific responses to different predator types, and vary responses to presentations in different sensory modes. In our playback experiments, sakis had weak responses to non-predator control vocalizations compared to jaguar growls and harpy eagle shrieks. In most predator playbacks, subjects' first glance corresponded to the direction from which simulated predators would typically attack (above vs. below). However, although sakis exhibited appropriate movement responses to harpy playbacks (i.e., descending canopy), they exhibited no clear movement patterns when presented with jaguar playbacks. In contrast, jaguar model experiments consistently elicited fast approaches, mobbing-style responses, and long alarm calling bouts. Thus, if we had relied on playbacks alone, we might have concluded that sakis have only generalized responses to terrestrial ambush predators. In fact, in all variables measured (e.g., latency, number of calls, and response duration), models of both predator species elicited stronger reactions than playbacks. Results indicate that bald-faced sakis can identify predators based solely on vocalizations, but do not exhibit predator-specific escape responses to terrestrial predators based on acoustic cues alone. The differential response to playbacks and models calls into question the reliability of using acoustic-only stimuli to assess the specificity of anti-predator behavior to predator hunting styles in some primate species.     

Ribosomal proteins as biomarkers for bacterial identification by mass spectrometry in the clinical microbiology laboratory

Whole-cell matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a rapid method for identification of microorganisms that is increasingly used in microbiology laboratories. This identification is based on the comparison of the tested isolate mass spectrum with reference databases. Using Neisseria meningitidis as a model organism, we showed that in one of the available databases, the Andromas database, 10 of the 13 species-specific biomarkers correspond to ribosomal proteins. Remarkably, one biomarker, ribosomal protein L32, was subject to inter-strain variability. The analysis of the ribosomal protein patterns of 100 isolates for which whole genome sequences were available, confirmed the presence of inter-strain variability in the molecular weight of 29 ribosomal proteins, thus establishing a correlation between the sequence type (ST) and/or clonal complex (CC) of each strain and its ribosomal protein pattern. Since the molecular weight of three of the variable ribosomal proteins (L30, L31 and L32) was included in the spectral window observed by MALDI-TOF MS in clinical microbiology, i.e., 3640–12000 m/z, we were able by analyzing the molecular weight of these three ribosomal proteins to classify each strain in one of six subgroups, each of these subgroups corresponding to specific STs and/or CCs. Their detection by MALDI-TOF allows therefore a quick typing of N. meningitidis isolates.