AnAnolis skin melanophore assay suitable for photoaffinity labeling studies with α-MSH

Summary Visual determination of MSH-induced pigment migration in melanophores of small pieces ofAnolis carolinensis skin is standardized by first measuring photoelectrometrically the change in reflection/transmission of the whole dorsal skin in response to different hormone concentrations. This method allows the rapid and precise recording of time-response curves after photoaffinity labeling of MSH receptors or of dose-response curves of large series of synthetic compounds.Acknowledgments. We wish to thank Ms V. Jäggin, Ms C. Schulthess and Ms G. van Hees for excellent technical assistance, Dr. R. Andreatta, Ciba-Geigy AG, Basel, for his generous gift of -MSH and Prof. A Pletscher for his continuous interest. This work was supported by the Swiss National Science Foundation.     

Changes in dopamineβ-hydroxylase activity of monkey plasma with age

Summary Plasma dopamine -hydroxylase activity of Japanese monkeys (Macaca fuscata fuscata) increased with age, and the developmental changes were similar to those of human beings. However, the adult level plasma DBH activity of various monkey species was much lower than that of human beings.We thank Dr.K. Nozawa, Dr.O. Takenaka and Mr.T. Shotake (Primate Research Institute, Kyoto University) for their help in collecting monkey blood samples.     

β-phenylethyl isothiocyanate-mediated apoptosis in hepatoma HepG2 cells

-Phenylethyl isothiocyanate (PEITC) is a promising chemoprotective compound that is routinely consumed in the diet as its glucosinolate precursor. Previous studies have shown that PEITC can inhibit phase I enzymes and induce phase II detoxification enzymes along with apoptosis in vitro. The detailed mechanisms involved in the apoptotic cascade, however, have not been elucidated. In the present study, we demonstrate that PEITC can induce apoptosis in hepatoma HepG2 cells in a concentration- and time-dependant manner as determined by TUNEL positive and SubG1 population analysis. Caspase-3-like activity and poly(ADP-ribosyl)polymerase cleavage increased during treatment with 20 µM PEITC; high concentrations, however, induced necrosis. Pre-treatment with Z-VAD-FMK and the caspase-3-specific inhibitor Ac-DEVD-CHO prevented PEITC-induced apoptosis, as determined by caspase-3-like activity and DNA fragmentation. Additional investigations also showed that at concentrations of 5-C10 µM PEITC, DNA synthesis was inhibited and G2/M phase cell cycle arrest occurred, correlating with an alteration in cyclin B1 and p34^cdc2 protein levels. Furthermore, we also demonstrate a concentration- and time-dependant burst of superoxide (O₂^-) in PEITC-treated cells. However, pre- and co-treatment with the free radical scavengers Trolox, ascorbate, mannitol, uric acid and the superoxide mimetic manganese (III) tetrakis (N-methyl-2-pyridyl) porphyrin failed to prevent PEITC-mediated apoptosis. Taken together, these results suggest that PEITC potently induces apoptosis and cell cycle arrest in HepG2 cells and that the generation of reactive oxygen species appears to be a secondary effect.Received 23 December 2002; accepted 22 April 2003     

Anti-bovineβ-lactoglobulin antibodies react with a human lactoferrin fragment and bovineβ-lactoglobulin present in human milk

Human milk samples react against anti-bovine-lactoglobulin rabbit antibodies, as measured by a competitive radioimmunoassay. Immunoreactivity was positive even in milk from mothers consuming a diet free of cow's milk. An increase with a diet rich in cow's milk proteins was detected by immunoelectrophoresis. The human milk fraction cross-reacting with anti-bovine-lactoglobulin antibodies corresponds to the 20 kDa fragment from the N-terminal end of human lactoferrin. Three regions of this fragment exhibit sequence homology with a sequence contained in cow's-lactoglobulin (between residues 124 and 141).     

Hepatic cyclophorase activity in rats treated with 1,2,3,4-tetrahydro-β-naphtylamine (THNA)

Riassunto Gli omogenati di fegato dei ratti sacrificati dopo 4 h dalla somministrazione di-tetraidronaftilammina (30 mg pro kg per via intraperitoneale) presentano una aumentata capacità di ossidare alcuni composti del Ciclo di Krebs, in rapporto ai controlli. Viene avanzata l'ipotesi che l'intensa glicogenolisi epatica e muscolare e l'aumentata acetilazionein vivo dell'acido para-aminobenzoico provocate nel ratto dalla somministrazione di-tetraidronaftilammina, siano causate da un esaltato metabolismo intermedio ossidativo.     

