Capping of the barbed ends of actin filaments by a high-affinity profilin-actin complex

Profilin, a ubiquitous 12 to 15-kDa protein, serves many functions, including sequestering monomeric actin, accelerating nucleotide exchange on actin monomers, decreasing the critical concentration of the barbed end of actin filaments, and promoting actin polymerization when barbed ends are free. Most previous studies have focused on profilin itself rather than its complex with actin. A high-affinity profilin-actin complex (here called profilactin) can be isolated from a poly-(L)-proline (PLP) column by sequential elution with 3 M and 7 M urea. Profilactin inhibited the elongation rate of pyrenyl-G-actin from filament seeds in a concentration- and time-dependent manner. Much greater inhibition of elongation was observed with spectrin-F-actin than gelsolin-F-actin seeds, suggesting that the major effect of profilactin was due to capping the barbed ends of actin filaments. Its dissociation constant for binding to filament ends was 0.3 microM; the on- and off-rate constants were estimated to be 1.7 x 10(3) M-1 s-1 and 4.5 x 10(-4) s-1, respectively. Purified profilin (obtained by repetitive applications to a PLP column and assessed by silver-stained polyacylamide gels) did not slow the elongation rate of pyrenyl-G-actin from filament seeds. Capping protein could not be detected by Western blotting in the profilactin preparation, but low concentrations of gelsolin did contaminate our preparation. However, prolonged incubation with either calcium or EGTA did not affect capping activity, implying that contaminating gelsolin-actin complexes were not primarily responsible for the observed capping activity. Reapplication of the profilactin preparation to PLP-coupled Sepharose removed both profilin and actin and concurrently eliminated its capping activity. Profilactin that was reapplied to uncoupled Sepharose retained its capping activity. Phosphatidylinositol-4,5-bisphosphate (PIP2) was the most potent phosphoinositol in reducing the capping activity of profilactin. Dissociation of the tight profilactin complex may serve as a unique mechanism by which profilin helps regulate actin filament growth.     

Adducin is an in vivo substrate for protein kinase C: phosphorylation in the MARCKS-related domain inhibits activity in promoting spectrin-actin complexes and occurs in many cells, including dendritic spines of neurons

Adducin is a heteromeric protein with subunits containing a COOH-terminal myristoylated alanine-rich C kinase substrate (MARCKS)-related domain that caps and preferentially recruits spectrin to the fast-growing ends of actin filaments. The basic MARCKS-related domain, present in alpha, beta, and gamma adducin subunits, binds calmodulin and contains the major phosphorylation site for protein kinase C (PKC). This report presents the first evidence that phosphorylation of the MARCKS-related domain modifies in vitro and in vivo activities of adducin involving actin and spectrin, and we demonstrate that adducin is a prominent in vivo substrate for PKC or other phorbol 12-myristate 13-acetate (PMA)-activated kinases in multiple cell types, including neurons. PKC phosphorylation of native and recombinant adducin inhibited actin capping measured using pyrene-actin polymerization and abolished activity of adducin in recruiting spectrin to ends and sides of actin filaments. A polyclonal antibody specific to the phosphorylated state of the RTPS-serine, which is the major PKC phosphorylation site in the MARCKS-related domain, was used to evaluate phosphorylation of adducin in cells. Reactivity with phosphoadducin antibody in immunoblots increased twofold in rat hippocampal slices, eight- to ninefold in human embryonal kidney (HEK 293) cells, threefold in MDCK cells, and greater than 10-fold in human erythrocytes after treatments with PMA, but not with forskolin. Thus, the RTPS-serine of adducin is an in vivo phosphorylation site for PKC or other PMA-activated kinases but not for cAMP-dependent protein kinase in a variety of cell types. Physiological consequences of the two PKC phosphorylation sites in the MARCKS-related domain were investigated by stably transfecting MDCK cells with either wild-type or PKC-unphosphorylatable S716A/S726A mutant alpha adducin. The mutant alpha adducin was no longer concentrated at the cell membrane at sites of cell-cell contact, and instead it was distributed as a cytoplasmic punctate pattern. Moreover, the cells expressing the mutant alpha adducin exhibited increased levels of cytoplasmic spectrin, which was colocalized with the mutant alpha adducin in a punctate pattern. Immunofluorescence with the phosphoadducin-specific antibody revealed the RTPS-serine phosphorylation of adducin in postsynaptic areas in the developing rat hippocampus. High levels of the phosphoadducin were detected in the dendritic spines of cultured hippocampal neurons. Spectrin also was a component of dendritic spines, although at distinct sites from the ones containing phosphoadducin. These data demonstrate that adducin is a significant in vivo substrate for PKC or other PMA-activated kinases in a variety of cells, and that phosphorylation of adducin occurs in dendritic spines that are believed to respond to external signals by changes in morphology and reorganization of cytoskeletal structures.     

