Automatic sample production by depositing solutions on filters for the organization of proficiency tests.

This article describes a device intended to produce replicas on filters by liquid deposition of anion or metal solutions. Schematically, the filters are housed in cassettes labelled automatically by means of a code. An automatic arm takes each cassette, reads the code, and deposits the amount of element required. Weighing before and after deposition allows the amount deposited to be accurately checked and determined. This automated system allows the production of replicas with high deposition regularity, replica dispersion for the most part being <1%. The samples produced can be used during proficiency tests where the assigned value is determined either by the participants or by the organizer.

Structure of transcripts from the homeotic Antennapedia gene of Drosophila melanogaster: two promoters control the major protein-coding region.

The Antennapedia (Antp) homeotic gene of Drosophila melanogaster regulates segmental identity in the thorax. Loss of Antp function results in altered development of the embryonic thoracic segments or can cause legs to be transformed into antennae. Certain combinations of Antp recessive lethal alleles complement to permit normal development. The structure of the Antp gene, analyzed by sequencing cDNA clones and exons and by transcript mapping, revealed some of the basis for its genetic complexity. It has two promoters governing two nested transcription units, one unit 36 and one 103 kilobase pairs (kb) long. Both units incorporated the same protein-coding exons, all of which are located in the 3'-most 13 kb of the gene. The two promoters resulted in the attachment of either of two long noncoding leader sequences (1.5 and 1.7 kb) to a 1.1-kb open reading frame. Both transcription units used the same pair of alternative polyadenylation sites 1.4 kb apart; the choice of sites was developmentally regulated. Some of the mutations that disrupt the larger transcription unit complemented a mutation affecting the smaller one. Dominant mutations that transform antennae into legs split the gene but left the coding exons intact. The encoded protein has unusually long runs of glutamine and a homeodomain near the C terminus.

Increase of spontaneous intrachromosomal homologous recombination in mammalian cells expressing a mutant p53 protein.

Homologous recombination plays an essential role in processes involved in genome stability/instability, such as molecular evolution, gene diversification, meiotic chromosome segregation, DNA repair and chromosomal rearrangements. p53 devoid cells exhibit predisposition to neoplasia, defects in G1 checkpoint and high genetic instability but a normal rate of point mutations. We investigated the effect of a p53 mutation, on spontaneous homologous recombination between intrachromosomal direct repeat sequences, in mouse L cells. In these cells, wild type for the p53 gene, we have overexpressed the mutant p53(175(Arg>His)) protein leading to a p53 mutant phenotype, as verified by the absence of a G1 arrest after gamma-irradiation. We show that the rate of spontaneous recombination is increased from five- to 20-fold in the mutant p53 lines. Moreover, this increase is observed in gene conversion as well as in deletion events. Our results provide new insights into the molecular mechanisms of genetic instability due to a defect of p53.

QRI8, a novel ubiquitin-conjugating enzyme in Saccharomyces cerevisiae.

We have cloned and sequenced the gene encoding a novel ubiquitin-conjugating enzyme in Saccharomyces cerevisiae. Disruption of this gene shows that it is not essential for cell viability.

Vectors for Drosophila P-element-mediated transformation and tissue culture transfection.

We describe nine P-element vectors that can be used to study gene regulation and function in Drosophila. These vectors were designed for use in germline transformation and cell culture transfection assays. One set consists of five P elements that can be used to study transcriptional regulatory sequences. These vectors contain several unique restriction sites for insertion of a foreign promoter upstream from either a cat or lacZ reporter gene. Two of the beta-galactosidase-coding vectors also require the insertion of a start codon for translation of the reporter enzyme and thus can be used to study translational regulatory sequences. The second set of P elements consists of four vectors that contain the Drosophila cytoplasmic actin 5C promoter and polyadenylation signals. Upon insertion of a foreign DNA segment, these vectors direct constitutive expression of the encoded RNA and protein.

Allergenicity and cross-reactivity of rye grass pollen extracts revealed by monoclonal antibodies.

Using monoclonal antibodies the immunogenic and allergenic characteristics of rye Group I were redefined by SDS-PAGE analysis and immunoblotting. The purified rye Group I from NIH possesses a major component of approximately 34000 Da against which most of the sera from grass-sensitive patients and none from non-atopic volunteers contain specific IgE antibodies. Monoclonal antibodies to rye Group I were raised and used to purify the antigen and to verify the cross-reactivity between grass extracts. The 3 monoclonal antibodies studied recognized different components of timothy grass and 2 of them recognized kentucky june grass but none recognized components of ragweed extracts.

