Interleukin-3 dependent c-myc protein expression during the cell cycle of murine mast cells.

Aim of the study was to evaluate the relationship between the mitogenic stimulus interleukin-3 to normal murine mast cells and the cell cycle dependent expression of the nuclear c-myc protein. In order to do that on a cell by cell basis, we measured the nuclear c-myc protein simultaneously by flow cytometry, via specific monoclonal antibodies, and the DNA content via the intercalating dye propidium iodide. When cells were deprived from interleukin-3 (IL-3), proliferation was inhibited and the majority of cells arrested in early G1 (G1A, characterized by low c-myc content). Readdition of IL-3 resulted in a slow transition of cells from G1A to late G1 (G1B, at higher c-myc content) before DNA synthesis started. G1A cells with low c-myc content do not undertake DNA synthesis. Using a stathmokinetic methodology we confirmed that the G1A cells are early postmitotic G1 phase cells. The low content of c-myc within these cells appears a direct consequence of reduced c-myc levels during mitosis. Cumulatively, the data suggest that c-myc protein levels of murine mast cells fall at mitosis and that these levels must rise before cells can traverse the G1 phase. Our data are compatible with a model in which c-myc protein content of G1 phase cells has to reach a critical threshold before the cells can move further into the cell cycle.     

Effects of maternal marijuana and cocaine use on fetal growth

To investigate the effects on infants of the use of marijuana and cocaine during pregnancy and to compare the importance of urine assays with that of interviews in ascertaining drug use, we prospectively studied 1226 mothers, recruited from a general prenatal clinic, and their infants. On the basis of either interviews or urine assays conducted prenatally or post partum, 27 percent of the subjects had used marijuana during pregnancy and 18 percent had used cocaine. When only positive urine assays were considered, the corresponding values were 16 percent and 9 percent, respectively. When potentially confounding variables were controlled for in the analysis, the infants whose mothers had positive urine assays for marijuana, as compared with the infants whose mothers were negative according to both interviews and urine assays, had a 79-g decrease in birth weight (P = 0.04) and a 0.5-cm decrement in length (P = 0.02). Women who had positive assays for cocaine, as compared with nonusers, had infants with a 93-g decrease in birth weight (P = 0.07), a 0.7-cm decrement in length (P = 0.01), and a 0.43-cm-smaller head circumference (P = 0.01). To compare our findings with those of other investigators who did not use urine assays, we repeated the analyses, considering only self-reported use of marijuana (23 percent) and cocaine (13 percent). There were no significant associations between such use as determined by interviews alone and any of the measures of outcome. We conclude that the use of marijuana or cocaine during pregnancy is associated with impaired fetal growth and that measuring a biologic marker of such use is important to demonstrate the association.     

A comparison of skinfold anthropometry and dual-energy X-ray absorptiometry for the evaluation of body fat in cirrhotic patients

BACKGROUND AND AIMS: Skinfold anthropometry has been used to evaluate the nutritional status in cirrhosis. Such estimates are based on the calculations which derive from healthy subjects and may not apply to cirrhotic patients. We aimed to calculate the limits of agreement between Skinfold anthropometry (SA) and dual-energy X-ray absorptiometry (DXA) in estimating body fat in cirrhotics. METHODS: Forty cirrhotic patients were studied by both methods. The limits of agreement were estimated by the Bland and Altman method. RESULTS: Percentage body fat was similar when measured by DXA and SA (29.6 +/- 9.2 vs 28.9 +/- 7.5 %). Body fat mass was also similar (20.3 +/- 8.4 vs 20.3 +/- 7.7 kg). The limits of agreement between DXA and SA measurements were -7.04 (95%CI: -9.55 to -5.2) +8.56 (95%CI: +10.7 to +6.4.) in the assessment of percentage body fat and -5.32 (95%CI: -6.77 to -3.87) +5.24 (95%CI: +3.79 to +6.69) in the assessment of fat mass. CONCLUSION: Percentage body fat can be evaluated by SA or DXA with a difference of less then 5% in the majority of cirrhotic patients without overt fluid retention. This result is important when considering the large applicability of SA.     

Role of potassium channels in the antinociception induced by agonists of alpha2-adrenoceptors

1. The effect of the administration of pertussis toxin (PTX) as well as modulators of different subtypes of K+ channels on the antinociception induced by clonidine and guanabenz was evaluated in the mouse hot plate test. 2. Pretreatment with pertussis toxin (0.25 microg per mouse i.c.v.) 7 days before the hot-plate test, prevented the antinociception induced by both clonidine (0.08-0.2 mg kg(-1), s.c.) and guanabenz (0.1-0.5 mg kg(-1), s.c.). 3. The administration of the K(ATP) channel openers minoxidil (10 microg per mouse, i.c.v.), pinacidil (25 microg per mouse, i.c.v.) and diazoxide (100 mg kg(-1), p.o.) potentiated the antinociception produced by clonidine and guanabenz whereas the K(ATP) channel blocker gliquidone (6 microg per mouse, i.c.v.) prevented the alpha2 adrenoceptor agonist-induced analgesia. 4. Pretreatment with an antisense oligonucleotide (aODN) to mKv1.1, a voltage-gated K+ channel, at the dose of 2.0 nmol per single i.c.v. injection, prevented the antinociception induced by both clonidine and guanabenz in comparison with degenerate oligonucleotide (dODN)-treated mice. 5. The administration of the Ca2+-gated K+ channel blocker apamin (0.5-2.0 ng per mouse, i.c.v.) never modified clonidine and guanabenz analgesia. 6. At the highest effective doses, none of the drugs used modified animals' gross behaviour nor impaired motor coordination, as revealed by the rota-rod test. 7. The present data demonstrate that both K(ATP) and mKv1.1 K+ channels represent an important step in the transduction mechanism underlying central antinociception induced by activation of alpha2 adrenoceptors.     

