Regulation of the actin cycle in vivo by actin filament severing

Cycling of actin subunits between monomeric and filamentous phases is essential for cell crawling behavior. We investigated actin filament turnover rates, length, number, barbed end exposure, and binding of cofilin in bovine arterial endothelial cells moving at different speeds depending on their position in a confluent monolayer. Fast-translocating cells near the wound edge have short filament lifetimes compared with turnover values that proportionately increase in slower moving cells situated at increasing distances from the wound border. Contrasted with slow cells exhibiting slow actin filament turnover speeds, fast cells have less polymerized actin, shorter actin filaments, more free barbed ends, and less actin-associated cofilin. Cultured primary fibroblasts manifest identical relationships between speed and actin turnover as the endothelial cells, and fast fibroblasts expressing gelsolin have higher actin turnover rates than slow fibroblasts that lack this actin-severing protein. These results implicate actin filament severing as an important control mechanism for actin cycling in cells.     

From the Cover: Archaeal actin from a hyperthermophile forms a single-stranded filament

The prokaryotic origins of the actin cytoskeleton have been firmly established, but it has become clear that the bacterial actins form a wide variety of different filaments, different both from each other and from eukaryotic F-actin. We have used electron cryomicroscopy (cryo-EM) to examine the filaments formed by the protein crenactin (a crenarchaeal actin) from Pyrobaculum calidifontis, an organism that grows optimally at 90 °C. Although this protein only has ∼20% sequence identity with eukaryotic actin, phylogenetic analyses have placed it much closer to eukaryotic actin than any of the bacterial homologs. It has been assumed that the crenactin filament is double-stranded, like F-actin, in part because it would be hard to imagine how a single-stranded filament would be stable at such high temperatures. We show that not only is the crenactin filament single-stranded, but that it is remarkably similar to each of the two strands in F-actin. A large insertion in the crenactin sequence would prevent the formation of an F-actin-like double-stranded filament. Further, analysis of two existing crystal structures reveals six different subunit–subunit interfaces that are filament-like, but each is different from the others in terms of significant rotations. This variability in the subunit–subunit interface, seen at atomic resolution in crystals, can explain the large variability in the crenactin filaments observed by cryo-EM and helps to explain the variability in twist that has been observed for eukaryotic actin filaments.Actin is one of the most highly conserved as well as abundant eukaryotic proteins. From chickens to humans, an evolutionary separation of ∼350 million years, there are no amino acid changes in the skeletal muscle isoform of actin (1). There are at least six different mammalian isoforms that are quite similar to each other, and all seem to have diverged from a common ancestral actin gene (2). In contrast, we now know that bacteria have actin-like proteins that share a common fold (3–5) but have vanishingly little sequence similarity both among themselves and to eukaryotic actin (6).Two recent crystal structures of a crenarchaeal actin, crenactin (7, 8), raise interesting questions about the structure of the crenactin filament and its evolutionary relationship to F-actin. In both crystals (with two different space groups) crenactin forms a single-stranded filament with eight subunits per ∼420-Å right-handed turn, with a rise and rotation per subunit, therefore, of ∼53 Å and 45°, respectively. In contrast, in F-actin there is a rise and rotation of ∼55 Å and ∼27° along each of the two long-pitch right-handed strands. It was stated (9) that outside of the crystal the crenactin filaments are double-stranded, based upon the suggestion (8) that a single-stranded filament would unlikely be stable and that power spectra from crenactin filaments showed a strong layer line at ∼1/(210 Å), half of the repeat in the crystals.We have been able to image and reconstruct at low resolution the filaments formed by crenactin. Surprisingly, these filaments contain only a single strand, as opposed to the two strands present in F-actin and in the filaments formed by a number of bacterial actin-like proteins (9–13). We show that the crenactin filament is consistent with the single strand seen in crystals (7, 8), and that this strand is quite similar to each of the two strands within F-actin. This supports arguments about the close phylogenetic proximity between eukaryotic actin and archaeal actin (14, 15) and highlights the substantial divergence that has taken place between the bacterial actin-like proteins and eukaryotic actin (6).     

