Thermoregulatory Activity of Dermorphin Fragments

We studied the effect of C-terminal truncation of the dermorphin (DM) molecule and analogs of its N-terminal tetrapeptide, [DOrn²]-DM_1–4, [DArg²]-DM_1–4, [DAla⁴]-DM_1–4, [DArg², DAla⁴]-DM_1–4, Arg-DM_1–4, Arg-[DArg²]-DM_1–4, Arg-[DAla⁴]-DM_1–4, and Arg-[DArg², DAla⁴]-DM_1–4, on the functional status of the thermoregulation system in rats at different ambient temperatures. For the first time, we demonstrate that the N-terminal tetrapeptide is the minimal fragment with the hypothermic effect. Only the N-terminal octapeptide exerted the vasomotor effect. Amino acid substitutions in the tetrapeptide affected its hypothermic effect. [DArg²]-DM_1–4 and [DArg², DAla⁴]-DM_1–4 had the greatest effect. Addition of Arg to the N-terminus of DM_1–4 analogs changed their thermoregulatory activity. The greatest thermoregulatory effect was observed for Arg-[DArg²]-DM_1–4 and Arg-[DArg², DAla⁴]-DM_1–4.     

Study of thermoregulatory activity of a fragment of natural dermorphin precursor Arg-Dermorphin and its analogs

We studied the effect of a fragment of natural dermorphin (DM) precursor Arg-DM and its analogs (Pro-DM, 4Amino-Pro-DM, Met-DM, Kre-DM, Arg-[DArg²]-DM, and Arg-[DAla⁴]-DM) after intraperitoneal administration on the functional status of the thermoregulation system in rats. The obtained data demonstrated that the hypothermic and vasomotor effects of Arg-DM were temperature-dependent and had the same pattern as DM (Emelyanova et al., 1996). At termoneutral and room temperatures, the peptide induced a two-phase vascular response. The first phase, vasodilation, was twice as strong as for DM and was not removed by naloxone preadministration. The second phase, vasoconstriction, was blocked by naloxone. Replacement of Arg with 4Amino-Pro, Met, and Kre as well as DAla² to DArg² or Gly⁴ to DAla⁴ replacements in the Arg-DM molecule affected the thermoregulatory activity of the peptide. For instance, only the vasodilatory response was observed for Arg-[DAla²]-DM and Arg-[DAla⁴]-DM while only the vasoconstrictive response was observed for 4Amino-Pro-DM.Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 1, 2005, pp. 47–54.Original Russian Text Copyright © 2005 by Emelyanova, Guzevatykh, Goryacheva, Andreeva, Alfeeva, Myasoedov.     

Effect of Dermorphin Analogs on Thermoregulation of Rats under Various Thermal Conditions

We studied the influence of dermorphin (dermorphin) analogs with stereochemical modification of the amino acid residue proline in position 6 (Pro6), Tyr-D-Ala-Phe-Gly-Tyr-Hyp-Ser-NH₂, Tyr-D-Ala-Phe-Gly-Tyr-[D-Pro]-Ser-NH₂, Tyr-D-Ala-Phe-Gly-Tyr-[dehydro-Pro]-Ser-NH₂, and Tyr-D-Ala-Phe-Gly-Tyr-[D-dehydro-Pro]-Ser-NH₂, after their intraperitoneal injection at 0.5 mg/kg dose in the cold (4–7°C), thermoneutral (27–28°C), and hot (31–33°C) environment. Stereochemical modifications of amino acid residue Pro6 proved to induce specific changes in the thermoregulatory effect of the peptide. Substitution of DPro6 for Pro6 has the most dramatic consequences: it considerably attenuates the thermoregulatory effect of dermorphin in the cold environment, cancels it in the hot environment, and inverts the dermorphin-specific thermoregulatory response in thermoneutral conditions. The data obtained indicate the important role of Pro6 residue in realization of this physiological activity of dermorphins.     

