Detailed analysis of mortality rates in the female progeny of 1,001 Holstein bulls allows the discovery of new dominant genetic defects.

Reducing juvenile mortality in cattle is important for both economic and animal welfare reasons. Previous studies have revealed a large variability in mortality rates between breeds and sire progeny groups, with some extreme cases due to dominant mutations causing various syndromes among the descendants of mosaic bulls. The purpose of this study was to monitor sire-family calf mortality within the French and Walloon Holstein populations, and to use this information to detect genetic defects that might have been overlooked by lack of specific symptoms. In a population of heifers born from 1,001 bulls between 2017 and 2020, the average sire-family mortality rates were of 11.8% from birth to 1 year of age and of 4.2, 2.9, 3.1, and 3.2% for the perinatal, postnatal, preweaning, and postweaning subperiods, respectively. After outlining the 5 worst bulls per category, we paid particular attention to the bulls Mo and Pa, because they were half-brothers. Using a battery of approaches, including necropsies, karyotyping, genetic mapping, and whole-genome sequencing, we described 2 new independent genetic defects in their progeny and their molecular etiology. Mo was found to carry a de novo reciprocal translocation between chromosomes BTA26 and BTA29, leading to increased embryonic and juvenile mortality because of aneuploidy. Clinical examination of 2 calves that were monosomic for a large proportion of BTA29, including an orthologous segment deleted in human Jacobsen syndrome, revealed symptoms shared between species. In contrast, Pa was found to be mosaic for a dominant de novo nonsense mutation of GATA 6 binding protein (GATA6), causing severe cardiac malformations. In conclusion, our results highlight the power of monitoring juvenile mortality to identify dominant genetic defects due to de novo mutation events.

The role of adenosine receptors in regulating production of tumour necrosis factor-alpha and chemokines by human lung macrophages.

BACKGROUND AND PURPOSE: Adenosine is a major endogenous regulator of macrophage function, and activates four specific adenosine receptors (A(1), A(2A), A(2B) and A(3)). Here, we have assessed in human lung macrophages the modulation of the expression of adenosine receptor mRNA by lipopolysaccharide (LPS), and the relative contributions of the different adenosine receptors to LPS-induced production of tumour necrosis factor (TNF)-alpha and chemokines. EXPERIMENTAL APPROACH: Lung macrophages isolated from resected lungs were stimulated with LPS and treated with adenosine receptor agonists or/and antagonists. Adenosine receptor expression was assessed with qRT-PCR. Cytokines were measured in lung macrophage supernatants with elisa. KEY RESULTS: LPS increased (about 400-fold) mRNA for A(2A) adenosine receptors, decreased mRNA for A(1) and A(2B), but had no effect on A(3) adenosine receptor mRNA. The adenosine receptor agonist NECA inhibited TNF-alpha production concentration dependently, whereas the A(1) receptor agonist, CCPA, and the A(3) receptor agonist, AB-MECA, inhibited TNF-alpha production only at concentrations affecting A(2A) receptors. NECA also inhibited the production of CCL chemokines (CCL2, CCL3, CCL4, CCL5) and CXCL chemokines (CXCL9 and CXCL10), but not that of CXCL1, CXCL8 and CXCL5. Reversal of NECA-induced inhibition of TNF-alpha and chemokine production by the selective A(2A) adenosine receptor antagonist ZM 241385, but not the A(2B) receptor antagonist, MRS 1754, or the A(3) receptor antagonist, MRS 1220, indicated involvement of A(2A) receptors. CONCLUSIONS AND IMPLICATIONS: LPS up-regulated A(2A) adenosine receptor gene transcription, and this receptor subtype mediated inhibition of the LPS-induced production of TNF-alpha and of a subset of chemokines in human lung macrophages.

Protocadherin 15 (PCDH15): a new secreted isoform and a potential marker for NK/T cell lymphomas.

Natural killer cells are well known to play an important role in immune defense against tumor development and viral infections. To further characterize new functionally relevant structures in these cells, we studied a series of monoclonal antibodies that we have raised against the NK cell line YT. One of these antibodies previously described as AY19, recognizes a 85 kD surface glycoprotein. Here we report the identification of a new secreted isoform of protocadherin 15, PCDH15C, which represents a potential associated protein for p85. Importantly, whereas protocadherins are absent from the surface of normal hematopoietic cells, we describe, for the first time, that PCDH15 is expressed in cytotoxic tumor-derived T- and NK-cell lines as well as in biopsies of nasal NK/T-cell lymphomas.

