Dynamic changes of microtubule and actin structures in CV-1 cells during electrofusion

To study the involvement of the cytoskeletal system in the fusion of animal cells, we examined the dynamic changes of cytoskeletal proteins during the various stages of cell fusion. CV-1 cells were fused by applying a radio-frequency electrical pulse. Structural changes of microtubules (MTs) and F-actin were monitored simultaneously by double-label fluorescence microscopy. It was observed that in a few minutes after the initiation of cell fusion, MT bundles began to extend into the cytoplasmic bridges which were formed by fusing the membranes of neighboring cells. Later, a network of parallel MT bundles appeared between the adjacent nuclei of the fusing cells; such MT bundles may provide the mechanical links that are responsible for nuclear aggregation. The structural changes of F-actin during cell fusion were more complicated. We observed many different patterns of actin distribution in the fusing cells, including some giant, ring-shaped structures. Reorganization of actin is unlikely to be involved in the nuclear aggregation process. Instead, actin bundles condensed at the cell edges may help to widen the cytoplasmic bridges to allow merging of cellular contents between the fusing cells.     

Partial characterization of microsomal sialyltransferase from chicken liver and hepatoma Mc-29: I. Effect of nucleotides and metal ions

CMP-N-acetylneuraminic acid: glycoprotein sialyltransferase activities were assayed in microsomal fractions from chicken liver and hepatoma, induced by the leukosis virus strain Mc-29, using asialofetuin as the substrate acceptor of N-acetylneuraminic acid. The effect of some nucleotides and metal ions on the enzyme activity was investigated. Kinetic studies revealed that the Km values toward asialofetuin at a saturation concentrations of CMP-N-acetylneuraminic acid for both liver and hepatoma enzymes are very closed, while V value was lower for the tumor enzyme. The liver and hepatoma enzymes have no exogenous Mn cations requirement and are inhibited by CTP, CMP and ATP. CMP was shown to act as a competitive inhibitor with an apparent Ki of 0.24 mM for the liver and 0.16 mM for hepatoma enzyme, respectively.     

Effects of hyperthermia and photodynamic therapy on BALB/c mice bearing subcutaneous tumors

The therapeutic effects of photodynamic therapy and hyperthermia on mice bearing subcutaneous tumors were investigated. Ehrlich ascites tumor cells (1 x 10(7)) were implanted subcutaneously into the femoral area of BALB/c mice. A total of 134 tumor-bearing mice were treated with photodynamic therapy, i.e., administration of laser irradiation (514.5 nm, 112.5 mW/cm2 for 11.12 min with a total energy 75 J/cm2) after injection (i.p.) of hematoporphyrin derivative (HPD, 7.5 and 10.0 mg/kg body weight) and/or hyperthermia (by electric heating needles to 44 and 45 degrees C for 30 min) once a day for three successive days. The results revealed that the therapeutic effects of the combination of photodynamic therapy and hyperthermia were improved when compared with photodynamic therapy or hyperthermia alone. A combination of photodynamic therapy (10.0 mg HPD/kg body weight and 75 J/cm2 of total laser irradiation energy) and hyperthermia (44 degrees C for 30 min) had the best therapeutic effect, indicating that the mortality rate within 120 days (MR120) was 12.5% and the mean survival time (MST120) was 113.8 days.     

Linkage disequilibrium studies in multiple endocrine neoplasia type 1 (MEN1)

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterised by tumours of the parathyroids, pancreas and anterior pituitary. The MEN1 gene has been localised to a 2-Mb region of chromosome 11q13 by meiotic mapping studies in MEN1 families. Such studies may have a limited resolution of approximately 1 cM (i.e. 1 Mb) and we have therefore investigated 96 MEN1 families (40 British, 17 French, 12 Finnish, 7 Swedish, 7 Dutch, 7 North American, 2 Australian, 1 New Zealand, 1 German, 1 Spanish and 1 Danish) for linkage disequilibrium, in order to facilitate a finer mapping resolution. We have utilised five microsatellite DNA sequence polymorphisms from the candidate region and have accurately determined their allele sizes, which ranged from 161 bp to 272 bp. The heterozygosity and number of alleles (given in brackets), respectively, at the loci were: D11S1883 (76%, 11), D11S457 (55%, 5), PYGM (94%, 18), D11S1783 (10%, 4) and D11S449 (87%, 16). Allelic association was assessed by Chi-square 2 ×n contingency tables, by Fisher exact 2 ×n contingency tables and by a likelihood-based approach. The results of haplotype analysis revealed 91 different affected haplotypes in the 96 families, an identical affected haplotype being observed in no more than two families. These results indicate the absence of an ancestral affected haplotype. Significant linkage disequilibrium (P < 0.005) could be established amongst the microsatellite loci but not between the loci and MEN1 in either the total population or in any of the geographical sub-populations. The absence of linkage disequilibrium between MEN1 and the polymorphic loci is probably the result of the occurrence of multiple different disease-causing mutations in MEN1. Received: 1 April 1997 / Accepted: 25 June 1997     

Low-level inversion of the L component of pseudorabies virus is not dependent on sequence homology.

Pseudorabies virus has a class 2 genome in which the S component is found in two orientations relative to the L component. The L component is bracketed by sequences that are partially homologous; it is found mainly in one orientation, but a small proportion is inverted (J. M. DeMarchi, Z. Lu, G. Rall, S. Kuperschmidt, and T. Ben-Porat, J. Virol. 64:4968-4977, 1990). We have ascertained the role of the patchy homologous sequences bracketing the L component in its inversion. A viral mutant, vYa, from which the sequences at the right end of the L component were deleted was constructed. Despite the absence of homologous sequences bracketing the L component in vYa, its L component inverted to an extent similar to that of the L component in the wild-type virus. These results show the following. (i) The low-frequency inversion of the L component of PrV is not mediated by homologous sequences bracketing this component. (ii) Cleavage of concatemeric DNA at the internal junction between the S and L components is responsible for the appearance of the minority of genomes with an inverted L component in populations of pseudorabies virus. (iii) The signals present near or at the end of the S component are sufficient to allow low-frequency cleavage of concatemeric DNA; the sequences at the end of the L component are not essential for cleavage, although they enhance it considerably.     

Root growth inhibition of the holorganic layer of moder humus under spruce (Picae abies KARST.) and beech (Fagus Silvatica L.)

A test of root growth inhibition of spruce and beech roots, according to Lynch's procedure (1977), shows the inhibitory effects of soil solution extracted from the holorganic layers (Of₂-Oh) under beech and spruce. Molecular gel filtration of soil solutions shows that the molecular weights vary over a wide range, from less than 100 to over 40,000 daltons. Chemical analysis, using CGC, HPLC and sometimes MS shows only negligible concentrations of simple aliphatic (C₁-C₅) and aromatic acids in the free state. Using the fraction scheme of Forsyth (1977) and the carbazole procedure, it is shown that uronic acids represent only a small percentage of the carboxylic acids, and have no inhibitory effects on root growth. By analogy with results of other authors, the presence of polycarboxylic acids in the soil solution are considered to be the main cause of root growth inhibition.