Effect of 3T3 plasma membranes on cells exposed to epidermal growth factor

Epidermal growth factor (EGF) induced DNA synthesis in non-confluent, G₀-arrested Swiss 3T3 fibroblasts is partially blocked by plasma membranes isolated from the EGF receptor deficient NR-6 Swiss 3T3 cell line. This inhibition could be due to either a steric block of the receptor by the membranes, a membrane induced down regulation of the EGF receptor, or a signal generated by membrane binding which is antagonistic towards the mitogenic signal generated by EGF. Binding measurements utilizing ¹²⁵I-labeled EGF demonstrated that membranes do not block either the EGF induced down regulation of the receptor or alter the number of receptors on the surface. These results suggest that the membranes exert their inhibitory effect via generation of a signal which is antagonistic to the EGF induced mitogenic signal, with the result expressed as a reduced mitogenic response.     

Regulatory anatomy of the murine interleukin-2 gene.

We have cloned the mouse IL2 gene and sequenced 2800 bp of 5' flanking DNA. Comparison to the previously reported human sequence revealed extensive identity (approximately 86%) between the two genes from +1 to -580 with additional small islands of homology further upstream. Proximal sites which have been shown to be important in regulation of the human IL2 gene are well conserved in sequence and location. Transfection experiments using hybrid gene constructs containing varying lengths of the mouse 5' flanking DNA linked to a CAT reporter gene have demonstrated the presence of several novel positive and negative regulatory elements. One negative regulatory region lying between -750 and -1000 consists primarily of alternating purines and pyrimidines and is absent from the human gene. The conserved region from -321 and -578, an upstream segment from -1219 to -1332, and another region of approximately 450 bp from -1449 to -1890, which contained a well-conserved sequence of 60 bp, were each associated with enhanced levels of expression. We found no evidence for intragenic or downstream enhancer elements in this gene. All the elements identified affect only the magnitude of the inducible response, for no region when deleted had the effect of altering either the need for induction, the kinetics of stimulation, or the cell-type specificity of expression. Deletion studies suggest a strong requirement for NFAT binding even in the presence of extensive 5' flanking sequence. Therefore we conclude that IL2 gene expression is controlled primarily through a central TH1-specific signaling pathway, which acts through proximal elements, while distal cis-elements exert a secondary modulating effect.     

The complete amino acid sequence of a human folate binding protein from KB cells determined from the cDNA

Two species of folate binding protein (FBP), an integral membrane-associated form and a soluble secreted form, have been previously purified from cultured human KB cells. The complete nucleotide sequence of the complementary DNA (cDNA) clone for the coding region of the mature membrane-associated FBP has now been determined, and the deduced amino acid sequence has been computer-analyzed for a prediction of the secondary structure of the protein. The clone has 857 nucleotides of which 678 comprise the coding region for 226 amino acids. The deduced amino sequence contains the identical sequence of the published 18 NH2-terminal amino acids of the purified FBP from KB cells and the published partial amino acid sequence of the human milk FBP except for 1 residue. There was also over 90% homology with the published amino acid sequence of the bovine milk FBP. A total of 16 cysteine residues has been conserved in the three proteins indicating that this amino acid may provide a tertiary structure which is required for its ligand binding function. Northern blot analysis using the cDNA probe identified a single band of 1.28-kilobase pair mRNA in KB cells which was 4.7-fold more intense in folate-depleted cells than in normal cells. These results indicate that the membrane FBP and the soluble FBP in the medium are translation products of the same gene. Computer analysis of the deduced amino acid sequence indicates that there is only one stretch of amino acids of sufficient hydrophobicity and length to span the lipid bilayer of the plasma membrane, but it lacked a predictable helical structure. Those regions of the sequence which did have a predictable helical structure lacked sufficient hydrophobicity required for a membrane anchor. Thus, it is likely that the fatty acids previously reported to be present in the membrane-associated FBP from these cells rather than a peptide sequence provide an important membrane anchoring function.     

