High-throughput human primary cell-based airway model for evaluating influenza, coronavirus, or other respiratory viruses in vitro.

Influenza and other respiratory viruses present a significant threat to public health, national security, and the world economy, and can lead to the emergence of global pandemics such as from COVID-19. A barrier to the development of effective therapeutics is the absence of a robust and predictive preclinical model, with most studies relying on a combination of in vitro screening with immortalized cell lines and low-throughput animal models. Here, we integrate human primary airway epithelial cells into a custom-engineered 96-device platform (PREDICT96-ALI) in which tissues are cultured in an array of microchannel-based culture chambers at an air-liquid interface, in a configuration compatible with high resolution in-situ imaging and real-time sensing. We apply this platform to influenza A virus and coronavirus infections, evaluating viral infection kinetics and antiviral agent dosing across multiple strains and donor populations of human primary cells. Human coronaviruses HCoV-NL63 and SARS-CoV-2 enter host cells via ACE2 and utilize the protease TMPRSS2 for spike protein priming, and we confirm their expression, demonstrate infection across a range of multiplicities of infection, and evaluate the efficacy of camostat mesylate, a known inhibitor of HCoV-NL63 infection. This new capability can be used to address a major gap in the rapid assessment of therapeutic efficacy of small molecules and antiviral agents against influenza and other respiratory viruses including coronaviruses.

COPD-related morbidity and mortality after smoking cessation: status of the evidence.

The evidence base for the benefit of quitting smoking as regards morbidity and mortality outcomes in patients with moderate-to-severe chronic obstructive pulmonary disease (COPD) is limited. The present article is a review of the existing literature. A systematic literature search in medical databases was performed until March 2006, and subsequently until September 1, 2007. The outcomes examined were COPD-related morbidity and mortality (including all-cause mortality) in COPD patients in connection with smoking cessation. A total of 21 and 27 published articles on morbidity and mortality, respectively, were identified and reviewed. For both outcomes, only a few of the studies included patients with severe COPD. Most of the studies reported a beneficial effect of smoking cessation compared with continued smoking, whereas a few found no improvement. Methodological problems, including small study sizes, poor data quality, possibility of reverse causality and incomplete ascertainment of cause of death, limit interpretation of some of the studies. The evidence as a whole supports the conclusion that, even in severe chronic obstructive pulmonary disease, smoking cessation slows the accelerated rate of lung function decline and improves survival compared with continued smoking.

Greatly reduced risk of EBV reactivation in rituximab-experienced recipients of alemtuzumab-conditioned allogeneic HSCT.

EBV-associated post-transplant lymphoproliferative disease (PTLD) remains an important complication of allogeneic haematopoietic stem cell transplantation (allo-HSCT). We retrospectively analysed the incidence and risk factors for EBV reactivation in 186 adult patients undergoing consecutive allo-HSCT with alemtuzumab T-cell depletion at a single centre. The cumulative incidence of EBV reactivation was 48% (confidence interval (CI) 41-55%) by 1 year, with an incidence of high-level EBV reactivation of 18% (CI 13-24%); 8 patients were concurrently diagnosed with PTLD. Amongst patients with high-level reactivation 31/38 (82%) developed this within only 2 weeks of first EBV qPCR positivity. In univariate analysis age⩾50 years was associated with significantly increased risk of EBV reactivation (hazard ratio (HR) 1.54, CI 1.02-2.31; P=0.039). Furthermore, a diagnosis of non-Hodgkin lymphoma (NHL) was associated with greatly reduced risk of reactivation (HR 0.10, CI 0.03-0.33; P=0.0001) and this was confirmed in multivariate testing. Importantly, rituximab therapy within 6 months prior to allo-HSCT was also highly predictive for lack of EBV reactivation (HR 0.18, CI 0.07-0.48; P=0.001) although confounding with NHL was apparent. Our data emphasise the risk of PTLD associated with alemtuzumab. Furthermore, we report the clinically important observation that rituximab, administered in the peri-transplant period, may provide effective prophylaxis for PTLD.

Identification and sequence analysis of a silent gene (ushA0) in Salmonella typhimurium.

