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The attention of most immunologists is held by the elaborate nonspecific and antigen-specific mechanisms of reactive immunity. There is, however, a more fundamental level of immunity to infection that is shared by all forms of life: constitutional resistance to infection. In this article, the nature and importance of constitutional immunity is illustrated using examples from population, cell and molecular biology.
A trypsin-like endopeptidase which cleaves the synthetic substrate Dansyl-Phe-Leu-Arg-Arg-Ala-Ser-Leu-Gly-COOH (Dansyl-Phe-Kemptide) primarily at the Arg4-Ala5 bond has been partially purified from bovine adrenal chromaffin granules, brain and liver. The enzyme appears to have a relatively homogeneous tissue distribution, although highest levels were found in brain regions such as the hippocampus and corpus striatum. Sucrose density gradient fractionation established that enzyme activity assayed at pH 8.5 is not associated with lysosomes. Purified enzyme displays a dimeric structure with subunit molecular weights of 40 kDa and 42 kDa and a native molecular weight of 85,000 Da. The endopeptidase has a neutral pH optimum, is sensitive to divalent cations and thiol reagents, and can cleave on either the amino or carboxyl side of some but not all internal basic amino acids.
A retro-convection enhanced delivery (R-CED) method has been developed to improve the entry of intravenously administered therapeutics within solid brain tumors. R-CED uses an osmotic gradient to withdraw brain interstitial fluid (ISF) in a controlled manner via an implanted microdialysis catheter. Withdrawal of ISF increases the local tissue specific gravity in normal brain and increases twofold the extravasation of intravenous Evans blue (EB) albumin in normal brain and in an orthotopic 9L tumor. R-CED also increases the extravasation of 70 nm fluorescent liposomes fivefold in the 9L tumor. Thus the transmembrane osmotic gradient induces movement of substances in the blood into the tissue parenchyma. Following probe removal, the magnitude of the R-CED effect on EB-albumin extravasation decreases to control values within 1.5 h in normal brain; however, the effect persists beyond 6 h in the tumor. There was no evidence of histologic damage to the neurons at either 6 h or 2 weeks after R-CED. These studies establish the feasibility of applying R-CED to increase the distribution of systemically administered drugs in both the normal tissue-tumor margin as well as in the central tumor core, holding forth the possibility of improved antitumor drug efficacy.
Molecular dynamics simulations were performed in order to study the transport and adsorption of a charged macromolecule (desmopressin) onto a charged solid surface in an electrolytic solution. The strong Coulombic interaction from the charged solid surface represents the major force for accelerating, orienting, entrapping in the electrical double layer, and adsorbing the macromolecule onto the charged solid surface. The macromolecule is flattened as it approaches the charged surface, giving rise to a stronger surface exclusion effect that shields surface sites. When adsorbed, the macromolecule is restrained by a surface interaction more than one hundred times stronger than the thermal energy, of which 99.8% results from the strong dominant Coulombic interaction, and trapped by a hydration layer adjacent to the surface. This leads to zero lateral displacement of the adsorbed macromolecule and indicates that surface diffusion is a physically implausible mechanism in similar systems. Explicit solvent is required for realistic representation of the macromolecular structure and the surface interaction energy. The adsorbed macromolecule also decreased the electrostatic potential gradient perpendicular to the charged solid surface and introduced additional electrostatic potential gradients laterally. The results obtained from the molecular dynamics simulations confirm the importance of electrophoretic migration and support the physical mechanisms used in a macroscopic continuum model that predicts an overshoot in the concentration of a charged macromolecule in the adsorbed phase under certain conditions of pH and ionic strength.
A series of amphiphilic macromolecules, amphiphilic scorpion-like macromolecules (AScMs) and amphiphilic star-like macromolecules (ASMs), were evaluated as potential drug delivery systems for intravenous administration. AScMs aggregate to form polymeric micelles; whereas the ASMs have a covalently bound core structure and behave as unimolecular micelles. Four structurally different AScMs and two ASMs were selected for further evaluation focusing on micellar stability and biocompatibility. AScMs were determined to have extremely low cmc values, indicating excellent thermodynamic stability compared to other polymeric micelle systems. Particle sizes of the AScM polymeric micelles and ASM unimolecular micelles were between 10 and 20 nm, and remained constant for up to 3 weeks storages as aqueous solutions at room temperature (approximately 23 degrees C) and 37 degrees C. The dissociation kinetics of the AScM polymeric micelles were slowed relative to small molecule surfactant micelles, again indicating enhanced kinetic stability. With respect to hemolytic activity, AScMs with longer acyl chains were hemolytic; whereas the ASMs had minimal hemolytic activity due to the covalently bound structure. Both ASM unimolecular micelles and AScM polymeric micelles have excellent micellar stability, but the ASMs are more suitable as injectable drug delivery systems due to their low hemolytic activity.
