Effects of profilin-annexin I association on some properties of both profilin and annexin I: modification of the inhibitory activity of profilin on actin polymerization and inhibition of the self-association of annexin I and its interactions with liposomes

We have previously shown that annexin I, a member of a family of calcium-dependent phospholipid and membrane binding proteins, interacts with profilin with high specificity and affinity. This finding further suggests that annexin I is involved through profilin in the regulation of membrane-cytoskeleton organization. We have investigated the consequences of a complex formed by these two proteins on the functions of both profilin and annexin I. Annexin I is able to modify the inhibitory effect of profilin on actin polymerization. This action is partial and the mechanism involved appears to be complex. On the other hand, the association between annexin I and profilin is sufficiently strong to inhibit the self-association of annexin I. The binding capacity of annexin I to liposomes containing phosphatidylserine, which mimics annexin I binding to membranes, is also decreased by profilin. This binding is nevertheless restored when phosphatidylinositol 4,5-biphosphate (PtdInsP2) is included in the liposomes. Finally, the capacity of annexin I to aggregate liposomes is also modified. It is worthwhile mentioning that the liposomes-binding and liposomes-aggregating activities of annexin I are independently regulated. The cell localization and functions of annexin I and profilin suggest that interaction between these two proteins may be directly implicated in the regulation of membrane-cytoskeleton. The phospholipid composition of membranes may be one of the modulating factors.     

Strains of Synechocystis sp. PCC 6803 with altered PsaC. II. EPR and optical spectroscopic properties of FA and FB in aspartate, serine, and alanine replacements of cysteines 14 and 51

A psaC deletion mutant of the unicellular cyanobacterium Synechocystis sp. PCC 6803 was utilized to incorporate site-specific amino acid substitutions in the cysteine residues that ligate the FA and FB iron-sulfur clusters in Photosystem I (PS I). Cysteines 14 and 51 of PsaC were changed to aspartic acid (C14DPsaC, C51DPsaC, C14D/C51DPsaC), serine (C14SPsaC, C51SPsaC), and alanine (C14APsaC, C51APsaC), and the properties of FA and FB were characterized by electron paramagnetic resonance spectroscopy and time-resolved optical spectroscopy. The C14DPsaC-PS I and C14SPsaC-PS I complexes showed high levels of photoreduction of FA with g values of 2.045, 1. 944, and 1.852 after illumination at 15 K, but there was no evidence of reduced FB in the g = 2 region. The C51DPsaC-PS I and C51SPsaC-PS I complexes showed low levels of photoreduction of FB with g values of 2.067, 1.931, and 1.881 after illumination at 15 K, but there was no evidence of reduced FA in the g = 2 region. The presence of FB was inferred in C14DPsaC-PS I and C14SPsaC-PS I, and the presence of FA was inferred in C51DPsaC-PS I and C51SPsaC-PS I by magnetic interaction in the photoaccumulated spectra and by the equal spin concentration of the irreversible P700(+) cation generated by illumination at 77 K. Flash-induced optical absorbance changes at 298 K in the presence of a fast electron donor indicate that two electron acceptors function after FX in the four mutant PS I complexes at room temperature. These data suggest that a mixed-ligand [4Fe-4S] cluster is present in the mutant sites of C14X-PS I and C51X-PS I (where X = D or S), but that the proposed spin state of S = 3/2 renders the resonances undetectable in the g = 2 region. The C14APsaC-PS I, C51APsaC-PS I and C14D/C51DPsaC-PS I complexes show only the photoreduction of FX, consistent with the absence of PsaC. These results show that only those PsaC proteins that contain two [4Fe-4S] clusters are capable of assembling onto PS I cores in vivo.     

