Insulinomimetic effect on glucose transport by epidermal growth factor when combined with a major histocompatibility complex class I-derived peptide

Peptides derived from the alpha 1-region of the murine H-2Dk molecule enhance glucose uptake in rat adipose cells above the maximum obtained with insulin stimulation alone (Stagsted, J., Reaven, G. M., Hansen, T., Goldstein, A., and Olsson, L. (1990) Cell 62, 297-307). We now describe that epidermal growth factor (EGF) in combination with the same peptides, Dk-(61-85) and Dk-(62-85), stimulates cellular glucose uptake 5-7 times over the basal level, i.e. to 30-50% of the maximal insulin effect. EGF alone increased glucose uptake by only approximately 50% above basal and the peptide alone by 100% above basal. Maximal effect of EGF and peptide was reached in 10-20 min with 30 microM peptide (EC50 10-15 microM) and 50 nM EGF (EC50 1-2 nM). The effect of EGF and peptide on glucose uptake was additive to that of insulin and peptide until the maximal level attained with insulin and peptide was reached. The combined effect of EGF plus peptide on glucose transport was associated with a recruitment of GLUT4 molecules to the plasma membrane. However, the phosphatidylinositol (PI) kinase which is activated by insulin was not activated by EGF plus peptide. Thus, the effect of EGF plus peptide on glucose uptake seems independent of the activity status of the insulin receptor. 125I-Labeled EGF bound specifically to rat adipose cells with an apparent affinity of approximately 2 nM and Bmax approximately 5 x 10(3). However, the major histocompatibility complex (MHC) peptides did not affect EGF-stimulated internalization of EGF receptor, in contrast to their effect on the insulin receptors. Transforming growth factor alpha had an effect similar to EGF on glucose uptake. Three other peptides derived from other parts of murine MHC class I had no effect on glucose uptake in combination with EGF. Thus, EGF in combination with certain MHC class I-derived peptides is insulinomimetic concerning glucose transport and this effect is independent of the insulin receptor activity.     

Detection of an anatoxin-a(s)-like anticholinesterase in natural blooms and cultures of cyanobacteria/blue-green algae from Danish lakes and in the stomach contents of poisoned birds

Ten natural bloom samples of cyanobacteria from the Danish lakes Knud s? (5), Ravn s? (4), and Salten Langs? (1) collected during 1993-1995 were assayed for toxicity by mouse bioassay, for acetylcholinesterase inhibiting activity by a colorimetric method, and for microcystins by enzyme-linked immunosorbent assay. In the mouse bioassay, seven samples were neurotoxic, two were non-toxic and one gave a protracted toxic response. One of the non-toxic and the single protracted toxic sample both contained anticholinesterase activity equivalent to 4 micrograms anatoxin-a(s) g-1. The neurotoxic samples contained equivalents to 20-3300 micrograms anatoxin-a(s) g-1. The highest anticholinesterase activities (equivalent to 2300 and 3300 micrograms anatoxin-a(s) g-1, respectively) were found in samples collected from Lake Knud s? in connection with bird-kills in 1993 and 1994. Small amounts of microcystins (0.1-0.9 microgram g-1) were detected in all samples but one. All Lake Knud s? and Lake Ravn s? samples were dominated by Anabaena lemmermannii, and the Lake Salten Langs? sample by several species of Anabaena. Gel filtration profiles indicated similarity between the toxic component from the Lake Knud s? 1994 bloom with registered bird-kills and anatoxin-a(s) isolated from Anabaena flos-aquae NRC-525-17. Anticholinesterase-producing cultures of A. lemmermannii were isolated from the Lake Knud s? 1993 bloom. These laboratory cultures produced anatoxin-a(s) equivalents of 29-743 micrograms g-1. Other cultures of A. lemmermannii isolated from Lake Knud s? and Lake Ravn s? were hepatotoxic or non-toxic. Dead birds collected from Lake Knud s? during the neurotoxic 1993 Anabaena bloom possibly died from cyanobacterial toxicosis. The stomach contents contained colonies and single trichomes of Anabaena, and anticholinesterase activities equivalent to 2.1-89.7 micrograms anatoxin-a(s) kg-1 body weight and microcystins (53-95 ng kg-1) were also detected.     

