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.     

The molecular basis for Duchenne versus Becker muscular dystrophy: Correlation of severity with type of deletion

About 60% of both Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) is due to deletions of the dystrophin gene. For cases with a deletion mutation, the "reading frame" hypothesis predicts that BMD patients produce a semifunctional, internally deleted dystrophin protein, whereas DMD patients produce a severely truncated protein that would be unstable. To test the validity of this theory, we analyzed 258 independent deletions at the DMD/BMD locus. The correlation between phenotype and type of deletion mutation is in agreement with the "reading frame" theory in 92% of cases and is of diagnostic and prognostic significance. The distribution and frequency of deletions spanning the entire locus suggests that many "in-frame" deletions of the dystrophin gene are not detected because the individuals bearing them are either asymptomatic or exhibit non-DMD/non-BMD clinical features.     

Purkyn?'s description of pressure phosphenes and modern neurophysiological studies on the generation of phosphenes by eyeball deformation

(a) When a subject indents one of his eyeballs in total darkness, he immediately perceives light extending slowly across the whole visual field of the indented eye. The appearance and the time course of these pressure or deformation phosphenes are described. (b) With simultaneous binocular indentation of the eyeballs a flickering patterned phosphene is observed. (c) A short history of the research on pressure phosphenes and its consequences for the theories of vision is presented. (d) Purkyn?'s observations of monocular deformation phosphenes are described. He repeatedly noted patterned light structures, which most observers only perceive with simultaneous binocular eyeball deformation. It is suggested that Purkyn?'s deviating observations were caused by amblyopia of one eye. (e) The neurophysiological basis of the monocular pressure phosphenes was investigated by means of microelectrode recordings from single optic tract fibers. The activity of single retinal ganglion cells (on-center, off-center neurons, latency class I [Y-neurons] or latency class II [X-neurons]), was recorded in anaesthetized cats. Eyeball deformation in total darkness led to an activation of the on-center ganglion cells, while the off-center ganglion cells were inhibited. The latency and strength of this activation or inhibition varied considerably between different neurons, but were fairly constant in the same neuron when the eyeball indentation was repeated after a pause of 1-3 min. The latency and strength of neuronal activation or inhibition seemed to be dependent mainly upon the neuron location relative to the point of eyeball indentation. Some on-center neurons also exhibited a short activation at "deformation off". (f) The antagonistic response type of on-center and off-center ganglion cells was also observed when the eyeball was deformed as a hydrostatic open system and the intraocular pressure was kept at 25 mm Hg basic pressure. (g) Dark adaptation up to 45 min affected the deformation responses of retinal neurons only to a small degree, if at all. This corresponds to the observation that deformation phosphenes in a human observer changed little during the course of dark adaptation. (h) We assume that the activation of on-center and inhibition of off-center ganglion cells by eyeball deformation are caused by retinal stretching, which also leads to horizontal cell stretch. Stretching the horizontal cell membrane probably generates an increase in membrane sodium conductivity and a depolarization of the membrane potential. This depolarization of the horizontal cell membrane potential is transmitted either directly or indirectly (via receptor synapses) from the horizontal to the bipolar cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Antheseabhängige UV-Muster in Blütenständen von Asteraceen

The pseudanthia ofHeliopsis scabra andRudbeckia vulgaris (Asteraceae) were examined during the anthesis for differences in their UV patterns. Distinct changes in the reflectance and absorbance properties could be observed. The results suggest a close correlation between different stages of floral development and pollinator attraction.

Herrn Prof. Dr.Lothar Geitler zum 90. Geburtstag gewidmet.     

Streptokinase mutations relieving Escherichia coli K-12 (prlA4) of detriments caused by the wild-type skc gene

A novel phenotype is described for Escherichia coli K-12 carrying the prlA4 allele determining a membrane component of the protein export mechanism. It is manifest as transformation deficiency for plasmids containing the cloned group C streptococcal streptokinase gene, skc. Streptokinase plasmid mutations relieving the prlA4 strain of this deficiency fell into three classes. Class 1 included skc::IS5 insertions, with IS5 integrated in a region encoding the Skc signal sequence and inactivating skc. Class 2 included IS1 insertions leaving skc intact but reducing skc expression, presumably by altering the function of the skc promoter as judged by an insertion site close to the -35 region. The most interesting class, 3, included skc deletions removing the entire signal sequence or a tetrapeptide from its hydrophobic core. The tetrapeptide deletion reduced the size, hydrophobicity, and predicted alpha-helicity of the central region of the Skc signal sequence but facilitated the export of mature Skc in both the wild type and the prlA4 mutant. These findings indicate that the incompatibility between prlA4 and skc is related to deleterious effects of the Skc signal sequence. The tetrapeptide deletion may function by altering the conformation of the signal sequence so as to render interaction with both the PrlA wild-type protein and the PrlA4 mutant protein less detrimental to the export mechanism. These findings also provide an explanation for the difficulties encountered in cloning streptokinase genes in E. coli plasmids and maintaining their structural stability.     

