Showing papers by "University of Perugia published in 1994"

Acute Antihyperglycemic Mechanisms of Metformin in NIDDM: Evidence for Suppression of Lipid Oxidation and Hepatic Glucose Production

Abstract: To establish the antihyperglycemic mechanisms of metformin in non-insulin-dependent diabetes mellitus (NIDDM) independently of the long-term, aspecific effects of removal of glucotoxicity, 21 NIDDM subjects (14 obese, 7 nonobese) were studied on two separate occasions, with an isoglycemic (plasma glucose approximately 9 mM) hyperinsulinemic (two-step insulin infusion, 2 h each, at the rate of 4 and 40 mUm-2min-1) clamp combined with [3-3H]glucose infusion and indirect calorimetry, after administration of either metformin (500 mg per os, at -5 and -1 h before the clamp) or placebo Compared with placebo, hepatic glucose production (HGP) decreased approximately 30% more after metformin (from 469 +/- 50 to 330 +/- 54 mumol/min), but glucose uptake did not increase Metformin suppressed free fatty acids (FFAs) by approximately 17% (from 042 +/- 004 to 035 +/- 004 mM) and lipid oxidation by approximately 25% (from 45 +/- 04 to 34 +/- 04 mumolkg-1min-1) and increased glucose oxidation by approximately 16% (from 162 +/- 14 to 193 +/- 13 mumolkg-1min-1) compared with placebo (P < 005), but did not affect nonoxidative glucose metabolism, protein oxidation, or total energy expenditure Suppression of FFA and lipid oxidation after metformin correlated with suppression of HGP (r = 070 and r = 051, P < 0001) The effects of metformin in obese and nonobese subjects were no different We conclude that the specific, antihyperglycemic effects of metformin in the clinical condition of hyperglycemia in NIDDM are primarily due to suppression of HGP, not stimulation of glucose uptake, and are mediated, at least in part, by suppression of FFA and lipid oxidation

Formation of Shc-Grb2 complexes is necessary to induce neoplastic transformation by overexpression of Shc proteins.

Abstract: The mammalian SHC gene encodes three overlapping proteins which all contain a carboxy-terminal SH2 domain. Shc proteins are phosphorylated on tyrosine by a variety of receptor and cytoplasmic tyrosine kinases. Phosphorylated Shc proteins form a complex with the SH2-SH3 containing Grb2 protein which is implicated in the regulation of Ras, suggesting that Shc is involved in the intracellular transmission of growth signals from activated tyrosine kinases to Ras. Overexpression of Shc proteins in cultured fibroblasts induces a transformed phenotype. We now report that, in vitro, the high affinity binding of Grb2 to Shc proteins requires phosphorylation of Shc at Tyr317, which lies within the high affinity binding motif for the Grb2 SH2 domain, pYVNV, where Asn at the +2 position is crucial for complex formation. In vivo, Tyr317 is the major, but not the only, site for Shc phosphorylation, and is the sole Shc high affinity binding site for Grb2. Mutant Shc proteins with substitution of the Tyr317 by Phe lose the capacity to be highly phosphorylated on tyrosine upon growth factor receptor activation, to bind Grb2 and to induce neoplastic transformation. In contrast, Shc proteins that have an extensive aminoterminal deletion, but retain the Tyr317 site and the SH2 domain conserve the capacity to be phosphorylated, to bind to Grb2 and to induce cell transformation. These data indicate that the formation of the Shc-Grb2 complex is a crucial event in the transformation induced by overexpression of Shc and support the notion that Shc proteins can deliver activation signals to RAS.

The L3 silicon microvertex detector

Abstract: The design and construction of the silicon strip microvertex detector (SMD) of the L3 experiment at LEP are described. We present the sensors, readout electronics, data acquisition system, mechanical assembly and support, displacement monitoring systems and radiation monitoring system of the recently installed double-sided, double-layered SMD. This detector utilizes novel and sophisticated techniques for its readout.

