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The majority of variants of unknown clinical significance (VUCS) in the CFTR gene are missense variants. While change on the CFTR protein structure or function is often suspected, impact on splicing may be neglected. Such undetected splicing default of variants may complicate the interpretation of genetic analyses and the use of an appropriate pharmacotherapy.We selected 15 variants suspected to impact CFTR splicing after in silico predictions on 319 missense variants (214 VUCS), reported in the CFTR-France database. Six specialized laboratories assessed the impact of nucleotide substitutions on splicing (minigenes), mRNA expression levels (quantitative PCR), synthesis and maturation (western blot), cellular localization (immunofluorescence) and channel function (patch clamp) of the CFTR protein. We also studied maturation and function of the truncated protein, consecutive to in-frame aberrant splicing, on additional plasmid constructs.Six of the 15 variants had a major impact on CFTR splicing by in-frame (n = 3) or out-of-frame (n = 3) exon skipping. We reclassified variants into: splicing variants; variants causing a splicing defect and the impairment of CFTR folding and/or function related to the amino acid substitution; deleterious missense variants that impair CFTR folding and/or function; and variants with no consequence on the different processes tested.The 15 variants have been reclassified by our comprehensive approach of in vitro experiments that should be used to properly interpret very rare exonic variants of the CFTR gene. Targeted therapies may thus be adapted to the molecular defects regarding the results of laboratory experiments.

Because CFTR gene studies now represent one of the most frequent genetic analyses routinely performed worldwide, the number of rare CFTR variants identified in various clinical situations, regularly increases. To provide appropriate diagnosis and prognosis to CF patients as well as appropriate genetic counseling to families, the clinical impact and the phenotypic spectrum of variants identified by diagnostic techniques need to be characterized. Three complementary locus specific databases, called CFTR1, CFTR2 and CFTR-France were developed to address these issues. Besides, the growing knowledge of the CF pathophysiology and the technical evolution in molecular biology allowed to identify candidate modifier genes, regulatory loci, epigenetic profiles and trans-regulators that could help to refine genotype-phenotype correlations at the individual level. These different factors may contribute to the large phenotypic variability between patients with CF, even when they carry identical CFTR variants, regarding lung function, meconium ileus susceptibility or the risk for developing CFTR-related diabetes and liver disease. Finally, the availability of new therapies that target the CFTR protein for numbers of CF patients led to the identification of 'good' and 'poor' responders, thus raising questions of pharmacogenetics factors that may influence treatment efficiency as a novel feature of the complexity of CF patients' management. © 2020 French Society of Pediatrics. Published by Elsevier Masson SAS. All rights reserved.

International audience; BACKGROUND:The CFTR genotype remains incomplete in 1% of Cystic Fibrosis (CF) cases, because only one or no disease-causing variants is detected after extended analysis. This fraction is probably higher in CFTR-Related Disorders (CFTR-RD). Deep-intronic CFTR variants are putative candidates to fill this gap. However, the recurrence, phenotypic spectrum and full molecular characterization of newly reported variants are unknown.METHODS:Minigenes and analysis of CFTR transcripts in nasal epithelial cells were used to determine the impact on CFTR splicing of intronic variants that we previously identified by next generation sequencing of the whole CFTR locus. Phenotypic data were collected in 19 patients with CF and CFTR-RD, in whom one of the deep intronic variants has been detected.RESULTS:Three deep-intronic variants promoted the inclusion of pseudo-exons (PE) in the CFTR transcript, hindering the synthesis of a functional protein. The c.2989-313A > T variant, detected in four patients with CF or CFTR-RD from three different families, led to the inclusion of a 118 bp PE. The c.3469-1304C > G variant promoted the inclusion of a 214 bp-PE and was identified in five patients with CF from four families. Haplotype analysis confirmed that this variant was associated with one CF chromosome of African origin. The most represented variant in our cohort was the c.3874-4522A > G, detected in 10 patients with various phenotypes, from male infertility to CF with pancreatic insufficiency.CONCLUSION:These three deep intronic CFTR variants are associated with a large phenotypic spectrum, including typical CF. They should be included in CF diagnostic testing and carrier screening strategies.

Resume Le depistage neonatal (DNN) de la mucoviscidose, grâce a une prise en charge multidisciplinaire tres precoce des nourrissons, est optimal en termes de pronostic pour les patients. Depuis 20 ans, il a connu une expansion internationale spectaculaire. Les performances du DNN national francais realise depuis 2002 sont en accord avec les standards europeens pour la valeur predictive positive (0,31 pour un minimum de 0,30) et la specificite (0,95 pour un minimum de 0,95) ; nous soulignons le nombre tres faible de cas non conclus, un pourcentage tres eleve de test de la sueur realises (95,5 %) et d’identification des mutations (96,6 %), un ratio de cas de mucoviscidose par rapport aux cas de diagnostic incertain de 6,3:1, ainsi qu’une strategie efficace pour reperer les faux negatifs. Une nouvelle organisation du DNN francais vient de se mettre en place, il est capital de maintenir l’efficacite du processus, du nouveau-ne en maternite jusqu’au diagnostic dans des centres de reference ou de competences de la mucoviscidose avec un recueil exhaustif des donnees et leur validation. Par ailleurs, une proposition de changement d’algorithme introduisant le dosage du polypeptide d’activation pancreatique est soumise aux nouvelles instances du DNN. L’annonce d’un diagnostic positif reste difficile et peu standardisee, si bien qu’une plateforme de simulation est en cours de mise en place. Nous detaillons ici les bonnes pratiques et les difficultes de realisation du test de la sueur, avec les seuils de chlorures recemment redefinis, ainsi que les modalites du conseil genetique.

