Interleukin 2-induced tyrosine phosphorylation. Interleukin 2 receptor beta is tyrosine phosphorylated

Interaction of interleukin 2 (IL2) with its high affinity membrane receptor complex (IL2R) is sufficient to induce proliferation of T lymphocytes. However, the biochemical mechanisms by which IL2 induces this process remain unresolved. The IL2R complex consists of at least two distinct polypeptides that bind IL2, a 75-kDa intermediate affinity subunit (IL2R beta) and a 55-kDa low affinity subunit (IL2R alpha). As indicated by Western blotting with anti-phosphotyrosine-specific antibodies and confirmed by phosphoamino acid analysis, we now demonstrate that interaction of the T cell growth factor interleukin 2 (IL2) with its high affinity receptor on IL2-sensitive human peripheral blood lymphoblasts induces tyrosine phosphorylation of proteins of 92, 80, 78, 70-75, and 57 kDa. IL2 induced tyrosine phosphorylation in YT 2C2 cells which express only the 75-kDa intermediate affinity IL2 binding molecule (IL2R beta) but not in cells which either express only the 55-kDa low affinity IL2 receptor molecule (IL2R alpha) or no IL2-binding sites. Therefore, IL2R beta, in the absence of IL2R alpha, appears sufficient to transduce the transmembrane signal leading to tyrosine phosphorylation. Two different antibodies reactive with phosphotyrosine specifically immunoprecipitated IL2R beta cross-linked to radiolabeled IL2. These findings suggest that IL2R beta is a substrate for the tyrosine kinase which is activated by IL2 binding to its receptor. Thus, like several other growth factor receptors, activation of the IL2R results in an increase in tyrosine phosphorylation with the receptor itself serving as one substrate.     

Isolation and characterisation of the Xenopus laevis albumin genes: loss of 74K albumin gene sequences by library amplification.

The blood of the frog X.laevis contains 2 albumins of 68,000 and 74,000 daltons which are encoded in the liver by two related mRNAs. When an amplified X.laevis DNA library was screened with cloned albumin cDNA only 68,000 dalton albumin gene sequences were isolated. Hybridisation of the albumin cDNA to Southern-blots of Eco R1 digested X.laevis DNA showed that the sequences present in the recombinants did not account for all the fragments which hybridised on the Southern-blots. This indicated that 74K albumin gene sequences exist but that they are not present in the amplified DNA library. A X.laevis genomic library was therefore constructed and screened for albumin genes without amplification. Both 68K and 74K albumin gene sequences were isolated. Recombinants containing 74K albumin gene sequences grew extremely poorly and this probably explains why the 74K albumin sequences were ot isolated from the amplified library. Characterisation of the cloned DNA indicates that there is one sequence coding for the 68K albumin but two different sequences coding for the 75K albumin.     

Biofeedback treatment of dysmenorrhea

Nine dysmenorrheic women were run in EMG and thermal biofeedback procedures with concurrent autogenic relaxation practice. Significant reductions in subjective estimates of symptomology associated with dysmenorrhea were noted in all subjects. EMG levels correlated positively with the reductions in symptoms. Thermal levels did not correlate with EMG. In fact no consistent patterns in thermal measures were noted. However, thermal biofeedback cannot be ruled out as an effective treatment for dysmenorrhea since reductions in symptoms occurred during thermal biofeedback training. Another significant aspect of the present study is the effectiveness of long treatment procedures. A six month period was employed and significant reductions in symptoms were noted following two months of biofeedback treatment. Finally, the importance of beginning biofeedback treatment prior to onset of menstrual symptoms is indicated.     

