Demonstration of a late amiloride-sensitive event as a necessary step in initiation of DNA synthesis by thrombin

Amiloride, a Na⁺ influx inhibitor, has been shown to inhibit initiation of DNA synthesis by thrombin in mouse embryo fibroblast-like cells. Long exoosures (24 hr) to high concentrations of amiloride inhibited incorporation of thymidine into the DNA of both thrombin-stimulated and nonstimulated cells, suggesting that this inhibition might not be specific for thrombin-initiated DNA synthesis. Fluorescence microscopy and spectrofluorimetry showed that amiloride was internalized with an apparent mitochondrial association and that the internalized amiloride was readily released from the cells after removing amiloride from the medium. Based on this reversibility, cells were exposed to amiloride for short periods of time during thrombin treatment to determine the temporal relationship between any amiloride-sensitive event(s) and initiation of DNA synthesis. The presence of amiloride (100 μM) during a 12-hr exposure to thrombin did not block thrombin-initiated DNA synthesis or cell division but did delay the onset of DNA synthesis and the peak of thymidine incorporation into DNA by approximately 3 hr, suggesting that early initiation events might proceed in the presence of amiloride. ⁸⁶Rb⁺ transport studies demonstrated that in this system ouabain-sensitive K⁺ uptake via the Na, K-ATPase was stimulated by thrombin during both an early and a late period. This stimulation was amiloride-sensitive under the same conditions used for growth experiments, suggesting that amiloride was inhibiting thrombin-stimulated Na⁺ transport in this system. Additional experiments showed that exposing cells to amiloride only during the first 8 hr after thrombin addition did not inhibit initiation. The presence of amiloride from 8–12 hr after thrombin addition maximally inhibited thrombin-stimulated DNA synthesis. Together these results demonstrate that amiloride inhibits thrombin-initiated DNA synthesis not by inhibiting an early event occurring during the first 8 hr, but rather by inhibiting some later event 8–12 hr after thrombin addition.     

Bombesin-like peptides and receptors in human tumor cell lines

Human cancer cell lines were assayed for bombesin-like peptides and receptors. Acid extracts derived from small cell lung cancer, but not other types of cancer had high levels of immunoreactive bombesin. Regardless of patient treatment, site of tumor origin (bone marrow, lymph node, or pleural effusion) or culture conditions, small cell lung cancer cell lines had high levels of bombesin-like peptides. Thus, bombesin levels in small cell lung, but not other types of human cancer, are routinely elevated. Also, small cell lung cancer lines in contrast to other cell lines have a high density of binding sites for a radiolabeled bombesin analogue. The presence of high concentrations of bombesin-like peptides and receptors suggests that bombesin may function as an important regulatory agent in human small cell lung cancer.     

Phylogenetic analysis of Alloglossidium Simer, 1929 (Digenea: Plagiorchiiformes: Macroderoididae) with discussion of the origin of truncated life cycle patterns in the genus.

Alloglossidium comprises 9 species of North American plagiorchiiform digeneans using ictalurid catfish, freshwater crustacea, and hirudinid leeches as definitive hosts. Two hypotheses about the evolution of this array of definitive hosts were examined using phylogenetic systematic analysis. Two most parsimonious trees, based on 15 homologous series derived from morphological data, each indicated the 2 species utilizing ictalurid catfish definitive hosts are basal members of the group, whereas the 2 species using freshwater crayfish definitive hosts and the 5 utilizing leech definitive hosts each comprise relatively derived monophyletic sister groups. The results suggest that species using crustaceans as definitive hosts are derived by life cycle truncation, whereas those using leeches as definitive hosts appear to be derived through a switch from crustaceans to leeches.     

Isolation and sequencing of cDNA clones encoding the Dictyostelium discoideum 30,000-dalton actin-bundling protein

The Dictyostelium 30,000-dalton protein is a calcium-regulated actin filament-bundling protein which has been suggested to contribute to the structure and reorganization of filopodia and pseudopodia accompanying cell movements. cDNAs encoding this protein were isolated using antibody and oligonucleotide probes to screen cDNA libraries in phage lambda. The sequence of the cDNA predicts a protein of 295 amino acids with a molecular weight of 33,355. The sequence reveals two EF-hand calcium-binding regions that provide a structural explanation for calcium regulation of the activity of this protein. The putative calcium-binding region of the 30,000-dalton protein has similarity to sequences of other calcium-regulated actin-binding proteins such as alpha-actin and fimbrin. One region of the sequence with similarity to both Dictyostelium gelation factor (ABP 120) and fructose bisphosphate aldolase is a potential actin-binding sequence. A highly charged region of the protein is similar to a sequence in human cytovillin that is repeated eight times in chicken gizzard caldesmon. No strong homology to previously identified actin-binding sequences of other actin-binding proteins is apparent. Results from Southern blot experiments indicate that the 30,000-dalton protein is encoded by a single gene in the Dictyostelium genome.     

Metabolism of proctolin, a pentapeptide neurotransmitter in insects

The in vitro metabolism of [tyrosyl-3, 5-3H]proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH) was studied in the following tissues from the American cockroach, Periplaneta americana: proctodeum, midgut, hemolymph, brain, terminal ganglion, and coxal depressor muscles. In all tissues assayed, the Tyr-Leu bond is the primary cleavage site, but scission of the Arg-Tyr bond is also significant. Greater than 90% of the degradative activity is found in the 100,000 X g supernatant from homogenates. In vivo studies with the tobacco hornworm, Manduca sexta, show that topically applied proctolin does not penetrate larval cuticle; proctolin is readily degraded to constituent amino acids (at least to Tyr) upon ingestion.     

Quantitative description of classic and variant small cell lung cancer cell lines by nuclear image cytometry

Six small cell lung cancer (SCLC) cell lines were examined using nuclear image analysis to find features characteristic of the classic and the variant type of SCLC. On the basis of their biochemical and biological properties three of these cell lines have been shown to represent the classic types, and three represent the variant type of SCLC. Using a combination of the image-derived run length, density, and geometric features, it was possible to distinguish between the classic and variant SCLC cell lines. The results of this study may be of help in assessing photometric features for the separation of the classic and variant subtypes of SCLC in solid tumors. Because of differences in treatment and prognosis between these two subtypes, such a separation may be of clinical value.     

Initiation of proliferative events by human alpha-thrombin requires both receptor binding and enzymic activity

To determine the role of thrombin high-affinity receptor occupancy and enzymic activity in thrombin initiation of cell proliferation, we have utilized thrombin derivatives which separate these functions. We previously showed that enzymically active gamma-thrombin stimulates ion fluxes without binding to high-affinity sites, whereas proteolytically inhibited DIP-alpha-thrombin which binds to high-affinity receptors does not. Since neither derivative initiates DNA synthesis by itself, this suggested that two separate sequences of events might be necessary for a complete initiation signal. We now report that the combination of DIP-alpha-thrombin and gamma-thrombin initiate DNA synthesis and cell proliferation to levels approaching the maximal initiation by native alpha-thrombin. This combinatory effect is dose-dependent for both gamma-thrombin and DIP-alpha-thrombin in the same concentration range as alpha-thrombin alone. Thus, these same concentrations of alpha-thrombin alone may be required to initiate each sequence of events. The combinatory stimulation could be achieved even if the derivatives were added individually up to 8 hr apart. Moreover, preincubation with either derivative shortened the lag period for initiation of DNA synthesis by native alpha-thrombin. These results indicate that both receptor occupancy and enzymic activity are necessary for thrombin initiation of cell proliferation and that each action initiates a sequence of early events which moves the cell forward toward entry into a proliferative cycle.