NSO myeloma cell death: Influence of bcl-2 overexpression

The productivity of recombinant mammalian cell lines growth in batch culture is often limited by the rapidity with which cells die on entry into the decline phase (the period of culture after the maximum cell density has been reached and where cell viability begins to fall). We examined the decline phase characteristics of the NSO myeloma cell line with a view to modulating the cell death that ensues. Examination of nuclear morphology during culture revealed that the onset of the decline phase was marked by a time-dependent increase in the percentage of cells that exhibited condensed and fragmented nuclei. Furthermore, these changes coincided with a fall in DNA integrity. High molecular weight DNA appeared to be degraded into oligonucleosomal fragments. Taken together, these observations indicated that NSO cells die by the process of apoptosis. The protein encoded by the bcl-2 gene has been shown to counter apoptosis induced by a large variety of stimuli and in a number of different cell types, but is not expressed in NSO cells. We examined whether overexpression of this protein could prevent/delay the onset of cell death seen during batch culture and also in response to serum limitation. Bcl-2 failed to affect the decline phase characteristics and serum dependence of NSO cells. In our search to explain these findings, we found that the NSO cell line expresses bax and also a high level of another Bcl-2 related protein, Bcl-x(L). Given that Bcl-X(L) is a sequence and functional homologue of Bcl-2, it is possible that Bcl-2 is redundant in the NSO cell background. These data therefore indicate that cells such as NSO, which are used in biotechnologically important processes such as generation of hybridomas and expression of recombinant proteins, may express only a subset of genes important in apoptotic regulation. Modulation of the death characteristics of such cells will need to take account of the expression profile of such genes and their regulatory interactions. (c) 1996 John Wiley & Sons, Inc.     

Nuclear pore clustering is a consistent feature of apoptosis in vitro

Two cell lines which show different patterns of DNA fragmentation have been examined for variations of their nuclear morphology during apoptosis. FDCP-Mix, a pluripotent murine haemopoietic stem cell line which undergoes typical internucleosomal cleavage of DNA when induced to apoptosis either by drugs or withdrawal of growth factor (IL-3) was compared with the human lymphoid leukemia cell line MOLT-4, a cell line which undergoes apoptosis without production of a typical DNA 'ladder'. The nuclear morphology of FDCP-Mix cells was consistent after apoptotic induction by drug or by growth factor withdrawal. Apoptotic nuclear morphology for MOLT-4 and FDCP-Mix showed variations in the distribution, density and texture of the electron dense nuclear marginations. Despite these differences, clustering of nuclear pore complexes (NPCs) after treatment with the topoisomerase II inhibitor etoposide was a common phenomenon for both cell lines. Moreover, pore clustering for FDCP-Mix nuclei occurred independently from the way in which apoptosis was induced, either by growth factor withdrawal or etoposide treatment. In a novel approach, we visualised the clustering of NPCs three-dimensionally by field emission in-lens scanning electron microscopy (FEISEM).     

A Novel Triazolopyridine-Based Spleen Tyrosine Kinase Inhibitor That Arrests Joint Inflammation

Autoantibodies and the immunoreceptors to which they bind can contribute to the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA). Spleen Tyrosine Kinase (Syk) is a non-receptor tyrosine kinase with a central role in immunoreceptor (FcR) signaling and immune cell functionality. Syk kinase inhibitors have activity in antibody-dependent immune cell activation assays, in preclinical models of arthritis, and have progressed into clinical trials for RA and other autoimmune diseases. Here we describe the characterization of a novel triazolopyridine-based Syk kinase inhibitor, CC-509. This compound is a potent inhibitor of purified Syk enzyme, FcR-dependent and FcR-independent signaling in primary immune cells, and basophil activation in human whole blood. CC-509 is moderately selective across the kinome and against other non-kinase enzymes or receptors. Importantly, CC-509 was optimized away from and has modest activity against cellular KDR and Jak2, kinases that when inhibited in a preclinical and clinical setting may promote hypertension and neutropenia, respectively. In addition, CC-509 is orally bioavailable and displays dose-dependent efficacy in two rodent models of immune-inflammatory disease. In passive cutaneous anaphylaxis (PCA), CC-509 significantly inhibited skin edema. Moreover, CC-509 significantly reduced paw swelling and the tissue levels of pro-inflammatory cytokines RANTES and MIP-1α in the collagen-induced arthritis (CIA) model. In summary, CC-509 is a potent, moderately selective, and efficacious inhibitor of Syk that has a differentiated profile when compared to other Syk compounds that have progressed into the clinic for RA.     

