SeedStor: A Germplasm Information Management System and Public Database.

SeedStor (https://www.seedstor.ac.uk) acts as the publicly available database for the seed collections held by the Germplasm Resources Unit (GRU) based at the John Innes Centre, Norwich, UK. The GRU is a national capability supported by the Biotechnology and Biological Sciences Research Council (BBSRC). The GRU curates germplasm collections of a range of temperate cereal, legume and Brassica crops and their associated wild relatives, as well as precise genetic stocks, near-isogenic lines and mapping populations. With >35,000 accessions, the GRU forms part of the UK's plant conservation contribution to the Multilateral System (MLS) of the International Treaty for Plant Genetic Resources for Food and Agriculture (ITPGRFA) for wheat, barley, oat and pea. SeedStor is a fully searchable system that allows our various collections to be browsed species by species through to complicated multipart phenotype criteria-driven queries. The results from these searches can be downloaded for later analysis or used to order germplasm via our shopping cart. The user community for SeedStor is the plant science research community, plant breeders, specialist growers, hobby farmers and amateur gardeners, and educationalists. Furthermore, SeedStor is much more than a database; it has been developed to act internally as a Germplasm Information Management System that allows team members to track and process germplasm requests, determine regeneration priorities, handle cost recovery and Material Transfer Agreement paperwork, manage the Seed Store holdings and easily report on a wide range of the aforementioned tasks.

Genetic diversity in European Pisum germplasm collections.

The distinctness of, and overlap between, pea genotypes held in several Pisum germplasm collections has been used to determine their relatedness and to test previous ideas about the genetic diversity of Pisum. Our characterisation of genetic diversity among 4,538 Pisum accessions held in 7 European Genebanks has identified sources of novel genetic variation, and both reinforces and refines previous interpretations of the overall structure of genetic diversity in Pisum. Molecular marker analysis was based upon the presence/absence of polymorphism of retrotransposon insertions scored by a high-throughput microarray and SSAP approaches. We conclude that the diversity of Pisum constitutes a broad continuum, with graded differentiation into sub-populations which display various degrees of distinctness. The most distinct genetic groups correspond to the named taxa while the cultivars and landraces of Pisum sativum can be divided into two broad types, one of which is strongly enriched for modern cultivars. The addition of germplasm sets from six European Genebanks, chosen to represent high diversity, to a single collection previously studied with these markers resulted in modest additions to the overall diversity observed, suggesting that the great majority of the total genetic diversity collected for the Pisum genus has now been described. Two interesting sources of novel genetic variation have been identified. Finally, we have proposed reference sets of core accessions with a range of sample sizes to represent Pisum diversity for the future study and exploitation by researchers and breeders.

Renal FMD may not confer a familial hypertensive risk nor is it caused by ACTA2 mutations.

Renal fibromuscular dysplasia (FMD) can cause hypertension, and previous reports suggest that FMD is familial. We hypothesized that, in families containing an individual with proven FMD, relatives of index cases would have an increased risk of hypertension. ACTA2 mutations cause a spectrum of extra-renal arteriopathy, leading to our second hypothesis that mutations are implicated in FMD. The blood pressure of first-degree relatives was measured using standard devices and, when indicated, with 24-h ambulatory monitoring. Leucocyte DNA was obtained from FMD index cases and ACTA2 sequenced. Thirteen unrelated index cases, aged 2-32 (median 15) years, were recruited. Blood pressure was assessed in 40 first-degree relatives, comprising 22 parents aged 28-58 (median 44) years and 18 siblings aged 3-30 (median 13) years. Hypertension was evident in six (27%) parents but in none of the eight adult siblings. Of the ten screened siblings aged less than 18 years, one teenager was pre-hypertensive (90th-95th centile), the remainder being normotensive. No ACTA2 mutations were found in 13 index cases. Hypertension was evident in 20% of all assessed adult first-degree relatives and is therefore not increased relative to 25% of the adult population. Although hypertensive parents did not undergo angiography to assign FMD status, this observation, together with the lack of hypertension in 18 siblings, indicates that FMD is unlikely to confer an excess hypertension risk in first-degree relatives up to middle-age. Furthermore, in our cohort, FMD was not caused by ACTA2 mutations.

Whole-genome linkage and association scan in primary, nonsyndromic vesicoureteric reflux.

