Role of cervical dendritic cell subsets,co-stimulatory molecules,cytokine secretion profile and beta-estradiol in development of sequalae to Chlamydia trachomatis infection

Background  

Chlamydia trachomatis infection of the female genital tract can lead to serious sequelae resulting in fertility related disorders. Little is known about the mechanism leading to Chlamydia induced pathology and factors responsible for it. As only some of the women develops reproductive disorders while majority of the women clears infection without any severe sequalae, mucosal immune response in women with or without fertility disorders was studied to identify factors which may lead to final clinical outcome of chlamydial infection.     

Possible degradation/biotransformation of lutein in vitro and in vivo: isolation and structural elucidation of lutein metabolites by HPLC and LC-MS (atmospheric pressure chemical ionization)

Metabolites of lutein are highly concentrated in the human macula and are known to provide protection against age-related macular degeneration. The aim of this investigation was to characterize the in vitro oxidation products of lutein obtained through photo-oxidation and to compare them with biologically transformed dietary lutein in intestine, plasma, liver, and eyes of rats. In vivo studies involved feeding rats a diet devoid of lutein for 2 weeks to induce deficiency. Rats were divided into two equal groups (n=6/group) and received either micellar lutein by gavage for 10 days or diet supplemented with fenugreek leaves as a lutein source for 4 weeks. Lutein metabolites/oxidation products obtained from in vivo and in vitro studies were characterized by HPLC and LC-MS (APCI) techniques to elucidate their structure. The characteristic fragmented ions resulting from photo-oxidation of lutein were identified as 523 (M(+)+H(+)-3CH(3)), 476 (M(+)+H(+)-6CH(3)), and 551 (M(+)+H(+)-H(2)O). In the eyes, the fragmented molecules resulting from lutein were 13-Z lutein, 13'-Z lutein, 13-Z zeaxanthin, all-E zeaxanthin, 9-Z lutein, 9'-Z lutein, and 3'-oxolutein. Epoxycarotenoids were identified in liver and plasma, whereas anhydrolutein was identified in intestine. This study emphasizes the essentiality of dietary lutein to maintain its status in the retina.     

Chitosan IFN-γ-pDNA Nanoparticle (CIN) Therapy for Allergic Asthma

Background  

Allergic subjects produce relatively low amounts of IFN-γ, a pleiotropic Th-1 cytokine that downregulates Th2-associated airway inflammation and hyperresponsiveness (AHR), the hallmarks of allergic asthma. Adenovirus-mediated IFN-γ gene transfer reduces AHR, Th2 cytokine levels and lung inflammation in mice, but its use would be limited by the frequency of gene delivery required; therefore, we tested chitosan/IFN-γ pDNA nanoparticles (CIN) for in situ production of IFN-γ and its in vivo effects.     

9‐bromonoscapine‐induced mitotic arrest of cigarette smoke condensate‐transformed breast epithelial cells

In the present investigation, we determined the chemotherapeutic efficacy of 9‐bromonoscapine (Br‐Nos), a more potent noscapine analog, on MCF10A, spontaneously immortalized human normal breast epithelial cells and MCF10A‐CSC3, cigarette smoke condensate (CSC)‐transformed cells. The results from cytogenetic analysis showed that Br‐Nos induced polyploidy and telomeric association in MCF10A‐CSC3 cells, while MCF10A cells remained unaffected. Our immunofluorescence data further demonstrated that MCF10A‐CSC3 cells were susceptible to mitotic catastrophe on exposure to Br‐Nos and failed to recover after drug withdrawal. MCF10A‐CSC3 cells exhibited Br‐Nos‐induced aberrant multipolar spindle formation, which irreversibly impaired the alignment of replicated chromosome to the equatorial plane and finally culminated in cell death. Although MCF10A cells upon Br‐Nos treatment showed bipolar spindles with some uncongressed chromosomes, these cells recovered fairly well after drug withdrawal. Our flow‐cytometry analysis data reconfirmed that MCF10A‐CSC3 cells were more susceptible to cell death compared to MCF10A cells. Furthermore, our results suggest that decreased levels of cdc2/cyclin B1 and cdc2 kinase activity are responsible for Br‐Nos‐induced mitotic cell arrest leading to cell death in MCF10A‐CSC3 cells. This study thus explores the underlying mechanism of Br‐Nos‐induced mitotic catastrophe in CSC‐transformed MCF10A‐CSC3 cells and its potential usefulness as a chemotherapeutic agent for prevention of cigarette smoke‐induced breast cancer growth. J. Cell. Biochem. 106: 1146–1156, 2009. © 2009 Wiley‐Liss, Inc.     

