Exploring the expression of Pseudomonas aeruginosa genes directly from sputa of cystic fibrosis patients.

UNLABELLED: Acquisition of Pseudomonas aeruginosa is known as a negative prognostic factor in patients with cystic fibrosis. We started a pilot study to evaluate Ps. aeruginosa gene expression directly from the sputum of infected patients. Total RNA was purified from 15 sputum samples collected from 10 patients, and the expression levels of five genes from Ps. aeruginosa were measured by RT-qPCR. Expression of algD, algR, antB, lasB and pqsA genes was determined in sputa that contained Ps. aeruginosa cells. The resultant data provided an overview of the expression of these genes in CF patients. Except for the correlation between algD expression and the mucoid phenotype, the gene expression profile could not be associated with the clinical status of patients. However, beyond the heterogeneity of the Ps. aeruginosa phenotype in sputum, we observed a correlation between the expression of antB and pqsA and a low level of lasB transcripts. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas aeruginosa infection leads to high morbidity and mortality in cystic fibrosis patients. The identification of Ps. aeruginosa-assigned factors is important to eradicate the colonization. We started a pilot study to evaluate the gene expression of Ps. aeruginosa directly from the sputum of infected patients. Preliminary results suggest that beyond the heterogeneity of the Ps. aeruginosa phenotype in sputum, we observe a correlation between the expression of antB and pqsA and a low level of lasB transcripts. This approach could shed some light on the behaviour of Ps. aeruginosa during pulmonary infection and may reveal some important elements for optimizing therapy.

Non-invasive tests for fibrosis and liver stiffness predict 5-year survival of patients chronically infected with hepatitis B virus.

BACKGROUND: Liver stiffness and non-invasive tests predict overall survival in chronic hepatitis C. However, in patients chronically infected with hepatitis B virus (HBV), only the association between liver stiffness and the risk of hepatocellular carcinoma has been published. AIM: To evaluate the 5-year prognostic value of liver stiffness, non-invasive tests of liver fibrosis, and liver biopsy, to predict overall survival in chronic hepatitis B. METHODS: In a consecutive cohort, we prospectively assessed fibrosis, with liver stiffness, FibroTest, APRI, FIB-4 and liver biopsy (if indicated). We examined death and liver transplantation during a 5-year follow-up, and factors associated with overall survival. RESULTS: A total of 600 patients (men 64%, mean age 42 years, inactive carriers 36%) with chronic hepatitis B were included. At 5 years, 25 patients were dead (13 liver-related deaths) and four patients had liver transplantation. Overall survival was 94.1% and survival without liver-related death 96.3%. No liver-related death was observed in inactive carriers. Survival was significantly decreased in patients diagnosed with severe fibrosis, whatever the non-invasive method used (P < 0.0001), or liver biopsy (P = 0.02). Patients' prognosis decreased as liver stiffness and FibroTest increased. In multivariate analysis, FibroTest and liver stiffness had the highest hazard ratio with survival. The association persisted after adjustment on age, necro-inflammatory histological activity presumed by ActiTest and treatment. CONCLUSIONS: Liver stiffness measurement or FibroTest can predict survival in chronic HBV infection. Thus, these tools may help physicians to early assess prognosis and discuss specific treatments, such as liver transplantation.

Prospective evaluation of a new automated nucleic acid extraction system using routine clinical respiratory specimens.

The aim of the study was to evaluate the MagNA Pure 96™ nucleic acid extraction system using clinical respiratory specimens for identifying viruses by qualitative real-time PCR assays. Three extraction methods were tested, that is, the MagNA Pure LC™, the COBAS Ampliprep™, and the MagNA Pure 96™ with 10-fold dilutions of an influenza A(H1N1)pdm09 sample. Two hundred thirty-nine respiratory specimens, 35 throat swabs, 164 nasopharyngeal specimens, and 40 broncho-alveolar fluids, were extracted with the MagNA Pure 96™ and the COBAS Ampliprep™ instruments. Forty COBAS Ampliprep™ positive samples were also tested. Real-time PCRs were used to identify influenza A and influenza A(H1N1)pdm09, rhinovirus, enterovirus, adenovirus, varicella zoster virus, cytomegalovirus, and herpes simplex virus. Similar results were obtained on RNA extracted from dilutions of influenza A(H1N1)pdm09 with the three systems: the MagNA Pure LC™, the COBAS Ampliprep™, and the MagNA Pure 96™. Data from clinical respiratory specimens extracted with the MagNA Pure 96™ and COBAS Ampliprep™ instruments were in 98.5% in agreement (P < 0.0001) for influenza A and influenza A(H1N1)pdm09. Data for rhinovirus were in 97.3% agreement (P < 0.0001) and in 96.8% agreement for enterovirus. They were in 100% agreement for adenovirus. Data for cytomegalovirus and HSV1-2 were in 95.2% agreement (P < 0.0001). The MagNA Pure 96™ instrument is easy-to-use, reliable, and has a high throughput for extracting total nucleic acid from respiratory specimens. These extracts are suitable for molecular diagnosis with any type of real-time PCR assay.