Angiotensin II-mediated expression of p27Kip1 and induction of cellular hypertrophy in renal tubular cells depend on the generation of oxygen radicals

Presentation of MHC class I antigens by professional antigen-presenting cells (APC) is an important pathway in priming cytotoxic T lymphocyte responses in vivo. This study sought to identify the nature of the professional APC responsible for indirect class I presentation by examining a special feature of professional APC, namely their ability to process exogenous forms of antigen for class I presentation. Incubation of highly purified bone marrow-derived precursor cells with chicken ovalbumin (OVA) led to the efficient presentation of the major class I-restricted OVA determinant by mature dendritic cells (DC), but not by macrophages (Mphi) derived from the precursor population. DC as well as macrophages were, however, able to mediate class II presentation of OVA, suggesting that macrophages were deficient in class I processing but not in capturing exogenous OVA. The majority of mature DC, i.e. over 80 %, generated from the precursor cells pulsed with OVA, presented the class I OVA epitope. Upon maturation, class I presentation of OVA by DC was greatly reduced, suggesting that class I processing of exogenous antigen is modulated during DC maturation in a manner similar to class II antigen processing. This study shows that bone marrow-derived DC/ME progenitors capture exogenous antigen for class I presentation, and that cells of the DC lineage can be functionally distinguished from cells of the macrophage lineage based on their ability to process exogenous antigen for class I presentation.     

The Status of Diagnostic Testing.

In this column, I wish to make a few observations on what I believe to be a rather serious problem in relation to diagnostic psychological testing. I cannot guarantee the generality of these observations, but I am led to believe, by my own experience and by that of others to whom I have talked, that the practices to which I shall refer are rather widespread. Apparently many staff psychologists regard diagnostic testing as a necessary, but at best tedious and relatively unimportant, responsibility. As a result, a disproportionate amount of the diagnostic testing is assigned to interns or trainees, thus reinforcing the notion among other professional people, and among the trainees themselves, that this is a second-rate, low level skill. I believe that, unless a fundamental change of attitude toward diagnostic testing is effected in many of our training centers, clinical psychology as a profession must eventually suffer. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

An x-ray study of the condensed and separated states of sciatic nerve myelin

Low-angle X-ray diffraction patterns of peripheral nerve myelin after modification by either rehydration in various solutions or by chemical treatment have been recorded. These X-ray patterns and the previously reported modified nerve myelin patterns demonstrate that nerve myelin has at least five different states: the normal state, condensed state I and II and separated state I and II. There are two membranes per unit cell in the normal state and in states II whereas there is one membrane per unit cell in states I. Under certain conditions normal nerve can go reversibly into either of states II. With continued treatment the nerve myelin structure moves irreversibly from state II to state I and, once in state I, the nerve myelin layers cannot return to the normal state. Our results demonstrate that there is a reversible transformation between condensed state I and separated state I. Fourier profiles of nerve myelin in the normal state, condensed state I and separated state I are presented.     

Characterization of the interaction between synapsin I and calspectin (brain spectrin or fodrin)

We characterized the properties of the interaction between synapsin I and calspectin using purified proteins. The binding assay in the native state using antibodies specific to the tail region of synapsin I revealed that the binding is a high affinity with Kd of 9 nM, which is almost comparable to that of synapsin I to synaptic vesicles and to F-actin. We demonstrated that the head-middle region of synapsin I binds the NH2-terminal domain of beta subunit of calspectin, which also contains an actin binding site. Furthermore, the interaction was significantly inhibited by phosphorylation of synapsin I by cAMP-dependent protein kinase or by Ca2+, calmodulin-dependent protein kinase II. These properties of the interaction between synapsin I and calspectin may help understanding of its modulatory roles in neurotransmitter release.     

NK cells can recognize different forms of class I MHC

NK recognition and lysis of targets are mediated by activation receptor(s) whose effects may be over-ridden by inhibitory receptors recognizing class I MHC on the target. Incubation of normal lymphoblasts with a peptide that can bind to their class I MHC renders them sensitive to lysis by syngeneic NK cells. By binding to class I MHC, the peptide alters or masks the target structure recognized by an inhibitory NK receptor(s). This target structure is most likely an "empty" dimer of class I heavy chain and beta2m as opposed to a "full" class I trimer formed by binding of specific peptide that is recognized by CTL.     