Development of a reverse enzymoallergosorbent test (REAST) to detect timothy-specific IgE antibodies. Comparison with RAST.

Radioallergosorbent test (RAST) for the measurement of IgE antibodies has been introduced more than 15 years ago and a number of technical modifications have since improved its sensitivity and reproducibility. The test has been applied to the diagnosis of allergy and to determine changes in the levels of IgE antibodies following immunotherapy. However, specific IgG antibodies are raised during such a therapy and can interfere with the RAST. We have developed a reverse enzymoallergosorbent test (REAST) where microtiter plates are first coated with a purified polyclonal anti-IgE antibody, then with the serum to test and finally with peroxidase-labeled antigen. This assay is antigen specific as shown by the significant inhibition of binding of the labeled antigen in presence of unlabeled specific antigen (greater than 95%) and the absence of inhibition in presence of irrelevant antigens. The values found in atopic patients (85 subjects) were significantly higher than in the non-atopic donors (35 subjects) (1.14 U +/- 1.20 vs. 0.01 U +/- 0.02, P less than 0.0005) and there was a good correlation with the Pharmacia RAST (P less than 0.0005). The levels of specific IgE by both REAST and RAST correlated well with the clinical symptomatology.

Three-month efficacy and safety of once-daily diltiazem in chronic stable angina pectoris.

The 3-month efficacy and safety of a once-daily controlled formulation of diltiazem (180 to 360 mg/day) were assessed in a study of 54 patients with angina pectoris. This multicenter study was a nonrandomized, placebo run-in, open-label, 3-month trial followed by a 1-week, double-blind, randomized period during which most patients (89%) received placebo. There were only minimal changes in the time to termination (mean change +/- SEM -5.8 +/- 9.6 seconds), time to onset of angina (10.5 +/- 12.2 seconds), and the time to 1 mm ST-segment depression (2.9 +/- 12.5 seconds) from the end of the titration phase to the end of the open-label study. There were, however, statistically significant differences between the end of the 3-month treatment phase and the end of the 1-week randomized placebo phase for those 3 efficacy parameters (-37.3 +/- 11.2, -58.6 +/- 13.6, and -45.6 +/- 16.4 seconds, respectively). Diltiazem significantly decreased the frequency of anginal attacks and nitroglycerin use at the end of the 3-month treatment phase compared with results at the end of the randomized double-blind placebo phase. No new or unusual adverse events were reported during treatment. The present results suggest that there is no loss of efficacy of once-a-day diltiazem when administered for a long period to patients with chronic stable angina pectoris.

Sex steroid regulation of beta-adrenergic sensitive adenylate cyclase in rabbit myometrial cells in primary culture.

Cyclic adenosine 3'-5'-monophosphate (cAMP) is the likely mediator of myometrial relaxation induced by agents like isoproterenol and relaxin. In vivo, the relaxation response to beta-adrenergic catecholamines is modulated by estradiol (E2) and progesterone (P) during the estrous cycle and pregnancy. However, it is still debated whether that regulation occurs via a direct action on myometrial cells or indirectly through modulation of adrenergic neurotransmitters. We have studied the properties of adenylate cyclase in primary cultures of smooth muscle cells isolated from rabbit myometrium. Adenylate cyclase activity increased with time in culture reaching a maximum on day 6. The cultured cells had basal activity which could be stimulated by guanosine triphosphate (GTP, 2.3 fold), its non-hydrolysable analogue guanylyl-imido-diphosphate (GPP[NH]p, 8.3 fold) and sodium fluoride (NaF, 15.7 fold). Isoproterenol (Iso) and prostaglandin E2 (PGE2) stimulated adenylate cyclase activity to the same level (4 fold) and required the presence of exogenous GTP. Sex steroid treatment did not affect basal activity or its stimulation by guanyl nucleotides, PGE2 or NaF. However, the adenylate cyclase response to Iso was significantly affected by E2 and/or P treatment. E2 treatment for 6 days diminished the sensitivity 10 fold (from 10 nM to 100 nM) while E2 for 3 days followed by P for 3 days increased it 10 fold (to 1 nM). Maximal response was not affected by treatment with E2 for 6 days but was diminished by 43% (p less than 0.001) when P was added alone and increased by 63% (p less than 0.001) when P was added following E2 pretreatment. Our results demonstrate that sex steroids can alter beta-adrenergic response in vitro by a direct action on myometrial cells.

The embryo influences adenylate cyclase activity and hormonal response in rabbit myometrium during early pregnancy.