KIF5C: A new binding partner for protein kinase CK2 with a preference for the CK2α’ subunit

Protein kinase CK2 is a highly conserved serine/threonine kinase that is ubiquitously expressed in eukaryotic cells. CK2 is a constitutively active tetrameric enzyme composed of two catalytic α and/or α’-subunits and two regulatory β-subunits. There is increasing evidence that the individual subunits may have independent functions and that they are asymmetrically distributed inside the cell. To gain a better understanding of the functions of the individual subunits, we employed a yeast-two-hybrid screen with CK2α and CK2α’. We identified the motor neuron protein KIF5C as a new binding partner for CK2. The interaction found in the yeast-two-hybrid screen was confirmed by co-sedimentation analysis on a sucrose density gradient and by co-immunoprecipitation analysis. Pull-down experiments and surface plasmon resonance spectrometry revealed a direct binding of KIF5C to CK2α’. Co-localization studies with neuroblastoma cells, bone marrow and with primary neurons confirmed the biochemical analysis that KIF5C preferentially bound to CK2α’. Received 8 August 2008; received after revision 3 November 2008; accepted 4 November 2008     

17β-estradiol in vitro affects Na-dependent and depolarization-induced Ca2+ transport in rat brain synaptosomes

Effects of 17-estradiol (E₂) in vitro on Na-dependent Ca²⁺ efflux from, and depolarization-induced Ca²⁺ uptake into, the nerve cell were studied with the use of synaptosomes isolated from the brain stem, mesencephalic reticular formation (MRF), caudate nucleus and the hippocampus of long-term ovariectomized adult female rats. It was found that E₂ (1) at a concentration of 10 nM or lower, stimulates Na-dependent Ca²⁺ efflux in the caudate nucleus and hippocampus, and does not affect the efflux in MRF and brain stem; (2) at concentrations above 10 nM has no effect on the Ca²⁺ efflux in any of the four structures investigated; and (3) produces a biphasic effect on the depolarization-induced Ca²⁺ uptake, increasing it in all structures except MRF at 10 nM concentration, and decreasing it at concentrations higher than 10 nM, irrespective of the structure investigated. These results suggest that E₂, acting at extranuclear sites, modulates synaptic transmission via alterations of Ca²⁺ transport mechanisms in nerve endings.     

β2-integrins mediate a novel form of chemoresistance in cycloheximide-induced U937 apoptosis

In this study with cycloheximide (CHX, an inhibitor of protein synthesis) and the human leukaemic cell line U937, a novel form of chemoresistance, which we termed sudden drug resistance (SDR), was identified using Hoechst33258 staining, Western blott and DNA Ladder. CHX^high (10–100 g/ml)-induced apoptosis can spontaneously subside after 4–6 h or can be inhibited by short-term preincubation with CHXlow (2.5 g/ml). Unlike typical multidrug resistance, SDR is not caused by reduced drug accumulation or altered protein expression, and may be associated with a non-P-glycoprotein mechanism. To uncover this underlying mechanism, we focused on U937 cell aggregation promoted by CHX, because cell adhesion has been suggested to influence cell survival and prevent apoptosis. EDTA, or anti-CD18 monoclonal antibody, but not EGTA, acetylsalicylic acid or RGDS tetrapeptide, abrogated this homotypic aggregation and greatly increased CHX-induced apoptosis in a time-dependent manner, while fibrinogen and soluble intercellular adhesion molecule-1 exerted opposite effects. These results establish that ₂-integrin engagement is a key mediator of SDR, although it may be non-exclusive. This finding supplements the classical basis of chemoresistance and may provide another opportunity for improved leukemia therapy.Received 15 April 2004; received after revision 18 May 2004; accepted 21 June 2004     

Einbau des 2′,4,4′,6′-Tetrahydroxy-chalkon-2′-glucosid-[β-14C] in Isoflavone

Summary 2,4,4,6-Tetrahydroxychalcone-2-glucoside-[-¹⁴C] is incorporated into biochanin-A in chana germ (Cicer arietinum L.) without randomization of the radioactivity. Possibly a very low incorporation into formononetin occurs also. After administration of the chalcone a large part of the activity is bound to the acid insoluble residue of the cicer shoots (lignin-like substances) in an (as yet) undetermined manner. This binding also occurs in heat denatured homogenates.