BACKGROUND: The clinical spectrum associated with cystic fibrosis transmembrane conductance regulator (CFTR) variant p.Arg117His is highly variable, ranging from full-blown cystic fibrosis (CF) in a small number of cases to CFTR-related disorders (CFTR-RDs) or no symptoms at all. Therefore, taking into account phenotype variability is essential for interpretation. External quality assessment (EQA) schemes can help laboratories to objectively assess the quality of genotyping and reporting by the laboratory. METHODS: We performed a retrospective longitudinal data analysis on laboratory performance regarding the interpretation of p.Arg117His during CF EQA scheme participation. Completeness and accuracy of reporting on two mock clinical cases were each compared over time (case 1: 2005, 2007 and 2012; case 2: 2015 and 2018). These cases concerned subjects compound heterozygous for p.Phe508del and p.Arg117His in cis with 7T, but with different clinical backgrounds (family planning (case 1) versus diagnostic testing for a child (case 2)). Furthermore, we analyzed the influence of previous participations, annual test volume, accreditation status and laboratory setting on overall performance. RESULTS: Overall performance improved over time, except during the 2007 CF EQA scheme. In addition, previous participations had a beneficial effect on laboratory performance. Accreditation status, annual test volume and laboratory setting did not significantly influence total interpretation scores. CONCLUSIONS: In general, laboratories performed well on both cases, although reporting on the variable clinical spectrum of p.Arg117His in cis with 7T and on the disease liability of individual CFTR variants can still improve. Moreover, this study underlined the educational role of CF EQA schemes. ispartof: JOURNAL OF CYSTIC FIBROSIS vol:19 issue:6 pages:969-974 ispartof: location:Netherlands status: published

International audience; Whole-exome/genome sequencing analyses lead to detect disease-causing variants that are unrelated to the initial clinical question. Irrespective of any actionable gene list, only pathogenic variants should be considered. The pathogenicity of 55 cystic fibrosis transmembrane conductance regulator (CFTR) variants of known various impacts was assessed by a group of experts by comparing data from specialized databases CFTR-France and CFTR2 with those of general clinical databases ClinVar and Human Gene Mutation Database (HGMD®) Professional and data aggregators VarSome and InterVar. The assessment of cystic fibrosis (CF) variants was correct with ClinVar and HGMD® Professional while less reliable with VarSome and InterVar. Conversely, the risk of overclassifying variants as CF-causing was up to 82% with HGMD® Professional. The concordance between data aggregators was only 50%. The use of general databases and aggregators is thus associated with a substantial risk of misclassifying variants. This evaluation may be extrapolated to other disease conditions and incites to remain cautious in interpreting and disclosing incidental findings.

Background: The CFTR gene is responsible for the autosomal recessive disease Cystic Fibrosis (CF) and CFTR-related disorders (CFTR-RD). More than 2000 variants have been reported. However, the CFTR genotype remains incomplete (with one or none disease-causing variants) in 1% of CF typical cases and even more in CFTR-RD. Aims and Objectives: To find the second disease causing-variant in unsolved cases of CF and CFTR-RD with new methods and assess their biological impact. Methods: We looked for deep intronic variants by studying the whole CFTR locus by Next Generation Sequencing. Impact of variants on splicing was confirmed by in vitro minigene experiments and analysis of CFTR transcripts from patients’ nasal cells. Imaging, spirometric, and biological data (sweat test values, trypsinemia in neonates) were collected. Results: We identified two novel deep intronic CFTR variants that create new splice sites leading to pseudo-exon inclusion and the apparition of a premature stop codon, in 14 patients from 11 families. Ten had bronchiectasis, of whom four had pulmonary infection with atypical germs. Two patients (>40 y/o) had pulmonary surgery. Five patients had normal respiratory function (FEV1% >100) including one with bronchiectasis and pancreatic insufficiency, the two youngest patients ( Conclusion: The two new deep intronic variants are associated with a large phenotypic spectrum, from male infertility to typical CF. These variants should be systematically tested in patients with intermediate or positive sweat tests who do not carry two disease-causing CFTR variants after routine analysis.