Gelsolin, a protein that caps the barbed ends and severs actin filaments, enhances the actin-based motility of Listeria monocytogenes in host cells

The actin-based motility of Listeria monocytogenes requires the addition of actin monomers to the barbed or plus ends of actin filaments. Immunofluorescence micrographs have demonstrated that gelsolin, a protein that both caps barbed ends and severs actin filaments, is concentrated directly behind motile bacteria at the junction between the actin filament rocket tail and the bacterium. In contrast, CapG, a protein that strictly caps actin filaments, fails to localize near intracellular Listeria. To explore the effect of increasing concentrations of gelsolin on bacterial motility, NIH 3T3 fibroblasts stably transfected with gelsolin cDNA were infected with Listeria. The C5 cell line containing 2.25 times control levels of gelsolin supported significantly higher velocities of bacterial movement than did control fibroblasts (mean +/- standard error of the mean, 0.09 +/- 0.003 micro(m)/s [n = 176] versus 0.05 +/- 0.003 micro(m)/s [n = 65]). The rate of disassembly of the Listeria-induced actin filament rocket tail was found to be independent of gelsolin content. Therefore, if increases in gelsolin content result in increases in Listeria-induced rocket tail assembly rates, a positive correlation between gelsolin content and tail length would be expected. BODIPY-phalloidin staining of four different stably transfected NIH 3T3 fibroblast cell lines confirmed this expectation (r = 0.92). Rocket tails were significantly longer in cells with a high gelsolin content. Microinjection of gelsolin 1/2 (consisting of the amino-terminal half of native gelsolin) also increased bacterial velocity by more than 2.2 times. Microinjection of CapG had no effect on bacterial movement. Cultured skin fibroblasts derived from gelsolin-null mice were capable of supporting intracellular Listeria motility at velocities comparable to those supported by wild-type skin fibroblasts. These experiments demonstrated that the surface of Listeria contains a polymerization zone that can block the barbed-end-capping activity of both gelsolin and CapG. The ability of Listeria to uncap actin filaments combined with the severing activity of gelsolin can accelerate actin-based motility. However, gelsolin is not absolutely required for the actin-based intracellular movement of Listeria because its function can be replaced by other actin regulatory proteins in gelsolin-null cells, demonstrating the functional redundancy of the actin system.     

Coronin promotes the rapid assembly and cross-linking of actin filaments and may link the actin and microtubule cytoskeletons in yeast

Coronin is a highly conserved actin-associated protein that until now has had unknown biochemical activities. Using microtubule affinity chromatography, we coisolated actin and a homologue of coronin, Crn1p, from Saccharomyces cerevisiae cell extracts. Crn1p is an abundant component of the cortical actin cytoskeleton and binds to F-actin with high affinity (Kd 6 x 10(-9) M). Crn1p promotes the rapid barbed-end assembly of actin filaments and cross-links filaments into bundles and more complex networks, but does not stabilize them. Genetic analyses with a crn1Delta deletion mutation also are consistent with Crn1p regulating filament assembly rather than stability. Filament cross-linking depends on the coiled coil domain of Crn1p, suggesting a requirement for Crn1p dimerization. Assembly-promoting activity is independent of cross-linking and could be due to nucleation and/or accelerated polymerization. Crn1p also binds to microtubules in vitro, and microtubule binding is enhanced by the presence of actin filaments. Microtubule binding is mediated by a region of Crn1p that contains sequences (not found in other coronins) homologous to the microtubule binding region of MAP1B. These activities, considered with microtubule defects observed in crn1Delta cells and in cells overexpressing Crn1p, suggest that Crn1p may provide a functional link between the actin and microtubule cytoskeletons in yeast.     