SSR591813, a novel selective and partial alpha4beta2 nicotinic receptor agonist with potential as an aid to smoking cessation.

(5aS,8S,10aR)-5a,6,9,10-Tetrahydro,7H,11H-8,10a-methanopyrido[2',3':5,6]pyrano[2,3-d]azepine (SSR591813) is a novel compound that binds with high affinity to the rat and human alpha4beta2 nicotinic acetylcholine receptor (nAChR) subtypes (Ki = 107 and 36 nM, respectively) and displays selectivity for the alpha4beta2 nAChR (Ki, human alpha3beta4 > 1000, alpha3beta2 = 116; alpha1beta1deltagamma > 6000 nM and rat alpha7 > 6000 nM). Electrophysiological experiments indicate that SSR591813 is a partial agonist at the human alpha4beta2 nAChR subtype (EC50 = 1.3 micro M, IA =19% compared with the full agonist 1,1-dimethyl-4-phenyl-piperazinium). In vivo findings from microdialysis and drug discrimination studies confirm the partial intrinsic activity of SSR591813. The drug increases dopamine release in the nucleus accumbens shell (30 mg/kg i.p.) and generalizes to nicotine or amphetamine (10-20 mg/kg i.p.) in rats, with an efficacy approximately 2-fold lower than that of nicotine. Pretreatment with SSR591813 (10 mg/kg i.p.) reduces the dopamine-releasing and discriminative effects of nicotine. SSR591813 shows activity in animal models of nicotine dependence at doses devoid of unwanted side effects typically observed with nicotine (hypothermia and cardiovascular effects). The compound (10 mg/kg i.p.) also prevents withdrawal signs precipitated by mecamylamine in nicotine-dependent rats and partially blocks the discriminative cue of an acute precipitated withdrawal. SSR591813 (20 mg/kg i.p.) reduces i.v. nicotine self-administration and antagonizes nicotine-induced behavioral sensitization in rats. The present results confirm important role for alpha4beta2 nAChRs in mediating nicotine dependence and suggest that SSR591813, a partial agonist at this particular nAChR subtype, may have therapeutic potential in the clinical management of smoking cessation.

Novel N-(arylalkyl)indol-3-ylglyoxylylamides targeted as ligands of the benzodiazepine receptor: synthesis, biological evaluation, and molecular modeling analysis of the structure-activity relationships.

A series of N-(arylalkyl)indol-3-ylglyoxylylamides (4-8) was synthesized as ligands of the benzodiazepine receptor (BzR) and tested for their ability to displace [(3)H]flumazenil from bovine brain membranes. The new compounds, bearing a branched (4) or a geometrically constrained benzyl/phenylethyl amide side chain (5-8), represent the continuation of our research on N-benzylindol-3-ylglyoxylylamides 1 (Da Settimo et al., 1996), N'-phenylindol-3-ylglyoxylohydrazides 2 (Da Settimo et al., 1998), and N-(indol-3-ylglyoxylyl)alanine derivatives 3 (Primofiore et al., 1989). A few indoles belonging to the previously investigated benzylamides 1 and phenylhydrazides 2 were synthesized and tested to enrich the SARs in these two series. The affinities and the GABA ratios of selected compounds for clonal mammalian alpha(1)beta(2)gamma(2), alpha(3)beta(2)gamma(2), and alpha(5)beta(3)gamma(2) BzR subtypes were also determined. It was hypothesized that the reduced flexibility of indoles 4-8 would both facilitate the mapping of the BzR binding cleft and increase the chances of conferring selectivity for the considered receptor subtypes. In the series of indoles 4, the introduction of a methyl group on the benzylic carbon with the R configuration improved affinity of the 5-substituted (5-Cl and 5-NO(2)) derivatives, whereas it was detrimental for their 5-unsubtituted (5-H) counterparts. All S enantiomers were less potent than the R ones. Replacement of the methyl with hydrophilic substituents on the benzylic carbon lowered affinity. The isoindolinylamide side chain was tolerated if the 5-position was unsubstituted (K(i) of 5a = 123 nM), otherwise affinity was abolished (5b, c). All the 2-indanylamides 6 and (S)-1-indanylamides 8 were devoid of any appreciable affinity. The 5-Cl and 5-NO(2) (R)-1-indanylamides 7b (K(i) 80 nM) and 7c (K(i) 28 nM) were the most potent among the indoles 5-8 geometrically constrained about the side chain. The 5-H (R)-1-indanylamide 7a displayed a lower affinity (K(i) 675 nM). The SARs developed from the new compounds, together with those collected from our previous studies, confirmed the hypothesis of different binding modes for 5-substituted and 5-unsubstituted indoles, suggesting that the shape of the lipophilic pocket L(1) (notation in accordance with Cook's BzR topological model) is asymmetric and highlighted the stereoelectronic and conformational properties of the amide side chain required for high potency. Several of the new indoles showed selectivity for the alpha(1)beta(2)gamma(2) subtype compared with the alpha(3)beta(2)gamma(2) and alpha(5)beta(3)gamma(2) subtypes (e.g.: 4t and 7c bind to these three BzR isoforms with K(i) values of 14 nM, 283 nM, 239 nM, and 9 nM, 1960 nM, 95 nM, respectively). The GABA ratios close to unity exhibited by all the tested compounds on each BzR subtype were predictive of an efficacy profile typical of antagonists.