The evolution of new or improved enzyme specificities in prokaryotes has been proposed to involve gene duplication, followed by silencing of one of the duplicates at the transcriptional or translational level. Such "silent gene intermediates" are distinct from "cryptic" genes, which are proposed to have a different role in evolution. We describe the identification in Salmonella typhimurium of a silent gene (ushA0) using the active (homologous) ushA gene (encoding UDP-sugar hydrolase) from Escherichia coli as a probe. The ushA0 gene has been cloned and, in the multicopy state, very weak expression can be detected; the gene product was shown to be immunologically and functionally related to the enzyme from E. coli. The sequence of the ushA0 gene was found to be highly homologous to the previously determined sequence of the ushA gene, and the respective promoter and ribosomal-binding sites are also very similar. However, a presumed strong rho-independent terminator in the ushA gene is absent from ushA0; although a weak stem-and-loop structure is present in the 3' region of ushA0, its structure is atypical of rho-independent terminators. The sequence analysis also revealed an insertion-sequence like sequence at the 3' end of ushA0 with a convergent open reading frame terminating 116 base-pairs from the ushA0 stop codon. A deletion of the 5' region of the open reading frame results in increased expression of ushA0, indicating that convergent transcription plays some role in the silencing of ushA0. S. typhimurium contains a UDP-sugar hydrolase, biochemically and genetically distinct from that in E. coli, encoded by the ushB gene. Our results indicate that ushB is not strongly sequence-related to ushA0, and its gene product is not immunologically related to the ushA gene product. ushB is hence a functional duplicate of ushA and provides a rationale for the silencing of ushA0. This situation, and the DNA sequence comparison of ushA and ushA0, strongly suggests that rather than being a cryptic gene, ushA0 has been silenced recently during the evolution of S. typhimurium.

A neuroendocrine peptide derived from the amino-terminal half of rat procalcitonin.

The sequence of rat procalcitonin reveals that calcitonin is located within the precursor's midregion, flanked by two potential polybasic cleavage sites that separate it from amino- and carboxyl-terminal domains. Cleavage at the polybasic sites during precursor processing to generate the 32-residue calcitonin should also generate 57- and 16-residue peptides from the amino- and carboxyl-terminal flanking regions. The carboxyl-terminal flanking hexadecapeptide and its coordinate secretion from C cells with calcitonin have been previously reported. In the present study we have focused on the predicted 57-residue amino-terminal procalcitonin cleavage peptide (N-proCT). We raised antisera to synthetic peptides homologous to the carboxyl- and amino-terminal regions of the putative 57-amino-acid N-proCT and screened calcitonin-rich neoplastic and nonneoplastic C-cells for these two immunoreactivities. A single species of 7.4 kilodaltons detected in C cells by gel filtration and reversed-phase HPLC analyses accounts for most of the carboxyl- and amino-terminal immunoreactivities and possesses the biochemical and biological features predicted for N-proCT. When C cell hyperplasia is induced by a high fat diet, thyroidal levels of calcitonin and N-proCT increase in parallel. In neoplastic C cell cultures, N-proCT and calcitonin concentrations are nearly equimolar in both cellular extracts and basal medium; dexamethasone increases both the cellular and secreted concentration of these peptides. Basal and dexamethasone-treated cultures show calcium-dependent, parallel secretion of N-proCT and calcitonin. Thus, the 57-residue N-proCT predicted from analysis of the procalcitonin sequence is a secretory peptide that appears to be present in equimolar amounts and coordinately regulated with calcitonin in vivo and in vitro.

Characteristics of NaF-induced differentiation of HL-60 cells.

Sodium fluoride (NaF) is known to stimulate osteoblastic bone formation, but little attention has been given to the possibility that NaF also affects bone resorption and the differentiation of osteoclastic progenitor cells. When human promyelocytic HL-60 cells were treated with NaF (0.5 mM, 0-4 days), cell proliferation was inhibited, and the addition of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) (10nM, 0-4 days) augmented this antiproliferative effect. NaF increased cellular reduction of nitroblue tetrazolium (NBT), and this effect was strongly augmented by 1,25(OH)2D3. In addition, NaF produced marked changes in cellular morphology, increased cellular adhesion to plastic, reduced the nuclear/cytoplasmic ratio, and increased cellular expression of chloroacetate esterase, but failed to alter cellular nonspecific esterase activity. Furthermore, NaF increased expression of CD11b and CD66b, and this stimulation was enhanced by adding 1,25(OH)2D3. The sum of these changes in classical promyelocytic cellular indices suggest: (1) that NaF stimulates the early stages of HL-60 differentiation toward a granulocyte-like cell and (2) that 1,25(OH)2D3 promotes these actions of NaF. Additional experiments aimed at further understanding the NaF-induced conversion of HL-60 cells identified further changes. NaF also increased cellular production of prostaglandin E2 (PGE2) and nitric oxide (NO) and induced expression of inducible nitric oxide synthase (iNOS); 1,25(OH)2D3 once again augmented these NaF-induced effects. Similarly, NaF stimulated the production of interleukin 1 alpha (IL-1 alpha), IL-6, and tumor necrosis factor-alpha, and 1,25(OH)2D3 again strongly enhanced these effects. Indomethacin completely blocked stimulation of NBT reduction, NO production, and iNOS expression induced by NaF plus 1,25(OH)2D3; adding exogenous PGE2 (0.1-10 ng/ml) to these indomethacin-blocked cultures dose-dependently restored NO production. These additional findings together with the observed slow onset (24-48 h) of NaF and 1,25(OH)2D3 interaction strongly suggest that 1,25(OH)2D3 acts as a cofactor with NaF primarily through interaction with an endogenous NaF-induced cyclo-oxygenase product(s), quite possibly PGE2 itself. Such a mechanism for NaF and 1,25(OH)2D3 interaction would be strongly analogous to the interaction we have recently demonstrated between 1,25(OH)2D3 and PGE1 on the differentiation of HL-60 cells.

