Interactions between a lymphoma membrane-associated guanosine 5'-triphosphate-binding protein and the cytoskeleton during receptor patching and capping

In this study we have used several complementary techniques to isolate and characterize a lymphoma membrane-associated 41-kDa protein that shares a number of structural and functional similarities with the alpha i subunit of the guanosine 5'-triphosphate (GTP)-binding protein (e.g., Gi alpha-like protein). In addition, using permeabilized lymphoma cells, we have found that: 1) GTP or GTP-tau-S augments, and pertussis toxin inhibits, phospholipase C (PLC) activity and receptor capping; and 2) the addition of lymphoma 41-kDa Gi alpha-like protein stimulates PLC activity and receptor patching/capping, and reverses the inhibitory effect of pertussis toxin on both activity and receptor patching/capping. Additional cytochemical and biochemical data indicate that the lymphoma 41-kDa protein is closely associated with several cytoskeletal proteins (e.g., actin, myosin, and fodrin) all of which colocalize under receptor cap structures. Furthermore, both the 41-kDa-mediated phospholipase C activity and receptor patching/capping are inhibited by cytochalasin D (a microfilament disrupting drug) and W-7 drug (a calmodulin inhibitor). Together, these data provide strong evidence for a functional association between the lymphoma membrane cytoskeleton and the 41-kDa (Gi alpha-like) protein. Specifically, this association appears to be required for the activation of phospholipase C that results in inositol triphosphate production, subsequent internal Ca2+ release, and finally surface receptor patching and capping.     

Combined in-beam electron impact-B/E-linked scan mass spectrometry of oxazoline derivatives for the structure determination of long-chain unsaturated fatty acids

Long-chain unsaturated fatty acids (UFA) having up to six double bonds are derivatized to 2-substituted 4,4-dimethyloxazolines (DMOX) and then analyzed by combined in-beam electron impact (IBEI)-B/E-linked scan mass spectrometry. This technique provides highly characteristic mass spectra and may serve as an auxiliary means for direct structure determination of individual UFA in mixtures.     

Characterization of recombinant thioesterase and acyl carrier protein domains of chicken fatty acid synthase expressed in Escherichia coli

Fatty acid synthase of animal tissue is a multifunctional enzyme comprised of two identical subunits, each containing seven partial activities and a site for the prosthetic group, 4'-phosphopantetheine (acyl carrier protein). We have recently isolated cDNA clones of chicken fatty acid synthase coding for the dehydratase, enoyl reductase, beta-ketoacyl reductase, acyl carrier protein, and thioesterase domains (Chirala, S.S., Kasturi, R., Pazirandeh, M., Stolow, D.T., Huang, W.Y., and Wakil, S.J. (1989) J. Biol. Chem. 264, 3750-3757). To gain insight into the structure and function of the various domains, the portion of the cDNA coding for the acyl carrier protein and thioesterase domains was expressed in Escherichia coli by using an expression vector that utilizes the phage lambda PL promoter. The recombinant protein was efficiently expressed and purified to near homogeneity using anion-exchange and hydroxyapatite chromatography. As expected from the coding capacity of the cDNA expressed, the protein has a molecular weight of 43,000 and reacts with antithioesterase antibodies. The recombinant thioesterase was found to be enzymatically active and has the same substrate specificity and kinetic properties as the native enzyme of the multifunctional synthase. Treatment of the recombinant protein with alpha-chymotrypsin results in the cleavage of the acyl carrier protein and thioesterase domain junction sequence at exactly the same site as with native fatty acid synthase. The amino acid composition of the purified recombinant protein revealed the presence of 0.6 mol of beta-alanine/mol of protein, indicating partial pantothenylation of the recombinant acyl carrier protein domain. These results indicate that the expressed protein has a conformation similar to the native enzyme and that its folding into functionally active domains is independent of the remaining domains of the multifunctional synthase subunit. These conclusions are consistent with the proposal that the multifunctional synthase gene has evolved from fusion of component genes.     

