Mechanism of Cl- sensitivity in internal ion receptors of the leech; an inward current gated off by Cl- in the nephridial nerve cells

Summary The nephridial nerve cells of the leech, Hirudo medicinalis, 34 sensory cells, each associated with one nephridium, are sensitive to changes in extracellular Cl^- concentration, an important factor in ion homeostasis. Using single-electrode current- and voltage clamp and ion substitution techniques, the specificity and mechanism of Cl^- sensitivity of the nephridial nerve cell was studied in isolated preparations. Increase of the normally low external Cl^- concentration leads to immediate and sustained hyperpolarization, decrease of the frequency of bursts and decrease of membrane conductance. The response is halogen specific: Cl^- can be replaced by Br^–, but not by organic mono- or divalent anions or inorganic divalent anions.At physiological Cl^- concentrations (36mM extra-cellular Cl^-), the nephridial nerve cell has a high resting conductance for Cl^- and the membrane potential is governed by Cl^-. In high extracellular Cl^- concentrations (110–130 mM), membrane conductance is low, most likely due to the gating off of Cl^- channels. Under these conditions, membrane potential is dominated by the K⁺ distribution and the nephridial nerve cell hyperpolarizes towards E_K.Abbreviations NNC nephridial nerve cell - V _m membrane potential - E _Cl(k) equilibrium potential for Cl (K) - IV-curve current-voltage relationship     

Parathyroid hormone-induced changes of the brush border topography and cytoskeleton in cultured renal proximal tubular cells

Summary In order to examine the possibility of parathyroid hormone-mediated ultrastructural rearrangements in target epithelium, isolated canine renal proximal tubular cells were grown on a collagen-coated semipermeable membrane in a defined medium. Scanning and transmission electron microscopy of these monolayers revealed abundant microvilli. Exposure of the proximal tubular cells to parathyroid hormone resulted in a biphasic changes involving: (1) dramatic shortening and rarefaction of microvilli within 1 min; and (2) recovery of microvillar topography after 5 min. A similar shortening of microvilli was observed following exposure to ionomycin, whereas incubation with cyclic AMP resulted in an elongation of microvilli. Parathyroid hormone stimulated cyclic AMP production and increased cytoplasmic free calcium concentration in cultured proximal tubular cells. Pretreatment of cells with a calmodulin inhibitor abolished the effect of parathyroid hormone on brush border topography. Shortening of microvilli was associated with a disappearance of microvillar core filaments. Staining of F-actin with fluoresceinphalloidin showed that parathyroid hormone resulted in fragmentation of stress fibers. It is concluded that parathyroid hormoneinduced cell activation involves cytoplasmic-free calcium, calmodulin, and the cytoskeleton.     

Voltage-dependent channel formation by rods of helical polypeptides

Summary The voltage-dependence of channel formation by alamethicin and its natural analogues can be described by a dipole flip-flop gating model, based on electric field-induced transbilayer orientational movements of single molecules. These field-induced changes in orientation result from the large permanent dipole moment of alamethicin, which adopts -helical conformation in hydrophobic medium. It was, therefore, supposed that the only structural requirement for voltage-dependent formation of alamethicin-type channels might be a rigid lipophilic helical segment of minimum length.In order to test this hypothesis we synthesized a family of lipophilic polypeptides—Boc-(Ala-Aib-Ala-Aib-Ala) _n -OMe,n=1–4—which adopt -helical conformation forn=2–4 and studied their interaction with planar lipid bilayers. Surprisingly, despite their large difference in chain length, all four polypeptides showed qualitatively similar behavior. At low field strength of the membrane electric field these polypeptides induce a significant, almost voltage-independent increase of the bilayer conductivity. At high field strength, however, a strongly voltage-dependent conductance increase occurs similar to that observed with alamethicin. It results from the opening of a multitude of ion translocating channels within the membrane phase.The steady-state voltage-dependent conductance depends on the 8^th–9^th power of polypeptide concentration and involves the transfer of 4–5 formal elementary charges. From the power dependences on polypeptide concentration and applied voltage of the time constants in voltage-jump current-relaxation experiments, it is concluded that channels could be formed from preexisting dodecamer aggregates by the simultaneous reorientation of six formal elementary charges. Channels exhibit large conductance values of several nS, which become larger towards shorter polypeptide chain length. A mean channel diameter of 19 Å is estimated corresponding roughly to the lumen diameter of a barrel comprised of 10 -helical staves. Similar to experiments with the N-terminal Boc-derivative of alamethicin we did not observe the burst sequence of nonintegral conductance steps typical of natural (N-terminal Ac-Aib)-alamethicin. Saturation in current/voltage curves as well as current inactivation in voltage-jump current-relaxation experiments are found. This may be understood by assuming that channels are generated as dodecamers but, while reaching the steady state, reduce their size to that of an octamer or nonamer. We conclude that the overall behavior of these synthetic polypeptides is very similar to that of alamethicin. They exhibit the same concentration and voltage-dependences but lack the stabilizing principle of resolved channel states characteristic of alamethicin.     

