Sequential modification of membrane currents with classical conditioning

Pavlovian conditioning of the nudibranch mollusc Hermissenda crassicornis was previously shown to produce long-lasting reduction of two K+ currents measured across the Type B photoreceptor soma membrane (Alkon et al., 1982a; Alkon et al., 1985). Pavlovian conditioning of the rabbit was also shown to be followed by persistent K+ current reduction (Disterhoft et al., 1986). Here we report the first evidence that Ca2+ currents can also be modified by conditioning. The amplitude of the currents rather than their voltage-dependence remains reduced at least 1-2 d after conditioning (but not control procedures). Conditioning-induced changes of both K+ and Ca2+ currents increased as a function of training, the Ca2+ currents only changing substantially with greater than or equal to 250 trials. The later changes of the Ca2+ current may function to limit the magnitude of excitability increases due to associative learning.     

Regulation of chicken pineal arylalkylamine-N-acetyl transferase by postsynaptic alpha 2-adrenergic receptors

The present investigation sought to characterize the adrenergic inhibition of arylalkylamine-N-acetyltransferase in cultured chicken pineal glands. Arylalkylamine-N-acetyltransferase, the melatonin rhythm generating enzyme, displays daily oscillations of activity that are driven by a circadian oscillator. Norepinephrine released at sympathetic nerve endings inhibits the enzyme and appears to play a role in maintaining a circadian rhythm of melatonin release. Chicken pineal glands were isolated in organ culture and the effects of adrenergic agents on the night time peak of N-acetyltransferase activity were studied. Norepinephrine and clonidine prevented 50 to 65% of the nocturnal rise of N-acetyltransferase activity. When applied at middark, norepinephrine and clonidine caused a 50 to 65% inhibition of N-acetyltransferase activity in 2 hours. Dose-response studies indicated clonidine was 100 times more potent than norepinephrine or cirazoline at inhibiting N-acetyltransferase activity. Inhibition of N-acetyltransferase activity was also observed, at micromolar concentration with epinephrine, UK 14,304 and alpha-methylnorepinephrine but not with phenylephrine, isoproterenol or dopamine. Epinephrine and clonidine actions were antagonized by yohimbine but not by prazosin. Destruction of the presynaptic compartment by bilateral superior cervical ganglionectomy did not affect the clonidine-induced inhibition of N-acetyltransferase and its reversal by yohimbine. It is concluded that the adrenergic inhibition of N-acetyltransferase activity in chicken pineal gland probably occurs via stimulation of postsynaptic alpha 2-adrenergic receptors.     

Indirect estimation of the maximal aerobic power of a male and female population from Brussels aged 6 to 23 years. Comparison with a direct technic for measuring maximal oxygen consumption

Validation of the maximal multistage 20 m shuttle run test with 1 min steps has been compared with a stepwise load increase on a bicycle ergometer among 201 male and female subjects ranging from 14 to 30 years. A slight underestimation of VO2 max (5.2%) amounting to 2.71 ml . min-1 . kg-1 was observed for the multistage shuttle test as compared to the bicycle test (r = 0.72). The analysis of the biological values collected after exercise does not show major differences between the two tests (plasma lactate, urinary total protein and albumin, creatinine). The renal handling of plasma proteins appears to be equally disturbed under the influence of exhaustive exercise. Maximal aerobic power regularly increases with age in both sexes, being more pronounced however for boys (1.16 to 3.37 l . min-1) than for girls (1.17 to 2.43 l . min-1) from 6 to 20 years old. Boys nearly sustain 50 ml . min-1 . kg-1 throughout childhood. On the contrary, from 8 years on girls progressively reduce their VO2 max down to 37.1 ml . min-1 . kg-1 at the age of 19. The decrease is more pronounced during the 11-16 years period. The present results constitute tentative norms on 1,025 brussels male and female subjects ranging from 6 to 23 years.     

