Visualization of neuropeptide-binding sites on individual telencephalic neurons of the rat

The identification of high-affinity binding sites for neuropeptides on individual target cells is a prerequisite when studying the sites of action and the manner in which peptides act as neuromediators. In situ and in vitro, this can be achieved using newly synthesized, biologically active conjugates of somatostatin or cholecystokinin (sulphated octapeptide) with colloidal gold. Labelled neurons show a peptide-specific, non-overlapping distribution in rat telencephalic structures; i.e, whereas the somatostatin-gold conjugate labels binding sites on neurons and glial cells, cholecystokinin-binding sites are restricted to neurons. Binding of either gold-labelled ligand can be competitively suppressed by excess amounts of the native peptide or its analogues. Neuronal somatostatin-binding sites are visualized on neurons in lamina III and, in particular, in lamina V/VI of the primary somatosensory cortex and in the magnocellular nucleus of the telencephalic cholinergic system. Cholecystokinin-binding sites are localized in the main olfactory bulb, on neurons in the cortical hindlimb and forelimb region, in the hippocampus, and in the cingulate and visual cortex.     

Theoretical studies on the necessary number of components in mixtures

Summary Theoretical studies on the necessary number of components in mixtures (for example multiclonal varieties or mixtures of lines) have been performed according to yielding ability. All theoretical investigations are based upon a Gram-Charlier frequency distribution of the component means with skewness ₁ and kurtosis ₂. The selected fraction p of the best components constitutes the mixture under consideration. The same selection differential S = S (p, ₁, ₂) can be realized by different parameter values of p, ₁ and ₂. Therefore, equal yield levels of the mixture can be achieved by different selected fractions p which implies different numbers of components in the mixture. Numerical results of S = S(p) for different values of ₁ and ₂ are presented and discussed. Of particular interest are the selected fractions p which lead to a maximal selection differential S. These results on S for large populations must be reduced in the case of finite population size. For this correction term we used an approximation B = B (p, n, ₁, ₂) given by Burrows (1972) where n = number of selected components. For given parameter values of ₁, ₂ and p, the necessary number n of components can be calculated by using the condition: Burrows-correction less than a certain percentage g of S — for example with g = 0.05 or g = 0.01. For given ₁ and ₂, the number n leading to a maximal selection differential S can be regarded as necessary number of components (necessary = maximum gain of selection under the given conditions). Numerical results are given for ₂ = 0 and for eight situations which are defined by linear relations ₂ = c ₁ between skewness and kurtosis. These cases will contain all possible numerical situations for ₁ and ₂, which may be relevant for practical applications. The necessary number of components turns out to be nearly independent of the numerical value of the kurtosis ₂. The n-intervals leading to selected fractions p from 0.01 to 0.20 approximately are: 2 n 4 for g = 0.05, 6 n 20 for g = 0.01 and 11 n 40 for g = 0.005, respectively. However, percentages g less than 0.01 would be unrealistically excessive. Therefore, following the assumptions and restrictions given in this paper one may conclude that n = 20 seems to be an appropriate upper bound for the necessary number of components in mixtures.     

Coordination dynamics of heme-copper oxidases. The ligand shuttle and the control and coupling of electron transfer and proton translocation

Results are presented which, taken with evidence developed by others, suggest a general mechanism for the entry and binding of exogenous ligands (including O₂) at the binuclear site (Cu_B Fe _a3) of the heme-copper oxidases. The mechanism includes a ligand shuttle wherein the obligatory waystation for incoming ligands is Cu_B and the binding of exogenous ligands at this site triggers the exchange and displacement of endogenous ligands at Fe _a3. It is suggested that these ligand shuttle reactions might be functionally important in providing a coupling mechanism for electron transfer and proton translocation. Scenarios as to how this might happen are delineated.     

