Encapsulation of viral vectors for gene therapy applications

In gene therapy, a number of viruses are currently being used as vectors to provide transient expression of therapeutic proteins. A drawback of using free virus is that it gives a potent immune response, which reduces gene transfer and limits re-administration. An alternative delivery system is to encapsulate the virus in poly(lactide-co-glycolide) (PLG) microspheres prior to administration. A recombinant adenovirus (Ad) expressing green fluorescent protein (GFP) was used to test the transduction efficiency of Ad encapsulated in microspheres on target cells. The number of infected cells that expressed GFP was measured by flow cytometry. It was demonstrated that encapsulated viral vectors could successfully transduce target cells with encapsulation efficiencies up to 23% and that the level of transduction could be controlled by varying both the quantity of microspheres and the amount of Ad in the microspheres. High transduction efficiencies and its recognized biocompatibility make PLG-encapsulated Ad an attractive alternative to the use of free virus in gene therapy applications. The infectivity of Ad was found to be significantly influenced by the processing conditions and changes in environmental factors. Free Ad and encapsulated Ad were able to infect both E1 complimenting cells (HEK 293) and non-complimenting cells (A549), with the viral expression in HEK 293 cells being 2.1 times greater than for A549 cells.     

Long-term changes in temperate marine fish assemblages are driven by a small subset of species

The species composition of plant and animal assemblages across the globe has changed substantially over the past century. How do the dynamics of individual species cause this change? We classified species into seven unique categories of temporal dynamics based on the ordered sequence of presences and absences that each species contributes to an assemblage time series. We applied this framework to 14,434 species trajectories comprising 280 assemblages of temperate marine fishes surveyed annually for 20 or more years. Although 90% of the assemblages diverged in species composition from the baseline year, this compositional change was largely driven by only 8% of the species' trajectories. Quantifying the reorganization of assemblages based on species shared temporal dynamics should facilitate the task of monitoring and restoring biodiversity. We suggest ways in which our framework could provide informative measures of compositional change, as well as leverage future research on pattern and process in ecological systems.     

An antitumor promoter from Moringa oleifera Lam.

In the course of studies on the isolation of bioactive compounds from Philippine plants, the seeds of Moringa oleifera Lam. were examined and from the ethanol extract were isolated the new O-ethyl-4-(α- -rhamnosyloxy)benzyl carbamate (1) together with seven known compounds, 4(α- -rhamnosyloxy)-benzyl isothiocyanate (2), niazimicin (3), niazirin (4), β-sitosterol (5), glycerol-1-(9-octadecanoate) (6), 3-O-(6′-O-oleoyl-β- -glucopyranosyl)-β-sitosterol (7), and β-sitosterol-3-O-β- -glucopyranoside (8). Four of the isolates (2, 3, 7, and 8), which were obtained in relatively good yields, were tested for their potential antitumor promoting activity using an in vitro assay which tested their inhibitory effects on Epstein–Barr virus-early antigen (EBV-EA) activation in Raji cells induced by the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). All the tested compounds showed inhibitory activity against EBV-EA activation, with compounds 2, 3 and 8 having shown very significant activities. Based on the in vitro results, niazimicin (3) was further subjected to in vivo test and found to have potent antitumor promoting activity in the two-stage carcinogenesis in mouse skin using 7,12-dimethylbenz(a)anthracene (DMBA) as initiator and TPA as tumor promoter. From these results, niazimicin (3) is proposed to be a potent chemo-preventive agent in chemical carcinogenesis.     