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Extensive changes in cytokeratin expression patterns in pathologically affected human gingiva

The stratified squamous epithelium of the oral gingiva and the hard palate is characterized by a tissue architecture and a cytoskeletal composition similar to, although not identical with, that of the epidermis and fundamentally different from that of the adjacent non-masticatory oral mucosa. Using immunocytochemistry with antibodies specific for individual cytokeratins, in situ hybridization and Northern blots of RNA with riboprobes specific for individual cytokeratin mRNAs, and gel electrophoresis of cytoskeletal proteins of microdissected biopsy tissue samples, we show changes in the pattern of expression of cytokeratins and their corresponding mRNAs in pathologically altered oral gingiva. Besides a frequently, although not consistently, observed increase in the number of cells producing cytokeratins 4 and 13 (which are normally found as abundant components in the sulcular epithelium and the alveolar mucosa but not in the oral gingiva) and a reduction in the number of cells producing cytokeratins 1, 10 and 11, the most extensive change was noted for cytokeratin 19, a frequent cytokeratin in diverse one-layered and complex epithelia. While in normal oral gingiva cytokeratin 19 is restricted to certain, sparsely scattered cells of --or near--the basal cell layer, probably neuroendocrine (Merkel) cells, in altered tissue of inflamed samples it can appear in larger regions of the basal cell layer(s) and, in apparently more advanced stages, also in a variable number of suprabasal cells. Specifically, our in situ hybridization experiments show that this altered suprabasal cytokeratin 19 expression is more extended at the mRNA than at the protein level, indicating that cytokeratin 19 mRNA synthesis may be a relatively early event during the alteration. These changes in cytokeratin expression under an external pathological influence are discussed in relation to other factors known to contribute to the expression of certain cytokeratins and with respect to changes occurring during dysplasia and malignant transformation of oral epithelia.     

A general method for highly selective cross-linking of unprotected polypeptides via pH-controlled modification of N-terminal alpha-amino groups

A method is described for the highly selective modification of the alpha-amino groups at the N-termini of unprotected peptides to form stable, modified peptide intermediates which can be covalently coupled to other molecules or to a solid support. Acylation with iodoacetic anhydride at pH 6.0 occurs with 90-98% selectivity for the alpha-amino group, depending on the N-terminal residue (as shown with a series of model hexapeptides containing a competing Lys residue). Although Cys residues must be protected (reversibly or irreversibly) before the anhydride reaction, there are no detectable side reactions of the alpha-amino moiety--of the reagent or of modified peptide--with the side chains of His, Met, or Lys. The reaction works well in denaturants, so that inhibitory effects of noncovalent structure can be minimized. In a second step the iodoacetyl-peptide can be reacted with a thiol group on a protein, on a solid chromatography matrix, on a spectroscopic probe, etc. This is illustrated by reaction of a series of N alpha-iodoacetyl-peptides with murine interferon-gamma, which contains a C-terminal Cys residue. Data are presented which suggest that this iodoacetic anhydride scheme is superior in selectivity for alpha-amino groups to conventional chemical approaches to cross-linking such as use of 2-iminothiolane or N-hydroxysuccinimide-activated carboxylic acid esters. The reaction is ideally suited for modifying peptide fragments, as pure species or as mixtures, derived from proteolytic or chemical fragmentation of proteins. Furthermore, polypeptides synthesized biosynthetically, for example via recombinant DNA techniques, can be cross-linked in this way.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nutrient additions by waterfowl to lakes and reservoirs: predicting their effects on productivity and water quality

Lakes and reservoirs provide water for human needs and habitat for aquatic birds. Managers of such waters may ask whether nutrients added by waterfowl degrade water quality. For lakes and reservoirs where primary productivity is limited by phosphorus (P), we developed a procedure that integrates annual P loads from waterfowl and other external sources, applies a nutrient load-response model, and determines whether waterfowl that used the lake or reservoir degraded water quality. Annual P loading by waterfowl can be derived from a figure in this report, using the days per year that each kind spent on any lake or reservoir. In our example, over 6500 Canada geese (Branta canadensis) and 4200 ducks (mostly mallards, Anas platyrhynchos) added 4462 kg of carbon (C), 280 kg of nitrogen (N), and 88 kg of P y^–1 to Wintergreen Lake in southwestern Michigan, mostly during their migration. These amounts were 69% of all C, 27% of all N, and 70% of all P that entered the lake from external sources. Loads from all external sources totaled 840 mg P m^–2 y^–1. Application of a nutrient load-response model to this concentration, the hydraulic load (0.25 m y^–1), and the water residence time (9.7 y) of Wintergreen Lake yielded an average annual concentration of total P in the lake of 818 mg m^–3 that classified the lake as hypertrophic. This trophic classification agreed with independent measures of primary productivity, chlorophyll-a, total P, total N, and Secchi disk transparency made in Wintergreen Lake. Our procedure showed that waterfowl caused low water quality in Wintergreen Lake.Contribution 824 of the National Fisheries Research Center-Great Lakes, 1451 Green Road, Ann Arbor, Michigan 48105, U.S.A. and 722 of the Kellogg Biological Station, Michigan State University.     