The effect of pseudopregnancy (PSPG; days: 0 (estrus), 1, 6.5, 9 and 15) and pregnancy (PG; days: 6.5, 9 and 15) on adenylate cyclase (AC) activity was verified in rabbit myometrium. During PSPG, there was a time related decline in basal activity from 71 +/- 16.2 (D 0) to 13.1 +/- 1.6 (PSPG-D9) pmoles cAMP formed/mg prot-min. Stimulation of the enzyme by GTP, Isoproterenol (ISO), Prostaglandin E2 (PGE2) or Sodium Fluoride (NaF) followed a similar pattern. AC activity was compared in myometrial tissues of pregnant animals (PG) separated into embryonic (ES) and interembryonic (IES) sites. On days 6.5 and 9, AC activity measured in tissues from PG rabbits (ES and IES) was always higher than that found in tissues from PSPG animals on corresponding days. On day 6.5, AC activity was slightly higher (p less than 0.01) in ES than in IES. This was confirmed on day 9 where basal as well as GTP, ISO and PGE2 stimulated activities were higher in ES than in IES (p less than 0.001). Dose response to ISO, expressed as % of GTP, were similar on D0, 1, 6.5 and 15 of PSPG. However, on day 9, there was a striking diminution in response reflected by a lower stimulation at suboptimal dose (0.1 microM; p less than 0.05) from 115 +/- 2 on day 0 to 104 +/- 4 on day 9. These results suggest that protein content which is increased during pseudopregnancy could be responsible for the decline of AC activity. However, results obtained on day 9 and 15 suggest that other factors are involved. Dose responses to ISO during PG showed an alteration in response on days 6.5 and 9 at ES. It was reflected by a higher stimulation at suboptimal (0.1 microM) and optimal doses (100 microM). These results suggest that myometrial AC activity could be regulated by the presence of the embryo.

Efficacy and Safety of Evening Administration of Controlled-Delivery Diltiazem Capsules in Chronic Stable Angina Patients.

The safety and efficacy of controlled-delivery (CD) once-a-day formulation of diltiazem administered in the evening, at a dose of 240 mg, was assessed in 37 patients with stable angina pectoris. A double-blind, placebo-controlled, randomized, crossover protocol was used. Following a 4-day washout period, patients entered a 5--7-day single-blind placebo run-in period during which qualification and reproducibility exercise treadmill tests (ETTs) were performed 24 h postdose. Eligible patients were randomized in a double-blind fashion, to either CD diltiazem or to placebo for a 7--10-day treatment period. They then entered a 5--7-day single-blind washout period, after which they received the alternate treatment for another 7 to 10 days. ETTs were performed at the end of each treatment period. Compared to placebo, evening administration of CD diltiazem produced a marked improvement of the time to ETT termination, time to onset of angina, and time to 1 mm ST depression. In addition, the number of angina attacks recorded in patient diaries was reduced compared to placebo. Incidence of adverse events was comparable with CD diltiazem and placebo. We conclude that evening administration of controlled-delivery diltiazem is highly effective and safe in the treatment of stable angina pectoris.

Reference-based pricing in British Columbia: implications for cardiologists--an analysis.

Under the reference-based pricing (RBP) policy, British Columbia will fund drug therapies based on the cost of the 'gold standard' therapy that meets the needs of the majority of patients with a specific condition. Hence, Pharmacare will pay for the lowest cost drug within a cluster of related but different drugs, regardless of the indication. When evaluating the impact of drugs on health care expenditure, one must consider that their costs are more than offset by the clinical and economic benefits they provide. Pharmaceutical expenditure accounts for a small proportion of health care expenditure and should be viewed as an essential and interactive component in the global health care budget rather than as an independent constituent. In that respect, insight should be gained from many countries in which RBP has been implemented A wealth of data converge to the same conclusion: price controls and restricted access to drugs do not reduce prescription drug expenditures but actually increase health care costs. Furthermore, cost containment being the main issue behind RBP in British Columbia, the contentious issue of therapeutic substitution has not been taken fully into consideration, nor has its impact on the quality of care of the patient. The case of diltiazem once-a-day versus diltiazem tablets for hypertensive and angina patients illustrates the important considerations that must be taken into account in writing the overall financial equation that drives the implementation of the RBP policy. If pharmacotherapy is to be an appropriate treatment to attain optimal cost effective health care, its benefit can only be optimized with a strategy that entails the right therapy, for the right patient, in the right dosage form and at the right time. Accordingly, RBP in British Columbia should be analyzed in light of patient welfare and appropriate use of collective resources.

The role of diltiazem in treating hypertension and coronary artery disease: new approaches to preventing first events.