Biphasic Modulation of GABAA Receptor Binding by Steroids Suggests Functional Correlates

Neuroactive steroids and other positive modulators of GABA_A receptors showed regional variation in both the efficacy and potency for modulation of [³⁵S]TBPS binding to rat brain membrane homogenates, with biphasic concentration-dependence. GABA present in the binding assays prevented the enhancement phase of the steroid concentration-dependence plot while the antagonists bicuculline and RU5135 prevented the inhibition phase. Using recombinant GABA_A receptors, expressed in insect cell line Sf9 using baculovirus, enhancement by steroids of [³⁵S]TBPS binding was sensitive to the presence of the 2 subunit and the nature of the subunit (122S > 12, 62, 622S, and 62). As in cerebellum, addition of RU5135 reduced the inhibitory phase and revealed a small enhancement of TBPS binding by neuroactive steroids. The subunit-dependent interactions of steroid and GABA site ligands are consistent with a three-state model in which the receptor mono-liganded by GABA or steroid has a different affinity for TBPS than the resting state, and the receptor biliganded by GABA, steroid, or both has little affinity for TBPS.     

Binding of benzo(α)pyrene, ellipticine, and cis-parinaric acid to β-lactoglobulin: Influence of protein modifications

The binding of benzo()pyrene, ellipticine, and cis-parinaric acid to native, esterified, and alkylated -lactoglobulin was followed by enhancement of the ligand fluorescence. Three studied ligands bind to native or modified -lactoglobulin in apparent molar ratios varying between 1/8 and 2/1, with apparent dissociation constants in the range of 10^–8 M for ligand/-lactoglobulin complexes. The studied, chemically modified -lactoglobulin derivatives display higher binding affinities for all studied ligands, cis-parinaric acid excluded. The reductive alkylation of -NH₂ lysyl residues of -lactoglobulin increases the apparent molar ratios of benzo()pyrene and cis-parinaric acid, and decreases it for ellipticine. The esterified and native -lactoglobulin complexed to the investigated ligands display similar stoichiometries. Dynamic light scattering study of ligand--lactoglobulin complexes in solution shows the formation of aggregates: the apparent hydrodynamic radius value of -lactoglobulin dimer (3.4 nm) reaches 49, 46, and 74 nm upon addition and binding of benzo()pyrene, ellipticine, and cis-parinaric acid, respectively.     

Liver and intestinal fatty acid binding proteins in control and TGFβ1 gene targeted deficient mice

The effect of transforming growth factor beta-1 (TGF1) expression on fatty acid binding proteins was examined in control and two strains of gene targeted TGF1-deficient mice. Homozygous TGF1-deficient 129 × CF-1, expressing multifocal inflammatory syndrome, had 25% less liver fatty acid binding protein (L-FABP) when compared to control mice. The decrease in L-FABP expression was not due to multifocal inflammatory syndrome since homozygous TGF1-deficient/immunodeficient C3H mice on a SLID background had 36% lower liver L-FABP than controls. This effect was developmentally related and specific to liver, but not the proximal intestine, where L-FABP is also expressed. Finally, the proximal intestine also expresses intestinal-FABP (1-FABP) which decreased 3-fold in the TGF1-deficient/immunodeficient C3H mice only. Thus, TGF1 appears to regulate the expression of L-FABP and I-FABP in the liver and the proximal intestine, respectively.Abbreviations L-FABP liver fatty acid binding protein - I-FABP intestinal fatty acid binding protein - TGF1 transforming growth factor beta-1 - TNF- tumor necrosis factor- - MIP- macrophage inflammatory protein- - PMSF phenylmethyl sulfonyl fluoride - PBS phosphate buffered saline     

Molecular Modeling Studies on Novel Open-chain and Cyclic Thia Compounds and their Ag(I) and Hg(II) Complexes

Molecular modeling techniques were used to generate structures of several HLA-DQ proteins associated with insulin-dependent diabetes mellitus (IDDM). A peptide fragment from glutamic acid decarboxylase (GAD), a known IDDM autoantigen, binds to certain HLA-DQ molecules positively associated with IDDM. Modeling studies were used to explore possible binding interactions between this GAD peptide and several HLA-DQ molecules. Based on the characterization of anchor pockets in the HLA-DQ binding groove and of peptide side chains, a novel binding mode was proposed. This binding mode predicts the GAD peptide is positioned in the binding groove in the direction opposite the orientation observed for class I proteins and the class II DR1, DR3, and I-E^k proteins. Peptide docking exercises were performed to construct models of the HLA-DQ/peptide complexes, and the resulting models have been used to design peptide binding experiments to test this reverse-orientation binding mode. A variety of experimental results are consistent with the proposed model and suggest that some peptide ligands of class II molecules may bind in a reversed orientation within the binding groove.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s0089460020205     

