Influence of pH on the uptake of 5-fluorouracil into isolated tumour cells

To investigate the possible dependence of 5-fluorouracil (5FU) uptake in tumours on the intra- (pHi) and extracellular (pHe) pH, a pH gradient (deltapH) was imposed across the plasma membrane of ascites tumour cells in vitro, similar to that known to occur in some solid tumours in vivo, by incubation in media of PHe 5-8. A > or = 2:1 (intracellular/extracellular) accumulation of radiolabelled 5FU occurred after 5 min incubation of the cells with 0.5 mM 5FU at pHe of 5.0, 5.5 or 6.0. 5FU metabolism is slow under these conditions, and 5FU uptake was not affected by longer incubations up to 20 min, nor by the absence of a sodium gradient. pHi was estimated from the distribution of the weak acid, 5.5-dimethyl-2,4-oxazolidione ([14C]DMO) across the cell membrane. There was significant correlation between the intracellular/extracellular 5FU ratio and pHe (from pHe 6-8), deltapH and pHi (P < 0.02). Similar results were obtained with HT29 cells. Incubation with a drug that made plasma membranes permeable to H+ significantly decreased 5FU uptake in Lettre cells. The co-transport of 5FU may occur on a proton symport using the proton motive force of the deltapH.     

Estimation of unit risk for coke oven emissions

We are investigating the hypothesis that biotin multimers can be used with streptavidin and monoclonal antibody conjugates in cancer pretargeting protocols to provide a method of increasing the amount of radioactivity bound on cancer cells in patients. As part of that investigation, a series of biotinylated Starburst dendrimers (BSBDs) have been prepared and evaluated in vitro and in vivo. In this study, a new biotinidase-stabilized, water-solubilizing biotinylation reagent was prepared and reacted with Starburst (PAMAM) dendrimers, generations 0, 1, 2, 3, and 4. The reaction conditions employed resulted in perbiotinylation of generation 0 (four biotin moieties conjugated), generation 1 (eight biotin moieties conjugated), generation 2 (16 biotin moieties conjugated), and generation 3 (32 biotin moieties conjugated). With generation 4, incomplete biotinylation was achieved resulting in the largest portion of that BSBD having 51 biotin moieties (of 64 possible) conjugated. The ability of each BSBD to cross-link streptavidin (SAv) was examined in an in vitro assay. In that assay, an assessment was made of the quantity of [125I]SAv bound with polystyrene-bound SAv after treatment with the synthesized BSBDs. All BSBDs cross-linked the polystyrene-bound SAv with [125I]SAv; however, the amount of [125I]SAv bound varied with the different BSBDs. Roughly 1 equiv of [125I]SAv was bound when Starburst dendrimers containing three or four biotin moieties (generation 0) were used. Two equivalents were bound with BSBD generation 1, and 4 equiv were bound with BSBDs generations 2, 3, and 4. To assess the distribution of BSBDs generations 0, 1, and 2 in mice (at 4 h postinjection), a method was developed for radioiodinating them using the NHS ester of p-[125I]iodobenzoate ([125I]PIB). It was found that the radioiodinated BSBDs had low blood concentrations (i.e., 0.13-0.20% ID/g) at the 4 h time point. In fact, most tissues examined had low concentrations of biotinylated dendrimers, except kidney and liver. Kidney had the highest concentration of [125I]-labeled BSBDs, and its concentration increased with increasing size and charge of dendrimer (e.g., 8-48% ID/g). On the basis of the increased radioactivity observed in the in vitro assay and the rapid clearance from blood in mice, additional in vivo studies with perbiotinylated Starburst dendrimer, generation 2, are planned.     

Pegylated peptides. IV. Enhanced biological activity of site-directed pegylated GRF analogs

Conditions have been developed for the site-specific pegylation (NH2-terminus, side-chain and carboxy-terminus) of a potent analog of growth hormone-releasing factor, [Ala15]-hGRF(1-29)-NH2. These pegylated peptides were prepared by solid-phase peptide synthesis using the Fmoc/tBu strategy, and were fully characterized by analytical HPLC, amino-acid analysis, 1H-NMR spectroscopy and laser desorption mass spectrometry. Biological activities of hGRF analogs were determined in vitro utilizing stimulation of growth hormone release by cultured rat pituitary cells as an index. GH-releasing potencies of the pegylated hGRF analogs were compared to a series of model analogs of [Ala15]-hGRF(1-29)-NH2 that were acetylated or protected as the ethylamides at the pegylation sites. It was found that acetylation at the NH2-terminus resulted in reduced potency, which was not further affected when the NH2-terminus was pegylated, regardless of the size of poly(ethyleneglycol) (PEG) employed (e.g. PEG2000 or PEG5000). Pegylation at Asp8 or Lys12 decreased biological potency, a situation which was exacerbated by increasing the molecular weight of PEG. Pegylation at Lys21 or Asp25 did not significantly affect biological activity. The C-terminal model peptide, [Ala15,Orn(Ac)30]-hGRF(1-29)-NH2, was the most potent analog identified in this series (ca. 4-5-fold that of hGRF(1-44)-NH2. The COOH-terminal pegylated analogs retained this increased level of biological activity independent of PEG molecular weight. These studies demonstrate that a biologically active peptide can be pegylated and retain the full in vitro potency of the peptide. However, the biological activity is highly dependent on the site of pegylation and, in some cases, the molecular weight of PEG (degree of pegylation) moiety used.     