Primary vesicoureteric reflux accounts for approximately 10% of kidney failure requiring dialysis or transplantation, and sibling studies suggest a large genetic component. Here, we report a whole-genome linkage and association scan in primary, nonsyndromic vesicoureteric reflux and reflux nephropathy. We used linkage and family-based association approaches to analyze 320 white families (661 affected individuals, generally from families with two affected siblings) from two populations (United Kingdom and Slovenian). We found modest evidence of linkage but no clear overlap with previous studies. We tested for but did not detect association with six candidate genes (AGTR2, HNF1B, PAX2, RET, ROBO2, and UPK3A). Family-based analysis detected associations with one single-nucleotide polymorphism (SNP) in the UK families, with three SNPs in the Slovenian families, and with three SNPs in the combined families. A case-control analysis detected associations with three additional SNPs. The results of this study, which is the largest to date investigating the genetics of reflux, suggest that major loci may not exist for this common renal tract malformation within European populations.

Catabolite repression of SOS-dependent and SOS-independent spontaneous mutagenesis in stationary-phase Escherichia coli.

Previous work in our laboratory established that a spontaneous mutagenesis process operating in stationary-phase Escherichia coli cells undergoing selection is subject to regulation by the global regulatory mechanism known as catabolite repression (formerly also called glucose-repression). Here, we set out to determine the identity of this hitherto unknown catabolite-repressible spontaneous mutation generation mechanism(s). We used two different spontaneous mutation detection assays, reversion of a Lac(-) (lacI33OmegalacZ) frameshift marker and forward mutation to valine-resistance, and tested the effects of varying the nature of the carbon source(s) present in the selective plating medium on the mutability of bacterial cells carrying known defects in the recA, umuDC and dinB genes, three well-known SOS response genes, whose products are important for mutagenesis in E. coli. Consistent with the results of our previous Lac(-)-->Lac(+) assay using otherwise SOS-proficient bacterial cells, we found that the overall numbers of spontaneous Lac(+)E. coli revertants were highest when the selective medium contained lactose and lowest when it contained lactose plus the non-utilizable but strongly catabolite-repressing glucose analogue, methyl-alpha-d-glucopyranoside (alphaMG). In contrast, we found that the numbers of Lac(+) revertants appearing on the lactose and lactose+alphaMG selection plates were greatly diminished and not significantly different when the bacterial cells concerned carried either a DeltarecA or DeltadinB mutation. Furthermore, introducing the DeltadinB mutant allele into bacterial cells over-expressing the recA gene reduced the numbers of Lac(+) mutations to those being recovered with the DeltadinB cells. These results appear to suggest that (i) the DinB-dependent mutation generation pathway is alone responsible for spontaneous reversion of the lacI33OmegalacZ frameshift marker, and (ii) the varying numbers of Lac(+) colonies that we recover on the lactose and lactose+alphaMG plates provide a direct measure of the differential effects of these particular carbon compounds on the overall expression of the dinB gene. Interestingly, the yields of spontaneous Val mutations arising in wild-type, DeltarecA, DeltadinB and DeltaumuDC cells were found to be similar, but always tended to be highest when the medium contained only a non-repressing carbon source (glycerol) and lowest when it had been supplemented with a strong catabolite repressor such as glucose or alphaMG. Together, our results would seem to establish that stationary-phase E. coli cells exposed to strong selection pressures can accumulate spontaneous mutations via SOS-dependent and SOS-independent mutation generation pathways whose levels of expression are regulated by catabolite repression.

UNIFOLIATA regulates leaf and flower morphogenesis in pea.

BACKGROUND: The vegetative phenotype of the pea mutant unifoliata (uni) is a simplification of the wild-type compound leaf to a single leaflet. Mutant uni plants are also self-sterile and the flowers resemble known floral meristem and organ identity mutants. In Antirrhinum and Arabidopsis, mutations in the floral meristem identity gene FLORICAULA/LEAFY (FLO/LFY) affect flower development alone, whereas the tobacco FLO/LFY homologue, NFL, is expressed in vegetative tissues, suggesting that NFL specifies determinacy in the progenitor cells for both flowers and leaves. In this paper, we characterised the pea homologue of FLO/LFY. RESULTS: The pea cDNA homologue of FLO/LFY, PEAFLO, mapped to the uni locus in recombinant-inbred mapping populations and markers based on PEAFLO cosegregated with uni in segregating sibling populations. The characterisation of two spontaneous uni mutant alleles, one containing a deletion and the other a point mutation in the PEAFLO coding sequences, predicted that PEAFLO corresponds to UNI and that the mutant vegetative phenotype was conferred by the defective PEAFLO gene. CONCLUSIONS: The uni mutant demonstrates that there are shared regulatory processes in the morphogenesis of leaves and flowers and that floral meristem identity genes have an extended role in plant development. Pleiotropic regulatory genes such as UNI support the hypothesis that leaves and flowers derive from a common ancestral sporophyll-like structure. The regulation of indeterminancy during leaf and flower morphogenesis by UNI may reflect a primitive function for the gene in the pre-angiosperm era.