Reproducible DNA fingerprinting with the random amplified polymorphic DNA (RAPD) method

Abstract: Much interest has recently arisen in methods for DNA fingerprinting based on the polymerase chain reaction (PCR). Among these, the Random Amplified Polymorphic DNA (RAPD) method, developed by Williams etal. (1), is currently receiving particular attention (2) because of its extreme simplicity and requirement for minimal amounts of genomic DNA. The basic strategy involves the PCR amplification of random fragments of genomic DNA with single or multiple (3) primers of arbitrary sequence. Polymorphism between individuals (or strains) is detected as differences between the pattern of DNA fragments amplified from the different DNAs using a given primer(s). Although RAPD PCR is an extremely powerful tool for such tasks as gene mapping, population and pedigree analysis, phylogenetic studies and bacterial strain identification, the reproducibility of RAPD fingerprints can be quite problematic (4-6). Since the pattern of fragments amplified is, in large part, a function of the sites on the template to which productive annealing of the oligonucleotide primer can occur, differences between DNA preparations that affect primer annealing could be one major source of irreproducibility of RAPD patterns. Here we show directly that ethanol precipitable contaminants in DNA are a major cause of irreproducibility. In preparing DNA for RAPD analysis using standard extraction techniques we found a perfect correlation between the procedure used to collect ethanol precipitated DNA and the reproducibility of the RAPD pattern. We always obtained highly reproducible patterns from DNAs that were wound on a glass rod, while patterns from DNAs collected by centrifugation displayed different degrees of variability. Since centrifugation of an ethanol precipitate of a previously wound DNA sample did not introduce variability, we concluded that winding the DNA must free it of the material that causes the variability exhibited by the centrifuged samples. The results in the figure demonstrate that the RAPD pattern of centrifuged DNA differs from that of wound DNA (lanes Al and Bl, respectively), that the pattern produced by wound DNA can be produced from centrifuged DNA if the latter is reprecipitated from ethanol but collected by winding on a glass rod (lanes A2), and that an altered pattern is obtained using wound DNA to which its supernatant material has been added back (lanes B2). Although RNase A digestion modified the RAPD pattern produced from centrifuged DNA, this treatment did not result in the same RAPD pattern produced with wound DNA (results not shown). Therefore, contaminating RNA may only be partly responsible for the variability observed with centrifuged samples. On the other hand, DNase treatment modified the RAPD pattern produced from wound DNA (results not shown), suggesting that either the presence of very short DNA fragments or shortened templates, or both, may lead to RAPD variability. These findings support the hypothesis that the ethanol precipitable contaminants include very low molecular weight DNA and/or RNA which, in some way, alter the formation of productive template/primer complexes. Whatever their nature, our results unequivocally show that ethanol precipitable contaminants in DNA extracted by standard techniques are a major, if not sole, source of variability in RAPD

Pentoxifylline prevents indomethacin induced acute gastric mucosal damage in rats: role of tumour necrosis factor alpha.

Abstract: Neutrophil adherence within the gastric microcirculation is thought to be a major step in the pathogenesis of gastric mucosal damage induced by indomethacin Pentoxifylline, a methylxanthine derivative, prevents leukocyte adherence to vascular endothelium and protects organs from shock by reducing tumour necrosis factor alpha (TNF alpha) concentrations Rats were treated with 20 mg/kg oral indomethacin, pretreated with vehicle or with four different doses of pentoxifylline intraperitoneally, and killed after three hours The gross gastric mucosal injury, neutrophil margination into the gastric microcirculation, mucosal concentrations of 6-keto-prostaglandin F1 alpha (PGF1 alpha), and PGE2 and serum TNF alpha values were measured Whether the pentoxifylline induced protection involved nitric oxide mediated pathways or gastric acid secretion was evaluated The data indicate that pentoxifylline reduces indomethacin induced mucosal damage and neutrophil margination in a dose dependent manner without exerting any effect on gastric mucosal prostaglandin concentrations The maximally effective dose (200 mg/kg) of pentoxifylline reduced gastric damage by 90% and slightly stimulated acid secretion The effect of pentoxifylline was not affected by pretreatment with the nitric oxide inhibitor Pentoxifylline prevented the indomethacin induced increase in TNF alpha concentrations in a dose dependent fashion Serum TNF alpha values were 305 (70) IU/ml (mean (SEM)) in rats treated with indomethacin alone and 50 (25) IU/ml (p < 001) in rats treated with indomethacin plus 200 mg/kg pentoxifylline Pentoxifylline, therefore, prevents the acute gastric mucosal damage and neutrophil margination induced by indomethacin and reduces indomethacin induced release of TNF alpha

