Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0026764 (multiple myeloma)
 36,148 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A calcium-binding immunoglobulin G (IgG1 lambda RUP) was identified in the serum of a patient with multiple myeloma, asymptomatic hypercalcemia, and a normal ionized serum calcium. Calcium binding by IgGRUP was confirmed by two-dimensional electrophoresis with calcium-45 and equilibrium dialysis. Amino acid analyses indicated an unusually high number of glutamic (or glutamine) residues in the L chain and Fab fragment but no detectable gamma-carboxyglutamic acid. As determined by equilibrium dialysis with 45Ca, the intact IgGRUP and its Fab fragments bound calcium at an optimum pH of 7.4. There was minimal binding of calcium to H chains and no binding by L chains or the Fc fragment. Recombination of H and L chains partially restored the binding activity. By Scatchard analysis, the binding affinity (Kd) of IgGRUP was 1.7 X 10(-3) M and the binding capacity was 4 mol of calcium/mol of IgG. The binding of 4 mol of calcium/mol of IgG is twice that reported previously for two other calcium-binding myeloma proteins and suggests unique properties of IgGRUP.
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PMID:A human myeloma immunoglobulin G binding four moles of calcium associated with asymptomatic hypercalcemia. 673 8

We have constructed an expression vector that leads to secretion of the whole Fc of human immunoglobulin E (hIgE-Fc) from mammalian cells at levels up to 100 mg/l of culture. Two surface glycosylation sites at Asn265 and Asn371 have been changed to glutamine, to obtain a more homogeneous preparation of hIgE-Fc for structural studies. Comparison of wild-type and mutant products revealed that Asn371 is rarely glycosylated in Chinese hamster ovary cells. Both the double mutant and wild-type hIgE-Fc bind to the high-affinity IgE receptor, Fc epsilon RI, with about the same affinity as myeloma IgE (Ka in the range 10(10)-10(11) M-1), and were able to sensitize isolated human basophils for anti-IgE triggering of histamine release. However, only the double mutant hIgE-Fc approached the affinity of myeloma IgE for the low-affinity receptor, Fc epsilon RII (Ka = 7.3 x 10(7) M-1), whereas the wild-type hIgE-Fc bound with a 10-fold lower affinity (Ka = 4.1 x 10(6) M-1).
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PMID:Secretion of recombinant human IgE-Fc by mammalian cells and biological activity of glycosylation site mutants. 754 6

Multiple myeloma (MM) is a late-stage B-cell cancer with an unknown etiology. Activating mutations of the N-ras and K-ras oncogenes occur with a high frequency in myeloma and, therefore, may play a role in the pathogenesis of the disease. To study the role of N-ras-activating mutations in the regulation of myeloma tumor growth, we introduced a constitutively active N-ras cDNA containing a glutamine to arginine (CAA-CGA) amino acid substitution at codon 61 into the interleukin 6 (IL-6)-dependent myeloma cell line ANBL6. Expression of the mutant N-ras cDNA resulted in significant IL-6-independent growth, as well as augmentation of growth at suboptimal concentrations of IL-6. The IL-6-independent growth pattern was not the result of activation of autocrine IL-6 production in the mutant N-ras-expressing population because neutralizing antibodies to the IL-6 receptor and to IL-6 had no effect on the rate of DNA synthesis in the absence of IL-6. Furthermore, mutant N-ras expression decreased the percentage of cells undergoing apoptosis in the absence of IL-6. These data suggest that activating mutations of the ras oncogenes may result in growth factor independence accompanied by a suppression of apoptosis in MM. Therefore, the use of therapies designed to block IL-6 action in MM may have less of an impact on tumors bearing activated ras mutations.
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PMID:Introduction of an activated N-ras oncogene alters the growth characteristics of the interleukin 6-dependent myeloma cell line ANBL6. 762 74

A model for transport of ammonia and ammonium ions across cell membranes is presented. The model suggests that ammonium ions compete with potassium ions for inward transport, over the cytoplasmic membrane, via potassium transport proteins like the Na+/K(+)-ATPase and the Na+K+2Cl(-)-cotransporter. It also explains the difference between the ammonia/ammonium that is added to the cells and which is formed by the cells during metabolism of amino acids, especially glutamine and glutamate. The ammonium transport and subsequent events lead to predictable intracellular and extracellular pH (pHe) changes. Experiments which verified the model and the predicted consequences were performed by measurements of the pHe in concentrated cell suspensions. Addition of ammonium ions caused a time-dependent pHe increase which was inhibited by potassium ions. The test system is not per se specific for transport measurements but the effect of potassium ions on the pHe strongly favors our suggested model. Simple diffusion of ammonium ions would not be counteracted by potassium ions. The results show that ammonium ion transport in the murine myeloma cell line (Sp2/0-Ag14) used is inhibited by an excess of potassium ions. Results from experiments with specific inhibitors of suggested transport proteins were not conclusive. It is postulated that one important toxic effect of ammonia/ammonium is an increased demand for maintenance energy, caused by the need to maintain ion gradients over the cytoplasmic membrane. The results also suggest that potassium ions can be used to detoxify ammonia/ammonium in animal cell cultivations.
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PMID:Mechanisms of ammonia and ammonium ion toxicity in animal cells: transport across cell membranes. 776 10