Glucocorticoids inhibit gonadotropin-releasing hormone by acting directly at the hypothalamic level

Glucocorticoids, the end-product of the hypothalamic-pituitary-adrenal (HPA) axis, suppress gonadotropin release by acting at the level of the pituitary gland. However, experimental evidence suggests that they may also act at the hypothalamic level to suppress gonadotropin-releasing hormone (GnRH) release. The lack of a direct demonstration of this assumption, prompted us to evaluate the effects of glucocorticoids on hypothalamic GnRH release from individually-incubated hemi-hypothalami explanted from male rats. Since testosterone (T), dihydrotestosterone (DHT), and progesterone suppress GnRH release and androgens potentiate the effects of glucocorticoids on GnRH release, we studied also the interaction of these steroids with glucocorticoids on GnRH release. Corticosterone (B), the main glucocorticoid of the rodents with greater affinity for the type I glucocorticoid receptor, and dexamethasone (DEX), a synthetic type II glucocorticoid receptor agonist, were able to suppress basal GnRH release in a concentration-dependent fashion. DEX induced a more profound suppression of GnRH release. Neither T (0.1 nM) nor DHT (0.01 nM) modulated the suppressive effects of low (10 nM) or high (100 nM) concentrations of B on GnRH release. On the other hand, progesterone counteracted the suppressive effect of low concentrations of B (10 nM) on GnRH release, but had no effect on the suppression caused by a higher concentration of B (100 nM). The ability of glucocorticoids to inhibit directly GnRH release suggests that these stress-responsive hormones act also at the hypothalamic level to suppress the reproductive function. The suppressive effect of B was not modulated by androgens, but it was neutralized by progesterone, at least when B was used at low concentrations. We speculate that this steroid "protects" the GnRH-secreting neuron only during basal, but not stress-induced, HPA axis activity when the concentrations of glucocorticoids are more elevated.     

Myosin-10 produces its power-stroke in two phases and moves processively along a single actin filament under low load