Comparative analysis of analgesic activities of dermorphin, [DPro<Superscript>6</Superscript>]-dermorphin,and their C-terminal tripeptides

Analgesic activities of dermorphin (DM), [DPro⁶]-DM, and their C-terminal tripeptides were comparatively studied. Analgesic activity was evaluated in tail flick, hot plate, tail pinch, formalin, and acetic acid writhing tests describing different levels of organization of pain sensitivity. Intraperitoneal administration of the peptides decreased the pain threshold in all these tests. The C-terminal tripeptides DM_5–7 and [DPro⁶]-DM_5–7 demonstrated analgesics activity comparable or sometimes higher than that of the full-length molecules. The effect of DM, [DPro⁶]-DM, and C-terminals fragments DM_5–7 and [DPro⁶]-DM_5–7 decreased after co-administration with naloxone, which points to the opioid nature of analgesic activity of the peptides.     

Simultaneous determination of 6β- and 6α-hydroxycortisols and 6β-hydroxycortisone in human urine by stable isotope dilution mass spectrometry

A capillary gas chromatographic–mass spectrometric method for the simultaneous determination of 6β-hydroxycortisol (6β-OHF, 6β,11β,17α,21-tetrahydroxypregn-4-ene-3,20-dione), 6α-hydroxycortisol (6α-OHF, 6α,11β,17α,21-tetrahydroxypregn-4-ene-3,20-dione) and 6β-hydroxycortisone (6β-OHE, 6β,17α,21-trihydroxypregn-4-ene-3,11,20-trione) in human urine is described. Deuterium-labelled compounds, 6β-[1,1,19,19,19-²H₅]OHF (6β-OHF-d₅), 6α-[1,1,19,19,19-²H₅]OHF (6α-OHF-d₅) and 6β-[1,1,19,19,19-²H₅]OHE (6β-OHE-d₅) were used as internal standards. Quantitation was carried out by selected-ion monitoring of the characteristic fragment ions ([M-31]⁺) of the methoxime–trimethylsilyl (MO–TMS) derivatives of 6β-OHF, 6α-OHF and 6β-OHE. The sensitivity, specificity, precision and accuracy of the method were demonstrated to be satisfactory for measuring 6β-OHF, 6α-OHF and 6β-OHE in human urine.     

Investigation of the effect of intracellular calcium on the cAMP-mediated increase in the calcium current

The experiments were conducted on intracellularly persfused neurons of the molluskHelix pomatia, in which a serotonin (5-HT)-induced increase in the calcium current (I_Ca), mediated by cAMP, is observed. It was established that desensitization of the cell to the action of 5-HT is due to an increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i). At [Ca²⁺]_i=10^–7 and 10^–6 M, the effect of 5-HT constituted 60 and 32% of this value in the control (in the presence of 10 mM EGTA in the intracellular solution), respectively; at [Ca²⁺]_i=10^–5 M, there was no effect of 5-HT at all. The addition of the calmodulin antagonist trifluoperazine (5·10^–5 mM) or blockers of phosphodiesterase (5 mM theophylline or 10^–4 M IBMX) to the perfusate sharply weakened the ability of calcium to inhibit the effect of 5-HT; at [Ca²⁺]_i=10^–5 M, in the presence of one of these compounds, the effect constituted 60–70% of its control value. It is concluded that the calcium-calmodulin-dependent phosphodiesterase is a key link in the interaction of the two-signal transduction pathway — Ca²⁺ and cAMP in modulation of the calcium-channel activity.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 3, pp. 306–313, May–June, 1991.     

Relative stability of α-melanotropin and related analogues to rat brain homogenates