International audience; Cystic Fibrosis is among the first diseases to have general population genetic screening tests and one of the most common indications of prenatal and preimplantation genetic diagnosis for single gene disorders. During the past twenty years, thanks to the evolution of diagnostic techniques, our knowledge of CFTR genetics and pathophysiological mechanisms involved in cystic fibrosis has significantly improved. Areas covered: Sanger sequencing and quantitative methods greatly contributed to the identification of more than 2,000 sequence variations reported worldwide in the CFTR gene. We are now entering a new technological age with the generalization of high throughput approaches such as Next Generation Sequencing and Droplet Digital PCR technologies in diagnostics laboratories. These powerful technologies open up new perspectives for scanning the entire CFTR locus, exploring modifier factors that possibly influence the clinical evolution of patients, and for preimplantation and prenatal diagnosis. Expert commentary: Such breakthroughs would, however, require powerful bioinformatics tools and relevant functional tests of variants for analysis and interpretation of the resulting data. Ultimately, an optimal use of all those resources may improve patient care and therapeutic decision-making.

This review is based partly on complete articles and partly on abstracts. Three of the 60 articles deal with the total uptake of elements in strawberry plant organs in two different strawberry production systems, both considered as optimal concerning amount and balance of elements. The effect on fruit quality may be dramatic if the level of a particular element is outside this range, but there may also be effects initiated by differences within the optimal range of elements. Most articles refer to product oriented quality, but some focus on consumer oriented quality, as discussed by Shewfelt (1999). The discussion here is on a general basis, so one should keep in mind that there are cultivar differences and that specification of nutrition ideally should mirror the needs of a single cultivar, or a group of cultivars with similar requirements. Also, to get a complete understanding of the subject future reviews should embrace a broader access of information including the effect on plant development ...

The Bakamjian-Thomas relativistic quark model, describing hadrons with a fixed number of constituents, yields in the heavy quark limit of QCD covariant Isgur-Wise functions and satisfies the whole tower of lowest moment sum rules (Bjorken-Uraltsev type sum rules). We first recall, as well as earlier results, the new formalism presented in our recent papers on Lorentz representations, which provide an elegant framework for the analysis of this model in the heavy quark limit and stress the results which have been already obtained in this direction. Then, we give some very explicit demonstrations of the fact that the Bakamjian-Thomas framework satifies the sum rules by considering simple cases of Isgur-Wise functions. In addition to the specific Bjorken and Uraltsev sum rules, an important sum rule that involves only heavy mesons with light cloud $j^P = {1 \over 2}^-$ and their radial excitations is demonstrated. This latter sum rule is phenomenologically interesting because it constrains the derivatives of the radially excited Isgur-Wise functions at zero recoil. On the other hand, we recall the limitations of the Bakamjian-Thomas scheme. At finite mass, current matrix elements with the current coupled to the heavy quark are no longer covariant, and higher moment sum rules that hold in the heavy quark limit of QCD are not satisfied.

The OPE treatment that has been so successful in describing inclusive $\bar B \to l \bar \nu X_c$ decays yields sum rules (in particular the Uraltsev sum rule and its higher moments) implying the dominance of the $P$ wave $j_q = 3/2$ charm states in $X_c$ over their $j_q=1/2$ counterparts. This prediction is supported by other general arguments as well as quark model calculations, which illustrate the OPE results, and by preliminary lattice findings. Its failure would indicate a significant limitation in our theoretical understanding of $\bar B \to l \bar \nu X_c$. Some experimental issues have been clarified since a preliminary version of this note had appeared, yet the verdict on the composition of the final states {\em beyond} $D$, $D^*$ and the two narrow $j_q = 3/2$ resonances remains unsettled. Establishing which hadronic configurations -- $D/D^* + \pi, D/D^* + 2 \pi, ...$ -- contribute, what their quantum numbers are and their mass distributions will require considerable experimental effort. We explain the theoretical issues involved and why a better understanding of them will be of significant value. Having significant contributions from a mass continuum distribution below 2.5 GeV raises serious theoretical questions for which we have no good answer. Two lists are given, one with measurements that need to be done and one with items of theoretical homework. Some of the latter can be done by employing existing theoretical tools, whereas others need new ideas.

The Bakamjian-Thomas relativistic quark model for hadron current matrix elements, while non-covariant at finite mass, is successful in the heavy quark limit : form factors are covariant and satisfy Isgur-Wise scaling and Bjorken-Uraltsev sum rules. Motivated by the so-called "1/2 vs. 3/2 puzzle" in B decays to positive parity D**, we examine the implications of the model at finite mass. In the elastic case 1/2^- -> 1/2^-, the HQET constraints for the O(1/m_Q) corrections are analytically fulfilled. A number of satisfying regularities is also found for inelastic transitions. We compute the form factors using the wave functions given by the Godfrey-Isgur potential. For 1/2^- \to 3/2^+ the departures from the heavy quark limit are small, but we find a strong enhancement in 1/2^- -> 1/2^+ (for 0^- -> 0^+). This enhancement is linked to a serious difficulty of the model at finite mass for the inelastic transitions, namely a violation of the HQET constraints at zero recoil formulated by Leibovich et al. These are nevertheless satisfied in the non-relativistic limit for the light quark. We conclude that these HQET rigorous constraints are crucial in the construction of a sensible relativistic quark model of inelastic form factors.