Inhibition of glioma cells in vitro and in vivo using a recombinant adenoviral vector containing an astrocyte-specific promoter.

Gene therapy using the herpes simplex virus thymidine kinase (HSV-TK) gene in combination with the drug ganciclovir (GCV) is a promising approach for the treatment of cancer-inducing gliomas, a tumor with a poor prognosis. In an attempt to limit the toxic effects on normal tissues, we constructed a recombinant adenoviral vector, Adgfa2TK, in which the HSV-TK gene is driven by the promoter for the gene encoding glial fibrillary acidic protein, an intermediate filament protein expressed primarily in astrocytes. Infection by Adgfa2TK of a glial cell line (C6) and a non-glial cell line (MDA-MB-231) revealed markedly increased expression of HSV-TK in glial cells as determined by Western blot. In comparison, high HSV-TK protein levels were produced in both cell lines after infection with a control virus, AdCMVTK, in which the constitutive cytomegalovirus viral promoter was used to direct HSV-TK expression. Infection of two glial cell lines (C6, U251) and two non-glial cell lines (HepG2, MDA-MB-231) with Adgfa2TK followed by GCV treatment revealed high toxicity in glial cell lines (50% growth inhibitory concentration: <2 microg/mL of GCV) with little or no toxicity (50% growth inhibitory concentration: >75 microg/mL) in the non-glial cell lines. In vivo, injection of Adgfa2TK into C6 tumors grown in nude mice followed by intraperitoneal GCV treatment significantly repressed tumor growth compared with the controls. Adgfa2TK may be useful for directing expression of the HSV-TK gene to gliomas.

Regulation of ATP-sensitive potassium channel mRNA expression in rat kidney following ischemic injury.

ATP-sensitive potassium (K(ATP)) channels are involved in the regulation of potassium homeostasis in kidneys. In the event of renal ischemia, they are thought to contribute to the important intracellular potassium loss observed in proximal tubules and thus to hypoxic injury. We have analyzed the transcriptional regulation of K(ATP) genes in rat kidney following transient renal ischemia. We observed that mRNA expression level was down-regulated for Kir1.1 and Kir4.1 potassium channels between 24 and 120 h after ischemia. In contrast, a strong increase in mRNA expression was observed for Kir6.1 shortly (2-6 h) after ischemia. Thus, renal ischemia followed by reperfusion provokes differential regulation of K(ATP) channel gene expression.

Diminution of nicotinic receptor alpha 3 subunit mRNA expression in aged rat brain.