α-Melanotropin (α-MSH) retains less than 1% of its original activity after a 60 min incubation with 10% rat brain homogenate. [Nle⁴, D-Phe⁷]-α-MSH is nonbiodegradable in rat serum (240 min incubation) and still maintains 10% of its original activity in 10% rat brain homogenate (240 min incubation). The related fragment analogue, Ac-[Nle⁴, D-Phe⁷]-α-MSH_4–10-NH₂, retains 50% of its activity after a 240 min incubation in rat brain homogenate, whereas Ac-[Nle⁴, D-Phe⁷]-α-MSH_4–11-NH₂ is totally resistant to inactivation by rat brain homogenate. Both [Nle⁴, D-Phe⁷]-fragments are resistant to degradation by rat serum, but [Nle⁴]-α-MSH, Ac-[Nle⁴]-α-MSH_4–10-NH₂ and Ac-[Nle⁴]-α-MSH_4–11-NH₂ are rapidly inactivated under both conditions. The cyclic melanotropin, [ ]-α-MSH, is inactivated in rat brain homogenate as is the shorter Ac-[ ]-α-MSH_4–10-NH₂ analogue, but neither cyclic melanotropin is inactivated upon incubation in serum from rats. Ac-[ ]-α-MSH_4–10-NH₂ is resistant to inactivation by either rat serum or a brain homogenate. Some of these melanotropin analogues may provide useful probes for the localization and characterization of putative melanotropin receptors in both the central nervous system and peripheral tissues.     

Validation of the use of the lipophilic thiocyanate anion for the determination of membrane potential in Ehrlich ascites tumor cells

Summary The utility of the lipophilic anion thiocyanate (SCN⁺) as a probe for the indirect estimation of the cell membrane potential (V _m ) in Ehrlich ascites tumor cells has been evaluated by comparison to direct electrophysiological measurements. SCN accumulation is consisten with first-order uptake into a single kinetically-identifiable cellular compartement, achieving steadystate distribution in 20–30 min at 22°C. The steady-state distribution ratio ([SCN^–] _c /[SCN^–] _e ) in physiological saline is 0.44±0.02. Treatment of the cells with proparanolol (0.13 mM), an activator of Ca²⁺ dependent K⁺ channels, reduces the steady-state distribution ratio to 0.19±0.02. Conversely, treatmetn with BACl₂ (10 mM), an antagonist of the pathway, increases the SCN^– distribution ratio to 0.62±0.01. The equilibrium potentials (V _SCN ) calculated under these conditions are virtually identical to direct electrophysiological measurements of theV _m made under the same conditions. The effect of varing extracellular [K⁺]([K⁺] _e ) in the presence of constant [Na⁺] _e =100 mM has also been tested. In control cells, elevation of [K⁺] _e from 6 to 60 mM reducesV _SCN from –20.6±1.0 to –13.2±1.2 mV. Again, microelectrode measurements give excellent quantitative agreement. Propranolol increases the sensitivity of the cells to varying [K⁺] _e , so that a 10-fold elevation reducesV _SCN by approximately 31 mV. BaCl₂ greatly reduces this reponse: a 10-fold elevation in [K⁺] _e yielding only a 4-mV rediction inV _SCN . It is concluded that the membrane potential of Ehrlich cells can be estimated accurately from SCN^– distribution measurements.     

Intracellular potassium activity and the role of potassium in transepithelial salt transport in the human reabsorptive sweat duct