Identification of the types of insulin-like growth factor-binding proteins that are secreted by muscle cells in vitro

We have previously shown that muscle cells secrete insulin-like growth factor-binding proteins. In the present study, BC3H-1 cells were shown to secrete one binding protein of Mr 32,000, whereas L6 cells secreted two binding proteins of Mr 31,000 and 24,000, as determined by ligand blotting. Subconfluent proliferating L6 cells secrete more of the Mr 24,000 binding protein, relative to the Mr 31,000 form. In contrast, differentiated L6 myotubes secreted similar quantities of the two forms. Insulin-like growth factor I preferentially stimulated secretion of the Mr 31,000 versus the Mr 24,000 binding protein from L6 cells and caused an increase in the secretion of the Mr 32,000 binding protein from BC3H-1 cells. The Mr 31,000 binding protein from L6 cells had a greater affinity for insulin-like growth factor II compared with insulin-like growth factor I, as did the Mr 32,000 binding protein of BC3H-1 cells. In contrast, the Mr 24,000 binding protein of L6 cells preferred insulin-like growth factor I. Neither porcine insulin nor relaxin competed for 125I-IGF-I binding. In conclusion, these muscle cell lines secrete only one or two forms of insulin-like growth factor-binding proteins. L6 cell differentiation is associated with a relative increase in the secretion of the Mr 31,000 binding protein compared with the Mr 24,000 form. Insulin-like growth factor I stimulates the secretion of its own binding proteins from muscle cells, and this may be an important mechanism for modulating cellular responsiveness to this growth factor.     

Cetacean relaxin. Isolation and sequence of relaxins from Balaenoptera acutorostrata and Balaenoptera edeni

The tendency toward extremely high variability among relaxins derived from purportedly closely related species has come to an abrupt end with the discovery of quasi-porcine relaxin in the minke whale (Balaenoptera acutorostrata) and the Bryde's whale (Balaenoptera edeni). An aqueous abstract of the corpora lutea of the two baleen whales contained significant amounts of relaxin-like activity as determined by a mouse bioassay and by cross-reactivity with anti-pig relaxin antibodies. The activity could be isolated and purified to homogeneity. Sequence analysis revealed that both whale relaxins differed from each other by about 3 residues, whereas the relaxin of B. edeni differed at only one position from that of pig relaxin. The similarity appears to include even the chain length heterogeneity observed at the C-terminal end of the B chain in porcine relaxin which is produced by a peculiar mode of connecting peptide removal from the pro-hormone. This finding may well represent one of the better documented challenges to the current paradigm of molecular evolution.     

Tumor necrosis factor signal transduction. Tissue-specific serine phosphorylation of a 26-kDa cytosolic protein

Binding of tumor necrosis factor-alpha (TNF-alpha) to its receptor on U937 cells results in rapid and TNF dose-dependent phosphorylation of a cytosolic protein with an apparent molecular mass of 26,000 kDa (p26) and an isoelectric point of 5.6. Half-maximal phosphorylation of p26 was achieved at concentrations of 1.8 ng/ml and was detectable within 20 s of TNF-alpha treatment. p26 is phosphorylated exclusively at serine residues. p26 phosphorylation occurs at 37 degrees C as well as at 14 degrees C, indicating that internalization of the TNF receptor is not required for serine kinase activation. Dephosphorylation of p26 starts 10 min after TNF-induced phosphorylation, suggesting a possible regulatory function of this cytosolic protein within the post-TNF receptor signaling system. p26 is also phosphorylated upon treatment with lymphotoxin. In contrast, both interferon-gamma and lipopolysaccharide fail to induce p26 phosphorylation. Whereas phosphorylated p26 was detected in the TNF-sensitive breast cancer cell line CRL1500, other TNF-responsive tumor cell lines investigated lacked enhanced phosphorylation of p26 in response to TNF, indicating that the 26-kDa phosphoprotein (pp26) may be a cell type-specific second messenger molecule involved in TNF signal transduction in some, but not all, target cells. p26 is also phosphorylated in a subclone of U937 (U937.C27) that responds to TNF-alpha with differentiation, yet is resistant to TNF-alpha-mediated growth inhibition. In contrast, p26 is not phosphorylated in another U937 derivative (U937.G3) that is resistant to both TNF-alpha-induced growth arrest and differentiation, suggesting that pp26 may play a role in the TNF signaling pathway linked to differentiation processes rather than to growth control.     