We show that the sum rule recently proved by Uraltsev in the heavy quark limit of QCD holds in relativistic quark models \`a la Bakamjian and Thomas, that were already shown to satisfy Isgur-Wise scaling and Bjorken sum rule. This new sum rule provides a {\it rationale} for the lower bound of the slope of the elastic IW function $\rho^2 \geq {3 \over 4}$ obtained within the BT formalism some years ago. Uraltsev sum rule suggests an inequality $|\tau_{3/2}(1)| > |\tau_{1/2}(1)|$. This difference is interpreted in the BT formalism as due to the Wigner rotation of the light quark spin, independently of a possible LS force. In BT models, the sum rule convergence is very fast, the $n = 0$ state giving the essential contribution in most of the phenomenological potential models. We underline that there is a serious problem, in the heavy quark limit of QCD, between theory and experiment for the decays $B \to D^*_{0,1}(broad) \ell \nu$, independently of any model calculation., Comment: 16 pages, Latex

Glycosylation represents an attractive target for protein engineering of novel antibiotics, because specific attachment of one or more deoxysugars is required for the bioactivity of many antibiotic and antitumour polyketides. However, proper assessment of the potential of these enzymes for such combinatorial biosynthesis requires both more precise information on the enzymology of the pathways and also improved Escherichia coli-actinomycete shuttle vectors. New replicative vectors have been constructed and used to express independently the dnmU gene of Streptomyces peucetius and the eryBVII gene of Saccharopolyspora erythraea in an eryBVII deletion mutant of Sac. erythraea. Production of erythromycin A was obtained in both cases, showing that both proteins serve analogous functions in the biosynthetic pathways to dTDP-L-daunosamine and dTDP-L-mycarose, respectively. Over-expression of both proteins was also obtained in S. lividans, paving the way for protein purification and in vitro monitoring of enzyme activity. In a further set of experiments, the putative desosaminyltransferase of Sac. erythraea, EryCIII, was expressed in the picromycin producer Streptomyces sp. 20032, which also synthesises dTDP-D-desosamine. The substrate 3-alpha-mycarosylerythronolide B used for hybrid biosynthesis was found to be glycosylated to produce erythromycin D only when recombinant EryCIII was present, directly confirming the enzymatic role of EryCIII. This convenient plasmid expression system can be readily adapted to study the directed evolution of recombinant glycosyltransferases.

We explicitly check quark-hadron duality to order $(m_b-m_c)\Lambda/m_b^2$ for $b \to c l\nu$ decays in the limit $m_b-m_c \ll m_b$ including ground state and orbitally excited hadrons. Duality occurs thanks to a new sum rule which expresses the subleading HQET form factor $\xi_3$ or, in other notations, $a_+^{(1)}$ in terms of the infinite mass limit form factors and some level splittings. We also demonstrate the sum rule, which is not restricted to the condition $m_b-m_c \ll m_b$, applying OPE to the longitudinal axial component of the hadronic tensor without neglecting the $1/m_b$ subleading contributions to the form factors. We argue that this method should produce a new class of sum rules, depending on the current, beyond Bjorken, Voloshin and the known tower of higher moments. Applying OPE to the vector currents we find another derivation of the Voloshin sum rule. From independent results on $\xi_3$ we derive a sum rule which involves only the $\tau_{1/2}^{(n)}$ and $\tau_{3/2}^{(n)}$ form factors and the corresponding level splittings. The latter strongly supports a theoretical evidence that the $B$ semileptonic decay into narrow orbitally-excited resonances dominates over the decay into the broad ones, in apparent contradiction with some recent experiments. We discuss this issue., Comment: 9 pages

Poly(A) polymerase I (PAP I) of Escherichia coli is a member of the nucleotidyltransferase (Ntr) superfamily that includes the eukaryotic PAPs and all the known tRNA CCA-adding enzymes. Five highly conserved aspartic acids in the putative catalytic site of PAP I were changed to either alanine or proline, demonstrating their importance for polymerase activity. A glycine that is absolutely conserved in all Ntrs was also changed yielding a novel mutant protein in which ATP was wastefully hydrolysed in a primer-independent reaction. This is the first work to characterize the catalytic site of a eubacterial PAP and, despite the conservation of certain sequences, we predict that the overall architecture of the eukaryotic and eubacterial active sites is likely to be different. Binding sites for RNase E, a component of the RNA degradosome, and RNA were mapped by North-western and Far-western blotting using truncated forms of PAP I. Additional protein-protein interactions were detected between PAP I and CsdA, RhlE and SrmB, suggesting an unexpected connection between PAP I and these E. coli DEAD box RNA helicases. These results show that the functional organization of PAP I is similar to the eukaryotic PAPs with an N-terminal catalytic domain, a C-terminal RNA binding domain and sites for the interaction with other protein factors.

