Composition and function of human cervical mucus

Human cervical mucus was collected from seven donors during the follicular, ovulatory and luteal phases of the ovulatory menstrual cycle. Individual mucus samples were solubilized and fractionated on Sepharose columns into excluded mucins and low-molecular-weight proteins. Mucin fractions were highly purified, as evidenced by the presence of a single N-terminal amino acid residue, threonine, and by the absence of contaminating plasma proteins. Amino acid compositions of mucins isolated during different menstrual phases of a single donor or from different donors were similar. Mucin carbohydrate compositions were also similar, except for the sialic acid-to-fucose ratio, which varied significantly between donors but not within the menstrual cycle of a single donor. An analysis of variance was applied to evaluate the contribution of mucin composition to viscoelasticity, as quantitated by microrheometry. Viscoelasticity was dependent on the donor, on the percent nondialyzable solids and on the mucin content, but not on the phase of the menstrual cycle during which the sample was collected. These findings suggest that mucus function (viscoelasticity) is reflected in carbohydrate composition and/or structure and that this menstrual relationship is unique for each donor. Furthermore, the absence of menstrual phase-dependent differences in mucins suggests that mucin concentration and not composition changes in response to alterations in the hormonal milieu.     

人细小病毒B19病毒样颗粒的制备

采用昆虫杆状病毒表达系统,制备人细小病毒B19病毒样颗粒(VLPs)。先通过PCR方法合成细小病毒B19衣壳蛋白基因VP2,将其克隆到pFastBac1质粒,然后转化含杆状病毒穿梭载体Bacmid的E.coliDH10Bac感受态细胞,获得重组杆状病毒表达质粒Bacmid-VP2。在脂质体介导下转染Sf9昆虫细胞,包装重组杆状病毒rBac-VP2。利用rBac-VP2感染Sf9细胞表达B19VP2蛋白,通过间接免疫荧光、Western blotting等方法鉴定目的蛋白表达。采用两次超速离心的方法对表达产物进行纯化,纯化产物在透射电镜下可见直径约22nm的VLPs。本研究成功制备了人细小病毒B19的VLPs,为B19感染血清学检测方法的建立提供了参考。     

Cyclic changes of sialidase in human cervical mucus

• 1.1. Sialidase activity is detectable in whole cervical mucus of normal women throughout the menstrual cycle and presents cyclic variations towards endogenous and exogenous substrates.
• 2.2. The level of sialic acid bound to the mucus increases progressively till mid-cycle and declines in the post-ovulatory phase.
• 3.3. The sialidase of the mucus probably derives from different sources and its role remains speculative.

     

Scanning electron microscope study of human cervical mucus

The ultrastructure of human cervical mucus was studied by scanning electron microscopy. In fertile women, naturally occurring midcycle cervical mucus showed an arrangement of parallel fibers oriented along the main axis of the mucus sample. Sperm migration through a column of cervical mucus in vitro was also studied. It was found that sperm present among the longitudinal fibers were oriented parallel to the them and to the main axis of the mucus sample.     

Structures of virus and virus-like particles

Virus structures continue to be the basis for mechanistic virology and serve as a paradigm for solutions to problems concerning macromolecular assembly and function in general. The use of X-ray crystallography, electron cryomicroscopy and computational and biochemical methods has provided not only details of the structural folds of individual viral components, but also insights into the structural basis of assembly, nucleic acid packaging, particle dynamics and interactions with cellular molecules.     

Molecular and structural characterization of fluorescent human parvovirus B19 virus-like particles

Although sharing a T=1 icosahedral symmetry with other members of the Parvoviridae family, it has been suggested that the fivefold channel of the human parvovirus B19 VP2 capsids is closed at its outside end. To investigate the possibility of placing a relatively large protein moiety at this site of B19, fluorescent virus-like particles (fVLPs) of B19 were developed. The enhanced green fluorescent protein (EGFP) was inserted at the N-terminus of the structural protein VP2 and assembly of fVLPs from this fusion protein was obtained. Electron microscopy revealed that these fluorescent protein complexes were very similar in size when compared to wild-type B19 virus. Further, fluorescence correlation spectroscopy showed that an average of nine EGFP domains were associated with these virus-like structures. Atomic force microscopy and immunoprecipitation studies showed that EGFP was displayed on the surface of these fVLPs. Confocal imaging indicated that these chimeric complexes were targeted to late endosomes when expressed in insect cells. The fVLPs were able to efficiently enter cancer cells and traffic to the nucleus via the microtubulus network. Finally, immunoglobulins present in human parvovirus B19 acute and past-immunity serum samples were able to detect antigenic epitopes present in these fVLPs. In summary, we have developed fluorescent virus-like nanoparticles displaying a large heterologous entity that should be of help to elucidate the mechanisms of infection and pathogenesis of human parvovirus B19. In addition, these B19 nanoparticles serve as a model in the development of targetable vehicles designed for delivery of biomolecules.     