Long-term recovery from unawareness, deficient counterregulation and lack of cognitive dysfunction during hypoglycaemia, following institution of rational, intensive insulin therapy in IDDM.

Abstract: Hypoglycaemia unawareness, is a major risk factor for severe hypoglycaemia and a contraindication to the therapeutic goal of near-normoglycaemia in IDDM. We tested two hypotheses, first, that hypoglycaemia unawareness is reversible as long as hypoglycaemia is meticulously prevented by careful intensive insulin therapy in patients with short and long IDDM duration, and that such a result can be maintained long-term. Second, that intensive insulin therapy which strictly prevents hypoglycaemia, can maintain long-term near-normoglycaemia. We studied 21 IDDM patients with hypoglycaemia unawareness and frequent mild/severe hypoglycaemia episodes while on “conventional” insulin therapy, and 20 nondiabetic control subjects. Neuroendocrine and symptom responses, and deterioration in cognitive function were assessed in a stepped hypoglycaemia clamp before, and again after 2 weeks, 3 months and 1 year of either intensive insulin therapy which meticulously prevented hypoglycaemia (based on physiologic insulin replacement and continuous education, experimental group, EXP, n=16), or maintenance of the original “conventional” therapy (control group, CON, n=5). At entry to the study, all 21 IDDM-patients had subnormal neuroendocrine and symptom responses, and less deterioration of cognitive function during hypoglycaemia. After intensive insulin therapy in EXP, the frequency of hypoglycaemia decreased from 0.5±0.05 to 0.045±0.02 episodes/patient-day; HbA1C increased from 5.83±0.18 to 6.94±0.13% (range in non-diabetic subjects 3.8–5.5%) over a 1-year period; all counterregulatory hormone and symptom responses to hypoglycaemia improved between 2 weeks and 3 months, with the exception of glucagon which improved at 1 year; and cognitive function deteriorated further as early as 2 weeks (p<0.05). The improvement in responses was maintained at 1 year. The improvement in plasma adrenaline and symptom responses inversely correlated with IDDM duration. In contrast, in CON, neither frequency of hypoglycaemia, nor neuroendocrine responses to hypoglycaemia improved. Thus, meticulous prevention of hypoglycaemia by intensive insulin therapy reverses hypoglycaemia unawareness even in patients with long-term IDDM, and is compatible with long-term near-normoglycaemia. Because carefully conducted intensive insulin therapy reduces, not increases the frequency of moderate/severe hypoglycaemia, intensive insulin therapy should be extended to the majority of IDDM patients in whom it is desirable to prevent/delay the onset/progression of microvascular complications.

Transformation by polyoma virus middle T-antigen involves the binding and tyrosine phosphorylation of Shc

Abstract: POLYOMA virus middle T-antigen converts normal fibroblasts to a fully transformed, tumorigenic phenotype1. It achieves this, at least in part, by binding and activating one of the non-receptor tyrosine kinases, pp60c-src, pp62c-yes or pp59c-fyn (reviewed in refs 2 and 3). As a result, middle T-antigen itself is phosphorylated on tyrosine residues4,5, one of which (Tyr 315) acts as a binding site for the SH2 domains of phosphatidylinositol-3'OH kinase 85K sub-unit6–8. Here we show that another tyrosine phosphorylation site in middle T-antigen (Tyr 250; refs 4, 5) acts as a binding region for the SH2 domain of the transforming protein Shc9. This results in Shc also becoming tyrosine-phosphorylated and binding to the SH2 domain of Grb2 (ref. 10). This probably stimulates p21ras activity through the mammalian homologue of the Drosophila guanine-nucleotide-exchange factor Sos (reviewed in ref. 11). We suggest that middle T-antigen transforms cells by acting as a functional homologue of an activated tyrosine kinase-associated growth-factor receptor.