Model for the alignment of actin filaments in endothelial cells subjected to fluid shear stress

Cultured vascular endothelial cells undergo significant morphological changes when subjected to sustained fluid shear stress. The cells elongate and align in the direction of applied flow. Accompanying this shape change is a reorganization at the intracellular level. The cytoskeletal actin filaments reorient in the direction of the cells' long axis. How this external stimulus is transmitted to the endothelial cytoskeleton still remains unclear. In this article, we present a theoretical model accounting for the cytoskeletal reorganization under the influence of fluid shear stress. We develop a system of integro-partial-differential equations describing the dynamics of actin filaments, the actin-binding proteins, and the drift of transmembrane proteins due to the fluid shear forces applied on the plasma membrane. Numerical simulations of the equations show that under certain conditions, initially randomly oriented cytoskeletal actin filaments reorient in structures parallel to the externally applied fluid shear forces. Thus, the model suggests a mechanism by which shear forces acting on the cell membrane can be transmitted to the entire cytoskeleton via molecular interactions alone.     

Peptides from the conserved ends of the rod domain of desmin disassemble intermediate filaments and reveal unexpected structural features: a circular dichroism, Fourier transform infrared, and electron microscopic study

Synthetic peptides representing the conserved ends of the rod domain of desmin are shown to disassemble preformed desmin filaments when added in moderate molar excess. This argues for a similar importance of both ends of the rod for filament stability. Recent structural models of intermediate filaments suggest close proximity of the ends and perhaps even an interaction (N. Geisler, J. Schünemann, and K. Weber, 1992, Eur. J. Biochem. 206, 841-852; P. M. Steinert, L. N. Marekov, R. D. B. Fraser, and D. A. D. Parry, 1993, J. Mol. Biol. 230, 436-452). Since the disassembling activity of the peptides, in addition to their sequences, should be related in some way to their secondary structure, we have investigated the structures of a number of related peptides which all arise from the ends of the rod using electron microscopic and spectroscopic methods. All peptides showed the expected alpha-helical structure at low concentrations in the presence of trifluoroethanol, as revealed by circular dichroism. At higher concentrations the peptides showed extensive self-aggregation into various types of filaments. The filaments contain the peptides in beta-sheet conformation as shown by Fourier transform infrared spectroscopy.     

Role of the ninaC proteins in photoreceptor cell structure: ultrastructure of ninaC deletion mutants and binding to actin filaments

The ninaC proteins are found in Drosophila photoreceptor cells. Their primary sequences suggest they are kinase/myosin chimeras, but their myosin head-like domain is the most divergent amongst all the myosin-like proteins described to date. To investigate possible roles of the ninaC proteins in cell structure, we examined the ultrastructure of the photoreceptor cells in various ninaC mutants, and tested the ability of the proteins to interact with actin filaments in a myosin-like manner. In flies lacking the larger ninaC protein, p174, an ultrastructural phenotype was evident before eclosion. The axial actin cytoskeleton of the rhabdomeral microvilli appeared either fragmented or as an isolated structure, without linkage to the microvillar membrane. Deletion of the myosin head-like domain or the calmodulin-binding domain of p174 resulted in a similar abnormal cytoskeleton. Breakdown of the rhabdomeres followed, although at different rates depending on the deletion. Lack of the smaller protein, p132, per se did not result in photoreceptor degeneration, but in older flies there was an abnormal accumulation of multivesicular bodies. Moreover, the presence of p132 retarded the degeneration that occurs in the absence of p174, even though the p132 remained outside the rhabdomere. Biochemical studies showed that both ninaC proteins bind actin filaments and cosediment with actin filaments in an ATP-sensitive manner. These results outline structural roles for the ninaC proteins, and are consistent with the notion, suggested by their amino acid sequences, that the proteins are actin-based mechanoenzymes.     

Desmin and actin filaments in membrane-cytoskeletal preparations of the electric tissue of Electrophorus electricus, L

OBJECTIVES: To assess the effect of undertaking custodial care of a grandchild on grandparents' depression levels and to determine what characteristics are associated with higher depression levels among caregiving grandparents. DESIGN: A longitudinal national probability panel study: the National Survey of Families and Households. The first wave of data (n= 13 008) was collected in 1987 and 1988, and the second wave of data (n=10008) was collected from 1992 through 1994. SETTING: The survey was conducted in respondents' households in the coterminous United States. PARTICIPANTS: The subsample for this study was composed of 3111 respondents who reported being grandparents during the 1992-1994 interviews and for whom complete depression information was available. Of these grandparents, 158 were the primary caregivers for their grandchildren in the 1990s. MAIN OUTCOME MEASURES: Depression was measured using a modified version of the Center for Epidemiological Studies Depression Scale. RESULTS: Those who provide primary care for a grandchild are almost twice as likely to have levels of depressive symptoms above the traditional Center for Epidemiological Studies Depression Scale cut point of 16 (25.1% vs 14.5%). Even when controlling for baseline depression and demographic variables known to affect depressive symptoms, undertaking the care of a grandchild was associated significantly with higher depression levels in a multivariate prospective analysis (P<.01). Among caregiving grandparents, those who recently assumed caregiving responsibilities (P<.05) and women (P<.10) were more depressed and older respondents (P<.10) and those in good health (P<.001) were less depressed. CONCLUSIONS: Undertaking the primary care of a grandchild is associated with an increase in levels of depression. Particularly in light of the recent dramatic increase in the prevalence of grandparent caregiving in the United States, physicians need to explore familial role changes with midlife and older patients who have symptoms of depression. Special attention should be paid to the most at-risk subsets of grandparent caregivers: those who are new caregivers, those in poor health, those who are younger, and women.     