Summary We have measured the intracellular potassium activity, [K⁺]_i and the mechanisms of transcellular K⁺ transport in reabsorptive sweat duct (RSD) using intracellular ion-sensitive microelectrodes (ISMEs). The mean value of [K⁺]_i in RSD is 79.8±4.1mm (n=39). Under conditions of microperfusion, the [K⁺]_i is above equilibrium across both the basolateral membrane, BLM (5.5 times) and the apical membrane, APM (7.8 times). The Na⁺/K⁺ pump inhibitor ouabain reduced [K⁺]_i towards passive distribution across the BLM. However, the [K⁺]_i is insensitive to the Na⁺/K⁺/2 Cl^– cotransport inhibitor bumetanide in the bath. Cl^– substitution in the lumen had no effect on [K⁺]_i. In contrast, Cl^– substitution in the bath (basolateral side) depolarized BLM from –26.0±2.6 mV to –4.7^*±2.4 mV (n=3;^* indicates significant difference) and decreased [K⁺]_i from 76.0±15.2mm to 57.7^* ±12.7mm (n=3). Removal of K⁺ in the bath decreased [K⁺]_i from 76.3±15.0mm to 32.3^*±7.6mm (n=4) while depolarizing the BLM from –32.5±4.1 mV to –28.3^*±3.0 mV (n=4). Raising the [K⁺] in the bath by 10-fold increased [K⁺]_i from 81.7±9.0mm to 95.0^*±13.5mm and depolarized the BLM from –25.7±2.4 mV to –21.3^*±2.9 mV (n=4). The K⁺ conductance inhibitor, Ba²⁺, in the bath also increased [K⁺]_i from 85.8±6.7mm to 107.0^*±11.5mm (n=4) and depolarized BLM from –25.8±2.2 mV to –17.0^*±3.1 mV (n=4). Amiloride at 10^–6 m increased [K⁺]_i from 77.5±18.8mm to 98.8^*±21.6mm (n=4) and hyperpolarized both the BLM (from –35.5±2.6 mV to –47.8^*±4.3 mV) and the APM (from –27.5±1.4 mV to –46.0^* ±3.5 mV,n=4). However, amiloride at 10^–4 m decreased [K⁺]_i from 64.5±0.9mm to 36.0^*±9.9mm and hyperpolarized both the BLM (from –24.7±1.4 mV to –43.5^*±4.2 mV) and APM (from –18.3±0.9 mV to –43.5^*±4.2 mV,n=6). In contrast to the observations at the BLM, substitution of K⁺ or application of Ba²⁺ in the lumen had no effect on the [K⁺]_i or the electrical properties of RSD, indicating the absence of a K⁺ conductance in the APM. Our results indicate that (i) [K⁺]_i is above equilibrium due to the Na⁺/K⁺ pump; (ii) only the BLM has a K⁺ conductance; (iii) [K⁺]_i is subject to modulation by transport status; (iv) K⁺ is probably not involved in carrier-mediated ion transport across the cell membranes; and (v) the RSD does not secrete K⁺ into the lumen.     

Simultaneous determination of tetrahydrocortisol and tetrahydrocortisone in human plasma and urine by stable isotope dilution mass spectrometry

A capillary gas chromatographic–mass spectrometric method for the simultaneous determination of tetrahydrocortisol (THF, 3α,11β,17α,21-tetrahydroxy-5β-pregnane-20-one), allo-tetrahydrocortisol (allo-THF, 3α,11β,17α,21-tetrahydroxy-5α-pregnane-20-one) and tetrahydrocortisone (THE, 3α,17α,21-trihydroxy-5β-pregnane-11,20-dione) in human plasma and urine is described. [1,2,3,4,5-²H₅]THF (THF-d₅), allo-[1,2,3,4,5-²H₅]THF (allo-THF-d₅) and [1,2,3,4,5-²H₅]THE (THE-d₅) were used as internal standards. A double derivatization (bismethylenedioxypentafluoropropionate, BMD-PFP) made possible the separation of the three tetrahydrocorticoids with good gas chromatographic behavior. Quantitation was carried out by selected-ion monitoring of the characteristic fragment ions ([M−30]⁺) of the BMD-PFP derivatives of THF, allo-THF and THE. The sensitivity, specificity, precision and accuracy of the method were demonstrated to be satisfactory for measuring low concentrations of THF, allo-THF and THE in human plasma and urine.     

Carbamylcholine-induced morphological changes and spatial dynamics of [Ca2+]c in Harderian glands of guinea pigs: calcium-dependent lipid secretion and contraction of myoepithelial cells