Neutralization of IL-10 up-regulates nitric oxide production and protects susceptible mice from challenge with Candida albicans.

Abstract: In contrast to several inbred strains of mice that develop Th1-associated anticandidal protection, DBA/2 mice are highly susceptible to systemic infection with Candida albicans cells of the attenuated variant PCA-2, and fatal outcome is observed in concurrence with sustained CD4+ cell production in vitro of IL-4 and IL-10. These Th2 cytokines were previously shown to inhibit nitric oxide (NO) production and yeast killing by activated macrophage cultures. We now show that macrophages from DBA/2 mice, either intact or infected with PCA-2, have lower capacity than resistant strains to synthesize NO in response to IFN-gamma. However, when treated with anti-IL-10 Abs at the time of infection, DBA/2 mice survived challenge and displayed increased production of NO in vitro after IFN-gamma activation. Cure was associated with the onset of footpad responses and durable protection, and higher frequencies of IFN-gamma-secreting cells were found in splenic CD4+ lymphocytes that expressed lower levels of IL-4 and IL-10 mRNA. Therefore, in DBA/2 mice, IL-10 contributes significantly to the selection of a Th2 response and lethality after PCA-2 challenge. An IL-10-induced defect in the activation and/or expansion of IFN-gamma-producing Th1 cells, IL-10 suppression of yeast killing, and the relative inability of DBA/2 macrophages to produce adequate levels of candidacidal NO may all contribute to the abnormal susceptibility of these mice to candidiasis.

Relative roles of insulin and hypoglycaemia on induction of neuroendocrine responses to, symptoms of, and deterioration of cognitive function in hypoglycaemia in male and female humans

Abstract: To assess the relative roles of insulin and hypoglycaemia on induction of neuroendocrine responses, symptoms and deterioration of cognitive function (12 cognitive tests) during progressive decreases in plasma glucose, and to quantitate glycaemic thresholds, 22 normal, non-diabetic subjects (11 males, 11 females) were studied on four occasions: prolonged fast (n=8, saline euglycaemia study, SA-EU), stepped hypoglycaemia (plasma glucose plateaus of 4.3, 3.7, 3 and 2.3 mmol/l) or euglycaemia during insulin infusion at 1 and 2 mU·kg−1·min−1 (n=22, high-insulin hypoglycaemia and euglycaemia studies, HI-INS-HYPO and HI-INS-EU, respectively), and stepped hypoglycaemia during infusion of insulin at 0.35 mU· kg−1·min−1 (n=9, low-insulin hypoglycaemia study, LO-INS-HYPO). Insulin per se (SA-EU vs HI-INS-EU), suppressed plasma glucagon (∼20%) and pancreatic polypeptide (∼30%), whereas it increased plasma noradrenaline (∼R10%, p<0.05). Hypoglycaemia per se (HI-INS-HYPO vs HI-INS-EU) induced responses of counterregulatory hormones (CR-HORM), symptoms and deteriorated cognitive function. With the exception of suppression of endogenous insulin secretion, which had the lowest glycaemic threshold of 4.44±0.06 mmol/l, pancreatic polypeptide, glucagon, growth hormone, adrenaline and cortisol had similar glycaemic thresholds (∼3.8-3.6 mmol/l); noradrenaline (3.1±0.0 mmol/l), autonomic (3.05±0.06 mmol/l) and neuroglycopenic (3.05±0.05 mmol/l) symptoms had higher thresholds. All 12 tests of cognitive function deteriorated at a glycaemic threshold of 2.45±0.06 mmol/l, but 7 out of 12 tests were already abnormal at a glycaemic threshold of 2.89±0.06 mmol/l. Although all CR-HORM had a similar glycaemic threshold, the lag time of response (the time required for a given parameter to increase) of glucagon (15±1 min) and growth hormone (14±3 min) was shorter than adrenaline (19±3 min) and cortisol (39±4 min) (p<0.05). With the exception of glucagon (which was suppressed) and noradrenaline (which was stimulated), insulin per se (HI-INS-HYPO vs LO-INS-HYPO) did not affect the responses of CR-HORM, and did not influence the symptoms or the cognitve function during hypoglycaemia. Despite lower responses of glucagon, adrenaline and growth hormone (but not thresholds) in females than males, females were less insulin sensitive than males during stepped hypoglycaemia.

