Quantitative determination of pectic substances as an example of a rhamnopolysaccharide assay

A quantitative enzymatic assay for rhamnopolysaccharides is described. The procedure is shown with pectic substances as an example. The test is based on the enzymatic degradation of the macromolecules to liberate L-rhamnose. This sugar can be quantitatively determined with the help of L-rhamnose dehydrogenase under concomitant reduction of NAD, thus allowing the quantitative evaluation of the original pectin.     

Evidence for a homolog of the yeast cell cycle regulatory gene product of cdc2+ in Physarum polycephalum

Evidence for the presence of a Cdc2-like protein in Physarum polycephalum has been obtained using a peptide antibody directed against a highly conserved amino acid sequence near the N-terminal end of Cdc2, Cdc28 and Cdc2HS. The antibody detected a 34 kDa cytoplasmic protein, similar in apparent size to Cdc2 in yeast and Cdc2Hs in HeLa cells. A 60 kDa nuclear band was also detected in Physarum but not in yeast or HeLa. Evidence is presented that this is not related to the 34 kDa protein nor is it found in HeLa nuclei or yeast cells. The Cdc2-like protein level did not fluctuate over more than 10 h of the naturally synchronous cell cycle of Physarum. Several heat-shock experiments using regimens that either: delayed mitosis and S-phase; prevented mitosis or uncoupled S-phase from mitosis were performed. None had any effect on the level of the Cdc2-like protein. The induction of spherulation by starvation was shown to have no effect on the levels of the 34 kDa Cdc2 analog. The invariant level of the 34 kDa protein during the cell cycle and starvation is consistent with previous results obtained with yeast. Three heat-shock regimens which either delay mitosis, eliminate S-phase or uncouple mitosis from S-phase in Physarum also had no effect on the level of the 34 kDa protein. This result emphasizes the stable nature of this protein.     

Reclustering of scattered Golgi elements occurs along microtubules

Depolymerization of the interphase microtubules by nocodazole results in the scattering and apparent fragmentation of the Golgi apparatus in Vero fibroblast cells. Upon removal of the drug, the interphase microtubules repolymerize, and the scattered Golgi elements move back to the region around the microtubule-organizing center (MTOC) within 40 to 60 min. Using a fluorescent lipid analogue (C6-NBD-ceramide) as a vital stain for the scattered Golgi elements, their relocation was visualized by video-enhanced fluorescence microscopy in Vero cells maintained at 20 degrees C. The NBD-labeled structures were identified as Golgi elements by their colocalization with galactosyltransferase in the fixed cells. During reclustering, NBD-labeled Golgi elements were observed to move by discontinuous saltations towards the MTOC with velocities of 0.1 to 0.4 micron/s. Paths along which Golgi elements moved were super-imposable on microtubules visualized by indirect immunofluorescence. Neither the collapse of intermediate filaments caused by microinjection of antibodies to vimentin nor the disruption of microfilaments by cytochalasin D had an effect on the reclustering of Golgi elements or the positioning of the Golgi apparatus. These data show that scattered Golgi elements move along microtubules back to the region around the MTOC, while neither intact intermediate filaments nor microfilaments are involved.     

Correction of lipid peroxidation in stress with the peptide bioregulator thymopentin

Experiments on rats subjected to acute stress have revealed protective effect of thymopentin pentapeptide on somatic disorders and the state of the antioxidation system and the processes of lipid peroxidation in blood and brain.     

The role of Na K ATPase in thiamine and lipoic acid interrelations during their absorption in the gastrointestinal tract of mice

35S-lipoic acid (20 mumol/kg) with different doses of thiamine or 35S-thiamine (50 mumol/kg) with unlabelled lipoic acid where administrated to the white mice stomach. It was established that absorption and entrance of both lipoic acid and thiamine when they were in 1:5 quantities in organs and tissues were maximal. The activity of Na+, K-ATPase determined in stomach, duodenum and small intestine was the maximal too.     

Spin label and microcalorimetric studies of the interaction of DNA with unilamellar phosphatidylcholine liposomes

High-molecular DNA from chicken erythrocytes interacts with 1,2-dipalmitoylphosphatidylcholine in unilamellar liposomes, both in the presence and absence of Mg2+ ions. This interaction results in a phase separation in liposome membranes. The new phase induced by DNA and Mg2+ has a higher gel-liquid crystal phase transition temperature as measured by microcalorimetry. In the liquid crystalline state, the 16- and 5-doxyl stearic acid spin labels indicate changed local bilayer properties at the label position in the new phase.     

Differential distribution of alpha-tubulin isotypes in Euplotes eurystomus determined by confocal immunofluorescence microscopy

Detergent permeabilized Euplotes eurystomus (a fresh water hypotrichous ciliate) was reacted with monoclonal and polyclonal antibodies specific for either detyrosinated or tyrosinated alpha-tubulin (Glu- or Tyr-tubulin). The isolated cytoskeleton-nuclear complex was examined by Western immunoblotting and by immunofluorescent and electron microscopic methods. Both Glu- and Tyr-tubulins were detected by immunoblot analysis. Immunofluorescent microscopy indicated that the alpha-tubulin isotypes are concentrated in different regions of permeabilized cells: Glu-tubulin is located primarily in cirri, membranelles, and surrounding the macro- and micronuclei. Tyr-tubulin is principally at the bases of cirri and membranelles. This differential distribution of alpha-tubulin isotypes is discussed in terms of current concepts concerning the correlation of tubulin post-translational modifications to microtubule stability. Confocal immunofluorescent imaging was of critical importance in clearly differentiating the Glu-tubulin isotype surrounding the macro- and micronuclei from a brilliantly fluorescent environment originating from cytoskeletal structures. In conjunction with conventional and stereo-electron microscopy, confocal optical microscopy provided convincing evidence for a "basket" of microtubules surrounding both nuclei.     

Method for rapid separation of 3,5,3'-triiodothyroacetic acid in human serum by fast protein liquid chromatography

The 3,5,3'-triiodothyroacetic acid (TRIAC) has been approved as a valuable agent in the management of hyperthyroidism secondary to inappropriate secretion of thyrotropin. We have developed a fast protein liquid chromatography (FPLC) method for separation and quantification of TRIAC. Serum samples charged with TRIAC were extracted with methanol/ammonium acetate, the supernatants were evaporated to dryness, reconstituted in NaOH and injected on a reversed phase column for chromatography. For separation an isocratic elution method (methanol water; 0.1% trifluoroacetic acid) was used. The area under the curve (ml%) was compared with those of the calibration curves. Recoveries were 70 +/- 10.8%. TRIAC was eluted in 2.33 ml. Conclusively, the present method shows that TRIAC can be measured by FPLC and may be applied to the measurement of TRIAC in pharmacological studies.     

Epstein-Barr virus episome-based promoter function in human myeloid cells.

Epstein-Barr virus (EBV) episomal replicons offer an expeditious means for amplifying transfected genes in human cells. A panel of EBV episomes was constructed to assess the relative utility of five distinct eukaryotic promoter elements for high level and inducible gene expression in stably transfected human myeloid leukemia cells. The Rous sarcoma virus 3' long terminal repeat (LTR) was most highly suited for EBV episome-based gene expression, whereas the lymphopapilloma virus and the SV40 early regulatory elements exhibited substantially lower activities. Chemically responsive promoter elements, such as the SV40 early, human metallothionein IIA and rat GRP78 gene promoters, retained their inducibility when EBV episome-based. Differences in gene expression obtained with the episomes reflected differential promoter activity rather than significant variations in episome copy numbers per cell. These observations provide guidelines for the optimal design of EBV episomal expression vectors for human expression work.