There has been increased interest in bacterial polyadenylation with the recent demonstration that 3′ poly(A) tails are involved in RNA degradation. Poly(A) polymerase I (PAP I) of Escherichia coli is a member of the nucleotidyltransferase (Ntr) family that includes the functionally related tRNA CCA-adding enzymes. Thirty members of the Ntr family were detected in a search of the current database of eubacterial genomic sequences. Gram-negative organisms from the β and γ subdivisions of the purple bacteria have two genes encoding putative Ntr proteins, and it was possible to predict their activities as either PAP or CCA adding by sequence comparisons with the E. coli homologues. Prediction of the functions of proteins encoded by the genes from more distantly related bacteria was not reliable. The Bacillus subtilis papS gene encodes a protein that was predicted to have PAP activity. We have overexpressed and characterized this protein, demonstrating that it is a tRNA nucleotidyltransferase. We suggest that the papS gene should be renamed cca , following the notation for its E. coli counterpart. The available evidence indicates that cca is the only gene encoding an Ntr protein, despite previous suggestions that B. subtilis has a PAP similar to E. coli PAP I. Thus, the activity involved in RNA 3′ polyadenylation in the gram-positive bacteria apparently resides in an enzyme distinct from its counterpart in gram-negative bacteria.

A 6-kb region from the chromosome of Streptomyces antibioticus, an oleandomycin producer, was cloned and sequenced. This region was located between the 3' end of the gene encoding the third subunit of the oleandomycin type I polyketide synthase and the oleP and oleB genes, which encode a cytochrome P450 monooxygenase and an oleandomycin resistance gene, respectively. Analysis of the nucleotide sequence revealed the presence of five genes encoding a cytochrome P450-like protein (oleP1), two glycosyltransferases (oleG1 and oleG2) involved in the transfer of the two 6-deoxysugars (L-oleandrose and D-desosamine) to the oleandomycin macrolactone ring, a methyltransferase (oleM1), and a gene (oleY) of unknown function. Insertional inactivation of this region by gene disruption generated an oleandomycin non-producing mutant which accumulated a compound that, according to mass spectrometry analysis, could correspond to the oleandomycin macrolactone ring (oleandolide), suggesting that the mutation affects oleandrosyl glycosyltransferase.

We study CP-violation in resonant three-body Bd→DPP decays, where PP stands for either D π , D K S , ππ or πKS. Analogously to the Bd→3π channel and the extraction of 2α, the first three channels are shown to measure cos2β in addition to sin2β, thus allowing to resolve the β→ π 2 −β ambiguity, while the DπKS final state leads to a measurement of 2β+γ. As cos2β and cos(2β+γ) are CP-conserving quantities, their determination is possible without tagging the flavor of the Bd meson. The Bd0(t)→D+D−π0 channel via the interference between D ∗∗ orbitally excited resonances is taken as an example, although this Cabibbo-suppressed decay suffers from irreducible penguin uncertainties. Then two penguin-free and Cabibbo-dominant modes are proposed: Bd0(t)→D+D−KS with the D s ∗∗ resonances, and Bd0(t)→D0CPπ+π− with the D ∗∗ plus the ρ. Finally, the Bd0(t)→D±π∓KS channel with the D s ∗∗ and K ∗ resonances provides a new clean method to measure the unusual angle 2β+γ. We present in all cases a crude estimate of the number of cos2β (respectively 2β+γ) sensitive events. We show that this number is an increasing function of the resonance mass, a favorable situation compared to the more extensively studied three-pion Dalitz plot. However, the poor detection efficiency of the D mesons could pose a problem. As an annex and speculative application of these Dalitz plot based methods, the penguin-dominated Bd0(t)→KSKSKL decay also measures 2β.

The gene cluster (ery) governing the biosynthesis of the macrolide antibiotic erythromycin A by Saccharopolyspora erythraea contains, in addition to the eryA genes encoding the polyketide synthase, two regions containing genes for later steps in the pathway. The region 5' of eryA that lies between the known genes ermE (encoding the erythromycin resistance methyltransferase) and eryBIII (encoding a putative S-adenosylmethionine-dependent methyltransferase), and that contains the gene eryBI (orf2), has now been sequenced. The inferred product of the eryBI gene shows striking sequence similarity to authentic beta-glucosidases. Specific mutants were created in eryBI, and the resulting strains were found to synthesise erythromycin A, showing that this gene, despite its position in the biosynthetic gene cluster, is not essential for erythromycin biosynthesis. A mutant in eryBIII and a double mutant in eryBI and eryBIII were obtained and the analysis of novel erythromycins produced by these strains confirmed the proposed function of EryBIII as a C-methyltransferase. Also, a chromosomal mutant was constructed for the previously sequenced ORF19 and shown to accumulate erythronolide B, as expected for an eryB mutant and consistent with its proposed role as an epimerase in dTDP-mycarose biosynthesis.