Modulation of the kynurenine pathway in search for new neuroprotective agents. Synthesis and preliminary evaluation of (m-nitrobenzoyl)alanine, a potent inhibitor of kynurenine-3-hydroxylase.

Abstract: The synthesis of (o-nitrobenzoyl)-, (m-nitrobenzoyl)-, and (p-nitrobenzoyl)alanine (o-, m-, and p-NBA), three new kynurenine analogues, and their evaluation as inhibitors of kynureninase and kynurenine-3-hydroxylase are reported. The most potent of these compounds, m-NBA, has an IC50 of 0.9 +/- 0.1 microM as an inhibitor of kynurenine-3-hydroxylase and of 100 +/- 12 microM as an inhibitor of kynureninase. When administered to rats, m-NBA significantly increases the concentration of kynurenine and kynurenic acid in the brain as well as in blood and in the liver. m-NBA has also been shown to increase the concentration of kynurenic acid in hippocampal extracellular fluid. This increase is associated with sedative and anticonvulsant activities, thus confirming the possibility of antagonizing L-glutamate-mediated effects by modulating the kynurenine pathway of L-tryptophan metabolism.

Velocity dependence of collisional alignment of oxygen molecules in gaseous expansions

Abstract: THE orientational dependence of molecular interactions has long been recognized as central to an understanding of reaction mechanisms and of collisions in the gas phase and at surfaces. Studies of orientation effects have recently become possible owing to the development of techniques for aligning molecules. 'Brute-force' methods using electric or magnetic fields can induce alignment of molecules with dipole moments1,2, and polarized-absorption approaches3 can be used in cases where there are suitable molecular transitions; but one of the simplest and most general methods involves the supersonic expansion of molecular beams seeded with molecules that induce rotational alignment—selection of specific rotational states—by collisions4–12. Here we use such an approach to induce strong rotational alignment of oxygen molecules in a beam seeded with various other gases at close to atmospheric pressure. Most significantly, we find that the degree of alignment depends on the velocity of the molecules in the supersonic expansion—fast molecules are much more highly aligned than slower ones, and the velocity of maximum alignment can be altered by changing the gas mixture. In this way, we can prepare rotationally aligned molecules with well defined velocities, opening up new possibilities for experiments in molecular dynamics.

Preventing Fungal Infection in Neutropenic Patients with Acute Leukemia: Fluconazole Compared with Oral Amphotericin B

Abstract: Objective: To compare the efficacy and tolerability of fluconazole and oral amphotericin B in preventing fungal infection in neutropenic patients with acute leukemia Design: A randomized, controll

Comparative Molecular Field Analysis Using GRID Force-Field and GOLPE Variable Selection Methods in a Study of Inhibitors of Glycogen Phosphorylase b