Calcitonin gene-related peptide stimulates potassium efflux through adenosine triphosphate-sensitive potassium channels and produces membrane hyperpolarization in osteoblastic UMR106 cells.

In previous studies, we have shown that calcitonin gene-related peptide (CGRP) acutely inhibits 45Ca2+ uptake in osteoblastic UMR106 cells, and we have proposed that ATP-sensitive potassium (K(ATP)) channels are involved in mediating this action of CGRP. To directly test this proposed mechanism, we have now examined the effects of CGRP on both membrane potential (Em) and K+ mobilization in UMR106 cells, using specific fluorometric dye assays. CGRP (0.01-100 nM) induced membrane hyperpolarization in a dose-dependent manner, with a half maximal effect (ED50) at approximately 0.2 nM and a maximal effect at 100 nM. Both pinacidil (Pina; a K(ATP) channel opener) and forskolin (FSK) induced similar membrane hyperpolarization, but the actions of these three agents could be easily distinguished: both CGRP and Pina actions were well antagonized by glibenclamide (Glib; a selective K(ATP) channel blocker), whereas FSK action was strongly attenuated only by tetraethylammonium (a K(Ca) channel blocker) or compound H-89 (an inhibitor of cAMP-dependent protein kinases). Cells pretreated with Pina no longer responded to CGRP, but they could still respond to FSK; furthermore, pretreatment with FSK failed to block successive treatment with either CGRP or Pina. In parallel with observed changes in Em, CGRP (0.01-100 nM) decreased intracellular K+ concentrations ([K+]i) in a dose-dependent manner, with an ED50 identical to that obtained for alterations in Em. This action of CGRP was sensitive to Glib and had only slight sensitivity to tetraethylammonium; this CGRP effect was mimicked by Pina but not by FSK. Interestingly, CGRP significantly elicited changes in cell shape by a Glib-sensitive mechanism that included notable decreases in cross-sectional cytoplasmic area. These observations strongly support our proposal that CGRP primarily stimulates K+ efflux via activation of K(ATP) channels and thereby induces membrane hyperpolarization in UMR106 cells. Furthermore, our data also suggest that this cascade of initial cellular events may result in rapid changes in cell morphology and decreases in cellular area of the type that are thought to act as triggers for proliferation and/or differentiation in many cellular phenotypes.

Calcitonin gene-related peptide rapidly inhibits calcium uptake in osteoblastic cell lines via activation of adenosine triphosphate-sensitive potassium channels.

In certain neurons, alternative RNA processing generates calcitonin gene-related peptide (CGRP) from the same gene that encodes the hormone calcitonin. As CGRP-containing nerve fibers are prominent in skeleton, we evaluated the effects of CGRP on osteoblasts. Because the vasodilatory effect of neural CGRP in smooth muscle probably involves inhibition of unstimulated Ca2+ uptake, we examined the acute effects of CGRP on this parameter in rat osteoblastic cells. CGRP inhibits 45Ca2+ uptake in both UMR 106 osteosarcoma and RCOB-3 osteoblastic cells. This inhibition is rapid (0.5 min), occurs with an EC50 of 1 nM, and cannot be demonstrated in the presence of 0.1 mM diltiazem, a blocker of voltage-dependent Ca2+ channels. Depolarization of bone cells with high extracellular potassium (K+) also blocks the effect of CGRP on 45Ca2+ uptake, suggesting a central role for K+ channels in mediating this action. In agreement with this hypothesis, the effect of CGRP is blocked by 1 microM glybenclamide, a specific inhibitor of ATP-sensitive potassium (K(ATP)) channels, or by pretreatment of cells with 1 mM iodoacetic acid to deplete intracellular ATP. Blocking Ca2+-activated potassium channels with 1 mM tetraethylammonium does not prevent CGRP's effect. Pinacidil, a specific activator of K(ATP) channels, mimics CGRP's effect. Both CGRP and pinacidil also produce a small significant stimulation of cellular Ca2+ efflux in UMR 106 cells. These data suggest that inhibition of diltiazem-sensitive Ca2+ channels occurs secondary to the hyperpolarization engendered by CGRP activation of K(ATP) channels in osteoblastic cells, an effect similar to that of CGRP on smooth muscle cells.