The excitatory amino acid glutamate (Glu) is a potent neurotransmitter in the central nervous system and exerts its action via a variety of glutamate receptors (GluRs). Because we had previously shown that a poly-glutamate (poly-Glu) peptide stimulates bone resorption in vitro, an effect specific to Glu (Raynal, C., Delmas, P. D., and Chenu, C. Bone sialoprotein stimulates in vitro bone resorption. Endocrinology 137:2347-2354; 1996), we investigated the possibility that bone cells express GluRs, and whether they may be important for osteoclast activity. Using immunocytochemistry on rat bone sections, we have shown that all mature bone cells (osteoblasts and osteoclasts) express GluRs, and that the ionotropic N-methyl-D-aspartate (NMDA) receptor 1 subunit (NMDAR1) appears most highly expressed. Osteoclasts isolated from rabbit long bones also possessed NMDAR1 GluRs. Bone resorption in vitro by isolated osteoclasts was inhibited by a monoclonal antibody directed against NMDAR1, as well as by two pharmacological antagonists of this receptor (D-AP5 and MK 801), which also antagonized poly-Glu stimulated bone resorption. These results suggest a possible new mechanism for regulating osteoclast activity and indicate that excitatory amino acids such as glutamate may be important local regulators of bone cell functions.

The production of erythromycin A by Saccharopolyspora erythraea requires the synthesis of dTDP-D-desosamine and dTDP-L-mycarose, which serve as substrates for the transfer of the two sugar residues onto the macrolactone ring. The enzymatic activities involved in this process are largely encoded within the ery gene cluster, by two sets of genes flanking the eryA locus that encodes the polyketide synthase. We report here the nucleotide sequence of three such ORFs located immediately downstream of eryA, ORFs 7, 8 and 9. Chromosomal mutants carrying a deletion either in ORF7 or in one of the previously sequenced ORFs 13 and 14 have been constructed and shown to accumulate erythronolide B, as expected for eryB mutants. Similarly, chromosomal mutants carrying a deletion in either ORF8, ORF9, or one of the previously sequenced ORFs 17 and 18 have been constructed and shown to accumulate 3-alpha-mycarosyl erythronolide B, as expected for eryC mutants. The ORF13 (eryBIV), ORF17 (eryCIV) and ORF7 (eryBII) mutants also synthesised small amounts of macrolide shunt metabolites, as shown by mass spectrometry. These results considerably strengthen previous tentative proposals for the pathways for the biosynthesis of dTDP-D-desosamine and dTDP-L-mycarose in Sac. erythraea and reveal that at least some of these enzymes can accommodate alternative substrates.

Once chosen the dynamics in one frame, the rest frame in this paper, the Bakamjian-Thomas method allows one to define relativistic quark models in any frame. These models have been shown to provide, in the infinite quark mass limit, fully covariant current form factors as matrix elements of the quark current operator. In this paper we use the rest frame dynamics fitted from the meson spectrum by various authors, already shown to provide a reasonable value for ${\ensuremath{\rho}}^{2}.$ From the general formulas for the scaling invariant form factors ${\ensuremath{\xi}}^{(n)}(w),$ ${\ensuremath{\tau}}_{1/2}^{(n)}(w),$ and ${\ensuremath{\tau}}_{3/2}^{(n)}(w),$ we predict quantitavely the $B$ semileptonic branching ratios to the ground state and orbitally excited charmed mesons $D,$ ${D}^{*},$ and ${D}^{**}.$ We check Bjorken's sum rule and discuss the respective contributions to it. We find $\ensuremath{\xi}(w)\ensuremath{\simeq}[2/(1+w){]}^{2},$ resulting from the fact that the ground state wave function is Coulomb-like. We also find ${\ensuremath{\tau}}_{3/2}\ensuremath{\simeq}0.5[2/(1+w){]}^{3}$ and ${\ensuremath{\tau}}_{1/2}(w)\ensuremath{\ll}{\ensuremath{\tau}}_{3/2}(w).$ Very small branching ratios into $j=1/2$ orbitally excited $D$'s results. The overall agreement with experiment is rather good within the present accuracy which is poor for the orbitally excited charmed mesons. We predict a ratio $B(B\ensuremath{\rightarrow}{D}_{2}^{*}l\ensuremath{\nu})/B(B\ensuremath{\rightarrow}{D}_{1}l\ensuremath{\nu})=1.55\ifmmode\pm\else\textpm\fi{}0.15$ as a mere consequence of the heavy quark symmetry. If some faint experimental indications that $B(B\ensuremath{\rightarrow}{D}_{1}l\ensuremath{\nu})\ensuremath{\simeq}B(B\ensuremath{\rightarrow}{D}_{2}^{*}l\ensuremath{\nu})$ were confirmed, it would indicate a sizable ${O(1/m}_{c})$ correction.