A new highly automated extraction system for quantitative real-time PCRs from whole blood samples: routine monitoring of opportunistic infections in immunosuppressed patients.

BACKGROUND: Rapid, high throughput extraction systems are needed to monitor viral infections in immunosuppressed patients. OBJECTIVES: Evaluate the performance of the MagNA Pure 96™ extraction system, and compare it to the COBAS Ampliprep™ for quantitative real-time PCR from whole blood samples. STUDY DESIGN: Compare the MagNA Pure LC™, COBAS Ampliprep™ and MagNA Pure 96™ using ten-fold dilutions of blood samples containing cytomegalovirus. Evaluate analytical performances of the MagNA Pure 96™ from test samples containing cytomegalovirus. Evaluate clinical performances from 209 blood samples collected prospectively, extracted with the COBAS Ampliprep™ and the MagNA Pure 96™ systems and tested for cytomegalovirus, Epstein-Barr, BK and JC viruses. RESULTS: All three extraction systems gave similar results with dilutions of a cytomegalovirus-positive sample. Analytical tests showed that the limit of detection was 500 copies/ml, specificity was 100%, with no cross-contamination. Quantification was linear from 3.0 to 6.0 log(10)copies/ml. Intra-assay variation was 8.3-0.9% and inter-assay variation 8.8-5.2%. Clinical specimens extracted with the MagNA Pure 96™ and COBAS Ampliprep™ instruments agreed well for cytomegalovirus (r=0.54; p=0.07), Epstein-Barr virus (0.69; p=0.0005) and BK virus (0.85; p=0.01). All 55 samples were negative for JC virus. Mean loads were similar for cytomegalovirus (0.17 log(10)copies/ml) and BK virus (-0.24 log(10)copies/ml) while that of Epstein-Barr virus was slightly lower (1.02 log(10)copies/ml). CONCLUSIONS: The MagNA Pure 96™ instrument is an easy-to-use, reliable high throughput platform for extracting nucleic acid from clinical whole blood specimens.

Seroprevalence of Toxoplasma gondii among Nunavik Inuit (Canada).

As a result of their intimate contact with the land and their nutritional habits, the Inuit of Nunavik are considered to be at risk from zoonotic infections. To better understand the risk factors for Toxoplasma gondii infection, a serosurvey was conducted in Nunavik, Québec, in September 2004. A representative sample of the Inuit adult population of Nunavik participated in this cross-sectional study (n = 917). Antibodies (IgG) against T. gondii were detected by immunoassay. Information on sociodemographic characteristics, traditional activities, domestic environment and nutrition was gathered by questionnaire and explored as variables explanatory of seropositive results. Associations found to be statistically significant in univariate analyses were assessed by multivariable logistic regression to control for confounding factors. Almost two thirds (59.8%) of the Inuit of Nunavik were found to be seropositive for T. gondii. In multivariate analyses, risk factors for seropositivity were: increasing age, gender (women > men), lower level of education, consumption of potentially contaminated water (determined by an index of risk from waterborne infections), frequent cleaning of water reservoirs, and consumption of seal meat and feathered game. There was some variation in seroprevalence between the Ungava Bay coast (52.3%) and the Hudson Bay coast (65.6%), the two main regions of Nunavik, but this variation was not significant in the multivariable logistic regression model. This cross-sectional study demonstrated high T. gondii seroprevalence in the Inuit population and revealed that age, gender, schooling and community of residence all influence serostatus in this population. Variables related to drinking water and food choices may also influence the risk of infection. These results raise important questions about T. gondii transmission in Nunavik including possible links between terrestrial and marine cycles.

Comparison of two highly automated DNA extraction systems for quantifying Epstein-Barr virus in whole blood.

