Killing of T lymphocytes by synthetic ceramide is by a nonapoptotic mechanism and is abrogated following mitogenic activation.

Ceramide induces apoptosis in leukemia cell lines and has been proposed as a potential therapeutic agent in malignancies refractory to conventional treatment. Here we show that synthetic N-acetyl-d-erythro-sphingosine (C2 ceramide) kills normal human T lymphocytes by a caspase-independent nonapoptotic mechanism. By contrast, T cells were induced to caspase-dependent apoptosis by okadaic acid. Furthermore, C2 ceramide treatment of the Jurkat leukemia cell line induced killing by apoptosis. Activation of T lymphocytes by phytohemagglutinin abrogated killing by C2 ceramide. The data here suggest that ceramide triggers caspase-dependent apoptosis in leukemia cells lines, but activates caspase-independent nonapoptotic killing of resting T lymphocytes which is abrogated following mitogenic activation.     

Engineering Chinese hamster ovary cells to maximize sialic acid content of recombinant glycoproteins.

We have engineered two Chinese hamster ovary cell lines secreting different recombinant glycoproteins to express high levels of human beta1,4-galactosyltransferase (GT, E.C. 2.4.1.38) and/or alpha2, 3-sialyltransferase (ST, E.C. 2.4.99.6). N-linked oligosaccharide structures synthesized by cells overexpressing the glycosyltransferases showed greater homogeneity compared with control cell lines. When GT was overexpressed, oligosaccharides terminating with GlcNAc were significantly reduced compared with controls, whereas overexpression of ST resulted in sialylation of >/=90% of available branches. As expected, GT overexpression resulted in reduction of oligosaccharides terminating with GlcNAc, whereas overexpression of ST resulted in sialylation of >/=90% of available branches. The more highly sialylated glycoproteins had a significantly longer mean residence time in a rabbit model of pharmacokinetics. These experiments demonstrate the feasibility of genetically engineering cell lines to produce therapeutics with desired glycosylation patterns.     

Characterization and in vitro expression of non-structural 1 protein of canine parvovirus (CPV-2) in mammalian cell line

Parvoviruses are small, 260-A-diameter, icosahedral, non-enveloped, single-stranded DNA viruses with a genome of approximately 5 kb. Non structural protein, (NS-1) is especially relevant, being both essential for virus replication and the main factor responsible for virus pathogenicity and cytotoxicity. This protein has also been reported to possess the property of killing of transformed cells. The present study was carried out to clone, characterize and express the NS-1 gene of canine parvovirus. NS-1 complete CDS 2020bp was amplified, cloned into eukaryotic expression vector pcDNA 3.1(+), sequenced and characterized by in vitro expression analysis. Functional activity of recombinant construct, pcDNA.cpv.NS-1, was evaluated by RT-PCR and flow cytometry for the expression of NS-1 specific mRNA and NS-1 protein, respectively, in transfected HeLa cells. This recombinant plasmid may serve as an important tool to evaluate the apoptotic potential of NS-1 protein of canine parvovirus in cultured HeLa cells.     

A Highlights from MBoC Selection: DynaMorph: self-supervised learning of morphodynamic states of live cells

A cell’s shape and motion represent fundamental aspects of cell identity and can be highly predictive of function and pathology. However, automated analysis of the morphodynamic states remains challenging for most cell types, especially primary human cells where genetic labeling may not be feasible. To enable automated and quantitative analysis of morphodynamic states, we developed DynaMorph—a computational framework that combines quantitative live cell imaging with self-supervised learning. To demonstrate the robustness and utility of this approach, we used DynaMorph to annotate morphodynamic states observed with label-free measurements of optical density and anisotropy of live microglia isolated from human brain tissue. These cells show complex behavior and have varied responses to disease-relevant perturbations. DynaMorph generates quantitative morphodynamic representations that can be used to compare the effects of the perturbations. Using DynaMorph, we identify distinct morphodynamic states of microglia polarization and detect rare transition events between states. The concepts and the methods presented here can facilitate automated discovery of functional states of diverse cellular systems.     

Tissue transglutaminase: an enzyme with a split personality.

