Fluorescence of squid axon membrane labelled with hydrophobic probes

Summary Extrinsic fluorescence changes in squid giant axons were examined under a variety of experimental conditions using 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and other fluorescent probes. Measurements of the degree of polarization of the fluorescent light (with the axis of the polarizer parallel to the longitudinal axis of the axon) indicated that the class of the TNS molecules in the axon membrane which participate in production of fluorescence signals have a definite orientation with their absorption and emission oscillators directed parallel to the long axis of the axon. Rectangular depolarizing voltage pulses produced a transient decrease in the fluorescent intensity, of which the early component is correlated tentatively with the rise in the membrane conductance. In response to hyperpolarizing pulses, there was an increase in fluorescence intensity which may be explained in terms of increased incorporation of TNS into the ordered structure in the membrane. Hyperpolarizing responses in KCl depolarized axons were accompanied by a change in fluorescent intensity. Tetrodotoxin appeared to suppress the initial component of the fluorescence signal produced by depolarizing clamping pulses. The technique for detecting these fluorescence changes and the physico-chemical properties of TNS are described in some detail.     

An approach to the study of intracellular proteins related to the excitability of the squid giant axon

The technique for covalently labeling proteins with 125I-labelled Bolton-Hunter reagent was used to determine the quantities of proteins released from the axoplasmic side of the squid axon membrane. The reagent could be introduced into the interior of the axon by the technique of intracellular perfusion, the radioiodination reaction being carried out in situ. Alternatively, the reaction could be carried out in vitro, i.e., by mixing the reagent with samples of proteins dissolved in the intracellular perfusion fluid collected from the axon. This technique was found to be sensitive enough to permit analysis of a large number of protein samples collected from a single axon. By the method of sodium dodecyl sulfate polyacrylamide gel electrophoresis, it was found that proteins of approx. 56 000 daltons were released into the perfusate when a solution of potassium chloride or potassium bromide was introduced into the interior of an axon. Suppression of axonal excitability was associated with this release of proteins. The significance of these findings in relation to the structure and function of the axon is discussed.     

BIOCHEMICAL STUDIES OF NERVE EXCITABILITY: THE USE OF PROTEIN MODIFYING REAGENTS FOR CHARACTERIZING SITES INVOLVED IN NERVE EXCITATION

A variety of protein modifying reagents were applied to crab nerves extracellularly while monitoring the action potentials. These reagents were also applied to squid giant axons intracellularly, while monitoring the action potentials, and for some compounds the membrane resistance and resting membrane potential. The effects of modifying sulfhydryl, imidazole, amino, and methionine groups on the nerve excitability were examined. These studies revealed that imidazole and sulfhydryl groups are present on the axoplasmic side of the axolemma and that chemical modification of these groups leads to block of nerve conduction. When various reagents which modify amino groups were applied either from the inside of squid axons or to the outside of crab nerves, the excitability was not usually affected. The suppression of excitability due to the effect of some of these amino reagents could be attributed to the modification of groups other than amino groups. The hydrolytic action of cyanogen bromide was found to be effective in bringing about conduction block, suggesting that there exist accessible methionine groups in protein molecules which must remain intact in order to maintain nerve conduction.     

Adaptive changes in translation initiation activities for rat pancreatic protein synthesis with feeding of a high-protein diet

We have previously demonstrated that dietary protein induced pancreatic hypergrowth in pancreaticobiliary diverted (PBD) rats. Dietary protein and dietary amino acids stimulate protein synthesis by regulating translation initiation in the rat skeletal muscle and liver. The aim of the present study was to determine whether feeding a high-protein diet induces activation of translation initiation for protein synthesis in the rat pancreas. In PBD rats in which the bile-pancreatic juice was surgically diverted to the upper ileum for 11-13 days, pancreatic dry weight and protein content were doubled compared with those in sham rats and further increased with feeding of a high-protein diet (60% casein diet) for 2 days. These pancreatic growth parameters were maintained at high levels for the next 5 days and were much higher than those of sham rats fed a high-protein diet. In both sham and PBD rats, feeding of a high-protein diet for 2 days induced phosphorylation of eukaryotic initiation factor 4E-binding protein 1 and 70-kDa ribosomal protein S6 kinase, indicating the activation of the initiation phase of translation for pancreatic protein synthesis. However, this increased phosphorylation returned to normal levels on Day 7 in PBD but not in sham rats. We concluded that feeding a high-protein diet induced pancreatic growth with increases in the translation initiation activities for pancreatic protein synthesis within 2 days and that prolonged feeding of a high-protein diet changed the initiation activities differently in sham and PBD rats.     

