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.     

The biological effect of sodium butyrate (NaBT) on SGC-7901 cells

Changes of (Na+-K+)-ATPase activity, cAMP and fibronectin (FN) content and cell surface microvilli were studied cytochemically, immunocytochemically and scanning electron microscopically on human stomach Glandular carcinoma (SGC-7901) cells treated with NaBT(2.5 mM). It was found that NaBT not only inhibited cell growth but also remarkably decreased the activity of cell surface (Na+-K+)-ATPase of SGC-7901 cells. Note worthy was that, in comparison with the untreated tumor cells, the increase of the intensity of intracellular cAMP and FN immunofluorescence in NaBT-treated tumor cells was striking. Moreover, in contrast to untreated tumor cells, the cell surface of NaBT-treated tumor cells showed more smooth and fewer microvilli under SEM. That NaBT may induce differentiation of SGC-7901 cells through inhibition of (Na+-K+)-ATPase activity and modulation of cellular cAMP and FN content was discussed.     

中枢多巴胺神经元放电活动的特点

中脑黑质和腹侧被盖区DA神经元自发放电活动的特点表现在:动作电位时程较宽(2～5ms),伴有上升相切迹;放电频率较慢(1～10spikes/s);有单放电(single firing)和爆发性放电(burst firing)两种型式,前者动作电位幅度无显著改变,后者动作电位幅度逐个减低,时程逐个加宽,并且动作电位间隔逐渐延长。DA受体激动剂或D_2亚型选择性激动剂抑制DA神经元放电活动,它能被DA受体拮抗剂所逆转。     

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.     

Circadian Rhythm of the Prokaryote Synechococcus sp. RF-1

The prokaryotic Synechococcus sp. RF-1 exhibited a nitrogen fixation circadian rhythm with characteristics remarkably similar to the circadian rhythm of eukaryotes. The rhythm had a free-running period of about 24 hours when the length of the preen-trained cycle did not differ too much from 24 hours, and it was insensitive to changes in temperature from 22°C to 33°C. Because the endogenous rhythm of nitrogen fixation was not affected by a phase-shift of its previous cycles, the circadian rhythm in Synechococcus sp. RF-1 was not considered to be controlled simply by a feedback mechanism.     

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.     

Antigenic nature of the chloride-stimulated cellobiosidase and other cellulases of Fibrobacter succinogenes subsp. succinogenes S85 and related fresh isolates.

Polyclonal and monoclonal antibodies to the Cl-stimulated cellobiosidase of Fibrobacter succinogenes subsp. succinogenes S85 reacted with numerous proteins of both higher and lower molecular weights from F. succinogenes subsp. succinogenes S85, but not with Escherichia coli proteins, and only one protein each from Butyrivibrio fibrisolvens and Ruminococcus albus. Different profiles were observed for Western blots (immunoblots) of peptide digests of both the purified enzyme from F. succinogenes and immunoreactive proteins of higher and lower molecular weights, demonstrating that they were different proteins. Therefore, F. succinogenes appeared to produce numerous proteins with one or more common antigenic determinants. However, with the exception of Cl-stimulated cellobiosidase, none were cellulases that have been characterized. An affinity-purified polyclonal antibody to Cl-stimulated cellobiosidase reacted with numerous proteins in cells of each of three fresh isolates of F. succinogenes subsp. succinogenes and one of F. succinogenes subsp. elongata when analyzed by Western blotting. Antibodies to periplasmic cellodextrinase, endoglucanase 2 (EG2), and EG3, when reacted in Western blots with the various cellulases, including Cl-stimulated cellobiosidase, revealed limited antigenic similarity among the different proteins and none with either B. fibrisolvens or R. albus proteins. The periplasmic cellodextrinase antibody reacted with an antigen with a size corresponding to cellodextrinase in each of the three F. succinogenes subsp. succinogenes isolates but not with any antigens from the F. succinogenes subsp. elongata isolate. The anti-EG2 antibody reacted with single antigens in each of the four isolates, while the anti-EG3 antibody reacted with only one of the four isolates.(ABSTRACT TRUNCATED AT 250 WORDS)