Low levels of high-density lipoprotein cholesterol are associated with vascular remodeling in cardiac transplant recipients.

BACKGROUND: Early atherosclerosis may be associated with compensatory vessel enlargement, termed positive remodeling. Enlarged brachial artery diameter has been reported in patients with risk factors for atherosclerosis and in individuals with coronary atherosclerosis, indicating that brachial artery enlargement is a marker for the presence of atherosclerotic changes. Cardiac transplant recipients often have abnormal lipid levels, but the effect of specific lipid abnormalities on vascular remodeling in this population has not been evaluated. This study examined the relationship between lipid levels and brachial artery diameter in cardiac transplant recipients. METHODS: Thirty-five stable cardiac transplant recipients underwent high-resolution brachial artery ultrasound to evaluate resting brachial artery diameter. Levels of high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglycerides were determined and the presence of other cardiac risk factors was assessed. RESULTS: Brachial artery diameter was larger (4.3 +/- 0.1 mm) in subjects with low levels of HDL-C (< 40 mg/dL, n = 11) compared to subjects with high HDL-C (> or = 40 mg/dL, n = 24), who had a mean brachial artery diameter of 3.7 +/- 0.1 mm (P = .006). Neither high LDL-C (> or = 100 mg/dL) nor high triglycerides (> or = 200 mg/dL) were associated with differences in brachial artery diameter. Multivariate analysis demonstrated that the relationship between low HDL-C and increased brachial artery diameter was independent of body surface area or statin use. CONCLUSIONS: Low levels of HDL-C are an independent predictor of brachial artery enlargement in stable cardiac transplant recipients. These findings suggest that suboptimal HDL-C levels may be associated with the development of vascular remodeling and atherosclerosis in this population.

Contexts Paired with Junk Food Impair Goal-Directed Behavior in Rats: Implications for Decision Making in Obesogenic Environments.

The high prevalence of obesity and related metabolic diseases calls for greater understanding of the factors that drive excess energy intake. Calorie-dense palatable foods are readily available and often are paired with highly salient environmental cues. These cues can trigger food-seeking and consumption in the absence of hunger. Here we examined the effects of palatable food-paired environmental cues on control of instrumental food-seeking behavior. In Experiment 1, adult male rats received exposures to one context containing three "junk" foods (JFs context) and another containing chow (Chow context). Next, rats were food-deprived and trained to perform instrumental responses (lever-press) for two novel food rewards in a third, distinct context. Contextual influences on flexible control of food-seeking behavior were then assessed by outcome devaluation tests held in the JF, chow and training contexts. Devaluation was achieved using specific satiety and test order was counterbalanced. Rats exhibited goal-directed control over behavior when tested in the training and chow-paired contexts. Notably, performance was habitual (insensitive to devaluation) when tested in the JF context. In Experiment 2 we tested whether the impairment found in the JF context could be ameliorated by the presentation of a discrete auditory cue paired with the chow context, relative to a second cue paired with the JF context. Consistent with the results of Experiment 1, the devaluation effect was not significant when rats were tested in the JF context with the JF cue. However, presenting the chow cue increased the impact of the devaluation treatment leading to a robust devaluation effect. Further tests confirmed that performance in the chow context was goal-directed and that sensory-specific satiety in the JF context was intact. These results show that environments paired with palatable foods can impair goal-directed control over food-seeking behavior, but that this deficit was improved by a cue paired with chow. This has promising implications for assisting individuals in controlling their eating behavior in environments designed to dysregulate it.

C-ski transcripts with and without exon 2 are expressed in skeletal muscle and throughout chick embryogenesis.

