Characterization of potassium channels in pancreatic beta cells from ob/ob mice

The patch-clamp technique in the cell-attached mode was used to study the K channels present in the membrane of cultured pancreatic beta cells from ob/ob mice. Three types of K+ channels were regularly observed, with conductances of 64, 20 and 146 pS. The conduction and kinetic properties of the 64 pS channel were similar to those of the ATP-sensitive potassium channel from normal beta cells. Furthermore, glucose blocked the activity of this channel at the same concentrations as that reported for normal cells. The 20 pS and the 146 pS were insensitive to glucose. The latter K+ channel appears to be similar to the large conductance voltage-activated potassium channels described in normal rodent beta cells. Thus, potassium channels in ob/ob pancreatic beta cells in culture are in most respects normal. Other factors may account for the abnormal electrical response to glucose of ob/ob pancreatic islets, such as reversible impairment of their function in vivo or defects not related to potassium permeability.     

DISCOVERY OF MONOGRAPTIDS IN BASAL PART OF LOWER SILURIAN FROM S. ANHUI WITH SPECIAL REFERENCE TO THEIR ORIGIN

For a long time, the evolutionary series of a idoraptids-dimorphograptids-monograptids has been generally recognized by graptolite researchers. In the past years, the akidograptids were found to appear in the Lower Silurian Parakidograptus acuminatus Zone, while the dimorphograptids in the P. acuminatus and Orthograpius vesiculosus Zones, but the monograptids appeared as late as in the O. vesiculosus zone; this evolutionary series can be easily accepted by other people. Chen Xu and Lin Yao-kun (1978) pu...     

Analysis of endo-beta-N-acetylglucosaminidase activity by high-pressure liquid chromatography on a silica-based chemically bonded octadecyl column

We have devised a new method for assaying the endo-β-N-acetylglucosaminidase activity by using the dansyl asparaginyl oligosaccharide, (Man)₅(GlcNAc)₂-Asn-DNS, as the substrate and analyzing the product, GlcNAc-Asn-DNS, by a reverse-phase high-pressure liquid chromatography using a silica-based chemically bonded octadecyl column (Waters μBondapack C₁₈). The column is eluted with 8% acetonitrile in 25 mm sodium borate buffer, pH 7.5, at 3 ml/min. The effluent is monitored by a Perkin-Elmer LC-75 uv monitor at 213 nm and a Perkin-Elmer LC-1000 fluorescence monitor (excitation, 313 nm; emission, 540 nm). Under these conditions, GlcNAc-Asn-DNS is well separated from (Man)₅(GlcNAc)₂-Asn-DNS and the analysis can be completed in 5 min. The peak height is used to quantify the dansyl derivatives. Under the conditions described above, the lower limit of detection is 0.1 nmol of dansyl glycopeptides.     

Synchronization of 9L rat brain tumor cells by centrifugal elutriation

Asynchronous 9L cells were separated into relatively homogeneously-sized populations using centrifugal elutriation with both a conventional collection method and a long collection method. A substantial increase in the homogeneity of the volume distributions and in the degree of synchrony of the separated fractions was obtained using the long collection method. Autoradiographic data indicated that fractions containing ≥97% G₁ cells, ≥80% S cells, and 70–75% G₂ cells could be routinely recovered with this procedure. Recovery in these fractions varied from 5 to 8% of the total number of cells elutriated. The colony forming efficiency (CFE) of cells from fractions representing each phase of the cell cycle was a constant 60–70%, which was comparable to the 60–80% usually found for asynchronous 9L cells. The percentage of cells in the G₁, S, and G₂ phases in the elutriated fractions was more accurately determined from the volume distribution than from computer fits of the DNA histogram obtained from flow cytometry. In general, the degree of synchrony was related to the coefficient of variation (CV) of the volume distributions of the elutriated fractions. The CV was about 14% for all elutriated fractions. When the ≥97% G₁ population was allowed to progress to S and G₂, the CVs were about 17 and 20.2%, respectively. Thus, the best nonperturbing method for obtaining synchronous 9L cells in the S or G₂ phases was direct elutriation with the long collection method.     