We elucidate the behavior of the operator (p2+m2)1/2−α/r near the critical value α=αc where it ceases to be bounded below, by obtaining a family H(z) of operators which is self-adjoint holomorphic in a domain including all real z>−αc−αc′, and such that H(αc−α)(α⩽αc) is just the operator (p2+m2)1/2−α/r or its Friedrich extension, while H(−αc−α)(αc′ αc). We show that these eigenvalues cannot vanish, but that the lowest eigenvalue of H(−αc−α) goes to zero when α→αc′. The L>0 eigenvalues are analytic in α at α=αc.

We discuss some specific problems involved in the determination of the CP angles α, β, γ of the B0d - B0d unitarity triangle, and some related theoretical issues in non-leptonic B decays.

The heavy mass limit of quark models based on the Bakamjian-Thomas cons\-truction reveals remarkable features. In addition to previously demonstrated properties of covariance and Isgur-Wise scaling, exact duality, leading to the Bjorken-Isgur-Wise sum rule, is proven, for the first time to our knowledge in relativistic quark models. Inelastic as well as elastic contributions to the sum rule are then discussed in terms of ground state averages of a few number of operators corresponding to the nonrelativistic dipole operator and various relativistic corrections., Comment: 14 pages

We consider the dominant $c\bar{c}$ contribution to $\Delta \Gamma$ for the $B_s^0$-$\bar{B}_s^0$ system in the heavy quark limit for both $b$ and $c$ quarks. In analogy with the Bjorken-Isgur-Wise sum rule in semileptonic heavy hadron decay, we impose duality between the parton model calculation of $\Delta \Gamma$ and its estimation by a sum over heavy mesons. Varying the mass ratio $m_c/m_b$ and assuming factorization and saturation by narrow resonances $(N_c \to \infty$), we obtain new sum rules that involve the Isgur-Wise functions $\xi^{(n)}(w)$ and $\tau^{(n)}_{1/2}(w)$ and the decay constants $f^{(n)}$, $f^{(n)}_{1/2}$ ($n$ stands for any radial excitation). Alternatively, we deduce the sum rules with another method free of the factorization hypothesis, from the saturation of the expectation value of a product of two currents by heavy hadrons and by the corresponding free quarks. The sum rules read $\sum\limits_{n}{f^{(n)} \over f^{(0)}} \xi^{(n)}(w) = 2 \sum\limits_{n}{f^{(n)}_{1/2} \over f^{(0)}} \tau^{(n)}_{1/2}(w) = 1$, valid for all $w$. Moreover, we obtain, in the heavy quark limit, $f^{(n)}_{3/2} = 0$. As a consequence, unlike the BIW sum rule, the slope of the elastic function $\xi (w)$ is related to radial excitations alone. These are generalizations, rigorous for QCD in the heavy quark limit, of results that have an easy understanding in the non-relativistic quark model., Comment: 15 pages

Once chosen the dynamics in one frame, for example the rest frame, the Bakamjian and Thomas method allows to define relativistic quark models in any frame. These models have been shown to provide, in the heavy quark limit, fully covariant current form factors as matrix elements of the quark current operator. They also verify the Isgur-Wise scaling and give a slope parameter $\rho^2>3/4$ for all the possible choices of the dynamics. In this paper we study the $L=1$ excited states and derive the general formula, valid for any dynamics, for the scaling invariant form factors $\tau_{1/2}^{(n)}(w)$ and $\tau_{3/2}^{(n)}(w)$. We also check the Bjorken-Isgur-Wise sum rule already demonstrated elsewhere in this class of models.

We have constructed a strain that overexpresses E coli poly(A) polymerase (PAP I). The recombinant protein was soluble, and a partially purified extract had high levels of poly(A) polymerising activity. An antiserum raised against the overexpressed PAP I has permitted two types of analysis: the identification of other E coli proteins that may interact with PAP I, and the search for PAP I-like proteins in other bacteria. Immunoprecipitation experiments suggest that PAP I is associated with a 48-kDa protein. This protein remains to be identified. Western blotting using the antiserum against E coli PAP I revealed related proteins in a variety of Gram-negative bacteria and in B subtilis. A comparison of the E coli protein with putative poly(A) polymerases recently identified in H influenza and B subtilis showed highly conserved sequences in the amino terminal and central portions of the proteins that may be important for enzyme activity.

