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.     

Hyperthermic treatment of chromomycosis with disposable chemical pocket warmers

A case of chromomycosis in which hyperthermia proved effective is reported. The patient was a 56-year-old male bean curd maker who, without any previous history of minor trauma, developed on the extensor side of the left upper arm an eczematous lesion that underwent gradual radial expansion. The lesion showed a well-defined, 7×10 cm infiltrated erythematous plaque with the central area healed and, at the upper and lower borders, adherent scales and crusts on the surface. Histological examination revealed granulomatous changes in the dermis, as well as sclerotic cells within giant cells and microabscesses. On culturing,Fonsecaea pedrosoi was isolated. The patient was treated with disposable chemical pocket warmers, which were secured over the lesion with a rather tight elastic bandage, so that they kept the affected area warm for 24 hours a day. After a month of such hyperthermic treatment, the erythema and infiltration had decreased considerably, and microscopic examination and culture of the crusts both yielded negative results. Examination of biopsy specimens of the lesion after the third month showed that it had cicatrized. The treatment was stopped after 4 months, and no relapse occurred. We also summarize the published results of local hyperthermic treatment of chromomycosis in Japan.     

Identification of HLA-B44 subtypes associated with extended MHC haplotypes

The HLA class I antigen B44 is found in each of two different extended major histocompatibility haplotypes (allele combinations of HLA-B, HLA-DR, and complement genes BF, C2, C4A, and C4B in linkage disequilibrium). Using isoelectric focusing, two variants of HLA-B44 were identified. The basic variant was found in all cell lines with the extended haplotype HLA-B44, DR7, FC31, and the acidic variant in all cell lines with the extended haplotype HLA-B44, DR4, SC30. The occurrence of each antigen variant with a unique extended haplotype explains previous observations concerning the nonrandom association of B44 variants with DR antigens.     

Heparin potentiates the action of plasma membrane-associated growth stimulatory activity

Plasma membranes prepared from mouse liver have been previously shown to contain growth stimulatory activity as determined with cultured mouse fibroblasts. This growth stimulatory activity, termed plasma membrane-associated growth stimulatory activity (PMGA), is highly mitogenic in the presence of platelet-poor plasma. We now demonstrate that the growth stimulatory action of PMGA is dramatically enhanced by the addition of heparin. The half-maximal effect of heparin was observed at 1-3 micrograms/ml. The synergistic effect was seen in two distinct assays; the stimulation of DNA synthesis in quiescent cells, and an increase of cell number over a 3-day culture period. Heparin, by itself, does not have any measurable influence on the growth of fibroblasts. The action of heparin is not unique to this glycosaminoglycan, as several other highly sulfated polysaccharides, including dextran sulfate, pentosan polysulfate, and fucoidan, also exhibited the highly synergistic effect. Among other glycosaminoglycans examined, chondroitin sulfate B and heparan sulfate had a small, but significant, effect on enhancing the growth stimulatory action of PMGA. Chondroitin sulfate A, chondroitin sulfate C, hyaluronic acid dextran, and poly-L-glutamic acid, however, had no detectable effect. Further experiments suggested that the effect of heparin is twofold, namely, both a potentiation of growth stimulatory activity and a protection of PMGA activity. The data presented here suggest that the association of various cell surface components, such as PMGA and specific proteoglycans, can modulate the growth potential of a cell.     

Carboxypeptidase Y digestion of band 3, the anion transport protein of human erythrocyte membranes

The exposure of the carboxyl-terminal of the Band 3 protein of human erythrocyte membranes in intact cells and membrane preparations to proteolytic digestion was determined. Carboxypeptidase Y digestion of purified Band 3 in the presence of non-ionic detergent released amino acids from the carboxyl-terminal of Band 3. The release of amino acids was very pH dependent, digestion being most extensive at pH 3, with limited digestion at pH 6 or above. The 55,000 dalton carboxyl-terminal fragment of Band 3, generated by mild trypsin digestion of ghost membranes, had the same carboxyl-terminal sequence as intact Band 3, based on carboxypeptidase Y digestion. Treatment of intact cells with trypsin or carboxypeptidase Y did not release any amino acids from the carboxyl-terminal of Band 3. In contrast, carboxypeptidase Y readily digested the carboxyl-terminal of Band 3 in ghosts that were stripped of extrinsic membrane proteins by alkali or high salt. This was shown by a decrease in the molecular weight of a carboxyl-terminal fragment of Band 3 after carboxypeptidase Y digestion of stripped ghost membranes. No such decrease was observed after carboxypeptidase Y treatment of intact cells. In addition, Band 3 purified from carboxypeptidase Y-treated stripped ghost membranes had a different carboxyl-terminal sequence from intact Band 3. Cleavage of the carboxyl-terminal of Band 3 was also observed when non-stripped ghosts or inside-out vesicles were treated with carboxypeptidase Y. However, the digestion was less extensive. These results suggest that the carboxyl-terminal of Band 3 may be protected from digestion by its association with extrinsic membrane proteins. We conclude, therefore, that the carboxyl-terminal of Band 3 is located on the cytoplasmic side of the red cell membrane. Since the amino-terminal of Band 3 is also located on the cytoplasmic side of the erythrocyte membrane, the Band 3 polypeptide crosses the membrane an even number of times. A model for the folding of Band 3 in the erythrocyte membrane is presented.