Tissue transglutaminase (tTG) belongs to the family of transglutaminase enzymes that catalyze the posttranslational modification of proteins via Ca(2+)-dependent cross-linking reactions. The catalytic action of tTG results in the formation of an isopeptide bond that is of great physiological significance since it is highly resistant to proteolysis and denaturants. Although tTG-mediated cross-linking reactions have been implicated to play a role in diverse biological processes, the precise physiological function of the enzyme remains unclear. Recent data, however, suggest that the protein polymers resulting from tTG-catalyzed reactions may play a role in commitment of cells to undergo apoptosis. On the same token, tTG-mediated formation of insoluble protein aggregates may underlie the markers of numerous pathological conditions, such as the senile plaques in Alzheimer's disease and the Lewy bodies in Parkinson's disease. In addition to catalyzing Ca(2+)-dependent cross-linking reactions, tTG can also bind and hydrolyze guanosine triphosphate and adenosine triphosphate. By virtue of this ability, tTG has been identified as a novel G-protein that interacts and activates phospholipase C following stimulation of the alpha-adrenergic receptor. The ability of tTG to mediate signal transduction may contribute to its involvement in the regulation of cell cycle progression. The following review summarizes the important features of this multifunctional enzyme that have emerged as a result of recent work from different laboratories.     

4-Chlorobutanol induces unusual reversible and irreversible thermal unfolding of ribonuclease A: thermodynamic, kinetic, and conformational characterization

The thermal denaturation of ribonuclease A has been studied by differential scanning calorimetry in the presence of 4-chlorobutan-1-ol. The thermal transitions were observed to be reversible at pH 5.5 in the presence of low concentration (up to 50 mM) of the alcohol, irreversible in the intermediate (50 mM < c < mM) and again reversible in the presence of 250 mM and higher concentrations of 4-chlorobutan-1-ol. In the presence of 50 mM 4-chlorobutan-1-ol, ribonuclease A is present in two conformational states unfolding at different temperatures. The reversible thermal transitions have been fitted to a two-state native-to-denatured mechanism. Irreversible thermal transitions have been analyzed according to two-state irreversible native-to-denatured kinetic model. Using the irreversible model, rate constant as a function of temperature and energy of activation of the irreversible process have been calculated. Circular dichroism and fluorescence spectroscopic results corroborate the DSC observations and indicate a protein conformation with poorly defined tertiary structure and high content of secondary structure in the presence of 50 mM 4-chlorobutan-1-ol at a temperature corresponding to the second transition. Similar results have been observed at pH 3.9.     

Rapid host defense against Aspergillus fumigatus involves alveolar macrophages with a predominance of alternatively activated phenotype

The ubiquitous fungus Aspergillus fumigatus is associated with chronic diseases such as invasive pulmonary aspergillosis in immunosuppressed patients and allergic bronchopulmonary aspergillosis (ABPA) in patients with cystic fibrosis or severe asthma. Because of constant exposure to this fungus, it is critical for the host to exercise an immediate and decisive immune response to clear fungal spores to ward off disease. In this study, we observed that rapidly after infection by A. fumigatus, alveolar macrophages predominantly express Arginase 1 (Arg1), a key marker of alternatively activated macrophages (AAMs). The macrophages were also found to express Ym1 and CD206 that are also expressed by AAMs but not NOS2, which is expressed by classically activated macrophages. The expression of Arg1 was reduced in the absence of the known signaling axis, IL-4Rα/STAT6, for AAM development. While both Dectin-1 and TLR expressed on the cell surface have been shown to sense A. fumigatus, fungus-induced Arg1 expression in CD11c(+) alveolar macrophages was not dependent on either Dectin-1 or the adaptor MyD88 that mediates intracellular signaling by most TLRs. Alveolar macrophages from WT mice efficiently phagocytosed fungal conidia, but those from mice deficient in Dectin-1 showed impaired fungal uptake. Depletion of macrophages with clodronate-filled liposomes increased fungal burden in infected mice. Collectively, our studies suggest that alveolar macrophages, which predominantly acquire an AAM phenotype following A. fumigatus infection, have a protective role in defense against this fungus.     

Improvement of stability and enzymatic activity by site-directed mutagenesis of E. coli asparaginase II

Bacterial asparaginases (EC 3.5.1.1) have attracted considerable attention because enzymes of this group are used in the therapy of certain forms of leukemia. Class II asparaginase from Escherichia coli (EcA), a homotetramer with a mass of 138 kDa, is especially effective in cancer therapy. However, the therapeutic potential of EcA is impaired by the limited stability of the enzyme in vivo and by the induction of antibodies in the patients. In an attempt to modify the properties of EcA, several variants with amino acid replacements at subunit interfaces were constructed and characterized. Chemical and thermal denaturation analysis monitored by activity, fluorescence, circular dichroism, and differential scanning calorimetry showed that certain variants with exchanges that weaken dimer–dimer interactions exhibited complex denaturation profiles with active dimeric and/or inactive monomeric intermediates appearing at low denaturant concentrations. By contrast, other EcA variants showed considerably enhanced activity and stability as compared to the wild-type enzyme. Thus, even small changes at a subunit interface may markedly affect EcA stability without impairing its catalytic properties. Variants of this type may have a potential for use in the asparaginase therapy of leukemia.