Uterine contractions at the time of embryo transfer alter pregnancy rates after in-vitro fertilization

To investigate the possible consequences of uterine contractions (UC) as visualized by ultrasound (US) on in-vitro fertilization (IVF)-embryo transfer outcome, we studied prospectively 209 infertile women undergoing 220 cycles of controlled ovarian stimulation. Inclusion criteria were age < or = 38 years, a morphologically normal uterus, and at least three good quality embryos transferred. Just before embryo transfer, women underwent 5 min digital recordings of the uterus using US image analysis software for UC assessment. Plasma progesterone and oestradiol concentrations were measured. Four groups were defined according to UC frequency: < or = 3.0 (n = 53), 3.1-4.0 (n = 50), 4.1-5.0 (n = 43), and > 5.0 (n = 74) UC/min respectively. Patients, controlled ovarian hyperstimulation and embryology characteristics were comparable in all groups. A stepwise decrease in clinical and ongoing pregnancy rates as well as in implantation rates occurred from the lowest to the highest UC frequency groups (53, 36, 21; 46, 32, 20; 23, 19, 10; and 14, 11, 4%; P < 0.001). Plasma progesterone and UC frequency were negatively correlated (r = -0.34, P < 0.001). Direction of UC did not affect embryo transfer outcome. As this study was controlled strictly for confounding variables and UC were assessed objectively by a computerized system, its results indicate that high frequency UC on the day of embryo transfer hinder IVF-embryo transfer outcome, possibly by expelling embryos out of the uterine cavity. The negative correlation between UC frequency and progesterone concentrations supports the uterine relaxing properties of progesterone.     

Colocalization of oestrogen receptor immunoreactivity and preproenkephalin mRNA expression to neurons in the superficial laminae of the spinal and medullary dorsal horn of rats

Glucose-dependent insulinotropic polypeptide (GIP) is a 42-amino acid peptide produced by K cells of the mammalian proximal small intestine and is a potent stimulant of insulin release in the presence of hyperglycemia. However, its relative physiological importance as a postprandial insulinotropic agent is unknown. Using LGIPR2 cells stably transfected with rat GIP receptor cDNA, GIP (1-42) stimulation of cyclic adenosine monophosphate (cAMP) production was inhibited in a concentration-dependent manner by GIP (7-30)-NH2. Competition binding assays using stably transfected L293 cells demonstrated an IC50 for GIP receptor binding of 7 nmol/liter for GIP (1-42) and 200 nmol/liter for GIP (7-30)-NH2, whereas glucagonlike peptide-1 (GLP-1) binding to its receptor on ++betaTC3 cells was minimally displaced by GIP (7-30)-NH2. In fasted anesthetized rats, GIP (1-42) stimulated insulin release in a concentration-dependent manner, an effect abolished by the concomitant intraperitoneal administration of GIP (7-30)-NH2 (100 nmol/ kg). In contrast, glucose-, GLP-1-, and arginine-stimulated insulin release were not affected by GIP (7-30)-NH2. In separate experiments, GIP (7-30)-NH2 (100 nmol/kg) reduced postprandial insulin release in conscious rats by 72%. It is concluded that GIP (7-30)-NH2 is a GIP-specific receptor antagonist and that GIP plays a dominant role in mediating postprandial insulin release.     

