Studies on the mechanism of action of a bilayer stabilizing inhibitor of protein kinase C: Cholesterylphosphoryldimethylethanolamine

Cholesterylphosphoryldimethylethanolamine is a zwitterionic compound which is a good bilayer stabilizer. As has been found with many other compounds having these properties, cholesterylphosphoryldimethylethanolamine is found to be a potent inhibitor of protein kinase C in both vesicle and micelle assay systems. The kinetics of the inhibition in Triton X-100 micelles was non-competitive with respect to ATP, histone, diolein, phorbol ester and Ca²⁺. It has a K_i of about 30 m. The inhibition kinetics as a function of phosphatidylserine concentration is more complex but suggestive of competitive inhibition. Cholesterylphosphoryldimethylethanolamine does not prevent the partitioning of protein kinase C into the membrane. This inhibitor lowers the Ca²⁺-phosphatidylserine-independent phosphorylation of protamine sulfate by protein kinase C and directly affects the catalytic segment of the enzyme generated by tryptic hydrolysis. Thus, this zwitterionic bilayer stabilizing inhibitor of protein kinase C both competes with the binding of phosphatidylserine as well as affects the active site of protein kinase C.Abbreviation CPD cholesterylphosphoryldimethylethanolamine     

Acute phase response to recombinant interleukin-6 and macrophage- and fibroblast-derived crude cytokine preparations in primary cultures of mouse hepatocytes

A number of acute phase proteins were determined by electroimmunoassay in media from CBA mouse hepatocytes cultured for 2 days with human recombinant IFN beta 2/IL-6, as well as with conditioned media from LPS-stimulated rat macrophages, and of murine L fibroblasts. It was found that human recombinant IL-6 caused three-fold increase in secretion of fibrinogen, while haptoglobin, complement C3 and transferrin were increased respectively, to 168 per cent, 151 per cent, and 145 per cent of the control. DEX(10(-7) M) in DMEM supplemented with 5 per cent FCS, enhanced the IL-6 effect on the three positive acute phase proteins. IL-6 elevated haptoglobin mRNA in mouse hepatocytes to a degree comparable with the concentration of the protein in the culture medium. The effect of conditioned media from murine fibroblasts and peritoneal rat macrophages was generally similar to that of recombinant IL-6. However, both natural preparations of the cytokines caused decrease in albumin and alpha-1-proteinase inhibitor secretion.     

Search for polythionates in cultures of Chromatium vinosum after sulfide incubation

Cultures of Chromatium vinosum, devoid of sulfur globules, were supplemented with sulfide and incubated under anoxic conditions in the light. The concentrations of sulfide, polysulfides, thiosulfate, polythionates and elemental sulfur (sulfur rings) were monitored for 3 days by ion-chromatography and reversed-phase HPLC. While sulfide disappeared rapidly, thiosulfate and elemental sulfur (S₆, S₇ S₈ rings) were formed. After sulfide depletion, the concentration of thiosulfate decreased fairly rapidly, but elemental sulfur was oxidized very slowly to sulfate. Neither polysulfides (S _x ^2– ), polythionates (S_nO ₆ ^2– , n=4–6), nor other polysulfur compounds could be detected, which is in accordance with the fact that sulfide-grown cells were able to oxidize polysulfide without lag. The nature of the intracellular sulfur globules is discussed.     

The Cellular Mechanism of Orcadian Rhythms-A View on Evidence, Hypotheses and Problems

A stable period length is a characteristic property of circadian oscillations. The question about whether higher frequency oscillators (0.5-8 hr) contribute to or establish the stable circadian periodicity cannot be answered at present. A sequential coupling of quantal subcycles appears possible on the basis of known “ultradian” oscillations. There is, however, no supporting evidence for such a concept. Phase response curves of the circadian clock derived from various perturbing pulses allow qualitative conclusions concerning the perturbed clock process. Deductions from computer simulations also allow conclusions about the phase of this oscillatory process.