To determine whether lipid-secreting cells have cytosolic Ca²⁺ concentration ([Ca²⁺]_c)-related secretory mechanisms, morphological changes and intracellular calcium dynamics of Harderian glands of guinea pigs stimulated by secretagogs were studied by electron microspy and Fura-2/AM digital image analysis. Control glandular cells contained large lipid vacuoles that were bordered by multi-layered membranes. Rough-surfaced endoplasmic reticulum, mitochondria, and smooth-surfaced endoplasmic reticulum may be involved in lipid vacuole formation. Myoepithelial cells surrounded alveoli. After carbamylcholine (CCh, 10^–6, 10^–5, and 10^–3 M) stimulation, lipid materials within the membranous structures were frequently discharged by an exocytotic mechanism. Conspicuous deformation of glandular cells caused by vigorous contraction of myoepithelial cells was observed in isolated alveoli after 10^–6M CCh stimulation, whereas the deformaties of glandular tissues perfused via vessels were small even after 10^–3M CCh stimulation. Connective tissue between glandular alveoli inhibited unbridled myoepithelial-cell contraction. Fura-2/AM digital imaging analysis revealed that CCh stimulation caused an increase in [Ca²⁺]_c in isolated alveoli. The morphological reactions and changes in [Ca²⁺]_c were prevented by atropine. When extracellular calcium ions were absent, enhanced extrusion of lipid vacuoles, myoepithelial-cell contraction, and a rise in [Ca²⁺]_c after CCh stimulation were not observed. Nicotine and catecholamines had no effect on the secretion or on the dynamics of [Ca²⁺]_c. It can be concluded that acetylcholine elicits exocytosis in glandular cells and contraction of the myoepithelial cells of Harderian glands, accompanied by an increase in [Ca²⁺]_c. The dynamics of [Ca²⁺]_c of the gland alveoli are mostly dependent on extracellular Ca²⁺.     

Functionalization of polyoxomolybdates: the example of nitrosyl derivatives

Nitrosyl derivatives of polyoxomolybdates have been synthesized and characterized by X-ray diffraction. Most of them contain the Mo^II(NO)³⁺ unit and their structures are related to the following structural types: Lindqvist, Keggin and decatungstate [W₁₀O₃₂]^4–. Reductive nitrosylation of (NBu₄)₄[-Mo₈O₂₆] by hydroxylamine in methanol yields (NBu₄)₂[Mo₅O₁₃(OMe)₄(NO){Na(MeOH)}]. 3MeOH, which is a versatile reagent yielding a variety of derivatives (i) by the transformation of [Mo₅O₁₃(OMe)₄(NO)]^3– into [Mo₆O₁₈(NO)]^3– in acetonitrile, (ii) by the formation of [PMo₁₂O₃₉(NO)]^4– by reaction of [Mo₅O₁₃(OMe)₄(NO)]^3– with [PMO₁₂O₄₀]^3– in basic condition and (iii) by the formation of mixed valence Mo^VI/Mo^V/Mo^II decamolybdates [Mo₁₀O₂₄(OMe)₇(NO)]^2–, [Mo₁₀O₂₅(OMe)₆(NO)]^– and [Mo₁₀O₂₀(OMe)₉(NO)₃]^2– by chemical reduction of [Mo₅O₁₃(OMe)₄(NO)]^3–; Mo^II is localized while Mo^V are delocalized in the first two species but localized in the third. The unique ligating properties of [Mo₅O₁₃(OMe)₄(NO)]^3– have been documented: this species acts as a tetradentate ligand in [Ce{Mo₅O₁₃(OMe)₄(NO)}₂]^2–, a symmetrically tetraligating ligand in [Rh₂Cp*₂(-Br){-Mo₅O₁₃(OMe)₄(NO)}] and a bidentate ligand in [Mo₅O₁₃(OMe)₄(NO){RhCp*(H₂O)}]^–. Some polyoxomolybdates of the type [Mo₅(NO)₂O₁₂{RC(NH₂)NHO}₂{RC(NH)NO}₂]^2–, which contain the Mo⁰(NO) ₂ ²⁺ unit, have also been characterized.     

Simultaneous determination of methionine and total homocysteine in human plasma by gas chromatography–mass spectrometry

A gas chromatographic–mass spectrometric method for the simultaneous determination of methionine and total homocysteine in human plasma is described. dl-[²H₄]Methionine and dl-[²H₈]homocystine were used as internal standards. The method involved reduction of the disulfide bond with dithiothreitol, purification by cation-exchange chromatography using a BondElut SCX cartridge and derivatization with isobutyl chlorocarbonate in water–ethanol–pyridine. Quantitation was performed by selected-ion monitoring of the quasi-molecular ions of N(O,S)-isobutyloxycarbonyl ethyl ester (IBC-OEt) derivatives for methionine and [²H₄]methionine, respectively, and the fragment ions ([M+H–COOisoBu–COOEt]⁺) for IBC-OEt derivatives for homocysteine and [²H₄]homocysteine, respectively. The sensitivity, specificity, accuracy and precision of the method were demonstrated to be satisfactory for measuring concentrations of methionine and total homocysteine in human plasma.     