BACKGROUND: Optimal automated molecular methods are needed to monitor Epstein-Barr virus (EBV) infections in transplant recipients. OBJECTIVES: To compare the extraction of EBV DNA from whole blood using the COBAS Ampliprep and the MagNA Pure instruments (Roche) for quantifying EBV DNA by real-time PCR. STUDY DESIGN: EBV DNA content was determined on clinical samples extracted by both systems. RESULTS: The detection limit was 2.16log(10)copies/mL using the COBAS Ampliprep extraction system. Specificity was 100% and we saw no cross-contamination. Extraction was linear from 2.60 to 5.60log(10)copies/mL. The intra-assay variation was 1.91% for 3.60, 2% for 4.60 and 4.51% for 5.60log(10)copies/mL; inter-assay variation was 4.88%. Sixty-six samples were tested: 26 were positive and 28 were negative by both methods. One sample was MagNA Pure positive/COBAS Ampliprep negative (virus load 3.15log(10)copies/mL) and 10 samples were MagNA Pure negative/COBAS Ampliprep positive (virus loads from 1.59 to 3.51log(10)copies/mL) (P<0.0001). Both methods gave similar quantitative results (average difference 0.07log(10)copies/mL) which were well correlated (r=0.73, P<0.001). CONCLUSIONS: The COBAS Ampliprep extraction system is comparable to the MagNA Pure and offers a high reliability for extracting EBV DNA from whole blood.

Follow-up of complete cytogenetic remission in patients with chronic myeloid leukemia after cessation of interferon alfa.

PURPOSE: A small proportion of patients with chronic myeloid leukemia (CML) achieve a complete cytogenetic response (CCR), defined as the disappearance of Philadelphia (Ph) chromosome-positive metaphases, after treatment with interferon alfa (IFN). In this population of patients, the question of whether treatment should then be withdrawn is not yet resolved. PATIENTS AND METHODS: In the present study, we followed 15 patients who stopped IFN after achieving CCR. In nine patients IFN was stopped in view of adverse reactions (n = 8) or patient's choice (n = 1). For the remaining six patients, the treatment was stopped because no BCR/ABL rearrangement could be detected by reverse transcriptase polymerase chain reaction (RT-PCR) in four successive analyses using peripheral-blood samples. RESULTS: Loss of CCR and survival were not statistically different (P =.48; P =.7) for the 15 patients who stopped IFN compared with 41 other CCR patients who continued IFN therapy in our institution. The median follow-up after discontinuation of IFN treatment was 36 months (range, 6 to 108 months). Seven patients (47%) (females, or CCR > 24 months and RT-PCR negative before IFN cessation; P <.0001) did not relapse. Eight other patients (53%) relapsed (lost CCR) within 3 to 33 months of treatment discontinuation. One of them relapsed in major cytogenetic remission (MCR) and was still in MCR 87 months after stopping therapy without any treatment. CONCLUSION: It is possible to stop IFN treatment at least in some patients with CML who achieve a prolonged period of CCR. This study also illustrates the hypothesis that persistence of low numbers of Ph-positive cells does not necessarily imply hematologic relapse.

Expression of interferon-alpha (IFN-alpha) receptor 2c at diagnosis is associated with cytogenetic response in IFN-alpha-treated chronic myeloid leukemia.

For the management of chronic myeloid leukemia (CML), prediction or early determination of the response to interferon-alpha (IFN-alpha) treatment is important for identifying nonresponder patients to whom alternative therapy may be proposed. In this study, the levels of expression of both BCR-ABL and subunit 2c of IFN-alpha receptor (IFN-alphaR2c) genes were analyzed at diagnosis in 74 patients with chronic phase CML treated with an IFN-alpha monotherapy. By using blood samples, real-time quantitative polymerase chain reaction was performed to quantify BCR-ABL, IFN-alphaR2c, and G6PDH mRNA as external control. The results were compared with hematologic and cytogenetic responses to IFN-alpha. A wide variation in the BCR-ABL/G6PDH ratio was observed at diagnosis (median, 6.68%; range, 0.18%-41.31%), but no significant association with response to IFN-alpha was observed. In contrast, the variation of IFN-alphaR2c/G6PDH ratio at diagnosis was significantly associated with the achievement of major cytogenetic response (MCR; 34% or lower Ph(+) metaphases). Median values of IFN-alphaR2c/G6PDH ratio for patients achieving MCR and for those who did not achieve it were 110.75% (range, 9.47%-612.30%) and 64.42% (range, 5.96%-425.40%), respectively (P =.037). In addition, this novel molecular factor, combined with the achievement of complete hematologic response at 3 months, makes it possible to predict MCR achievement with high probability by Kaplan-Meier analysis (91% +/- 17% at 24 months; P =.0001). (Blood. 2001;97:3568-3573)

Comparative methodologic study of NFkappaB activation in cultured endothelial cells.