Insulin suppresses hepatitis B surface antigen expression in human hepatoma cells

The human hepatoma Hep3B cells contain integrated hepatitis B viral genome and continually secret hepatitis B surface antigen (HBsAg). The production of HBsAg (but not alpha-fetoprotein) was suppressed by addition of low concentrations (0.1-1 nM) of insulin into serum-free medium. In addition, the suppression of HBsAg production by insulin was paralleled with the decrease in HBsAg mRNA abundance. Insulin also cause a rapid rate of disappearance of HBsAg mRNA (t 1/2, 2 h) in Hep3B cells. The Hep3B cells carry specific receptor with high affinity for insulin (Kd = 1.8 nM). The receptor showed an insulin-dependent protein tyrosine kinase activity. The half-maximal insulin concentration for the activation of the receptor kinase was about 5 nM. Only very high concentrations of insulin-like growth factor I and human proinsulin can compete for the insulin receptor binding and suppress HBsAg production, this suggests that insulin may act through its receptor binding to suppress HBsAg expression in human hepatoma Hep3B cells.     

Cytomegalovirus infection stimulates expression of monocyte-associated mediator genes

Monocytes and tissue macrophages play important roles in host defense against virus infections and, in the case of human cytomegalovirus (HCMV) and HIV, may also be the reservoir for latent disease. Because these cells can also rapidly respond to most infections by secretion of inflammatory mediators, we were interested in determining if HCMV infection could have a direct activating effect on macrophage cytokine production. To do this, we primarily investigated the influence of HCMV infection on IL-1 beta-mRNA expression in peripheral blood monocytes and the promyelocytic cell line, ML-3 as well as the inflammatory response genes TNF-alpha, MAD-9, MAD-6, and MAD-2 in the promyelocytic ML-3 cell line. Exposure of ML-3 cells to the virus prior to induction of differentiation had little influence on mediator gene expression. However, induction of the macrophage phenotype by pretreatment of ML-3 cells with the phorbol ester, PMA, followed by HCMV challenge, resulted in a greatly extended period of expression of IL-1 beta, TNF-alpha, MAD-9, and CSF-1 but not MAD-6 and MAD-2. Constitutively expressed genes such as lysozyme and actin were not similarly modulated. Both RNA dot-blot and in situ hybridization studies demonstrated that infection of human peripheral blood monocytes with HCMV leads to sustained expression of IL-1 beta mRNA for up to 96 h, which contrasted markedly with mock-infected or LPS-stimulated monocytes. Flow cytometric analysis of the intracellular levels of IL-1 beta protein in ML-3 cells indicated that not only was there more protein produced in infected cells, but that the majority of the cells had responded. Enhanced levels of the intracellular form of IL-1 beta in monocytes was confirmed by Western blot analysis. Cotransfection experiments were performed using IL-1 beta-CAT chimeric plasmids together with plasmids encoding HCMV-immediate-early gene region products. Transactivation of the IL-1 beta gene by region 2 of the immediate-early gene was observed in ML-3 cells that had been induced to differentiate prior to transfection. No stimulation of IL-1 beta promoter activity was observed in ML-3 cells that were undifferentiated prior to transfection. In summary, HCMV infection, although not leading to productive infection, nonetheless may contribute to the pathology of the infection through enhancement of monocyte inflammatory mediator gene expression with subsequent stimulation of protein synthesis.     

Breast and subscapular pain following submuscular placement of breast prostheses

The technique of placing the breast prosthesis beneath the pectoralis major and the serratus anterior muscles appears to minimize the incidence of the firm breast following breast reconstruction commonly seen with other techniques. However, in 8 of 146 individuals I have noted a problem with pain in the lateral aspect of the breast mound and the subscapular area, along with a depressed deformity superomedially and an unsightly bulge inferolaterally and/or laterally. Surgical exploration of the breast mound showed no abnormalities within the submuscular compartment. However, in all instances, the serratus anterior muscles were found to be detached from the ribcage all the way to the point beyond the posterior axillary line. While continuous pressure exerted on the serratus muscles by the implant appears to play an important role in the pathogenesis of this clinical entity, the onset of problems was usually delayed. Removal of the implants or repair of the cavity defects is necessary for patients who have developed this problem.     