Intraplantar injection of hyaluronic acid at low pH into the rat hindpaw produces tissue acidosis and enhances withdrawal responses to mechanical stimuli

Application of buffers covering a range of acidic pH values activates and sensitizes nociceptors and produces pain. The purpose of this study was to determine whether a range of acidic pH in tissue produces mechanical hyperalgesia. Tissue acidosis was produced in the hindpaw of the rat by intraplantar injections of hyaluronic acid (HA) adjusted to pH 7.4, 6.0, 5.0, 4.0 or 3.0. Mechanical hyperalgesia was assessed by evaluating responses to application of a von Frey monofilament to the plantar surface before and after injection of HA. In separate experiments, magnitude of tissue acidosis produced by injection of HA was determined by measuring pH of intraplantar tissue using a pH microelectrode. Although needle stick alone produced mechanical hyperalgesia, intraplantar injections of HA at pH 6.0 or 5.0 produced significantly greater mechanical hyperalgesia. In contrast, mechanical hyperalgesia produced by injection of HA at pH 7.4, 4.0 or 3.0 was not different from that produced by needle stick. Although injection of HA at low pH produced tissue acidosis in a pH dependent manner, only a narrow range of tissue acidosis (pH = 6.38-6.00) produced mechanical hyperalgesia. Our data suggest that tissue acidosis induces mechanical hyperalgesia; however, the range of tissue pH that produces this effect is limited.     

Responses of an Amazonian teleost, the tambaqui (Colossoma macropomum), to low pH in extremely soft water

Our goal was to compare the internal physiological responses to acid challenge in an acidophilic tropical teleost endemic to dilute low-pH waters with those in nonacidophilic temperate species such as salmonids, which have been the subjects of most previous investigations. The Amazonian tambaqui (Colossoma macropomum), which migrates between circumneutral water and dilute acidic "blackwater" of the Rio Negro, was exposed to a graded low-pH and recovery regime in representative soft water (Na+ = 15, Cl- = 16, Ca2+ = 20 mumol L-1). Fish were fitted with arterial catheters for repetitive blood sampling. Water pH was altered from 6.5 (control) to 5.0, 4.0, 3.0, and back to 6.5 (recovery) on successive days. Some deaths occurred at pH 3.0. Throughout the regime, there were no disturbances of blood gases (O2 and CO2 tensions and contents) or lactate levels, and only very minor changes in acid-base status of plasma and red cells. However, erythrocytic guanylate and adenylate levels increased at pH's less than or equal to 5.0. Down to pH 4.0, plasma glucose, cortisol, and total ammonia levels remained constant, but all increased at pH 3.0, denoting a stress response. Plasma Na+ and Cl- levels declined and plasma protein concentration increased at pH 3.0, indicative of ionoregulatory and fluid volume disturbance, and neither recovered upon return to pH 6.5. Cortisol and ammonia elevations also persisted. Transepithelial potential changed progressively from highly negative values (inside) at pH 6.5 to highly positive values at pH 3.0; these alterations were fully reversible. Experimental elevations in water calcium levels drove the transepithelial potential positive at circumneutral pH, attenuated or prevented changes in transepithelial potential at low pH, and reduced Na+ and Cl- loss rates to the water during acute low-pH challenges. In general, tambaqui exhibited responses to low pH that were qualitatively similar but quantitatively more resistant than those previously documented in salmonids.     

Prolactin and beta-endorphin responses to hypoglycemia are reduced in well-controlled insulin-dependent diabetes mellitus