A family of mammalian E3 ubiquitin ligases that contain the UBR box motif and recognize N-degrons

A subset of proteins targeted by the N-end rule pathway bear degradation signals called N-degrons, whose determinants include destabilizing N-terminal residues. Our previous work identified mouse UBR1 and UBR2 as E3 ubiquitin ligases that recognize N-degrons. Such E3s are called N-recognins. We report here that while double-mutant UBR1(-/-) UBR2(-/-) mice die as early embryos, the rescued UBR1(-/-) UBR2(-/-) fibroblasts still retain the N-end rule pathway, albeit of lower activity than that of wild-type fibroblasts. An affinity assay for proteins that bind to destabilizing N-terminal residues has identified, in addition to UBR1 and UBR2, a huge (570 kDa) mouse protein, termed UBR4, and also the 300-kDa UBR5, a previously characterized mammalian E3 known as EDD/hHYD. UBR1, UBR2, UBR4, and UBR5 shared a approximately 70-amino-acid zinc finger-like domain termed the UBR box. The mammalian genome encodes at least seven UBR box-containing proteins, which we propose to call UBR1 to UBR7. UBR1(-/-) UBR2(-/-) fibroblasts that have been made deficient in UBR4 as well (through RNA interference) were significantly impaired in the degradation of N-end rule substrates such as the Sindbis virus RNA polymerase nsP4 (bearing N-terminal Tyr) and the human immunodeficiency virus type 1 integrase (bearing N-terminal Phe). Our results establish the UBR box family as a unique class of E3 proteins that recognize N-degrons or structurally related determinants for ubiquitin-dependent proteolysis and perhaps other processes as well.     

Chronic hypertriglyceridemia in young watanabe heritable hyperlipidemic rabbits impairs endothelial and medial smooth muscle function

Several studies have suggested that hypertriglyceridemia is a common risk factor for coronary heart disease. Although increasing serum levels of triglyceride correlate with hypercoagulability, little is known about the contribution of hypertriglyceridemia to vascular function. We successfully segregated two lines of rabbits with genetically-determined severely high (TGH; 2764 +/- 413 mg/dl) and moderately high (TGL; 191 +/- 12 mg/dl) levels of triglyceride, but with comparable levels of total cholesterol, from Watanabe heritable hyperlipidemic rabbits. To determine whether hypertriglyceridemia was involved in alterations of vascular function, we conducted isometric tension studies and analyzed protein expression on thoracic aortic rings isolated from young (3-4 month) TGH, TGL and Japanese White rabbit (JW). No difference in percentage of plaque area in the thoracic aorta was found between TGH and TGL. Relaxing responses, evoked by sodium nitroprusside were similar in JW, TGL and TGH, but endothelium-dependent relaxation to acetylcholine was impaired in TGH compared with JW or TGL (maximal relaxation in JW; 83.5 +/- 2.7%, TGL; 79.9 +/- 5.3%, TGH; 59.1 +/- 5.7%, p<0.05). Relaxation to A23187 was also attenuated in TGH compared with JW, but not significantly different between TGL and JW. Endothelium-independent relaxation elicited by isoproterenol in TGH was significantly decreased compared with JW or TGL (maximal relaxation in JW; 95.2 +/- 2.6% TGL; 91.0 +/- 4.9%, TGH; 75.1 +/- 5.2%, p<0.05). Protein expression of angiotensin II type-1 receptor was increased in TGH and that of nitric oxide synthases-3 was attenuated in TGH compared with TGL. This is the first study showing that endothelium-dependent and -independent vascular relaxation under the condition of combined hyperlipidemia was severely impaired as compared to that under only hypercholesterolemia. These results suggest that hypertriglyceridemia aggravates functional impairment induced by hypercholesterolemia in endothelial and smooth muscle cells.     

Chaperone-Independent Peripheral Quality Control of CFTR by RFFL E3 Ligase

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1-Benzyl-1,2,3,4-Tetrahydroisoquinoline as a Parkinsonism-Inducing Agent: A Novel Endogenous Amine in Mouse Brain and Parkinsonian CSF

Abstract: 1-Benzyl-1,2,3,4-tetrahydroisoquinoline (1BnTIQ) was detected as a novel endogenous amine in mouse brain and parkinsonian CSF by using the gas chromatography-selected ion-monitoring method. The level of 1BnTIQ was very high in CSF of some parkinsonian patients compared with that of controls with other neurological diseases, the mean value being three times higher (parkinsonians: 1.17 ± 0.35 ng/ml of CSF, n = 18; vs. controls: 0.40 ± 0.10 ng/ml of CSF, n = 11; mean ± SEM, not significantly different). The pole test, a toxicological examination to evaluate behavior abnormalities related to Parkinson's disease, was used to examine the pharmacological effect of 1BnTIQ in mice. Repeated administration of 1BnTIQ induced behavior abnormalities, which pretreatment with 1-methyl-1,2,3,4-tetrahydroisoquinoline could prevent. We suggest that 1BnTIQ may be related to the idiopathic Parkinson's disease.     

Electrophysiological Studies of the Antrum Muscle Fibers of the Guinea Pig Stomach

The membrane potentials of single smooth muscle fibers of various regions of the stomach were measured, and do not differ from those measured in intestinal muscle. Spontaneous slow waves with superimposed spikes could be recorded from the longitudinal and circular muscle of the antrum. The development of tension was preceded by spikes but often tension appeared only when the slow waves were generated. Contracture in high K solution developed at a critical membrane potential of -42 mv. MnCl₂ blocked the spike generation, then lowered the amplitude of the slow wave. On the other hand, withdrawal of Na⁺, or addition of atropine and tetrodotoxin inhibited the generation of most of the slow waves but a spike could still be elicited by electrical stimulation. Prostigmine enhanced and prolonged the slow wave; acetylcholine depolarized the membrane without change in the frequency of the slow waves. Chronaxie for the spike generation in the longitudinal muscle of the antrum was 30 msec and conduction velocity was 1.2 cm/sec. The time constant of the foot of the propagated spike was 28 msec. The space constants measured from the longitudinal and circular muscles of the antrum were 1.1 mm and 1.4 mm, respectively.