Trends and variations in neonatal length of in-hospital stay in Canada

The objectives of this study were to evaluate the possible mechanisms involved in prolongation of bleeding time in pre-eclamptic patients receiving a magnesium sulfate infusion to prevent convulsions. Eighteen pre-eclamptic patients near term or at term (4 cases 33 to 35 weeks; the remainder > 36 weeks) were studied. Fifteen of them received magnesium sulfate infusion; 3 did not and served as controls. Bleeding time (modified Ivy method with Surgicutt), platelet count, platelet aggregation pattern, as well as serum arachidonic acid metabolites [thromboxane B2 (TxB2) and 6-Keto-prostaglandin F1 alpha (6-Keto-PGF1 alpha)] werde done on admission to the labor floor (before magnesium infusion) and repeated at discontinuation of the infusion, 12-24 hours postpartum; the controls received the second test 24 hours postpartum. Thirteen of 15 patients receiving magnesium sulfate had an increase in bleeding time from an average of 6 minutes 31 seconds to 11 minutes 56 seconds, an 82% rise (p < 0.004). In 2 there was a decrease. Among the 3 controls the averages were 6 minutes 38 seconds and 6 minutes 3 seconds. The total magnesium given ranged from 52.5 to 145 grams. Platelet counts averaged 251,000/mm3 (range 145,000-519,000). Platelet aggregation pattern done in 11 patients and was normal and unchanged after magnesium in 10 of the patients with increased bleeding time and one control. TxB2 and 6-Keto-PGF1 alpha levels did not change significantly either after magnesium administration (688 and 135 pgm/ml, to 654 and 117) or in controls (695 and 230 pgm/ml, to 445 and 225). Likewise, the ratio of these 2 substances did not change in either group (6.3 to 6.6, and 4.2 to 2.2). There was no correlation between duration of infusion or total magnesium given and directions of small changes observed. This study confirms a prior preliminary observation that magnesium sulfate infusion, as currently used to prevent eclamptic convulsions, induces a significant prolongation of bleeding time. This effect is mediated neither by changes in platelets count or aggregation pattern, nor by changing the level or ratios of serum arachidonic acid metabolites (TxB2 and 6-Keto-PGF1 alpha). Further studies are needed to clarify the mechanism of this clinically important observation of increased bleeding following magnesium sulfate infusion.     

Mutational analysis of the three cysteines and active-site aspartic acid 103 of ketosteroid isomerase from Pseudomonas putida biotype B

In order to clarify the roles of three cysteines in ketosteroid isomerase (KSI) from Pseudomonas putida biotype B, each of the cysteine residues has been changed to a serine residue (C69S, C81S, and C97S) by site-directed mutagenesis. All cysteine mutations caused only a slight decrease in the k(cat) value, with no significant change of Km for the substrate. Even modification of the sulfhydryl group with 5,5'-dithiobis(2-nitrobenzoic acid) has almost no effect on enzyme activity. These results demonstrate that none of the cysteines in the KSI from P. putida is critical for catalytic activity, contrary to the previous identification of a cysteine in an active-site-directed photoinactivation study of KSI. Based on the three-dimensional structures of KSIs with and without dienolate intermediate analog equilenin, as determined by X-ray crystallography at high resolution, Asp-103 was found to be located within the range of the hydrogen bond to the equilenin. To assess the role of Asp-103 in catalysis, Asp-103 has been replaced with either asparagine (D103N) or alanine (D103A) by site-directed mutagenesis. For D103A mutant KSI there was a significant decrease in the k(cat) value: the k(cat) of the mutant was 85-fold lower than that of the wild-type enzyme; however, for the D103N mutant, which retained some hydrogen bonding capability, there was a minor decrease in the k(cat) value. These findings support the idea that aspartic acid 103 in the active site is an essential catalytic residue involved in catalysis by hydrogen bonding to the dienolate intermediate.     