The distinction between processes (a) essential to the clock mechanism, (b) maintaining and controlling the clock (inputs) and (c) depending on the clock (outputs) on the basis of “oscillatory” and “change of φ or τ after perturbation” seems to be useful but not stringent. Protein synthesis may be an essential or input process. Oscillatory changes of this process may be due to periodic translational control or RNA-supply. Circadian changes in protein concentration and/or activity may depend on periodic synthesis, proteolysis, covalent modifications or aggregations. Specific essential proteins have not been identified conclusively. The large overlap between the group of agents and treatments that phase shift the clock and the group that induces stress proteins suggest that the latter may play a role in the controlling (input) or essential domain.

The role of membranes in the clock mechanism is not clear: concepts assuming an essential function are based on circumstantial evidence. The membrane potential as well as Ca²⁺ may be involved in either input or essential function. Ca²⁺ -calmodulin may also be important as concluded from inhibitor experiments. It is tempting to assume that a calmodulin-dependent kinase is part of a periodic protein phosphorylation process, yet it is not clear whether the periodic protein phosphorylation that has been observed is essential or is just another output process.     

Biological effects of synthetic AKH in Manduca sexta and estimates of the amount of AKH in corpora cardiaca

Dose-response curves were measured with synthetic Manduca adipokinetic hormone (AKH) for glycogen phosphorylase activation in larvae and for lipid mobilization in adults. Both responses are known hormonal functions in Manduca sexta. In ligated larvae, full activation of glycogen phosphorylase was achieved with 0.1 pmol and half-maximal activation with 0.03-0.04 pmol. Maximal lipid mobilization in adults required 10 pmol and half-maximal mobilization 0.15 to 0.2 pmol, respectively. An estimate of AKH content of corpora cardiaca from M. sexta was gained by comparing the dose-response curves for synthetic Manduca AKH with curves from gland extracts. Corpora cardiaca extracts were also quantitated by high performance liquid chromatography. According to both estimates corpora cardiaca of adults contain 10-20 pmol AKH per pair, while a pair of larval corpora cardiaca contains 0.7-2 pmol.     

Interactions of chiral molecules with an (r)-n-(3,5-dinitrobenzoyl) phenylglycine HPLC stationary phase

Forty different chiral molecules were studied by liquid chromatography with a Pirkle-type, (R)-N-(3,5-dinitrobenzoyl) phenylglycine (DNBPG), chiral stationary phase column. The dramatic effect of a small molecular change on chiral recognition was demonstrated using DL-amino acid derivatives. The inductive effect on chiral recognition was also studied using trifluoro-, trichloro-, dichloro-, monochloroacetyl, and acetyl derivatives of four different chiral amines. The study of the enantiomer separation of 11 different crown ethers of 2,2′-binaphthyldiyl showed that the rigidity of the chiral center can be an additional parameter in chiral recognition for the DNBPG phase but not for a β-cyclodextrin bonded chiral phase. It is apparent from this study that steric effects, inductive effects, and molecular rigidity play important roles in chiral recognition with DNBPG chiral stationary phases.     

Temperature-dependent osmotic permeability in glycoproteion containing liposomes

The osmotic water outflow of large multilamellar liposomes containing ₁-acid glycoprotein was measured at a temperature near the lipid's phase transition temperature. The liposomes were formed from a mixture of DPPC, cholesterol and glycoprotein in molar ratios 100:20:1, by continuous sucrose density gradient centrifugation. These liposomes captured 35% of the radiolabeled glycoprotein. The temperature-dependent experiments showed that near phase transition temperature the initial rate of water outflow increased drastically in comparison with glycoprotein free liposomes incubated in buffer containing glycoprotein. We suggested that eventual a channel mechanism may be involved due to spontaneous incorporation of glycoprotein into the bilayer.     

Menstrual-cycle phase and sexual behavior in semi-free-ranging stumptail macaques (Macaca arctoides)

The sexual behavior and female reproductive cycles of a group of island-dwelling stumptail macaques (Macaca arctoides)were monitored over a 6-month period, yielding 530 observation hr and 268 copulations. Compared to nondominant males, the dominant male copulated at a relatively high rate throughout the cycle, but largely with one high-ranking female. The non-dominant males copulated most frequently at midcycle. Female presenting was highest at midcycle, but only to the dominant male. Cross-study discrepancies may be due to different observation methods and restricted environmental conditions that mask female-initiated sexual behavior. The more naturalistic setting of this study allowed for a fuller expression of proceptivity. Contrary to some previous conclusions, present findings suggest that both hormonal and socioenvironmental factors influence the patterns of sexual behavior found in stumptail macaque colonies.     

