The syntaxin Tlg1p mediates trafficking of chitin synthase III to polarized growth sites in yeast

Tlg1p and Tlg2p, members of the syntaxin family of SNAREs in yeast, have been implicated in both endocytosis and the retention of late Golgi markers. We have investigated the functions of these and the other endocytic syntaxins Pep12p and Vam3p. Remarkably, growth is possible in the absence of all four proteins. In the absence of the others, Pep12p and Tlg1p can each create endosomes accessible to the endocytic tracer dye FM4-64. However, although Pep12p is required for the ligand-induced internalization of the alpha factor receptor and its passage via Pep12p-containing membranes to the vacuole, Tlg1p is not. In contrast, Tlg1p is required for the efficient localization of the catalytic subunit of chitin synthase III (Chs3p) to the bud neck, a process that involves endocytosis and polarized delivery of Chs3p. In wild-type cells, internalized Chs3p cofractionates with Tlg1p and Tlg2p, and in a strain lacking the other endocytic syntaxins, either Tlg1p or Tlg2p is sufficient for correct localization of the enzyme. Pep12p is neither necessary nor sufficient for this process. We conclude that there are two endocytic routes in yeast that can operate independently and that Tlg1p is located at the junction of one of these with the polarized exocytic pathway.     

The Rho-GEF Rom2p localizes to sites of polarized cell growth and participates in cytoskeletal functions in Saccharomyces cerevisiae

Rom2p is a GDP/GTP exchange factor for Rho1p and Rho2p GTPases; Rho proteins have been implicated in control of actin cytoskeletal rearrangements. ROM2 and RHO2 were identified in a screen for high-copy number suppressors of cik1 delta, a mutant defective in microtubule-based processes in Saccharomyces cerevisiae. A Rom2p::3XHA fusion protein localizes to sites of polarized cell growth, including incipient bud sites, tips of small buds, and tips of mating projections. Disruption of ROM2 results in temperature-sensitive growth defects at 11 degrees C and 37 degrees C. rom2 delta cells exhibit morphological defects. At permissive temperatures, rom2 delta cells often form elongated buds and fail to form normal mating projections after exposure to pheromone; at the restrictive temperature, small budded cells accumulate. High-copy number plasmids containing either ROM2 or RHO2 suppress the temperature-sensitive growth defects of cik1 delta and kar3 delta strains. KAR3 encodes a kinesin-related protein that interacts with Cik1p. Furthermore, rom2 delta strains exhibit increased sensitivity to the microtubule depolymerizing drug benomyl. These results suggest a role for Rom2p in both polarized morphogenesis and functions of the microtubule cytoskeleton.     

The cytoplasmic tail of rhodopsin acts as a novel apical sorting signal in polarized MDCK cells

All basolateral sorting signals described to date reside in the cytoplasmic domain of proteins, whereas apical targeting motifs have been found to be lumenal. In this report, we demonstrate that wild-type rhodopsin is targeted to the apical plasma membrane via the TGN upon expression in polarized epithelial MDCK cells. Truncated rhodopsin with a deletion of 32 COOH-terminal residues shows a nonpolar steady-state distribution. Addition of the COOH-terminal 39 residues of rhodopsin redirects the basolateral membrane protein CD7 to the apical membrane. Fusion of rhodopsin's cytoplasmic tail to a cytosolic protein glutathione S-transferase (GST) also targets this fusion protein (GST-Rho39Tr) to the apical membrane. The targeting of GST-Rho39Tr requires both the terminal 39 amino acids and the palmitoylation membrane anchor signal provided by the rhodopsin sequence. The apical transport of GST-Rho39Tr can be reversibly blocked at the Golgi complex by low temperature and can be altered by brefeldin A treatment. This indicates that the membrane-associated GST-Rho39Tr protein may be sorted along a yet unidentified pathway that is similar to the secretory pathway in polarized MDCK cells. We conclude that the COOH-terminal tail of rhodopsin contains a novel cytoplasmic apical sorting determinant. This finding further indicates that cytoplasmic sorting machinery may exist in MDCK cells for some apically targeted proteins, analogous to that described for basolaterally targeted proteins.     