Inhibition of the high-affinity glucose transporter GLUT 1 affects the sensitivity to glucose in a hamster-derived pancreatic beta cell line (HIT)

Summary HIT is a hamster-derived beta-cell line which in contrast to normal beta cells that only express the high K_m GLUT-2 glucose transporter, also expresses the low K_m glucose transporter GLUT 1. In HIT cells the abnormal glucose transport mechanism is associated with a marked shift to the left of the glucose-induced insulin release dose-response curve. We have used this cell model to investigate whether changes in glucose transport affect the glucose-induced insulin release. HIT cells were first incubated with a concentration of cytochalasin B (0.4 mol/l) that selectively inhibits the GLUT-1 but not the GLUT-2 transporter. The consequences of blocking glucose phosphorylation and insulin release were studied. Exposure to 0.4 mol/l cytochalasin B for 1 h caused a selective loss of the low K_m transport: the calculated V_max of GLUT 1 was reduced from 1726±98 to 184±14 pmol · mg protein^–1 5 min^–1 (mean±SEM, n=6, p<0.005), while no major difference in the high K_m (GLUT-2) transport was observed. In cytochalasin B exposed HIT cells the glucose phosphorylating activity (due to hexokinase and glucokinase) was unaffected. In these cells, however, the dose-response curve of glucose-induced insulin release was significantly shifted to the right: the 50% of maximal response (increment over baseline) was reached at an average glucose concentration of 2.9±0.2 mmol/l (vs 0.6±0.01 mmol/l in control HIT cells mean±SE, n=5, p<0.05) and the maximal effect was reached at 11.0 mmol/l glucose (vs 2.8 mmol/l in control HIT cells p<0.005). These results are consistent with the hypothesis that the affinity of the glucose transport system may contribute to determination of the glucose threshold concentration that triggers insulin secretion.     

Comparative effects of L-methionine, S-adenosyl-L-methionine and 5'-methylthioadenosine on the growth of preneoplastic lesions and DNA methylation in rat liver during the early stages of hepatocarcinogenesis.

Male Wistar rats, initiated with diethylnitrosamine (DENA), were subjected to a selection treatment, according to the "resistant hepatocyte" model, followed or not followed by phenobarbital (PB). Rats received, for 3 weeks after selection, 4 i.m. doses (96 mmol/kg) of L-methionine, S-adenosyl-L-methionine (SAM), or 5'-methylthioadenosine (MTA), a SAM catabolite formed during polyamine synthesis or by spontaneous splitting of SAM at physiologic temperature and pH. They were then killed. In some rats, SAM and MTA treatments were started 20 weeks after initiation. The animals were killed 3 weeks later and persistent (neoplastic) nodules (PN) were collected. Some rat groups received 1/2 and 1/4 of the above SAM and MTA doses, or 1/8 of the above MTA dose. SAM and MTA, but not methionine, caused a dose-dependent decrease in number and surface area of gamma-glutamyltranspeptidase (GGT)-positive foci, and in labeling index (LI) of focal cells, coupled with remodeling. SAM and MTA liver contents, SAM/S-adenosylhomocysteine (SAH) ratio and overall methylation of liver DNA were low during the development of GGT-positive foci. SAM, but not methionine, caused a dose-dependent recovery of SAM content and DNA methylation, and a partial reconstitution of liver MTA pool. Exogenous MTA only induced the reconstitution of MTA pool, without affecting SAM level and DNA methylation. Recovery of SAM and MTA pool and DNA methylation was found in the rats subjected to SAM plus MTA, indicating the absence of inhibition of DNA methyltransferases in vivo by MTA. MTA also inhibited liver reparative growth in partially hepatectomized rats, without modifying SAM content and DNA methylation of regenerating liver (RL). A high activity of ornithine decarboxylase (ODC) was found in the liver, during the development of preneoplastic foci, and in PN. This activity was inhibited by SAM and MTA treatments. Although MTA was more effective than SAM, the decrease in ODC activity was coupled with a larger fall in DNA synthesis in SAM-treated than in MTA-treated rats. Thus the antipromotion effect of SAM could not merely depend on its (spontaneous) transformation into MTA. Although MTA production may play a role in the SAM antipromotion effect, other mechanisms could be involved. A role of DNA methylation in the inhibition of growth by SAM is suggested. MTA is a potential chemopreventive agent for liver carcinogenesis.     

Epidermal growth-factor receptor and erbb2 protein in colorectal tissue - comparison between cancer and normal mucosa

Epidermal growth factor receptor (EGFr) and p185neu protein were measured in 55 samples of carcinoma and 55 of normal colorectal mucosa from the same patient, using a ligand binding assay and an ELISA method respectively. The binding characteristics of EGFr were similar in cancer and normal tissue. The concentrations of both EGFr and p185 showed gaussian distribution and were not significantly different between normal and cancer tissue, although a trend toward higher levels of EGFr in normal mucosa was found. Moreover, no significative variations were found in the ratios between cancer and normal tissue after desaturation of the EGFr. No correlations were found between EGFr and p185 and the main clinopathological parameters.