<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">ex vivo</Emphasis> effect of hyaluronic acid on erythrocyte flow properties

Background  

Hyaluronic acid (HA) is present in many tissues; its presence in serum may be related to certain inflammatory conditions, tissue damage, sepsis, liver malfunction and some malignancies. In the present work, our goal was to investigate the significance of hyaluronic acid effect on erythrocyte flow properties. Therefore we performed in vitro experiments incubating red blood cells (RBCs) with several HA concentrations. Afterwards, in order to corroborate the pathophysiological significance of the results obtained, we replicated the in vitro experiment with ex vivo RBCs from diagnosed rheumatoid arthritis (RA) patients, a serum HA-increasing pathology.     

Variation of physico-chemical parameters along a river transect through the Okavango Delta,Botswana

The Okavango Delta depends on water quantity and quality to sustain its ecosystem services. Whereas many studies have been carried out on its hydrology, few have been done on water quality in the delta. Water pH, electrical conductivity (EC), dissolved oxygen (DO), turbidity, total suspended solids (TSS) and dissolved organic carbon (DOC) were monitored at 10 sites along the Okavango–Boro–Thamalakane–Lake Ngami system almost fortnightly from June 2008 to June 2010. Water quality in the delta was generally good, despite high evapotranspiration rates which would normally produce very saline waters. Electrical conductivity and water temperature increased with distance from Mohembo to Lake Ngami, the former most likely due to evapoconcentration. In contrast, pH, DO, turbidity and TSS decreased with distance from Mohembo to Boro at the lower end of the seasonal floodplain, before increasing again to Lake Ngami. Dissolved oxygen and TSS most likely declined due to biological uptake and particle sedimentation, respectively. Strong and significant relationships were observed between TSS and turbidity and between DOC and EC, indicating that turbidity and EC could be useful proxies for routine estimations of TSS and DOC, respectively, in the delta.     

Ribosomal RNA sequence phylogeny is not congruent with ascospore morphology among species in Ceratocystis sensu stricto

The genus Ceratocystis sensu stricto includes important fungal pathogens of woody and herbaceous plants. This genus is distinguished from species in Ceratocystis sensu lato by the presence of Chalara anamorphs. Ascospore shape has been used extensively in delineating Ceratocystis taxa, which show a large variety of ascospore shapes. Sequence analysis of one region of he 18S ribosomal RNA subunit and two regions of the 28S ribosomal RNA subunit showed that there was a majority of multiple substitutions at nucleotide sites and that there was a low transition/transversion ratio, T = 0.72. Both of these results suggest that these are well established, old species. Ascospore morphology, for the most part, was not congruent with the molecular phylogeny, and the use of morphological characters may be misleading in the taxonomy of these species.      

Chemical caspase inhibitors enhance cell culture viabilities and protein titer

Mammalian cell cultures are integral to the production of therapeutic and diagnostic proteins. A common problem encountered in culturing these cell lines, however, is a loss in viability at later stages of the cell culture process. In this study the effects of three newly synthesized chemical caspase inhibitors were investigated for their capacity to inhibit cell death. Findings show that these protease inhibitors were successful in prolonging cellular viabilities among anchorage-dependent CHO-K1 and HEK-293 cells lines. Cells treated with one inhibitor, 7312, performed as well or better when compared with the commercially known caspase inhibitor, zVAD.fmk. Suspension CHO cells producing an IgG were used to investigate the capacity of 7312 to improve protein production. Treatment of cells with 7312 increased integrated cell densities by 33% with treated cells having a higher maximum cell density and higher viability. As a result, monoclonal antibody titers increased by 20% and higher in spinner flask experiments. Increasing productivity in mammalian cell cultures has key implications for the pharmaceutical and biotechnology sectors, which are presently focused on developing methods to enhance cell performance in bioreactor environments.     