Antibody response to Candida albicans cell wall antigens

The cell wall of Candida albicans is not only the structure where many essential biological functions reside but is also a significant source of candidal antigens. The major cell wall components that elicit a response from the host immune system are proteins and glycoproteins, the latter being predominantly mannoproteins. Both carbohydrate and protein moieties are able to trigger immune responses. Proteins and glycoproteins exposed at the most external layers of the wall structure are involved in several types of interactions of fungal cells with the exocellular environment. Thus, coating of fungal cells with host antibodies has the potential to profoundly influence the host-parasite interaction by affecting antibody-mediated functions such as opsonin-enhanced phagocytosis and blocking the binding activity of fungal adhesins to host ligands. In this review we examine various members of the protein and glycoprotein fraction of the C. albicans cell wall that elicit an antibody response in vivo. Some of the studies demonstrate that certain cell wall antigens and anti-cell wall antibodies may be the basis for developing specific and sensitive serologic tests for the diagnosis of candidiasis, particularly the disseminated form. In addition, recent studies have focused on the potential of antibodies against the cell wall protein determinants in protecting the host against infection. Hence, a better understanding of the humoral response triggered by the cell wall antigens of C. albicans may provide the basis for the development of (i) effective procedures for the serodiagnosis of disseminated candidiasis, and (ii) novel prophylactic (vaccination) and therapeutic strategies to control this type of infections.     

alpha3beta1 integrin modulates neuronal migration and placement during early stages of cerebral cortical development

We show that alpha3 integrin mutation disrupts distinct aspects of neuronal migration and placement in the cerebral cortex. The preplate develops normally in alpha3 integrin mutant mice. However, time lapse imaging of migrating neurons in embryonic cortical slices indicates retarded radial and tangential migration of neurons, but not ventricular zone-directed migration. Examination of the actin cytoskeleton of alpha3 integrin mutant cortical cells reveals aberrant actin cytoskeletal dynamics at the leading edges. Deficits are also evident in the ability of developing neurons to probe their cellular environment with filopodial and lamellipodial activity. Calbindin or calretinin positive upper layer neurons as well as the deep layer neurons of alpha3 integrin mutant mice expressing EGFP were misplaced. These results suggest that alpha3beta1 integrin deficiency impairs distinct patterns of neuronal migration and placement through dysregulated actin dynamics and defective ability to search and respond to migration modulating cues in the developing cortex.     

Glutamine and the maintenance of meiotic arrest in mouse oocytes: influence of culture medium,glucose, and cumulus cells

The selection of culture media and supplements therein has a tremendous impact on the regulation of oocyte maturation in vitro. In the present study, we have evaluated how altering the levels of glutamine in the presence or absence of glucose affects meiotic arrest in cumulus cell-enclosed oocytes (CEO) and denuded oocytes (DO) when cultured in either the simple medium M16 or the more complex Eagle's minimum essential medium (MEM). We have also tested the effectiveness of follicle-stimulating hormone (FSH) in triggering germinal vesicle breakdown (GVB) and purine de novo synthesis in differing MEM culture conditions. When DO were cultured 17-18 hr in hypoxanthine (HX)- or dbcAMP-supplemented M16 medium, neither glucose nor glutamine had any effect on oocyte maturation, with dbcAMP the more effective inhibitor. In the absence of glutamine, cumulus cells promoted meiotic resumption, since significantly lower levels of meiotic arrest were maintained in CEO than in DO by either HX or dbcAMP, but addition of the amino acid dose-dependently decreased the maturation percentage in CEO below that observed in DO. In MEM, glutamine and glucose again had little effect on the maturation of DO, although the percentage of maturing DO in HX-supplemented medium was about 20% lower than that in M16 medium. In the absence of glucose, high levels of maturation were observed in CEO in glutamine-free medium that were dose-dependently lowered by the amino acid. However, when glucose was present, CEO were as effectively arrested as DO when glutamine was absent, with no further effect of the amino acid. This inhibitory action of glucose was dependent on the essential amino acids present in MEM. The effects of glutamine were not due to changes in metabolic coupling between the oocyte and cumulus cells. Measurement of purine de novo synthesis indicated that the maintenance of meiotic arrest as well as FSH induction of meiotic resumption were associated with increases in purine synthesis. We conclude that glucose and glutamine act cooperatively to promote the synthesis of new purine compounds within the somatic compartment and that the timing and duration of such synthesis determines whether meiotic resumption will be suppressed or promoted.     