OBJECTIVE: To review the role of diltiazem in treating and preventing a group of cardiovascular diseases, including painful and silent cardiac ischemia, stroke, nonfatal myocardial infarction and sudden cardiac death, by modulating certain physiological causes that they appear to share. DATA SOURCES: A MEDLINE search was conducted for all clinical articles on the use of diltiazem for hypertension and coronary artery disease. When clinical data were not available, basic research findings were reviewed. DATA EXTRACTION AND SYNTHESIS: Because many cardiovascular events show a marked daily periodicity--which appears to coincide with circadian peaks in the ability of platelets to aggregate, sympathetic activity, coronary tone, blood pressure, heart rate and hematocrit, and a trough in fibrinolytic activity--the impact of diltizazem on these physiological changes was assessed. CONCLUSIONS: Diltiazem influences many of these events by increasing myocardial bloodflow, and reducing myocardial oxygen demand and cardiac workload. However, it differs from other calcium antagonists in its mild negative inotropic and moderate negative dromotropic effects, without apparent stimulation of cardiac performance or contractility. In addition, it inhibits platelet aggregation, decreases catecholamine release, diminishes coronary tone and blocks the vasoconstrictive actions of endothelin-1. This appears to translate into a beneficial effect on ischemia, thrombolysis, arrhythmias, infarct parameters, atherosclerosis and hypertension. Diltiazem has a relatively favourable safety and tolerability profile, and is available in a once-daily dosage form. The most common adverse effects are related to vasodilation (eg, edema and headache), and the most frequent serious adverse event is atrioventricular block, which occurs rarely. In summary, diltiazem appears to be well suited to preventing the first occurrence of cardiovascular events and may even have a role in preventing certain types of secondary events. The data accumulated so far indicate the need for a large scale random clinical trial addressing these outcomes.

Respiratory symptoms and bronchial reactivity among pig and dairy farmers.

OBJECTIVES: This study assessed the prevalence of respiratory manifestations among French pig and dairy farmers and determined the relationship between bronchial reactivity and respiratory manifestations. METHODS: The pig farmers included 102 men working more than half-time inside swine confinement buildings. There were 51 male dairy farmers and 81 male referents. The demographic characteristics of the three groups were similar except for more smokers among the referents. Each subject completed a standardized questionnaire. Pulmonary function tests were performed before and after a methacholine challenge (cumulative doses 80, 240, and 560 micrograms). Airborne dust, ammonia, and carbon dioxide were measured inside 28 swine confinement buildings. RESULTS: The pig farmers were exposed to a total dust level of 2.41 mg.m-3. The respirable particle concentration was low. The pig and dairy farmers had a significantly higher prevalence of cough and morning phlegm than the referents. Before the methacholine challenge, the dairy farmers had nonsignificantly lower mean lung function values than the other groups. Among the subjects with no history of asthma, nonspecific bronchial hyperreactivity was significantly higher among the pig and dairy farmers than among the referents. There was a fall in the forced expiratory volume in 1 s (FEV 1.0) that was greater than 10% in 6.7% of the referents, 17.9% of the swine workers, and 35.6% of the dairy farmers. This result was unchanged after adjustment for the initial FEV1.0. CONCLUSIONS: The prevalence of respiratory symptoms was significantly higher among the pig farmers without base-line lung function impairment. However, both the pig and the dairy farmers had increased bronchial reactivity.

Possible involvement of the yeast POLIII DNA polymerase in induced gene conversion.

In Saccharomyces cerevisiae, three different DNA polymerase complexes, POLI, POLII and POLIII, are known to be involved in DNA replication. The catalytic subunit of POLIII is encoded by the essential CDC2 gene. The existence of different thermosensitive noncomplementing mutants of CDC2 offers the possibility of using a genetic approach to investigate the involvement of POLIII in induced gene conversion. When cdc2 heteroallelic cells were irradiated and incubated under restrictive conditions, almost no induction of thermoresistant cells could be detected, suggesting an essential role for POLIII in mitotic gene conversion events.

Targeted disruption of hoxc-4 causes esophageal defects and vertebral transformations.