Tumor Necrosis Factor-α (TNF-α), Interferon-γ, and Interleukin-6 but Not TNF-β Induce Differentiation of Neuroblastoma Cells: The Role of Nitric Oxide

Abstract: Tumor necrosis factor-a (TNF-α), interferon-γ (IFN-7), and interleukin-6 (IL-6), but not TNF-β, can induce the in vitro differentiation of the neuroblastoma cell line N103 in a dose-dependent manner. Differentiation of N103 was accompanied by the arrest of cell growth and neurite formation. The induction of neuroblastoma cell differentiation by TNF-α and IFN-γ can be specifically inhibited by a nitric oxide (NO) synthase inhibitor, l -N^G-monomethylarginine. In contrast, the differentiation of N103 cells by IL-6 was not affected by l -N^G-monomethylarginine. These results indicate that TNF-α and IFN-γ, but not IL-6, induce the differentiation of neuroblastoma cells via NO. This is confirmed by the finding that the culture super- natants of N103 cells induced by TNF-α and IFN-γ, but not that by IL-6, contained high levels of NO₂⁻, the production of which was inhibited by l - N ^G-monomethylarginine. Furthermore, the differentiation of N103 cells can be induced directly in a dose-dependent manner by the addition of nitroprusside, a generator of NO, into the culture medium. These data therefore indicate that NO may be an important mediator in the induction of neuronal cell differentiation by certain cytokines such as TNF-α and IFN-γ and that neuronal cells, in addition to the macrophagelike brain cells, can be induced by immunological stimuli to produce large quantities of NO.     

Reconstruction of ionic currents in a molluscan photoreceptor.

Two-microelectrode voltage-clamp measurements were made to determine the kinetics and voltage dependence of ionic currents across the soma membrane of the Hermissenda type B photoreceptor. The voltage-dependent outward potassium currents, IA and ICa(2+)-K+, the inward voltage-dependent calcium current, ICa2+ and the light-induced current, IIgt, were then described with Hodgkin-Huxley-type equations. The fast-activating and inactivating potassium current, IA, was described by the equation; IA(t) = gA(max)(ma infinity[1-exp(-t/tau ma)])3 x (ha infinity [1-exp(-t/tau ha)] + exp(-t/tau ha)) (Vm-EK), where the parameters ma infinity, ha infinity, tau ma, and tau ha are functions of membrane potential, Vm, and ma infinity and ha infinity are steady-state activation and inactivation parameters. Similarly, the calcium-dependent outward potassium current, ICa(2+)-K+, was described by the equation, ICa(2+)-K+ (t) = gc(max)(mc infinity(VC)(1-exp[-t/tau mc (VC)]))pc (hc infinity(VC) [1-exp(-t/tau hc)] + exp(-t/tau hc(VC)])pc(VC-EK). In high external potassium, ICa(2+)-K+ could be measured in approximate isolation from other currents as a voltage-dependent inward tail current following a depolarizing command pulse from a holding potential of -60 mV. A voltage-dependent inward calcium current across the type B soma membrane, ICa2+, activated rapidly, showed little inactivation, and was described by the equation: ICa2+ = gCa(max) [1 + exp](-Vm-5)/7]-1 (Vm-ECa), where gCa(max) was 0.5 microS. The light-induced current with both fast and slow phases was described by: IIgt(t) = IIgt1 + IIgt2 + IIgt3, IIgti = gIgti [1-exp(- ton/tau mi)] exp(-ton/tau hi)(Vm-EIgti) (i = 1, 2).(ABSTRACT TRUNCATED AT 250 WORDS)