Losses in nicotinic acetylcholine receptors (nAChRs) have been linked to a decline in cognitive function in patients with neurodegenerative diseases, but the impact of normal aging on the different neuronal nicotinic receptor subunits has yet to be fully characterized. The expression pattern of nine nAChR subunits mRNA (alpha2-7 and beta2-4) was investigated in this study in young and aged rat brains, 5 weeks and 30 months old, respectively. Microtissue samples were dissected from brain slices and nAChR subunit mRNA expression was analyzed by reverse transcription polymerase chain reaction (RT-PCR) from eight different brain areas. In several regions, a loss of PCR signal was found for the alpha3, and to a lesser extent, for alpha2 subunit mRNA in aged rat brain. A relative quantification of alpha3 and alpha4 mRNA expression was then carried out in four of these brain regions. A significant diminution of alpha3 expression level was observed in all regions tested while, in comparison, much less modification in alpha4 mRNA was detected. This decrease in alpha3 subunit mRNA may represent a selective degradation of neurons expressing the alpha3 subunit or a diminution of alpha3-containing nAChR subtypes in those neurons during aging.

Structural elements of the gamma-aminobutyric acid type A receptor conferring subtype selectivity for benzodiazepine site ligands.

gamma-aminobutyric acid type A (GABAA) receptors comprise a subfamily of ligand-gated ion channels whose activity can be modulated by ligands acting at the benzodiazepine binding site on the receptor. The benzodiazepine binding site was characterized using a site-directed mutagenesis strategy in which amino acids of the alpha5 subunit were substituted by their corresponding alpha1 residues. Given the high affinity and selectivity of alpha1-containing compared with alpha5-containing GABAA receptors for zolpidem, mutated alpha5 subunits were co-expressed with beta2 and gamma2 subunits, and the affinity of recombinant receptors for zolpidem was measured. One alpha5 mutant (bearing P162T, E200G, and T204S) exhibited properties similar to that of the alpha1 subunit, notably high affinity zolpidem binding and potentiation by zolpidem of GABA-induced chloride current. Two of these mutations, alpha5P162T and alpha5E200G, might alter binding pocket conformation, whereas alpha5T204S probably permits formation of a hydrogen bond with a proton acceptor in zolpidem. These three amino acid substitutions also influenced receptor affinity for CL218872. Our data thus suggest that corresponding amino acids of the alpha1 subunit, particularly alpha1-Ser204, are the crucial residues influencing ligand selectivity at the binding pocket of alpha1-containing receptors, and a model of this binding pocket is presented.

Secretion of lecithin:cholesterol acyltransferase by brain neuroglial cell lines.

The ability of different human and rat brain cell lines (neuronal and gliomal) to secrete lecithin:cholesterol acyltransferase (LCAT) was examined. Of these, the strongly secreting human gliomal (U343 and U251) cell lines were selected for a detailed study of enzymatic and structural properties of the secreted LCAT. Both plasma- and brain-derived enzymes are inhibited by DTNB (90%) and are activated by apolipoprotein A-I. LCAT mRNA was measured in these cell lines at levels similar to that found in HepG2 cells. In contrast, apoA-I, apoE, and apoD mRNAs were undetectable in these cell lines. The presence of functional LCAT secreted by cultured nerve cells provides an in vitro model to study the expression and function of LCAT in the absence of others factors of plasma cholesterol metabolism.

Development of an inducible NMDA receptor stable cell line with an intracellular Ca2+ reporter.

Cytotoxicity associated with NMDA receptor activation has impeded the establishment of cell lines expressing recombinant subtypes of this ligand-gated ion channel class. To circumvent this toxicity, we describe in this report the use of a potent inducible promoter in the construction of a cell line stably expressing the NR1a/NR2A subtype of the NMDA receptor. Western blot analysis using subunit selective antibodies revealed that NR2A subunits were constitutively expressed in this cell line, whereas expression of NR1a subunits was tightly regulated by tetracycline. Upon tetracycline removal, electrophysiological recordings using the patch clamp technique indicated the expression of functional receptors with biophysical and pharmacological properties corresponding to those expected of the NR1a/NR2A subtype. In addition, we utilized this cell line with the recombinant membrane targeted Ca2+ reporter, aequorin, in a functional assay of NMDA receptor activation. An evaluation of the coupling efficiency of NMDA receptor activation and aequorin response, as well as the pharmacological profile of this assay, illustrates the suitability of this cell line and the Ca2+ reporter assay to functionally identify novel NMDA receptor antagonists.