Modeling the cucumber fermentation: Growth ofLactobacillus plantarum

Summary Specific growth rate models of product-inhibited cell growth exist but are rarely applied to fermentations beyond ethanol and large-scale antibiotic production. The present paper summarizes experimental data and the development of a model for growth of the commercially important bacterium,Lactobacillus plantarum, in cucumber juice. The model provides an excellent correlation of data for the influence on bacterial growth rate of NaCl, protons (H⁺), and the neutral, inhibitory forms of acetic acid and the fermentation product, lactic acid. The effects of each of the variables are first modeled separately using established functional forms and then combined in the final model formulation.Nomenclature [C] inhibitory component concentration, mM - [C]_max concentration of the inhibitory component where the specific growth rate is zero, mM, determined by model fitting - [H⁺] hydrogen ion concentration, mM - [HLa] undissociated lactic acid concentration, mM - [La^–] dissociated lactic acid concentration, mM - [La_t] total lactic acid ([HLa]+[La^–]) concentration, mM - [HAc] undissociated acetic acid concentration, mM - [Ac^–] dissociated acetic acid concentration, mM - [Ac_t] total acetic acid ([HAc]+[Ac^–]) concentration, mM - [NaCl] sodium chloride concentration, %, w/v - specific growth rate, h^–1 - _max maximum specific growth rate, h^–1 - ₀ specific growth rate, h^–1, at 0 concentration of additive - K _ij inhibition coefficient - , ,K _m coefficients determined by model fitting Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the US Department of Agriculture or North Carolina Agricultural Research Service, nor does it imply approval to the exclusion of other products that may be suitable.     

Effects of potassium on the anion and cation contents of primary cultures of mouse astrocytes and neurons

Inastrocytes, as [K⁺]_o was increased from 1.2 to 10 mM, [K⁺]_i and [Cl^–]_i were increased, whereas [Na⁺]_i was decreased. As [K⁺]_o was increased from 10 to 60 mM, intracellular concentration of these three ions showed no significant change. When [K⁺]_o was increased from 60 to 122 mM, an increase in [K⁺]_i and [Cl^–]_i and a decrease in [Na⁺]_i were observed.Inneurons, as [K⁺]_o was increased from 1.2 to 2.8 mM, [Na⁺]_i and [Cl^–]_i were decreased, whereas [K⁺]_i was increased. As [K⁺]_o was increased from 2.8 to 30 mM, [K⁺]_i, [Na⁺]_i and [Cl^–]_i showed no significant change. When [K⁺]_o was increased from 30 to 122 mM, [K⁺]_i and [Cl^–]_i were increased, whereas [Na⁺]_i was decreased. Inastrocytes, pH_i increased when [K⁺]_o was increased. Inneurons, there was a biphasic change in pH_i. In lower [K⁺]_o (1.2–2.8 mM) pH_i decreased as [K⁺]_o increased, whereas in higher [K⁺]_o (2.8–122 mM) pH_i was directly related to [K⁺]_o. In bothastrocytes andneurons, changes in [K⁺]_o did not affect the extracellular water content, whereas the intracellular water content increased as the [K⁺]_o increased. Transmembrane potential (E_m) as measured with Tl-204 was inversely related to [K⁺]_o between 1.2 and 90 mM, a ten-fold increase in [K⁺]_o depolarized the astrocytes by about 56 mV and the neurons about 52 mV. The E_m values measured with Tl-204 were close to the potassium equilibrium potential (E_k) except those in neurons at lower [K⁺]_o. However, they were not equal to the chloride equilibrium potential (E_Cl) at [K⁺]_o lower than 30 mM in both astrocytes and neurons. Results of this study demonstrate that alteration of [K⁺]_o produced different changes in [K⁺]_i, [Na⁺]_i, [Cl^–]_i, and pH_i in astrocytes and neurons. The data show that astrocytes can adapt to alterations in [K⁺]_o, in such a way to maintain a more suitable environment for neurons.     

