The m1 muscarinic acetylcholine receptor transactivates the EGF receptor to modulate ion channel activity

Intracellular tyrosine kinases link the G protein-coupled m1 muscarinic acetylcholine receptor (mAChR) to multiple cellular responses. However, the mechanisms by which m1 mAChRs stimulate tyrosine kinase activity and the identity of the kinases within particular signaling pathways remain largely unknown. We show that the epidermal growth factor receptor (EGFR), a single transmembrane receptor tyrosine kinase, becomes catalytically active and dimerized through an m1 mAChR-regulated pathway that requires protein kinase C, but is independent of EGF. Finally, we demonstrate that transactivation of the EGFR plays a major role in a pathway linking m1 mAChRs to modulation of the Kv1.2 potassium channel. These results demonstrate a ligand-independent mechanism of EGFR transactivation by m1 mAChRs and reveal a novel role for these growth factor receptors in the regulation of ion channels by G protein-coupled receptors.     

The L5178Y/tk+/- mouse lymphoma specific gene and chromosomal mutation assay a phase III report of the U.S. Environmental Protection Agency Gene-Tox Program

The L5178Y/tk+/- (-)3.7.2C mouse lymphoma assay (MLA) which detects mutations affecting the heterozygous thymidine kinase (tk) locus is capable of responding to chemicals acting as clastogens as well as point mutagens. Improvements in the assay to enhance detection of this spectrum of genetic events are summarized, and criteria for evaluating the data are defined. Using these criteria, the Phase III Work Group reviewed and evaluated literature containing MLA results published from 1976 through 1993. The data base included 602 chemicals of which 343 were evaluated as positive, 44 negative, 18 equivocal, 54 apparently inappropriate for evaluation in this test system with the published protocols, and 142 that were inadequately tested, and thus a definitive call could not be made. The overall performance of the assay is summarized by chemical class, and the outcome of testing 260 chemicals in the MLA is compared with Gene-Tox and National Toxicology Program evaluations of rodent carcinogenesis bioassay results for the same chemicals. Based on the Work Group's evaluation of published MLA data for chemicals that were considered adequately tested, it is concluded that for most chemicals the L5178Y/tk+/- mouse lymphoma assay is eminently well suited for genotoxicity testing and for predicting the potential for carcinogenicity.     

Regenerating sciatic nerve does not utilize circulating cholesterol

Vascular endothelial growth factor (VEGF) is a major contributor to retinal neovascularization. The possible participation of VEGF and its high-affinity tyrosine kinase receptors, flk-1 and flt-1, in early background diabetic retinopathy was studied in the streptozotocin-induced diabetic rat model of experimental retinopathy using in situ hybridization, blotting techniques, and immunohistochemistry. Diabetic retinopathy was assessed by quantitative morphometry of retinal digest preparations. The number of acellular capillaries increased 2.7-fold in diabetic animals with diabetes' duration of 6 months compared with nondiabetic controls. VEGF expression was not detectable by in situ hybridization in nondiabetic rats but was highly increased in the ganglion cell layer and in the inner and outer nuclear layers of retinas from diabetic animals. VEGF protein was extractable only from diabetic retinas, and a strong immunolabeling was detected in vascular and perivascular structures. Increased flk-1 and flt-1 mRNA levels were also found in the ganglion cell and both nuclear layers of diabetic samples only. Dot blot and Western blot analyses confirmed the increase in flk-1 mRNA and protein in diabetic retinas. Also, flk-1 immunoreactivity was associated with vascular and nonvascular structures of the inner retinas from diabetic animals. These data obtained from a rodent model in which retinal neovascularization does not occur support the concept that the VEGF/VEGF receptor system is upregulated in early diabetic retinopathy.     

Expression studies of delta-globin gene alleles associated with reduced hemoglobin A2 levels in Greek Cypriots

We previously identified five delta-globin gene alleles associated with reduced hemoglobin (Hb) A2 (Trifillis, P., Ioannou, P., Schwartz, E., and Surrey, S. (1991) Blood 78, 3298-3305). We have now evaluated functional consequences of the changes after expression in COS-1 cells to monitor effects on RNA splicing. In addition, variant Hb A2 tetramers were expressed in yeast to assess effects of amino acid changes on oxygen binding and stability to heat and mechanical agitation. The G --> T change at codon 27 and the A --> G change in IVS-2 both affect RNA splicing, whereas the C --> T change at codon 97 and the AT deletion in IVS-2 have no effect. Oxygen equilibrium curves of the Hb A2 variants expressed in yeast were similar to that of wild type Hb A2. None of the three variant Hb A2 tetramers (Thr --> Ile at codon 4 (Hb deltaT4I), Ala --> Ser at codon 27 (Hb deltaA27S), and Arg --> Cys at codon 116 (Hb deltaR116C)) showed decreased heat stability compared with Hb A2, whereas the Hb deltaT4I variant showed highest instability to mechanical agitation. Co-expression in yeast of alpha-globin chain and the delta-chain variant containing a Leu --> Pro change at codon 141 yielded no identifiable tetramers, suggesting lack of assembly or severe tetramer instability. These studies show the probable cause for decreased Hb A2 for two alleles is due to defective splicing, whereas decreased protein stability, increased tetramer association with red cell membranes, increased interdisulfide bond formation of delta-chains, which inhibits assembly with alpha-chains, and/or reduced assembly is suggested for the other three alleles.     