We have developed several approaches to create cell lines with improved characteristics in cell culture. In some cases it has been possible to isolate natural variants with useful properties. Cholesterol independent variants of the mouse NSO myeloma cell line were isolated by cloning in a selective medium. A glutamine independent variant of a hyridoma was isolated by continuous (chemostat) culture under glutamine limited conditions in the presence of glutamate. Choline independent cells were isolated from a choline limited chemostat. In an alternative approach to modifying cell behaviour, we have used recombinant DNA techniques to introduce the glutamine synthetase (GS) gene to a hybridoma. This resulted in glutamine independence and increased productivity.
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PMID:Selecting and designing cell lines for improved physiological characteristics. 776 22

It is now well documented that apoptosis represents the prevalent mode of cell death in hybridoma cultures. Apoptotic or programmed cell death occurs spontaneously in late exponential phase of batch cultures. Until lately, no specific triggering factors had been identified. Recently, we observed that glutamine, cystine or glucose deprivation induced apoptosis in both hybridoma and myeloma cell lines whereas accumulation of toxic metabolites induced necrotic cell death in these cells. Other triggering factors such as oxygen deprivation might also be responsible for induction of apoptosis. In the present study, induction of cell death by exposure to anoxia was examined in batch culture of the SP2/0-derived hybridoma D5 clone. The mode of cell death was studied by morphological examination of acridine orange-ethidium bromide stained cells in a 1.5 L bioreactor culture grown under anoxic conditions for 75 hours. Under such conditions, viable cell density levelled off rapidly and remained constant for 25 hours. After 45 hours of anoxia, cell viability had decreased to 30% and the dead cell population was found to be 90% apoptotic. In terms of cellular metabolism, anoxia resulted in an increase in the utilization rates of glucose and arginine, and in a decrease in the utilization rate of glutamine. The lactate production rate and the yield of lactate on glucose increased significantly while the MAb production rate decreased. These results demonstrate that glycolysis becomes the main source of energy under anoxic conditions. Cells incubated for 10 hours or less under anoxic conditions were able to recuperate almost immediately and displayed normal growth rates when reincubated in oxic conditions whereas cells incubated for 22 hours or more displayed reduced growth rates. Nonetheless, even after 22 h or 29 h of anoxia, cells reincubated in oxic conditions showed no further progression into apoptosis. Therefore, upon removal of the triggering signal, induction of apoptosis ceased.
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PMID:Induction of apoptosis in oxygen-deprived cultures of hybridoma cells. 776 24

The variability in expression of recombinant proteins has been analyzed with regard to (a) comparison of clones from the same transfection experiment; (b) comparison of the same genetic construct in different cell lines; (c) the effect of the culture system used (free suspension, aggregate suspension, and microcarrier); and (d) physiochemical parameters in long-term (100d) culture in a macroporous fixed bed bioreactor (FBR). Differences in product expression between clones were accompanied by differences in growth rates, metabolic kinetics, and ability to grow in suspension as opposed to attached culture. The single most important factor affecting product expression when comparing constructs (for SEAP and IgG), cell lines (BHK 21 and myeloma), and culture systems was whether cells were grown in an attached or suspension mode. Thus key factors could be related to cell morphology (suspension versus monolayer), the presence of microenvironments and physiological stress to control growth rate. The relationship of key process parameters to volumetric and specific rAb productivity of the FBR was investigated in a partial factorial experiment with a rBHK cell line. The highest productivity levels are associated with a combination of the highest values tested for re-cycle (195 ml min-1) and dilution rates (1 d-1) and glutamine concentration (2.5 mmol 1-1), plus the lowest values for bead size (2 mm) and inoculum density (10(7) m1-1). Together with data from fluidised bed cultures, these results suggest that higher productivity is not primarily the result of greater cell numbers within the system but more the physicochemical definition of the system.
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PMID:Cultural and physiological factors affecting expression of recombinant proteins. 776 43