Overexpression of v-ski or c-ski cDNAs has a pronounced effect on proliferation, morphological transformation and myogenic differentiation in cells in culture and in transgenic animals. Yet, little is known about expression of the c-ski locus or the relationship between c-ski cDNAs and alternatively spliced c-ski transcripts in chicken tissues, particularly in skeletal muscle or during embryogenesis. We developed a series of probes and oligonucleotide primers specific for the eight coding exons and the long 3' noncoding region found in chicken c-ski mRNAs. The most abundant chicken c-ski mRNAs in a vast array of tissues are 8.5 kb, with additional, but less abundant, mRNAs of 7.5, 6.5 and 4.4 kb. Steady-state levels of c-ski mRNAs, indistinguishable from transcripts in other tissues, accumulate in skeletal muscle from embryonic, newly hatched, and adult chicks. Only exon 2, a small exon of 111 bp, was found to be alternatively spliced in c-ski mRNAs. Transcripts with and without exon 2 appear in all tissues, in somites, and from the earliest stages of chick embryogenesis. Thus, c-ski cDNA sequences, which extend about 4.3 kb, represent either the least abundant form of c-ski mRNAs in tissues or a severely truncated form of the major 8.5 kb transcripts.

Collaborative study for the establishment of the WHO 3(rd) International Standard for Endotoxin, the Ph. Eur. endotoxin biological reference preparation batch 5 and the USP Reference Standard for Endotoxin Lot H0K354.

An international collaborative study was organised jointly by the World Health Organization (WHO)/National Institute for Biological Standards and Control (NIBSC), the United States Pharmacopeia (USP) and the European Directorate for the Quality of Medicines & HealthCare (EDQM/Council of Europe) for the establishment of harmonised replacement endotoxin standards for these 3 organisations. Thirty-five laboratories worldwide, including Official Medicines Control Laboratories (OMCLs) and manufacturers enrolled in the study. Three candidate preparations (10/178, 10/190 and 10/196) were produced with the same material and same formulation as the current reference standards with the objective of generating a new (3(rd)) International Standard (IS) with the same potency (10 000 IU/vial) as the current (2(nd)) IS, as well as new European Pharmacopoeia (Ph. Eur.). and USP standards. The suitability of the candidate preparations to act as the reference standard in assays for endotoxin performed according to compendial methods was evaluated. Their potency was calibrated against the WHO 2(nd) IS for Endotoxin (94/580). Gelation and photometric methods produced similar results for each of the candidate preparations. The overall potency estimates for the 3 batches were comparable. Given the intrinsic assay precision, the observed differences between the batches may be considered unimportant for the intended use of these materials. Overall, these results were in line with those generated for the establishment of the current preparations of reference standards. Accelerated degradation testing of vials stored at elevated temperatures supported the long-term stability of the 3 candidate preparations. It was agreed between the 3 organisations that batch 10/178 be shared between WHO and EDQM and that batches 10/190 and 10/196 be allocated to USP, with a common assigned value of 10 000 IU/vial. This value maintains the continuity of the global harmonisation of reference materials and unitage for the testing of endotoxins in parenteral pharmaceutical products. Based on the results of the collaborative study, batch 10/178 was established by the European Pharmacopoeia Commission as the Ph. Eur. Endotoxin Biological Reference Preparation (BRP) batch 5. The same batch was also established by the Expert Committee on Biological Standardisation (ECBS) of WHO as the WHO 3(rd) IS for Endotoxin. Batch 10/190 was adopted as the USP Endotoxin Reference Standard, lot H0K354 and vials from this same batch (10/190) will serve as the United States Food and Drug Administration (USFDA) Endotoxin Standard, EC-7.

Infusion of a monoamine oxidase inhibitor into the locus coeruleus can prevent stress-induced behavioral depression.

