Quiescence in 9L cells and correlation with radiosensitivity and PLD repair

The onset of quiescence, changes in X-ray sensitivity, and changes in capacity for potentially lethal damage (PLD) repair of unfed plateau-phase 9L44 cell cultures have been systematically investigated. The quiescent plateau phase in 9L cells was the result of nutrient deprivation and was not a cell contact effect. Eighty-five to 90% of the plateau-phase cells had a G1 DNA content and a growth fraction less than or equal to 0.15. The cell kinetic shifts in the population were temporally correlated with a developing radioresistance, which was characterized by a larger shoulder in the survival curve of the quiescent cells (Dq = 5.71 Gy) versus exponentially growing cells (Dq = 4.48 Gy). When the quiescent plateau-phase cells were refed, an increase in radiosensitivity resulted which approached that of exponentially growing 9L cells. Delayed plating experiments after irradiation of exponentially growing cells, quiescent plateau-phase cells, and synchronized early to mid-G1-phase cells indicated that while significant PLD repair was evident in all three populations, the quiescent 9L cells had a higher PLD repair capacity. Although data for immediate plating indicated that 9L cells may enter quiescence in the relatively radioresistant mid-G1 phase, the enhanced PLD repair capacity of quiescent cells cannot be explained by redistribution into G1 phase. When the unfed quiescent plateau-phase 9L cells were stimulated to reenter the cell cycle by replating into fresh medium, the first G1 was extended by 6 h compared with the G1 of exponentially growing or refed plateau-phase 9L cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning of a lymphocyte homing receptor reveals a lectin domain

Lymphocytes express cell surface molecules, termed homing receptors, that mediate their selective attachment to specialized high endothelial venules found within secondary lymphoid organs. Previous work has demonstrated that the adhesive interaction between lymphocytes and the endothelium of peripheral lymph nodes appears to involve a lectin-like activity. Moreover, MEL-14, a monoclonal antibody that blocks lymphocyte-peripheral lymph node binding and presumably recognizes the homing receptor mediating this adhesive interaction, appeared to detect the lectin-like receptor. In this paper we describe the cloning of a murine cDNA that encodes the antigen recognized by the MEL-14 antibody. Characterization of the cDNA encoding the putative mouse peripheral lymph node-specific homing receptor shows that it contains a lectin domain that appears to be involved in the binding of lymphocytes to peripheral lymph node endothelium, thus defining a new type of cellular adhesion molecule. This result supports a novel mechanism for the distribution of lymphocyte populations to various lymphoid organs.     

Characteristics of intracellular calcium changes required for augmentation of phorbol ester-stimulated pancreatic enzyme secretion

Previous studies demonstrated that Ca2+ ionophores augment the pancreatic enzyme secretion caused by phorbol esters. The present study was performed to determine the nature of the cellular Ca2+ effects responsible for the augmentation. Relatively low concentrations (0.3-1.0 microM) of the nonfluorescent Ca2+ ionophore, 4-bromo-A23187 (Br-A23187), did not measurably increase free cytosolic Ca2+ ([Ca2+]i) and caused little or no enzyme release from guinea pig pancreatic acini. However, these concentrations of Br-A23187 augmented the amylase release caused by the phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA). This augmentation occurred in the absence of extracellular Ca2+ as long as the intracellular agonist-sensitive pool contained Ca2+. Greater concentrations of Br-A23187 (3-10 microM) alone caused transient increases in [Ca2+]i and transient increases in amylase release. Although not resulting in an increase in [Ca2+]i, the low concentrations of Br-A23187 caused release of Ca2+ from the intracellular agonist-sensitive pool. These results suggest that Ca2+ mediates enzyme release by two distinct mechanisms in the pancreatic acinar cell. First, an increase in [Ca2+]i alone mediates enzyme release. Second, Ca2+ release from the agonist-sensitive pool not resulting in a measurable increase in [Ca2+]i augments enzyme release stimulated by a phorbol ester. The second effect of Ca2+ may be due to a small localized change in cell Ca2+ or an induction of cytosolic Ca2+ oscillations.     

Acetyl-cholinesterase and fluoride-resistant acid phosphatase activities in dorsal root ganglia in the rat

Acetylcholinesterase and fluoride-resistant acid phosphatase activities were contrasted in alternative serial sections of rat dorsal root ganglia. The morphometric analysis demonstrated no correlation between cellular size and enzymatic activity. Differences with previous works in this area are discussed.     