Charge clusters and the orientation of membrane proteins

Summary Although hydrophobic forces probably dominate in determining whether or not a protein will insert into a membrane, recent studies in our laboratory suggest that electrostatic forces may influence the final orientation of the inserted protein. A negatively charged hepatic receptor protein was found to respond totrans-positive membrane potentials as though electrophoresing into the bilayer. In the presence of ligand, the protein appeared to cross the membrane and expose binding sites on the opposite side. Similarly, a positively charged portion of the peptide melittin crosses a lipid membrane reversibly in response to atrans-negative potential. These findings, and others by Date and co-workers, have led us to postulate that transmembrane proteins would have hydrophobic transmembrane segments bracketed by positively charged residues on the cytoplasmic side and negatively charged residues on the extra-cytoplasmic side. In the thermodynamic sense, these asymmetrically placed charge clusters would create a compelling preference for correct orientation of the protein, given the inside-negative potential of most or all cells. This prediction is borne out by examination of the few transmembrane proteins (glycophorin, M13 coat protein, H-2K^b, HLA-A2, HLA-B7, and mouse Ig heavy chain) for which we have sufficient information on both sequence and orientation.In addition to the usual diffusion and pump potentials measurable with electrodes, the microscopic membrane potential reflects surface charge effects. Asymmetries in surface charge arising from either ionic or lipid asymmetries would be expected to enhance the bias for correct protein orientation, at least with respect to plasma membranes. We introduce a generalized form of Stern equation to assess surface charge and binding effects quantitatively. In the kinetic sense, dipole potentials within the membrane would tend to prevent positively charged residues from crossing the membrane to leave the cytoplasm. These considerations are consistent with the observed protein orientations. Finally, the electrostatic and hydrophobic factors noted here are combined in two hypothetical models of translocation, the first involving initial interaction of the presumptive transmembrane segment with the membrane; the second assuming initial interaction of a leader sequence.     

Physical and Epitope Analysis of a Recombinant Human T-Cell Receptor Vα/Vβ Construct Support the Similarity to Immunoglobulin

The genetic organization and protein structure of T-cell receptors (TCR) and immunoglobulins (Ig) are remarkably similar. Through recombinant, physical, and peptide-based immunological studies we demonstrated that rabbit antisera generated against a recombinant single-chain TCR (scTCR) react with defined peptide epitopes of their constituent TCR and chains. These antisera cross-react with the light-chain Mcg as well as with peptides duplicating its covalent structure. Conversely, rabbit antisera generated to human light chains cross-reacted with the recombinant scTCR. Rabbit anti- antibodies purified on an scTCR affinity column bound to T-cell lines and to T and B lymphocytes from peripheral blood. Circular dichroism analysis demonstrated plots characteristic of -sheets for both Mcg and recombinant scTCR. Antisera directed against TCR -chain synthetic peptides reacted with scTCR, Mcg light-chain protein, synthetic peptides from regions of sequence homology in -chains, and Mcg. Based upon this homology and the serological cross-reactions which reflect conformational determinants, we suggest that the V/V antigen-binding domain of this particular monoclonal scTCR construct is substantially similar to the conformational structure of light chains.     

