The larval midgut of the cassava hornworm (Erinnyis ello)

Summary Columnar cells of the larval midgut of the cassava hornworm, Erinnyis ello, display microvilli with vesicles pinching off from their tips (anterior and middle midgut) or with a large number of double membrane spheres budding along their length (posterior midgut). Basal infoldings in columnar cells occur in a parallel array with many openings to the underlying space (posterior midgut) or are less organized with few openings (anterior and middle midgut). Goblet cells have a cavity, which is formed by invagination of the apical membrane and which occupies most of the cell (anterior and middle midgut) or only its upper part (posterior midgut). The infolded apical membrane shows modified microvilli, which sometimes (posterior midgut) or always (anterior and middle midgut) contain mitochondria. The cytoplasmic side of the membrane of the microvilli that contain mitochondria are studded with small particles. The results suggest that the anterior and middle region of the midgut absorbs water, whereas the posterior region secretes it. This results in a countercurrent flux of fluid, which is responsible for the enzyme recovery from undigested food before it is expelled. Intermediary and final digestion of food probably occur in the columnar cells under the action of plasma membrane-bound and glycocalix-associated enzymes.     

Versatile Solid Supports for Oligonucleotide Synthesis that Incorporate Urea Bridge

The universal solid support, USIII, representing a new and improved version of commercial USII, as well as 2 ′-deoxynucleoside and 2 ′-deoxy-2 ′-fluoronucleoside bound supports, incorporating a labile phenoxyacetyl fragment, was synthesized by an aminomethyl polystyrene carbamoylation with corresponding azides in the presence of aqueous triethylammonium bicarbonate. All three solid phases incorporate a stable urea tether, thus bridging the polymer and functional linker. These new matrices proved to be potent solid phases for the synthesis of DNA, RNA, or modified oligonucleotides as well as randomized mixed 2 ′-ribo/2 ′-deoxy-2 ′-fluoro-RNA libraries and/or DNA libraries, randomized with trinucleotides (codons).     

Multi-dimensional Co-separation Analysis Reveals Protein–Protein Interactions Defining Plasma Lipoprotein Subspecies