Myosin-10 is an actin-based molecular motor that participates in essential intracellular processes such as filopodia formation/extension, phagocytosis, cell migration, and mitotic spindle maintenance. To study this motor protein’s mechano-chemical properties, we used a recombinant, truncated form of myosin-10 consisting of the first 936 amino acids, followed by a GCN4 leucine zipper motif, to force dimerization. Negative-stain electron microscopy reveals that the majority of molecules are dimeric with a head-to-head contour distance of ∼50 nm. In vitro motility assays show that myosin-10 moves actin filaments smoothly with a velocity of ∼310 nm/s. Steady-state and transient kinetic analysis of the ATPase cycle shows that the ADP release rate (∼13 s⁻¹) is similar to the maximum ATPase activity (∼12–14 s⁻¹) and therefore contributes to rate limitation of the enzymatic cycle. Single molecule optical tweezers experiments show that under intermediate load (∼0.5 pN), myosin-10 interacts intermittently with actin and produces a power stroke of ∼17 nm, composed of an initial 15-nm and subsequent 2-nm movement. At low optical trap loads, we observed staircase-like processive movements of myosin-10 interacting with the actin filament, consisting of up to six ∼35-nm steps per binding interaction. We discuss the implications of this load-dependent processivity of myosin-10 as a filopodial transport motor.Myosins are a superfamily of actin-based molecular motors comprising ∼35 distinct classes (1, 2), responsible for a wide variety of cellular functions. They convert the chemical energy derived from ATP hydrolysis into directed motion along actin and are responsible for a wide variety of intracellular motilities and for muscle contraction. Mammalian myosin-10 was originally found to localize to the tips of filopodia (3, 4) and is known to be important for filopodial formation and extension (4–6), phagocytosis (7), cell migration (8), and mitotic spindle length maintenance and anchoring (9). It is an ∼240-kDa protein consisting of an N-terminal, consensus motor region (which binds actin, hydrolyses ATP, and produces force and movement), a light chain binding domain (LCBD) with three IQ motifs (10), and a stable single alpha-helix (SAH) followed by a tail region containing, an antiparallel coiled-coil (APCC) region, a proline, glutamate, serine, and threonine rich (PEST) domain, three pleckstrin homology (PH) domains, a myosin tail homology 4 (MyTH4) domain, and a band 4.1, ezrin, radixin, moesin (FERM) domain (Fig. 1A). Recently, Umeki et al. (11) expressed native full-length myosin-10 in an Sf9/baculovirus expression system, and electron micrographs of this protein indicate that most molecules are single-headed monomers and that the tail region can fold back onto the head, switching off the ATPase and actin-binding activity. ATPase activity was found to be regulated both by Ca²⁺ and phospholipids. Knight et al. (12) also found that a heavy meromyosin (HMM)-like fragment terminating at residue 953, which would include the SAH domain and the APCC region, was mostly monomeric, with only about 10% of the molecules clearly dimerized.Open in a separate windowFig. 1.Myosin-10 heavy meromyosin-like (M10HMM) construct design. (A) Illustration of the full-length myosin-10 (Upper) and the M10HMM construct (Lower) structural organization. (B) Image of a typical SDS-polyacrylamide gel electrophoretogram (4–20%) of M10HMM after elution from an anti-FLAG resin. Approximately 0.4–0.8 mg of protein was purified per preparation. (C) Collage of M10HMM dimers negatively stained with 1% uranyl acetate (Each window = 92 × 92 nm). From electron micrographs it was determined that 87.2% of heavy chains were dimerized (N_observations = 1,089). (Scale bar for all panels, 20 nm.) (D) An example M10HMM image from a panel from C with identification of regions, including motor domain (MD)/3 IQ motifs, the proximal coiled-coil region, and regions that most likely represents the stable SAH domains. Window size = 92 × 92 nm. (E) Images of M10HMM bound to actin in the presence of 1 μM ATP. Both single-headed binding and double-headed binding are seen. Examples can be seen in which the two heads of M10HMM span the actin pseudorepeat. (Scale bar for all panels, 20 nm.) (F) Histogram showing the contour length of M10HMM dimers (black bins), measured from the tip of one motor domain to the tip of the other [black line = Gaussian fit; peak = 51 ± 5.5 nm (SD); N_obs = 795; R² = 0.99]. As a comparison, the contour length of myosin-5a-HMM (M5aHMM) (red bins) are shown [red line = Gaussian fit; peak = 60 ± 3.8 nm (SD); N_obs= 221; R² = 0.96]. (G) Histogram showing the distribution of head-head angular variation of M10HMM (black bins; N_obs = 782) and M5aHMM (red bins; N_obs = 230) dimers. Gaussian fits showed peaks at 115 ± 2.7° (SEM) for M10HMM (black line; R² = 0.89) and 110 ± 3.1° (SEM) (red line; R² = 0.86) for M5aHMM.Solution kinetic studies of truncated recombinant myosin-10, which resembles the soluble subfragment-1 produced by proteolytic cleavage of muscle myosin (i.e., S1-like), have shown that release of product, ADP, is slow relative to the overall ATPase cycle time, making it a high duty cycle ratio motor, spending a significant fraction of its ATPase cycle tightly bound to actin (13, 14). Consistent with these kinetic studies, single molecule fluorescence imaging studies using artificially dimerized GFP-tagged myosin-10 (15–19) constructs that resemble HMM (i.e., HMM-like) shows that it moves processively along filamentous actin (F-actin), presumably using its two heads to take one step after another. Using surface-immobilized F-actin, Nagy et al. (15) showed that myosin-10 is more processive (i.e., walks further before detaching) on fascin-bundled F-actins compared with single F-actins, implying that the two heads might move along by binding alternately on neighboring F-actins, described as a “waddling” movement. Sun et al. (18) found that processive motion along a single F-actin is increased if it is held suspended in midsolution, allowing the myosin molecule to take a left-handed helical path around the filament as it moves along, representing a “whirling” movement around the F-actin. Each ATP-driven step taken by such truncated chimeras of myosin-10 has been reported to be in the range of 18–34 nm long (16–19). Furthermore, live cell fluorescence imaging studies (20, 21) using a full-length GFP-tagged myosin-10 construct showed that single molecules move in a highly directed manner within the filopodia. The fact that myosin-10 moves processively along actin filaments is perhaps surprising because, although the motor has the correct kinetic properties, the lever arm region, initially thought of to be composed of only the three IQ binding motifs (3), might be expected to produce a power stroke that falls short of the F-actin helix pseudorepeat of 36 nm (22). However, recent structural, biochemical, and molecular modeling studies (12, 23, 24) indicate that myosin-10 has a 70-amino acid residue-long (amino acids 813–882) SAH domain immediately following its three IQ motifs, which might serve to extend the lever arm. A structural study (25) using a peptide corresponding to amino acid residues 883–934 showed this later region can form a relatively low affinity (K_d = 0.6 μM) APCC. The authors postulated that the APCC region might mediate dimerization of myosin-10 heavy chains within the filopodia, where the local concentration is highest. Rotary-shadowed electron micrographs of a truncated myosin-10 genetic construct that encoded the first 953 amino acids (12) showed a minority of dimeric (two-headed) molecules (∼10%), consistent with the notion that myosin-10 has a weak propensity to dimerize at low protein concentration. Although a picture is emerging about how myosin-10 might be regulated and how it may switch between monomeric and dimeric states (26), little is known about its mechanical properties or how it generates force and movement. To study the mechano-chemistry of dimeric myosin-10, we generated a recombinant protein with a leucine zipper motif appended after amino acid residue 936 to augment dimerization. This protein construct gave rise to a soluble (HMM-like) protein that formed a majority of monodispersed, two-headed (dimeric) molecules at submicromolar protein concentrations. This protein allowed us to perform bulk biochemical and single molecule mechanical experiments to probe the kinetic and mechanical properties of the two-headed myosin-10. Using optical tweezers, we found that the myosin-10 construct produced a power stroke of ∼17 nm, which occurred in two distinct phases (15 + 2-nm displacements). The actin-attached lifetimes measured at different ATP concentrations showed Michaelis–Menten rate dependence with a limiting detachment rate of ∼13 s⁻¹, similar to both the ATP turnover and ADP release rates measured in bulk biochemical studies. Together, these data imply that both mechanical and biochemical cycles are most likely limited by the rate of ADP release. Finally, under conditions of low load, myosin-10 moved processively along a single actin filament, taking ∼36-nm steps in the single molecule optical tweezers assays.     