Arp2/3 is an actin binding complex that is enriched in the peripheral lamellipodia of fibroblasts, where it forms a network of short, branched actin filaments, generating the protrusive force that extends lamellipodia and drives fibroblast motility. Although it has been assumed that Arp2/3 would play a similar role in growth cones, our studies indicate that Arp2/3 is enriched in the central, not the peripheral, region of growth cones and that the growth cone periphery contains few branched actin filaments. Arp2/3 inhibition in fibroblasts severely disrupts actin organization and membrane protrusion. In contrast, Arp2/3 inhibition in growth cones minimally affects actin organization and does not inhibit lamellipodia protrusion or de novo filopodia formation. Surprisingly, Arp2/3 inhibition significantly enhances axon elongation and causes defects in growth cone guidance. These results indicate that Arp2/3 is a negative regulator of growth cone translocation.
Uptake of fluorescein isothiocynate-dextran (FITC-dextran) by Chinese hamster ovary cells was studied after exposure to ultrasonic standing wave (USW) in presence of Optison, an ultrasound contrast agent. Confluent Chinese hamster ovary cells were harvested and suspended in phosphate-buffered saline + 0.1% bovine serum albumin containing FITC-dextran (10, 40, and 500 kDa) at 10 microM final concentration. The suspension was seeded with contrast agent (75 microL/mL) and exposed to a 1.5 MHz USW system at acoustic pressures ranging from 0.98 to 4.2 MPa. Macromolecular uptake was assessed by fluorescent microscopy and quantified by flow cytometry 10 min after exposure. FITC-dextran positive cells, as assessed by flow cytometry, were 1 +/- 0.05% and 2.58 +/- 0.27% for acoustic pressures of 1.96 and 4.2 MPa, respectively (p = 0.006). Fluorescent microscopy indicated a degree of macromolecular loading at 0.98 MPa with 46% of peripherally FITC-dextran- and/or propidium iodide-stained cells coincident with the appearance of significant frequency (f0/2 and 2 f0) emission signals. At higher pressures, high macromolecular loading with 6% peripherally stained cells at 1.96 MPa was associated with lower order emission signals and white noise. The study conclusively demonstrates macromolecular loading in an USW, a significantly higher macromolecular loading at higher pressures and indicates potential of emission signals for a feedback loop to control the acoustic power outputs and fine-tune the biologic effects associated with sonoporation.
We have examined the influence of free metal ions on the affinity of structurally-defined proteins and peptides for model surface-immobilized metal ions. The model proteins chosen differed widely in both the type and quantity of surface-accessible electron donor groups. Metal ion affinity chromatography and equilibrium binding analyses demonstrated that the presence of excess free Cu(II) ions did not measurably affect either the affinity or the binding capacity of lysozyme for immobilized iminodiacetate-Cu(II). Similarly, the presence of excess free Cu(II) ions did not detectably affect the chromatographic behavior or measured affinity of either copper-saturated lactoferrin or iron-saturated lactoferrin for the immobilized Cu(II) ions. Its binding capacity however, was diminished. The affinities of small peptides for immobilized Cu(II) ions was found to be related to their number of His residues. Peptides with 0, 1, 2 and 3 His residues were resolved by high-performance immobilized Cu(II) affinity chromatography in both the presence and absence of added Cu(II) ions. In the presence of excess free Cu(II) ions, however, retention (affinities) of these peptides by immobilized Cu(II) ions was increased in relation to their number of His residues. These data demonstrate that protein surface binding sites for free and immobilized metal ions are functionally distinct. The presence of free and/or protein surface-bound metal ions does not preclude interaction with the same immobilized metal ions. Stationary phase immobilized metal ions can be a useful model system through which we can better understand the influence of macromolecular surface-immobilized metal ions on macromolecular recognition events. The significance of these findings are also important to the design of other site-specific and domain-specific affinity reagents involving metal ions.
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Dendrimers have emerged as promising multifunctional nanomaterials for drug delivery due to their well-defined size and tailorability. We compare two schemes to obtain methylprednisolone (MP)-polyamidoamine dendrimer (PAMAM-G4-OH) conjugate. Glutaric acid (GA) was used as a spacer to facilitate the conjugation. In scheme A, PAMAM-G4-OH was first coupled to GA and then further conjugated with MP to obtain PAMAM-G4-GA-MP conjugates. This scheme yields a lower conjugation ratio of MP, presumably because of lower reactivity and steric hindrance for the steroid at the crowded dendrimer periphery. In scheme B, this steric hindrance was overcome by first preparing the MP-GA conjugate, which was then coupled to the PAMAM-G4-OH dendrimer. The (1)H NMR spectrum of the conjugate from scheme B indicates a conjugation of 12 molecules of MP with the dendrimer, corresponding to a payload of 32 wt %. In addition, conjugates were further fluorescent-labeled with fluoroisothiocynate (FITC) to evaluate the dynamics of cellular entry. Flow cytometry and UV/visible spectroscopic analysis showed that the conjugate is rapidly taken up inside the cell. Fluorescence and confocal microscopy images on A549 human lung epithelial carcinoma cells treated with conjugates show that the conjugate is mostly localized in cytosol. MP-GA-dendrimer conjugate showed comparable pharmacological activity to free MP, as measured by inhibition of prostaglandin secretion. These conjugates can potentially be further conjugated with a targeting moiety to deliver the drugs to specific cells in vivo.