A Y526Q Mutation in the Newcastle Disease Virus HN Protein Reduces Its Functional Activities and Attenuates Virus Replication and Pathogenicity

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is a multifunctional protein that plays a crucial role in virus infectivity. In this study, using the mesogenic strain Beaudette C (BC), we mutated three conserved amino acids thought to be part of the binding/catalytic active site in the HN protein. We also mutated five additional residues near the proposed active site that are nonconserved between BC and the avirulent strain LaSota. The eight recovered NDV HN mutants were assessed for effects on biological activities. While most of the mutations had surprisingly little effect, mutation at conserved residue Y526 reduced the neuraminidase, receptor binding, and fusion activities and attenuated viral virulence in eggs and young birds.Newcastle disease virus (NDV) is an avian pathogen of the genus Avulavirus in the family Paramyxoviridae (10). The envelope of NDV contains two surface glycoproteins, the fusion (F) protein and the HN (hemagglutinin-neuraminidase [NA]) protein. The F protein mediates viral penetration and requires cleavage-activation by host protease. Cleavability of the F protein is a major determinant of virulence. However, other viral proteins, including HN, also contribute to virulence (5). HN is a multifunctional glycoprotein. It recognizes sialic acid-containing receptors on cell surfaces; promotes the fusion activity of F protein, thereby allowing the virus to penetrate the cell surface; and acts as an NA that removes sialic acid from progeny virus particles to prevent viral self-aggregation (9).HN is a type II homotetrameric glycoprotein with a monomer length of 577 amino acids for most NDV strains (14). The ectodomain of the HN protein consists of a 95-amino-acid stalk region supporting a 428-amino-acid terminal globular head. Although mutations in the transmembrane and stalk regions of the HN protein can affect the structure and activities of the protein (11, 15), the antigenic, receptor recognition, and NA active sites are all localized in the globular head (12, 16). The X-ray crystal structure of the globular head of the NDV HN protein has identified residues that appear to contribute to receptor recognition, NA, and fusion activities (4). Previous studies have proposed that conserved residues R174, I175, D198, K236, R416, R498, Y526, and E547 are important in receptor recognition and NA activities and that residues R174 and E547 influence the fusion promotion activity of the HN protein (3, 4, 6). Although transfection studies using plasmids expressing HN mutants of NDV have highlighted the importance of these residues in different biological functions of the HN protein, their contribution to NDV biology and pathogenesis in the context of the complete virus was not known.In this study, we examined the roles of three of the above-named conserved residues, R416, R498, and Y526 (all located near the sialic acid binding site), in the biological activities and pathogenesis of the HN protein of NDV in the context of infectious virus. In addition, comparison of the HN protein sequence between the avirulent strain LaSota and the moderately virulent strain Beaudette C (BC) identified 12 amino acid differences in the globular head region of the HN protein (H203, T214, I219, S228, L269, A271, E293, G310, S494, E495, T502, and N568, named according to the BC amino acid assignment). We also examined five of these nonconserved residues, T214, I219, S494, E495, and N568, located in close proximity to residues identified earlier by crystal structure studies, to determine whether these might affect HN function and contribute to the difference in pathogenicity between the LaSota and BC strains (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Three-dimensional structure of the NDV HN protein showing the positions of amino acid residues that were substituted in the present study. The residues are shown in space-filling mode and represented in different colors. The MacPymol (DeLano Scientific) software was used to generate the model of the globular domain of the NDV HN monomer. The structure was derived from the crystal structure of the NDV HN protein reported by Crennell et al. (4).We used site-directed mutagenesis (2) to introduce individual amino acid substitutions into a cDNA of the HN gene of strain BC. For the conserved residues, we changed arginine at positions 416 and 498 and tyrosine at position 526 to polar glutamine. For the nonconserved residues, the assignments T214, I219, S494, E495, and N568 of strain BC were altered to the corresponding assignments of strain LaSota: S214, V219, G494, V495, and D568, respectively. Each mutagenized HN gene was then inserted into a full-length cDNA clone of the BC antigenome. These clones were transfected into HEp2 cells, and mutant viruses were recovered as previously described (8). These viruses were designated according to the substitutions introduced: T214S, I219V, R416Q, S494G, E495V, R498Q, Y526Q, and N568D. The HN genes from recovered viruses were sequenced. This confirmed the presence of each introduced mutation and the lack of adventitious mutations in the HN gene. To determine the stability of each HN mutation, the recovered viruses were passaged five times in 9-day-old embryonated chicken eggs and five times in chicken embryo fibroblast DF-1 cells. Sequence analysis of the HN gene of the mutant viruses at each passage showed that the introduced mutations were unaltered (data not shown). To rule out the possibility that change in the HN protein sequence could be compensated for by a mutation in the F protein, the F gene from each recovered virus was sequenced. No compensatory mutations in the F gene were observed (data not shown). The HN protein content of each mutant virus, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie staining, was very similar to that of the parental BC virus (pBC) (Table ​(Table1).1). The multicycle growth kinetics of the recombinant HN mutant viruses in DF-1 cells (Fig. ​(Fig.2)2) showed that the replication kinetics of all of the HN mutant viruses were similar to those of pBC, with the exception of the Y526Q mutant, which showed delayed growth and had a lower virus yield (1.5 to 2.0 log₁₀ PFU/ml) than the parental and other mutant viruses. In addition, the Y526Q mutant produced syncytia at 72 h, whereas the parental and other mutant viruses initiated syncytia at 24 h postinfection. These studies showed the importance of amino acid residue Y526 at the active site of the HN protein of NDV.Open in a separate windowFIG. 2.Multicycle growth kinetics of HN mutants of NDV in chicken embryo fibroblast (DF-1) cells. Cells were infected with the indicated parental or mutant virus at an multiplicity of infection of 0.01. Supernatant samples were collected at 8-h intervals until 64 h postinfection, and virus titers were determined at different time points by plaque assay. Values are averages from three independent experiments.