Self-organization of actin filament orientation in the dendritic-nucleation/array-treadmilling model

The dendritic-nucleation/array-treadmilling model provides a conceptual framework for the generation of the actin network driving motile cells. We have incorporated it into a 2D, stochastic computer model to study lamellipodia via the self-organization of filament orientation patterns. Essential dendritic-nucleation submodels were incorporated, including discretized actin monomer diffusion, Monte-Carlo filament kinetics, and flexible filament and plasma membrane mechanics. Model parameters were estimated from the literature and simulation, providing values for the extent of the leading edge-branching/capping-protective zone (5.4 nm) and the autocatalytic branch rate (0.43/sec). For a given set of parameters, the system evolved to a steady-state filament count and velocity, at which total branching and capping rates were equal only for specific orientations; net capping eliminated others. The standard parameter set evoked a sharp preference for the +/-35 degree filaments seen in lamellipodial electron micrographs, requiring approximately 12 generations of successive branching to adapt to a 15 degree change in protrusion direction. This pattern was robust with respect to membrane surface and bending energies and to actin concentrations but required protection from capping at the leading edge and branching angles >60 degrees. A +70/0/-70 degree pattern was formed with flexible filaments approximately 100 nm or longer and with velocities < approximately 20% of free polymerization rates.     

Torsional rigidity of single actin filaments and actin–actin bond breaking force under torsion measured directly by in vitro micromanipulation

Knowledge of the elastic properties of actin filaments is crucial for considering its role in muscle contraction, cellular motile events, and formation of cell shape. The stiffness of actin filaments in the directions of stretching and bending has been determined. In this study, we have directly determined the torsional rigidity and breaking force of single actin filaments by measuring the rotational Brownian motion and tensile strength using optical tweezers and microneedles, respectively. Rotational angular fluctuations of filaments supplied the torsional rigidity as (8.0 ± 1.2) × 10⁻²⁶ Nm². This value is similar to that deduced from the longitudinal rigidity, assuming the actin filament to be a homogeneous rod. The breaking force of the actin–actin bond was measured while twisting a filament through various angles using microneedles. The breaking force decreased greatly under twist, e.g., from 600–320 pN when filaments were turned through 90°, independent of the rotational direction. Our results indicate that an actin filament exhibits comparable flexibility in the rotational and longitudinal directions, but breaks more easily under torsional load.     