Injury and recovery of Bacillus megaterium from mild chlorhexidine treatment

Treatment of Bacillus megaterium cell suspensions with 12 /μmol/1 chlorhexidine diacetate for 5 min led to an approximate 50%, reduction in viability when plated onto tryptone soya agar (TSA). Fifty percent of the surviving fraction were unable to form colonies on TSA containing 5.5% w/v KCI. Such loss of KCl tolerance is indicative of membrane damage, and was recovered within 30 min of incubation in tryptone soya broth (TSB). Multiplication of the damaged organisms did not recommence in this medium until after 60 min. Inclusion of inhibitors of respiration, and of protein, RNA and DNA synthesis in the TSB recovery medium did not significantly affect either the rate or extent of the recovery of KCl tolerance by damaged organisms.     

Reversed-phase high-performance liquid chromatography analysis of 6-thioguanine applicable to pharmacologic studies in humans

6-Thioguanine (6TG) and its metabolites were analyzed in human plasma with a reversed-phase high-performance liquid chromatographic method. 6TG and related compounds were extracted from plasma with an equal volume of 2 N perchloric acid at a 50–100% recovery efficiency. The neutralized extracts were chromatographed on a μBondapak C₁₈ column by two separate isocratic conditions. 6TG, 6-thiouric acid, 6-thioxanthine, 6-thioguanosine, and 6-methylthiouric acid were analyzed with 0.01 M sodium acetate, pH 3.5–10% methanol as the mobile phase and 340 nm for detection. 6-Methylthioguanine and three unknown metabolites were separated with acetate—25% methanol and 310 nm detection. One of the unknowns was identified as 6-methylthioguanosine. External standard calibration was used for quantitation. The 6TG detection limit was 0.8 nmol/ml in plasma.     

The preparation of bile acid amides and oxazolines, II, the synthesis of the amides and oxazolines of ursodeoxycholic acid, deoxycholic acid, hyodeoxycholic acid and cholic acid

Bile acid amides and oxazolines were synthesized by a sequence of steps involving the reaction of the free bile acid with formic acid to yield the formyloxy derivative, preparation of the formyloxy acid chloride, condensation of the acid chloride with 2-amino-2-methyl-1-propanol to give the amide and, finally, cyclization of the amide with thionyl chloride to give the oxazoline. The oxazolines were characterized by physical constants, thin layer and gas-liquid chromatography and identified by elemental analysis and gas-liquid chromatography-mass spectrometry. Some of the bile acid oxazoline derivatives alter the activity of bacterial 7-dehydroxylases $\text{in vitro}$, and inhibit the growth of certain anaerobic bacteria in pure culture.     

Structural variation in the heterochromatin of rye chromosomes in triticales

Summary Although Giemsa C-banding techniques have been used extensively for assaying cereal heterochromatin, a more specific technique for analyzing cereal heterochromatin has been developed recently with the isolation of DNA sequences present in heterochromatin and their employment in in situ hybridization to cereal chromosomes. A number of triticales were examined for the occurrence of modified rye chromosomes using the in situ hybridization technique. With a heterogeneous sequence probe the amount of rye heterochromatin appears to be relatively constant in wheat backgrounds but when a specific sequence probe was employed variation was observed. Whether this variation reflects polymorphism in rye or whether it is a result of adaption of the rye genome to coexistence with the wheat genome in triticales is discussed. — The triticale Rosner was examined in detail and it was established that the rye chromosome 2R had been replaced by the wheat chromosome 2D.     

Measurement of membrane potential inBacillus subtilis: A comparison of lipophilic cations,rubidium ion,and a cyanine dye as probes