N-myc suppresses major histocompatibility complex class I gene expression through down-regulation of the p50 subunit of NF-kappa B

In neuroblastoma, N-myc suppresses the expression of major histocompatibility complex (MHC) Class I antigens by reducing the binding of a nuclear factor to the enhancer-A element in the MHC Class I gene promoter. We show here that the p50 subunit of NF-kappa B is part of this complex and that expression of p50 mRNA is suppressed by N-myc. Transfection of a p50 expression vector in neuroblastoma cells that express N-myc at a high level leads to restoration of factor binding to the MHC Class I gene enhancer, restores enhancer activity and leads to re-expression of MHC Class I antigens at the cell surface. These data indicate that the p50 subunit of NF-kappa B is involved in the regulation of MHC Class I antigen expression and that N-myc down-regulates MHC Class I gene expression primarily through suppression of p50 expression.     

Effects of angiotensin I of the American bullfrog Rana catesbeiana on amphibian tissues

1. The effect of bullfrog angiotensin I [Asp1, Val5, Asn9] angiotensin I, (AT I) on short-circuit current (SCC) on isolated toad skin and aorta contractility was examined. 2. AT I increased SCC in toad skin, the effect was partially inhibited by angiotensin-converting enzyme inhibitor (ACEI) teprotide. 3. AT I induced contractile responses in isolated rings of toad aorta. This effect was partially inhibited by captopril and completely blocked by the peptide antagonist [Sar1, Ile8] angiotensin II. 4. Present results indicate that this homologue AT I would act in amphibian tissues by conversion to AT II.     

Dual effect of nitric oxide on the hyperpolarization-activated inward current (I(f)) in sino-atrial node cells of the rabbit

Using the whole-cell voltage-clamp technique, we have investigated the effect of nitric oxide (NO) donor (sodium nitroprusside, SNP) on hyperpolarization-activated inward current, I(f), in isolated rabbit sinoatrial node (SAN) cells. I(f) in the basal state increased when NO was applied but decreased when I(f) was pre-stimulated by isoproterenol (ISO) or by adding cAMP to the pipette solution. Both the stimulatory and the inhibitory effects of NO were abolished by guanylyl cyclase inhibitor, methylene blue (MB), suggesting that the effect of NO is mediated by cGMP. The inhibitory effect of NO was abolished when I(f) was pre-stimulated by 3-isobutyl-1-methylxanthine (IBMX), which is a phosphodiesterase (PDE) inhibitor, or by adding 8Br-cAMP (which is resistant to PDE) to the pipette solution. An analogue of cGMP, 8Br-cGMP, which is a potent stimulator of cGMP-dependent protein kinase (PKG) but has little effect on PDE, did not inhibit I(f) when I(f) was pre-stimulated by ISO. In its basal state, I(f) was still increased by 8Br-cGMP, and this effect was not prevented by the pretreatment with H-7, PKG inhibitor. The effect of acetylcholine (ACh) was not identical to that of NO: I(f) decreased when pre-stimulated not only by ISO, but also by IBMX. The above results suggest that via cGMP, NO exerts a dual effect on I(f): the inhibitory effect is mediated by cGMP-stimulated PDE, and the stimulatory effect may be attributable to direct binding of cGMP to I(f) channels.     

Role of unphosphorylated, newly synthesized I kappa B beta in persistent activation of NF-kappa B

Stimulation with inducers that cause persistent activation of NF-kappa B results in the degradation of the NF-kappa B inhibitors, I kappa B alpha and I kappa B beta. Despite the rapid resynthesis and accumulation of I kappa B alpha, NF-kappa B remains induced under these conditions. We now report that I kappa B beta is also resynthesized in stimulated cells and appears as an unphosphorylated protein. The unphosphorylated I kappa B beta forms a stable complex with NF-kappa B in the cytosol; however, this binding fails to mask the nuclear localization signal and DNA binding domain on NF-kappa B, and the I kappa B beta-NF-kappa B complex enters the nucleus. It appears therefore that during prolonged stimulation, I kappa B beta functions as a chaperone for NF-kappa B by protecting it from I kappa B alpha and allowing it to be transported to the nucleus.     