Isolation and characterization of two species of receptor which bind intrinsic factor

The intrinsic factor receptor from guinea pig ilea has been characterized following purification by affinity chromatography. The purified receptor complexed to intrinsic factor-cobalamin (holo-receptor) had an anhydrous molecular weight of 680,000, a Stokes radius of 10.9 nm, and a sedimentation coefficient of 15.1 S. In contrast, unsaturated receptor (apo-receptor) resolved into distinct "large" and "small" molecular species having, respectively, (i) molecular weights of 700,000 and 350,000; (ii) Stokes radii of 11.1 and 7.06 nm; (iii) sedimentation coefficients of 15.5 S and 11.9 S; (iv) association constants for binding the intrinsic factor-cobalamin complex of 7.3 and 2.5 X 10(10) liters mol-1; and (v) two isoproteins for the larger species (pI's 4.05 and 4.80), and one isoprotein for the smaller species (pI 4.90). A rabbit anti-receptor serum gave only one precipitation line in immunodiffusion against either the large or the small receptor, and each one of these two lines fused completely with the one of two lines which formed with the purified preparation containing both receptor species. Autoradiography of the precipitin lines obtained when the receptor was coupled to intrinsic factor-cyano[57Co]cobalamin demonstrated that both species of receptor were functional. The reaction of complete immunologic identity, the similar electrical properties and similar kinetics for binding to intrinsic factor, and the observed molecular weight differences indicate that the small and large apo-receptors are chemically interrelated, and suggest that the large receptor may consist of two small functional proteins.     

A simplified radioenzymatic assay for dihydrofolate reductase using [3H]dihydrofolate

This report describes a simple method to measure the activity of dihydrofolate reductase using the substrate [³H]dihydrofolate, which is generated by preincubation of [³H]folic acid for 10 min with dithionite before the enzymatic reaction. The procedure then measures the direct reduction of [³H]dihydrofolate to [³H]tetrahydrofolate by coprecipitating the unreduced substrate with excess unlabeled folic acid and acidified zinc sulfate. The advantage of this method is that [³H]dihydrofolate, which is not commercially available, can be generated from high specific activity [³H]folic acid, which is commercially available, immediately before initiating the enzymatic reaction. By this modification, the two important advantages of radioenzymatic assays for dihydrofolate reductase can be more easily exploited; namely, increased sensitivity because much less substrate need be used, and the ability to measure enzyme activity in crude tissue preparations without interference by precipitating proteins or nucleotide oxidases.     

In vitro maintenance of differentiation marker synthesis by subpopulations of mouse thymocytes

Mouse thymocyte populations enriched in functionally incompetent, “immature” cells on the one hand, or in competent “mature” cells on the other hand, express different steady-state levels of certain surface antigens and marker enzymes. In the cases of the glycoproteins H-2 (K and D), Qa, and TL, and the DNA polymerase terminal deoxynucleotidyl transferase (TdT), these levels reflect different rates of de novo synthesis in the two populations. Thus each population appears to manifest a characteristic pattern of synthetic rates for the various products relative to total protein synthesis. To investigate the maintenance of these patterns, enriched pools of “immature” and “mature” thymocytes were incubated in vitro for 24 h, and the rates of product synthesis before and after culture were compared. H-2 synthesis, initially most rapid in the mature cells, continued to be made at the highest rate in this population. TdT synthesis, a characteristic activity of the immature cells, was not induced in the mature cells, but proceeded at an increased relative rate in the immature population. Therefore, the differences between the rates of H-2 and TdT synthesis were stable properties of the two thymocyte populations. Another marker of immature cells, TL, did not continue to be produced in parallel with TdT. Rather, its synthesis was selectively curtailed in relation to the continuing protein synthesis in the immature cultures. This non-coordinate regulation of TL and TdT production in immature thymocytes may be due to several mechanisms. These are discussed with regard to their implications for pathways of thymocyte maturation.     

The control of relative humidity with aqueous solutions of sodium hydroxide

Using values contained in the International Critical Tables (1928) a gravimetric method, based on one used by Solomon (1951), is described to produce a stock solution of sodium hydroxide, from which graded aqueous solutions can be prepared to give relative humidities in steps of 10 per cent between 0° C and 40° C.

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Zusammenfassung In der vorliegenden Zusammenstellung werden die Verdünnungsgrade wäßriger Natrium-hydroxyd-Lösungen angegeben, die bei Temperaturen zwischen 0° und 40° C bestimmte Stufen relativer Luftfeuchtigkeit in 10%-Intervallen auf Grund der entsprechenden spezifischen Gewichte der Lösungen bedingen. Die Werte sind den internationalen kritischen Tafeln (1928) entnommen und beruhen auf den gravimetrischen Methoden von Solomon (1951).