Production and characterization of antibodies cross-reactive with major aflatoxins

Antibodies cross-reactive with 4 major aflatoxins were demonstrated three weeks after immunization of rabbits with an immunogen which was prepared by conjugating aflatoxin B₃ to bovine serum albumin. Aflatoxin B₃ was first converted to its hemisuccinate before conjugation to the protein. Tritiated aflatoxin B₁ (AFB₁) was used as the marker ligand both for antibody titer determination as well as for analysis of antibody specificity. Competitive RIA revealed that the antibodies have good cross-reactivity with aflatoxins B₁, B₂, G₁, and G₂ when tritiated AFB₁ was used as the marker ligand. The concentrations causing 50% inhibition of binding of³H-AFB₁ to the antibodies by unlabeled aflatoxins B₁, B₂, G₁, G₂ and B₃ were found to be 0.25, 3.34, 0.32, 4.0 and 0.53 ng/assay, respectively. The antibodies could be used for simultaneous analysis of aflatoxins B₁ and G₁, two of the most important toxic metabolites produced byAspergillus flavus andA. parasiticus.     

Arrest of somatic cells at G2 by Sevin (pesticide)

Summary The treatment of onion root meristems with the pesticide Sevin [carbaryl (1-naphthyl n-methyl carbamate)] induces an accumulation of interphase nuclei with a larger diameter, and a subsequent decrease in the index of smaller nuclei. It is concluded that Sevin depresses mitosis by arresting cells at G₂ without affecting DNA synthesis.The authors are grateful to Prof. K. S. Bilgrami, Head of the Postgraduate Department of Botany, Bhagalpur University, for providing facilities and to Prof. B.N. Mookerjee for his valued suggestions.     

Insulin activates Gαil,2 protein in rat hepatoma (HTC) cell membranes

Insulin action is initiated by binding to its cognate receptor, which then triggers multiple cellular responses by activating different signaling pathways. There is evidence that insulin receptor signaling may involve G protein activation in different target cells. We have studied the activation of G proteins in rat hepatoma (HTC) cells. We found that insulin stimulated binding of guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-³⁵S) to plasma membrane proteins of HTC cells, in a dose-dependent manner. This effect was completely blocked by pertussis toxin treatment of the membranes, suggesting the involvement of G proteins of the Gα _i/Gα _o family. The expression of these Gα proteins was checked by Western blotting. Next, we used blocking antibodies to sort out the specific Gα protein activated by insulin stimulation. Anti-Gα _il,2 antibodies completely prevented insulin-stimulated GTP binding, whereas anti-Gα _o,i3 did not modify this effect of insulin on GTP binding. Moreover, we found physical association of the insulin receptor with Gα _i1,2 by copurification studies. These results further support the involvement of a pertussis toxin-sensitive G protein in insulin receptor signaling and provides some evidence of specific association and activation of Gα _i1,2 protein by insulin. These findings suggest that Gα _i1,2 proteins might be involved in insulin action. Received 23 September 1998; received after revision 23 November 1998; accepted 25 November 1998     

The cell cycle of an established line ofDrosophila melanogaster cells in vitro

Riassunto La durata media delle fasi del ciclo cellulare (G₁, S e G₂) e del tempo di generazione totale (G _T ) in una linea stabilizzata di cellule diDrosophila melanogaster risulta essere rispettivamente di 1.8, 10.0, 7.2 e 18.8 ore.     

Nuclear fusion and irregular cytokinesis in binucleate and tetraploid cells ofVicia faba after caffeine treatment

Summary By means of 3 h treatment with 0.2% caffeine solution, binucleate and tetraploid cells were obtained in the lateral root meristem ofVicia faba. During recovery changing rates of fused interphases were noticed. Cell walls were formed in the equatiorial plane of the preceeding division of binucleate and tetraploid cells at interphase and in the course of bimitosis or 4n-mitosis at prophase or metaphase; during bitelophase a constriction of the fused nuclei could be seen. The conclusion is that the basic requirements of cytokinesis are not affected by caffeine.     