Polymorphism in anhydrous theophylline--implications on the dissolution rate of theophylline tablets

The objects of this investigation were (i) to prepare and characterize a new anhydrous theophylline phase that is metastable under ambient conditions, and (ii) to prepare model tablet formulations containing either this metastable anhydrate (I*) or stable anhydrous theophylline (I), store them under different relative humidity (RH) conditions, and compare their dissolution behavior. I* was prepared by dehydration of theophylline monohydrate (II). Variable temperature X-ray powder diffractometry of II revealed the following series of transitions: II-->I*-->I. The metastable anhydrate, I*, which has not yet been reported in the literature, appears to be related monotropically to I. It was characterized by ambient and variable temperature X-ray powder diffractometry, Karl Fischer titrimetry, and thermoanalytical techniques (differential scanning calorimetry and thermogravimetric analysis). Tablet formulations containing either I* or I were prepared and stored at 33 and 52% RH (room temperature). The solid state of the drug was monitored by X-ray powder diffractometry and the tablets were subjected to the USP dissolution test. In tablets, I* completely converted to I in < or = 10 days when stored at either 33 or 52% RH. Scanning electron microscopy provided direct visual evidence of recrystallization. This recrystallization was accompanied by a decrease in the dissolution rate of the stored formulations that was so pronounced in the formulations stored at 52% RH that they failed the USP dissolution test. The in situ solid-state transition appears to be responsible for the decrease in dissolution rate observed following storage. Stored tablets containing I showed neither a phase transition nor an alteration in their dissolution behavior.     

Alternative models for describing the acid unfolding of the apomyoglobin folding intermediate

The acid-induced unfolding of the pH 4 intermediate of apomyoglobin (I) is described by either of two models: (1) a Monod-Wyman-Changeux-based model (MWC) where salt bridges perturb the pKa values of specific ionizable side chains, causing unfolding of I as these salt bridges are broken at low pH, and (2) the Linderstrom-Lang smeared charge model (L-L), which attributes acid unfolding of I to charge repulsion caused by the accumulation of positive charge on the surface of the protein. Both models fit earlier acid unfolding data well, but they make differing predictions about the effects of electrostatic mutants, which have been made and tested. Deletions of positive charge within I are found to stabilize I, but disruptions of potential salt bridges have little effect. These results show that the acid unfolding of I (I<-->U) is largely caused by generalized charge effects rather than by the loss of specific salt bridges. Acid unfolding of the native form, which is caused largely by a single histidine with a severely depressed pKa, is a sensitive indicator of changes in stability produced by mutations. In contrast, the I <--> U transition is caused by a number of groups with smaller pKa perturbations and both models predict that the pH midpoint of the I right harpoon over left harpoon U transition is an insensitive indicator of stability. This result reconciles previous conflicting results, in urea and acid unfolding studies of hydrophobic contact mutants, by showing that changes in the stability of I are poorly detected by acid unfolding.     

Inhibition of a snake venom hemorrhagic metalloproteinase by human and rat alpha-macroglobulins

Jararafibrase I is a hemorrhagic metalloproteinase purified from Bothrops jararaca venom, which induces local hemorrhage by degrading the basement membrane components. The present study was undertaken to investigate the inhibition of jararafibrase I by human and rat serum proteinase inhibitors. The proteolytic activity of jararafibrase I was completely inhibited by human and rat sera. In particular, rat serum displayed a greater inhibitory capacity. The inhibitory capacities of both sera were dependent on alpha-macroglobulins. SDS-PAGE analysis revealed that jararafibrase I formed complexes with alpha-macroglobulins that were present in normal sera. The proteolytic activity of jararafibrase I was completely inhibited by alpha1-macroglobulin and murinoglobulin in rat serum, and by human alpha2-macroglobulin. The inhibition molar ratios of alpha-macroglobulin/jararafibrase I were 1.5 for rat alpha1-macroglobulin and human alpha2-macroglobulin, and 2.4 for rat murinoglobulin. SDS-PAGE under reducing conditions demonstrated that the bait region of human alpha2-macroglobulin and rat murinoglobulin was cleaved by jararafibrase I. The bait region cleavage sites were identified as being situated at the 696Arg-697Leu peptide bond in human alpha2-macroglobulin, and at the 686Ala-687Val peptide bond in rat murinoglobulin.     