Agonist-induced phosphorylation of the angiotensin AT1a receptor is localized to a serine/threonine-rich region of its cytoplasmic tail

Autosomal recessive juvenile parkinsonism (AR-JP) is a distinct clinical and genetic entity characterized by selective degeneration of nigral dopaminergic neurons and young-onset parkinsonism with remarkable response to levodopa. Recently, we mapped the gene locus for AR-JP to chromosome 6q25.2-q27 by linkage analysis and we identified a novel large gene, Parkin, consisting of 12 exons from this region; mutations of this gene were found to be the cause of AR-JP in two families. Now we report results of extensive molecular analysis on 34 affected individuals from 18 unrelated families with AR-JP. We found four different homozygous intragenic deletional mutations, involving exons 3 to 4, exon 3, exon 4, and exon 5 in 10 families (17 affected individuals). In addition to the exonic deletions, we identified a novel one-base deletion involving exon 5 in two families (2 affected individuals). All mutations so far found were deletional types in which large exonic deletion accounted for 50% (17 of 34) and the one-base deletion accounted for 6% (2/34); in the remaining, no homozygous mutations were found in the coding regions. Our findings indicate that loss of function of the Parkin protein results in the clinical phenotype of AR-JP and that subregions between introns 2 and 5 of the Parkin gene are mutational hot spots.     

Molecular identification and functional characterization of a novel protein that mediates the attachment of erythroblasts to macrophages

We have previously identified a novel protein that mediates the attachment of erythroblasts to macrophages in vitro. This attachment promotes terminal maturation and enucleation of erythroblasts (Hanspal and Hanspal, Blood 84:3494, 1994). This protein is referred to here as Emp for erythroblast macrophage protein. Two immunologically related isoforms of Emp with apparent molecular weights of 33 kD and 36 kD were detected in macrophage membranes. The complete amino acid sequence of the larger isoform of Emp was deduced from the nucleotide sequence of a full-length 2.0-kb cDNA that was isolated from a human macrophage cDNA library using affinity-purified anti-Emp antibodies. Of the 2,005 bp, 1,185 bp encode for 395 amino acids representing 43 kD (the sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] molecular mass is 36 kD). Northern blot analysis of human macrophage poly(A) RNA detected a message for Emp of 2.1 kb. The deduced amino acid sequence contains a putative transmembrane domain near the N-terminus. To investigate the structure/function relationships of Emp, recombinant fusion proteins of full-length and truncated Emp were produced in bacteria, COS-7, and HeLa cells. Cell binding assays showed that the N-terminus is exposed on the cell surface. The recombinant Emp functions as a cell attachment molecule when expressed in heterologous cells. Furthermore, we showed that the demise of erythroblasts in the absence of Emp-mediated erythroblast-macrophage association is accompanied by apoptosis. We postulate that Emp-mediated contact between erythroblasts and macrophages promotes terminal maturation of erythroid cells by suppressing apoptosis.     

The trkA receptor mediates growth cone turning toward a localized source of nerve growth factor

We have developed an in vitro system for studying the interaction of chick dorsal root ganglion neuronal growth cones with a localized source of nerve growth factor (NGF) covalently conjugated to polystyrene beads. Growth cones rapidly turned and migrated under NGF-coated beads in a process that involved the initial formation of persistent contact with a bead, followed by directed flow of cytoplasm toward the point of contact. A role for the local activation of the high-affinity NGF receptor trkA was suggested by a strong inhibition of the turning response by (1) the addition of an antibody against the extracellular portion of trkA, (2) the elevation of the background concentration of NGF to saturate trkA, or (3) the presence of a concentration of the drug K252a that inhibits trkA activation. NGF binding to the pan-neurotrophin receptor p75 is also involved but is not required for turning. These data show a new role for both the trkA and the p75 receptors: the mediation of local events in the guidance of nerve growth cones.     

Lmb, a protein with similarities to the LraI adhesin family, mediates attachment of Streptococcus agalactiae to human laminin

Streptococcus agalactiae is a leading cause of neonatal sepsis and meningitis. Adherence to extracellular matrix proteins is considered an important factor in the pathogenesis of infection, but the genetic determinants of this process remain largely unknown. We identified and sequenced a gene which codes for a putative lipoprotein that exhibits significant homology to the streptococcal LraI protein family. Mutants of this locus were demonstrated to have substantially reduced adherence to immobilized human laminin. The nucleotide sequence of the gene was subsequently designated lmb (laminin binding) and shown to be present in all of the common serotypes of S. agalactiae. To determine the role of Lmb in the adhesion of S. agalactiae wild-type strains to laminin, a recombinant Lmb protein harboring six consecutive histidine residues at the C terminus was cloned, expressed, and purified from Escherichia coli. Preincubation of immobilized laminin with recombinant Lmb significantly reduced adherence of the wild-type strain O90R to laminin. These results indicate that Lmb mediates the attachment of S. agalactiae to human laminin, which may be essential for the bacterial colonization of damaged epithelium and translocation of bacteria into the bloodstream.     