Cell cycle determination of phytohemagglutinin-stimulated lymphocytes from the opossum,Didelphis virginiana

Résumé La courbe PLM (pourcentages indiqués de mitoses) pour les lymphocytes d'opossum in vitro a fait preuve d'unT _c (temps générateur) que n'atteignait pas la somme de G₂, S, et M (mitosis). Tandis que les calculations pour les phases de G₂ et S sont représentées avec exactitude sur la courbe, il y a des variations dans le G₁ parmi les population de lymphocytes au fonctionnement divers (au nombre de deux ou peut-être plus parmi cellesci) qui sont responsables de ce désaccord apparent.     

Binding of aflatoxins B1 and G1 to human serum proteins

Zusammenfassung In vitro Experimente mit¹⁴C-markiertem, gereinigtem Aflatoxin zur Untersuchung der Bindung von Aflatoxin B₁ und G₁ an verschiedene Serumproteine ergaben, dass Aflatoxin B₁ hauptsächlich mit-Globulin, G₁ dagegen vorwiegend mit Albumin bindet.

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This work was supported by part by a grant from the China Medical Board of New York, Inc., and was performed during one of us (S.S.L.) received a class C. research award from the National Science Council, Republic of China.     

Cation inhibition of DNA synthesis in mammary epithelial cells in vitro

Zusammenfassung Lithium- und Ammoniumionen verhindern den Anfang der DNA-Synthese im Milchdrüsenepithel in vitro. Die Replikation von DNA, nach dem Anfang derS-Periode, wird jedoch nicht verhindert. Diese spezifische Verhinderung der Dauer derG ₁-Periode durch die ionale Umgebung zeigt, dass Mechanismen, die für die Regulierung der Zellproduktion wichtig sind, während derG ₁-Periode intervenieren.

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This work was supported in part by grant No. CA 10268 from the U.S. Public Health Service.     

Monoclonal antibodies to L-asparaginase

Summary Five hybridomas secreting monoclonal antibody toE. coli L-asparaginase were isolated. These monoclonal antibodies were classified into 3 different subclasses; Ig G₁ (1 clone), Ig G₂ (2 clones) and Ig G₃ (2 clones). One of them possessed anti-L-asparaginase neutralizing activity. Four antibodies examined demonstrated a linear Langmuir binding plot and binding affinities, with equilibrium dissociation constant (K_d) ranging between 2.5×10^–9M and 6.3×10^–10 M. The monoclonal antibodies should be useful probes for investigation of the enzyme activity.     

Der Mitosezyklus im diploiden und triploiden Wurzelmeristem vonScilla sibirica

Summary In meristematic root tip cells ofScilla sibirica (2n=12 and 3n=18) the following results were obtained with the aid of autoradiography: 1. The average duration of the mitotic cycle (2n=12) is 69.5 h. The G₁-phase lasts 36.5 h, the DNA synthetic phase 17.5 h, the G₂-phase 8 h and mitosis 7.5 h. 2. There are no marked differences in the lengths of the cell cycles nor in the duration of the various phases between diploid and triploid plants.     

Radical yields in X-irradiated aqueous solutions

Résumé La valeur deGh+Goh peut être obtenue en utilisant des solutions aqueuses de certains composés organiques contenant Cu²⁺ et/ou Fe³⁺. Les ions métalliques sont réduits par H et par les radicaux produits par l'oxydation des composés organiques par OH. A concentration convenable des réactifs, on a;G _Fe ²⁺ =G _H+G _OH.     

