Regulation of ornithine decarboxylase activity by spermidine and the spermidine analogue N1N8-bis(ethyl)spermidine.

Polyamine biosynthesis in intact cells can be exquisitely controlled with exogenous polyamines through the regulation of rate-limiting biosynthetic enzymes, particularly ornithine decarboxylase (ODC). In an attempt to exploit this phenomenon as an antiproliferative strategy, certain polyamine analogues have been identified [Porter, Cavanaugh, Stolowich, Ganis, Kelly & Bergeron (1985) Cancer Res. 45, 2050-2057] which lower ODC activity in intact cells, have no direct inhibitory effects on ODC, are incapable of substituting for spermidine (SPD) in supporting cell growth, and are growth-inhibitory at micromolar concentrations. In the present study, the most effective of these analogues, N1N8-bis(ethyl)SPD (BES), is compared with SPD in its ability to regulate ODC activity in intact L1210 cells and in the mechanism(s) by which this is accomplished. With respect to time and dose-dependence of ODC suppression, both polyamines closely paralleled one another in their response curves, although BES was slightly less effective than SPD. Conditions of minimal treatment leading to near-maximal ODC suppression (70-80%) were determined and found to be 3 microM for 2 h with either SPD or BES. After such treatment, ODC activity was fully recovered within 2-4 h when cells were re-seeded in drug-free media. By assessing BES or [3H]SPD concentrations in treated and recovered cells, it was possible to deduce that an intracellular accumulation of BES or SPD equivalent to less than 6.5% of the combined cellular polyamine pool was sufficient to invoke ODC regulatory mechanisms. Decreases in ODC activity after BES or SPD treatment were closely paralleled by concomitant decreases in ODC protein. Since cellular ODC mRNA was not similarly decreased by either BES or SPD, it was concluded that translational and/or post-translational mechanisms, such as increased degradation of ODC protein or decreased translation of ODC mRNA, were probably responsible for regulation of enzyme activity. Experimental evidence indicated that neither of these mechanisms seemed to be mediated by cyclic AMP or ODC-antizyme induction. On the basis of the consistent similarities between BES and SPD in all parameters studied, it is concluded that the analogue most probably acts by the same mechanisms as SPD in regulating polyamine biosynthesis.     

Specific Inhibition of Cell Proliferation In the Mouse Intestine By an Aqueous Extract of Rabbit Colon

Aqueous extracts from rabbit colon, kidney, testis and small intestinal mucosa were prepared by homogenization and centrifugation at 105,000 g. After precipitation with ammonium sulphate. the 0–50 fraction (F₁) and the supernatant (F₂) were collected, dialysed against a phosphate buffer and tested on mice in vivo. 1 hr after a single injection of F₁ (15 mg content) from colon, the uptake of tritiated thymidine was decreased in jejunal and colonic DNA in mice. This effect, maximal after 3 hr and totally reversible after 7 hr, was found in neither the kidney nor the testis. the F₁ fractions of non-digestive organs (kidney, testis) were also found to exert a significant inhibition on thymidine incorporation into intestinal DNA in vivo. F₁ fractions of intestinal contents, prepared under the same conditions, exerted no significant effects on DNA synthesis in mouse intestine. Conversely, the colon F₂ fraction did not inhibit the synthesis of jejunal and colonic DNA in vivo. A slowing of cellular migration was also noticed in the jejunum and colon of mice injected with colon or small intestine F₁, as ascertained radioautographically by determining the position of the leading edge of the labelled cells in jejunal or colonic F₁-injected mice. Our results suggest that the F₁ fraction of the aqueous extract of rabbit colon contains one or more substances, which may act either on intestinal DNA synthesis or on the G₁-S transition of the cellular cycle in the mouse intestine. This reversible and tissue-specific intestinal action appears to inhibit cell proliferation and presents several of the characteristics defining a chalone, as does the action of small intestinal F₁ previously reported (Sassier & Bergeron, 1977). However, because of a relative lack of origin specificity of this effect, the physiological significance of our data remains to be ascertained.     

The future of new oral antibiotics including the quinolones.

It is estimated that more than 110 million dollars'' worth of oral antibiotics will have been sold in Canada in 1987. In the next few years several new oral antimicrobial agents will reach the market, including beta-lactamase inhibitors, cephalosporins, monobactams, erythromycins and quinolones. Most of these new agents have a broader spectrum of antibacterial activity than the presently available oral antibiotics. A few have a longer half-life and can be administered once a day. The new oral drugs, especially the quinolones and possibly beta-lactams, will now be used to treat infections that in the past could be treated only parenterally. Exacerbations of pulmonary infections due to Pseudomonas aeruginosa in cystic fibrosis can now be successfully treated at home with the new quinolones. Osteomyelitis, arthritis, pneumonia and pyelonephritis will most likely be treated at home in the future. In severe infections patients will be admitted to hospital for short courses of parenteral therapy, followed by oral treatment. If used appropriately the new oral agents may lead to new approaches to the treatment of infectious diseases.     

