THE SEPARATION OF DIFFERENT CELL CLASSES FROM LYMPHOID ORGANS : III. The Purification of Erythroid Cells by pH-Induced Density Changes

1. Mammalian erythrocytes swell as the pH of the isotonic suspending medium is lowered, as a direct consequence of the specialized permeability properties of the erythrocyte membrane. Lymphocytes and granulocytes from a variety of sources did not exhibit this property. 2. The behaviour of mouse bone marrow erythroid cells at various stages of differentiation was studied by using a change in buoyant density with pH as an index of swelling. The ability to swell with a pH drop was acquired while the cell was still nucleated. All non-nucleated cells showed swelling. Most small erythroblasts shared this property, whereas most large erythroblasts did not. 3. The density shift with pH was used to provide a purification scheme specific for erythroid cells. The bone marrow cells were first centrifuged to equilibrium in an isotonic albumin density gradient at neutral pH. Regions of the gradient containing the erythroid cells were collected, and the cells were recovered and redistributed in an albumin gradient at acid pH. The erythroid cells showed a specific density shift which removed them from contaminants. Preparations containing 90–97% erythroblasts were obtained by this technique. 4. Differentiation within the erythroid series was accompanied by a general increase in cell buoyant density at neutral pH. This density increase may have been a discontinuous process, since erythroid cells appeared to form a number of density peaks. 5. The pH shift technique, in association with established density distribution and sedimentation velocity procedures, provides a range of cell separation techniques for biological or biochemical studies of erythroid cell differentiation in the complex cell mixtures in bone marrow or spleen.     

Establishment and characterization of a human malignant schwannoma cell line (HKMS)

The malignant schwannoma cell line (HKMS) was established from the subcutaneous tumor of Axilla region of a 48-year-old Japanese woman. The HKMS line has the following biological properties. 1. The HKMS cells were spindle in shape and showed neoplastic and pleomorphic features. The monolayer sheet of HKMS cells showed the resemble cell-arrangement with that of the original tumor tissue. 2. The cells showed a stable growth and the serial passages were successively carried out 150 times within 3 years. Their population doubling time is about 40 hours. 3. The chromosome number varied widely, and the modal number was stable at the 78-80. The marker chromosomes were present. 4. The cells were transplanted into the subcutis of nude mice and produced the malignant schwannoma.     

Establishment and characterization of human neuroblastoma cell line (HSNB)

We cultured an aspiration fluid of the sternal bone marrow of the patient having adrenal neuroblastoma and established a neuroblastoma cell line (HSNB). The HSNB line has the following biological properties. 1. They are small round in shape and proliferate in flotation while forming cell aggregate, and often they attach the bottom of plastic dish and process the nerve-like fibers. A rough-endoplasmic reticulum are poorly developed, however, a lot of free ribosomes are scattered in the cytoplasm. In the peripheral area of the cells, small spherical secretory granules (60-140 nm in diameter) are existed. One characteristic of this cell is existence of microtubules in the cell-projections. 2. They show a stable growth and the doubling time is about 50 hours. 3. Their chromosome number varied widely and the mode is 46. The double minute chromosomes were present in 50% of cells. 4. When they are transplanted in the cheek pouch of hamster, they produced the neuroblastoma. 5. They produce neuron specific enolase. 6. N-myc gene was amplified ca 250 folds.     

Mechanism of arachidonic acid liberation in platelet-activating factor-stimulated human polymorphonuclear neutrophils