Summary Two of the commonly used probes for measuring membrane potential—lipophilic cations and the cyanine dye diS-C₃(5)—indicated nominally opposite results when tetraphenylarsonium ion was added as a drug to suspensions of metabolizingBacillus subtilis cells. [³H]-Triphenylmethylphosphonium uptake was enhanced by the addition, indicating hyperpolarization, yet fluorescence of diS-C₃(5) was also enhanced, indicating depolarization. Evidence is presented that both effects are artifactual, and can occur without any change in membrane potential, as estimated by⁸⁶Rb⁺ uptake in the presence of valinomycin. The fluorescence studies suggest that tetraphenylarsonium ion displaces the cyanine dye from the cell envelope, or other binding site, into the aqueous phase.The uptake characteristics of the radiolabeled lipophilic cations were quite unusual: At low concentrations (e.g., less than 10 m for triphenylmethylphosphonium) there was potential-dependent uptake of the label to a stable level, but subsequent addition of nonradioactive lipophilic cation caused further uptake of label to a new stable level. Labeled triphenylmethylphosphonium ion taken up to the first stable level could be displaced by 10mm magnesium ion, whereas⁸⁶Rb⁺ uptake was unperturbed. Association of the lipophilic cations with the surface of de-energized cells was concentration-dependent, but there was no evidence for cooperative binding. This phenomenon of stimulated uptake inB. subtilis (which was not seen inEscherichia coli cells or vesicles) is consistent with a two-compartment model with access to the second compartment only being possible above a critical cation concentration. We tentatively propose such a model, in which these compartments are the cell surface and the cytoplasm, respectively.Triphenylmethylphosphonium up to 0.5mm exhibited linear binding to de-energized cells; binding of tetraphenylphosphonium and tetraphenylarsonium was nonlinear but was not saturated at the highest concentration tested (1mm). The usual assumption, that association of the cation with cell surfaces is saturated and so can be estimated on de-energized cells, therefore leads to undercorrected estimates of cytoplasmic uptake inB. subtilis, and hence to overestimates of membrane potential. We describe a more realistic procedure, in which the estimate of extent of binding is based on a mean aqueous concentration related both to the external concentration and to the much higher internal concentration that exists in energized cells. Using this procedure we estimate the membrane potential inB. subtilis to be 120 mV, inside-negative. The procedure is of general applicability, and should yield more accurate estimates of membrane potential in any system where there is significant potential-dependent binding.Work performed while on sabbatical leave from Department of Biology, Ben-Gurion University of the Negev, Beer-Sheva, Israel.     

Effect of abscisic acid on CO2 exchange in Lemna gibba

The effects of abscisic acid (ABA) on photosynthesis, dark respiration, and photorespiration were studied in Lemna gibba L. plants. The initial concentration of ABA in the nutrient solution was 10⁻⁷M and in a few experiments, 10⁻⁶M. The cultures were grown in the same solution for time periods ranging from one hour to 12 days. Net photosynthesis, measured as CO₂ uptake by infrared gas analyser technique, was inhibited after four hours of ABA treatment and reached a minimum after four to seven days depending on the time of the year. After 12 days a substantial recovery of photosynthesis was observed. Dark respiration was significantly stimulated after two to seven days of ABA treatment but then returned to the control level. The transient effects of ABA on photosynthesis and dark respiration corresponded to the previously measured time course of [¹⁴C]-ABA uptake by Lemna . Photorespiration measured as oxygen inhibition of photosynthesis was not affected by ABA.     

Critical fields in cell fusion and dielectrophoresis

It is increasingly recognized that exposure to electrical fields can reversibly increase the electrical permeability and conductivity of the cellular membrane. It is therefore worthwhile to determine under what circumstances the critical fields capable of affecting the cellular membrane can be reached. We have evaluated the field intensities E and the gradients of E² in the neighborhood of the more common field electrode shapes, the sphere-sphere and cylinder-cylinder electrodes, and put these in terms of the values useful for the practical electro-fusion and dielectrophoresis (DEP) of cells. The calculations were performed using the charge-image technique. From this it was observed that gross errors (underestimations of up to 1000-fold) of the field would be made if neglect of the electrode polarization were made as in simple electrostatic calculation. The dielectric forces (in terms of (E)²) show interesting spatial character.The electrical breakdown of cellular membranes affects the exchange of information and materials between the cell and its environment, and can be used to fuse cells of the same type or of differing types. It is moreover of importance in handling cells during DEP while obtaining their spectral characteristics or in cell-sorting. It is expected that the results here will aid in the more proper use of electrical fields for such ends.