Several pituitary hormones, including corticotropin (ACTH), growth hormone (GH), prolactin, and beta-endorphin (but not thyrotropin, follicle-stimulating hormone, or luteinizing hormone), are released in response to hypoglycemia in normal subjects. In patients with insulin-dependent diabetes mellitus (IDDM), the degree of glycemic control is known to alter ACTH and GH responses to hypoglycemia. The current study was performed to examine the effect of glycemic control on prolactin and beta-endorphin responses to hypoglycemia in subjects with IDDM. We performed 3-hour stopped hypoglycemic-hyperinsulinemic clamp studies (12 pmol/kg/min) during which plasma glucose was decreased from 5.0 mmol/L to 2.2 mmol/L in steps of 0.6 mmol/L every 30 minutes in 20 subjects with uncomplicated IDDM (12 males and eight females; age, 26 +/- 2 years; IDDM duration, 10 +/- 1 years; body mass index, 23.6 +/- 0.6 kg/m2) and 10 healthy subjects (five males and five females aged 30 +/- 1 years). The 10 diabetic subjects in good glycemic control (mean hemoglobin A1 [HbA1], 7.5% +/- 0.3%; normal range, 5.4% to 7.4%) were compared with the 10 poorly controlled patients (mean HbA1, 12.6% +/- 0.5%; P < .001 v well-controlled diabetic group). During hypoglycemia, prolactin levels in the well-controlled diabetic group did not change (7 +/- 1 microgram/L at plasma glucose 5.0 mmol/L to 9 +/- 2 micrograms/L at plasma glucose 2.2 mmol/L), whereas prolactin levels increased markedly in the poorly controlled diabetic group (7 +/- 2 micrograms/L to 44 +/- 17 micrograms/L) and healthy volunteers (12 +/- 2 micrograms/L to 60 +/- 19 micrograms/L, P < .05 between IDDM groups). The plasma glucose threshold required for stimulation of prolactin secretion was 2.2 +/- 0.1 mmol/L in well-controlled IDDM, 3.0 +/- 0.4 mmol/L in poorly controlled IDDM, and 2.4 +/- 0.1 mmol/L in healthy subjects (P < .05 between IDDM groups). Responses in males and females were similar. The increase in beta-endorphin levels was also attenuated in well-controlled IDDM patients (4 +/- 1 pmol/L at plasma glucose 5.0 mmol/L to 11 +/- 4 pmol/L at plasma glucose 2.2 mmol/L) versus poorly controlled IDDM patients (5 +/- 1 pmol/L to 26 +/- 7 pmol/L) and healthy subjects (8 +/- 1 pmol/L to 56 +/- 13 pmol/L). The plasma glucose threshold required for stimulation of beta-endorphin release was again lower in well-controlled IDDM versus poorly controlled IDDM patients (2.2 +/- 0.1 v 3.0 +/- 0.3 mmol/L) and healthy subjects (2.5 +/- 0.4 mmol/L, P < .05 between IDDM groups). In conclusion, prolactin and beta-endorphin responses to a standardized hypoglycemic stimulus (plasma glucose, 2.2 mmol/L) are reduced and plasma glucose levels required to stimulate release of prolactin and beta-endorphin are lower in well-controlled IDDM compared with poorly controlled IDDM and healthy subjects. Thus, stress hormones not previously considered to have a primary role in plasma glucose recovery from hypoglycemia are affected by glycemic control, suggesting a more generalized alteration of hypothalamic-pituitary responses to hypoglycemia in IDDM patients with strict glycemic control.     

Macromolecular recognition: effect of multivalency in the inhibition of binding of yeast mannan to concanavalin A and pea lectins by mannosylated dendrimers

The synthesis and binding properties of a new family of high affinity alpha-D-mannopyranoside ligands are described. The synthesis of the new multivalent ligands is based on the scaffolding of multiantennary branches of L-lysine residues having electrophilic N-chloroacetylated end groups as core structures. An alpha-D-mannopyranoside with p-substituted aryl aglycon ending with a thiol group was prepared and covalently attached to each of the branches of the dendritic structures. The resulting glycodendrimers with 2 (12), 4 (14), 8 (16), and 16 (18) mannoside residues were tested for their relative inhibitory potency by solid-phase enzyme-linked lectin assays (ELLA) using methyl and p-nitrophenyl alpha-D-mannopyranosides as standards. Concentrations necessary for 50% inhibition (IC50s) of binding of yeast mannan to Jack bean phytohemagglutinin (Canavalia ensiformis, concanavalin A) and to pea lectin (Pisum sativum) were determined. Analogous mannosylated copolyacrylamides were also prepared for comparison. The IC50 values were also plotted as a function of dendrimer valencies. The inhibitions showed 16-mer 18 to be approximately 600- and 2000-fold more potent than methyl alpha-D-mannopyranoside, and 66- and 1383-fold more potent than p-nitrophenyl alpha-D-mannopyranosides with Con A and pea lectins, respectively. Even when these numbers are expressed relative to single mannopyranoside residues per dendrimers, the relative potencies against the aromatic mannoside are still 4- and 86-fold better against Con A and pea lectins. These results unequivocally indicate that the optimum inhibitory binding properties of the new mannosylated dendrimers vary with both dendrimers and lectin valencies.     

5-fluorouracil/leucovorin-induced inhibition of thymidylate synthase in normal tissues of mouse and man

We evaluated the effects of 5-fluorouracil (5FU) and leucovorin (LV) on thymidylate synthase (TS) in normal rapidly dividing tissues, which may contribute to toxic side-effects of treatment with 5FU and LV. TS levels were determined in biopsies of human liver and colon mucosa and murine bone marrow, liver and intestinal mucosa at several time points after administration of therapeutic doses of 5FU or LV/5FU. In murine liver, after treatment with 100 mg/kg 5FU, TS inhibition was significantly higher than after LV/5FU administration (P < 0.001). A similar trend was observed in human liver tissue. Murine intestinal mucosa had TS levels below the limit of detection after 5FU or LV/5FU treatment. In human colon mucosa samples, administration of 500 mg/m2 5FU resulted in a large extent of TS inhibition but the small number of samples did not allow a time- or 5FU-LV/5FU-related evaluation. TS activity in murine bone marrow cells was strongly inhibited to 10% of the control value during 48 h. LV/5FU administration resulted in a slightly higher inhibition. No human bone marrow was available to measure TS levels. Both in mice and humans the most pronounced TS inhibition occurred in the tissue that was involved in dose-limiting toxicity. Therefore it is very likely that TS inhibition in normal tissues contributes to the toxic side-effects of 5FU treatment.     