The distribution of circulating lipoprotein particles affects the risk for cardiovascular disease (CVD) in humans. Lipoproteins are historically defined by their density, with low-density lipoproteins positively and high-density lipoproteins (HDLs) negatively associated with CVD risk in large populations. However, these broad definitions tend to obscure the remarkable heterogeneity within each class. Evidence indicates that each class is composed of physically (size, density, charge) and compositionally (protein and lipid) distinct subclasses exhibiting unique functionalities and differing effects on disease. HDLs in particular contain upward of 85 proteins of widely varying function that are differentially distributed across a broad range of particle diameters. We hypothesized that the plasma lipoproteins, particularly HDL, represent a continuum of phospholipid platforms that facilitate specific protein–protein interactions. To test this idea, we separated normal human plasma using three techniques that exploit different lipoprotein physicochemical properties (gel filtration chromatography, ionic exchange chromatography, and preparative isoelectric focusing). We then tracked the co-separation of 76 lipid-associated proteins via mass spectrometry and applied a summed correlation analysis to identify protein pairs that may co-reside on individual lipoproteins. The analysis produced 2701 pairing scores, with the top hits representing previously known protein–protein interactions as well as numerous unknown pairings. A network analysis revealed clusters of proteins with related functions, particularly lipid transport and complement regulation. The specific co-separation of protein pairs or clusters suggests the existence of stable lipoprotein subspecies that may carry out distinct functions. Further characterization of the composition and function of these subspecies may point to better targeted therapeutics aimed at CVD or other diseases.Lipoproteins are circulating emulsions of protein and lipid that play important roles, both positive and negative, in cardiovascular disease (CVD).¹ Historically defined by their density as separated by ultracentrifugation, the major lipoprotein classes include the neutral lipid ester-rich very low-density and low-density lipoproteins (VLDLs and LDLs, respectively), which function to transport triglyceride and cholesterol from the liver to the peripheral tissues. Significant epidemiological evidence, in vitro studies, animal experiments, and human clinical trials have shown that high-LDL cholesterol is a bona fide causative factor in CVD (1). In contrast, protein- and phospholipid-rich high-density lipoproteins (HDLs) are thought to mediate the reverse transport of cholesterol from the periphery to the liver for catabolism and to perform anti-oxidative and anti-inflammatory functions (reviewed in Refs. 2 and 3). A host of human epidemiology and animal studies indicate that HDLs are atheroprotective (4). However, recent clinical trials of therapeutics that generically raise HDL, at least as measured by its cholesterol levels, have failed to confer the expected CVD protections (5–7).Although these traditional density-centric definitions have been used for nearly 40 years, accumulating evidence indicates that they are not particularly reflective of lipoprotein compositional and functional complexity. With respect to most physical traits (size, charge, lipid content, protein content, etc.), one can demonstrate significant heterogeneity within each density class. This suggests that particle subspecies exist with unique functions and effects on disease. For example, LDL can be resolved into large, buoyant and small, dense forms (8), with subjects carrying more cholesterol in the small, dense LDL exhibiting a greater CVD risk (9). HDL is particularly noted for heterogeneity, as it can be separated into numerous subfractions by density (10), diameter (11), charge (12), and major apolipoprotein content (13). Most strikingly, recent applications of soft-ionization mass spectrometry (MS) have identified upward of 85 HDL proteins with functions that go well beyond the structural apolipoproteins, lipid transport proteins, and lipid-modifying enzymes known from previous biochemical studies (14, 15). Many of these proteins imply functions as diverse as complement regulation, acute phase response, protease inhibition, and innate immunity (16). Individual HDL subspecies can apparently draw from this palette of proteins to produce distinct particles of distinct function. One well-defined HDL subfraction, termed trypanosome lytic factor, contains apolipoprotein apoA-I, haptoglobin-related protein, and apoL-I. Working together, these proteins enter the trypanosome brucei brucei and kill it via lysosomal disruption (17). There are numerous other instances of on-particle protein cooperation in HDL related to CVD (reviewed in Ref. 15). Furthermore, two-dimensional electrophoresis studies by Asztalos and colleagues (18), as well as our own work (11, 19), strongly support the concept that certain apolipoproteins segregate among different HDL particles. These observations present the intriguing possibility that the phospholipids of HDLs act as an organizing platform that facilitates the assembly of specific protein complexes (20). Such subspecies could have important functional implications in the context of CVD protection, inflammation, or even innate immune function. Furthermore, this subspeciation may explain why therapeutics that raise HDL cholesterol levels across the board have not yet shown promise with regard to CVD.To address this hypothesis, we began to think of lipoproteins as a continuum of phospholipid platforms that support the assembly of specific protein complexes analogous to those in cells that perform coordinated biological functions (i.e. ribosomes, centrosomes, etc.). Two common methods for characterizing protein complexes are tandem affinity purification (21) and immunoprecipitation. Both rely on the specific pull-down of a target protein (by either an introduced affinity tag or an antibody) followed by the identification of co-precipitated proteins via MS. Unfortunately, tandem affinity purification strategies are impractical in humans, and we have found that immunoprecipitation experiments with human plasma lipoproteins result in a high false-positive rate due to the low abundance of most of these proteins, particularly those in HDLs. Therefore, we took an alternative approach called co-separation analysis, a method based on the principle that stable protein complexes can be identified by tracking their co-migration as they undergo biochemical separation by multiple orthogonal approaches (22). Native proteins are analyzed in an unbiased manner without affinity tags or antibodies, and purification to homogeneity is not necessary for the identification of putative protein complexes.Most current studies of the lipoprotein proteome utilize samples isolated via density ultracentrifugation because contaminating lipid-unassociated lipoproteins, which can be highly abundant and obscure the identification of targeted lipid-associated proteins, are thus removed prior to the analysis. In previous work, we characterized the use of a calcium silica hydrate (CSH) resin that allowed the specific isolation of phospholipid-associated proteins and their subsequent MS identification without ultracentrifugation (11). This advance enabled the use of a variety of non-density-based separation methods for the study of plasma lipoproteins. Here, we take advantage of this to analyze the proteome of human plasma lipoproteins separated via three separation techniques that exploit different physicochemical properties: (i) gel filtration chromatography (size), (ii) anion exchange chromatography (charge interaction), and (iii) isoelectric focusing. By tracking the co-migration of specific proteins across these separations (Fig. 1), we identified a host of putative protein pairings, including the previously known trypanosome lytic factor HDL fraction, for further biochemical verification and characterization.Open in a separate windowFig. 1.Overview of the multi-dimensional separation co-migration analysis used in this study (see “Experimental Procedures” for details).     