Behavioral depression produced by exposing animals to a stressor that they cannot control (uncontrollable shock) was reversed by infusion of the monoamine oxidase (MAO) inhibitor pargyline into the locus coeruleus (LC) region of the brain stem. Following exposure to uncontrollable shock, rats were infused through bilateral cannulas implanted in the LC region with either pargyline or vehicle. At 110 min after infusion, animals were tested for behavioral activity in a swim tank. Immediately following the behavioral test, animals were sacrificed for determination of the monoamines [norepinephrine (NE), dopamine (DA), serotonin (5-HT)], as well as 5-hydroxy-indoleacetic acid (5-HIAA) in various brain regions. The results showed that animals exposed to uncontrollable shock and then infused with vehicle exhibited significantly less activity in the swim test than animals not exposed to shock and similarly infused with vehicle; thus, the usual behavioral depression following exposure to uncontrollable shock was observed. On the other hand, shocked animals infused with pargyline did not show reduced activity in the swim test. Unshocked animals infused with pargyline showed no more activity than did shocked animals infused with pargyline or unshocked animals infused with vehicle, which demonstrated that the infusion of pargyline into shocked animals did not eliminate the shock-induced depression of activity simply by generally stimulating motor activity. Measurement of the concentration of NE, DA, 5-HT, and 5-HIAA present in seven brain regions at the conclusion of the swim test showed that pargyline infusion into the LC eliminated the large depletion of NE in the LC that is normally observed after exposure to uncontrollable shock while having no effect on NE levels in the other brain regions examined. The level of 5-HT in the LC was also raised by infusion of pargyline into the LC, but again, there was no effect of pargyline infusion on 5-HT levels in any of the other brain regions. In conclusion, infusion of pargyline into the LC region of the brain eliminated both the large depletion of NE in the LC region and the behavioral depression that otherwise results from exposure of animals to uncontrollable shock.

Infusion of adrenergic receptor agonists and antagonists into the locus coeruleus and ventricular system of the brain. Effects on swim-motivated and spontaneous motor activity.

These studies examined how pharmacological stimulation and blockade of alpha receptors would affect active motor behavior in rats. In experiment I, alpha-2 receptor antagonists (piperoxane, yohimbine) and agonists [clonidine, norepinephrine (NE)] were infused into various locations in the ventricular system of the brain, including the locus coeruleus region, and motor activity was measured. Activity was measured principally in a swim test but spontaneous (ambulatory) activity was also recorded while drugs were being infused. When infused into the locus coeruleus region, small doses of the antagonists piperoxane and yohimbine depressed activity in the swim test while infusion of the agonists clonidine and NE had the opposite effect of stimulating activity. These effects were highly specific to the region of the locus coeruleus, since infusions of these drugs into other nearby locations in the ventricular system or use of larger doses had different, often opposite effects. This was especially true of clonidine and NE which profoundly depressed activity when infused posterior to the locus coeruleus, particularly over the dorsal vagal complex. Infusion of small doses of these drugs into the lateral ventricle had effects similar to infusion into the locus coeruleus region, though less pronounced. Changes in spontaneous motor activity were also observed, but this measure differentiated the groups less well than did the swim test. In experiment II, the predominantly postsynaptic receptor agonists isoproterenol (beta agonist) and phenylephrine (alpha-1 agonist) were infused into the ventricular system. Since infusions of piperoxane and yohimbine into the locus coeruleus that decreased activity in experiment I increase the release of NE by blocking alpha-2 inhibitory receptors on cell bodies and dendrites of the locus coeruleus, experiment II tested whether ventricular infusion of predominantly postsynaptic receptor agonists would also decrease activity in the swim test. Both isoproterenol and phenylephrine produced this effect, but did so selectively with respect to dose and location of infusion in the ventricular system. These findings are consistent with recent results relating to the mechanism that underlies stress-induced depression of active behavior.

Reversal of behavioral depression by infusion of an alpha-2 adrenergic agonist into the locus coeruleus.

This experiment demonstrated that behavioral depression produced by exposure of rats to strong uncontrollable shocks could be reversed by infusion of the alpha-2 adrenergic agonist clonidine into the region of the locus coeruleus (LC). A 20-min infusion, through bilateral cannulae, into the locus coeruleus of clonidine, piperoxane (alpha-2 antagonist) or inactive vehicle (0.85% saline), was given beginning 70 min after the animals were removed from the stress situation. The dose and volume of drug given in the infusion (0.16 microgram/microliter, 0.1 microliter/min) had been previously shown to produce effects specific to the locus coeruleus (Weiss, Simson, Hoffman, Ambrose, Cooper and Webster, 1986; Neuropharmacology 25: 367-384). At the conclusion of the infusion, active behavior of animals was measured in a 15-min swim test. Results showed that stressed animals infused with vehicle exhibited significantly less active behavior in the swim test than did non-stressed animals infused with vehicle, thereby showing the usual behavioral depression seen after exposure to an uncontrollable stress. Stressed animals infused with clonidine showed no difference in active behavior in comparison to non-stressed animals infused with vehicle and showed significantly more activity than did the stressed animals infused with vehicle. Stressed animals infused with piperoxane showed no significant difference in activity in comparison to the stressed animals infused with vehicle and were significantly less active than either the non-stressed animals infused with vehicle or the stressed animals infused with clonidine. Thus, infusion into the locus coeruleus of the alpha-2 agonist clonidine, but not the alpha-2 antagonist piperoxane, eliminated behavioral depression.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the anti-respiratory syncytial virus activity and toxicity of papaverine hydrochloride and pyrazofurin in vitro and in vivo.