Uterine contractions at the time of embryo transfer: a hindrance to implantation?

OBJECTIVES: To investigate the hormonal control and the possible consequences of uterine contractions (UC) on IVF-ET outcome. MATERIALS AND METHODS: We studied prospectively 220 controlled ovarian hyperstimulation (COH) cycles for IVF-ET. Just before ET, women underwent 5-minute digital recordings of the uterus using US image analysis software for UC assessment. Plasma progesterone (P) and estradiol were measured. Four groups were defined according to UC frequency: < or = 3.0 (n = 53), 3.1 to 4.0 (n = 50), 4.1 to 5.0 (n = 43), and > 5.0 (n = 74) UC/minute, respectively. RESULTS: Patients, COH and embryology characteristics were comparable in all groups. Notwithstanding estradiol levels were not associated with UC characteristics, plasma P and UC frequency were negatively correlated (r = -0.34, P < 0.001). A stepwise decrease in clinical and ongoing pregnancy as well as implantation rates occurred from the lowest to the highest UC frequency groups (53%, 36%, 21%; 46%, 32%, 20%; 23%, 19%, 10%; and 14%, 11%, 4%; P < 0.001). Direction of UC did not affect ET outcome. CONCLUSIONS: The negative correlation between UC frequency and P levels supports the utero-relaxing properties of P. High frequency UC on the day of ET hinder IVF-ET outcome, possibly by expelling embryos out of the uterine cavity.     

Murine cytomegalovirus replication in the lungs of athymic BALB/c nude mice

Uridine diphosphoglucose (UDPG) is a precursor of uridine that can be used as a rescuing agent from 5-fluorouracil (5FU) toxicity. Four doses of UDPG (2000 mg/kg i.p. or p.o. at 2, 6, 24, and 30 h after 5FU bolus) allowed the escalation of a weekly bolus of 5FU from 100 mg/kg (5FU100) to 150 mg/kg (5FU150) in healthy and tumor-bearing BALB/c, C57/BI, and CD8F1 (BALB/c x DBA/8) mice. 5FU150 without rescuing agents is not tolerated by the animals. When followed by UDPG, on the contrary, it is possible to increase the dose of 5FU even when it is modulated by leucovorin. Toxicity was the same for 5FU100 and 5FU150 + UDPG, and the nadir values (expressed as a percentage of pretreatment values) were 83 and 85% for weight, 45 and 45% for hematocrit, and 45 and 61% for leukocytes, respectively. Platelets were not affected by treatment. A protective effect was also shown for the gastrointestinal tract. The enzymes thymidine kinase, maltase, and sucrase were measured in the intestinal mucosa at different times after 5FU treatment with or without UDPG rescue. Even if the nadir values in enzyme activities were similar in mice receiving or not receiving UDPG, the pattern of recovery showed that cell repopulation was more rapid in the group treated with UDPG. 5FU150 + UDPG had enhanced antitumor activity against CD8F1 mammary carcinoma and against the resistant tumor Colon 26 (tumor doubling time 1.9 days for controls, 8.5 days for 5FU100, 13.7 days for 5FU150 + UDPG, and 15.9 days for 5FU150 + leucovorin + UDPG). We demonstrated that UDPG administered at 2, 24, and 30 h after 5FU100 does not reduce the antitumor activity of 5FU in two sensitive tumors (Colon 38 and Colon 26-10). In conclusion, UDPG is a promising rescuing agent for 5FU; it reduces the toxic side effects and increases the therapeutic index.     

Synthesis and antitumor evaluation of bis[(pivaloyloxy)methyl] 2'-deoxy-5-fluorouridine 5'-monophosphate (FdUMP): a strategy to introduce nucleotides into cells

