Existence of a commitment program for mitosis in early G1 in tumour cells

The proliferation of normal non-tumourigenic mouse fibroblasts is stringently controlled by regulatory mechanisms located in the postmitotic stage of G₁ (which we have designated G₁ pm). Upon exposure to growth factor depletion or a lowered de novo protein synthesis, the normal cells leave the cell cycle from G₁ pm and enter G₀. The G₁ pm phase is characterized by a remarkably constant length (the duration of which is 3 h in Swiss 3T3 cells), whereas the intercellular variability of intermitotic time is mainly ascribable to late G₁ or pre S phase (G₁ ps) (Zetterberg & Larsson (1985) Proc. Natl. Acad. Sci. USA 82 , 5365). As shown in the present study two tumour-transformed derivatives of mouse fibroblasts, i.e. BPA31 and SVA31, did not respond at all, or only responded partially, respectively, to serum depletion and inhibition of protein synthesis. If the tumour cells instead were subjected to 25-hydroxycholesterol (an inhibitor of 3-hydroxy-3 methyglutaryl coenzyme A reductase activity), their growth was blocked as measured by growth curves and [³H]-thymidine uptake. Time-lapse analysis revealed that the cells were blocked specifically in early G₁ (3-4h after mitosis), and DNA cytometry confirmed that the arrested cells contained a G₁ amount of DNA. Closer kinetic analysis revealed that the duration of the postmitotic phase containing cells responsive to 25-hydroxycholesterol was constant. These data suggest that transformed 3T3 cells also contain a ‘G₁ pm program’, which has to be completed before commitment to mitosis. By repeating the experiments on a large number of tumour-transformed cells, including human carcinoma cells and glioma cells, it was demonstrated that all of them possessed a G₁ pm-like stage. Our conclusion is that G₁ pm is a general phenomenon in mammalian cells, independent of whether the cells are normal or neoplastic.     

Defective regulation of the cytosolic Ca2+ activity in parathyroid cells from patients with hyperparathyroidism

The parathyroid hormone (PTH) release and cytosolic Ca²⁺ activity were determined in normal bovine parathyroid cells and parathyroid cells obtained from patients with hyperparathyroidism (HPT). There was a sigmoid relation between the cytosolic Ca²⁺ activity and the extracellular calcium concentration between 0.5 and 6.0 mmol/l. The PTH release was inhibited in parallel with the rise in the cytosolic Ca²⁺ activity. Both the hormone release and the cytosolic Ca²⁺ activity were lower in cells from human adenomas and hyperplastic glands~ and in comparison with the bovine preparations these ceils had higher set points for the cytosolic Ca²⁺ activity and PTH release. There was a close correlation between the individual set points for the cytosolic Ca²⁺ activity and PTH release in a material containing both normal and pathological cells. The results indicate that the abnormal PTH release characteristic of HPT is due to a defective regulation of the cytosolic Ca²⁺ activity.     

Inhibition of Escherichia coli fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate

Fructose 2,6-bisphosphate, a potent inhibitor of fructose-1,6-bisphosphatases, was found to be an inhibitor of the Escherichia coli enzyme. The substrate saturation curves in the presence of inhibitor were sigmoidal and the inhibition was much stronger at low than at high substrate concentrations. At a substrate concentration of 20 μM, 50% inhibition was observed at 4.8 μM fructose 2,6-bisphosphate. Escherichia coli fructose-1,6-bisphosphatase was inhibited by AMP (K_j = 16 μM) and phosphoenolpyruvate caused release of AMP inhibition. However, neither AMP inhibition nor its release by phosphoenolpyruvate was affected by the presence of fructose 2,6-bisphosphate. The results obtained, together with previous observations, provide further evidence for the fructose 2,6-bisphosphate-fructose-1,6-bisphosphatase active site interaction.     