Mechanism of the actomyosin ATPase: effect of actin on the ATP hydrolysis step.

The Lymn-Taylor model for the actomyosin ATPase suggests that during each cycle of ATP hydrolysis the complex of myosin subfragment 1 (S-1) with actin must dissociate into S-1.ATP plus actin before ATP hydrolysis can occur. In the present study we tested whether such a mandatory detachment step occurs by measuring the effect of actin on the rate and magnitude of the ATP hydrolysis step (initial Pi burst) and on the steady-state ATPase rate. We find that the rate of the initial Pi burst markedly increases at high actin concentration although the Lymn-Taylor model predicts the rate should remain nearly constant or decrease. In addition, at high actin concentration, the magnitude of the initial Pi burst is much larger than is predicted by the Lymn-Taylor model. Finally, at 360 microM actin, at which more than 90% of the S-1.ATP is bound to actin, there is no inhibition of the steady-state ATPase activity although the Lymn-Taylor model predicts that 70% inhibition should occur. We conclude that the acto-S-1 complex is not dissociated by ATP during each cycle of ATP hydrolysis; in fact, the rate of the initial Pi burst appears to be even faster when S-1.ATP is bound to actin than when it is dissociated.     

Unique single molecule binding of cardiac myosin binding protein-C to actin and phosphorylation-dependent inhibition of actomyosin motility requires 17 amino acids of the motif domain

Cardiac myosin binding protein-C (cMyBP-C) has 11 immunoglobulin or fibronectin-like domains, C0 through C10, which bind sarcomeric proteins, including titin, myosin and actin. Using bacterial expressed mouse N-terminal fragments (C0 through C3) in an in vitro motility assay of myosin-generated actin movement and the laser trap assay to assess single molecule actin-binding capacity, we determined that the first N-terminal 17 amino acids of the cMyBP-C motif (the linker between C1 and C2) contain a strong, stereospecific actin-binding site that depends on positive charge due to a cluster of arginines. Phosphorylation of 4 serines within the motif decreases the fragments' actin-binding capacity and actomyosin inhibition. Using the laser trap assay, we observed individual cMyBP-C fragments transiently binding to a single actin filament with both short (~20 ms) and long (~300 ms) attached lifetimes, similar to that of a known actin-binding protein, α-actinin. These experiments suggest that cMyBP-C N-terminal domains containing the cMyBP-C motif tether actin filaments and provide one mechanism by which cMyBP-C modulates actomyosin motion generation, i.e. by imposing an effective viscous load within the sarcomere.     

Molecular architecture of the Spire-actin nucleus and its implication for actin filament assembly

The Spire protein is a multifunctional regulator of actin assembly. We studied the structures and properties of Spire-actin complexes by X-ray scattering, X-ray crystallography, total internal reflection fluorescence microscopy, and actin polymerization assays. We show that Spire-actin complexes in solution assume a unique, longitudinal-like shape, in which Wiskott-Aldrich syndrome protein homology 2 domains (WH2), in an extended configuration, line up actins along the long axis of the core of the Spire-actin particle. In the complex, the kinase noncatalytic C-lobe domain is positioned at the side of the first N-terminal Spire-actin module. In addition, we find that preformed, isolated Spire-actin complexes are very efficient nucleators of polymerization and afterward dissociate from the growing filament. However, under certain conditions, all Spire constructs--even a single WH2 repeat--sequester actin and disrupt existing filaments. This molecular and structural mechanism of actin polymerization by Spire should apply to other actin-binding proteins that contain WH2 domains in tandem.     

Use of high throughput sequencing to observe genome dynamics at a single cell level

With the development of high throughput sequencing technology, it becomes possible to directly analyze mutation distribution in a genome-wide fashion, dissociating mutation rate measurements from the traditional underlying assumptions. Here, we sequenced several genomes of Escherichia coli from colonies obtained after chemical mutagenesis and observed a strikingly nonrandom distribution of the induced mutations. These include long stretches of exclusively G to A or C to T transitions along the genome and orders of magnitude intra- and intergenomic differences in mutation density. Whereas most of these observations can be explained by the known features of enzymatic processes, the others could reflect stochasticity in the molecular processes at the single-cell level. Our results demonstrate how analysis of the molecular records left in the genomes of the descendants of an individual mutagenized cell allows for genome-scale observations of fixation and segregation of mutations, as well as recombination events, in the single genome of their progenitor.     