We reexamine the calculation of the flavor-changing neutral effective vertices {ital bsZ}{sup 0}, {ital bs}{chi}{sup 0}, and {ital bsH} ({chi}{sup 0} is the unphysical Higgs boson and {ital H} is the physical Higgs boson) at one loop in dimensional regularization in the `t Hooft--Veltman (`tHV) method, comparing to naive dimensional regularization (NDR). We show that the {ital differences} between the Green functions in both schemes satisfy {ital simplified} Slavnov-Taylor identities (STI`s). The `tHV scheme breaks chiral invariance, and hence gauge symmetry. Moreover, this scheme leaves room for ambiguities in the extension of the current to {ital D} dimensions; for some choices, the regularized bare quantities may even violate Hermiticity. However, we show that the STI`s can be satisfied in the `tHV scheme by specific finite counterterms. In both methods, `tHV and NDR, the flavor-changing neutral counterterms are uniquely determined through the STI`s and the on-shell renormalization conditions, fixing the renormalized Green functions on and off shell. A difference between both schemes is that in the physical amplitude the sum of all counterterms vanishes for NDR, while for the `tHV scheme the sum of all counterterms does not vanish, and cancels the terms of the regularized vertices that arise from themore » violation of chiral invariance (and, in some cases, Hermiticity) in this scheme. Also, as expected, any finite difference between renormalization schemes that satisfy the STI`s leads to the same {ital S}-matrix elements. Although the renormalization algorithm needs neutral flavor-changing counterterms, the elecroweak theory is completely predictive in the flavor-changing neutral sector in both renormalization schemes.« less

A major problem in the determination of the the CP angle $\alpha$, that should be measured through modes of the type $B_d, \bar{B}_d \to \pi \pi , \cdots ,$ is the uncertainty coming from Penguin diagrams. We consider the different ground state modes $\pi \pi$, $\pi \rho$, $\rho \rho$, and, assuming the FSI phases to be negligible, we investigate the amount of uncertainty coming from Penguins that can be parametrized by a dilution factor $D$ and an angle shift $\Delta \alpha$. The parameter $D$ is either 1 or very close to 1 in all these modes, and it can be measured independently, up to a sign ambiguity, by the $t$ dependence. Assuming factorization, we show that $\Delta \alpha$ is much smaller for the modes $\rho \pi$ and $\rho \rho$ than for $\pi \pi$, and we plot their allowed region as a function of $\alpha$ itself. Moreover, we show that most of the modes contribute to the asymmetry with the same sign, and define for their sum an effective $D_{eff}$ and an effective $\Delta \alpha_{eff}$, an average of $\Delta \alpha$ for the different modes. It turns out that $D_{eff}$ is of the order of 0.9, $\Delta \alpha_{eff}$ is between 5 $\%$ and 10 $\%$ and, relative to $\pi \pi$, the statistical gain for the sum is of about a factor 10. Finally, we compute the ratios $K\pi /\pi \pi , \cdots$ that test the strength of the Penguins and depend on the CP angles, as emphasized by Silva and Wolfenstein and by Deandrea et al., Comment: 18 pages

We point out that, if we assume the factorization hypothesis, current estimates of form factors fail to explain the nonleptonic decays B\ensuremath{\rightarrow}\ensuremath{\psi}K(${\mathit{K}}^{\mathrm{*}}$) and that the combination of data on the semileptonic decays D\ensuremath{\rightarrow}K(${\mathit{K}}^{\mathrm{*}}$)l\ensuremath{\nu} and on the nonleptonic decays B\ensuremath{\rightarrow}\ensuremath{\psi}K(${\mathit{K}}^{\mathrm{*}}$) (in particular, recent polarization data) severely constrain the form (normalization and ${\mathit{q}}^{2}$ dependence) of the heavy-to-light-meson form factors. From a simultaneous fit to B\ensuremath{\rightarrow}${\mathit{K}}^{(\mathrm{*})}$\ensuremath{\psi} and D\ensuremath{\rightarrow}${\mathit{K}}^{(\mathrm{*})}$l\ensuremath{\nu} data we find that strict heavy quark limit scaling laws do not hold when going from D to B and must have large corrections that make softer the dependence on the masses. We find that ${\mathit{A}}_{1}$(${\mathit{q}}^{2}$) should increase slower with ${\mathit{q}}^{2}$ than ${\mathit{A}}_{2}$,V,${\mathit{f}}_{+}$. We propose a simple parametrization of these corrections based on a quark model or on an extension of the heavy-to-heavy scaling laws to the heavy-to-light case, complemented with an approximately constant ${\mathit{A}}_{1}$(${\mathit{q}}^{2}$). We analyze in the light of these data and theoretical input various theoretical approaches (lattice calculations, QCD sum rules, quark models) and point out the origin of the difficulties encountered by most of these schemes. In particular we check the comptability of several quark models with the heavy quark scaling relations.

It is shown that the Herbst equation, i.e., a Coulomb potential wave equation including a free relativistic kinetic energy, presents a rather nontrivial series expansion of its eigenvalues in powers of α. In fact, in contrast to Klein-Gordon and Dirac equations, it presents not only odd powers of α but also nonanalytic 1n α terms. The first orders (α4, α5) are obtainable by a standard perturbation method on the Sommerfeld correction. A much more effective and systematic method is proposed to get higher orders (α7). To appreciate the resulting expansion, we compare it to the known results coming from other approaches to relativistic bound states, namely Klein-Gordon and Dirac equations in an external field and for the two-body problem, the Breit and Sucher-type equations, and the positronium QED result.