Heterogeneity in the distribution of actin filaments in the endothelial cells of arteries and arterioles in the rat kidney

The effect of two types of electrical stimulation designed to induce long-lasting plasticity of the Schaffer/commissural inputs to CA1 pyramidal neurons was investigated using in vitro hippocampal slices made from young (3-6 month) and old (24-27 month) Fischer 344 rats. The first stimulation paradigm, primed burst (PB) stimulation, consisted of a total of five physiologically patterned stimuli: a single priming pulse followed 170 ms later by a burst of four pulses at 200 Hz. The second stimulation paradigm, long-term potentiation (LTP) stimulation, consisted of a 200 Hz/1 second train (a total of 200 stimuli). Primed burst and LTP stimulation were equally effective at inducing a lasting increase in the population spike recorded from slices made from young rats. However, the enhancement of population spike amplitude produced by PB, but not LTP, stimulation was significantly less in slices made from old rats. These results suggest that the capacity of the hippocampus to demonstrate long-lasting synaptic plasticity is not altered with age, but that engaging plasticity-inducing mechanisms becomes more difficult. Furthermore, these data suggest that physiologically patterned paradigms for inducing long-lasting synaptic plasticity may more accurately assess the functional status of hippocampal memory encoding mechanisms than does conventional LTP stimulation.     

Insulin receptor substrate-2 binds to the insulin receptor through its phosphotyrosine-binding domain and through a newly identified domain comprising amino acids 591-786

We compared the interaction between the insulin receptor (IR) and the IR substrate (IRS) proteins IRS-1 and IRS-2) using the yeast two-hybrid system. Both IRS proteins interact specifically with the cytoplasmic portion of the IR and the related insulin-like growth factor-I receptor, and these interactions require receptor tyrosine kinase activity. Alignment of IRS-1 and IRS-2 revealed two conserved domains at the NH2 terminus, called IH1PH and IH2PTB, which resemble a pleckstrin homology (PH) domain and a phosphotyrosine binding (PTB) domain, respectively. The IH2PTB binds to the phosphorylated NPXY motif (Tyr-960) in the activated insulin receptor, providing a specific mechanism for the interaction between the receptor and IRS-1. Although the IH2PTB of IRS-2 also interacts with the NPEY motif of the insulin receptor, it is not essential for the interaction between the insulin receptor and IRS-2 in the yeast two-hybrid system. IRS-2 contains another interaction domain between residues 591 and 786, which is absent in IRS-1. This IRS-2-specific domain is independent of the IH2PTB and does not require the NPEY motif; however, it requires a functional insulin receptor kinase and the presence of three tyrosine phosphorylation sites in the regulatory loop (Tyr-1146, Tyr-1150, and Tyr-1151). Importantly, this novel domain mediates the association between IRS-2 and insulin receptor lacking the NPXY motif and may provide a mechanism by which the stoichiometry of regulatory loop autophosphorylation enhances IRS-2 phosphorylation.     

Epidermal growth factor-induced activation and translocation of c-Src to the cytoskeleton depends on the actin binding domain of the EGF-receptor