Positive evidence on effectiveness of selected smoking prevention programs in the United States.

Various smoking intervention approaches have been demonstrated to successfully alter smoking behavior among individual smokers, but it is difficult to demonstrate the benefit of these individual cessation approaches across the population of smokers. In contrast, efforts that concentrate on altering the social and economic environment within which the smoker smokes, most notably the media, taxation, and changing the social acceptability of smoking, have been linked to substantial shifts in the smoking behavior of the US population. Attacking tobacco use as a form of sociological carcinogenesis, rather than focusing on the individual smoker, allows alteration at the root of smoking behavior, ie, its personal, social, and psychological utility for the smoker.

Positive selection vectors: a small plasmid vector useful for the direct selection of Sau3A-generated overlapping DNA fragments.

A positive selection plasmid vector ( pLA7 ), containing a unique BclI site, and its use in the facile selection of a library of overlapping DNA fragments, generated by a partial digest with Sau3A, is described. Selection depends on the 5-fluorouracil + 5' AMP resistance of upp- ush - [ pLA7ush ::cloned DNA] cells, whereas upp- ush - [ pLA7ush +] cells are sensitive under the same conditions.

Characterization of the ush gene of Escherichia coli and its protein products.

The Escherichia coli ush gene has been subcloned and the coding sequence delineated using BAL31 nuclease digestion. Synthesis of proteins encoded by the ush gene have been examined in "maxicells"; two proteins are made, one of which corresponds in Mr (61000) to purified uridine diphosphoglucose hydrolase and the other, less abundant, has an Mr of 43 000. A deletion at the 3' end of the gene introduced by restriction endonuclease digestion, results in the synthesis of a truncated protein of the expected Mr of about 43 000. Precursors of all these proteins are observed in maxicells under conditions known to inhibit processing of secreted proteins. Whereas the precursor of the major ush-encoded protein is retained in the cytoplasm-plus-membrane fraction, unexpectedly the precursor of the truncated protein is secreted. The mature forms of both the normal and truncated proteins are secreted.

Rare codons in E. coli and S. typhimurium signal sequences.

Codon usage has been examined in the signal sequences of 27 genes encoding proteins which possess leader peptides, and are inner-membrane located or exported. The results have been compared with codon usage in the corresponding coding sequences of most of the mature proteins. A bias is observed in the usage of rare codons for two of the three hydrophobic amino acids for which there are rare codons. Since hydrophobic residues are predominant in leader peptides, we suggest that a resulting concentration of rare codons in the signal sequence may play a role (or have played a role in the evolutionary past) in the secretion process by delaying translation.

Diverse actions of calcitonin gene-related peptide on intracellular free Ca2+ concentrations in UMR 106 osteoblastic cells.

Calcitonin gene-related peptide (CGRP) was examined for its effects on intracellular free Ca2+ concentration ([Ca2+]i) in UMR 106 osteoblast-like cells. Cells loaded with the Ca2+ dye FURA-2 dose-dependently responded to CGRP (1-100 nM) with transient two-fold increases in [Ca2+]i. An intracellular source for this Ca2+ transient was suggested by the failure of membrane depolarization with high extracellular K+ or acute depletion of extracellular Ca2+ ([Ca2+]e) with EGTA to attenuate this response. After cells were incubated for 45 min with 0.1 mM extracellular Ca2+ to deplete intracellular Ca2+ stores, CGRP produced a 25-30% decrease in [Ca2+]i rather than a transient increase. This calcium decrease was mimicked by membrane depolarization or by pinacidil, a specific activator of adenosine triphosphate (ATP)-sensitive potassium (KATP) channels, and blocked by glybenclamide, a specific blocker of KATP channels. Our data suggest that CGRP has diverse Ca2+ regulatory effects in UMR 106 cells, mobilizing Ca2+ from intracellular stores via classical signaling while possibly promoting cellular Ca2+ efflux or inhibiting uptake through voltage-dependent Ca2+ channels via KATP-mediated hyperpolarization.