Experimental Human Pneumococcal Carriage Augments IL-17A-dependent T-cell Defence of the Lung

Pneumococcal carriage is both immunising and a pre-requisite for mucosal and systemic disease. Murine models of pneumococcal colonisation show that IL-17A-secreting CD4⁺ T-cells (Th-17 cells) are essential for clearance of pneumococci from the nasopharynx. Pneumococcal-responding IL-17A-secreting CD4⁺ T-cells have not been described in the adult human lung and it is unknown whether they can be elicited by carriage and protect the lung from pneumococcal infection. We investigated the direct effect of experimental human pneumococcal nasal carriage (EHPC) on the frequency and phenotype of cognate CD4⁺ T-cells in broncho-alveolar lavage and blood using multi-parameter flow cytometry. We then examined whether they could augment ex vivo alveolar macrophage killing of pneumococci using an in vitro assay. We showed that human pneumococcal carriage leads to a 17.4-fold (p = 0.007) and 8-fold (p = 0.003) increase in the frequency of cognate IL-17A⁺ CD4⁺ T-cells in BAL and blood, respectively. The phenotype with the largest proportion were TNF⁺/IL-17A⁺ co-producing CD4⁺ memory T-cells (p<0.01); IFNγ⁺ CD4⁺ memory T-cells were not significantly increased following carriage. Pneumococci could stimulate large amounts of IL-17A protein from BAL cells in the absence of carriage but in the presence of cognate CD4⁺ memory T-cells, IL-17A protein levels were increased by a further 50%. Further to this we then show that alveolar macrophages, which express IL-17A receptors A and C, showed enhanced killing of opsonised pneumococci when stimulated with rhIL-17A (p = 0.013). Killing negatively correlated with RC (r = −0.9, p = 0.017) but not RA expression. We conclude that human pneumococcal carriage can increase the proportion of lung IL-17A-secreting CD4⁺ memory T-cells that may enhance innate cellular immunity against pathogenic challenge. These pathways may be utilised to enhance vaccine efficacy to protect the lung against pneumonia.     

Synthesis of Analogues of 3′-Deoxypsicothymidine

Abstract

Preparation of 3′-deoxypsicothymidines bearing a tether group at O1′ is described. Selective protection of the primary hydroxy functions of the starting nucleoside is briefly discussed.     

The effect of nisin on Listeria monocytogenes in culture medium and long-life cottage cheese

M.A.S.S. FERREIRA AND B.M. LUND. 1996. The sensitivity to nisin of 27 strains of Listeria monocytogenes , four of L. innocua and one of L. ivanovii was estimated at pH 6.8 and pH 5.5. Strains of L. monocytogenes showed differences in sensitivity which were not correlated with serotype. Strains of L. innocua were as resistant as the most resistant strains of L. monocytogenes , whereas the strain of L. ivanovii was relatively sensitive. Two of the most resistant strains of L. monocytogenes multiplied in aerated liquid medium adjusted to pH 5.0 with HCl, incubated at 20°C; nisin, 500 IU ml^-1, prevented multiplication and caused death. Following inoculation of a resistant strain into long-life cottage cheese, pH 4.6–4.7, the number of viable L. monocytogenes decreased approximately 10-fold during storage at 20°C for 7 d; addition of nisin, 2000 IU g^-1, to the cottage cheese increased the rate of inactivation to approximately a 1000-fold decrease in 3 d.     

Spontaneous mutagenesis is enhanced in Apex heterozygous mice

Germ line DNA directs the development of the next generation and, as such, is profoundly different from somatic cell DNA. Spermatogenic cells obtained from young adult lacI transgenic mice display a lower spontaneous mutant frequency and greater in vitro base excision repair activity than somatic cells and tissues obtained from the same mice. However, spermatogenic cells from old lacI mice display a 10-fold higher mutant frequency. This increased spontaneous mutant frequency occurs coincidentally with decreased in vitro base excision repair activity for germ cell and testicular extracts that in turn corresponds to a decreased abundance of AP endonuclease. To directly test whether a genetic diminution of AP endonuclease results in increased spontaneous mutant frequencies in spermatogenic cell types, AP endonuclease heterozygous (Apex(+/-)) knockout mice were crossed with lacI transgenic mice. Spontaneous mutant frequencies were significantly elevated (approximately twofold) for liver and spleen obtained from 3-month-old Apex(+/-) lacI(+) mice compared to frequencies from Apex(+/+) lacI(+) littermates and were additionally elevated for somatic tissues from 9-month-old mice. Spermatogenic cells from 9-month-old Apex(+/-) lacI(+) mice were significantly elevated twofold compared to levels for 9-month-old Apex(+/+) lacI(+) control mice. These data indicate that diminution of AP endonuclease has a significant effect on spontaneous mutagenesis in somatic and germ line cells.