Characterization of tumor-associated fucogangliosides from PC 12 pheochromocytoma cells

PC 12h pheochromocytoma cells were subcutaneously transplanted into rat. We found the transplanted tumors accumulated some fucogangliosides associated with PC 12 cells. These gangliosides were isolated and purified by DEAE-Sephadex A-25 and Iatrobeads column chromatographies. Their structures were determined by fast atom bombardment mass spectrometry, proton nuclear magnetic resonance spectrometry, permethylation study, and sequential degradation using various exoglycosidases and mild acid hydrolysis. Two tumor-associated fucogangliosides were found to possess the blood group B determinant as follows: G6: IV2Fuc alpha, IV3Gal alpha, II3NeuAc, GgOse4Cer; G11: IV2Fuc alpha, IV3Gal alpha, II3 (NeuAc)2, GgOse4Cer. A ganglioside with the similar structure as ganglioside G6 was isolated from rat hepatoma cells (Holmes, E.H., and Hakomori, S-I. (1982) J. Biol. Chem. 257, 7698-7703). However, ganglioside G11 has not previously been reported in the literature. These fucogangliosides reacted with the monoclonal antibody prepared by immunizing mice with PC 12h cells. Other fucogangliosides were also found to accumulate in the transplanted tumor tissues. They were identified as fucosyl-GM1 and fucosyl-GDlb. These fucogangliosides did not react with the monoclonal antibody against PC 12h cells.     

Structure-activity relationship of gramicidin S analogues on membrane permeability

The previous study of the action of gramicidin S on bacteria (Katsu, T., Kobayashi, H. and Fujita, Y. (1986) Biochim. Biophys. Acta 860, 608-619) prompted us to investigate further the structure-activity relationship of the gramicidin S analogues on membrane permeability. Two types of the gramicidin S analogues were used in the present study: (1) cyclo(-X-D-Leu-D-Lys-D-Leu-L-Pro-)2, where X = Gly, D-Leu and D-cyclohexylalanine (D-cHxAla); (2) N,N'-diacetyl derivative of gramicidin S (diacetyl-gramicidin S) which lacks a cationic moiety of gramicidin S. All the analogues have a beta-sheet conformation as gramicidin S. The following cellular systems were used: Staphylococcus aureus as Gram-positive bacteria, Escherichia coli as Gram-negative bacteria, human erythrocytes, rat liver mitochondria and artificial liposomal membranes. It was found that gramicidin S and one of the type 1 analogues having X = D-cHxAla induced the efflux of K+ through the cytoplasmic membrane of all types of the cells. In addition, these two peptides had the ability to lower the phase transition temperature of dipalmitoylphosphatidylcholine. Accordingly, it was concluded that, if peptides can expand greatly the membrane structure of neutral lipids which constitute main parts of the biological membrane, they can stimulate the permeability of cells without any selectivity. The action of the type 2 peptide, diacetyl-gramicidin S, was strongly cell dependent. Although this peptide stimulated the efflux of K+ from mitochondria, it did not do so efficiently, if at all, from S. aureus, E. coli and erythrocytes. In experiments using liposomes, diacetyl-gramicidin S increased markedly the permeability of liposomes composed of egg phosphatidylcholine. The presence of egg phosphatidylethanolamine or cholesterol reduced its activity. These results on liposomes explained well the low sensitivity of diacetyl-gramicidin S against E. coli and erythrocytes in terms of lipid constituents of the membranes. The mechanism of action of diacetyl-gramicidin S was discussed from the formation of a boundary lipid induced by this peptide.     

Vasoactive-intestinal-polypeptide (VIP)-like immunoreactive cells in the skull base of rats

Summary We investigated the distribution of vasoactive intestinal polypeptide (VIP)-like immunoreactive cell bodies in relation to the major cerebral and internal carotid arteries at the skull base in rats. Acetylcholinesterase (AChE) histochemistry was also applied to investigate the localization of this enzyme. VIP staining revealed a few positive cell bodies in nerves close to the internal carotid artery at the base of the skull as well as in the cerebral arterial wall. Ganglion-like cell bodies were detectable within the greater superficial petrosal (GSP) nerve. AChE activity was observed in VIP-like immunoreactive cell bodies along the whole of the GSP nerve. These cell bodies in the GSP nerve may give rise to at least some of the perivascular VIP-and AChE-containing nerves of the internal carotid arteries at the base of the skull.     

Enhancement of the actin-activated ATPase activity of myosin from canine cardiac ventricle by purealin

The effects of purealin isolated from the sea sponge, Psammaplysilla purea, on the enzymatic properties of myosin and natural actomyosin (a complex of myosin, actin, tropomyosin and troponin) from canine cardiac ventricle were studied. Purealin increased the ATPase activity of natural actomyosin and the actin-activated ATPase activity of myosin, and accelerated the superprecipitation of natural actomyosin. The Ca2+- and Mg2+-ATPase activities of myosin were inhibited by purealin, whereas the K+-EDTA-ATPase activity was increased. These results suggest that purealin binds to the myosin portion involved in actin-myosin interaction and increases the actin-activated ATPase activity of myosin.     