The transcriptional regulatory protein nuclear factor kappaB (NFkappaB) participates in the control of gene expression of many modulators of the inflammatory and immune responses. Various activators trigger NFkappaB release and nuclear translocation after phosphorylation and proteolytic degradation of IkappaB. This study evaluated the abilities of fluorescence and confocal microscopies, laser scanning cytometry (LSC), electrophoretic mobility-shift assay (EMSA), and Western blotting to detect NFkappaB activation in endothelial cells (ECs) and to investigate the role of homocysteine (Hcy) in NFkappaB activation. ECs were treated with interleukin-1B (10 ng/mL) or Hcy thiolactone (1 and 5 mmol/L) as NFkappaB activators. Hcy, a thiol-containing amino acid, has been shown to directly damage ECs in vitro. Experimental evidence suggests that the atherogenic propensity associated with hyperhomocysteinemia results from EC dysfunction. When ECs were pretreated with an inhibitor (pyrrolidine dithiocarbamate, 100 micromol/L) or with staurosporine (5 microL/mL), no NFkappaB activation was observed. NFkappaB activation in ECs could be detected with all five techniques, clearly showing NFkappaB translocation from the cytoplasm to the nuclei. Confocal microscopy was more sensitive and less subjective than immunofluorescence microscopy. LSC was even more sensitive, specific, and reproducible. EMSA, the reference method, has the disadvantages of being radioactive, expensive, and time consuming. Western blot analysis detected the NFkappaB p50 subunit implicated in NFkappaB activation. The techniques usually used to detect NFkappaB activation in ECs are immunofluorescence microscopy and confocal microscopy, LSC, EMSA, and Western blot analysis, but none of them is ready for routine use.

[Study of the incidence of giardiasis in Quebec (Canada) and association with drinking water source and quality]. / Etude de l'incidence de la giardiase au Québec (Canada) et de l'association avec la source et la qualité de l'eau potable.

BACKGROUND: We analyzed data from the notifiable diseases data base in Québec to document the incidence of giardiasis. The objectives were to perform a descriptive analysis of the cases of giardiasis and to verify the relation between their incidence and the quality of drinking water. METHODS: The Québec notifiable diseases data-base contained 4273 cases of giardiasis declared between January 1st, 1990 and December 31st, 1995. Incidence rates were adjusted for age and calculated monthly. The sources and kinds of treatment of drinking water permitted to elaborate a vulnerability scale for classifying contamination by Giardia sp. into four categories. Incidence of giardiasis was examined in relation with this vulnerability scale. Other socioeconomic indicators possibly associated with the incidence of giardiasis were also analyzed. RESULTS: Analysis showed that there were few annual variations in the incidence of giardiasis and that there were no epidemic peaks during the study period. According to age, the incidence follows a bimodal pattern with a peak for young children and young adults. The incidence rates showed an increase of the cases at the end of summer and at the beginning of fall, with a higher relative risk for males. Even if no relation was found between the incidence of giardiasis and the vulnerability of the drinking water source, incidence rates were lower for people living in communities that use the St. Lawrence River as a drinking water source than for those using other sources of surface water. CONCLUSION: This study allowed us to obtain a good description of the cases of giardiasis declared in Québec and to formulate hypothesis about their causes. The lower incidence of giardiasis in communities that use the St. Lawrence river as their drinking water source is possibly related to a lower contamination of this source. However, considering the limits of this work, case-control studies should be considered to understand variables, which influence the incidence of giardiasis in Québec.

RGD1 genetically interacts with MID2 and SLG1, encoding two putative sensors for cell integrity signalling in Saccharomyces cerevisiae.