The effect of charge reversal mutations in the α-helical region of liver fatty acid binding protein on the binding of fatty-acyl CoAs,lysophospholipids and bile acids

Liver fatty acid binding protein (LFABP) is unique among the various types of FABPs in that it can bind a variety of ligands in addition to fatty acids. LFABP is able to bind long chain fatty acids with a 2:1 stoichiometry and the crystal structure has identified two fatty acid binding sites in the binding cavity. The presumed primary site (site 1) involves the fatty acid binding with the carboxylate group buried in the cavity whereas the fatty acid at site 2 has the carboxylate group solvent-exposed within the ligand portal region and in the vicinity of -helix II. The -helical region contains three cationic residues, K20, K31, K33 and modelling studies suggest that K31 on -helix II could make an electrostatic contribution to anionic ligands binding to site 2. The preparation of three charge reversal mutants of LFABP, K20E, K31E and K33E has allowed an investigation of the role of site 2 in ligand binding, particularly those ligands with a bulky anionic head group. The binding of oleoyl CoA, lysophosphatidic acid, lysophosphatidylcholine, lithocholic acid and taurolithocholate 3-sulphate to LFABP has been studied using the -helical mutants. The results support the concept that such ligands bind at site 2 of LFABP where solvent exposure allows the accommodation of their bulky anionic group.     

Solution structure of the human BTK SH3 domain complexed with a proline-rich peptide from p120cbl

X-linked agammaglobulinemia (XLA), an inherited disease, is caused by mutations in the Bruton's tyrosine kinase (BTK). The absence of functional BTK leads to failure of B cell differentiation which incapacitates antibody production in XLA patients leading to, sometimes lethal, bacterial infections. Point mutation in the BTK gene that leads to deletion of C-terminal 14 aa residues of BTK SH3 domain was found in one patient family. To understand the role of BTK in B cell development, we have determined the solution structure of BTK SH3 domain complexed with a proline-rich peptide from the protein product of c-cbl protooncogene (p120^cbl). Like other SH3 domains, BTK SH3 domain consists of five -strands packed in two -sheets forming a -barrel-like structure. The rmsd calculated from the averaged coordinates for the BTK SH3 domain residues 218–271 and the p120^cbl peptide residues 6–12 of the complex was 0.87 Å (±0.16 Å) for the backbone heavy atoms (N, C, and C) and 1.64 Å (±0.16 Å) for all heavy atoms. Based on chemical shift changes and inter-molecular NOEs, we have found that the residues located in the RT loop, n-Src loop and helix-like loop between 4 and 5 of BTK SH3 domain are involved in ligand binding. We have also determined that the proline-rich peptide from p120^cbl binds to BTK SH3 domain in a class I orientation. These results correlate well with our earlier observation that the truncated BTK SH3 domain (deletion of 4, 5 and the helix-like loop) exhibits weaker affinity for the p120^cbl peptide. It is likely that the truncated SH3 domain fails to present to the ligand the crucial residues in the correct context and hence the weaker binding. These results delineate the importance of the C-terminus in the binding of SH3 domains and also indicate that improper folding and the altered binding behavior of mutant BTK SH3 domain likely lead to XLA.     

Preparations of Ψ-peptide bond and peptide-aldehyde inhibitors of atrial granule serine proteinase, a candidate processing enzyme of pro-atrial natriuretic factor

Pseudo-peptide bond inhibitors (-bond inhibitors) and peptide-aldehyde inhibitors of atrial granule serine proteinase, the candidate processing enzyme of pro-atrial natrieuretic factor, are prepared in high yield and purity by novel synthetic routes. The -bond compounds retain essential residues for enzyme binding, but place the enzyme inhibition site in the midst of the peptide sequence. Thus, Bz-APR--LR and Bz-APR--SLRR can be considered readthrough inhibitors of atrial granule serine proteinase. The most potent -peptide, Bz-APR--SLRR (IC₅₀=250 M), is about fivefold less potent than the best peptide-aldehyde inhibitor (EACA-APR-CHO), and both the -bond and peptide-aldehyde compounds are competitive, reversible inhibitors of the enzyme. The -bond peptides containing two C-terminal Arg residues are three-to tenfold more potent than the analogous compounds containing only one C-terminal Arg residue, confirming the importance of both Arg residues in the enzyme processing recognition site. As expected, because of their moderate potencies, the -peptides are not useful affinity ligands for purification of atrial granule serine proteinase, but both peptide aldehydes are effective affinity ligands [Damodaran and Harris (1995),J. Protein Chem., this issue].Abbreviations AGSP atrial granule serine proteinase - ANF atrial natriuretic factor - Bz benzoyl - DIEA diisopropylethylamine - DIPCDI diisopropylcarbodiimide - DMF dimethylformamide - DMSO dimethylsulfoxide - EACA 6(e)-aminocaproic acid - EtOAc ethyl acetate - HEPES N-2-hydroxyethylpiperazine-N-propanesulfonic acid - HOBt N-hydroxybenzotriazole - HPLC high-performance liquid chrornatography - NMR nuclear magnetic resonance - PEG polyethylene glycol-3350 - PyBOP benzotriazole-1-yl-oxy-trispyrrolidino-phosphonium-hexafluorophospate - TEA triethylamine - TFA trifluoroacetic acid - THF tetrahydrofuran - TLC thin-layer chromatography - UV ultraviolet - pseudo-peptide bond -CH₂-NH-. Single-letter abbreviations are used to denote amino acids     