The effect of nicotinamide on spontaneous and induced activity in smooth and skeletal muscle.

BACKGROUND AND PURPOSE: Nicotinamide (NA) is currently undergoing clinical trials as a tumour radiosensitizer. The dose that can be administered is currently 80 mg/kg per day, but this may be restricted to 60 mg/kg per day by the high incidence of nausea and vomiting. To investigate some of NA's underlying mechanisms of action, we have used an ex vivo system to study the direct effect of this drug, over a wide range of concentrations, on isolated spontaneously active rat ileum. Effects on the gut were compared with the action of NA on skeletal and vascular smooth muscle. MATERIALS AND METHODS: Isolated rat ileum rings were perfused with oxygenated Krebs' solution in an organ bath. NA (1 microM to 10 mM) was introduced to the perfusate and the change in amplitude of spontaneous peristaltic activity recorded. Dissected frog sartorius muscle was bathed in modified oxygenated Ringer's solution in an organ bath. The muscle was electrically stimulated to generate isometric contractions. Tension was then measured before and after the addition of a range of NA concentrations (8.2-24.6 mM) to the organ bath. RESULTS: NA inhibited peristalsis in the ileum in a dose-dependent manner. At a drug concentration of 1 mM the amplitude of contractions was reduced to <50% of the initial control value. NA had no effect on the electrically induced contractions in the isolated frog sartorius muscle. CONCLUSIONS: Gut smooth muscle is highly sensitive to the relaxant effect of NA producing 50% relaxation at a concentration approximately 10 fold lower than that required in rat arterial smooth muscle, while having no effect on non-mammalian skeletal smooth muscle. This may provide explanations for the occurrence of emesis in patients undergoing combined nicotinamide therapies and highlight possible alternatives available to counter this unwanted side-effect.

Contractile properties of human renal cell carcinoma recruited arteries and their response to nicotinamide.

BACKGROUND AND PURPOSE: The manipulation of tumour blood supply and thus oxygenation is a potentially important strategy for improving the treatment of solid tumours by radiation. Increased knowledge about the characteristics that distinguish the tumour vasculature from its normal counterparts may enable tumour blood flow to be more selectively modified. Nicotinamide (NA) causes relaxation of preconstricted normal and tumour-supply arteries in rats. It has also been shown to affect microregional blood flow in human tumours. Direct effects of NA on human tumour supply arteries have not previously been reported. This paper describes our evaluation of the effects of NA on two parameters: 'spontaneous', oscillatory contractile activity and agonist (phenylephrine)-induced constriction in the arteries supplying human renal cell carcinomas. MATERIALS AND METHODS: Isolated renal cell carcinoma feeder vessels were perfused in an organ bath with the alpha(1)-adrenoceptor agonist phenylephrine (PE). When the arteries had reached a plateau of constriction, nicotinamide (8.2 mM) was added to the perfusate and changes in perfusion pressure were measured. RESULTS: PE (10 microM) induced a sustained constriction in the majority of the renal cell carcinoma feeder vessels examined, demonstrating that they retain contractile characteristics, at least in response to this alpha(1)-adrenoceptor agonist. In combination with NA (8.2 mM) the constriction was significantly attenuated in half of the preparations. In addition, seven arteries exhibited spontaneous contractile activity which was significantly attenuated by NA in six of them. CONCLUSIONS: NA can significantly attenuate both 'spontaneous' and agonist-induced constrictions in tumour-recruited human arteries, though not all arteries are sensitive.

Glutamine synthetase mRNA in cultured 3T3-L1 adipocytes. Complexity, content and hormonal regulation.

Glutamine synthetase (GS) activity increases more than 100-fold during adipocyte differentiation of cultured 3T3-L1 cells. We now find that Northern hybridization analysis of RNA from 3T3-L1 adipocytes with a rat GS cDNA clone (pGSRK-1) yields two hybridizable GS RNAs of length 3.2 and 1.6 kilobases (kb). Densitometric analyses of autoradiographs of the Northern blots probed with pGSRK-1 indicate that the 3.2 kb GS-specific RNA is at least 4- to 5-fold more abundant than the 1.6 kb GS RNA. Analyses of both total and poly(A+)RNA from 3T3-L1 adipocytes yielded similar results. (It is noteworthy that an mRNA of 1.2 kb would be sufficient to encode the 42 500 Da GS subunit.) Quantitative dot-blot hybridization analysis indicates that dexamethasone increases GS mRNA while both insulin and dibutyryl cAMP decrease GS mRNA and/or prevent the dexamethasone-mediated increase. Our data suggest that there are at least two GS mRNAs in 3T3-L1 adipocytes and that they are regulated in parallel by dexamethasone, insulin and dibutyryl cAMP.