TABLE 1.

Biological activities of HN mutants of NDV

Micropropagation of Three Varieties of Caralluma adscendens via Nodal Explants

A procedure for in vitro propagation of pharmaceutically valuable varieties of Caralluma adscendens from nodal explant, is described. The highest shoot multiplication with 80% frequency was achieved within one month on Murashige and Skoog’s medium supplemented with 8.87 μM BA. Shoot multiplication occurred in subsequent subcultures in culture bottles on MS medium. Regenerated shoots were rooted on half strength MS medium supplemented with NAA (0.54 μM) in all the three varieties. The rooted plants were hardened for establishment in soil.     

Topographic mapping and compression elasticity analysis of skinned cardiac muscle fibers in vitro with atomic force microscopy and nanoindentation

Surface topography and compression elasticity of bovine cardiac muscle fibers in rigor and relaxing state have been studied with atomic force microscopy. Characteristic sarcomere patterns running along the longitudinal axis of the fibers were clearly observed, and Z-lines, M-lines, I-bands, and A-bands can be distinguished through comparing with TEM images and force curves. AFM height images of fibers had shown a sarcomere length of 1.22±0.02 μm (n=5) in rigor with a significant 9% increase in sarcomere length in relaxing state (1.33±0.03 μm, n=5), indicating that overlap moves with the changing physiological conditions. Compression elasticity curves along with sarcomere locations have been taken by AFM compression processing. Coefficient of Z-line, I-band, Overlap, and M-line are 25±2, 8±1, 10±1, and 17±1.5 pN/nm respectively in rigor state, and 18±2.5, 4±0.5, 6±1, and 11±0.5 pN/nm respectively in relaxing state. Young's Modulus in Z-line, I-band, Overlap, and M-line are 115±12, 48±9, 52±8, and 90±12 kPa respectively in rigor, and 98±10, 23±4, 42±4, and 65±7 kPa respectively in relaxing state. The elasticity curves have shown a similar appearance to the section analysis profile of AFM height images of sarcomere and the distance between adjacent largest coefficient and Young's Modulus is equal to the sarcomere length measured from the AFM height images using section analysis, indicating that mechanic properties of fibers have a similar periodicity to the topography of fibers.