An Anti-β-Amyloid Vaccine for Treating Cognitive Deficits in a Mouse Model of Down Syndrome

In Down syndrome (DS) or trisomy of chromosome 21, the β-amyloid (Aβ) peptide product of the amyloid precursor protein (APP) is present in excess. Evidence points to increased APP gene dose and Aβ as playing a critical role in cognitive difficulties experienced by people with DS. Particularly, Aβ is linked to the late-life emergence of dementia as associated with neuropathological markers of Alzheimer’s disease (AD). At present, no treatment targets Aβ–related pathogenesis in people with DS. Herein we used a vaccine containing the Aβ 1–15 peptide embedded into liposomes together with the adjuvant monophosphoryl lipid A (MPLA). Ts65Dn mice, a model of DS, were immunized with the anti-Aβ vaccine at 5 months of age and were examined for cognitive measures at 8 months of age. The status of basal forebrain cholinergic neurons and brain levels of APP and its proteolytic products were measured. Immunization of Ts65Dn mice resulted in robust anti-Aβ IgG titers, demonstrating the ability of the vaccine to break self-tolerance. The vaccine-induced antibodies reacted with Aβ without detectable binding to either APP or its C-terminal fragments. Vaccination of Ts65Dn mice resulted in a modest, but non-significant reduction in brain Aβ levels relative to vehicle-treated Ts65Dn mice, resulting in similar levels of Aβ as diploid (2N) mice. Importantly, vaccinated Ts65Dn mice showed resolution of memory deficits in the novel object recognition and contextual fear conditioning tests, as well as reduction of cholinergic neuron atrophy. No treatment adverse effects were observed; vaccine did not result in inflammation, cellular infiltration, or hemorrhage. These data are the first to show that an anti-Aβ immunotherapeutic approach may act to target Aβ-related pathology in a mouse model of DS.     

The purification and properties of pig spleen phosphofructokinase.

Pig spleen phosphofructokinase has been purified 800-fold with a yield of 17%. Two isoenzymes that appear to be kinetically identical can be separated by DEAE-cellulose column chromatography. In common with the enzyme from other mammalian sources, the spleen enzyme has a pH optimum of 8.2. At pH 7.0 it displays sigmoidal kinetics with respect to fructose 6-phosphate concentration but its co-operative behaviour is very dependent on pH, protein concentration and the concentration of MgATP. MgGTP and MgITP can replace MgATP as phosphate donors but, unlike MgATP, these nucleotides do not cause significant inhibition. Mn2+ and Co2+ (as the metal ion-ATP complexes) act as cofactors and in the free form are far more inhibitory than free Mg2+. The spleen enzyme responds to a wide variety of potential effector molecules: ADP, AMP, cyclic AMP, aspartate, NH4+, fructose 6-phosphate, fructose 1,6-diphosphate and Pi all act as either activators or protectors, whereas Mg-ATP, Mg2+, citrate, phosphoenol-pyruvate and the phosphoglucerates are inhibitors.     

Bacteriological Evaluation of an Ultra-Pure Water-Distilling System

A prototype distillation and storage system with recycle for producing ultrapure water was monitored for bacteriological contamination during a period of 24 months. Naturally occurring Pseudomonas aeruginosa and P. cepacia were found to grow rapidly to levels of about 10⁵/ml in water taken from the storage reservoir and also in commercially prepared distilled water. The system was found to eliminate bacterial contaminants introduced into the still with the feed water, but the reservoir, once contaminated, remained contaminated during prolonged recycle. After a single treatment with free chlorine, the entire system remained uncontaminated until accidental or purposeful shutdown.     

OBSERVATIONS ON THE STRUCTURE OF RHODOSPIRILLUM RUBRUM

Sections of Rhodospirillum rubrum cells from cultures of different ages have been examined to obtain information on the development of chromatophores in this organism. Cells from the 12-hour cultures studied contain neither distinct invaginations of the cytoplasmic membrane nor distinct chromatophores. The first structures that can be related to chromatophore development occur peripherally in the cells, are relatively few in number, relatively high in density, and have an indistinct membrane. In cells from 26-hour cultures numerous distinct invaginations of the cytoplasmic membrane are present, and all layers of the cytoplasmic membrane are involved in the formation of each invagination. As the invaginations become more numerous, the ends of the invaginations become constricted to form one or more structures similar to the chromatophores previously described in this organism. Cells of R. rubrum, therefore, develop a structural continuum which initially consists of invaginations of the cytoplasmic membrane, and later of the chromatophores produced by and attached to these invaginations. The presence of this continuum, however, does not necessarily exclude the existence of discrete chromatophores within these cells. Several other structures previously reported in this organism are described in greater detail.     

Structure and Photochemical Activity of Chlorophyll-Containing Particles from Rhodospirillum rubrum

Comparative studies on isolated chromatophores and on sectioned cells of the photosynthetic bacterium Rhodospirillum rubrum confirm the assumption expressed in earlier investigations that the photochemically active chromatophores isolated from disrupted cells represent structural chlorophyll-bearing components of the protoplast. Actively growing cells from light-grown cultures about 12 hours old do not release chromatophores when disrupted in dilute buffers, but do release smaller, chlorophyll-containing structures about 25 mµ in diameter. Sections of such cells do not reveal chromatophores, but contain in the ground cytoplasm numerous particles somewhat smaller in size than the 25 mµ chlorophyll-containing particles released from disrupted cells. Similar particles are obtained by the sonication of isolated chromatophores obtained from cells of 1-day-old cultures. The small, subchromatophore particles described here appear to be functionally complete units which are photochemically active in photo-oxidation, photoreduction, and photophosphorylation, and it is postulated that they represent the basic biochemical and structural components of the chromatophore.