Testosterone modulates growth hormone secretion at the hypothalamic but not at the hypophyseal level in the adult male rhesus monkey

We investigated a possible modulation of growth hormone (GH) secretion by testosterone by measuring the growth hormone releasing hormone (GHRH)-stimulated and N-methyl-d,l-aspartic acid (NMA)-induced GH secretion in adult rhesus monkeys. Intact, orchidectomized and testosterone-substituted (testosterone enanthate 125 mg/week, i.m. for 5 weeks) orchidectomized monkeys (n=5) were used in the study. GHRH (25 microg/kg body weight) or NMA (15 mg/kg body weight) was infused through a Teflon cannula implanted in the saphenous vein. Sequential blood samples were collected 30-60 min before and 60 min after the injection of the neurohormone or the drug at 10-20-min intervals. All bleedings were carried out under ketamine hydrochloride anaesthesia (initial dose 5 mg/kg body weight i.m., followed by 2.5 mg/kg at 30-min intervals). The plasma concentrations of GH, testosterone and oestradiol (E(2)) were determined by using specific assay systems. Administration of GHRH elicited a significant increase in GH secretion in all three groups of animals. There was no significant difference in the responsiveness of pituitary somatotrophs to exogenous GHRH challenges between intact and orchidectomized monkeys and testosterone replacement in orchidectomized animals did not significantly alter the GHRH-induced GH response. The responsiveness of hypothalamic GHRH neurones apparently did undergo a qualitative change after orchidectomy, as GH response to NMA was less in orchidectomized animals than in intact monkeys. The responsiveness of GHRH neurones to exogenous NMA was restored and even potentiated when orchidectomized monkeys were treated with testosterone. Taken together, these findings suggest that testosterone does not affect the sensitivity of the pituitary somatotrophs to GHRH but stimulates the secretion of GH by modulation of the NMDA drive to GHRH neurones.     

Binding of a single divalent cation directly correlates with the blue-to-purple transition in bacteriorhodopsin

We have characterized a unique divalent cation binding site on bacteriorhodopsin which controls the blue-to-purple transition in the purple membrane of Halobacterium halobium. To identify this site we first showed the correlation between the binding of one Ca2+ per bacteriorhodopsin and the amount of blue membrane converted to purple membrane. When the free Ca2+ was reduced below 1 microM, and the pH was set below 5.0 with 0.5 mM citrate, only binding to this high-affinity site was observed, and we could separate its effect from the effect of other divalent cations binding to the membrane under other conditions. Second, the titration of purple membrane showed that protons are taken up in two distinct steps, about 13 with a pKa of 4-5 and an additional 2 protons with a pKa of 2.75, in 5 mM MgSO4. The latter is identical to the pKa for the purple-to-blue transition in 5 mM MgSO4. Taken together, these observations strongly suggest a direct role for cations in the regulation of the bacteriorhodopsin color under normal conditions. We have also found that the intrinsic pKa for the purple-to-blue transition is about 2.05, suggesting this is the pKa of the group or groups that, when protonated, lead to the blue membrane. Previously published data can now be interpreted to suggest that the cation regulates an active site near the retinal chromophore. A binding site for the divalent cation that includes Asp-212 and interactions with the protonated Schiff base, Asp-85, Tyr-57, Tyr-185, and Arg-82 is proposed.     

Phagocytic dendritic cells from myelomas activate tumor-specific T cells at a single cell level

Antigen-presenting cells (APCs) from subcutaneous mouse MOPC315 plasmacytoma phagocytosed immunoglobulin G-coated magnetic beads, enabling efficient isolation within 2 hours by magnetic separation (APC-MB). Cell morphology was heterogeneous, with some of the cells having dendrites. The surface phenotype of purified tumor APCs-MB was CD11b(+), CD11c(+), CD40(+), CD80(+), CD86(+), and MHC class II(+). Tumor APCs-MB expressed messenger RNA for fractalkine and ABCD-1 chemokines, and for CC-type chemokine receptors CCR5 and CCR7, indicating the presence of mature dendritic cells (DCs). Visualized at a single cell level within 4 hours after disruption of the tumor, APCs-MB induced rapid Ca(++) mobilization in MHC class II-restricted tumor idiotype (Id)-specific cloned CD4(+) T cells. In long-term assays, tumor APCs-MB induced proliferation of naive T cells from Id-specific T-cell receptor transgenic mice. The results suggest that tumor APCs-MB represent a heterogeneous cell population that includes myeloid-derived DCs of various stages of maturation. A considerable fraction (> or = 15%) of DCs is spontaneously primed with tumor-specific antigen.     