Harzkonservierte fossile Vogelfedern aus der untersten Kreide

Zusammenfassung Harzkonservierte Fossilien ermöglichen bei Anwendung adäquater Methoden die morphologische Analyse der Feinmerkmale bis zur Auflösungsgrenze des Lichtmikroskops, Beobachtung in verschiedenen Ebenen und Richtungen, und somit konkrete Rückschlüsse auf die Wirkung und Bedeutung der Einzelelemente und des Gesamtgefüges.Eine so eingehende funktionsmorphologische Analyse mit Berücksichtigung der Positionsvariation (graduell verschiedene Gestaltung in gesetzmäßiger Abhängigkeit von der Lage innerhalb der Gesamtfeder) der Einzelelemente wie Abzweigungs-, Knick-, Neigungswinkel, Krümmung, Länge, Dicke, Querschnitt, Dichte, Differenzierungsgrad der verschiedenen Abschnitte von Rhachis, Rami, Radii inklusive Häkchen und Cirren wird erstmals für fossile Vogelfedern geliefert (hier als Abriß zu einer dokumentarisch und thematisch ausführlicheren Darstellung in Stuttgarter Beiträge zur Naturkunde).Diese Federn entstammen der untersten Unterkreide und sind damit nur relativ wenig jünger alsArchaeopteryx. Sie weisen extrem differenzierten Aufbau auf, der auf hohe flugtechnische und wärmeisolierende Leistungsfähigkeit schließen läßt.Die hier vorgelegten funktionsmorphologischen Ermittlungen an fossilen Körperkonturfedern mögen auch zu einer intensiveren Analyse der bis jetzt stark vernachlässigten Untersuchung ganz normaler Körperfedern rezenter Vögel anregen. Erst dann, nach umfassender Kenntnis ihrer Ausgestaltung innerhalb der verschiedensten rezenten Vogelgruppen, läßt sich überzeugend begründen, ob und wieweit die hier vorgelegten Federn dieses Unterkreide-Vogels noch ursprüngliche Elemente (Plesiomorphien) oder ihnen eigene Sonderbildungen (Autapomorphien) aufweisen; das gilt sowohl für morphologische wie für funktionelle Elemente der Gesamtstruktur.

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Resin-preserved fossil bird's feathers from the Lowermost Cretaceous

Summary Parts of some feathers, originating from a single bird, were discovered in our collections of Lower Cretaceous amber from the Lebanon mountains — which, in general, contains the oldest terrestrial microfossils preserved with all morphological details.These contour feathers of the trunk, which are nearly as old as Archaeopteryx (Lowermost Cretaceous: Neocomian/Uppermost Jurassic: Kimmeridigian) were studied with magnifications of 500–900 in several levels by a special technique. (In normal fossils, i.e., impressions, the granulation of the sediment and the fossil's bulky carbon remainders cause a blurred image even at a magnification of merely 100).Special emphasis was laid on the study of the individual elements' gradual variation, depending on the respective position within the total feather (position variation). Where appropriate, an analysis of lengths, quantity, degree of differentiation, angle of inclination, break, and branching, cross-sectional view, curvature, etc. of the rhachis, rami, distal and proximal radii, barbicles, hooklets, etc. were undertaken. [Through measurements of the depth of details the effects caused by a sloping position (apparent variation) may be precisely separated from the real variation.]On the basis of such a detailed knowledge of structure and relative position a thorough functional analysis of the single elements as well as the total system is given.Principal features: The production of plain stability in the feather's center, and of flexibility in its apical and lateral rims; dispersion of forces in case of pressure or a pulling load; function of the hooklets (which donot serve as an interlocking mechanism while the feather is in the normal resting position, but function with increasing braking action only when a neighboring ramus diverges to a precisely defined extent from its resting position) including the mechanism of their unhooking; devices for the avoidance of harmful hooking into contacted parts of other feathers; production of maximal stability by minimal air resistance, and of minute chambers (<0,00001 mm³) with still air for optimal heat isolation.Apart from this abstract, further information, accompanied by numerous figures, will be given in a later paper in Stuttgarter Beiträge zur Naturkunde.