Immunologic identification of the aromatase enzyme system in human endometrium

Confluent human endometrial stromal cells were cultured in medium with no hormone or supplemented with medroxyprogesterone acetate (MPA), estradiol (E2), and porcine relaxin (RLX) for 5 days. These stromal cells were then labeled with [35S]methionine for 3 h. The radioactive proteins in the particulate fraction of cell homogenate were extracted by detergent and incubated with antisera to purified placental aromatase cytochrome P-450 (P-450arom) and NADPH-cytochrome P-450 reductase to isolate the radio-labeled aromatase enzyme components. Analysis of the radio-labeled protein, isolated by antibody to the cytochrome P-450arom from different preparations (P45FBIII or R-8-2) showed a major band at molecular weight 54k on SDS polyacrylamide gel electrophoresis (SDS-PAGE). The intensity of 54k band was stronger in hormone treated stromal cells than that of control in parallel with the increase of aromatase activity. The radio-labeled protein isolated by anti-NADPH cytochrome P-450 reductase, REDFBIV, showed a major band at the molecular weight 73k on SDS-PAGE with comparable intensity in control and hormone treated samples. Thus, the apparent molecular weights of endometrial cytochrome P-450arom and cytochrome P-450 reductase were identical to placental aromatase enzyme system. When a secretory endometrium and a decidua were labeled with [35S]methionine, the cytochrome P-450arom was detected only in the decidua. NADPH cytochrome P-450 reductase was detected both in the endometrium and the decidua. These results show that antisera to placental aromatase enzyme system cross reacts with the endometrial aromatase enzyme components. The synthesis of cytochrome P-450arom was stimulated by MPA, E2 and RLX while the synthesis of the NADPH-cytochrome P-450 reductase aromatase component was not affected by the hormone.     

Characterization of a protease with alpha- and beta-fibrinogenase activity from the Western diamondback rattlesnake, Crotalus atrox.

A metalloprotease from the rattlesnake Crotalus atrox venom was isolated and purified from multiple-step chromatographies including anion-exchange chromatography, gel permeation and reversed-phase HPLC. The fraction was shown to be homogeneous as judged by SDS-gel electrophoresis. It also showed a high proteolytic activity against alpha- and beta-chains of fibrinogen molecules. Further characterization of the purified fraction with fibrinogenase activity indicated that it is a single-chain protease with a molecular mass of about 24 kDa and an acidic isoelectric point. It is relatively heat stable up to about 65 degrees C, inhibited by EDTA, beta-mercaptoethanol, but not by phenylmethanesulfonyl fluoride, N alpha-p-tosyl-L-phenylalanine chloromethyl ketone and N alpha-p-tosyl-L-lysine chloromethyl ketone, soybean trypsin inhibitor and aprotinin. Amino acid analysis showed that the enzyme possesses an amino acid composition very similar to some metalloproteases characterized before from the closely related rattlesnake venoms. N-Terminal sequence analysis of the enzyme corroborated some similarity between this enzyme and the reported sequences of these enzymes characterized from the Crotalidae snake family. This study indicated the presence of a novel fibrinogenase (termed Catroxase) with N-terminal sequence different from the metalloprotease with hemorrhagic activity isolated from the same Western diamondback rattlesnake.     

Phospholipase D activity in nontransformed and transformed fibroblasts.

Rat embryo fibroblasts (REF52 cells) and the simian virus 40 transformed derivative (WT6 Ag6) were employed to characterize phospholipase D (PLD) activity in normal and transformed cells. In cells prelabeled with [3H]myristic acid or [3H]glycerol and treated with 12-O-tetradecanoylphorbol-13-acetate (TPA, 50 ng/ml medium) or vasopressin (VP, 100 ng/ml medium) in the presence of ethanol, the formation of labeled phosphatidylethanol (PEt) was 3- to 5-fold higher in REF52 cells than in the transformed cells. The transphosphatidylation of phosphatidylcholine (PC) to PEt was further examined in cell-free assay systems. Results demonstrated that the formation of PEt in the cell-free assays was dependent on the mode of substrate presentation and the source of the PC. With endogenous membrane-bound substrate, the formation of [3H]myristoyl-PEt was 5-fold higher in homogenates derived from normal cells as compared to transformed cell homogenates. In experiments using exogenous labeled PC isolated from either REF52 or transformed cells as substrate, cell-free PLD activity differed greatly with regard to the source of the PC. The formation of PEt from REF52-derived PC was approx. 4-fold higher as compared to PEt formed with PC derived from the transformed cells, irrespective of enzyme source. The results demonstrate that PLD in intact nontransformed fibroblasts is activatable by TPA and VP to a greater extent than in the transformed counterpart. The results from cell-free assays suggest that PLD activity is more dependent on the type of PC substrate than on the source of the enzyme.