Calmodulin controls adaptation of mechanoelectrical transduction by hair cells of the bullfrog's sacculus

Deflection of the mechanically sensitive hair bundle atop a hair cell opens transduction channels, some of which subsequently reclose during a Ca2+-dependent adaptation process. Myosin I in the hair bundle is thought to mediate this adaptation; in the bullfrog's hair cell, the relevant isozyme may be the 119-kDa amphibian myosin I beta. Because this molecule resembles other forms of myosin I, we hypothesized that calmodulin, a cytoplasmic receptor for Ca2+, regulates the ATPase activity of myosin. We identified an approximately 120-kDa calmodulin-binding protein that shares with hair-bundle myosin I the properties of being photolabeled by vanadate-trapped uridine nucleotides and immunoreactive with a monoclonal antibody raised against mammalian myosin I beta. To investigate the possibility that calmodulin mediates Ca2+-dependent adaptation, we inhibited calmodulin action and measured the results with two distinct assays. Calmodulin antagonists increased photolabeling of hair-bundle myosin I by nucleotides. In addition, when introduced into hair cells through recording electrodes, calmodulin antagonists abolished adaptation to sustained mechanical stimuli. Our evidence indicates that calmodulin binds to and controls the activity of hair-bundle myosin I, the putative adaptation motor.     

Autophosphorylation-independent activation of Acanthamoeba myosin I heavy chain kinase by plasma membranes

The three isoforms of Acanthamoeba myosin I (non-filamentous myosin with only a single heavy chain) express actin-activated Mg(2+)-ATPase activity only when phosphorylated at a single site by myosin I heavy chain kinase. The kinase is activated by autophosphorylation that is greatly stimulated by acidic phospholipids. Substantial fractions of the three myosins I and the kinase are associated in situ with membranes, and all four enzymes bind to purified membranes in vitro. We now report that when kinase and myosin I are incubated together with phosphatidylserine vesicles not only does the kinase autophosphorylate more rapidly than soluble kinase in the absence of phosphatidylserine but that, probably as a result, the kinase phosphorylates myosin I more rapidly than soluble kinase phosphorylates soluble myosin I. Similarly, plasma membrane-bound kinase phosphorylates membrane-bound myosin I and activates its actin-activated Mg(2+)-ATPase activity more rapidly than soluble kinase phosphorylates and activates soluble myosin I in the absence of membranes. However, the enhanced activity of membrane-bound kinase (which is comparable to the activity of kinase in the presence of phosphatidylserine) is not due to autophosphorylation of the membrane-bound kinase, which is very much slower than for kinase activated by phosphatidylserine vesicles.     

Distribution of annexins I, II, and IV in bovine mammary gland

Annexins belong to a family of proteins that are characterized by their ability to bind phospholipids in a Ca(2+)-dependent manner that is thought to be involved in a variety of biological processes. The present study determined the localization of annexins in subcellular fractions, nuclei in particular, of cow mammary gland by immunoblot analysis using monoclonal antibodies to annexins I, II, IV, and VI. The analysis revealed that annexins I, II, and IV were present in cytosol, but VI was not. Annexins I and IV were found in the nuclear fraction, but annexin II was only faintly present. Annexin VI was also undetectable in this fraction. Cytosolic annexin I had a molecular mass of 36 kDa. The 36-kDa annexin I was also found in the nuclear fraction. A 38-kDa annexin I was additionally detected in nuclei. The cytosolic and nuclear 36-kDa annexin I and the nuclear 38-kDa annexin I showed different isoelectric points, as revealed by two-dimensional PAGE. Annexin IV from cytosolic and nuclear fractions had similar molecular masses and isoelectric points.