The effects of the glucose supply on growth and metabolism of an SP2/0 derived recombinant myeloma cell line were studied in chemostat culture during growth on IMDM medium at a fixed dilution rate of 0.032 h-1. Lowering of the feed medium glucose concentration from 25.0 to 1.4 mmol/L resulted in a decrease of steady-state viable cell concentration from 1.9 x 10(9) to 1.0 x 10(9) L-1, whereas viability remained above 90%. Mass balances indicated that only a minor amount of glucose was utilized via the TCA cycle irrespective of the glucose concentration in the feed medium. The apparent biosynthetic yield of cells from ATP was independent of the ratio between the specific glucose and glutamine consumption rate. It is concluded that the primary role of glucose is the provision of intermediates for anabolic reactions. In addition, glucose may play an indirect catabolic role in the process of glutaminolysis by providing the pyruvate for the transamination of glutamate to alanine and alpha-ketoglutarate. At low glucose concentrations in the feed medium, glutamine is probably the sole energy source for this myeloma in chemostat culture.
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PMID:Effects of glucose supply on myeloma growth and metabolism in chemostat culture. 782 30

Human gelatinase A, a member of the matrix metalloproteinase family, is secreted from cells as the M(r) 72,000 latent precursor, progelatinase A. The autolytic removal of an N-terminal propeptide generates the M(r) 66,000 active form. Mutants of recombinant progelatinase A, altered such that the proposed active site glutamic acid residue (E375) was replaced by either an aspartic acid (proE375-->D), an alanine (proE375-->A) or a glutamine (proE375-->Q), were purified from medium conditioned by transfected NS0 mouse myeloma cells. Like wild-type progelatinase A, the mutant proenzymes were inactive and could bind tissue inhibitor of metalloproteinases (TIMP)-2 but not TIMP-1 to their C-terminal domains. Their rates of autolytic processing induced by the organomercurial (4-aminophenyl) mercuric acetate, however, were markedly slower and, of the three M(r) 66,000 forms so produced, only E375-->D displayed any proteolytic activity against either a synthetic substrate (kcat/Km = 10% that of the wild-type enzyme) or denatured type I collagen (specific activity = 0.9% that of the wild-type enzyme). ProE375-->A and proE375-->Q could be more rapidly processed to their M(r) 66,000 forms by incubation with a deletion mutant of gelatinase A that has full catalytic activity but lacks the C-terminal domain [delta (418-631) gelatinase A]. These two M(r) 66,000 forms displayed low activity on a gelatin zymogram (approximately 0.01% that of the wild-type enzyme) but, like E375-->D were able to bind TIMP-1 with an affinity equal to that of the activated wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mutation of the active site glutamic acid of human gelatinase A: effects on latency, catalysis, and the binding of tissue inhibitor of metalloproteinases-1. 791 25

Pigpen is a 67-kDa Sepharose-binding molecule isolated from mammalian endothelial and retinal pigmented epithelial cells. The protein is distributed nonhomogeneously in the nucleus, exhibiting diffuse staining throughout (excluding nucleoli), together with a small number of intensely stained focal points, or granules, and punctate staining along the nuclear envelope. Pigpen was absent or greatly attenuated in the nonepithelial cell types we examined, including fibroblasts, myeloma, and astroglia. cDNA sequence analysis revealed a positively charged molecule with an RNP-CS RNA-binding domain, 19 RGG repeats, and a consensus tyrosine phosphorylation site in the C-terminus. The amino terminal portion of the molecule is characterized by 7 glutamine-rich hexapeptide repeats similar to those found in the transactivation domain of known transcription activators. Pigpen has a high level of identity with the FUS gene product, TLS (Translocated in Liposarcoma; Crozat et al, 1993; Rabbits et al., 1993), a new member of the EWS family of proteins. Expression of pigpen is regulated during the transition between active and quiescent endothelial cell phenotypes. Both mRNA and overall protein levels are maintained at a steady level in actively growing cells. The number of nuclear granules increases as cultures approach confluency. When cells reach confluency, overall expression is sharply reduced and the number of nuclear focal points declines gradually. We observed that reactivation of endothelial cells locally by wounding of confluent cultures resulted in a spatially restricted reactivation of pigpen expression. This pattern of expression, taken together with structural data, suggests that pigpen may function in the growth and differentiation of endothelial cells during angiogenesis.
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PMID:A nuclear protein regulated during the transition from active to quiescent phenotype in cultured endothelial cells. 863 1


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