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Veränderte Fassung eines am 11. 10. 1971 gehaltenen Vortrages auf der 83. Jahresversammlung der Deutschen Ornithologengesellschaft in Bonn.     

Upregulation of NMDA Receptors in Hippocampus and Cortex in the Pentylenetetrazol-Induced “Kindling” Model of Epilepsy

Kindling is a phenomenon of epileptogenesis, which has been widely used as an experimental model of temporal lobe epilepsy. At the present work we investigated the contribution of NMDA receptors in the Pentylenetetrazol-induced kindling model in the mouse brain, by using quantitative autoradiography and the radioactive ligands [³H]MK801 and [³H]L-glutamate (NMDA-sensitive component). One week after establishment of kindling, a small but significant increase in [³H]MK801 as well as NMDA-sensitive [³H]glutamate binding was seen, being restricted to the molecular layer (ML) of the dentate gyrus (DG) and the CA3 region of the hippocampus. These binding augmentations persisted one month after establishment of kindling. A significant increase of NMDA receptor binding was also observed in the cortex-somatosensory and temporal one week after acquisition of the kindled state. The upregulation of NMDA receptors seen in DG and CA3 region of the hippocampus could be associated with the kindling process of this model especially with its maintenance phase, since it persists at long term, is area-specific and consistent with electrophysiological data. The increase of NMDA receptors seen in the cortex of the kindled animals could underlie the hyperexcitability detected by electrophysiological studies in this area.     

Signalling by protein kinase C isoforms in the heart

Understanding transmembrane signalling process is one of the major challenge of the decade. In most tissues, since Fisher and Krebs's discovery in the 1950's, protein phosphorylation has been widely recognized as a key event of this cellular function. Indeed, binding of hormones or neurotransmitters to specific membrane receptors leads to the generation of cytosoluble second messengers which in turn activate a specific protein kinase. Numerous protein kinases have been so far identified and roughly classified into two groups, namely serine/threonine and tyrosine kinases on the basis of the target amino acid although some more recently discovered kinases like MEK (or MAP kinase kinase) phosphorylate both serine and tyrosine residues.Protein kinase C is a serine/threonine kinase that was first described by Takai et al. [1] as a Ca- and phospholipid-dependent protein kinase. Later on, Kuo et al. [2] found that PKC was expressed in most tissues including the heart. The field of investigation became more complicated when it was found that the kinase is not a single molecular entity and that several isoforms exist. At present, 12 PKC isoforms and other PKC-related kinases [3] were identified in mammalian tissues. These are classified into three groups. (1) the Ca-activated -, -,and -PKCs which display a Ca-binding site (C2); (2) the Ca-insensitive -, -, -, -, and -PKCs. The kinases that belong to both of these groups display two cystein-rich domains (C1) which bind phorbol esters (for recent review on PKC structure, see [4]). (3) The third group was named atypical PKCs and include , , and -PKCs that lack both the C2 and one cystein-rich domain. Consequently, these isoforms are Ca-insensitive and cannot be activated by phorbol esters [5]. In the heart. evidence that multiple PKC isoforms exist was first provided by Kosaka et al. [6] who identified by chromatography at least two PKC-related isoenzymes. Numerous studies were thus devoted to the biochemical characterization of these isoenzymes (see [7] for review on cardiac PKCs) as well as to the identification of their substrates.This overview aims at updating the present knowledge on the expression, activation and functions of PKC isoforms in cardiac cells. (Mol Cell Biochem 157: 65–72, 1996)     

