PubMed · 1237009
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S Inutsuka, S Araki, I Kusaba, J Fujimoto, M Tsuchida. 1975. [Note on leukocyte separation (author's transl)].. https://pubmed.ncbi.nlm.nih.gov/1237009/
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We describe a temperature-control system for solutions in free flow, suitable for electrophysiological or optical studies of isolated cells, natural epithelia or cell culture monolayers. The system is small enough to be located close to the preparation and was designed specifically to be coupled to the inlets of a modified, continuous-flow Ussing chamber, allowing rapid change of the solutions bathing tissue surfaces. The system consists of a highly compact monoblock heating unit and a control circuit. Solutions from different reservoirs, kept at room temperature or lower (from an ice bath), can be rapidly switched at the inlet of the heating unit by manually or electrically actuated microvalves without affecting the temperature of the fluid leaving the heating unit. The control unit consists of a bead thermistor firmly placed close to the heating unit outlet and an electronic circuit which is basically a proportional controller. This unit continuously regulates the electric current through the Ni-Cr heater, keeping the temperature of the fluid leaving the heating unit constant at a preset value. The system allows control of fluid temperature (normally 37 degrees C) for flow rates in the range of 1.0 ml/min to 12 ml/min. However, the temperature can be set at any value above that of the incoming fluid.
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In driving T cell proliferation, IL-2 stimulates a new program of gene expression that includes proliferating cell nuclear antigen (PCNA), a requisite processivity factor for DNA polymerase delta. PCNA transcription is regulated in part through tandem CRE sequences in the promoter and CRE binding proteins; IL-2 stimulates CREB phosphorylation in the resting cloned T lymphocyte, L2. After culturing L2 cells for greater than 91 days, we consistently isolate a stable variant that exhibits constitutive CREB phosphorylation. L2 and L2 variant cells were tested for IL-2 responsiveness and rapamycin sensitivity with respect to specific kinase activity, PCNA expression and proliferation. In L2 cells, IL-2 stimulated and rapamycin inhibited the following: cAMP-independent CREB kinase activity, PCNA expression and proliferation. In L2 variant cells, CREB kinase activity was constitutively high; IL-2 stimulated and rapamycin blocked PCNA expression and proliferation. These results indicate that IL-2 induces a rapamycin-sensitive, cAMP-independent CREB kinase activity in L2 cells. However, phosphorylation of CREB alone is not sufficient to drive PCNA expression and L2 cell proliferation in the absence of IL-2.