The influence of extracellular calcium on the response of fly photoreceptors

Summary This paper presents a systematic investigation of the influence of the extracellular concentration of calcium ([Ca²⁺]₀) on the electrophysiological response of the fly's photoreceptors (R1–R6) to light. The hemisected heads of flies were perfused with a standard medium containing 10^–4 mol/1 CaCl₂ and in this medium the intracellularly recorded response of the cell was virtually identical to the normal response obtained in vivo. All the effects of changing the [Ca²⁺]₀ could be reversed within 5 min by perfusing the eye with the standard medium.Changing the [Ca²⁺]₀ did not influence the frequency with which quantum bumps occurred or the resting membrane potential, but did lead to changes in the latency and amplitude of the response and, most significantly, in the repolarization time (t _r). The plot oft _r versus the [Ca²⁺]₀ revealed that the value oft _r changes significantly in two distinct regions representing a [Ca²⁺]₀ of between 2×10^–8 and 10^–7 mol/l and 10^–4 and 10^–2 mol/l, respectively. Lowering the [Ca²⁺]₀ did not affect the amplitude of the response but did lead to a drastic increase int _r which was accompanied by an increase in latency and peak time. Raising the [Ca²⁺]₀ led to a reduction in the duration and amplitude of the response. The latter effect is evidence of reduction in the sensitivity of the photoreceptor cell which is dependent on the [Ca²⁺]₀.It is postulated that two types of binding site for calcium exist, high affinity binding sites (HABS) and low affinity binding sites (LABS), which modulate the functioning of ion channels in the cell membrane that are activated as a consequence of light absorption. The results indicate that the sensitivity of the photoreceptor cell is determined by the degree of saturation of the LABS.     

Ion fluxes inAcetabularia: Vesicular shuttle

Summary Ion flux relations in the unicellular marine algaAcetabularia have been investigated by uptake and washout kinetics of radioactive tracers (²²Na⁺,⁴²K⁺,³⁶Cl^– and⁸⁶Rb⁺) in normal cells and in cell segments with altered compartmentation (depleted of vacuole or of cytoplasm). Some flux experiments were supplemented by simultaneous electrophysiological recordings. The main results and conclusions about the steady-state relations are: the plasmalemma is the dominating barrier for translocation of K⁺ with influx and efflux of about 100 nmol·m^–2·sec^–1×K⁺ passes three- to sevenfold more easily than Rb⁺ does. Under normal conditions, Cl^– (the substrate of the electrogenic pump, which dominates the electrical properties of the plasmalemma in the resting state) shows two efflux components of about 17 and 2 mol·m^–2·sec^–1, and a cytoplasmic as well as vacuolar [Cl^–] of about 420mm ([Cl^–] _o =529mm). At 4°C, when the pump is inhibited, both influx and efflux, as well as the cellular [Cl^–], are significantly reduced. Na⁺ ([Na⁺] _i : about 70mm, [Na⁺] _o : 461mm), which is of minor electrophysiological relevance compared to K⁺, exhibits rapid and virtually temperature-insensitive (electroneutral) exchange (two components with about 2 and 0.2 mol·m^–2·sec^–1 for influx and efflux). Some results with Na⁺ and Cl^– are inconsistent with conventional (noncyclic) compartmentation models: (i) equilibration of the vacuole (with the external medium) can be faster than equilibration of the cytoplasm, (ii) absurd concentration values result when calculated by conventional compartmental analysis, and (iii) large amounts of ions can be released from the cell without changes in the electrical potential of the cytoplasm. These observations can be explained by the particular compartmentation of normalAcetabularia cells (as known by electron micrographs) with about 1 part cytoplasm, 5 parts central vacuole, and 5 parts vacuolar vesicles. These vesicles communicate directly with the central vacuole, with the cytoplasm and with the external medium.     

