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.     

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.     

Leptin deficiency enhances myocardial necrosis and lethality in a murine model of viral myocarditis

To investigate the role of leptin in the development of viral myocarditis and cardiac necrosis, we used a murine model of viral myocarditis. We intraperitoneally injected encephalomyocarditis virus (500 plaque-forming units/mouse) for wild type C57 BL/6 mice (WT) and leptin-deficient ob/ob mice (OB) (n = 20 for each). Ten-day survival rate was 25% in OB, whereas it was 95% in WT. Heart weights on day 10 were significantly elevated in OB compared with those in WT (107.2 +/- 9.4 vs. 96.6 +/- 7.9 mg, n = 4 for each). Thymus weights were significantly diminished in OB compared with those in WT on days 6 and 10. Histological score (grade 1 to 4 according to the size of involved area) for myocardial necrosis were significantly higher in OB than in WT (1.5 +/- 0.5 vs. 0.8 +/- 0.5, n = 4 for each). On day 4, viral titer in hearts was significantly elevated in OB compared with that in WT (3.3 +/- 0.5 vs. 1.9 +/- 0.2 TCID50/mg, n = 3 for each). Comparative expression of TNF-alpha mRNA in hearts from OB were significantly increased compared with those in WT on day 7 (n = 3 for each). Natural killer cell activities in spleens from OB were significantly lower than from WT on day 4 (27 +/- 5 vs. 42 +/- 8%, n = 4 for each). Thus, leptin deficiency could enhance severity of myocardial necrosis and mortality due to viral myocarditis.     

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.     

Population stability in relation to resource availability in an introduced population of an herbivorous lady beetle

Mechanisms responsible for population stability in relation to resource availability were studied in an introduced herbivorous lady beetle,Epilachna niponica. The introduced population was relatively constant over a seven-year study period. Egg density was related to the variation in host-plant abundance in different years, and was highly stabilized during the period from reproductive adult to egg stage. Two density-dependent processes were identified in the reproductive season: (1) density-dependent reduction in fecundity and (2) density-dependent increase in female mortality and/or emigration, all of which operated early in the season. As a result, temporal variability in cumulative egg density was greatly reduced by mid-May, by which time approximately40% of total eggs were laid. A field cage experiment demonstrated that egg-laying of individual females was largely limited by resource availability even at low levels of leaf herbivory. Since movement activity of ovipositing females increased in a density-dependent manner, inter-plant movement is more likely to cause density-dependent reduction in fecundity and female loss, due to enhanced energy expendiditure. The introduced population was less stable than the source population, probably because of decreased inter-plant movement of females and the unlikelihood of egg resorption, both of which contribute significantly to the temporal stability ofE. niponica population densities.     

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.     

Demographic attributes of an introduced herbivorous lady beetle

Demographic attributes of the adults of an introduced herbivorous lady beetleEpilachna niponica (Coleoptera: Coccinellidae) were investigated from 1975 to 1981 in the Botanical Garden of Kyoto University. Population growth rate varied from 4.8 to 16.8 throughout the study period. Fecundity and mortality in the late larval period contributed most to annual changes in the population growth rate. Population growth rate was negatively correlated with the density of overwintering adults. Adult survival from emergence to the reproductive season, which varied from 0.03 to 0.36 throughout the study, was almost completely determined by survival during the pre-hibernation period. Adult survival to the preproductive season changed in a size- and sex-dependent manner. Larger adults survived better than smaller individuals; male-biased mortality occurred from adult emergence to the reproductive age. Severe intraspecific competition among late instar larvae due to host plant defoliation produced a higher proportion of small-sized adults, resulting in lower adult survival to hibernation. The introduced population had a higher population growth rate and a lower adult survival to the reproductive season than the source population.     

The effect of a novel leukotriene C4/D4 antagonist, BAY-x-7195, on experimental allergic reaction

The effects of a novel leukotriene (LT) C₄/D₄ antagonist, BAY-x-7195 on experimental allergic reactions in airway and skin were compared to that of ONO-1078. BAY-x-7195 showed an antagonistic action to LTD₄-induced bronchoconstriction in vitro and in vivo. In in vitro experiments, BAY-x-7195 inhibited LTD₄-induced contraction of isolated guinea pig tracheal muscle (pA2=8.03). BAY-x-7195 at doses of 3 – 30 mg/kg clearly inhibited LTD₄-induced increases in respiratory resistance (Rrs) in guinea pigs. In contrast, BAY-x-7195 inhibited significantly U-46619-induced increases in Rrs at a dose of 30 mg/kg in guinea pigs. BAY-x-7195 at doses of 3 — 30 mg/kg inhibited the aerosolized antigen-induced biphasic increase in Rrs in guinea pigs. Moreover BAY-x-7195 inhibited repeated aeroantigen-induced airway hyperreactivity in guinea pigs. In mice, aeroantigen-induced airway inflammation were clearly inhibited by BAY-x-7195. These results show the efficacy of BAY-x-7195 against the antigen-induced increase in airway resistance and antigen-induced airway hyperreactivity in guinea pigs and mice, probably due to anti-LTD₄ antagonistic action and the inhibition of antigen-induced airway inflammation.