Cell killing by various monofunctional alkylating agents in Chinese hamster ovary cells

Cell killing by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), and methyl methanesulfonate (MMS) was measured in Chinese hamster ovary (CHO) cells using the colony-formation assay. Cell killing by these agents was determined in exponentially growing asynchronous cells, in synchronous cells as a function of cell-cycle position and in nondividing cells. Distinct differences in the cytotoxic effect of the 4 alkylating agents were found in respect to dose-response, cell cycle phase-sensitivity and growth state. MNNG and MNU showed the same biphasic dose-survival relationship in exponentially growing cells, with an initial steep decline followed by a shallow component. The shallow component disappeared in growth-arrested cells. MNNG and MNU differed, however, in the cell-cycle age response. No cell-cycle phase difference was seen with MNNG, whereas cells in G1 seemed more sensitive to MNU than cells in S phase. MMS and ENU both showed shouldered dose-response curves for exponentially growing asynchronous cells, and the same cell-cycle pattern for synchronous cultures with cells in early S phase being the most sensitive. However, survival of nondividing cells versus dividing cells was reduced much more by MMS than by ENU. Caffeine, which interferes with the regulation of DNA synthesis and is known to modify cell killing by DNA-damaging agents, enhanced cell killing by all agents. It is concluded that there must be a number of factors which contribute to cell killing by monofunctional alkylating agents, and that besides alkylation of DNA reaction with other cellular macromolecules should be considered.     

Seed Dormancy in Red Rice : VI. Monocarboxylic Acids: A New Class of pH-Dependent Germination Stimulants

The addition of an elicitor (glucan) to Phaseolus vulgaris cell suspension cultures increased the formation of the phytoalexin phaseollin. Intracellular pH and phosphate concentrations were studied with ³¹P nuclear magnetic resonance spectroscopy on elicitor-treated cells which were aerated during the nuclear magnetic resonance measurement. The pH of the vacuole and to a lesser extent the pH of the cytoplasm were affected at 10 minutes after elicitor addition; a decrease in pH from 5.3 to 4.8 was noted in the vacuole and from 7.46 to 7.28 in the cytoplasm. The ratio between the amount of Pi in the vacuole to that in the cytoplasm also changed within 10 minutes after elicitor addition. The signal for ATP (β-ATP) was low after elicitor addition and was high again 23 hours after elicitation. Forty-eight hours after elicitor addition, vacuolar and cytoplasmic pH had almost returned to their initial values. The rapid change in vacuolar and cytoplasmic pH may cause the change of metabolism that occurs in elicitor-treated P. vulgaris cells.     

Specific and nonspecific effects of nucleotides on hormone-induced phosphoinositide turnover in permeabilized human pituitary tumour cells (Flow 9000)

Previous studies have shown that agonist-induced inositol phosphate formation in the human embryonic pituitary cell line Flow 9000 is regulated by guanine nucleotides, and it is likely that a guanine nucleotide-binding protein is involved in coupling receptors to phosphoinositidase C (PIC) [(1986) Biochem.Soc.Trans. 14, 1135-1136]. We have now tested the specificity of various nucleotides in regulating PIC activity in the absence or presence of the hormone cholecystokinin (CCK-8) in saponin-permeabilized [3H]inositol-labelled Flow 9000 cells. We found that all nucleotides tested (i.e. CTP, UTP, ITP, TTP, GTP, GppNHp, GTP[S], ATP, AppNHp and ATP[S]) stimulated total [3H]inositol phosphate ([3H]IP) formation in a dose-dependent manner with similar potency and efficacy. However, only guanine nucleotides significantly enhanced CCK-8 stimulation of [3H]IP production. These results indicate a physiological role for guanine nucleotides in regulating hormone-induced phosphoinositide turnover. In addition, the effects of nucleotides on calcium-dependent PIC activity are discussed.     

Rec dependence of mu transposition from P22-transduced fragments.

Derivatives of bacteriophage Mu carrying a lac operon and a selectable drug resistance element (Mu d phages) are frequently used tools of bacterial genetics. Mu d prophages used in this way can be treated as transposons, in that the inserted material can be transduced from one strain to another by general transducing phages, such as P1 and P22. When a Mu d prophage is transduced into a new recipient by P1 or P22, the Mu d element can transpose from the transduced fragment into the bacterial chromosome. Transposition of the Mu d element from a P22-transduced fragment shows several striking differences from transposition of a Mu d genome injected by a Mu virion. First, the frequency of transposition from a transduced fragment is greatly enhanced by a P22 helper genome. Second, transposition requires the host recA, B, and C functions. Transposition of Mu following injection by a Mu virion is rec independent. While the basis of these observations is not understood, we suggest that the Mu X protein, a 65-kilodalton protein injected by a Mu virion and required for Mu transposition, may not be packaged by P22. We suggest that the effects seen reflect the behavior of a Mu genome in the absence of the X protein.     

