Structural characterization of lipid A component of Erwinia carotovora lipopolysaccharide

The chemical structure of the lipid A component of lipopolysaccharide excreted into the liquid medium by the plant pathogenic enterobacterium Erwinia carotovora FERM P-7576 was characterized. It consists of a -1, 6-linked glucosamine disaccharide which carries ester-and amide-bound fatty acids and phosphate similar to the lipid A from other gram-negative bacteria. The lipid A preparation was not uniform in the number and composition of the fatty acids linked to the disaccharide. Four prominent lipids A were involved, they were composed of five to seven residues of fatty acid. Among them the major component was hexa-acyl lipid A, in which the hydroxyl group at position 3 and the amino group of the non-reducing glucosamine unit carry 3-dodecanoyl-oxytetradecanoyl residues. Positions 2 and 3 of the reducing glucosamine unit were substituted by 3-hydroxytetradecanoic acid. In the hepta-acyl lipid A, an additional hexadecanoic acid was linked to the hydroxyl group of the 3-hydroxytetradecanoyl residue at position 2 of the hexa-acyl lipid A. Two penta-acyl lipids A were the homologs of the hexa-acyl lipid A with decreasing acylation. Dodecanoic acid was missing from one, and 3-hydroxytetradecanoic acid from another. 3-Dodecanoyloxytetradecanoyl residue at position 3 differentiates E. carotovora lipid A from that of other gram-negative bacteria.Abbreviations LPS lipopolysaccharide - GlcN glucosamine - KDO 3-deoxy-d-manno-octulosonic acid - FAB-MS fast atom bombardment mass spectrometry - u atomic mass unit     

Distribution of Leu 7 (HNK-1) antigen in human digestive organs: an immunohistochemical study with monoclonal antibody

Summary Leu 7 immunoreactivity was demonstrated with the indirect peroxidase-labelled antibody method on frozen and paraffin-embedded tissue sections of human digestive organs. Anti-Leu 7 monoclonal antibody, which allegedly detects mononuclear cells with natural killer or killer activity, recognized lymphoid cells among intestinal epithelial cells and in the germinal centres of solitary lymphoid follicles of small and large intestine, and a few in gallbladder, liver and the lamina propria of the intestine. In addition, peripheral nerve fibres, endocrine cells in the gut and pancreas and carcinoid and islet cell tumours were also positively stained. At the ultrastructural level, Leu 7 antigen was localized on the plasma membrane of granulated lymphoid cells in the gut mucosa and on the secretory granules of intestinal endocrine cells. In normal pancreas, Leu 7 immunoreactivity was demonstrated in most cells containing pancreatic polypeptide and in many cells containing somatostatin or glicentin. Insulin-containing cells, however, lacked Leu 7 immunoreactant. These findings were obtained in both frozen sections and paraffin-embedded sections. The possible cross-reactivities of monoclonal antibodies are discussed as they raise an important caveat in immunohistochemical studies using these antibodies.     

Involvement of colchicine-sensitive cytoplasmic element in premitotic nuclear positioning ofAdiantum protonemata

Summary When the red-light grown protonema ofAdiantum capillus-veneris was transferred to the dark, the nucleus ceased its migration ca. 5 hours before cell plate formation (Mineyuki andFuruya 1980). To see whether the nucleus was held by some cytoplasmic structure during nuclear positioning, protonemata were treated with various centrifugal forces at different stages of the cell cycle. Nuclei of G₁ phase were easily displaced by centrifugation at 360×g for 15 minutes, but those of G₂ or M phase were not displaced by it, suggesting that the nuclei were held by some cytoplasmic elements in G₂ or M phase. This nuclear anchoring was not detectable in protonemata that were treated with 5mM colchicine. With this treatment, the nucleus did not stop its migration at late G₂ and moved even in prophase. And the retardation of organelle movement which was observed in cytoplasm on the lateral side of the nucleus after the cessation of premitotic nuclear migration (Mineyuki andFuruya 1984) was not observed in the presence of colchicine. Thus the nuclei appear to be held by colchicine-sensitive structure in cytoplasm between the lateral surface of the nucleus and cell wall during the premitotic nuclear positioning. Electron micrographs showing cytoplasmic microtubules were consistent with the idea.Abbreviations PPN Premitotic positioning of the nucleus - L region Cytoplasm between the lateral surface of the nucleus and cell wall (seeMineyuki et al. 1984)     

W-heterochromatin of chicken; its unusual DNA components,late replication,and chromatin structure

About 65% of DNA in the chicken W chromosome has been shown to consist ofXhoI andEcoRI family repetitive sequences. These sequences showed remarkable delay in the electrophoretic mobility at low temperature on a polyacrylamide gel. Three dimensional structures of the 0.7-kbXhoI and the 1.2-kbEcoRI family repeating units were estimated to be irregular solenoids using a computer program based on wedge angles of all the 16 dinucleotide steps. Fluorescencein situ hybridization demonstrated that these two family sequences were localized in a major heterochromatic body in an interphase nucleus. Incorporation of bromodeoxyuridine into the W chromosome in the synchronous culture of MSB-1 cells occurred about 1 h later than the peak of S phase. The chromatin structure formed alongXhoI andEcoRI family sequences was suggested to be different from the total chromatin or chromatin containing the β-actin gene sequence in that the linker DNA lengths of the former were significantly longer. Fractionation of theHaeIII-digested MSB-1 nuclei yielded a chromatin fraction in whichXhoI family sequences were partially enriched. Several DNA-binding proteins showing higher affinity for theXhoI family sequence were present in this fraction.     