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VI. Mitt. Zur Biogenese der Isoflavone. V. Mitt.H. Grisebach undG. Brandner, Biochim. biophys. Acta60, 51 (1962).     

TNF-α upregulates adenosine 2b (A2b) receptor expression and signaling in intestinal epithelial cells: a basis for A2bR overexpression in colitis

Adenosine is an endogenous signaling molecule upregulated during inflammatory conditions. Acting through the A2b receptor (A2bR), the predominant adenosine receptor in human colonic epithelia, adenosine has been directly implicated in immune and inflammatory responses in the intestine. Little is known about expression and regulation of A2bR during inflammation. Tumor necrosis factor alpha (TNF-α) is highly upregulated during chronic and acute inflammatory diseases. This study examined the expression of A2bR during colitis and studied effects of TNF-α on A2bR expression, signaling and function. Results demonstrated that A2bR expression increases during active colitis. TNF-α pretreatment of intestinal epithelial cells increased A2bR messenger RNA and protein expression. TNF-α significantly increased adenosine-induced membrane recruitment of A2bR and cyclic adenosine monophosphate downstream signaling. Further, TNF-α potentiated adenosine-induced shortcircuit current and fibronectin secretion. In conclusion, we demonstrated that TNF-α is an important regulator of A2bR, and during inflammation, upregulation of TNF-α may potentiate adenosine-mediated responses. Received 21 July 2005; received after revision 22 August 2005; accepted 19 September 2005 †These authors contributed equally to this work.     

Exact test for opposite trends of probability changes in one or several 3×2 tables

Zusammenfassung Der «Likelihood»-Quotiententest wird auf einer 3×2-Kontigenztafel mit unbekannten Wahrscheinlichkeitenp _ij (i=1,2,3;j=1,2) angewandt, um die Hypothese H₀:p _i1=p _i2 (i=1,2,3) gegen die alternative Hypothese H₁:p ₁₁>p ₁₂ p ₃₁<p ₃₂ zuprüfen, und zwar wenn kleine Stichproben vorhanden sind. Ausserdem wird die Kombination solcher Tests behandelt. Als Beispiel wird diese Testtheorie beim Wahlverhalten des Kabeljaus im Dressurversuch über akustische Lokalisation in zwei Stimulussituationen verwendet.

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The close cooperation with Drs.E. Meelis, Institute of Theoretical Biology Leyden, The Netherlands, in applying the likelihood ratio test to this case is gratefully acknowledged.

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The author is greatly indebted to Dr.J. Oosterhoff, Mathematical Institute, University of Nijmegen, The Netherlands, for introducing the subclasses 1b and 1c in the computation of vector and for the explicit expressions in these cases which eliminated the need for a numerical optimization. Further more valuable remarks regarding the combination problem were obtained.     

Isotrinervi-2β-ol. Structural isomers in the defense secretions of allopatric populations of the termiteTrinervitermes gratiosus

Summary The defense secretions of 3 allopatric populations of the nasute termiteTrinervitermes gratiosus were analyzed. One population afforded a new trinervitene, isotrinervi-2-ol, a missing link in the hypothetical biosynthesis of the trinervitenes. Populations could be readily distinguished on the basis of the chromatographic profiles of their major and minor soldier frontal gland secretions.These studies were initiated (1975–76) with partial financial support by the International Centre of Insect Physiology and Ecology, Nairobi, Kenya and NIH Postdoctoral Fellowship AI-05076. Support by the Department of Chemistry of the State University of New York at Stony Brook and the technical assistance of Mr J. Engstrom allowed completion of this investigation.     

How do Shp2 mutations that oppositely influence its biochemical activity result in syndromes with overlapping symptoms?