Complementation analysis of the inorganic carbon concentrating mechanism of Chlamydomonas reinhardtii

Summary Six independently isolated mutants of Chlamydomonas reinhardtii that require elevated CO₂ for photoautotrophic growth were tested by complementation analysis. These mutants are likely to be defective in some aspect of the algal concentrating mechanism for inorganic carbon as they exhibit CO₂ fixation and inorganic carbon accumulation properties different from the wild-type. Four of the six mutants defined a single complementation group and appear to be defective in an intracellular carbonic anhydrase. The other two mutations represent two additional complementation groups.Abbreviations HS high salt medium which has 13 mM phosphate at pH 6.8 - HSA high salt plus 36 mM acetate medium - YA high salt medium with 4 g yeast extract per L and 36mM acetate - Arg arginine - cia^- CO₂ accumulation mutants that cannot grow on low CO₂ - C_i inorganic carbon (CO₂+HCO _- ³ ) - CA carbonic anhydrase - mt mating type Supported in part by the McKnight Foundation and by NSF grant PCM 8005917 and published as journal article 11924 from the Michigan State Agriculatural Experiment Station     

烟草愈伤组织器官发生过程中外源激素的作用

近年来,利用愈伤组织系统在与器官发生有关的形态学、生理学和生物化学研究方面已经取得了一些进展(Thorpe 1980,刘涤 1983)。对烟草愈伤组织系统的研究明确了外源激素对器官发生类型的调节作用(Skoog 1971,Engelke等1973,刘涤等1980)。但是,这些研究仅考虑到外源激素的作用,而对器官发生过程中的其它变化并不了解。最近,Kamada和Harada(1981)比较了胡萝卜体细胞胚发育过程中内源IAA和ABA含量的变化,Noma等(1982)证实形成胚的和不形成胚的细胞间GA_3含     

Rat hypothalamic GRF elicits its biologic action in GH3 cells by interaction with VIP- preferring receptor site(s)

GH3 cells can be used effectively to study the in vitro mechanism of action of GRF. In these cells, there is a time and concentration-dependent release of cAMP into the medium. Rat hypothalamic GRF, (rGRF) is 7 to 10 fold more active than human hypothalamic GRF (hGRF). VIP, a peptide which is structurally homologous to GRF, stimulates cAMP efflux in GH3 cells, with a higher affinity than hGRF or rGRF. We propose that in contradistinction to the normal rat pituitary, the stimulation of cAMP release by GRF in GH3 cells occurs via activation of VIP-preferring receptors and that GRF (rGRF in particular) behaves as a partial VIP agonist.     