A serious limitation that precludes utilization of single-tailed, pH-sensitive detergents for the cytosolic delivery of macromolecules is their low limit of incorporation in stable liposomal formulations. To address this issue, we have prepared two Gemini surfactants or 'bis-detergents' by cross-linking the headgroups of single-tailed, tertiary amine detergents through oxyethylene (BD1) or acid-labile acetal (BD2) moieties. The membrane-bound pK(a) of the twin tertiary amine headgroups was determined to be 6.37 +/- 0.36 using a fluorescence-based assay. As evidenced by thin-layer chromatography, BD2was hydrolyzed under acidic conditions (pH 5.0) with an approximate half-life of 3 h at 37 degrees C, while BD1 remained stable. Low pH-induced collapse of liposomes containing acid-labile BD2 into micelles was more facile than that of BD1. With BD1, the process appeared to be reversible in that aggregation of micelles was observed at basic pH. The irreversible lamellar-to-micellar transition observed with BD2-containing liposomes can possibly be attributed to acid-catalyzed hydrolysis of the acetal cross-linker, which generates two detergent monomers within the bilayer. Liposomes composed of 75 mol % bis-detergent and 25 mol % phosphatidylcholine were readily prepared and could entrap macromolecules such as polyanionic dextran of MW 40 kDa with moderate efficiency. The ability of BD2-containing liposomes to promote efficient cytosolic delivery of antisense oligonucleotides was confirmed by (a) their diffuse intracellular distribution seen in fluorescence micrographs, and (b) the up-regulation of luciferase in an antisense functional assay. The low pH-responsive, bis-detergent constructs described herein are suitable for triggered release strategies targeted to acidic intracellular or interstitial environments.
Flower- or cake-shaped particles with uniform particle size ranging from nanometers to micrometers were prepared from the stereo multiblock copoly(rac-lactide)s (smb-PLAs) by precipitating the polymer from its solution in methylene chloride/ethanol via three different methods: slowly lowering the solution temperature, slowly evaporating the solvent, and slowly adding a nonsolvent. Under the same condition, sheet-shaped crystals in 10 mum size but not particles were obtained from the pure PLLA with almost the same molecular weight. Electron diffraction and WAXD data demonstrated that the stereocomplex particles belonged to the monoclinic system. All three methods resulted in particles with identical morphology and almost the same particle size. At a given stereoregularity of 88%, as the molecular weight of the polymer increased from 8700 to 23,200 Da, the crystallinity decreased, the particle morphology changed from flower-shaped to cake-shaped, and the diameter and height of the particles increased from 0.8 and 0.45 to 3.6 microm and 2.0 microm, respectively. The initial concentration of the polymer solution influenced the particle size slightly but affected the morphology markedly. On the basis of the above experimental observations, it was proposed that the smb-PLA particles of flower- or cake-shape were formed in four steps: (1) complexation in solution of the smb-PLA chains; (2) particle nucleation; (3) particle growth in the width direction; and (4) particle growth in the height direction. The curvature of the paired smb-PLA chains and the inner stress governed the particle size, and the interconnection between the neighboring particles determined the layered structure and the package density of the particles formed.
Quantitative tools to assess vascular macromolecular distributions have been limited by low signal-to-noise ratios, reduced spatial resolution, postexperimental motion artifact, and the inability to provide multidimensional drug distribution profiles. Fluorescence microscopy offers the potential of identifying exogenous compounds within intact tissue by reducing autofluorescence, the process by which endogenous compounds emit energy at the same wavelength as fluorescent labels. A new technique combining fluorescence microscopy with digital postprocessing has been developed to address these limitations and is now described in detail. As a demonstration, histologic cross-sections of calf carotid arteries that had been loaded endovascularly with FITC-Dextran (20 kD) ex vivo were imaged at two different locations of the electromagnetic spectrum, one exciting only autofluorescent structures and the other exciting both autofluorescent elements and exogenous fluorescent labels. The former image was used to estimate the autofluorescence in the latter. Subtraction of the estimated autofluorescence resulted in an autofluorescence-corrected image. A standard curve, constructed from arteries that were incubated until equilibrium in different bulk phase concentrations of FITC-Dextran, was used to convert fluorescent intensities to tissue concentrations. This resulted in a concentration map with spatial resolution superior to many of the previous methods used to quantify macromolecular distributions. The transvascular concentration profiles measured by quantitative fluorescence microscopy compared favorably with those generated from the proven en face serial sectioning technique, validating the former. In addition, the fluorescence method demonstrated markedly increased spatial resolution. This new technique may well prove to be a valuable tool for elucidating the mechanisms of macromolecular transport, and for the rational design of drug delivery systems.