Dehydroepiandrosterone (DHEA) modulates GHRH, somatostatin and angiotensin II action at the pituitary level

In view of the present controversy related to the potential beneficial effects of clinical dehydroepiandrosterone (DHEA) treatments, and considering our own previous results that reveal an influence of this steroid in pituitary hyperplasia development in vivo in rats, we decided to evaluate the role of DHEA in prolactin and GH secretion, as well as in second messengers involved, in cultured rat anterior pituitary cells. DHEA (1 x 10(-5) to 1 x 10(-7) M) did not modify basal GH or prolactin release, and a prolactin inhibitory effect was observed only for androstenediol, a metabolite of DHEA. DHEA partially prevented dopamine (1 x 10(-6) M)-induced prolactin inhibition and facilitated the prolactin-releasing effect of 10(-8) M Ang II, without modifying the resulting Ca2+(i) mobilization. Furthermore, DHEA potentiated the GH release and cAMP production induced by 1 x 10(-8) M GHRH. Finally, DHEA partially reversed the inhibitory effect of 1 x 10(-8) M somatostatin on GH, but not prolactin, release. We conclude that DHEA in vitro, directly or indirectly through conversion into metabolites, is able to modulate the hormonal response of the pituitary to hypothalamic regulators. It can enhance pituitary prolactin release and induce GH secretion. These effects could help explain some of the side effects observed in prolonged DHEA treatments in vivo and should be taken into account when considering its use in human clinical trials.     

In men, peripheral estradiol levels directly reflect the action of estrogens at the hypothalamo-pituitary level to inhibit gonadotropin secretion

CONTEXT: Estradiol inhibits gonadotropin release in men by an action at the hypothalamus and pituitary. Because of the tissue-specific regulation of aromatase, peripheral estradiol levels may not reflect brain estradiol concentrations. OBJECTIVE: We evaluated whether local aromatization of testosterone in the hypothalamus or pituitary is important for gonadotropin release and to what extent circulating estrogens affect gonadotropin levels and peripheral testosterone levels. DESIGN, SUBJECTS, AND INTERVENTIONS: We suppressed aromatase activity in 10 young healthy men with letrozole 2.5 mg once daily, restored plasma estradiol levels with estradiol patches (100 microg/d for the first week, 50 microg/d the second week, 25 microg/d the third week, and no estradiol patch the fourth week) and measured plasma testosterone, estradiol, LH, FSH, and SHBG levels. RESULTS: The mean estradiol and testosterone levels during the study ranged between 68.6 +/- 38.3 and 12.6 +/- 7.21 pg/ml for estradiol and 179 +/- 91 and 955 +/- 292 ng/dl (mean +/- sd) for testosterone. Levels of testosterone, LH, and FSH were inversely related to peripheral estradiol levels. During letrozole use, the mean plasma estradiol level needed to restore testosterone, LH, and FSH levels to baseline levels was not significantly different from the baseline mean estradiol level. CONCLUSIONS: Local aromatization of testosterone in the hypothalamo-pituitary compartment is not a prerequisite for expression of the inhibitory action of estrogens on gonadotropin secretion in men. Peripheral estradiol levels directly reflect the inhibitory tone exerted by estrogens on gonadotropin release and are a major determinant of peripheral testosterone, LH, and FSH levels.     