Ultrastructural analysis of pathologic lesions in sterol-deficient Nippostrongylus brasiliensis larvae

A variety of cellular lesions were manifested by the free-living larval stages of Nippostrongylus brasiliensis cultured axenically in medium lacking cholesterol. Pathologic changes developed rapidly and were most apparent in intestinal cells which displayed generalized degradation of membranous organelles. Mitochondria, endoplasmic reticulum, and Golgi complexes became disassociated and vacuolated. Autophagosomes appeared within intestinal cells and contained a wide variety of cellular components. By the 5th day gross vacuolization and degeneration of intestinal cells occurred and the hypodermis and lateral cords displayed lysed cytoplasmic regions. The latter structures are concerned with synthesis of cuticle and their degeneration correlates with the suppression of molting and the abnormal molts that occurred.     

Evidence for titratable gating charges controlling the voltage dependence of the outer mitochondrial membrane channel,VDAC

Summary A voltage-dependent anion-selective channel, VDAC, is found in outer mitochondrial membranes. VDAC's conductance is known to decrease as the transmembrane voltage is increased in either the positive or negative direction. Charged groups on the channel may be responsible for this voltage dependence by allowing the channel to respond to an applied electric field. If so, then neutralization of these charges would eliminate the voltage dependence. Channels in planar lipid bilayers which behaved normally at pH 6 lost much of their voltage dependence at high pH. Raising the pH reduced the steepness of the voltage dependence and raised the voltage needed to close half the channels. In contrast, the energy difference between the open and closed state in the absence of a field was changed very little by the elevated pH. The groups being titrated had an apparent pK of 10.6. From the pK and chemical modification, lysine epsilon amino groups are the most likely candidates responsible for VDAC's ability to respond to an applied electric field.     

Separation of Neuropeptides by HPLC: Evaluation of Different Supports, with Analytical and Preparative Applications to Human and Porcine Neurophysins, β-Lipotropin, Adrenocorticotropic Hormone, and β-Endorphin

The separation of 38 hypophyseal or hypothalamic peptides and proteins by reversed-phase HPLC is described. The efficacy of several supports is discussed: large-pore reversed-phase silica supports turned out to be most effective in the separation of neuropeptides and proteins. The use of high sodium phosphate and phosphoric acid in the eluent resulted in high yields and better separation. On the basis of this procedure, beta-lipotropin, beta-endorphin, and adrenocorticotropic hormone were prepared from porcine pituitary glands, and human neurophysins from human pituitary posterior lobes. Moreover, this system is used for the analysis of the perifusate of pituitary posterior lobes, showing the release of micro-heterogeneous neurophysins.     

The biological relevance of HPLC-purified vasoactive intestinal polypeptide monoiodinated at tyrosine 10 or tyrosine 22

Three major forms of monoiodinated VIP (M125I-VIP) were isolated after chloramine-T iodination and HPLC purification. The iodinated tyrosine residue was located in each form of M125I-VIP using arginase C and trypsin digestion for obtaining defined fragments containing only one tyrosine residue. The HPLC isolated iodinated fragments thus obtained were used for HPLC comigration studies with iodinated synthetic C and N terminal VIP fragments and for amino acid analysis. The first two eluting peaks 1 and 2 are (M125I-Tyr10-VIP); peak 1 has an oxidized methionine; peak 3 is a (M125I-Tyr22-VIP) which also has an oxidized methionine. A reduced counterpart of peak 3 named peak 4 was isolated by further HPLC analysis. The ability of the different species of M125I-VIP to stimulate adenosine cyclic 3',5'-phosphate (cAMP) production in transformed colonic cells in culture (HT-29) was compared to that of native VIP. The mean potencies of the M125I-VIP species expressed as a percentage relative to the potency of native VIP were, peak (1): 0.98; (2): 0.84; (3): 1.38; (4): 1.48, in the range of concentrations tested (2-60 pM). The M125I-Tyr22-VIP are significantly more active than native VIP (P less than 0.01). Oxidation of methionine or iodination of tyrosine 10 does not significantly modify the biological activity of VIP. We conclude that iodination of Tyr-22 located in the apolar helical COOH-terminal of VIP increases the effectiveness of VIP interaction with its receptors. Thus the tyrosyl residue and the localized hydrophobic features of VIP are critically involved in the function of this neurotransmitter.