High and low molecular weight rabbit secretory components. Evidence for the deletion of the second and third domains in the smaller polypeptide

Rabbit secretory components exist in two forms which differ in apparent mass by about 25 kDa. Each of these two forms were reduced, carboxymethylated, and extensively digested with trypsin. The resulting peptides were purified by reverse-phase high performance liquid chromatography and characterized by NH2- and COOH-terminal sequence determination and/or amino acid analysis. They were aligned with the protein sequence predicted from the cDNA nucleotide sequence encoding the rabbit poly(Ig) receptor (Mostov, K. E., Friedlander, M., and Blobel, G. (1984) Nature 308, 37-43). All peptides belonging to the fourth and fifth domains except one (positions 488-496) were accounted for in both forms. In addition, limited tryptic proteolysis of the native low Mr secretory components produced the intact 18-kDa NH2-terminal domain (positions 1-117) and the 30-kDa fragment encompassing the fourth and fifth domains. These results suggest that the smaller polypeptide derives from the larger secretory component form by the deletion of the second and third domains.     

Rat hepatocytes in serum-free primary culture elaborate an extensive extracellular matrix containing fibrin and fibronectin

Adult rat hepatocytes cultured on type IV collagen, fibronectin, or laminin and maintained in serum-free medium were examined by indirect immunofluorescence using polyclonal antibodies against extracellular matrix proteins. An extensive fibrillar matrix containing fibronectin and fibrin was detected in all hepatocyte cultures irrespective of the exogenous matrix substratum used to support cell adhesion. Fibrils radiated from the cell periphery and covered the entire culture substratum. In addition, thicker fibers or bundles of fibers were localized on top of hepatocytes. This matrix did not contain laminin or the major types of collagen found in the liver biomatrix (types I, III, and IV). Isolation of the fibrillar matrix and analysis on polyacrylamide gels under reducing conditions demonstrated a major 58-kD polypeptide, derived from beta-fibrinogen as indicated by immunoblotting and two-dimensional peptide mapping. Plasmin rapidly dissolved the matrix. Deposition of the fibrin matrix in hepatocyte cultures was arrested by hirudin, by specific heparin oligosaccharides that potentiate thrombin inhibition by antithrombin III, and by dermatan sulfate, an activator of heparin cofactor II-mediated inhibition of thrombin. The results indicate that hepatocytes in culture synthesize and activate coagulation zymogens. In the absence of inhibitory and fibrinolytic mechanisms, a fibrin clot is formed by the action of thrombin on fibrinogen. Fibronectin attaches to this fibrin clot but fails to elaborate a fibrillar matrix on its own in the presence of coagulation inhibitors.     

Rabbit secretory components: identification of a third allotype, t63

A third allotype of rabbit secretory component has been identified. The allotype previously referred to as t62 by our laboratory can now be subdivided into two allotypes, t62 and t63, with alloantisera capable of discriminating between the two. Results of family studies are consistent with a three allele system (t61, t62 and t63) at the t-locus. By SDS PAGE, electrophoretic mobilities of the multiple SC bands for each of the three allotypes are characteristic of the allotype; the apparent molecular sizes of the bands of the t62 allotype are 2 to 3 kDa lower than those for the t61 allotype. The banding patterns of the t61 and t63, although similar, are not identical to each other. Results of serologic cross-reaction studies and of tryptic peptide mapping studies suggest multiple structural differences between the allotypes as well as a closer relationship between t62 and t63 than between either of these allotypes and t61.     

The morphogenesis of the human Paneth cell. An immunocytochemical ultrastructural study

In human duodenal mucosa Paneth cells originate away from the base of crypts and migrate towards the base during maturation. The earliest cells in the Paneth cell lineage could be identified by labelling of lysozyme in the Golgi apparatus. Specific labelling for lysozyme was present in the rough endoplasmic reticulum, Golgi apparatus, condensing vacuoles, granules and many lysosomes of mature Paneth cells. The maturation of the Paneth cell is accompanied by an increase in the content of lysozyme in the secretory granules and with senescence lysozyme diffuses into the cytoplasm.