The high affinity calcium-binding sites in the epidermal growth factor module region of vitamin K-dependent protein S

Vitamin K-dependent protein S, a cofactor of the anticoagulant enzyme-activated protein C, has four epidermal growth factor (EGF)-like modules, all of which have one partially hydroxylated Asp (EGF 1; beta-hydroxyaspartic acid) or Asn (EGF 2, 3, and 4; beta-hydroxyasparagine) residue. The three C-terminal modules have a typical Ca2+ binding sequence motif that is usually present in EGF modules with hydroxylated Asp/Asn residues. Using the chromophoric Ca2+ chelators Quin 2 and 5,5'-Br2BAPTA, we have now determined the Ca2+ affinity of recombinant fragments containing EGF modules 1-3, 1-4, 2-3, and 2-4. EGF modules 1-4 and 2-4 each contains two very high affinity Ca2+-binding sites, i.e. with dissociation constants ranging from 10(-10) to 10(-8) M in the absence of salt and from 10(-8) to 10(-6) M in the presence of 0.15 M NaCl. In contrast, in EGF 1-3 and EGF 2-3, the Ca2+ affinity is 2-4 orders of magnitude lower. EGF 4 thus appears to have the highest Ca2+ affinity, and furthermore it seems to influence the Ca2+ affinity of its immediate N-terminal neighbor EGF 3 by a factor of approximately 230. In addition, EGF 4 seems to influence the Ca2+ affinity of EGF 2 by a factor of approximately 25. The Ca2+ affinity of the binding sites in EGF modules 3 and 4 in fragments EGF 1-4 and EGF 2-4 is 10(3)-10(5)-fold higher than in the corresponding isolated modules, implying important contributions to the Ca2+ affinity of each module from interactions with neighboring modules. This difference is much higher than the approximately 10-fold difference previously found in similar comparisons of EGF modules from fibrillin. However, the modules studied in protein S and fibrillin appear to have the similar Ca2+ ligands. The structural basis for the difference in Ca2+ affinity is not yet understood.     

The plant cDNA LCT1 mediates the uptake of calcium and cadmium in yeast

Nonessential metal ions such as cadmium are most likely transported across plant membranes via transporters for essential cations. To identify possible pathways for Cd2+ transport we tested putative plant cation transporters for Cd2+ uptake activity by expressing cDNAs in Saccharomyces cerevisiae and found that expression of one clone, LCT1, renders the growth of yeast more sensitive to cadmium. Ion flux assays showed that Cd2+ sensitivity is correlated with an increase in Cd2+ uptake. LCT1-dependent Cd2+ uptake is saturable, lies in the high-affinity range (apparent KM for Cd2+ = 33 microM) and is sensitive to block by La3+ and Ca2+. Growth assays demonstrated a sensitivity of LCT1-expressing yeast cells to extracellular millimolar Ca2+ concentrations. LCT1-dependent increase in Ca2+ uptake correlated with the observed phenotype. Furthermore, LCT1 complements a yeast disruption mutant in the MID1 gene, a non-LCT1-homologous yeast gene encoding a membrane Ca2+ influx system required for recovery from the mating response. We conclude that LCT1 mediates the uptake of Ca2+ and Cd2+ in yeast and may therefore represent a first plant cDNA encoding a plant Ca2+ uptake or an organellar Ca2+ transport pathway in plants and may contribute to transport of the toxic metal Cd2+ across plant membranes.     