Activating and inactivating mutations of SHP-2 are responsible, respectively, for the Noonan (NS) and the LEOPARD (LS) syndromes. Clinically, these developmental disorders overlap greatly, resulting in the apparent paradox of similar diseases caused by mutations that oppositely influence SHP-2 phosphatase activity. While the mechanisms remain unclear, recent functional analysis of SHP-2, along with the identification of other genes involved in NS and in other related syndromes (neurofibromatosis-1, Costello and cardio-facio-cutaneous syndromes), strongly suggest that Ras/MAPK represents the major signaling pathway deregulated by SHP-2 mutants. We discuss the idea that, with the exception of LS mutations that have been shown to exert a dominant negative effect, all disease-causing mutations involved in Ras/MAPK-mediated signaling, including SHP-2, might lead to enhanced MAPK activation. This suggests that a narrow range of MAPK signaling is required for appropriate development. We also discuss the possibility that LS mutations may not simply exhibit dominant negative activity. Received 30 November 2006; received after revision 8 February 2007; accepted 13 March 2007     

Dopamine-β-hydroxylase,adrenaline, noradrenaline and dopamine in the venous blood of adrenal gland of man: A comparison with levels in the periphery of the circulation

Summary In 10 human subjects plasma dopamine--hydroxylase activity was found in the adrenal vein blood to be as high as in the periphery of the circulation. Adrenaline concentration in the adrenal vein blood was in the mean 170 times, noradrenaline concentration 11 times and dopamine concentration little higher than levels in the periphery.This study was supported by the Deutsche Forschungsgemeinschaft.     

Preferential hydrophobic interactions are responsible for a preference of D-amino acids in the aminoacylation of 5′-AMP with hydrophobic amino acids

We have studied the chemistry of aminoacyl AMP to model reactions at the 3 terminus of aminoacyl tRNA for the purpose of understanding the origin of protein synthesis. The present studies relate to the D, L preference in the esterification of 5-AMP. All N-acetyl amino acids we studied showed faster reaction of the D-isomer, with a generally decreasing preference for D-isomer as the hydrophobicity of the amino acid decreased. The -branched amino acids, Ile and Val, showed an extreme preference for D-isomer. Ac-Leu, the -branched amino acid, showed a slightly low D/L ratio relative to its hydrophobicity. The molecular basis for these preferences for D-isomer is understandable in the light of our previous studies and seems to be due to preferential hydrophobic interaction of the D-isomer with ademine. The preference for hydrophobic D-amino acids can be decreased by addition of an organic solvent to the reaction medium. Conversely, peptidylation with Ac-PhePhe shows a preference for the LL isomer over the DD isomer.     

Effect of glucose and phloretin-2′-β-D-glucose (phloridzin) on in vitro fertilization of mouse ova

Summary The fertilization ratio of mouse ova in vitro decreased when glucose concentration in the medium was lowered. However, the addition of phloretin-2--D-glucose (phloridzin), known as a glucose uptake inhibitor, restored the fertilization ratio back to the control level. The glucose moiety of the phloridzin seemed to be responsible for this effect.     

Fluid shear stress-induced TGF-β/ALK5 signaling in renal epithelial cells is modulated by MEK1/2

Renal tubular epithelial cells are exposed to mechanical forces due to fluid flow shear stress within the lumen of the nephron. These cells respond by activation of mechano-sensors located at the plasma membrane or the primary cilium, having crucial roles in maintenance of cellular homeostasis and signaling. In this paper, we applied fluid shear stress to study TGF-β signaling in renal epithelial cells with and without expression of the Pkd1-gene, encoding a mechano-sensor mutated in polycystic kidney disease. TGF-β signaling modulates cell proliferation, differentiation, apoptosis, and fibrotic deposition, cellular programs that are altered in renal cystic epithelia. SMAD2/3-mediated signaling was activated by fluid flow, both in wild-type and Pkd1 ^?/? cells. This was characterized by phosphorylation and nuclear accumulation of p-SMAD2/3, as well as altered expression of downstream target genes and epithelial-to-mesenchymal transition markers. This response was still present after cilia ablation. An inhibitor of upstream type-I-receptors, ALK4/ALK5/ALK7, as well as TGF-β-neutralizing antibodies effectively blocked SMAD2/3 activity. In contrast, an activin-ligand trap was ineffective, indicating that increased autocrine TGF-β signaling is involved. To study potential involvement of MAPK/ERK signaling, cells were treated with a MEK1/2 inhibitor. Surprisingly, fluid flow-induced expression of most SMAD2/3 targets was further enhanced upon MEK inhibition. We conclude that fluid shear stress induces autocrine TGF-β/ALK5-induced target gene expression in renal epithelial cells, which is partially restrained by MEK1/2-mediated signaling.