Isolation of a genetic locus associated with metronidazole resistance in Helicobacter pylori

We examined the molecular mechanism of metronidazole resistance by constructing a lambda-Zap II phagemid expression library with genomic DNA from a metronidazole-resistance strain of Helicobacter pylori. Twenty-two clones were found to have elevated MTZ resistances in XLOLR strain of E. coli. Phagemids belonging to the twenty two clones were extracted and then retransformed into the XLOLR strain of E. coli. After MTZ selection, five clones could confer metronidazole resistance consistently. According to Southern hybridization and DNA sequencing, the five clones contained a same locus, recA. In addition, transforming the five clones into BL21 strain of E. coli produced a higher resistance to MTZ. Interestingly, electroporation of one of the five phagemid clones into two MTZ sensitive H. pylori yielded MTZ resistant strains. Comparing amino acid sequence in MTZ resistant with sensitive isolates revealed two point mutations at this locus. Above results suggest that mutation in recA may be associated with metronidazole resistance of H. pylori.     

Carcinogenic polycyclic aromatic hydrocarbons increase intracellular Ca2+ and cell proliferation in primary human mammary epithelial cells

A method was developed to determine in eggs 2 components [4,6-dimethyl-2-hydroxypyrimidine and 1,3-bis(4-nitrophenyl)urea] of the anticoccidial drug nicarbazin, used to treat poultry. Samples were extracted with acetonitrile, and the extracts were washed with hexane and evaporated to dryness before analysis by liquid chromatography/mass spectrometry with atmospheric pressure chemical ionization. By switching from positive to negative ion monitoring and using gradient elution, both components were measured within one run. The limit of quantitation of the assay was 10 ng/g for each component. The results of a preliminary feeding trial in which chickens were fed contamination levels of the drug are also reported.     

Prenatal diagnosis of partial monosomy 13q associated with occipital encephalocoele in a fetus

The pre- and postnatal findings of a fetus with a de novo del(13)(pter-->q21:) and an occipital encephalocoele are described. Maternal serum alpha-fetoprotein (AFP) screening at 19 weeks' gestation demonstrated a high level of 2.5 multiples of the median (MOM) and ultrasonography at 27 weeks' gestation showed severe intrauterine growth retardation, cardiomegaly, an occipital encephalocoele, and a calvarial defect. Genetic amniocentesis revealed a karyotype of 46,XX,del(13)(pter-->q21:). The proband postnatally displayed additional abnormalities such as microphthalmia, hypertelorism, large low-set ears, and micrognathia. We discuss the association of central nervous system (CNS) malformations with 13q deletions and emphasize that pregnancies with neural tube defects warrant cytogenetic analysis, especially when additional fetal abnormalities and neonatal dysmorphism are observed.     

Tramadol, M1 metabolite and enantiomer affinities for cloned human opioid receptors expressed in transfected HN9.10 neuroblastoma cells

Tramadol hydrochloride is a centrally acting synthetic analgesic in widespread clinical use. Despite different degrees of opioid-like characteristics in preclinical tests, it is characterized by lack of full naloxone reversibility or naloxone-precipitated withdrawal in humans. To investigate this apparent discrepancy, the present study measured the affinity of tramadol (and its enantiomers) and an active O-desmethyl metabolite (M1) (and its enantiomers) to cloned human opioid receptors of the mu, delta and kappa type stably expressed in HN9.10 neuroblastoma cells. At mu sites, the Ki values for tramadol, its (+) and (-) enantiomers, M1, and its (+) and (-) enantiomers were 17000, 15700, 28800, 3190, 153 and 9680 nM, respectively, compared to 7.1 nM for morphine. These results are consistent with the suggestion of a non-opioid contribution to the clinical profile of tramadol.