Distribution of attachment events relative to actin binding sites as evidenced in a bidirectional actomyosin interaction model

Optical trapping is one of the most evolving technologies that measures biophysical quantities and provides insights into some of the fundamental questions in the study of molecular motor proteins such as myosin. Several laboratories have successfully used this technique to observe and score nanometre-size displacements produced by myosin on interacting with actin. We have studied the distribution of attachment events for two myosin molecules with different orientations interacting with an actin filament within the framework of a Langevin-type bidirectional mathematical model. When myosin is detached from actin, our model predicts Brownian displacements centred at 0 +/- 8 nm (mean +/- SD, n = 251,058). When attached, the time-averaged displacements of the actin filament system produced step sizes with peaks of 8 +/- 6 nm (mean +/- SD, n = 22,174) (forward displacements) and -8 +/- 6 nm (mean +/- SD, n = 26,769) (reverse displacements). We infer from our results that the population distribution of attachment events is strongly dependent on (i) the magnitude of the Brownian displacements, (ii) the location of the actin binding sites relative to the myosin molecules, (iii) the orientation of the myosin molecules, and (iv) the relative kinetics (rate constants) for the forward and reverse displacement events.     

Optimization of a polarized photometric detector equipped with a split-type flow cell and its analytical application to oligo-saccharides

A novel, non-modulated polarimeter called a polarized photometric detector (PPD) was previously described by the authors. The PPD enables the measurement of the optical rotation of chiral compounds as a change in absorbance by placing two linear polarizers on either side of a flow cell of a conventional photometric detector. The present study describes the optimization of the conditions of PPD for highly sensitive detection of saccharides. To maximize the light intensity, the light balancing filter and slit were removed from the detector (Shimadzu model SPD-10AV). These modifications resulted in an approximately 15-fold increase in the incident light intensity when the maximum current was applied to the lamp. When this intense light was transmitted through the polarizers, the signal intensity followed the theoretical equation for phase angles up to around 1 rad. If the energy of the transmitted light was less than 700 mV, however, the baseline noise was too great to determine the chiral analyte accurately. Setting the phase angle between two polarizers at 50 degrees and the detection wavelength at 400 nm provided the most suitable conditions. This detector was applicable for the determinations of oligosaccharides in foodstuffs separated by HPLC using gradient elution.     

The renunciation of love: Dismissive attachment and its treatment.

The developmental insights of attachment theory as applied to children and adults suggest that insecure attachment correlates with relational difficulties over the course of time. Specifically, individuals with an avoidant attachment style who have been rebuffed by caregivers in childhood will be defensively constricted and unable to love in adulthood. These patients present particular challenges in treatment because they have become organized around avoidance of affect and relationship. Theories of treatment and technique that seem related to the successful resolution of such difficulties are discussed. Curative factors include a focus on defenses against relational longings, interpretation of and provision for certain selfobject needs, and a relatively high level of therapist self-disclosure. It is suggested that an integration of the findings of attachment research with relational theories that focus on treatment has potential to advance psychoanalytic thinking. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The emotional dog and its rational tail: A social intuitionist approach to moral judgment.

Research on moral judgment has been dominated by rationalist models, in which moral judgment is thought to be caused by moral reasoning. The author gives 4 reasons for considering the hypothesis that moral reasoning does not cause moral judgment; rather, moral reasoning is usually a post hoc construction, generated after a judgment has been reached. The social intuitionist model is presented as an alternative to rationalist models. The model is a social model in that it deemphasizes the private reasoning done by individuals and emphasizes instead the importance of social and cultural influences. The model is an intuitionist model in that it states that moral judgment is generally the result of quick, automatic evaluations (intuitions). The model is more consistent than rationalist models with recent findings in social, cultural, evolutionary, and biological psychology, as well as in anthropology and primatology. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The in vitro permeability of yeast peroxisomal membranes is caused by a 31 kDa integral membrane protein

A major 31 kDa integral peroxisomal membrane protein (PMP31) of Hansenula polymorpha was purified to homogeneity from isolated peroxisomal membranes by FPLC after solubilization by Triton X-100. Biochemical analysis indicated that this protein, which showed cross-reactivity with antibodies against the 31 kDa porin of the mitochondrial outer membrane of Saccharomyces cerevisiae, had pore-forming properties. Firstly, proteoliposomes composed of asolectin and purified PMP31 showed selective permeability, determined as the [14C]sucrose/[3H]dextran leakage ratios. Furthermore, the generation of a delta psi by potassium diffusion gradients was negatively affected by the presence of PMP31 in asolectin liposomes. A similar effect was observed in proteoliposomes containing purified cytochrome c oxidase as a delta psi generating system. Control experiments confirmed that the observed leakage is significant and introduced by the incorporation of PMP31 protein. Selective sucrose leakage was abolished in samples pretreated with glutaraldehyde; an identical effect of glutaraldehyde was, however, not observed for the membrane potential measurements.     

