Isolation and characterization of a 37,000-dalton protein associated with the erythrocyte membrane

We have purified a 37,000-dalton polypeptide (p37) from the red cell membrane that was found in previous studies to undergo a lineage-specific alteration in its membrane association. Our data suggest that p37 associates with the red cell membrane through electrostatic interactions that are resistant to 0.5 M NaCl or 10 mM EDTA. Conditions found to elute p37 from red cell ghosts include H2O at pH 12, 0.1 N NaOH + 1 mM ethanol and 1.0% Triton X-100. p37 was purified substantially from ghosts by Triton X-100 solubilization followed by sequential DEAE-Sephadex and CM-Sephadex chromatography. When p37 was analyzed by two-dimensional gel electrophoresis, a family of isoelectric focusing variants was detected ranging in pI from 7.0 to 7.8. All of the isoelectric focusing variants showed homology to one another when compared serologically with anti-p37 antibodies or by limited peptide mapping using Staphylococcus aureus V8 protease. The isoelectric focusing variants appear to represent distinct, yet related polypeptides rather than degrees of post-translational modifications to a single species, inasmuch as all of the variants are present in anti-p37 immunoprecipitates prepared from in vitro translations programmed with p37 mRNA.     

Dysfunction of calcium handling by smooth muscle in hypertension

Dysfunction of ion handling, including binding and fluxes (passive and active transport) of physiologically important ions such as potassium, sodium, calcium, and magnesium, by vascular smooth muscle cell membranes has repeatedly been reported to be associated with the pathophysiology of hypertension. The specific purpose of this review is to summarize and evaluate the evidence for alterations of calcium ion (Ca2+) handling by vascular smooth muscle in various forms of hypertension in the animal model on the basis that regulation of cytoplasmic Ca2+ concentration is a complex and yet vitally important process for a normal function of vascular smooth muscle and that derangement of such a regulation may result in excessive retention of cytoplasmic Ca2+, contribute toward increase of total peripheral resistance, and ultimately lead to elevation of blood pressure. Emphasis is placed upon the consideration of the usefulness of the subcellular membrane fractionation technique in studies of binding and transport of Ca2+ by vascular and nonvascular smooth muscle membranes from genetic as well as experimental hypertensive rats. The limitations of the interpretation of data using such an approach are also considered. Decreased active transport of Ca2+ across isolated plasma membrane vesicles from large and small arteries occurs in several but not all forms of hypertension. This membrane abnormality also occurs in nonvascular smooth muscles and other tissues or cells not confined to the cardiovascular system in genetic hypertension, but not in experimental hypertension. A hypothesis of general membrane defects in spontaneous hypertension is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of proliferation without affecting the generation of cytotoxicity in the human mixed lymphocyte reaction

Phosphoramide mustard (PM) is considered to be the major tumoricidal metabolite of cyclophosphamide in vivo. The effects of this metabolite in vitro on several immune functions of human lymphocytes have been investigated. Very low concentrations (10(-7) to 10(-9) M) of PM added to lymphocyte cultures inhibited proliferation of the lymphocytes in response to mitogens and alloantigens. At these concentrations, inhibition of proliferation appeared to be due to a direct action of PM on the proliferative cells. Thus, concanavalin A-stimulated lymphocytes still acquired IL-2 receptors (Tac antigen) normally in the presence of PM (10(-6) to 10(-9) M). Only exceedingly high concentrations of PM (10(-5) M or greater) prevented the acquisition of Tac antigen. Similarly, the inhibition of proliferation was probably not related to endogenous IL-2 levels: addition of exogenous IL-2 to PM-containing cultures did not result in any restoration of proliferation. Further evidence that PM directly affected proliferative cells was that low concentrations of PM inhibited the proliferation of T cells continuously growing in IL-2. The exposure time to PM necessary for inhibition was essentially identical to those for lymphoproliferative responses to mitogens and alloantigens. Paradoxically, however, the generation of cytotoxic lymphocytes in mixed lymphocyte reactions (MLRs) and mixed lymphocyte tumor cell cultures (MLTCs) was very resistant to PM. In parallel MLRs and MLTCs the cytotoxic responses were resistant to approximately 1000-fold more PM than were the proliferative responses. Only at 10(-5) M PM were these inhibited. These data suggest that clonal expansion of cytotoxic lymphocytes or their precursors by proliferation is not an absolute requirement for the generation of cytolytic activity.     

A method to quantitate Coomassie blue-stained proteins in cylindrical polyacrylamide gels

A method for the quantitation of Coomassie blue-stained proteins in cylindrical polyacrylamide gels is described. It involves an elution of the dye with an 80% methanol solution in a sealed Pyrex tube at 100 degrees C for 3 h and a measurement of its concentration at 585 nm. Using a 6.5% polyacrylamide gel and bovine serum albumin as a protein standard, the curve of absorbance of the dye solution as a function of the amount of protein was observed to be linear up to 30-40 micrograms of protein and as little as 0.8-1.0 micrograms of protein could be measured. The validity of the method was indicated by the values obtained for the relative proportions of the human erythrocyte membrane proteins. Using this method, the color yields of several proteins varying widely with respect to their size, amino acid composition, and carbohydrate content were determined in a 6.5% polyacrylamide gel. The results showed that they were generally the same except for proteins having a high carbohydrate content which were significantly lower.     