Diffusion of molecules and macromolecules in thylakoid membranes

The survival and fitness of photosynthetic organisms is critically dependent on the flexible response of the photosynthetic machinery, harbored in thylakoid membranes, to environmental changes. A central element of this flexibility is the lateral diffusion of membrane components along the membrane plane. As demonstrated, almost all functions of photosynthetic energy conversion are dependent on lateral diffusion. The mobility of both small molecules (plastoquinone, xanthophylls) as well as large protein supercomplexes is very sensitive to changes in structural boundary conditions. Knowledge about the design principles that govern the mobility of photosynthetic membrane components is essential to understand the dynamic response of the photosynthetic machinery. This review summarizes our knowledge about the factors that control diffusion in thylakoid membranes and bridges structural membrane alterations to changes in mobility and function. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components.     

Role of Ebola virus secreted glycoproteins and virus-like particles in activation of human macrophages

Ebola virus, a member of the family Filoviridae, causes one of the most severe forms of viral hemorrhagic fever. In the terminal stages of disease, symptoms progress to hypotension, coagulation disorders, and hemorrhages, and there is prominent involvement of the mononuclear phagocytic and reticuloendothelial systems. Cells of the mononuclear phagocytic system are primary target cells and producers of inflammatory mediators. Ebola virus efficiently produces four soluble glycoproteins during infection: sGP, delta peptide (Delta-peptide), GP(1), and GP(1,2Delta). While the presence of these glycoproteins has been confirmed in blood (sGP) and in vitro systems, it is hypothesized that they are of biological relevance in pathogenesis, particularly target cell activation. To gain insight into their function, we expressed the four soluble glycoproteins in mammalian cells and purified and characterized them. The role of the transmembrane glycoprotein in the context of virus-like particles was also investigated. Primary human macrophages were treated with glycoproteins and virus-like particles and subsequently tested for activation by detection of several critical proinflammatory cytokines (tumor necrosis factor alpha, interleukin-6 [IL-6], and IL-1 beta) and the chemokine IL-8. The presentation of the glycoprotein was determined to be critical since virus-like particles, but not soluble glycoproteins, induced high levels of activation. We propose that the presentation of GP(1,2) in the rigid form such as that observed on the surface of particles is critical for initiating a sufficient signal for the activation of primary target cells. The secreted glycoproteins do not appear to play any role in exogenous activation of these cells during Ebola virus infection.     

Purification and immunogenicity study of human papillomavirus 58 virus-like particles expressed in Pichia pastoris

Two human papillomavirus (HPV) prophylactic vaccines are currently available in the market: Gardasil and Cervarix. These two vaccines work against tumor high-risk subtypes HPV 16 and HPV 18. However, they do not include other high-risk subtypes such as HPV 58. Epidemiological research in China shows that HPV 58 is a prevalent high-risk subtype, second only to HPV 16 and HPV 18. Thus, for cervical cancer prevention in China, developing a vaccine against HPV 58 is necessary. In this study, HPV 58 virus-like particles (VLPs) were expressed in the Pichia pastoris, and subsequently purified through pretreatment and a three-step purification process consisting of strong cation exchange chromatography, size-exclusion chromatography, and hydroxyapatite chromatography. The highly purified HPV 58 VLPs were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, electron microscopy, dynamic laser scattering, and ultracentrifugation. The purified VLPs were used to immunize mice to test their ability to induce humoral immunity. Enzyme-linked immunosorbent assays were performed on the sera of the immunized mice and significantly high anti-HPV 58 VLP antibody titers were observed. The immunogenicity study demonstrates that the purified HPV 58 VLPs are HPV vaccine candidates.     