The stimulating effect of a cold, dark pretreatment on the etioplast/chloroplast transformation of angiosperms. I. The stimulating effect of cold predarkening on different stages of greening under white light

Schönbohm, E., Stute, U., Thienhaus, P. and Werner, U. 1988. The stimulating effect of a cold, dark pretreatment on the etioplast/chloroplast transformation of angiosperms I. The stimulating effect of cold predarkening on different stages of greening under white light. - Physiol. Plant. 72: 541–546.
The etioplast/chloroplast transformation in angiosperms is controlled by light; most of the processes are mediated by phytochrome. We have shown that in the primary leaves of etiolated seedlings of wheat ( Triticum aestivum L. cv. Kolibri), fire-bean ( Phaseolus multiflorus L. cv. Preisgewinner) and in the cotyledons of etiolated sun flower seedlings ( Helianthus annuus L. cv. macrocarpa) the chlorophyll accumulation in the phase after the end of the lag phase can be greatly stimulated by a cold predarkening period. This effect is not necessarily coupled with a red preirradiation. Furthermore the lag phase can be dramatically shortened by the cold, dark pretreatment, whereas the amount of photoconvertible protochlorophyll(ide) in the darkness remains unaffected by the cold, dark pretreatment. The stimulating effect of a cold, predarkening period on greening is fully reversible by a warm, dark phase that is placed between the cold period and the onset of the continuous white light phase. These findings cannot be generalized: We could demonstrate that in the tropical plant Momordica charantia greening under white light was not affected by different temperature pretreatments during predarkening. The stimulating effect of a cold, predarkening period on greening is assumed to have ecological relevance.     

ATPase activity in plasmalemma-rich vesicles isolated by aqueous two-phase partitioning from Vicia faba mesophyll and epidermis: Characterization and influence of abscisic acid and fusicoccin

Plasmalemma-rich microsomal vesicles were prepared from whole leaf and acid-washed epidermal tissue of Vicia faba L. cv. Osnabrücker Markt by aqueous two-phase partitioning in dextran T-500 and polyethylenglycol 1350 aqueous phases. These vesicles were tightly sealed and predominantly right-side out, and contained a K⁺ -stimulated, mg²⁺-dependent and vanadate-sensitive ATPase. The enzyme from both tissues exhibited nearly identical properties: pH optimum 6.4, K_m for ATP 0.60 mM(whole leaf) and 0.67 mM (epidermis). V_max -480 nmol (mg protein)¹ min¹ (whole leaf) and 510 nmol (mg protein)¹ min¹ (epidermis), I₅₀ (Na₃,VO₄) 7.5 μM (whole leaf) and 15 μM (epidermis). The enzyme was not inhibited by NO₃(50 mM)or sodium azide (I mM). DCCD (20 μM) reduced enzyme activity to 50% (whole leaf) and 58% (epidermis), gramicidin S (20 μM) to 36% (whole leaf) and 41%(epidermis). Ca²⁺ inhibited the ATPase [I₅₀, C²⁺: 0.5 mM(whole leaf) and 0.8 mM(epidermis)]. Ca²⁺ inhibited the ATPase [I₅₀, C²⁺ 0.5 mM(whole leaf) und 0.8 (epidermis)]. The vanadate-sensitive ATPase from whole leaf and epidermal tissue was slightly but significantly stimulated by fusicoccin (FC) at a concentration (0.13 μM) promoting stomatal opening. The stimulation was not seen in the solubilized ATPase. Stomata of the cultivar used here were insensitive lo (±)ABA up to 2 μM level which is effective in most other cultivars and species. Likewise, at this concentration no effect of ABA on the activity of the epidermal ATPase was observed. The data are discussed with respect to the interaction of FC and ABA with the ATPase.