High levels of soluble IFN γ receptor α chain in the plasma of rheumatoid arthritis patients

Soluble receptors for hormones and cytokines have beendescribed. They can serve as natural blockers of theirrespective ligands. The natural soluble interferongamma receptor (sIFNR) has been isolated andcharacterized only in urine. Chromatography of human(hu) plasma from rheumatoid arthritis (RA) patientsand controls on immobilized hu IFN orantibodies against IFN R chainpermitted us to isolate the sIFNR. Thereceptor isolated from one control is a protein witha molecular weight between 60-67 kDa depending on thepresence of reducing agents. We detected asignificantly higher level of plasma sIFNR inpatients with rheumatoid arthritis than in apparentlyhealthy subjects.     

Nucleotide regulation of vasoactive intestinal peptide binding to bovine thyroid plasma membranes

The specific binding of vasoactive intestinal peptide (VIP) to bovine thyroid plasma membranes is inhibited by guanine nucleotides. Guanosine 5-triphosphate (GTP) and the non-hydrolyzable GTP analogs guanosine 5-,-imidotriphosphate (Gpp(NH)p) and guanosine 5-O-(3-thiotriphosphate) (GTP--S) inhibited markedly the binding of VIP to its receptors. This inhibition was higher with GTP than with Gpp(NH)p and GTP--S and was due to an increase of the rate of dissociation of peptide bound to membranes. Other nucleotides did not show any effect.     

Improved in situ β-galactosidase staining for histological analysis of transgenic mice

The present study describes a novel method for the histochemical demonstration of -galactosidase activity on tissue sections. We have replaced 5-bromo-4-chloro-3-indolyl--D-galactoside (X-Gal) with 5-bromoindolyl--o-galactopyranoside (Bluo-Gal) as a chromogenic substrate for the bacterial -galactosidase (lacZ). After -galactosidic cleavage, Bluo-Gal precipitates in form of fine birefringent crystals, whereas X-gal gives rise to an amorphous precipitate. Upon microscopic examination under polarized light, the crystals emit a strong signal consisting of yellow reflected light. This property of Bluo-Gal results in greatly enhanced sensitivity of the staining method for -galactosidase and allows for optimal morphological resolution. To exemplify the applications of this technique, the expression is demonstrated in transgenic mice of -galactosidase driven by a fragment of the human tissue-type plasminogen activator promoter.     

Development of an L6 myoblast in vitro model of moniliformin toxicosis

L6 myoblasts were used as an in vitro model to investigate the role of moniliformin and its interaction with monensin in turkey knockdown syndrome and sudden death syndromes in poultry. Cell viability and microscopic and ultrastructural alterations noted in L6 myoblasts cultured in the presence of moniliformin (0.0–0.3 g/l) were compared to those observed in parallel cultures also containing one of the following compounds: selenium (0–0.004 ng/l), thiamine (0–0.3 g/l), or pyruvate (0–0.46 g/l). Marked dilation of the RER, membranous whorls, glycogen deposition, membrane-bound cytoplasmic inclusions and necrosis were observed in myoblasts exposed to 0.03/2-0.30 g moniliformin/l medium. Supplementation of medium with thiamine and pyruvate, or selenium, provided significant protection to cells exposed to 0.0–0.3 g/l or 0.0–0.15 g moniliformin/l, respectively. Dose-dependent differences in protein and ATP production were not detected. Myoblasts grown in medium containing 0–0.15 g moniliformin/l and 7.5–50.0 M A23187, beauvericin or monensin had degrees of cytotoxicity similar to parallel cultures receiving only an ionophore. L6 myoblasts were a useful model of moniliformin toxicosis. The findings of this study suggest cytotoxicity due to moniliformin in L6 myoblasts may be due in part to oxidative damage and altered pyruvate metabolism, and that moniliformin does not predispose myoblasts to ionophore toxicosis. This study supports the results of in vivo investigations in poultry that moniliformin and monensin do not act synergistically to induce knockdown or monensin toxicosis.