Histopathology of virus-like particles in Heliothis spp

Larvae of Heliothis spp. collected from cotton, soybean, and peanut fields in South Carolina were found to be infected with virus-like particles (VLPs). Infected larvae became pale and swollen, stopped feeding, and remained alive for 2–3 weeks. Hemolymph from these larvae was milky and contained numerous spherical bodies ranging in diameter from 2 to 10 μm. The hemolymph also contained VLPs which were oval and measured 375 × 125 nm. Infectivity tests with crude saline extracts of infected larvae demonstrated that the pathogen could be transmitted by injection but not per os. The spherical bodies contained VLPs (387 × 149 nm) surrounded by two envelopes and packed together in clusters. These VLPs were also found in fat body cells, cuticular epithelial cells, tracheal cells, and connective tissue associated with the body wall and the gut. They were not found in muscle tissue or in midgut epithelial cells. Similar VLPs have been found in Heliothis zea from Mississippi and Trichoplusia ni from California, but a positive identification of the VLPs has not been made in any of these studies. Morphologically they appear to be distinct from any other previously described insect viruses.     

A quantitative description of in vitro assembly of human papillomavirus 16 virus-like particles

The full-length human papillomavirus 16 major capsid protein L1 is expressed in Saccharomyces cerevisiae as virus-like particles (VLPs). However, yeast-expressed human papillomavirus 16 particles are irregular in shape and are prone to aggregate. When disassembled and reassembled, the resulting particles have improved stability and solubility. We have examined VLP dissociation and reassembly to define the important features of the assembly mechanism. We found that the VLPs rapidly disassemble at pH 8.2 and low ionic strength in the presence of low concentrations of reducing agents. The pH dependence of assembly kinetics and extent of assembly under reducing conditions were differentially sensitive to ionic strength. Assembly at pH 5.2 was very fast and led to heavily aggregated particles. This sort of kinetic trap is expected for overinitiated assembly. We observed that reassembly at pH 6.2, 7.2, and 8.2 yielded regular particles over a broad range of ionic strength. At these three pH values, assembly was quantitative at 1 M NaCl. At pH 7.2, much more than at pH 6.2 or pH 8.2, assembly decreased monotonically with ionic strength. The free energy of association ranged from − 8 to − 10 kcal/mol per pentamer. The effect of pH on assembly was further investigated by examining dissociation of reassembled particles. Though indistinguishable by negative stain electron microscopy, particles assembled at pH 7.2 disassembled slower than pH 5.2, 6.2, or 8.2 VLPs. We hypothesize that pH 7.2 assembly reactions lead to formation of particles with conformationally different interactions.     

Cyclic AMP in cervical mucus

Cyclic adenosine monophosphate normally stimulates motility of spermatozoa. Its concentration in cervical mucus was studied by an isotopic competitive method in 15 normal women aged between 20 and 50 years. Values were very high, particularly in the periovulatory period, with a mean (+/-SD) value of 167.90 +/- 154.96 nmol/l. These are very high when compared with values in other biological fluids (blood serum and urine).     

Evidence for the tight binding of human mucus proteinase inhibitor to highly glycosylated macromolecules in sputum

Two extraction procedures of non-purulent sputum for the isolation of human mucus proteinase inhibitor (MPI) in its free and bound forms have been assayed. The dissociating procedure involved sputum homogenization in 1M NaCl and 4% (w/v) trichloroacetic treatment. When the soluble material was applied to a CM-Trisacryl column, a non-negligible, MPI-related inhibitory activity was recovered with the highly glycosylated constituents not retained on the column; the amount of MPI released in a free form was retained and eluted from the column according to the basic character of this inhibitor. The non-dissociating procedure consisted in a high water dilution (1:12) of sputum, known to bring into solution the macromolecular, fibrillar constituents, which was followed by ultrafiltration on selected Mr cut-off membranes. All the inhibitory activity was recovered with the high Mr (greater than 100,000) fraction which was shown on SDS-PAGE to be essentially composed of strongly glycosylated material; on electrophoretic analysis under non-reducing conditions, the MPI activity was visualized as three bands which corresponded to the inhibitor released from this high Mr fraction in the presence of SDS. As mucin-type molecules are the major, highly glycosylated constituents of bronchial secretions, it is suggested that they are responsible for the entrapping of MPI within their macromolecular network; it would appear that, as well as for lysozyme, electrostatic interactions occur between the acid charges of mucins and the basic charges of MPI. The possible in vivo consequences of these interactions on MPI activity are discussed.