Sulfinimine-mediated asymmetric synthesis of 1,3-disubstituted tetrahydroisoquinolines: a stereoselective synthesis of cis- and trans-6,8-dimethoxy-1,3-dimethyl-1,2,3,4-tetrahydroisoquinoline.

[reaction: see text] The highly diastereoselective addition of lateral lithiated o-tolunitriles to sulfinimines followed by treatment of the resulting sulfinamide with MeLi, hydrolysis, and reduction represents a concise new methodology for the asymmetric synthesis of 1,3-disubstituted tetrahydroisoquinolines.

Carboxyfluorescein and biotin neuromedin C analogues: synthesis and applications.

Two neuromedin C (NC) analogues were constructed by Fmoc synthesis and in situ coupling of 4(5)-carboxyfluorescein or biotin to the N-terminus. Both displayed full agonism in an amylase release assay and cross-reacted fully with a NC-specific antiserum. Biotin NC functioned in a streptavidin-capture ELISA. Carboxyfluorescein NC was used to probe receptor localization in rat stomach. Specific NC binding sites, which did not interact with substance P, angiotensin I, or neurokinin A, were labeled in the antrum. Identity of NC binding sites was confirmed by microautoradiography. The specifically labeled cells were all found in the lamina propria and at least some of cells were identified as eosinophils.

Silent genes in bacteria: the previously designated 'cryptic' ilvHI locus of 'Salmonella typhimurium LT2' is active in natural isolates.

Gene ilvG in Escherichia coli K-12 and ilvI in 'Salmonella typhimurium LT2' (S. enterica serotype Typhimurium, strain LT2) are inactive due to frameshift or nonsense mutations, respectively. These inactive genes have been suggested to be part of 'cryptic' genetic systems which are defined as being of long-term regulatory and evolutionary significance. We have shown that the nonsense mutation in ilvI is present only in derivatives of the laboratory strain 'S. typhimurium LT2'. All natural isolates of Salmonella examined have an arginine codon at the corresponding location of their ilvI sequences. Further, two randomly selected natural isolates of serotype Typhimurium are shown to each have an active ALS III isozyme. Our findings strongly suggest that the only Salmonella strains which lack a functional ilvHI locus are LT2 isolates. We suggest that the mutations leading to inactivation of both ilvI in 'S. typhimurium LT2' and ilvG in E. coli K-12 are more likely to have been acquired during laboratory storage and/or cultivation, rather than representing cryptic systems of gene regulation.

UDP-sugar hydrolase isozymes in Salmonella enterica and Escherichia coli: silent alleles of ushA in related strains of group I Salmonella isolates, and of ushB in wild-type and K12 strains of E. coli, indicate recent and early silencing events, respectively.

Escherichia coli contains a single periplasmic UDP-glucose hydrolase (5'-nucleotidase) encoded by ushA. Salmonella enterica, serotype Typhimurium, also contains a single UDP-glucose hydrolase but, in contrast to E. coli, it is membrane-bound and is encoded by the non-homologous ushB gene; Salmonella enterica (Typhimurium) also contains a silent allele of the ushA gene (ushA0). In this report, we show that nearly all natural isolates of Salmonella contain both UDP-sugar hydrolases, i.e. they are UshA+ UshB+. The only exceptions are all from sub-group I (S. gallinarum, S. pullorum, and most Typhimurium strains), are UshA- UshB+, and several have been shown to contain an ushA0 allele. These data, together with the fact that these latter strains are closely related genetically, strongly suggests a recent silencing mutation(s). We also report the presence in E. coli K-12, and in natural isolates of E. coli, of a DNA sequence which is homologous to the ushB gene of Salmonella; since E. coli does not contain UshB activity, we tentatively refer to this sequence as ushB0. Since all E. coli strains investigated are UshB-, we conclude that the silencing mutation(s) occurred relatively early following the divergence of Escherichia coli and Salmonella from a common ancestor that was ushA+ ushB+.