Ga<Subscript>q</Subscript> proteins: molecular pharmacology and therapeutic potential

Seven transmembrane G protein-coupled receptors (GPCRs) have gained much interest in recent years as it is the largest class among cell surface receptors. G proteins lie in the heart of GPCRs signalling and therefore can be therapeutically targeted to overcome complexities in GPCR responses and signalling. G proteins are classified into four families (G_i, G_s, G_12/13 and G_q); G_q is further subdivided into four classes. Among them G_αq and G_αq/11 isoforms are most crucial and ubiquitously expressed; these isoforms are almost 88% similar at their amino acid sequence but may exhibit functional divergences. However, uncertainties often arise about G_αq and G_αq/11 inhibitors, these G proteins might also have suitability to the invention of novel-specific inhibitors for each isoforms. YM-254890 and UBO-QIC are discovered as potent inhibitors of G_αq functions and also investigated in thrombin protease-activated receptor (PAR)-1 inhibitors and platelet aggregation inhibition. The most likely G protein involved in PAR-1 stimulates responses is one of the G_αq family isoforms. In this review, we highlight the molecular structures and pharmacological responses of G_αq family which may reflect the biochemical and molecular role of G_αq and G_αq/11. The advanced understanding of G_αq and G_αq/11 role in GPCR signalling may shed light on our understanding on cell biology, cellular physiology and pathophysiology and also lead to the development of novel therapeutic agents for a number of diseases.     

Änderungen der Zellzyklusparameter chinesischer Hamsterfibroblasten unter chronischer Hypoxie,gemessen am Impulszytophotometer

Summary By means of a pulse cytophotometer, changes in the distribution of chronically hypoxic Chinese hamster cells in vitro in the different phases of the cell cycle, were measured. Within a few hours, the fraction of cells in G₂ phase and late S phase decreases to a value which is no longer measurable, indicating a partial synchronization in G₁ and early S phase.

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Wir danken Herrn Dr.W. Göhde, Münster, für wertvolle Hinweise zur Interpretation der impulszytophotometrischen Histogramme.     

Mitotic inhibition induced in human kidney cells by methylglyoxal and kethoxal

Zusammenfassung Synchronisierte menschliche Nieren-T-Zellen zeigen eine Mitoseverzögerung, wenn sie in der G₁-, S- oder G₂-Phase des Zyklus Methylglyoxal oder Kethoxal ausgesetzt werden. Die Mitoseverzögerung wird durch 5mM Cysteamin eliminiert.     

Resolution of small G1 and G2 populations of L1210 leukemia by flow microfluorometric analysis aided by velocity sedimentation

Summary L1210 leukemic cells grown in vitro were subjected to kinetic analysis using a flow microfluorometer. A single broad peak was found for the DNA content distribution if unfractionated cells were used; prior fractionation using lg velocity sedimentation allowed the separation of small peaks with smaller (G₁) and larger (G₂) DNA contents from the dominant S phase peak with intermediate DNA content.This work was supported by grant number 5P01CA13053 awarded by the National Cancer Institute, DHEW USA, and by grant num ber RBI 76-1 from the Queen Wilhelmina Fund, The Netherlands.     

Analysis of <Emphasis Type="Italic">Dictyostelium</Emphasis> TACC reveals differential interactions with CP224 and unusual dynamics of <Emphasis Type="Italic">Dictyostelium</Emphasis> microtubules

We have localized TACC to the microtubule-nucleating centrosomal corona and to microtubule plus ends. Using RNAi we proved that Dictyostelium TACC promotes microtubule growth during interphase and mitosis. For the first time we show in vivo that both TACC and XMAP215 family proteins can be differentially localized to microtubule plus ends during interphase and mitosis and that TACC is mainly required for recruitment of an XMAP215-family protein to interphase microtubule plus ends but not for recruitment to centrosomes and kinetochores. Moreover, we have now a marker to study dynamics and behavior of microtubule plus ends in living Dictyostelium cells. In a combination of live cell imaging of microtubule plus ends and fluorescence recovery after photobleaching (FRAP) experiments of GFP-α-tubulin cells we show that Dictyostelium microtubules are dynamic only in the cell periphery, while they remain stable at the centrosome, which also appears to harbor a dynamic pool of tubulin dimers.     