The bis[(pivaloyloxy)methyl] [PIV2] derivative of 2'-deoxy-5- fluorouridine 5'-monophosphate (FdUMP) was synthesized as a potential membrane-permeable prodrug of FdUMP. The compound was designed to enter cells by passive diffusion and to revert to FdUMP after removal of the PIV groups by hydrolytic enzymes. The most convenient preparation of PIV2FdUMP was by condensation of 2'-deoxy-5-fluorouridine (FUdR) with PIV2 phosphate in the presence of triphenylphosphine and diethyl azodicarboxylate (the Mitsunobo reagent). PIV2FdUMP was stable in the pH range 1.0-4.0 (t1/2 > 100 h). It was also fairly stable at pH 7.4 (t1/2 = 40.2 h). In 0.05 M NaOH solution, however, it was rapidly degraded (t1/2 < 2 min). In the presence of hog liver carboxylate esterases, PIV2FdUMP was converted quantitatively to the mono-[(pivaloyloxy)methyl] [PIV1] analogue PIV1FdUMP. After a 24 h incubation, only trace amounts of FdUMP (1-3%) were observed, indicating that PIV1FdUMP is a poor substrate for carboxylate esterases. In mouse plasma, PIV2FdUMP was rapidly metabolized, first to PIV1FdUMP and then to FdUMP. With continued incubation, FUdR was formed, presumably due to further catabolism of FdUMP by plasma phosphatases or 5'-nucleotidases. Since PIV1FdUMP is a poor substrate for carboxylate esterase, the cleavage of the second PIV group is most likely mediated by plasma phosphodiesterases. The rate of degradation of PIV2FdUMP in the presence of acid and alkaline phosphatase, 5'-nucleotidase, or spleen phosphodiesterase was the same as that in buffer controls, indicating that the compound is not a substrate for these nucleotide catabolizing enzymes. The concentration of PIV2FdUMP and its 3'-O-acetyl ester (PIV2 3'-O-Ac-FdUMP) required to inhibit the growth of Chinese hamster ovary (CHO) cells in vitro to less than 50 cells per colony was 5 x 10(-6) M, the same as that required for 5-fluorouracil (FU). Both nucleotide prodrugs showed the same growth-inhibitory potency against a mutant CHO cell line that was 20-fold resistant to FU (CHO/FU). Administered intraperitoneally at optimal dosage for 5 consecutive days, PIV2FdUMP and PIV2 3'-O-Ac-FdUMP were as effective as FU at prolonging the life spans of mice bearing intraperitoneally implanted P388 leukemia. Both prodrugs retained full therapeutic activity against a P388 subline resistant to FU. Collectively, these data indicate that PIV2FdUMP and PIV2 3'-O-Ac-FdUMP are effective membrane-permeable prodrugs of FdUMP.     

In vitro myotoxicity of selected cationic macromolecules used in non-viral gene delivery

PURPOSE: Cationic lipid/DNA complexes have been proposed as a method of in vivo gene delivery via intravenous or intramuscular injection. A concern with using these polycationic molecules is whether they are associated with tissue toxicity at the injection site. Therefore, the objective of these studies was to investigate the myotoxic potential of selected non-viral gene delivery macromolecules (e.g., cationic lipids and polymers) with and without plasmid DNA (pDNA) in vitro. METHODS: Myotoxicity was assessed by the cumulative release of creatine kinase (CK) over 90 minutes from the isolated rodent extensor digitorum longus muscle into a carbogenated balanced salt solution (BBS, pH 7.4, 37 degrees C) following a 15 microL injection of the test formulation. Phenytoin (Dilantin) and normal saline served as positive and negative controls, respectively. RESULTS: The myotoxicity of plasmid DNA (pDNA, approximately 5000bp, 1 mg/ml) was not statistically different from normal saline. However, the myotoxicity of Dilantin was 16-times higher than either normal saline or pDNA (p < 0.05). Cationic liposomes were found to be less myotoxic than polylysine and PAMAM dendrimers. Polylysine's myotoxicity was found to be dependent upon concentration and molecular weight. The myotoxicity of formulations of cationic liposomes(s), lower molecular weight polylysine (25,000) and higher concentration of PAMAM dendrimers with pDNA were found to be statistically less significant than those formulations without pDNA. CONCLUSIONS: The cationic liposomes were less myotoxic compared to the dendrimers and polylysine. Myotoxicity was dependent upon the type of cationic lipid macromolecule, concentration, molecular weight and the presence of pDNA. A possible explanation for this reduced tissue damage in cationic lipids complexed with pDNA is that the formation of complex reduces the overall positive charge of the injectable system resulting in less damage.     

Synthesis and biological activity of chelator-peptide T conjugates

The solid phase procedure was used to prepare two peptide T derivatives in which the 4-[(1,4,8,11-tetraazacyclotetradec-1-yl)methyl]benzoyl unit is linked to their N-terminus. In a human monocyte chemotaxis assay, both chelator-peptide conjugates showed a high binding property to the CD4 receptor, comparable to the parent H-D-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-NH2 and its pentapeptide fragment T(4-8)-NH2. These encouraging results make the above cyclam-oligopeptides candidates for the development of the CD4 receptor imaging agents.     