[3H]t-butylbicycloorthobenzoate binding to recombinant alpha1beta2gamma2s GABA(A) receptor.

The interaction of several selected compounds with the binding of the cage convulsant t-[3H]butylbicycloorthobenzoate ([3H]TBOB) to membranes isolated from human embryonic kidney (HEK) 293 cells stably transfected with alpha1beta2gamma2s subtype of GABA(A) receptors was studied. Scatchard analysis of binding data revealed the existence of a single type of binding site for [3H]TBOB with a Kd of 47.06+/-4.06 nM and a Bmax value of 6.72+/-0.52 pmol/mg protein. GABA, thiopental, TBOB, picrotoxin and the neurosteroid dehydroepiandrosterone sulfate displaced concentration-dependently the binding of [3H]TBOB to this recombinant receptor. Dehydroepiandrosterone sulfate reversed the 5 microM GABA-induced inhibition of specific [3H]TBOB binding. It is concluded that membranes isolated from HEK 293 cells stably transfected with alpha1beta2gamma2s subunits exhibit specific high-affinity [3H]TBOB binding. The potency of drugs to inhibit [3H]TBOB binding mainly corresponded to that observed for the inhibition of the binding of cage convulsants to the native receptors or to transiently transfected HEK 293 cells.

Nicotinic acetylcholine subunit mRNA expression in dopaminergic neurons of the rat substantia nigra and ventral tegmental area.

The molecular composition of the nicotinic acetylcholine receptors (nAChRs) located on dopaminergic neurons and modulating their activity is unclear. Using the reverse transcriptase-polymerase chain reaction we have analyzed the mRNA for nAChR subunits expressed in the substantia nigra (SN) and ventral tegmental area (VTA) following unilateral 6-hydroxydopamine lesion of the dopaminergic system. In contrast to the unlesioned hemisphere, no signal was found in the lesioned hemisphere for alpha3, alpha5, alpha6 and beta4 subunits in the SN nor for alpha2, alpha3, alpha5, alpha6, alpha7 and beta4 subunits in the VTA, indicating the expression of these subunits in dopaminergic neurons. mRNA for alpha4, beta2 and beta3 subunits (and alpha7 in the SN) were still detected after lesion, suggesting that they are expressed in GABAergic neurons and interneurons of these brain areas. These results demonstrate the selective localisation of a number of nAChR subunit mRNA within dopaminergic neurons, strongly suggesting that a heterogenous population of nAChRs play a role in modulating dopaminergic neuronal activity.

Selective killing of glioma cell lines using an astrocyte-specific expression of the herpes simplex virus-thymidine kinase gene.

Gene therapy using the herpes simplex virus thymidine kinase gene (HSV-TK) is a promising new approach for the treatment of gliomas, a tumor type with a poor prognosis. To limit the toxic effects of this procedure, it is desirable to restrict expression of the HSV-TK gene to the target cells. This can be accomplished by use of the promoter of the glial fibrillary acidic protein gene, an intermediate filament protein expressed primarily in astrocytes. A plasmid containing the HSV-TK gene, driven by the human glial fibrillary acidic protein promoter gfa2, was lipofected into glioma cell lines and into an ovarian cancer cell line. Treatment with ganciclovir showed efficient killing of glioma cells, with no effect on the ovarian cells. Thus, the gfa2 promoter is a promising candidate for directing expression of toxic genes to gliomas.

Antagonist properties of eliprodil and other NMDA receptor antagonists at rat NR1A/NR2A and NR1A/NR2B receptors expressed in Xenopus oocytes.

We have studied the effects of a variety of N-methyl-D-aspartate (NMDA) antagonists acting at different sites of the NMDA receptor complex on NMDA-induced currents in Xenopus oocytes expressing heteromeric NR1A/NR2 and NR1A/NR2B receptors. The polyamine site antagonists eliprodil (IC50 = 3.0 microM) and ifenprodil (IC50 = 0.27 microM) antagonized NMDA responses at NR1A/NR2B receptors but not at NR1A/NR2A receptors (IC50 > 100 microM). The channel blockers dizocilpine, memantine and phencyclidine (PCP) were equally potent antagonists at both receptor subtypes whereas dextromethorphan was four times more potent at NR1A/NR2A receptors. The glycine site antagonists L-689,560 and 7-Cl-kynurenate were 10 times more potent at NR1A/NR2A than at NR1A/NR2B receptor subtypes. The selectivity of eliprodil and ifenprodil for the NR1A/NR2B receptor subtype may, at least partially, explain their favorable side effects profile.