Glucocorticoids suppress cystathionine gamma-lyase expression and H2S production in lipopolysaccharide-treated macrophages

Hydrogen sulfide (H₂S) plays an important role in inflammation. We showed that macrophages expressed the H₂S-forming enzyme cystathionine gamma-lyase (CSE) and produced H₂S. Lipopolysaccharide (LPS) stimulated the CSE expression and H₂S production rate. l-cysteine reduced LPS-induced nitric oxide (NO) production. CSE inhibitor blocked the inhibitory effect of l-cysteine. CSE knockdown increased, whereas CSE overexpression decreased LPS-induced NO production. Dexamethasone suppressed LPS-induced CSE expression and the H₂S production rate as well as NO production. l-arginine increased, whereas N^G-nitro-l-arginine methyl ester (l-NAME) decreased LPS-induced CSE expression and H₂S production. Dexamethasone plus l-NAME significantly decreased LPS-induced CSE expression and H₂S production compared to l-NAME. Our results suggest that macrophages are one of the H₂S producing sources. H₂S might exert anti-inflammatory effects by inhibiting NO production. Dexamethasone may directly inhibit CSE expression and H₂S production, besides the NO-dependent way. Inhibition of H₂S and NO production may be a mechanism by which glucocorticoids coordinate the balance between pro- and anti-inflammatory mediators during inflammation.     

Functional interaction between TRPC1 channel and connexin-43 protein: a novel pathway underlying S1P action on skeletal myogenesis

We recently demonstrated that skeletal muscle differentiation induced by sphingosine 1-phosphate (S1P) requires gap junctions and transient receptor potential canonical 1 (TRPC1) channels. Here, we searched for the signaling pathway linking the channel activity with Cx43 expression/function, investigating the involvement of the Ca²⁺-sensitive protease, m-calpain, and its targets in S1P-induced C2C12 myoblast differentiation. Gene silencing and pharmacological inhibition of TRPC1 significantly reduced Cx43 up-regulation and Cx43/cytoskeletal interaction elicited by S1P. TRPC1-dependent functions were also required for the transient increase of m-calpain activity/expression and the subsequent decrease of PKCα levels. Remarkably, Cx43 expression in S1P-treated myoblasts was reduced by m-calpain-siRNA and enhanced by pharmacological inhibition of classical PKCs, stressing the relevance for calpain/PKCα axis in Cx43 protein remodeling. The contribution of this pathway in myogenesis was also investigated. In conclusion, these findings provide novel mechanisms by which S1P regulates myoblast differentiation and offer interesting therapeutic options to improve skeletal muscle regeneration.     

Effect of prostaglandins on skin tumorigenesis

Summary Concomitant administration of prostaglandins E₂ (PGE₂) and F₂ a(PGF₂ a) with a carcinogen, 3-methylcholanthrene (MCA) to mice for 2 months markedly enhanced the occurrence of squamous cell carcinomas. Only epidermal cell hyperplasia occurred in mice treated with MCA alone by that time. Radioactivity measurements and electron microscopic autoradiography revealed that prostaglandins stimulate DNA, RNA and protein synthesis in neoplastic cells. These findings indicate that PGE₂ and PGF₂ a can act as cocarcinogens on skin tumorigenesis.     

Sphingosine 1-phosphate increases glucose uptake through trans-activation of insulin receptor

Sphingosine 1-phosphate (S1P) is a bioactive lipid that acts through a family of G-protein-coupled receptors. Herein, we report evidence of a novel redox-based cross-talk between S1P and insulin signaling pathways. In skeletal muscle cells S1P, through engagement of its S1P₂ receptor, is found to produce a transient burst of reactive oxygen species through a calcium-dependent activation of the small GTPase Rac1. S1P-induced redox-signaling is sensed by protein tyrosine phosphatase-1B, the main negative regulator of insulin receptor phosphorylation, which undergoes oxidation and enzymatic inhibition. This redox-based inhibition of the phosphatase provokes a ligand-independent trans-phosphorylation of insulin receptor and a strong increase in glucose uptake. Our results propose a new role of S1P, recognizing the lipid as an insulin-mimetic cue and pointing at reactive oxygen species as critical regulators of the cross-talk between S1P and insulin pathways. Any possible implication of S1P-directed insulin signaling in diabetes and insulin resistance remains to be established.