Succession in the southern part of the Canadian boreal forest

Forest succession following fire in a forest mosaic of northwestern Quebec has been studied in order to: (1) describe the successional pathways using communities of different ages and (2) evaluate convergence of successional pathways and possible effect of fire suppression on the establishment of steady-state communities. As a first step, ordination and classification techniques were used in order to remove changes in forest composition which are related to abiotic conditions. Then, ordinations based on tree diameter distributions were used to study shifts in species composition in relation to time since the last fire.Even under similar abiotic conditions, successional pathways are numerous. However, regardless of forest composition after fire, most stands show convergence toward dominance of Thuja occidentalis and Picea mariana on xeric sites and dominance of Abies balsamea and Thuja occidentalis on more mesic sites. Stable communities of >300 yr occur on xeric sites while on mesic sites directional succession still occurs after 224 yr. Nearly all species involved in succession are present in the first 50 yr following fire. Only Abies balsamea and Thuja occidentalis increase significantly in frequency during succession. Following initial establishment, successional processes can generally be explained by species longevity and shade tolerance. Early successional species may be abundant in the canopy for more than 200 yr while the rapid decrease of Picea glauca, a late successional species could be related to spruce budworm outbreaks. Considering the short fire rotation observed (about 150 yr), a steady-state forest is unlikely to occur under natural conditions, though it may be possible if fire is controlled.     

Pharmacological doses of insulin equalize insulin receptor phosphotyrosine content but not tyrosine kinase activity in plasmalemmal and endosomal membranes.

Following insulin administration to intact rats, the insulin receptor kinase activity of subsequently isolated cell fractions was significantly augmented. Of interest was the observation that the endosomal insulin receptor tyrosine kinase displayed four- to six-fold greater autophosphorylation activity than that of plasma membrane. Surprisingly, the endosomal insulin receptor tyrosine kinase displayed a decrease in beta-subunit phosphotyrosine content compared with that seen in the plasma membrane. These observations prompted the suggestion that insulin receptor tyrosine kinase phosphotyrosine dephosphorylation mediated by an endosome-specific phosphotyrosine phosphatase(s) yields activation of the endosomal insulin receptor tyrosine kinase. In a previous study we examined the effect of subsaturating doses of injected insulin. In this work we evaluated insulin receptor tyrosine kinase activity and phosphotyrosine content in plasma membrane and endosomes after a receptor-saturating pharmacological dose of insulin (150 micrograms/100 g body weight). At this dose the phosphotyrosine content per receptor was reduced compared with that seen earlier at insulin doses of 1.5 and 15 micrograms/100 g body weight. Endosomal insulin receptor tyrosine kinase was greater than that seen at the lower nonsaturating insulin doses. Furthermore, endosomal insulin receptor tyrosine kinase activity exceeded that of the plasma membrane, despite retaining about the same phosphotyrosine content per receptor. These data are consistent with the view that insulin receptor tyrosine kinase activity may be regulated by a particular pattern of phosphotyrosine content on the beta-subunit wherein both activating and inhibitory phosphotyrosine residues play a role.     

Differential and analytical subfractionation of rat liver components internalizing insulin and prolactin

Receptor-mediated endocytosis of 125I-insulin and 125I-prolactin into liver parenchymal cells has been studied by quantitative subcellular fractionation. Differential centrifugation yielded three particulate fractions, N (nuclear), ML (large granule), and P (microsomes), and a final supernatant (S). Quantitative differences in the extent and rates of accumulation of 125I-insulin and 125I-prolactin into the fractions were observed. The acidotropic agent chloroquine and the microtubule disrupting agent colchicine were administered separately to rats. The agents increased significantly the T 1/2 of hormone clearance from the liver and augmented the accumulation of both ligands in the low-speed ML fraction. However, differences in the rates of accumulation of insulin and prolactin into all cell fractions were still maintained. Analytical centrifugation of each of the particulate fractions was carried out in order to determine if different endocytic components were specific to insulin or prolactin internalization. This was not the case. An "early" endosomal component of density 1.11 was identified in microsomes. A "late" endosome of density 1.10 was identified in the large granule (ML) fraction. Both endosomal components appeared to accumulate insulin and prolactin but at different rates. Marker enzyme analysis identified the presumed plasma membrane component in microsomes (density approximately 1.155). This component showed a significant difference in the rate of loss of 125I-insulin (T 1/2 approximately 4.1 min) as compared to that of 125I-prolactin (T 1/2 approximately 12.7 min). A further difference in the handling of the ligands was observed in early endosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential kinetics and sensitivity to chloroquine of receptor-mediated insulin and prolactin endocytosis in liver parenchymal cells

Systemically injected [125I]prolactin or [125I]insulin was accumulated and cleared from rat liver at different rates. Quantitative subcellular fractionation indicated a predominant accumulation of [125I]insulin in liver microsomes while [125I]prolactin was found in both the light-mitochondrial and microsomal fractions. The acidotropic agent chloroquine diminished the rate and extent of loss of each ligand from liver homogenates. In chloroquine treated rats, radiolabeled insulin accumulated in both the light-mitochondrial and the microsomal fractions. Subfraction of microsomes on discontinuous sucrose gradients revealed "early' endosomes in which ligand uptake was maximal at 2-5 min. In contrast, comparable subfraction of the of light mitochondrial fraction revealed "late' endosomes in which ligand uptake was maximal at 10-20 min. Chloroquine-treated rats showed a more marked enhancement of insulin compared to prolactin uptake in the "early' endosomes. It is suggested that "early' endosomes found in the Golgi-intermediate and -heavy fractions floated from parent microsomes may selectively degrade insulin but not prolactin. This could account for the apparently different kinetics of insulin and prolactin uptake into liver parenchyma.     

Method for improving the detection of viruses in fecal samples.

A method is described that improved the detection of viruses in fecal samples by electron microscopy. The virus particles were concentrated, and much of the background debris was removed by adsorption of viruses on meat protein added to the fecal sample at a low pH and a low salt concentration. Viruses were eluted by raising the pH and the salt concentration. Further concentration was achieved by acid precipitation and vacuum dialysis.