Aven and Bcl-xL enhance protection against apoptosis for mammalian cells exposed to various culture conditions

A balance between proliferation and cell death is critical for achieving desirable high cell densities in mammalian cell culture. In this study, we evaluate a recently discovered anti-apoptotic gene, aven, and examine its effectiveness alone and in combination with a member of the Bcl-2 family, bcl-xL. The commercially popular cell line, Chinese hamster ovary (CHO), was genetically modified to constitutively express aven, bcl-xL, and the two genes in combination. Cells were exposed to several model insults that simulate severe bioreactor environments, including serum deprivation, spent medium, and Sindbis virus infection, as well as staurosporine, a known chemical inducer of apoptosis. CHO cells exhibited DNA fragmentation, a hallmark of apoptosis, after exposure to these model insults. After exposure to serum deprivation, 4- and 5-day spent medium, and staurosporine, cells expressing Aven provided limited protection against cell death when compared with the protection afforded by cells expressing Bcl-xL alone. However, the highest survival levels for all insults were achieved when Aven was expressed in combination with Bcl-xL. In fact, Aven appeared to act synergistically to enhance the protective function of Bcl-xL for several insults, because the protective function of the two genes expressed together in one cell line often exceeded the additive protective levels of each anti-apoptosis gene expressed alone. Surprisingly, Aven expression provided a mildly pro-apoptotic response in CHO isolates infected with Sindbis virus. However, CHO cells expressing both Bcl-xL and Aven showed protection against Sindbis virus infection due to the inhibitory properties of the bcl-xL anti-apoptosis gene. This study shows that combinatorial anti-apoptosis cell engineering strategies may be the most effective mechanisms for providing extended protection against cell death in mammalian cell culture.     

Engineering sialic acid synthetic ability into insect cells: identifying metabolic bottlenecks and devising strategies to overcome them

Previous studies have indicated negligible levels of both sialylation and the precursor N-acetylneuraminic acid (Neu5Ac) in a number of insect cell lines grown in serum-free medium. The overexpression of the human sialic acid 9-phosphate synthase (SAS) in combination with N-acetylmannosamine (ManNAc) feeding has been shown to overcome this limitation. In this study we evaluated the potential bottlenecks in the sialic acid synthesis pathway in a Spodoptera frugiperda (Sf9) insect cell line and devised strategies to overcome them by overexpression of the enzymatic pathway enzymes combined with appropriate substrate feeding. Coexpression of SAS and UDP-GlcNAc 2-epimerase/ManNAc kinase, the bifunctional enzyme initiating sialic acid biosynthesis in mammals, resulted in Neu5Ac synthesis without use of any external media supplementation to demonstrate that Neu5Ac could be generated intracellularly in Sf9 cells using natural metabolic precursors. N-Acetylglucosamine (GlcNAc) feeding in combination with this coexpression resulted in much higher levels of Neu5Ac compared to levels obtained with ManNAc feeding with SAS expression alone. The lower Neu5Ac levels obtained with ManNAc feeding suggested limitations in the transport and phosphorylation of ManNAc. The bottleneck in phosphorylation was likely due to utilization of GlcNAc kinase for phosphorylation of ManNAc in insect cells and was overcome by expression of ManNAc kinase. The transport limitation was addressed by the addition of tetra-O-acetylated ManNAc, which is easily taken up by the cells. An alternative sialic acid, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), could also be generated in insect cells, suggesting the potential for controlling not only the production of sialic acids but also the type of sialic acid generated. The levels of KDN could be increased with virtually no Neu5Ac generation when Sf9 cells were fed excess GlcNAc. The results of these studies may be used to enhance the sialylation of target glycoproteins in insect and other eukaryotic expression systems.     

Expression of a functional Drosophila melanogaster N-acetylneuraminic acid (Neu5Ac) phosphate synthase gene: evidence for endogenous sialic acid biosynthetic ability in insects

In this study, we report the first cloning and characterization of a N-acetylneuraminic acid phosphate synthase gene from Drosophila melanogaster, an insect in the protostome lineage. The gene is ubiquitously expressed at all stages of Drosophila development and in Schneider cells. Similar to the human homologue, the gene encodes an enzyme with dual substrate specificity that can use either N-acetylmannosamine 6-phosphate or mannose 6-phosphate to generate phosphorylated forms of both the sialic acids, N-acetylneuraminic acid and 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid, respectively, when expressed in either bacterial or baculoviral expression systems. The identification of a functional sialic acid synthase in Drosophila indicates that insects have the biosynthetic capability to produce sialic acids endogenously. Although sialylation is widely distributed in organisms of the deuterstome lineage, genetic evidence concerning the presence or absence of sialic acid metabolism in organisms of the protostome lineage has been lacking. Homology searches of the Drosophila genome identified putative orthologues of other genes required for sialylation of glycoconjugates.