塑料模具钢大型预硬化模块的研究

系统研究了LF+VD精炼工艺、钢锭模设计、氩气保护浇注工艺、大型钢锭高温扩散工艺、锻造工艺、淬火工艺及高温回火工艺等因素对718钢大型预硬化模块材料纯净度和硬度均匀性的影响规律。结果显示：LF和VD精炼过程中炉渣碱度分别保持在3.5～4.0和3.0～3.5,精炼后钢中w[S]控制到0.003%,T[O]为12×10-6;28t钢锭模尾锥形状和入口倒角优化设计后,尾部夹杂废品率由6.81%降低到1.55%;通过试验确定氩气流量按4～8 m3/h控制;大型钢锭高温扩散后,模块低倍组织的偏析和硬度偏差分别由≤3.0级和≤5.0HRC提高到≤2.0级和≤3.5HRC;采用FM法代替平砧拔长,模块低倍组织疏松和超声波探伤检验分别由≤3.0级和C/d提高到≤2.0级和D/d;采用水-空控时淬火和电加热回火炉回火对规格为650 mm×1 080 mm的模块进行预硬化处理,模块横断面硬度偏差≤3.5HRC。     

PCL reconstruction. In vitro biomechanical comparison of ''isometric'' versus single and double-bundled ''anatomic'' grafts

The analgesic tramadol inhibits the neuronal reuptake of norepinephrine and 5-hydroxytryptamine, facilitates 5-hydroxytryptamine release, and activates mu-opioid receptors. Each of these actions is likely to influence thermoregulatory control. We therefore tested the hypothesis that tramadol inhibits thermoregulatory control. Eight volunteers were evaluated on four study days, on which they received no drugs, tramadol 125 mg, tramadol 250 mg, and tramadol 250 mg with naloxone, respectively. Skin and core temperatures were gradually increased until sweating was observed and then decreased until vasoconstriction and shivering were detected. The core temperature triggering each response defined its threshold. Tramadol decreased the sweating threshold by -1.03 +/- 0.67 degrees C microgram-1.mL (r2 = 0.90 +/- 0.12). Tramadol also decreased the vasoconstriction threshold by -3.0 +/- 4.0 degrees C microgram-1.mL (r2 = 0.94 +/- 0.98) and the shivering threshold by -4.2 +/- 4.0 degrees C microgram-1.mL(r2 = 0.98 +/- 0.98). The sweating to vasoconstriction interthreshold range nearly doubled from 0.3 +/- 0.4 degree C to 0.7 +/- 0.6 degree C during the administration of large-dose tramadol (P = 0.04). The addition of naloxone only partially reversed the thermoregulatory effects of tramadol. The thermoregulatory effects of tramadol thus most resemble those of midazolam, another drug that slightly decreases the thresholds triggering all three major autonomic thermoregulatory defenses. In this respect, both drugs reduce the "setpoint" rather than produce a generalized impairment of thermoregulatory control. Nonetheless, tramadol nearly doubled the interthreshold range at a concentration near 200 ng/mL. This indicates that tramadol slightly decreases the precision of thermoregulatory control in addition to reducing the setpoint. IMPLICATIONS: The authors evaluated the effects of the analgesic tramadol on the three major thermoregulatory responses: sweating, vasoconstriction, and shivering. Tramadol had only slight thermoregulatory effects. Its use is thus unlikely to provoke hypothermia or to facilitate fever.     

Cellular interactions of 5-fluorouracil and the camptothecin analogue CPT-11 (irinotecan) in a human colorectal carcinoma cell line

CPT-11 (irinotecan) is a DNA topoisomerase I inhibitor active against metastatic colorectal carcinoma. We investigated, in a human colon carcinoma cell line, HT-29, the effects of CPT-11 and 5-fluorouracil (5FU) combinations. A strong synergism between CPT-11 and 5FU was observed after sequential exposure and only additivity or antagonism after simultaneous exposure. When cells were first exposed to 5FU, the product of cellular CPT-11 concentrations versus time (CxT) was 6895 +/- 1020 pmol x hr/10(6) cells, while it was 3875 +/- 121 pmol x hr/10(6) cells with CPT-11 alone (p < 0.01). The same phenomenon was observed with SN-38: 148.2 +/- 49.5 versus 83.4 +/- 23.6 pmol x hr/10(6) cells (p < 0.05). Consequently, the formation of protein-DNA complexes was 1.4 times greater with 5FU pretreatment than with CPT-11 alone (p = 0.03). Moreover, the incorporation of 5FU derivatives into DNA was multiplied by a factor of 1.5 24 hr after CPT-11 exposure. When cells were first incubated with CPT-11, the decrease in thymidylate synthase (TS) activity was identical to that obtained after 5FU exposure (1.09 to 0.023 pmol/min/mg protein), but this decrease persisted for 24 hr (0.014 pmol/min/mg protein) (p = 0.035). At the same time, a 1.8-fold increase in the incorporation of 5FU derivatives into DNA and a 2-fold increase in DNA-protein complex formation were evidenced. With the two sequential associations, we observed a persistent S-phase arrest, as compared with CPT-11 alone. These results suggest that CPT-11 and 5FU combinations are of clinical interest, and mechanisms of interaction between the two drugs seem to be multifactorial.     