Upon stimulation of human polymorphonuclear neutrophils with platelet-activating factor (PAF), arachidonic acid (AA) is released from membrane phospholipids. The mechanism for AA liberation, a key step in the synthesis of biologically active eicosanoids, was investigated. PAF was found to elicit an increase in the cytoplasmic level of free Ca2+ as monitored by fluorescent indicator fura 2. When [3H] AA-labeled neutrophils were exposed to PAF, the enhanced release of AA was observed with a concomitant decrease of radioactivity in phosphatidylinositol and phosphatidylcholine fractions. The inhibitors of phospholipase A2, mepacrine and 2-(p-amylcinnamoyl)-amino-4-chlorobenzoic acid, effectively suppressed the liberation of [3H]AA from phospholipids, indicating that liberation of AA is mainly catalyzed by the action of phospholipase A2. The extracellular Ca2+ is not required for AA release. However, intracellular Ca2+ antagonists, TMB-8 and high dose of quin 2/AM drastically reduced the liberation of AA induced by PAF, indicating that Ca2+ is an essential factor for phospholipase A2 activation. PAF raised the fluorescence of fura 2 at concentrations as low as 8 pM which reached a maximal level about 8 nM, whereas more than nM order concentrations of PAF was required for the detectable release of [3H]AA. Pretreatment of neutrophils with pertussis toxin resulted in complete abolition of AA liberation in response to PAF. However, the fura 2 response to PAF was not effectively inhibited by toxin treatment. In human neutrophil homogenate and membrane preparations, guanosine 5'-O-(thiotriphosphate) stimulated AA release and potentiated the action of PAF. Guanosine 5'-O-(thiodiphosphate) inhibited the effects of guanosine 5'-O-(thiotriphosphate). These results suggest several points: 1) PAF stimulates human polymorphonuclear neutrophils to liberate AA mainly by the action of phospholipase A2; 2) Ca2+ mobilization alone is not sufficient to stimulate AA release, although Ca2+ is the important factor for phospholipase A2 activation; and 3) a pertussis toxin-sensitive GTP-binding protein may be implicated in activation of phospholipase A2.     

Regionally selective alterations in enzymatic activities and metabolic fluxes during thiamin deficiency

To further elucidate the molecular basis of the selective damage to various brain regions by thiamin deficiency, changes in enzymatic activities were compared to carbohydrate flux through various pathways from vulnerable (mammillary bodies and inferior colliculi) and nonvulnerable (cochlear nuclei) regions after 11 or 14 days of pyrithiamin-induced thiamin deficiency. After 11 days,large decreases (–43 to –59%) in transketolase (TK) occurred in all 3 regions; 2-ketoglutarate dehydrogenase (KGDHC) declined (–45%), but only in mammillary bodies; pyruvate dehydrogenase (PDHC) was unaffected. By day 14, TK remained reduced by 58%–66%; KGDHC was now reduced in all regions (–48 to –55%); PDHC was also reduced (–32%), but only in the mammillary bodies. Thus, the enzyme changes did not parallel the pathological vulnerability of these regions to thiamin deficiency.¹⁴CO₂ production from¹⁴C-glucose labeled in various positions was utilized to assess metabolic flux. After 14 days, CO₂ production in the vulnerable regions declined severely (–46 to 70%) and approximately twice as much as those in the cochlear nucleus. Also by day 14, the ratio of enzymatic activity to metabolic flux increased as much as 56% in the vulnerable regions, but decreased 18 to 30% in the cochlear nuclei. These differences reflect a greater decrease in flux than enzyme activities in the two vulnerable regions. Thus, selective cellular responses to thiamin deficiency can be demonstrated ex vivo, and these changes can be directly related to alterations in metabolic flux. Since they cannot be related to enzymatic alterations in the three regions, factors other than decreases in the activity of these TPP-dependent enzymes must underlie selective vulnerability in this model of thiamin deficiency.Abbreviations KGDHC 2-ketoglutarate dehydrogenase complex EC 1.2.4.2., EC 2.3.1.61, EC 1.6.4.3. - PDHC pyruvate dehydrogenase complex EC 1.2.4.2., EC 2.3.1.12, EC 1.6.4.3 - TK transketolase (EC 2.2.1.1) - TPP thiamin pyrophosphate     

Identification of cellular differentiation-dependent nuclear factors that bind to a human gene for thymidylate synthase.