The RGD1 gene was identified during systematic genome sequencing of Saccharomyces cerevisiae. To further understand Rgd1p function, we set up a synthetic lethal screen for genes interacting with RGD1. Study of one lethal mutant made it possible to identify the SLG1 and MID2 genes. The gene SLG1/HCS77/WSC1 was mutated in the original synthetic lethal strain, whereas MID2/SMS1 acted as a monocopy suppressor. The SLG1 gene has been described to be an upstream component in the yeast PKC pathway and encodes a putative cell surface sensor for the activation of cell integrity signalling. First identified by viability loss of shmoos after pheromone exposure, and since found in different genetic screens, MID2 was recently reported as also encoding an upstream activator of the PKC pathway. The RGD1 gene showed genetic interactions with both sensors of cell integrity pathway. The rgd1 slg1 synthetic lethality was rescued by osmotic stabilization, as expected for mutants altered in cell wall integrity. The slight viability defect of rgd1 in minimal medium, which was exacerbated by mid2, was not osmoremediated. As for mutants altered in PKC pathway, the accumulation of small-budded dead cells in slg1, rgd1 and mid2 after heat shock was prevented by 1 M sorbitol. In addition, the rgd1 strain also displayed dead shmoos after pheromone treatment, like mid2. Taken together, the present results indicate close functional links between RGD1, MID2 and SLG1 and suggest that RGD1 and MID2 interact in a cell integrity signalling functionally linked to the PKC pathway.

Characterization of the ORF YBR264c in Saccharomyces cerevisiae, which encodes a new yeast Ypt that is degraded by a proteasome-dependent mechanism.

We identified the ORF YBR264c during the systematic sequencing of the Saccharomyces cerevisiae genome. It encodes a putative protein of 218 amino acids. We demonstrate here that the gene is indeed expressed and encodes a new Ypt in yeast. This protein specifically binds guanine nucleotides and interacts via its C-terminal end with the unique Rab GDP Dissociation Inhibitor (RabGDI). In accordance with a recent proposal, the gene is now designated YPT10. No mutant phenotype could be associated with inactivation of the gene. However, overexpression of YPT10 resulted in defects in growth; microscopic examination of such cells revealed an overabundance of vesicular and tubular structures, suggesting some alteration in the function of the Golgi apparatus. In addition, degradation of the Ypt10 protein, which possesses a PEST sequence, is shown to be dependent on proteasome activity.

Molecular confirmation of hepatitis A virus from well water: epidemiology and public health implications.

An outbreak of hepatitis A in a rural river-island community was found to be associated with consumption of contaminated well water. Specimens from case-patients, the implicated well, and a cesspool suspected to be the source of contamination were all positive for hepatitis A virus (HAV) RNA by immunocapture reverse-transcription polymerase chain reaction. All isolates were identical over about 400 bases from two capsid-encoding regions of the genome, identifying the chain of transmission. Other wells up to 60 m from the cesspool also contained HAV RNA. In addition, HAV RNA was detected in the contamination source well 6 months after the initial contamination, when fecal coliform bacteria were no longer present. These findings demonstrate the utility of viral detection techniques to evaluate contaminated ground water.

First characterization of the gene RGD1 in the yeast Saccharomyces cerevisiae.

We identified the ORF YBR260c during systematic sequencing of one region of chromosome II of Saccharomyces cerevisiae. This ORF encodes a putative protein of 666 aa, of which the C-terminal part of the deduced amino acid sequence resembles human and yeast Rho/Rac GTPase activating proteins (GAP). An initial study is reported in the paper. This gene was expressed in haploid and diploid cells and was called RGD1 for related GAP domain 1. Inactivation of RGD1 was carried out and phenotypic analysis of the mutant strain revealed only a slight viability defect when cells grown in minimal medium were close to stationary phase. Northern and western analyses showed that the RGD1 transcript and the corresponding protein were still abundant in cells cultivated in YNB during the stationary phase. No functional link seems to exist with the highly conserved GTPase Cdc42 involved in cytoskeletal polarization and cell polarity.

Complete DNA sequence of yeast chromosome II.