Use of electrophoretic karyotyping and DNA-DNA hybridization in yeast identification

The behavioral disturbances of 16 nursing home patients with a history of sundowning behaviour and dementia were treated with light therapy instead of psychopharmacological medication. After the withdrawal of the before established medication a very individual, heterogenous deterioration of the symptoms was noted. A significant improvement of the behaviour was not found by light therapy in any patient. The problems in practical handling and possible consequences of further investigation are discussed.     

Circulating angiotensin II and renal sodium handling in man: a dose-response study

1. Animal studies have shown that angiotensin II has a biphasic effect on urinary sodium excretion. To examine whether this is also true in man, we studied seven salt-replete male subjects in a single-blind placebo-controlled manner. 2. While undergoing maximum diuresis, subjects were infused with 0, 1, 2, 5 or 10 ng of angiotensin II min-1 kg-1 over 80 min. Subjects were studied while seated, and stood every 20 min for urine collection. 3. Angiotensin II produced a dose-dependent antidiuretic effect. The urine flow rate, in ml/min expressed as the change from baseline with increasing dose of angiotensin, was: +3.4 +/- 1.77, -1.26 +/- 0.49 (P < 0.05), -2.75 +/- 1.23 (P < 0.05), -4.21 +/- 0.82 (P < 0.05) and -6.51 +/- 1.07 (P < 0.01). 4. In contrast, the effect of angiotensin II on sodium excretion showed a flat dose-response curve beyond 5 ng min-1 kg-1. The urinary sodium excretion, in mumol/min expressed as the change from baseline with increasing dose of angiotensin, was: 9.5 +/- 21.2, -18.9 +/- 29.6, -37.0 +/- 11.6 (P < 0.05), -67.7 +/- 19.6 (P < 0.01) and -63.8 +/- 14.3 (P < 0.01). 5. The fractional distal reabsorption of sodium, determined by using the lithium clearance technique, showed a rise with all doses of angiotensin II used and reached statistical significance with the top two doses. 6. Unlike antidiuresis, antinatriuresis after graded doses of angiotensin II in human subjects showed a flat dose-response curve beyond 5 ng min-1 kg-1. Pressor doses of angiotensin II also have a significant effect on the distal tubule in promoting sodium reabsorption.     

Prenatal exposure to methylmercury alters locomotor activity of male but not female rats

In the present study the neurotoxic effects of a low dosage (0.5 mg/kg per day) of methylmercury (MeHg) on the developing nervous system were investigated. Pregnant rats were treated with MeHg from day 7 of pregnancy to day 7 of lactation. Locomotor activity (locomotion, rearing, and motility) and spatial learning ability were tested in the offspring at 6 months of age. The expression of tyrosine hydroxylase (TH) was examined by immunohistochemistry and in situ hybridization. A significant decrease in spontaneous motility and rearing was observed only in the MeHg-treated male rats. After administration of a low dose of d-amphetamine (0.5 mg/kg) no differences could be observed between control and MeHg-treated male rats, suggesting that changes in dopaminergic transmission were involved. However, no change in TH messenger RNA expression was observed. No changes in spatial learning acquisition or memory were shown in MeHg-treated rats. Taken together, these findings show that during development a very low dosage of MeHg exerts neurotoxic effects detectable in adulthood, and that susceptibility is gender-dependent.     

Adverse environmental conditions in the respiratory and medical ICU settings

Sleep deprivation and fragmentation occurring in the hospital setting may have a negative impact on the respiratory system by decreasing respiratory muscle function and ventilatory response to CO2. Sleep deprivation in a patient with respiratory failure may, therefore, impair recovery and weaning from mechanical ventilation. We postulate that light, sound, and interruption levels in a weaning unit are major factors resulting in sleep disorders and possibly circadian rhythm disruption. As an initial test of this hypothesis, we sampled interruption levels and continuously monitored light and sound levels for a minimum of seven consecutive days in a medical ICU, a multiple bed respiratory care unit (RCU) room, a single-bed RCU room, and a private room. Light levels in all areas maintained a day-night rhythm, with peak levels dependent on window orientation and shading. Peak sound levels were extremely high in all areas representing values significantly higher than those recommended by the Environmental Protection Agency as acceptable for a hospital environment. The number of sound peaks greater than 80 decibels, which may result in sleep arousals, was especially high in the intensive and respiratory care areas, but did show a day-night rhythm in all settings. Patient interruptions tended to be erratic, leaving little time for condensed sleep. We conclude that the potential for environmentally induced sleep disruption is high in all areas, but especially high in the intensive and respiratory care areas where the negative consequences may be the most severe.     

Quantitating Fluorescence Intensity from Fluorophore: The Definition of MESF Assignment

The quantitation of fluorescence radiance may at first suggest the need to obtain the number of fluorophore that are responsible for the measured fluorescence radiance. This goal is beset by many difficulties since the fluorescence radiance depends on three parameters 1) the probability of absorbing a photon (molar extinction), 2) the number of fluorophores, and 3) the probability of radiative decay of the excited state (quantum yield). If we use the same fluorophore in the reference solution and the analyte then, to a good approximation, the molar extinction drops out from the comparison of fluorescence radiance and we are left with the comparison of fluorescence yield which is defined as the product of fluorophore concentration and the molecular quantum yield. The equality of fluorescence yields from two solutions leads to the notion of equivalent number of fluorophores in the two solutions that is the basis for assignment of MESF (Molecules of Equivalent Soluble Fluorophore) values. We discuss how MESF values are assigned to labeled microbeads and by extension to labeled antibodies, and how these assignments can lead to the estimate of the number of bound antibodies in flow cytometer measurements.