Enzyme-entrapping behaviors in alginate fibers and their papers

Enzyme immobilization in the form of fiber and paper was easily achieved by wet spinning of aqueous admixture of sodium alginate and enzymes into divalent metallic ion solution as a coagulating bath, followed by paper making of resultant shortly cut fibers. Entrapment yields of enzymes used, e.g., glucoamylase, cyclodextrin glucanotransferase, endo-polygalacturonase, and protease, were always higher in calcium alginate fibers and their papers than those in corresponding beads. It was found that the yields increased with an increase of the discharge rate through the spinning nozzle because the higher discharge rate could provide more highly oriented metal-chelate linear polymer molecules along the fiber axis for preventing leakage of entrapped enzymes. Divalent metallic ions affected greatly the entrapment of glucoamylase in alginate fibers, the order of which followed roughly the ionotropic series of Thiele. Entrapment of glucoamylase in bicomponent systems comprising alginate and other water-soluble polymers was also investigated.     

Regional assignment of the gene for diphtheria toxin sensitivity using subchromosomal fragments in microcell hybrids

Human x mouse microcell hybrids resistant to G418 were constructed between mouse hepatoma cells and human x mouse whole cell hybrids containing only intact human chromosome 5 and 22 with an integrated neo ^r-gene. Among these, microcell hybrid BG15 produced four subclones, BG15-4, BG15-6, BG15-7 and BG15-9, which contained variously sized complements of human chromosome 5. BG15-6 contained an intact human chromosome 5, BG15-7 a deleted human chromosome 5 (5pter-q22) and BG15-4 and BG15-9 a translocation between parts of human chromosome 5 (pter-qter? and pter-q23, respectively) and a mouse chromosome. Southern DNA blot analysis showed that the human dihydrofolate reductase (DHFR) gene was present in all four subclones, whereas the human homolog of the v-fms gene was present in BG15-4 and 15-6, but absent from BG15-7 and 15-9. BG15-4, 15-6 and 15-9 were sensitive to diphtheria toxin, and only BG15-7 was resistant to the toxin. We used these microcell hybrids to restrict further the regional location of the gene for diphtheria toxin sensitivity to the q23 region of human chromosome 5.     

Regional assignment of five genes on human chromosome 19

A human-mouse hybrid segregant HM76Dd40-6 with new characteristics was derived from the hybrid cell line HM76Dd containing human chromosome 19 as the only human chromosome. Three virus sensitivities located on human chromosome 19 (PVS, E11S and RDRC) were lost in HM76Dd40-6, while six other genes (C3, LDLR, EF2, GPI, PEPD and MANB) were retained. Cytogenetic analysis and in situ hybridization using human or mouse repeated sequences as probes showed that the region q13.1-qter of human chromosome 19 had been replaced by a fragment of mouse chromosome. Our results permit further regional assignment for the following five genes on human chromosome 19: GPI in the region cen-q12, MANB in p13.2-q12, E11S and RDRC in q13.1-qter, and EF2 in pter-q12.     

Quantitative Analysis of Endogenous Gibberellins in Normal and Dwarf Cultivars of Rice

Endogenous levels of gibberellins in shoots and ears of twodwarf rice (Oryza sativa L.) cultivars, Tan-ginbozu (dx mutant)and Waito-C (dy mutant), were analyzed and compared with thoseof normal rice cultivar, Nihonbare. The endogenous levels of13-hydroxylated gibberellins in Tan-ginbozu were much lowerthan those in Nihonbare. In Waito-C, the levels of GA₁₉ andGA₂₀ in the shoots were higher but that of GA₁ was lower thanthe levels of these gibberellins in Nihonbare. These resultssupport the hypothesis that the dy gene controls the 3ß-hydroxylationof GA₂₀ to GA₁ while the dx gene controls a much earlier stepin the gibberellin biosynthesis. Our results indicate that GA₁is the active gibberellin that regulates the vegetative growthof rice. The endogenous levels of GA₄ in the ears of the twodwarf cultivars of rice were higher than the level of GA₄ inthe ears of the normal cultivar, Nihonbare suggesting that thebiosynthesis of gibberellin is not blocked in the anthers ofthe dwarf rice. (Received April 27, 1989; Accepted July 12, 1989)     

Cardiovascular changes associated with decreased aerobic capacity and aging in long-distance runners

Fifty-five male runners aged between 30 to 80 years were examined to determine the relative roles of various cardiovascular parameters which may account for the decrease in maximal oxygen uptake (VO2max) with aging. All subjects had similar body fat composition and trained for a similar mileage each week. The parameters tested were VO2max, maximal heart rate (HRmax), cardiac output (Q), and arteriovenous difference in oxygen concentration (Ca-Cv)O2 during graded, maximal treadmill running. Average body fat and training mileage were roughly 12% and 50 km.week-1, respectively. The average 10-km run-time slowed significantly by 6.0%.decade-1 [( 10-km run-time (min) = 0.323 x age (years) + 24.4] (n = 49, r = 0.692, p less than 0.001]. A strong correlation was found between age and VO2max [( VO2max (ml.kg-1.min-1) = -0.439 x age + 76.5] (n = 55, r = -0.768, p less than 0.001]. Thus, VO2max decreased by 6.9%.decade-1 along with reductions of HRmax (3.2%.decade-1, p less than 0.001) and Q (5.8%.decade-1, p less than 0.001), while no significant change with age was observed in estimated (Ca-Cv)O2. It was concluded that the decline of VO2max with aging in runners was mainly explained by the central factors (represented by the decline of HR and Q in this study), rather than by the peripheral factor (represented by (Ca-Cv)O2).