Some Electrical Properties of a Nuclear Membrane Examined with a Microelectrode

Electrical potential and resistance were measured with microelectrodes in in situ and isolated nuclei of gland cells of Drosophila flavorepleta. The nucleus-cytoplasm boundary was found to be rather impermeable to ion diffusion. It presents a resistance of the order of 1 Ω cm² and sustains a "resting" potential, the nucleoplasm being about 15 mv negative with respect to the cytoplasm. Both the resistance and potential appear to be associated with the nuclear membrane: the potential declines to zero and the resistance to a fraction of its original value, when the membrane is perforated experimentally.     

Changes in Organelle Movement in the Nuclear Region during the Cell Cycle of Adiantum Protonema

Movements of organelles in the nuclear region as the cell cycleprogresses in single-celled protonemata of Adiantum capillus-veneriswere examined by digital image processing techniques and microscopyof particle movement. Organelles in the nuclear region werenot very crowded and moving directionally along the longitudinalaxis of the filamentous cell in the G₁ and S phases. They beganto gather and accumulate in the nuclear region in early G₂ phase,after which directional movement changed to undirectional Brownianmotion-like movement in late G₂ phase. Movement of organelleslocated on the lateral surface of the nucleus slowed after premitoticpositioning of nucleus and lasted until the nucleolus disappeared.Movement of organelles in the cytoplasm surrounding the nucleoplasmresumed just after the nucleolus disappeared, whereas organelleslocated in the outer regions of the apical and basal surfacesof the nucleus moved rapidly during prophase but did not moveduring metaphase, movement being resumed after chromosome separation.Thus, organelle movement in the nuclear region showed temporaland spatial change during the cell cycle. (Received August 24, 1983; Accepted December 28, 1983)     

Development of two cloned epithelial cell lines from normal adult mouse and rat ventral prostates

Summary Two epithelial cell lines were established, one from adult C3H mouse and one from adult Fischer rat ventral prostate. These cell lines were obtained from explant cultures, using Ham's F12 medium supplemented with HEPES, insulin, testosterone, hydrocortisone, epidermal growth factor, and 7.5% fetal bovine serum. A low concentration of trypsin and EDTA in Ca⁺⁺-and Mg⁺⁺-free phosphate buffer was used for passaging the cells. The rat cell line was established following implantation of prostate tissue in nude mice. These cell lines stained positively for acid phosphatase and were dependent upon epidermal growth factor for growth. Morphological studies, including electron microscopy, revealed a highly characteristic epithelial morphology of both cell lines. These cell lines have hypotetraploid chromosome numbers and are capable of metabolizing benzo(a)pyrene. We propose the application of these cells as models for the study of prostate carcinogenesis. This work was supported in part by Grant CA-21, 746, and by the Electron Microscope Core Facility on Grant CA-14,089, from the National Cancer Institute, National Institutes of Health, Bethesda, MD.     

Reduced growth of Ehrlich ascites tumor cells in creatine depleted mice fed β-guanidinopropionic acid

The effect of implantation of Ehrlich ascites tumor (EAT) cells of creatine distribution was investigated. It was also studied how depletion of creatine by feeding creatine-analogue β-guanidinopropionic acid (β-GPA) affects the growth of EAT cells in mice. Enhanced mobilization of creatine from host tissues to EAT cells against a greater concentration gradient was observed. The creatine (but not creatinine) level in blood plasma was lowered to 22% of the normal value by β-GPA feeding alone and assimilation of ¹⁴C-creatine into EAT cells was inhibited. The growth of EAT cells was significantly reduced and the duration of survival of mice after implantation of EAT cells was extended when the creatine concentration was decreased. A decrease in daily food consumption and the degree of muscle atrophy after implantation of EAT cells was less in β-GPA than control groups. In the creatine-depleted mice, the rate of increase in total EAT cell number and the volume of abdominal ascites were approximately half of the control values, and more dead EAT cells were observed. These results suggest that supplementation of β-GPA inhibits creatine transfer to EAT cells and reduces the growth of cancer cells.     

The changes of nuclear position and distribution of circumferentially aligned cortical microtubules during the progression of cell cycle inAdiantum protonemata

The intracellular positions of the nucleus and of cortical, circumferentially aligned microtubules (CCAM) in filamentous, single-celled protonemata ofAdiantum capillus-veneris were determined throughout the cell cycle in the dark. When apical growth continued at G₁ phase, the nucleus migrated keeping a constant distance from the tip. When the apical growth stopped at late S or G₂ phase, the nucleus stopped moving forward and then slightly moved backward to the site of cytokinesis. The CCAM were found only in the dome of protonemal tip when growing under continuous red light; they increased in number after dark incubation for 12 hr and then decreased after 20th hr in the dark. The CCAM were usually observed in the region between the nucleus and the tip at 28 hr in the dark. They were located around the nuclear region at pre-prophase and prophase, but then totally disappeared at metaphase and thereafter.