Spectroscopic studies of isopenicillin N synthase. A mononuclear nonheme Fe2+ oxidase with metal coordination sites for small molecules and substrate

The nonheme iron oxidase isopenicillin N synthase catalyzes the formation of two new internal bonds in the tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to form the beta-lactam and thiazolidine rings of isopenicillin N. Concomitantly, O2 is reduced to 2 H2O. The recombinant enzyme from Cephalosporium acremonium (Mr = 38,400), expressed as an apoenzyme in Escherichia coli, binds 1 g atom of Fe2+/mol of enzyme to reconstitute full activity. M?ssbauer spectra of the 57Fe-enriched enzyme exhibit parameters (delta = 1.30 mm/s, delta EQ = 2.70 mm/s) which unambiguously show that the active site iron is high spin Fe2+. Anaerobic binding of ACV causes a substantial decrease in the isomer shift parameter delta (delta = 1.10 mm/s, delta EQ = 3.40 mm/s) showing that the substrate perturbs the iron site and makes its coordination environment much more covalent. Nitric oxide (NO) binds to the EPR silent active site iron to give an EPR active species (g = 4.09, 3.95, 2.0; S = 3/2) similar to those of the nitrosyl complexes of many other mononuclear Fe2+-containing enzymes. The rhombicity of the EPR spectrum is increased (g = 4.22, 3.81, 1.99) by anaerobic addition of ACV suggesting that the substrate binds to or near the iron without displacing NO. Interestingly, the enzyme.ACV.NO complex displays an optical spectrum similar to that of ferric rubredoxin in which the iron has only thiol coordination. This suggests that the Fe2+ of the enzyme.ACV.NO complex acquires Fe3+ character and that the cysteinyl thiol moiety of ACV coordinates to the iron. Similar substrate thiol coordination to the iron of the enzyme.ACV complex is the most probable explanation for the large decrease in isomer shift observed. These results provide the first evidence for the direct involvement of iron in this unique O2-dependent reaction and suggest novel roles for iron and oxygen in biological catalysis.     

Visualization of the polarity of isolated titin molecules: a single globular head on a long thin rod as the M band anchoring domain?

TII, the extractable form of titin, was purified from myofibrils and separated by high resolution gel permeation chromatography into two fractions (TIIA and TIIB). Novel specimen orientation methods used before metal shadowing and EM result in striking pictures of the two forms. Molecules layered on mica become uniformly oriented when subjected to centrifugation. TIIB comprises a very homogeneous fraction. All molecules reveal a single globular head at one end on a long and very thin rod of uniform diameter. The lengths of the rods have a very narrow distribution (900 +/- 50 nm). TIIA molecules seem lateral oligomers of TIIB, attached to each other via the head regions. While dimers are the predominant species, trimers and some higher oligomers can also be discerned. Mild proteolysis destroys the heads and converts TIIA and TIIB into TIIB-like rods. Similar molecules also result from titin purified from myofibrils by certain established purification schemes. Headless titin molecules show in gel electrophoresis only the TII band, while head bearing molecules give rise to two additional polypeptides at 165 and 190 kD. Immunoelectron microscopy of myofibrils identifies both titin-associated proteins as M band constituents. We speculate that in the polar images of TII the globular head region corresponds to the M band end of the titin molecules. This hypothesis is supported by immunoelectron micrographs of TIIB molecules using titin antibodies of known epitope location in the half sarcomere. This proposal complements our previous immunoelectron microscopic data on myofibrils. They showed that epitopes present only on the nonextractable TI species locate to the Z line and its immediately adjacent region (Fürst, D. O., M. Osborn, R. Nave, and K. Weber. 1988. J. Cell Biol. 106:1563-1572). Thus, the two distinct ends of the titin molecule attach to Z and M band material respectively.