Myristoylation and differential palmitoylation of the HCK protein-tyrosine kinases govern their attachment to membranes and association with caveolae

The human proto-oncogene HCK encodes two versions of a protein-tyrosine kinase, with molecular weights of 59,000 (p59hck) and 61,000 (p61hck). The two proteins arise from a single mRNA by alternative initiations of translation. In this study, we explored the functions of these proteins by determining their locations within cells and by characterizing lipid modifications required for the proteins to reach those locations. We found that p59hck is entirely associated with cellular membranes, including the organelles known as caveolae; in contrast, only a portion of p61hck is situated on membranes, and none is detectable in preparations of caveolae. These distinctions can be attributed to differential modification of the two HCK proteins with fatty acids. Both proteins are at least in part myristoylated, p59hck more so than p61hck. In addition, however, p59hck is palmitoylated on cysteine 3 in the protein. Palmitoylation of the protein requires prior myristoylation and, in turn, is required for targeting to caveolae. These findings are in accord with recent reports for other members of the SRC family of protein-tyrosine kinases. Taken together, the results suggest that HCK and several of its relatives may participate in the functions of caveolae, which apparently include the transduction of signals across the plasma membrane to the interior of the cell.     

Betaglycan can act as a dual modulator of TGF-beta access to signaling receptors: mapping of ligand binding and GAG attachment sites

Betaglycan, also known as the TGF-beta type III receptor, is a membrane-anchored proteoglycan that presents TGF-beta to the type II signaling receptor, a transmembrane serine/threonine kinase. The betaglycan extracellular region, which can be shed by cells into the medium, contains a NH2-terminal domain related to endoglin and a COOH-terminal domain related to uromodulin, sperm receptors Zp2 and 3, and pancreatic secretory granule GP-2 protein. We identified residues Ser535 and Ser546 in the uromodulin-related region as the glycosaminoglycan (GAG) attachment sites. Their mutation to alanine prevents GAG attachment but does not interfere with betaglycan stability or ability to bind and present TGF-beta to receptor II. Using a panel of deletion mutants, we found that TGF-beta binds to the NH2-terminal endoglin-related region of betaglycan. The remainder of the extracellular domain and the cytoplasmic domain are not required for presentation of TGF-beta to receptor II; however, membrane anchorage is required. Soluble betaglycan can bind TGF-beta but does not enhance binding to membrane receptors. In fact, recombinant soluble betaglycan acts as potent inhibitor of TGF-beta binding to membrane receptors and blocks TGF-beta action, this effect being particularly pronounced with the TGF-beta 2 isoform. The results suggest that release of betaglycan into the medium converts this enhancer of TGF-beta action into a TGF-beta antagonist.     

The quality of mother–infant attachment and its relationship to toddlers' initial sociability with peers.

37 age- and sex-matched toddler dyads were observed between ages 20 and 23 mo in a 30-min play session with mothers present. Assignment to dyad was determined by attachment classification, assessed at 18 mo in the M. D. Ainsworth et al (1978) Strange Situation procedure. Ss classified as securely attached (SAs), anxiously avoidant (AAs), or anxiously resistant (ARs) were paired with SAs. In the play situation, SAs were more sociable and more positively oriented toward both mother and peer. Mothers of SAs were more supportive and appropriately directive and played an important role in successful outcomes for their children. AAs participated actively in the play session but were rated as more negative in their orientation toward both mother and peer. ARs appeared highly stressed by this situation; they ignored most peer offers and were most negative toward their mothers. (24 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Radio-restoration by yeast s-RNA and its nucleotides of plant tissues cultured in vitro. Restorative activity of yeast s-RNA and a mixture of its nucleotides on the growth of Sorconera crown-gall tissue cultures

Gamma RNA extracts from yeast restore the growth of Scorsonere Crown gall tissues which were subjected to gamma radiation from sigma cobalt 60. A mixture of nucleotides obtained from enzymatic hydrolysis of s-RNA by a ribonuclease T1 has the same stimulatory effect.