Evidence for anterograde transport of secretory granules in processes of gastric paracrine (somatostatin) cells

Certain disseminated endocrine-like cells have previously been found to give off long cytoplasmic processes which end with small bulbous expansions on the membranes of other cell types. It is believed that the process-carrying cells control the functions of the receiving cells by local and directed (paracrine) secretion of messenger molecules (peptides, biogenic monoamines) through their processes. Following injections of amine precursors paracrine cells take up and convert these to the corresponding amines, which can be cytochemically visualized by the Falck-Hillarp formaldehyde-induced fluorescence technique. As the amines are stored in the cytoplasmic (secretory) granules of the cells, they form useful markers for studies of granule turnover and transport. By injecting, at different time intervals, two different precursors (L-5-hydroxytryptophan and L-3,4-dihydroxyphenylalanine), resulting in amines giving different fluorescence colours in the Falck-Hillarp procedure, we have been able to separately label old and new secretory granule fractions in different fluorescence colours. Examination of such double-labelled paracrine cells (mostly gastric somatostatin cells) indicates that their secretory granules are transported in a proximo-distal direction in the paracrine cell processes (" paraxons "). This finding strongly supports the concept that paracrine cells control the functions of the cells they contact by way of directed, process-mediated delivery of their secretory products.     

Modulation of protein synthesis during the growth cycle of a culture of scarlet rose.

Dilution of a stationary phase culture of Scarlet Rose results in an increased rate of protein synthesis. This study compares the time course of this increase with the changes in polyribosome content and the levels of adenine and guanine nucleotides. During the first two hours after dilution, protein synthesis increases 2- to 3-fold; much of the large monoribosome pool that characterizes the stationary state disappears and a steady state situation is reached in which 70% of the ribosomes are in polyribosomes. Between two and eight hours, there is no further change in polyribosome content although the rate of protein synthesis increases an additional 2- to 3-fold. During this initial 8-hour period there is little change in the levels of ATP and GTP. An explanation consistent with these observations is that the initial activation (within the first 2 hours), characterized by the monoribosome to polysome transition, is at the level of a component(s) of the initiation system, and that between two and eight hours, since neither mRNA availability nor energy level are primary determinants, protein synthesis is augmented by the activation of a translational component, perhaps an elongation factor. After 24 hours, there is a proliferative phase characterized by the onset of ribosome accumulation. By day 5, maximum ribosome levels, 5-fold that of 24-hour cells, are reached, but the rate of protein synthesis increases only 2.5-fold during this period. The lack of quantitative coincidence between the changes in polyribosome content and the rates of protein synthesis again suggests that factors other than mRNA availability are involved in determining the overall rate of protein synthesis. Finally at days 6–8, while the growth of the culture is still in the exponential phase, the rate of protein synthesis per unit fresh weight drops markedly concomitant with a decline in ribosome content. At days 11–12, the monoribosome to polysome ratio begins to change with the monoribosome pool increasing. Presence of either actinomycin D or cordycepin inhibits increased protein synthesis in direct relation to the ability of these compounds to inhibit RNA synthesis. This suggests that the protein synthetic processes occurring after dilution require either the synthesis of the mRNA that is being translated or of an RNA functioning in a closely linked reaction.     

Paradoxical activation of Leptomonas NAD-linked alpha-glycerophosphate dehydrogenase by ethidium and antrycide.

Antrycide and ethidium bromide — 2 cationic trypanocides — inhibited NAD-linked α-glycerophosphate dehydrogenase from $\text{Leptomonas}$ sp. The kinetics of enzyme inhibition was determined by Lineweaver-Burk, Dixon, or direct linear plots. Inhibition by Antrycide was noncompetitive for dihydroxyacetone phosphate in the presence of saturating Mg²⁺ or spermidine. With dihydroxyacetone phosphate at saturation, Antrycide inhibition was also noncompetitive with respect to Mg²⁺ (K_i = 115 μM) and spermidine (K_i = 85 μM). Inhibition by ethidium in the presence of saturating dihydroxyacetone phosphate, was noncompetitive for Mg²⁺ (K_i = 400 μM) but mixed for spermidine (K_i = 495 μM); inhibition was noncompetitive for dihydroxyacetone phosphate in the presence of saturating Mg²⁺ or spermidine. Rabbit-muscle α-glycerophosphate dehydrogenase was inhibited at all concentrations of Antrycide and ethidium tested, but the $\text{Leptomonas}$ enzyme was stimulated up to 3.5-fold by low concentrations of inhibitors in the absence of polyamine. New chemotherapeutic possibilities may thus be opened and an evolutionary distinction between trypanosomatid and mammalian enzyme.     