A method of analyzing procoagulant activity in monocytes at single cell level

BACKGROUND AND OBJECTIVES: Procoagulant activity (PCA) of monocytes is known to play a pivotal role in a variety of physiologic and pathophysiologic processes, such as disseminated intravascular coagulation, atherosclerosis, arterial and venous thromboembolism, cancer-related hypercoagulability and immunopathologies. Until now, PCA has been studied by clotting assays of a whole cell population or at single cell level by analyzing tissue factor antigen, the protein that initiates PCA but does not always correlate with it. Here, we describe a new simple flow cytometric method that allows the PCA of monocytes to be studied at a single cell level by quantifying the fibrin formed around the cells in suspension. DESIGN AND METHODS: Purified fibrinogen was tagged with FITC and added to a recalcified developer plasma containing suitable amounts of heparin in order to inhibit the expansion of clotting, thus limiting the formation of fibrin to the surface of cells with PCA. With appropiate amounts of heparin, in 10 min, large sea urchin-like cells with fibrin needles around some monocytes were formed and, after fixation, cytofluorimetrically analyzed. RESULTS: Blood mononuclear cells isolated and immediately analyzed showed less than 0.1% sea urchin cells. Adherence alone, lipopolysaccharides or ionomycin stimulated expression of PCA in a dose- and time-dependent relationship: after 30 min, 1-3% of the MNC showed PCA, and after 20 h this reached 5-10%. Density separation of monocytes showed that different stimulators act on different maturation stages. Subjects with diabetes express more monocytes with PCA than normal subjects after 30 min stimulation. INTERPRETATION AND CONCLUSIONS: This method allows PCA analysis of monocytes at single cell level and requires only a low number of cells. The signal produced by the fluorescent fibrin is strong and easily analyzed by flow cytometry. The method is suitable for analyzing blood from patients with different pathologies and many conditions under different stimuli.     

Oligosaccharyltransferase directly binds to ribosome at a location near the translocon-binding site

Oligosaccharyltransferase (OT) transfers high mannose-type glycans to the nascent polypeptides that are translated by the membrane-bound ribosome and translocated into the lumen of the endoplasmic reticulum through the Sec61 translocon complex. In this article, we show that purified ribosomes and OT can form a binary complex with a stoichiometry of ≈1 to 1 in the presence of detergent. We present evidence that OT may bind to the large ribosomal subunit near the site where nascent polypeptides exit. We further show that OT and the Sec61 complex can simultaneously bind to ribosomes in vitro. Based on existing data and our findings, we propose that cotranslational translocation and N-glycosylation of nascent polypeptides are mediated by a ternary supramolecular complex consisting of OT, the Sec61 complex, and ribosomes.     

Morphine directly modulates the release of stimulated corticotrophin-releasing factor-41 from rat hypothalamus in vitro

The actions of opioids and opiates on the hypothalamo-pituitary-adrenal axis are currently controversial. In the rat, morphine is reported to both stimulate and inhibit ACTH and corticosterone secretion, but the precise sites and mechanisms of these effects have remained unclear. To analyze further the hypothalamic actions of morphine, we have investigated its effect on hypothalamic fragments in vitro and measured the major CRF, CRF-41, by a specific RIA. The acute effects of morphine on both basal and stimulated ACTH release from dispersed pituitary cells were also investigated. Morphine (10(-8)-10(-6) M) did not significantly alter the basal secretion of CRF-41. However, similar concentrations of morphine inhibited CRF-41 release stimulated by norepinephrine in a dose-dependent manner. Similarly, morphine (10(-6) M) inhibited acetylcholine (10(-9) M)- and serotonin (10(-7) M)-stimulated CRF-41 release. The stimulatory effect on CRF-41 release induced by veratridine (10(-6) M) was inhibited by approximately 50% in the presence of morphine. KCl (28 nM)-mediated CRF-41 release was also significantly inhibited by morphine. Naloxone (10(-7)-10(-5) M) had no significant effect on either basal or norepinephrine-induced CRF-41 release, but reversed the inhibitory effect of morphine on norepinephrine-induced CRF-41 secretion in a dose-dependent manner. Morphine (10(-6)-10(-5) M) had no effect on either basal or CRF-41-stimulated ACTH release from dispersed pituitary cells. These data suggest that the predominant effect of morphine on hypothalamic CRF-41 release in vitro is suppression of the release induced by a variety of putative neurotransmitters and depolarizing agents. This inhibitory effect is reversed by naloxone, suggesting that it is mediated by opiate receptors, presumably situated directly on CRF-41 neurons.