Abstract: A primary goal in any drug design strategy is to predict the activity of new compounds. Comparative molecular field analysis (CoMFA) has been used in drug design and three-dimensional quantitative structure/activity relationship (3D-QSAR) methods. The CoMFA approach permits analysis of a large number of quantitative descriptors and uses chemometric methods such as partial least squares (PLS) to correlate changes in biological activity with changes in chemical structure. One of the characteristics of the 3D-QSAR method is the large number of variables which are generated in order to describe the nonbonded interaction energies between one or more probes and each drug molecule. Since it is difficult to know a priori which variables affect the biological activity of the compounds, much effort has been devoted to developing methods that optimize the selection of only those variables of importance. This work focuses on some of the aspects involved in the selection of such variables, applied to a series of glucose analogue inhibitors of glycogen phosphorylase b, using the program GRID to describe the molecular structures and using a method of generating optimal partial least squares estimations (program GOLPE) as the chemometric tool. This data set, consisting of over 30 compounds in which the three-dimensional ligand-enzyme bound structures are known, is well suited to study the effect of different data pretreatment procedures on the final model used for the prediction of new drug molecules. By relying on our knowledge of the real physical problem (i.e., using the combined crystallographic and kinetic results), it has been shown that suitable data pretreatment and variable selection have been found that does not result in a significant loss of relevant information. Moreover, by using an appropriate scaling procedure, GOLPE variable selection minimizes the risk of overfitting and overpredicting.

IL-12 is both required and prognostic in vivo for T helper type 1 differentiation in murine candidiasis.

Abstract: In murine models of systemic candidiasis, healing and nonhealing patterns of disease are associated with preferential expansion of Th1 and Th2 cells, respectively. As previous studies have shown IL-12 to be expressed transcriptionally in healer mice and to be required for Th1 development in vitro, this cytokine might play a role in Candida-driven Th1 cell differentiation in vivo. In the present study, IL-12-neutralizing Abs or recombinant IL-12 were administered to mice with healing or progressive candidiasis, respectively, and the animals were monitored for mortality, resistance to reinfection, serum levels of specific Abs, and IFN-gamma, IL-4, and IL-10 message/protein expression by CD4+ cells. In self-limiting infection by a yeast vaccine strain, neutralization of IL-12 ablated development of acquired anticandidal resistance and led to appearance of Candida-specific IgE and IL-4-producing cells. In mice with progressive systemic disease as well as in a mucosal infection model, administration of IL-12 did not result in therapeutic activity under conditions of yeast infection that would instead be resolved by serologic ablation of IL-4 or IL-10. Yet, in systemically infected mice cured by anti-IL-4 or anti-IL-10 therapy, the emergence of a Th1 response correlated with the detection of high levels of circulating IL-12 and splenic IL-12 transcripts. Although exogenous IL-12 may not be sufficient for Th conversion in the presence of an overwhelming IL-4/IL-10 response, endogenous production of IL-12 may be both required and prognostic for Th1 differentiation in vivo.

Anorectal manometric abnormalities and colonic propulsive impairment in patients with severe chronic idiopathic constipation

Abstract: Idiopathic chronic constipation is a frequent and disabling symptom, but its pathophysiological grounds are still poorly understood. In particular, there is little knowledge about the relationships between distal (anorectal area) and proximal (colonic area) motor abnormalities in this condition, especially concerning high-amplitude propagated colonic activity. For this purpose, we studied 25 patients complaining of severe idiopathic constipation and categorized them as normal- or slow-transit constipation according to colonic transit time. Twenty-five age-matched controls were also studied. Investigations included standard anorectal motility testing and prolonged (24-hr) colonic motility studies. Analysis of results showed that both groups of constipated patients displayed significantly different (P < 0.05) minimum relaxation volumes of the internal anal sphincter, defecatory sensation thresholds, and maximum rectal tolerable volumes with respect to controls. Patients with normal-transit constipation also showed lower internal anal sphincter pressure with respect to slow-transit constipation and controls (P < 0.001 and P < 0.02, respectively). The daily number of high-amplitude propagated contractions (mass movements) as well as their amplitude and duration, was significantly reduced in both subgroups of constipated patients (P < 0.02 vs controls). We conclude that (1) in normal-transit constipation, motor abnormalities are not limited to the anorectal area; (2) patients with slow-transit constipation probably have a severe neuropathic rectal defect; (3) prolonged colonic motility studies may highlight further the functional abnormalities in constipated subjects; and (4) an approach taking into account proximal and distal colon motor abnormalities might be useful to understand pathophysiological grounds of chronic constipation and lead to better therapeutic approaches.