Frequency of Dividing Cells, a New Approach to the Determination of Bacterial Growth Rates in Aquatic Environments

Frequency of dividing cells is suggested to be an indirect measure of the mean growth rate of an aquatic bacterial community. Seasonal changes in frequency of dividing cells were found which covariated with the bacterial uptake of ¹⁴C-labeled phytoplankton exudates. Batch and continuous culture growth experiments, using brackish water bacteria in pure and mixed enrichment cultures, were performed to establish a relationship between frequency of dividing cells and growth rate. An improved technique for bacterial direct counts, using fluorescent staining and epifluorescence microscopy, is presented. Based on a 6-month survey in a coastal area of the Baltic Sea, the bacterial production in the photic zone is estimated. Compared to the total primary production in the area, the bacterial population during this period utilized approximately 25% of the amount of carbon originally fixed by the primary producers.     

Comparison between (RS)-nipecotic acid and GABA transport in cultured astrocytes: Coupling with two sodium ions

The sodium ion dependency of the uptake of (RS)-nipecotic acid into astrocytes in primary cultures has been studied by performing kinetic analysis at different sodium ion concentrations (16–151 mM).V _max of the saturable component of the astroglial (RS-nipecotic acid uptake is clearly affected by the sodium ion concentration whereasK _m surprisingly remains unaffected. At high (RS)-nipecotic acid concentrations (50 M), uptake rates as a function of the sodium ion concentration were clearly sigmoid. This sigmoid shape was not obvious at lower concentrations of (RS)-nipecotic acid. The calculated Hill coefficients corresponding to all (RS)-nipecotic acid concentrations studied were approximately two. From these results it is concluded that (RS)-nipecotic acid uptake into astrocytes in primary cultures, like astroglical GABA uptake, requires the binding of at least two sodium ions per (RS)-nipecotic acid molecule transported.     

Studies on the molecular basis of H+ translocation by cytochromec oxidase

We report here studies which characterize further the interaction ofN,N-dicyclohexylcarbodiimide with cytochromec oxidase leading to inhibition of H⁺ translocation by the enzyme. Further evidence is presented to show that the inhibition results from a real interaction of DCCD with the enzyme and cannot be accounted for by uncoupling and, contrary to recent criticisms, this interaction occurs specifically with subunit III of the enzyme even at relatively high inhibitor-to-enzyme stoichiometries. Use of a spin-label analogue of DCCD has enabled us to demonstrate that the carbodiimide-binding site is highly apolar and may not lie on the pathway of electron transfer.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - NCCD N-(2, 2, 6, 6-tetramethylpiperidyl-1-oxyl)-N-(cyclohexyl)carbodiimide - Hepes 2-(N-2-hydroxyethylpiperazin-N-yl) ethane sulfonate - TMPD N,N,N,N-tetramethylphenylenediamine     

Comparative immunocytochemical localization of putative opioid ligands in the central nervous system

We report a detailed comparative immunocytochemical mapping of enkephalin, CCK and ACTH/beta-endorphin immunoreactive nerves in the central nervous system of rat and guinea pig. Enkephalin immunoreactivity was detected in many groups of nerve cell bodies, fibers and terminals in the limbic system, basal ganglia, hypothalamus, thalamus, brain stem and spinal cord. beta-endorphin and ACTH immunoreactivity was limited to a single group of nerve cell bodies in and around the arcuate nucleus and in fibers and terminals in the midline areas of the hypothalamus, thalamus and mesencephalic periaqueductal gray with lateral extensions to the amygdaloid area. Cholecystokinin immunoreactive nerve fibers and terminals displayed a distribution similar to that of enkephalin in many regions; but striking differences were also found. An immunocytochemical doublestaining technique, which allowed simultaneous detection of two different peptides in the same tissue section, showed that enkephalin-, CCK- and ACTH/beta-endorphin-immunoreactive nerves although closely intermingled in many brain areas, occurred separately. The distributions of nerve terminals containing these neuropeptides showed striking overlaps and also paralleled the distribution of opiate receptors. This may suggest that enkephalin, CCK, ACTH and beta-endorphin may interact with each other and with opiate receptors.