In the framework of the EU genome-sequencing programmes, the complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome II (807 188 bp) has been determined. At present, this is the largest eukaryotic chromosome entirely sequenced. A total of 410 open reading frames (ORFs) were identified, covering 72% of the sequence. Similarity searches revealed that 124 ORFs (30%) correspond to genes of known function, 51 ORFs (12.5%) appear to be homologues of genes whose functions are known, 52 others (12.5%) have homologues the functions of which are not well defined and another 33 of the novel putative genes (8%) exhibit a degree of similarity which is insufficient to confidently assign function. Of the genes on chromosome II, 37-45% are thus of unpredicted function. Among the novel putative genes, we found several that are related to genes that perform differentiated functions in multicellular organisms of are involved in malignancy. In addition to a compact arrangement of potential protein coding sequences, the analysis of this chromosome confirmed general chromosome patterns but also revealed particular novel features of chromosomal organization. Alternating regional variations in average base composition correlate with variations in local gene density along chromosome II, as observed in chromosomes XI and III. We propose that functional ARS elements are preferably located in the AT-rich regions that have a spacing of approximately 110 kb. Similarly, the 13 tRNA genes and the three Ty elements of chromosome II are found in AT-rich regions. In chromosome II, the distribution of coding sequences between the two strands is biased, with a ratio of 1.3:1. An interesting aspect regarding the evolution of the eukaryotic genome is the finding that chromosome II has a high degree of internal genetic redundancy, amounting to 16% of the coding capacity.

Sequence analysis of a 31 kb DNA fragment from the right arm of Saccharomyces cerevisiae chromosome II.

The nucleotide sequence of a 31,352 bp fragment from chromosome II of Saccharomyces cerevisiae has been determined and analysed. The fragment originates from the right arm of chromosome II, located between the GAL7,10,1 and the PHO3,5 loci, at a distance of about 130 kb from the centromere. The sequence contains a tRNA tandem repeat and 17 open reading frames (ORFs) larger than 100 amino acids. One of them extends into adjacent DNA and is incomplete. The two tRNA genes, coding for a tRNA(asp) and a tRNA(arg), and three of the ORFs, had been sequenced previously, i.e. HSP26, SEC18, and UBC4. Four other ORFs showed similarity with yeast genes; amino acid transporter genes, the RAD54, SNF2 and STH1 family, the SPS2 gene and the bromodomain of SPT7, respectively. Two showed homology with sequences from other organisms, i.e. with a Plasmodium falciparum gene encoding a surface antigen and with a gene from Saimirine herpes virus respectively. Three ORFs, YBR0726, YBR0735 and YBR0740 are completely contained in YBR0727, YBR0734 and YBR0739 respectively, and thus probably do not represent real genes. Two ORFs, YBR0727 and YBR0745 most likely contain an intron.

Identification of 'cystic fibrosis protein' as a complex of two calcium-binding proteins present in human cells of myeloid origin.

Cystic fibrosis protein is a serum protein characterized by a pI close to 8.4 and present with a higher concentration in serum and plasma of cystic fibrosis carriers than in controls. This protein was found immunologically indistinguishable from the cystic fibrosis antigen isolated from granulocytes and presenting a sequence analogous to that of MRP-8, a calcium-binding protein expressed in the myeloid cell lineage. Using antibodies directed against MRP-8 and its closely associated calcium-binding protein, MRP-14, we demonstrate here that cystic fibrosis protein purified from serum is a complex of the two proteins MRP-8 and MRP-14.

Isolation of the "cystic fibrosis protein" from serum.

"Cystic fibrosis protein" (CFP), a minor serum protein marker of the cystic fibrosis allele, was isolated from serum from patients with cystic fibrosis by use of the "FPLC" high-resolution chromatography system and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. CFP currently is characterized by its isoelectric point (8.4) on isoelectric focusing. However, after the first purification steps, we identified the protein, which was present in a very low concentration, by the immunosorbent assay of Hayward et al. (J Immunol Methods 1986;91:117-22), by virtue of its immunological relationship with the "CF antigen," a protein characterized in granulocytes by these same authors [Nature (London) 1985;315:513-5]. CFP, a protein of low molecular mass, about 14 kDa, appears to be strongly associated with IgG in serum. Using the same procedure with control serum permits us to assume that CFP normally is present in serum in trace amounts.

Cystic fibrosis proteins detected by electrophoretic techniques.

For the detection of the cystic fibrosis protein (CFP) in serum of cystic fibrosis (CF) carriers, thin-layer polyacrylamide gel isoelectric focusing proved inappropriate as a diagnostic test, but was useful for screening fractions on purification of CFP by chromatofocusing on a Mono P column. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis an Mr 12,000 protein (P12) was found in most CFP-positive sera, indicating good correlation between these two CF-associated proteins. Detection of the P12 protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was well reproducible and less delicate than IEF. The technique was also used to purify P12 from serum by two successive preparative electrophoresis steps in a 7.5-15% gradient and 15% homogeneous gel. The use of silver staining revealed that P12, which was present in all sera of CF patients and carriers with variable intensities, was also present in trace amounts in normal sera.