Functional dissection of lupus susceptibility loci on the New Zealand black mouse chromosome 1: evidence for independent genetic loci affecting T and B cell activation

In previous work, we demonstrated linkage between a broad region on New Zealand Black (NZB) chromosome 1 and increased costimulatory molecule expression on B cells and autoantibody production. In this study, we produced C57BL/6 congenic mice with homozygous NZB chromosome 1 intervals of differing lengths. We show that both B6.NZBc1(35-106) (numbers denote chromosomal interval length) and B6.NZBc1(85-106) mice produce IgG anti-nuclear autoantibodies, but B6.NZBc1(35-106) mice develop significantly higher titers of autoantibodies and more severe renal disease than B6.NZBc1(85-106) mice. Cellular analysis of B6.NZBc1(85-106) mice revealed splenomegaly and increased numbers of memory T cells. In addition to these features, B6.NZBc1(35-106) mice had altered B and T cell activation with increased expression of CD69, and for B cells, costimulatory molecules and MHC. Introduction of an anti-hen egg white lysozyme Ig transgene, as a representative nonself-reactive Ig receptor, onto the B6.NZBc1(35-106) background corrected the B cell activation phenotype and led to dramatic normalization of splenomegaly and T cell activation, but had little impact on the increased proportion of memory T cells. These findings indicate that there are multiple lupus susceptibility genes on NZB chromosome 1, and that although B cell defects play an important role in lupus pathogenesis in these mice, they act in concert with T cell activation defects.     

Casein-derived bioactive phosphopeptides: role of phosphorylation and primary structure in promoting calcium uptake by HT-29 tumor cells

Casein phosphopeptides beta-CN(1-25)4P and alpha(s1)-CN(59-79)5P, from beta- and alpha(s1)-casein, respectively, both carrying the characteristic 'acidic motif' Ser(P)-Ser(P)-Ser(P)-Glu-Glu, were chemically synthesized and administered to HT-29 cells differentiated in culture, which are a used model of intestinal epithelium for absorption studies. Both casein phosphopeptides caused an increase of [Ca(2+)](i) due to influx of extracellular Ca(2+). The response was quantitatively higher with beta-CN(1-25)4P than alpha(s1)-CN(59-79)5P. The synthetic peptide corresponding to the 'acidic motif' was ineffective and the dephosphorylated form of beta-CN(1-25)4P almost inactive. The lack of the N-terminally located five amino acids, or sequence modifications within the N-terminal segment of beta-CN(1-25)4P, caused a total loss of activity, whereas the lack of the C-terminal segment preserved activity. In conclusion, the influx of calcium into HT-29 cells caused by beta-CN(1-25)4P appears to depend on the phosphorylated 'acidic motif' and the preceding N-terminal region.     

Role of nuclear PKC delta in mediating caspase-3-upregulation in Jurkat T leukemic cells exposed to ionizing radiation

The suppressive role of endogenous regucalcin (RC), which is a regulatory protein of calcium signaling, in the enhancement of protein phosphatase activity (PPA) in the cytosol and nucleus of kidney cortex in calcium-administered rats was investigated. Calcium content in the kidney cortex was significantly increased at 0.5-5 h after a single intraperitoneal administration of calcium chloride solution (10 mg Ca/100 g body weight) to rats. The analysis with Western blotting of RC protein showed that RC levels in the cytosol and nucleus were significantly increased 0.5-5 h after the administration of calcium (10 mg/100 g). PPA toward phosphotyrosine, phosphoserine, and phosphothreonine was found in the cytosol and nucleus of kidney cortex. PPA toward three phosphoamino acids in the cytosol and nucleus was significantly increased by the administration of calcium (10 mg/100 g). The presence of anti-RC monoclonal antibody (25 ng/ml) in the enzyme reaction caused a significant increase in PPA toward phosphotyrosine, phosphoserine, and phosphothreonine in the cytosol and nucleus of kidney cortex in normal rats. The effect of anti-RC monoclonal antibody (25 ng/ml) in increasing PPA toward three phosphoamino acids in the cytosol and nucleus was significantly enhanced in calcium-administered rats. The effect of anti-RC monoclonal antibody (25 ng/ml) in increasing PPA in the cytosol and nucleus of normal rats and calcium-administered rats was completely abolished by the addition of RC (10(- 6) M) in the enzyme reaction mixture. The present study suggests that endogenous RC suppresses the enhancement of PPA in the cytosol and nucleus of kidney cortex in calcium-administered rats.