Spectrin, the major protein of the subcortical actin network in erythrocytes, contains two non-identical subunits, alpha and beta (refs 1, 2). Spectrin is indirectly associated with the transmembrane anion transporter through the binding of beta-spectrin to an extrinsic protein, ankyrin. In chicken embryo erythroid cells, alpha-spectrin is synthesized in a threefold excess relative to beta-spectrin, although the two subunits are assembled in equimolar amounts. To investigate the regulation of assembly of equimolar amounts of spectrin, an in vitro system from chicken embryo erythroid cells has now been developed where synthesis and assembly of spectrin can be uncoupled and studied separately. Following the in vitro translation of threefold more alpha- than beta-spectrin, 95% of the beta-spectrin and equimolar amounts of alpha-spectrin bind post-translationally to spectrin-depleted rabbit red blood cell membranes, and the excess alpha-spectrin remains unbound. This alpha-spectrin cannot bind spectrin-depleted plasma membranes subsequently added to the lysate. The assembly of alpha-spectrin is, therefore, limited by the availability of beta-spectrin, and both subunits assemble post-translationally.
High-throughput methods for detecting protein interactions, such as mass spectrometry and yeast two-hybrid assays, continue to produce vast amounts of data that may be exploited to infer protein function and regulation. As this article went to press, the pool of all published interaction information on Saccharomyces cerevisiae was 15,143 interactions among 4,825 proteins, and power-law scaling supports an estimate of 20,000 specific protein interactions. To investigate the biases, overlaps, and complementarities among these data, we have carried out an analysis of two high-throughput mass spectrometry (HMS)-based protein interaction data sets from budding yeast, comparing them to each other and to other interaction data sets. Our analysis reveals 198 interactions among 222 proteins common to both data sets, many of which reflect large multiprotein complexes. It also indicates that a "spoke" model that directly pairs bait proteins with associated proteins is roughly threefold more accurate than a "matrix" model that connects all proteins. In addition, we identify a large, previously unsuspected nucleolar complex of 148 proteins, including 39 proteins of unknown function. Our results indicate that existing large-scale protein interaction data sets are nonsaturating and that integrating many different experimental data sets yields a clearer biological view than any single method alone.
The pore-forming subunits of many ion channels exist in the membrane as one component of a regulatory protein complex, which may also contain one or more signaling proteins that contribute to the modulation of channel properties. Here I review this field, with emphasis on several different kinds of neuronal potassium channels for which the evidence for ion channel signaling complexes is most compelling. A key challenge for the future is to determine the roles of such signaling protein complexes in neuronal physiology and behavior.
In the present study, we show the isolation and characterization of the protein haemoporin, which constitutes the second most abundant protein fraction in the haemolymph of the marine gastropod Aplysia californica. Although Aplysia is commonly used to investigate the molecular basis of learning, not much is known about the proteins in its haemolymph, which is in contact with the neurons owing to the open circulatory system of molluscs. In the native state, haemoporin is a macromolecular complex forming a cylinder with a central solvent-filled pore. The native complex most probably is a homopentamer made up from 70 kDa subunits with a molecular mass of 360 kDa and a sedimentation coefficient of 11.7 S. Prediction of the secondary structure by CD spectroscopy revealed that haemoporin contains 36% alpha-helices and 19% beta-strands. An absorption band in the 300-400 nm region indicates that haemoporin probably contains a bound substance. Haemoporin also contains a below average amount of tryptophan as evident from absorption and fluorescence spectra. The specific absorption coefficient at 280 nm (a (280 nm, 1 mg/ml)) varies between 0.42 and 0.59 l x g(-1) x cm(-1) depending on the method. The function of the protein is not yet known, but there are structural parallels between haemoporin and a pore protein reported previously in the haemolymph of another marine gastropod Megathura crenulata. The alanine-rich N-terminal sequence (AAVPEAAAEATAEAAPVSEF) is unique among protein sequences and indicates an alpha-helical structure. Whereas one side of the helix is hydrophobic and faces the interior of the protein, the other side contains a glutamic cluster, which may form the channel of the pore in the quaternary structure. Thus both proteins might belong to a new class of haemolymph proteins present in the haemolymph of marine gastropods.