Mice carrying a nonfunctional allele of hoxc-4 have been generated by gene targeting. The phenotype of mice homozygous for this mutation is strikingly different from those reported in mice lacking the paralogous genes hoxa-4, hoxb-4, and hoxd-4. In contrast to the mutants of the paralogous family members, hoxc-4 homozygotes do not manifest abnormalities in the cervical vertebrae, but instead show vertebral defects that extend from the second thoracic vertebra (t2) to t11. Therefore, defects do not correspond to the anterior limit of expression of hoxc-4, but rather begin within the region of strong hoxc-4 expression in the prevertebral anlagen (i.e., pv7-14). While hoxc-4 mutant homozygotes that reach adulthood are fertile and appear outwardly normal, most die before weaning age. The high lethality appears to result from partial or complete blockage of the lumen of the esophagus over a large portion of its length, as well as disorganization of the esophageal musculature. Although the Drosophila homolog of hoxc-4, Deformed, is autoregulated, mutation of the hoxc-4 gene does not affect transcription of its paralogous family members. However, in hoxc-4 mutant embryos, transcription of both the hoxc-5 and hoxc-6 genes is altered. Employment of cissolidustrans analysis showed that the hoxc-4 mutation acts in cis to affect the pattern of hoxc-5 expression. Therefore, this mutation is likely to cause a reduction of hoxc-5 function as well as complete loss of hoxc-4 function.

Preparation and characterization of rabbit myometrial cells in primary culture: influence of estradiol and progesterone treatment.

Myometrial cells were obtained following a three-step enzymatic digestion of uterine horns from Day 1 pseudopregnant rabbits. Isolated cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), whole or steroid depleted (FBS-DC) at a plating density of 0.5 X 10(6) cells/ml. The cells reached confluency on Day 6 to 7 with whole serum and on Day 7 to 8 with DC serum. The process yielded myometrial cells at a purity level of at least 80% as assessed by indirect immunofluorescence using desmin antibody on confluent cultures. The addition of increasing doses of 17 beta-estradiol (E2) (0.1 nM to 1 microM) to the culture medium resulted in an increase in total protein and DNA content (1.5-fold at 1 nM). Similar treatment with progesterone (P) resulted in a 25% inhibition of protein and DNA content at 10 nM. Pretreatment of cells with E2 (1 nM) for 3 d followed by P (10 nM) for 3 d resulted in a 1.8-fold stimulation of protein with a higher protein: DNA ratio indicating that the increase was due to cellular hypertrophy. Analysis of desmin by polyacrylamide gel electrophoresis showed that this cytoskeleton protein was not affected by steroid treatment. Our results indicate that PR can generate two different responses depending on cell pretreatment. In as much as myometrial cells grown in primary culture respond differentially to E2 and P they should provide a useful model to study the regulation of myometrial contractility.

Control elements of the P2 promoter of the Antennapedia gene.

Antennapedia (Antp), a homeotic gene of Drosophila required for proper differentiation of the thorax of the fly, is expressed in complex spatial patterns during development. The gene is greater than 100 kb long and has two independently regulated promoters. To characterize cis-acting regulatory elements responsible for the expression pattern, fusions of the Antp promoter 2 cap site and upstream sequences to an Adh-lacZ gene were introduced into flies. A 10-kb sequence directs beta-galactosidase production in a pattern that closely resembles the endogenous P2 pattern. Transcription from the 10-kb fusions is regulated by three genes that regulate Antp transcription. Control elements, including a target of action of homeo-domain-containing proteins, were mapped by deleting parts of the 10-kb sequence.

Gene conversion at different points in the mitotic cycle of Saccharomyces cerevisiae.

In the Saccharomyces cerevisiae mitotic cycle, the timing of radiation-induced gene conversion has been studied using thermosensitive cell division cycle mutants. The cells were found to perform conversion at different G1 or post-replication steps. A lower yield in induction is found during the G2 phase and is explained by the competition for recombinational repair between sister chromatids and homologous chromosomes. The results are discussed in relation to repair.

(CA/GT)(n) microsatellites affect homologous recombination during yeast meiosis.

One of the most common microsatellites in eukaryotes consists of tandem arrays of the dinucleotide GT. Although the study of the instability of such repetitive DNA has been extremely fruitful over the last decade, no biological function has been demonstrated for these sequences. We investigated the genetic behavior of a region of the yeast Saccharomyces cerevisiae genome containing a 39-CA/GT dinucleotide repeat sequence. When the microsatellite sequence was present at the ARG4 locus on homologous chromosomes, diploid cells undergoing meiosis generated an excess of tetrads containing a conversion of the region restricted to the region of the microsatellite close to the recombination-initiation double-strand break. Moreover, whereas the repetitive sequence had no effect on the frequency of single crossover, its presence strongly stimulated the formation of multiple crossovers. The combined data strongly suggest that numerous recombination events are restricted to the initiation side of the microsatellite as though progression of the strand exchange initiated at the ARG4 promoter locus was impaired by the repetitive sequence. This observation corroborates in vitro experiments that demonstrated that RecA-promoted strand exchange is inhibited by CA/GT dinucleotide tracts. Surprisingly, meiotic instability of the microsatellite was very high (>0.1 alterations per tetrad) in all the spores with parental and recombinant chromosomes.