Involvement of Src‐homology/collagen (SHC) proteins in signaling through the insulin receptor and the insulin‐like‐growth‐factor‐I‐receptor

Abstract: Src homology/collagen (SHC) proteins are thought to participate in signaling through both receptor tyrosine kinases, such as the insulin receptor and the EGF (epidermal growth factor) receptor, and cytoplasmic tyrosine kinases, such as v-src and v-fps. Here we approached the insulin-induced and the insulin-like-growth-factor-I-induced (IGF-I-induced) phosphorylation of SHC proteins, and the possible role of these proteins in insulin and IGF-I signaling. First, we showed that SHC proteins are phosphorylated on tyrosine residues upon insulin and IGF-I treatment of fibroblasts transfected with a SHC cDNA construct. More important, ligand-activated insulin and IGF-I receptors phosphorylate SHC proteins in vitro, indicating that SHC proteins could be direct substrates for insulin and IGF-I receptors. Further, insulin or IGF-I treatment of SHC-transfected fibroblasts leads to immunoprecipitation of SHC proteins with insulin-receptor substrate 1 (IRS-1). We next looked at the possible effect of SHC proteins on biological responses in SHC-transfected fibroblasts. We found that the expression of exogenous SHC proteins results in an increased basal MEK (MAPK/ERK-activating kinase) activity. Further, neither the basal nor the insulin-induced or IGF-I-induced PtdIns-3-kinase activity were modified by expression of exogenous SHC proteins. These results illustrate that SHC proteins are implicated in the MAP(mitogen-activated protein)-kinase pathway, but not in that of PtdIns-3-kinase. Finally, we show that SHC-transfected cells, unlike control cells, are able to advance into the early phases of the cell cycle, and are more sensitive to the growth-promoting effect of insulin. In conclusion, SHC proteins are substrates for insulin and IGF-I receptors, and would appear to function as early post-receptor signaling components.

Pharmacokinetics, pharmacodynamics and glucose counterregulation following subcutaneous injection of the monomeric insulin analogue [Lys(B28),Pro(B29)] in IDDM

Abstract: The aim of these studies was to compare the pharmacokinetics, pharmacodynamics, counterregulatory hormone and symptom responses, as well as cognitive function during hypoglycaemia induced by s. c. injection of 0.15 IU/kg of regular human insulin (HI) and the monomeric insulin analogue [Lys(B28),Pro (B29)] (MI) in insulin-dependent-diabetic (IDDM) subjects. In these studies glucose was infused whenever needed to prevent decreases in plasma glucose below 3 mmol/l. After MI, plasma insulin increased earlier to a peak (60 vs 90 min) which was greater than after HI (294±24 vs 255±24 pmol/l), and plasma glucose decreased earlier to a 3 mmol/l plateau (60 vs 120 min) (p<0.05). The amount of glucose infused to prevent plasma glucose falling below 3 mmol/l was ∼three times greater after MI than HI (293±26 vs 90±25 μmol · kg−1 · 60–375 min−1, p<0.05). After MI, hepatic glucose production was more suppressed (0.7±1 vs 5.9±0.54 μmol · kg−1 · min−1) and glucose utilization was less suppressed than after HI (11.6±0.65 vs 9.1±0.11μmol · kg−1 · min−1) (p<0.05). Similarly, plasma NEFA, glycerol, and β-OH-butyrate were more suppressed after MI than HI (p<0.05), whereas plasma lactate increased only after MI, but not after HI. Responses of counterregulatory hormones, symptoms and deterioration in cognitive function during plasma glucose plateau of 3 mmol/l were superimposable after MI and HI (p=NS). Post-hypoglycaemia hyperglycaemia was greater after MI than HI (at 480 min 12.1±1 vs 11±1 mmol/l) because of greater hepatic glucose production during insulin waning which occurred at least 135 min earlier with MI as compared to HI (p<0.05). It is concluded that counterregulatory hormones, symptoms and deterioration in cognitive function during hypoglycaemia respond similarly after MI and HI. The biological effect of MI appears greater than that of HI for at least 4 h after the s.c. injection and appears as a good candidate for achieving optimal post-prandial glucose control in IDDM.