The relationship between retinal arterial (Pra) and aortic (Pa) pressures is unknown, and the relationship between retinal vein (Prv) pressure and intraocular pressure (IOP) is not clear. Also unclear is the effect of cerebrospinal fluid pressure (CSFp) upon retinal venous pressure. We aimed to measure the relationships among Pra, Prv, Pa, IOP, and CSFp. Dogs were anesthetized while IOP, CSFp, and Pa were monitored. Pipettes with 2.5-micron diameter tips, connected to a servonulling pressure transducer, were used to record pressures from the retinal arteries and veins. Across a range of IOP (16-22 mmHg), CSFp (0-21 mmHg), and Pa (23-195 mmHg) the Pra = 0.72 Pa + 4.3 (r = 0.99, n = 61, P < 0.01), which suggests that the relationship between Pra and Pa is linear over a broad range of systemic blood pressures. The correlation coefficient between Prv and IOP was greater than 0.96 (P < 0.01) at all venous sites and whether IOP was greater than or less than CSFp. The transmural pressure varied along the retinal vein from 1.3 +/- 0.3 mmHg (+/-95% CI, n = 30) at 1 disk diameter from the optic disk rim to 0.3 +/- 0.2 mmHg (n = 66) at the optic disk, with a 0.9-mmHg/mm pressure gradient. These are the first measurements demonstrating a retinal vein transmural pressure close to zero.     

Identification of a novel mechanism of regulation of the adherens junction by E1A, Rac1, and cortical actin filaments that contributes to tumor progression

Transformation progression toward more malignant behavior often results from a loss of epithelial cell behavior, especially cell-cell adhesion. E1A cooperates with ras to transform primary epithelial cells such that they maintain epithelial cell differentiation, including the proper localization of adherens junctions (AJs). Second exon mutants of E1A 12S cooperate with ras to produce a more aggressively transformed phenotype, termed hypertransformation, that includes the loss of adhesion. Such hypertransformation can also be achieved by the addition of activated Rac1 to cells expressing wild-type E1A and ras, suggesting that actin reorganization may be important for the hypertransformed phenotype. Primary epithelial cells expressing hypertransforming mutants of E1A or V12Rac1 exhibit the loss of cortical actin filaments. In these cells, AJ complexes do not incorporate alpha-catenin, fail to associate with the cytoskeleton, and fail to localize to the plasma membrane, resulting in the destabilization of the AJ components and a loss of function. Loss of these epithelial cell characteristics predisposes these cells to a more malignant phenotype due to the loss of cell-cell adhesion. Taken together, these results suggest a novel mechanism of regulation of AJ function in tumor progression that involves the correct targeting of the AJ components, and this is affected by the status of cortical actin, which can be differentially affected by E1A or Rac1.     

Area V1 in macaque monkeys projects to multiple histochemically defined subdivisions of the inferior pulvinar complex

To investigate how visuotopic connections relate to chemoarchitecture of the inferior pulvinar (PI) complex in macaques, neuroanatomical tracers were placed into known portions of the visual representation in V1. Separate foci of label associated with both the upper and lower visual quadrants occupied neurochemically defined medial, central, lateral, and lateral-shell subdivisions, PIM, PIC, PIL, and PIL-S. Visuotopic connection patterns thus supported the concept of a larger PI that includes portions of three classically defined 'nuclei' [C. Gutierrez, A. Yaun and C.G. Cusick, Neurochemical subdivisions of the inferior pulvinar in macaque monkeys, J. Comp. Neurol., 363 (1995) 545-562.], and corresponds to the topographically organized V1 projection zone.     

Comparative characterization of a wild type and transmembrane domain-deleted fatty acid amide hydrolase: identification of the transmembrane domain as a site for oligomerization

Fatty acid amide hydrolase (FAAH) is an integral membrane protein responsible for the hydrolysis of a number of primary and secondary fatty acid amides, including the neuromodulatory compounds anandamide and oleamide. Analysis of FAAH's primary sequence reveals the presence of a single predicted transmembrane domain at the extreme N-terminus of the enzyme. A mutant form of the rat FAAH protein lacking this N-terminal transmembrane domain (DeltaTM-FAAH) was generated and, like wild type FAAH (WT-FAAH), was found to be tightly associated with membranes when expressed in COS-7 cells. Recombinant forms of WT- and DeltaTM-FAAH expressed and purified from Escherichia coli exhibited essentially identical enzymatic properties which were also similar to those of the native enzyme from rat liver. Analysis of the oligomerization states of WT- and DeltaTM-FAAH by chemical cross-linking, sedimentation velocity analytical ultracentrifugation, and size exclusion chromatography indicated that both enzymes were oligomeric when membrane-bound and after solubilization. However, WT-FAAH consistently behaved as a larger oligomer than DeltaTM-FAAH. Additionally, SDS-PAGE analysis of the recombinant proteins identified the presence of SDS-resistant oligomers for WT-FAAH, but not for DeltaTM-FAAH. Self-association through FAAH's transmembrane domain was further demonstrated by a FAAH transmembrane domain-GST fusion protein which formed SDS-resistant dimers and large oligomeric assemblies in solution.     