Aerobic exercise and normotensive adults: a meta-analysis

Using the meta-analytic approach, the purpose of this study was to examine the effects of aerobic exercise on resting systolic (SYS) and diastolic (DIA) blood pressure in normotensive adults: The results of 35 human clinical training studies published in English-language journals between 1963 and 1992 and representing 1,076 subjects (800 exercise, 276 control) met criteria for inclusion. Across all categories and designs, statistically significant post-exercise reductions were found for both SYS and DIA blood pressure (mean +/- SD, SYS: -4.4 +/- 6.6 mm Hg, 95% CI, -6.2 to -2.6 mm Hg; DIA: -3.2 +/- 3.2 mm Hg, 95% CI, -4.0 to -2.2 mm Hg). When partitioned according to type of study: 1) (randomized controlled trials (RCT), 2) controlled trials (CT), and 3) no controls (NC), the following changes were noted: RCT, SYS: -4.5 +/- 7.2 mm Hg, 95% CI, -7.1 to -1.2 mm Hg; DIA: -3.8 +/- 2.9 mm Hg, 95% CI, -5.0 to -2.6 mm Hg; CT, SYS: -2.8 +/- 6.9 mm Hg, 95% CI, -10.0 to 4.4 mm Hg; DIA: -5.0 +/- 3.7 mm Hg, 95% CI, -8.9 to -1.1 mm Hg; NC, SYS: -4.7 +/- 6.1 mm Hg, 95% CI, -7.5 to 1.9 mm Hg; DIA: -1.7 +/- 3.0 mm Hg, 95% CI, -3.2 to -0.36 mm Hg. We concluded that aerobic exercise results in small reductions in resting SYS and DIA blood pressure among normotensive adults.     

5-Fluorouracil plus interferon alpha-2a compared to 5-fluorouracil alone in the treatment of advanced colon carcinoma: a multicentric randomized study

Biochemical modulation is one of the most interesting fields in cancer chemotherapy. Interferon-alpha (IFNalpha) is a cytokine that is able to influence the pharmacodynamics of 5-fluorouracil (5FU) through a number of mechanisms. With the aim of confirming some data emerging from the literature, we initiated a multicentric randomized study comparing the combination of 5FU and IFNalpha-2a with 5FU alone in the treatment of advanced or metastatic colon cancer. A group of 205 colon cancer patients (104 in the 5FU arm and 101 in the 5FU + IFNapha-2a arm) were included in the final intention-to-treat analysis. Rectal cancers were not considered eligible. All patients had measurable disease, were aged 75 years or less, had a Karnofsky index of at least 60 and had good bone marrow, renal, liver and cardiac functions. No previous chemo-immunotherapy was allowed. The treatment was 750 mg/m2 5FU (4 h i.v. infusion) on days 1 5 and then i.v. bolus weekly, starting from day 12, with or without IFNalpha-2a given s.c. three times weekly (starting dose 3 x 10(6) IU rising to 9 x 10(6) IU, if tolerated). Patients were treated until progression or, if responsive, for a maximum of 48 weeks and then observed for a period of 2 years. The primary end-point of the study was objective clinical response (OR); secondary parameters were time to progression, overall survival, and time to death after progression. WHO criteria were used for both clinical response and toxicity measurements. Dose reduction was planned a priori in the event of significant toxicity due to 5FU, IFNalpha-2a or both. Association between primary and secondary end-points and treatment was studied by univariate and multivariate analysis. Altogether, 47 patients achieved a documented response. A 25% OR was observed in the combination arm while a 21% OR was seen in the 5FU arm; this difference is not statistically significant (P = 0.6). Patients with a small tumour burden (below 5 cm2) showed a higher probability of response in both arms. Patients in the experimental arm had a higher but not statistically significant cumulative progression-free probability. Median survival was 47.1 weeks overall, while it was 43.7 and 48.5 weeks in the control and experimental arms, respectively. The combination was clearly more toxic than 5FU alone, leukopenia being the most frequent side-effect in the experimental arm and nausea and vomiting in the control arm. In conclusion these results are quite disappointing and 5FU + IFNalpha-2a can not be considered a standard treatment for advanced colon cancer.