Three nuclear factors were identified that interact with sequences in the 5'-upstream region of the human thymidylate synthase gene. Two of these factors interact with a sequence around the initiation codon of the thymidylate synthase gene. The amounts of these two factors changed dramatically as human promyelocytic leukemia HL-60 cells differentiated into macrophage-like cells by the treatment with 1,25-dihydroxyvitamin D3. The change was closely correlated with the decrease in the amount of thymidylate synthase mRNA during the differentiation. These findings suggest that the specific nuclear factors are involved in the regulation of the expression of human thymidylate synthase gene during the differentiation of HL-60 cells.     

Enhancing effect of wortmannin on muscarinic stimulation of phospholipase D in rat pheochromocytoma PC12 cells.

Wortmannin, a specific inhibitor of myosin light chain kinase (MLCK), enhanced carbachol-induced formation of [3H]phosphatidylethanol ([3H]PEt), a marker of phospholipase D (PLD) activity, in [3H]palmitic acid-labeled PC12 cells. The apparent EC50 value was 1.5 microM, and the effect was maximal at 3 microM and slightly attenuated at higher concentration. Wortmannin alone had no significant effect on [3H]PEt formation. The enhancing effect of wortmannin was observed at the initial increasing phase of [3H]PEt formation but not at the subsequent plateau phase. Wortmannin enhanced also phorbol ester-induced PLD activation. Although the precise mechanism remains to be clarified, these results suggest that MLCK may be involved in PLD regulation in PC12 cells.     

Production of a herbicide, 5-aminolevulinic acid,by Rhodobacter sphaeroides using the effluent of swine waste from an anaerobic digestor

Summary For the production of a herbicide, 5-amino-levulinic acid (ALA), from anaerobic digestion liquor, the utilization of the photosynthetic bacterium, Rhodobacter sphaeroides was examined. This bacterium could produce ALA extracelularly from this liquor with the addition of levulinic acid (LA), an inhibitor of ALA dehydratase (ALAD), and glycine, a precursor of ALA biosynthesis in the Shemin pathway. Succinate (another precursor) addition was unnecessary for ALA production. When repeated additions of LA were made together with glycine ALA production was significantly enhanced. However, above three additions of LA, ALA production was not further enhanced. The maximum value of ALA production attained was 4.2 mM (0.63 g/ 1), which was over double that of other ALA producers such as Chlorella vulgaris. Propionic acid was predominantly utilized compared with other lower fatty acids, suggesting that this might be converted to ALA via succinyl-coenzyme A (CoA) in the methylmalonyl-CoA pathway.Offprint requests to: Y. Nishizawa     

Membrane enzyme reactor with simultaneous separation using electrophoresis

A membrane enzyme reactor with simultaneous separation was investigated. Enzymes, urease and aspartase, were immobilized by a porous polytetrafluoroethylene membrane. Electrical field was applied in the medium while the reaction was carried out. Products with electrical charge could be separated through the membrane from the reaction medium as they were formed. Reaction behavior was analyzed by a simple model considering both pore-migration and reaction in the skelton of the membrane. According to the analysis the inherent reaction rate of the immobilized enzymes decreases significantly. This is probably caused by the structural variation of enzymes. For the case of urease, the change of pH inside the membrane may also cause the decrease of the reaction rate. The model analysis showed that the enzyme content in the membrane and the residence time of the substrate in the membrane governed overall extent of reaction.List of Symbols e g (dm³)^–1 enzyme concentration in the membrane - L cm membrane thickness - K _m mM Michaelis constant - Rate mmol · min^–1 · g^–1 rate of product formation per unit weight of enzyme - S mM substrate concentration - S _in mM inlet substrate concentration - S _out mM outlet substrate concentration - u cm · min^–1 migration rate - V V voltage between the electrodes - V _m mmol · min^–1 · g^–1 maximum reaction rate - X conversion - z cm distance from the surface inside the membrane - void fraction of the porous membrane - tortuosity of the membrane - min space time