Development of stable cell lines expressing different subtypes of GABAA receptors.

The experiments reported here were motivated by our interest to express in stably-transfected cells large amounts of recombinant rat GABAA receptors. For this, we developed an original two step selection strategy, in which the first step consisted of transfecting HEK 293 cells with rat GABAA receptor alpha and beta subunits. G 418 resistant colonies isolated at this step were screened for [3H] muscimol binding to select for those that coexpressed alpha- and beta-subunits. The best alpha and beta subunit expressing colony was then supertransfected with a plasmid coding for the gamma rat GABAA receptor subunit and a mutant DHFR gene. After a second round of selection, this time in presence of methotrexate, those colonies that coexpressed ternary alpha beta gamma GABAA receptor combinations were distinguished using [3H] flumazenil as a probe. This strategy was applied to the isolation of 3 GABAA receptor clones, alpha 1 beta 2 gamma 2s, alpha 3 beta 2 gamma 2s and alpha 5 beta 3 gamma 2s, that expressed relatively high levels of these proteins. These 3 cell lines exhibited pharmacological and functional properties similar to cells transiently-transfected with equivalent subunit combinations. These cell lines therefore provide attractive models with which to evaluate the intrinsic activity and potency of compounds at recombinant GABAA receptor subtypes.

Pharmacological profile of benzodiazepine site ligands with recombinant GABAA receptor subtypes.

Using [3H]flumazenil as a probe we investigated how benzodiazepine site pharmacology of alpha beta gamma ternary combinations of GABAA receptors can be influenced upon expression of different isoforms of alpha, beta and gamma subunits. The nature of the beta subunit did not alter the pharmacology of this site in that the affinities of alpha 5-containing GABAA receptors for various benzodiazepine modulatory ligands were essentially unchanged upon a comparison of different beta-variant forms (alpha 5 beta 1 gamma 2, alpha 5 beta 2 gamma 2 and alpha 5 beta 3 gamma 2). In contrast, both alpha and gamma variants contributed to notable differences in benzodiazepine site pharmacology. Thus alpha 1 beta 2 gamma 2, alpha 3 beta 2 gamma 2 and alpha 5 beta 2 gamma 2 receptors showed high, intermediate and low affinities for zolpidem, respectively. Exchanging gamma 2 for gamma 3 reduced the affinities of alpha 1 beta 2 gamma and alpha 3 beta 2 gamma receptors for zolpidem by factors of > 150 and > 5.8, respectively. The alpha 1 beta 2 gamma 3, alpha 3 beta 2 gamma 3 and alpha 5 beta 2 gamma 3 receptors exhibited, in contrast, higher affinity for CL218872 than their corresponding gamma 2 receptors. The information on these different recombinant GABAA receptor pharmacological profiles should help in the elucidation of native GABAA receptor subtype diversity.

Antagonist properties of the stereoisomers of ifenprodil at NR1A/NR2A and NR1A/NR2B subtypes of the NMDA receptor expressed in Xenopus oocytes.

The NMDA receptor antagonist ifenprodil contains two asymmetric centres which give rise to four stereoisomeric forms of this molecule. The inhibitory effects of each of these stereoisomers on recombinant NMDA receptors expressed from NR1A/NR2A and NR1A/NR2B subunit combinations were studied in Xenopus oocytes by voltage-clamp recording. All four ifenprodil stereoisomers were potent antagonists at NR1A/NR2B (IC50 < 0.8 microM), but weak antagonists at NR1A/NR2A receptors (IC50 > 100 microM). In heteromeric NR1A/NR2B receptors, (+) erythro- and (-) threo-ifenprodil (IC50 0.21 and 0.22 microM, respectively) were about 4 times more potent than (-) erythro- and (+) threo-ifenprodil (IC50 0.81 and 0.76, respectively). These results show that the stereoisomers of ifenprodil exhibit a weak though significant stereoselectivity at the NR1A/NR2B NMDA receptor subtype.