Based on reports describing their broad antiviral activity, the toxicity and antiviral efficacy of papaverine hydrochloride and pyrazofurin against respiratory syncytial virus (RSV) infection were tested in vitro in tissue culture cells and in vivo in cotton rats. Papaverine inhibited RSV replication in vitro; however, the median minimal toxic dose-median minimal inhibitory concentration ratios (MTD50:MIC50) in vitro and in vivo for papaverine were less than 4. Further work with this compound was discontinued. In contrast, pyrazofurin inhibited RSV replication in vitro (a mean MIC50 of 0.04 microgram/ml was obtained) and in vivo (RSV pulmonary titers were significantly reduced consistently in cotton rats given daily 10 mg/kg doses compared to untreated control animals). However, some toxic effects were observed in both the in vitro and in vivo tests of this compound. The remaining potential of pyrazofurin as an anti-RSV compound is discussed.

The antiviral activity of SP-303, a natural polyphenolic polymer, against respiratory syncytial and parainfluenza type 3 viruses in cotton rats.

SP-303, a naturally occurring polyphenolic polymer (average M.W. = 2100 Da), was tested in cotton rats (Sigmoden hispidus) for antiviral activity against respiratory syncytial (RSV) and parainfluenza type 3 (PIV3) viruses, and for acute toxicity. Significant reductions in pulmonary RSV titers, compared to pulmonary RSV titers in comparably treated control animals, were seen in cotton rats given 1-10 mg SP-303/kg/day intraperitoneally (i.p.) on days 1 through to 3, after experimental inoculation with RSV. The minimum efficacious dose of SP-303 against PIV3, when given i.p. for 3 days, was 3 mg/kg/day. Higher doses of SP-303 could not be given i.p., as doses > or = 30 mg/kg/day given once daily by this route for 3 or more consecutive days caused both significant weight loss and death in infected or uninfected animals. Although no toxicity was observed following oral administration of up to 270 mg of SP-303 daily for 3 days, this compound had variable antiviral activity when given by this route.

Further studies with short duration ribavirin aerosol for the treatment of influenza virus infection in mice and respiratory syncytial virus infection in cotton rats.

Ribavirin aerosol administration has been shown to be effective in the treatment of respiratory syncytial virus (RSV) infections in infants and in influenza A and B virus infections in young adults. Long treatment schedules and potential for environmental contamination have stimulated the search for alternative dosing schedules. Thus, we attempted to determine the length of time of ribavirin aerosol necessary for effective treatment of influenza and RSV. In RSV-infected cotton rats, aerosolization for just 30 min with high-dose ribavirin (HDR:60 mg ribavirin/ml in reservoir), 3 times daily, reduced viral lung titers/gm of tissue by 1.1 log10. In influenza virus-infected mice, 15 min of aerosolized HDR, 3 times daily, was effective in reducing both mortality and pulmonary virus titers (1.1 log10 reduction). When the intervals between aerosol administration each day were equally divided (i.e., q.8 h), the treatments were most effective. Treatment for 45 min, once daily, was not as effective as divided doses. Calculations of ribavirin concentrations in respiratory secretions following 15 min treatment in mice with HDR indicated that drug levels dropped below the ED50 for influenza viruses after about 9 h. A daily dosage of ribavirin, estimated to be 8-15 mg/kg, was effective for the treatment of influenza and RSV infections.

Evaluation of the toxicity and antiviral activity of carbocyclic 3-deazaadenosine against respiratory syncytial and parainfluenza type 3 viruses in tissue culture and in cotton rats.