Proliferation inhibition of murine pluripotent haemopoietic stem cells by interferon or poly I:C

The effect of mouse serum interferon (IF) in vitro and an inducer in vivo on the proliferation of a pluripotent stem cell population with high turnover rate was studied. Proliferation rate was characterized by the number of CFUs in the S phase of the cell cycle. Increased proliferation of bone marrow stem cell populations was produced either by irradiating the donor mice with 3.36 Gy (336 rad) 60Co-gamma rays 7 days before the experiment or by incubating normal bone marrow cells with 10(-11) M concentration of isoproterenol. IF considerably reduced the number of CFUs in S phase in both cases without reducing the CFUs content of the samples. Injection of IF inducer (4 mg/kg poly I:C) into regenerating mice also inhibited the proliferation of CFUs without decreasing the femoral CFUs level. Regeneration kinetics of CFUs from irradiated poly I:C-treated mice ran parallel with that of irradiated untreated animals but showed a characteristic delay corresponding to approximately one CFUs doubling. A transient, non-cytotoxic proliferation inhibitory effect of IF or IF inducer is, therefore, proposed.     

Modification of cell membrane in variants of chinese hamster cells resistant to abrin

Stable and heritable variants of Chinese hamster ovary (CHO) cells which are resistant to different levels (0.1, 1.0 and 10 μg/ml) of the toxin abrin have been isolated and characterized. The frequency of resistant colonies to abrin was increased with the concentration of a chemical mutagen. There was no effect of cell density or cross-feeding on the recovery of variants. In experiments using fluorescein-labeled abrin and ricin which bind to terminal (non-sialylated) galactose residues of cell-surface oligosaccharides, parental cells exhibited strong binding toward both toxins, whereas no fluorescence was observed in the resistant clones. A fluorescein-conjugated lectin, BS II, which is specific for terminal N-acetyl- -glucosaminyl residues, did not interact with the parental cells, but did with the resistant clones. This suggests that on the surface of resistant cells the number of terminal galactosyl residues of oligosaccharide chains in glycoproteins was reduced, exposing the penultimate N-acetyl- -glucosaminyl residues. The number of available endogenous acceptor sites for galactosyl transferase in the abrin-resistant clones was directly proportional to the degree of resistance. In the presence of great excess of exogenous acceptor, the rates of galactosyl transfer were similar in all the abrin-resistant cell types tested, with levels ranging from 1.4 to 1.7 times parental cell values. Studies with tetraploid cell hybrids reveal that resistance was a recessive trait. Fluctuation analysis showed that abrin resistance occurred in CHO cell populations at a rate of 4−7 × 10⁻⁸/cell/generation. The system may serve as a new marker for quantitative mutagenesis studies.     

Structures and fatty acid compositions of neutral glycosphingolipids of human plasma

Major neutral glycosphingolipids were isolated from human plasma and their structures and fatty acid compositions studied. The four neutral glycosphingolipids of plasma were characterized as Glc beta(1 leads to 1)ceramide, Gal beta(1 leads to 1)- ceramide, Gal beta(1 leads to 4) Glc beta (1 leads to 1)ceramide, Gal alpha(1 leads to 4) Gal beta(1 leads to 4) Glc beta(1 leads to 1)ceramide and GalNAc beta(1 leads to 3) Gal (1 leads to 4) Gal (1 leads to 4) Glc beta(1 leads to 1)-ceramide. The glycosphingolipids contained mostly short chain fatty acids of which most prominent was C16. Erythrocyte glucosylceramide and lactosylceramide exhibited similar fatty acid compositions as their plasma counterparts. Triglycosylceramide and globoside of erythrocytes contained almost exclusively long-chain fatty acids. In lactosylceramide obtained from "p" erythrocytes, an accumulation of long-chain fatty acids was found; this accumulation was not observed, however, in lactosylceramide isolated from "p" plasma. It was concluded that plasma and erythrocyte glycosphingolipids are synthesized at separate sites where short- and long-chain fatty acids, respectively, are available. Plasma and erythrocyte glucosylceramide, and probably a fraction of lactosylceramide, exchange between plasma and erythrocyte pools. The latter conclusion is discussed in the light of the relative roles of carbohydrate and lipid moieties of the glycosphingolipids in maintaining their association with erythrocyte membranes.     

The structure of monellin and its relation to the sweetness of the protein.

The sweet protein monellin [1-3] has been shown to consist of two non-identical subunits of 50 and 42 amino acid residues, which were separated electrophoretically and chromatographically. Automatic sequential Edman degradation gave the complete sequence of the longer subunit, and a partial sequency of the shorter one. It was found that the sweetness of monellin requires the undissociated molecule. The individual subunits were not sweet, neither did they block the sweet sensation of sucrose or monellin. Blocking of the single SH of monellin abolished its sweetness as did reaction of the single methionyl residue with CNBr. Since the cysteinyl and methionyl residues appear to be adjacent, it is suggested that this part of the molecule is essential for its sweetness.