Subfamily of submaxillary gland-specific Mup genes: chromosomal linkage and sequence comparison with liver-specific Mup genes

Mouse major urinary proteins (MUPs) are encoded by a family of ca. 35 genes that are expressed in a tissue-specific manner in several secretory organs; in the liver, in the submaxillary, sublingual, parotid and lachrymal glands, and in the skin sebaceous glands. In this paper we describe the isolation of a Mup gene, Mup-1.5a, which is expressed predominantly in the submaxillary gland of BALB/c mice. We show that Mup-1.5a is a member of a subfamily consisting of two closely related genes, both of which are closely linked to the Mup-1 locus on mouse chromosome 4. Mup-1 is the locus of a class of Mup genes (Group 1) expressed in the liver. The complete nucleotide sequence of Mup-1.5a has been determined, and was compared to a previously sequenced Group 1 Mup gene. The comparison shows that the differentially expressed Mup genes are uniformly divergent in exons, introns and in their flanking sequences. The regions of homology extend at least 5 kb into the 5' flanking region of Mup genes.     

Subunit structure and higher order assembly of the hemocyanins of five members of the Naticidae family of marine gastropods: Calinaticina oldroydii (Dall), Euspira heros (Say), Neverita duplicata (Say), Polinices draconis (Dall), and Polinices lewisii (Gould)

1. The hemocyanins of the Naticidae family, E. heros, N. duplicata, P. draconis, P. lewisii and C. oldroydii were investigated by sedimentation velocity and scanning transmission electron microscopy. 2. At pH 8.0, 0.05 M Mg2+ E. heros hemocyanin is found to be predominantly in the tri-decameric state with a sedimentation coefficient (So20,w) of 131.3 (+/- 0.6) S. While the hemocyanin of N. duplicata is also mainly in the 130 S form, the hemocyanin of C. oldroydii is largely in the di-decameric form with a sedimentation coefficient close to 100 S. Other Naticidae hemocyanins, those of P. lewisii and P. draconis, have mixtures of the 100 S and 130 S di- and tri-decamers, and minor amounts of 150 S and faster sedimenting components. 3. The average particle masses based on STEM measurements are 8.85 x 10(6), 1303 x 10(6), and 17.1 x 10(6) da for the di-, tri-, and tetra-decameric assemblies of hemocyanin. 4. The subunit mol. wts of C. oldroydii hemocyanin and the published values for E. heros hemocyanin at alkaline pHs and in the presence of 8.0 M urea range from 4.2 x 10(5) to 4.8 x 10(5), suggesting the same decameric organization of the sub-assemblies of the Naticidae hemocyanins as for other molluscan hemocyanins. 5. The appearance of the larger hemocyanin particles in the electron micrographs support the hypothesis for their assembly that was based on similar studies of the hemocyanins of the Melongenidae family. According to this scheme the formation of higher aggregates is accomplished by the tail-to-head addition of each decameric unit to a central di-decamer which itself has the tail-to-tail Mellema and Klug arrangement of decamers. In this model all the higher aggregates terminate from either end with the same "collar" ends.     

Yeast myosin heavy chain mutant: maintenance of the cell type specific budding pattern and the normal deposition of chitin and cell wall components requires an intact myosin heavy chain gene

Recent studies with myosin heavy chain mutants in the slime mold Dictyostelium discoideum and the yeast Saccharomyces cerevisiae indicate that the myosin heavy chain gene is not essential for cell survival under laboratory growth conditions. However, cells lacking a normal myosin heavy chain gene demonstrate substantial alterations in growth and cell division. In this study, we report that a disruption mutant in the rod portion of the yeast myosin heavy chain gene, MYO1, produces abnormal chitin distribution and cell wall organization at the mother-bud neck in a high proportion of dividing cells. It is suggested that this phenotype is the cause of the cell division defect and the osmotic sensitivity of yeast MYO1 mutants. In the absence of a normal MYO1 polypeptide, yeast cells alter their cell type specific budding pattern. It is concluded that an intact myosin heavy chain gene is required to maintain the cell type specific budding pattern and the correct localization and deposition of chitin and cell wall components during cell growth and division.     

Purification and characterization of the restriction endonuclease RsrI, an isoschizomer of EcoRI

Rhodobacter sphaeroides strain 630 produces restriction enzyme RsrI which is an isoschizomer of EcoRI. We have purified this enzyme and initiated a comparison with the EcoRI endonuclease. The properties of RsrI are consistent with a reaction mechanism similar to that of EcoRI: the position of cleavage within the -GAATTC-site is identical, the MgCl2 optimum for the cleavage is identical, and the pH profile is similar. Methylation of the substrate sequence by the EcoRI methylase protects the site from cleavage by the RsrI endonuclease. RsrI cross-reacts strongly with anti-EcoRI serum indicating three-dimensional structural similarities. We have determined the sequence of 34 N terminal amino acids for RsrI and this sequence possesses significant similarity to the EcoRI N terminus.