The toxicity and antiviral efficacy of carbocyclic 3-deazaadenosine (Cc3Ado) against respiratory syncytial (RSV) and parainfluenza type 3 (PIV3) virus infections were tested in tissue culture and in cotton rats. The mean median efficacious dose (ED50) of Cc3Ado in HEp2 cells against RSV and PIV3 was 9 and 14 micrograms/ml, respectively. These values were 85- and 55-fold less than the median inhibitory (toxic) dose (ID50) of Cc3Ado in this cell line (750 micrograms/ml), and similar to values obtained for ribavirin. Cc3Ado exhibited no significant antiviral activity against influenza A, influenza B, adeno type 5 or adeno type 7 viruses (all ED50 were greater than 1000 micrograms/ml). In cotton rats, animals given greater than or equal to 1 mg/kg/day Cc3Ado intraperitoneally on days 1, 2 and 3 after experimental challenge with virus, consistently had significant reductions in pulmonary RSV and PIV3 titers compared to pulmonary virus titers in comparably treated control animals. The minimum efficacious dose of ribavirin given under the same conditions was 30 mg/kg/day. Cc3Ado was also efficacious in cotton rats when given orally by gavage, or when different administration schedules were used. The median efficacious dose of Cc3Ado when given orally was 10 mg/kg/day. No significant toxic effects were noted in cotton rats, even in animals given 20 mg/kg daily for eight consecutive days.

Toxicity and antiviral activity of LY253963 against respiratory syncytial and parainfluenza type 3 viruses in tissue culture and in cotton rats.

LY253963, the sodium salt of 1,3,4-thiadiazol-2-ylcyanamide, was evaluated in tissue culture and in cotton rats for toxicity and antiviral efficacy against respiratory syncytial (RSV) and parainfluenza type 3 (PIV3) viruses. The selective index (ratio of the median toxic dose: median efficacious dose) of LY253963 in HEp2 tissue culture cells was greater than 100 against both RSV and PIV3. When given intraperitoneally to cotton rats, the minimum protective dose of LY253963 against both of these viruses was between 1 and 3 mg/kg/day. In contrast, doses of LY253963 as high as 30 mg/kg/day, administered orally after experimental inoculation of virus, did not significantly reduce pulmonary virus titers in treated animals compared to control animals given placebo. No toxic effects were noted in cotton rats, even in those given 20 mg/kg/day for eight consecutive days.

Expression of the c-ski proto-oncogene during cell cycle arrest and myogenic differentiation.

Although the ski oncogene plays a role in cell proliferation, morphological transformation, and myogenic differentiation, the myogenic activities of the proto-oncogene c-ski have yet to be elucidated. c-ski is expressed within myoblasts during embryogenesis. Transcripts from the proto-oncogene can be detected in somites early in myogenic commitment, as well as in terminally differentiated skeletal muscle. However, c-ski mRNAs expressed in cells of the myogenic lineage are indistinguishable from c-ski transcripts in other cell types, raising the possibility that muscle-specific c-ski transcripts are expressed transiently. Avian cell lines QM7 and QM5 were used as a model to analyze changes in expression and alternative exon usage of c-ski during synchronous muscle differentiation. Upon serum deprivation, QM7 cells undergo myogenic differentiation. In contrast, QM5 cells cease proliferation but do not differentiate. Results show that levels of expression and alternative splicing of c-ski transcripts remain unchanged during cell cycle arrest or myogenic differentiation.

Corticosteroids increase lipocortin I in BAL fluid from normal individuals and patients with lung disease.

Lipocortin I is a corticosteroid-inducible protein that has potent anti-inflammatory activity. To determine whether lipocortin I is present on the epithelial surface of the human lung, we used a specific polyclonal antibody by the technique of Western blotting to evaluate bronchoalveolar lavage (BAL) fluid of normal individuals and patients with idiopathic pulmonary fibrosis. Lipocortin I was a normal constituent of the epithelial surface of the normal lung and comprised 0.23 +/- 0.03% of BAL fluid proteins. Four separate immunoreactive species were detected, at 37, 36, 34, and 33 kDa, consistent with previously published results. Corticosteroids increased the amounts of lipocortin present in normal volunteers and in patients with idiopathic pulmonary fibrosis. These results demonstrate that lipocortin I is normally present in the human lung and further suggest that lipocortin I may be an important modulator of the anti-inflammatory effects of corticosteroids in the lung.