Cure of Murine Candidiasis by Recombinant Soluble Interleukin-4 Receptor

Abstract: Neutralization of interleukin (IL)-4 by specific antibody exerts therapeutic activity in a murine model of systemic candidiasis characterized by strong T helper type 2 (Th2) responses. To investigate whether recombinant soluble IL-4 receptor (sIL-4R) could be used to block IL-4 action in vivo, mice treated with pharmacologic doses of sIL-4R at the time of infection were examined for progression of disease, development of footpad responses, serum IgE levels, and cytokine production in vitro by CD4+ lymphocytes. Following sIL-4R treatment, persistent ablation of circulating IL-4 detected by ELISA was associated with a cure rate of > 90% in otherwise lethally infected mice, onset of durable protection, and a shift from a predominant Th2 to a Th1 pattern of reactivity. In addition, when administered to genetically susceptible adult mice with gastrointestinal yeast colonization, the sIL-4R stimulated Th1-associated anticandidal resistance.

Hyaluronate is costimulatory for human T cell effector functions and binds to CD44 on activated T cells.

Abstract: Lymphohematopoiesis, cell matrix adhesion, homing of leukocytes, T cell activation, and tumor metastasis are mediated through the CD44 family of cell surface receptors. We have recently shown that anti-CD44 mAb trigger protein tyrosine kinase-dependent activation of T cell effector functions. Here, we show that hyaluronate (HA), a CD44 ligand, in conjunction with CD3/TCR-mediated stimuli, is costimulatory for human peripheral blood T cell proliferation, for IL-2 production by Th clones, and for release of trypsin-like esterase by cytolytic T cell clones. A human T cell line, HUT-78, was found to bind HA and on HA coating it was used as a target for cytolytic T cell clones. After anti-CD3 stimulation, CD3+/CD8+ clones acquire the ability of lysing HA-coated HUT-78 cells more efficiently than the same HA-uncoated targets. Resting peripheral blood T cells and T cell clones do not adhere to HA-coated plates. However, 24-h anti-CD3 mAb stimulation gives them the transient ability to bind HA. HA adhesion of activated T cells and T cell clones, as well as that of T cell lines, is blocked by one anti-CD44 mAb (J-173). Two other anti-CD44 mAbs induce a 10-fold increase in HA adhesiveness of anti-CD3-stimulated peripheral blood T cells. This impressive HA adhesiveness is also readily blocked by J-173 anti-CD44 mAb. These data indicate that 1) HA is costimulatory for human T cell effector functions in conjunction with CD3/TCR-mediated stimuli, 2) the capacity to bind HA is acquired by resting T cells and T cell clones after anti-CD3 stimulation, and 3) HA binding occurs via specific interaction with CD44 molecules expressed on activated T cells.

Effects of the herbicide imazethapyr on soil microbial biomass and various soil enzyme activities

Abstract: The effects of imazethapyr, an imidazolinone derivative on the soil microbial biomass was investigated in a clay loam soil. Imazethapyr was applied in a field trial at the recommended field rate for soybean weeding, 50 g a.i. ha-1. In laboratory experiments the herbicide was incorporated at the field rate and 10-fold and 100-fold higher. In both the field trials and the laboratory experiments, the field rate of imazethapyr had no adverse effects on the microbiological processes tested. But at 10-fold and 100-fold higher rates, the herbicide decreased the biomass-C content and dehydrogenase activity and increased the hydrolytic capacity and protease and catalase activity. These findings suggest that there may be a risk in monocultural practices that require repeated herbicide treatments.