Selection of a support for immobilization of a microbial lipase for the hydrolysis of triglycerides

Baterial lipase from Staphylococcus carnosus (pLipMut2) has been immobilized on various supports in order to determine a suitable immobilization technique in terms of activity and stability, when utilized for the hydrolysis of tributyrin. The hydrophobic materials PBA Eupergit and PBA Eupergit 250L prooved to be appropriate supports, when the enzyme was crosslinked with glutaraldehyde after adsorption. No desorption of the immobilized enzyme occured during operation. The pore size of the support has a strong effect on the activity but does not influence stability.The initial activity for immobilized and soluble lipase is found to follow the Arrhenius equation at low temperature, where mass transfer does not affect reaction kinetics. Activation energies for soluble and immobilized lipase were evaluated to be 21.7 kJ mol^–1 and 60.8 kJ mol^–1, respectively.Operational stability was studied in a packed bed recirculation reactor. Thermal desactivation followed first order kinetics with a half-life of 1340 h at 10°C. Model calculations for productivity showed, that optimal temperatures for high productivity are well below the temperature of maximal activity.List of Symbols E _a [kJ mol^–1] activation energy - E _d [kJ mol^–1] activation energy of desactivation - H [–] half-number - k _d [h^–1] desactivation constant - k _d, [h^–1] constant - k _N [–] desactivation constant (number) - N [–] number of runs - p [mol dm^–3] productivity - t [h] time - t _0.5 [h] half-life - T [K] absolute temperature - V [U ml^–1] activity - V(N) [Uml^–1] activity exhibited in the n-th run - V _s,O [U ml^–1] initial activity of supernatant - V _s, [U ml^–1] activity of supernatant after immobilization - V _O [U ml^–1] initial activity - V [U ml^–1] constant - _imm [–] activity yield - [ml ml^–1] ratio of volume of support to volume of supernatant Financial support of this work by the Deutsche Forschungsgemeinschaft (SFB 145, A15) is gratefully acknowledged.     

The effect of N-terminal substitutions on the biological activity of MSH fragments

In order to study the role of N-terminal substitutions of peptide sequences related to the active site of α-melanotropin, [Glp⁵]α-MSH(5–10), [Glp⁵, -Phe⁷]α-MSH(5–10), [Sar⁵, -Phe⁷]α-MSH(5–10), [Nle⁴, -Phe⁷]α-MSH(4–10), [N-carbamoyl]α-MSH(5–10), and formyl and acetyl derivatives of α-MSH(5–10), [Gly⁵]α-MSH(5–10) and [Gly⁵, -Phe⁷]α-MSH(5–10), were synthesized in solution. The N-terminal acylations enhance by 2 to 10 times the melanin-dispersing activity of the unsubstituted sequences. Alkylation of the N-terminus does not change the biological activity of the parent peptide, suggesting the necessity of a carbonyl group for increasing the hormonal effect.     

An Interaction Between ATP and High K+: Mutual Impairment of ATP- and High K+-Evoked [Ca2+]i Increase in NG 108–15 Cells

The interaction between ATP- and high K⁺-evoked increase in intracellular free calcium concentration ([Ca²⁺]_i) was investigated to gain an insight into the mechanism of interaction of ATP with voltage-sensitive calcium channels. [Ca²⁺]_i was measured in the neuronal model, neuroblastoma × glioma hybrid cells (NG 108–15), using the fluorescence indicator fura-2. In the presence of 1.8 mM extracellular Ca²⁺, ATP induced a rapid, concentration-dependent increase in [Ca²⁺]_i. High K⁺ (50 mM) evoked a [Ca²⁺]_i rise from 109 ± 11 nM to 387 ± 81 nM (n = 16). The application of either of these two [Ca²⁺]_i-increase provoking agents in sequence with the other caused impairment of the latter effect. The mutual desensitization of the responses to ATP and high K⁺ strongly suggests that both agents rely at least in part on the same source of Ca²⁺ for elevation of [Ca²⁺]_i in NG 108–15 cells.