Male-like behaviour in an all-female lizard: relationship to ovarian cycle

A quantitative analysis of the relationship between pseudocopulatory behaviour and the ovarian cycle in the parthenogenetic lizard Cnemidophorus uniparens indicates (1) that this behaviour is frequently and regularly expressed by captive individuals, and (2) that the sexual role, either male-like or female-like, exhibited by an animal is correlated with its ovarian state. The expression of female-like behaviour patterns was associated with and primarily limited to the vitellogenic stage of the cycle. Male-like behaviour patterns occurred most frequently during post-ovulatory stages but was not limited to these stages. Neither behavioural role was ever expressed by non-reproductive individuals. Reproductive individuals often alternated in assuming the female-like and male-like roles during the progress of the ovarian cycle. These observations suggest that pseudosexual behaviour is hormonally activated in this species. However, it also appears that the prevailing social situation is an important factor determining which behavioural role is taken. This work strengthens the hypothesis that pseudosexual behaviour in all-female lizards occurs as the result of natural selection.     

Isolation and characterization of neuropeptide Y from porcine intestine

The isolation and primary structure of intestinal neuropeptide Y (NPY) is described. The peptide was purified from porcine intestinal extracts using a chemical assay and radioimmunoassay for NPY. The amino acid sequence of this peptide is: Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Leu-Ala- Arg-Tyr-Tyr- Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH2. This the structure of intestinal NPY is identical to the NPY of brain origin.     

Differential effects of abrin on normal and tumor cells

The effects of the plant toxin abrin on normal mouse embryonic fibroblasts (MEF), an untransformed mouse cell line (NIH 3T3), and two mouse tumor cell lines (LMTK- and S-180) were studied. Measurements of cell growth and colony formation showed that MEF and S-180 cells were more sensitive to abrin intoxication than NIH 3T3 and LMTK- cells. Also, the effects of abrin on the inhibition of [3H]leucine and [3H]thymidine incorporation were more evident in MEF and S-180 cells. The basis for these varying responses to abrin by the four different cells was examined. The number of abrin binding sites per cell was determined from [125I]abrin binding studies: NIH 3T3 and LMTK- cells had significantly fewer abrin binding sites than MEF and S-180 cells. The fate of the [125I]abrin after internalization was examined by gel electrophoresis and autoradiography. A pattern of time-dependent degradation was observed, degradation being more rapid in NIH 3T3 and S-180 cells than in LMTK- and MEF cells. We conclude that the varying responses of different cells to the toxin abrin may be due to several factors, including the relative number of abrin binding sites on the cell surface and the rate of degradation of the toxin once internalized. The results also show that the sensitivities of the cells to abrin do not necessarily correlate with their normal or neoplastic state.     

Influence of accessory cell and T cell surface antigens on mitogen-induced IL 2 receptor expression

We have assessed the inhibitory effects of various monoclonal antibodies on the expression of the IL 2 receptor. Anti-LFA-1, but not anti-Ly-2, markedly inhibited the induction of the IL 2 receptor on the Ly-2+ subset. T-depleted spleen cells, L cells, and B lymphoma cells all functioned as potent accessory cells (AC) for the induction of the IL 2 receptor on L3T4+ T cells. Anti-LFA-1 inhibited the induction of the IL 2 receptor irrespective of the type of AC used. Anti-L3T4 only inhibited the induction of IL 2 receptor expression when L cells were the source of AC. The inhibitory capacity of anti-L3T4 was not related to the expression of Ia on the AC population, because the magnitude of inhibition was comparable in cultures containing either Ia+ or Ia- L cells, whereas no inhibition was seen with either Ia+ or Ia-B lymphoma cells. We conclude from these studies that LFA-1 plays a critical role in mitogen-induced activation of both T cell subsets by promoting both T-AC and T-T interactions. Although anti-L3T4 can inhibit T cell activation in the absence of the recognition of Ia, the mechanism of inhibition and the proposed target molecule for L3T4 on the AC or the T cell have not been determined in our studies. A number of different models for the function of this cell surface antigen are discussed.     

Alterations in immunolocalization of the phosphoprotein B23 in HeLa cells during serum starvation

Bright nucleolar immunofluorescence was observed in HeLa S3 cells by immunostaining with a monoclonal antibody to the nucleolar phosphoprotein B23 (MW 37 kD/pI 5.1). After 48 h of incubation in a serum-free medium, the nucleolar fluorescence was diminished and a general nuclear immunofluorescence was observed. This change in localization of fluorescence indicated that protein B23 had migrated out of the nucleoli. No gross morphological change in nucleoli was observed by light microscopy and the immunolocalization of another nucleolar phosphoprotein, C23, was unaffected by serum deprivation. Relocation of protein B23 in nucleoli was observed after refeeding with serum-containing medium. This re-entry process was not observed after treatment with actinomycin D (50 ng/ml-5 micrograms/ml), but the process was unaffected by cycloheximide (0.2 mM). Quantitation of protein B23 in the nucleoli of the control (fed) or starved HeLa cells was done by ELISA immunoassay. A marked decrease in the amount of protein B23 occurred in the nucleoli of the starved cells (11.8 micrograms B23/mgDNA) as compared with the control nucleoli (20.8 micrograms B23/mgDNA). The amount of protein B23 in the nucleoplasm (excluding nucleoli) was 70% higher in the starved cells. Protein B23 was analysed by one- and two-dimensional PAGE. Three components of protein B23 with slightly different molecular weights and pIs (37 kD/5.1, 35 kD/5.1 and 35 kD/5.3) were observed in nucleoli. The lower molecular weight components were predominantly found in the nucleoplasm.