Modulation of actin affinity and actomyosin adenosine triphosphatase by charge changes in the myosin motor domain

The effects of mutations in an actin-binding surface loop of myosin (loop 2) are described. Part of loop 2, the segment between myosin residues 618 and 622, was replaced with sequences enlarged by the introduction of positively charged GKK or neutral GNN motifs. Constructs with loops carrying up to 20 additional amino acids and charge variations from -1 to +12 were produced. Steady-state and transient kinetics were used to characterize the enzymatic behavior of the mutant motor domains. Binding of nucleotide was not affected by any of the alterations in loop 2. In regard to their interaction with actin, constructs with moderate charge changes (-1 to +2) displayed wild-type-like behavior. Introduction of more than one GKK motif led to stronger coupling between the actin- and nucleotide-binding sites of myosin and an up to 1000-fold increased affinity for actin in the absence of ATP and at zero ionic strength. In comparison to the wild-type construct M765, constructs with 4-12 extra charges displayed an increased dependence on ionic strength in their interaction with actin, a 2-3-fold increase in kcat, a more than 10-fold reduction in Kapp for actin, and a 34-70-fold increase in catalytic efficiency.     

Both microtubules and actin filaments are required for efficient postendocytotic traffic of the polymeric immunoglobulin receptor in polarized Madin-Darby canine kidney cells

It has been postulated that membrane traffic in polarized epithelial cells requires both actin filaments and microtubules. We have tested this hypothesis by analyzing the effect of cytochalasin D (cytoD; an actin-disrupting agent), by itself or in combination with nocodazole (a microtubule depolymerizing agent), on postendocytic traffic in Madin-Darby canine kidney cells. CytoD treatment inhibited basolateral to apical transcytosis of IgA in polymeric immunoglobulin receptor-expressing cells by approximately 45%, but had little effect on basolateral recycling of transferrin. Apical recycling of IgA was also inhibited by approximately 20%. Like nocodazole, cytoD acted at an early step in transcytosis, and inhibited translocation of IgA between the basolateral early endosomes and the apical recycling endosome. There was little inhibition of the subsequent release of IgA from the apical recycling endosome of cytoD- or nocodazole-treated cells. Order-of-addition experiments suggest that the cytoD-sensitive step preceded the nocodazole-sensitive step. Treatment with both cytoD and nocodazole inhibited transcytosis 95%. These results suggest that in addition to microtubules, efficient postendocytic traffic in polarized epithelial cells also requires actin filaments.     

Characterization of a 50-kDa polypeptide in cytoplasmic dynein preparations reveals a complex with p150GLUED and a novel actin

Earlier work identified a series of accessory polypeptides of 150, 74, 59, 57, 55, 53, 50, and 45 kDa copurifying with cytoplasmic dynein. In the present study immunoprecipitation of the 50-kDa polypeptide from bovine brain cytosol with a specific monoclonal antibody revealed coprecipitating components of 150, 135, 62, and 45 kDa, which were completely distinct from the polypeptides immunoprecipitated using an antibody to the well established 74-kDa cytoplasmic dynein subunit. The 150- and 135-kDa polypeptides reacted with an antibody to p150Glued, the mammalian homologue of the Drosophila Glued gene. N-terminal microsequencing of tryptic peptides of the major 45-kDa component of the complex revealed it to be the alpha-isoform of centractin, a novel form of actin. Immunoblotting of sucrose gradient-fractionated brain cytosol revealed p150Glued, p50, and centractin to cosediment exclusively at 20 S. Immunofluorescence microscopy using antibody to p150Glued revealed centrosomal staining, which was abolished by microtubule depolymerization. Together these results reveal the 50-kDa polypeptide to be part of a cytosolic complex distinct from cytoplasmic dynein. However, the immunolocalization data indicate an association with microtubule minus ends, suggesting a possible interaction with cytoplasmic dynein in the cell.     

Prevalence of immunity to hepatitis A in recruits to the British Army and Royal Air Force

Between 1989 and 1992, 92% of a sample of 2790 Service recruits aged between 17 and 35 years (mean age 19 years 7 months) were found not to be immune to infection by hepatitis A virus. The proportion of males with immunity was consistently greater than that for females. There was a significantly increased probability of immunity if individuals originated from Northern England, the Midlands and Scotland, in particular the suburbs. Among male recruits there were significantly increased probabilities of immunity associated with travel to Southern and Eastern Europe or to the Tropics, and for females with travel to North West Europe or to Southern and Eastern Europe.