Minisequencing on oligonucleotide microarrays: comparison of immobilisation chemistries.

In the microarray format of the minisequencing method multiple oligonucleotide primers immobilised on a glass surface are extended with fluorescent ddNTPs using a DNA polymerase. The method is a promising tool for large-scale single nucleotide polymorphism (SNP) detection. We have compared eight chemical methods for covalent immobilisation of the oligonucleotide primers on glass surfaces. We included both commercially available, activated slides and slides that were modified by ourselves. In the comparison the differently derivatised glass slides were evaluated with respect to background fluorescence, efficiency of attaching oligonucleotides and performance of the primer arrays in minisequencing reactions. We found that there are significant differences in background fluorescence levels among the different coatings, and that the attachment efficiency, which was measured indirectly using extension by terminal transferase, varied largely depending on which immobilisation strategy was used. We also found that the attachment chemistry affects the genotyping accuracy, when minisequencing on microarrays is used as the genotyping method. The best genotyping results were observed using mercaptosilane-coated slides attaching disulfide-modified oligonucleotides.

A gene in Paracoccus for subunit III of cytochrome oxidase.

The region of Paracoccus denitrificans chromosome where the genes coding for cytochrome oxidase (cytochrome aa3) subunits are located has been cloned. DNA sequencing revealed an open reading frame that codes for a protein homologous to the subunit III of the eukaryotic, mitochondrial enzyme. This subunit is absent from the isolated Paracoccus oxidase. It now seems that it is part of the native enzyme in the bacterial cytoplasmic membrane. This may explain the observed discrepancies in the function of the isolated enzyme.

Are there isoenzymes of cytochrome c oxidase in Paracoccus denitrificans?

We have used a gene replacement strategy to delete the previously isolated gene [(1987) EMBO J. 6, 2825-2833] for the cytochrome c oxidase subunit I from Paracoccus denitrificans. The resulting mutant was still able to synthesize active cytochrome c oxidase. This led us to look for another locus which could completely suppress the mutation. In this study we report the isolation of a second gene encoding subunit I. An open reading frame coding for cytochrome c 550 was found upstream from this gene. We suggest that there are isoenzymes of cytochrome c oxidase (cytochrome aa3) in this bacterium.

Clodronate is effective in preventing corticosteroid-induced bone loss among asthmatic patients.

Clodronate is a novel drug used for inhibiting osteoclastic activity. The aim of the present double-blind study was to evaluate the efficacy and tolerability of clodronate (Leiras, Finland) in corticosteroid-induced bone loss among asthmatic patients. Seventy-four adult patients (41 women and 33 men, mean age 57.3 years) having a long history (mean 8.1 years) of oral and inhaled corticosteroid therapy were randomized to four parallel treatment groups: clodronate 800, 1600, or 2400 mg/day, or an identical placebo. The bone mineral density (BMD) of the lumbar spine (L2-4), femoral neck, and trochanter were assessed using dual-energy X-ray absortiometry at entry, 6 months, and 12 months. The baseline BMDs did not differ significantly between the study groups. In the lumbar spine, the mean BMD increased significantly between the baseline and 12-month visit in the clodronate groups of 1600 and 2400 mg/day, 2.6% (0.02 g/cm2, p < 0.02) and 3.0% (0.03 g/cm2, p < 0.01), respectively, but not in the placebo and clodronate 800 mg/day groups. The test for a linear trend (BMD percent change for L2-4) at 12 months was significant (p < 0.02), indicating a dose response to clodronate. The mean BMD values of the femoral neck increased significantly in the 2400 mg/day group, 4.3% (0.03 g/cm2, p < 0.0001), as well as in the trochanter region 2.8% (0.02 g/cm2, p < 0.02). Gastric irritation was the most common adverse effect noted on a clodronate dose of 2400 mg/day. We conclude that oral clodronate is effective in preventing bone loss or increasing bone mass in asthmatic patients having a long history of continuous peroral and inhaled corticosteroid administration.

Fuel chip-induced hypersensitivity pneumonitis caused by penicillium species.

Two cases of Penicillium-induced hypersensitivity pneumonitis in farmers handling fuel chips are presented. Attention should be paid to safe handling of all types of solid fuel (wood, chips, and peat) and other materials in which mold may grow.

Nucleotide sequence of the gene coding for cytochrome oxidase subunit I from the thermophilic bacterium PS3.

The gene coding for cytochrome oxidase subunit I (COI) was isolated from a genomic DNA library of the thermophilic bacterium PS3 and sequenced. The N-terminal of the COI protein was also sequenced to verify the initiation site of the reading frame. The deduced amino acid sequence of COI protein is composed of 536 amino acid residues and its molecular mass is 59,510. The protein is clearly homologous to the corresponding subunit in the mitochondrial cytochrome oxidase and similarly appears to have 12 trans-membrane segments. The proposed ligands to two hemes (cytochrome aa3) and a copper atom (CuB) in this protein (Holm et al. (1987) EMBO J. 6, 2819-2823) are conserved in the sequence.

Is the risk of occupational tuberculosis higher for young health care workers?

SETTING: Health care workers in Finland. OBJECTIVE: Occupational tuberculosis (TB) was studied separately in nurses, assistant nurses, physicians, psychiatric nurses, medical laboratory workers and radiographers during the period 1971-1995. DESIGN: All 447 notified cases between 20 and 59 years of age were included. Incidence ratios by age and occupation were compared to the corresponding general population. RESULTS: A common profile, with a higher rate of TB towards older age, was seen in the general population but not among health care workers. Among nurses, assistant nurses and physicians, the incidence ratio was higher among those aged 20-39 than among those aged 40-59 years. Compared to the controls, the risk of TB was significantly smaller in the older group of nurses during 1971-1990 and in assistant nurses during 1971-1995. The risk among the older group of physicians was significantly lower during 1971-1980, and among the younger group it was significantly higher during 1976-1990. CONCLUSIONS: We speculate that in certain fields of health care, young workers are at the greatest risk of TB.

Tuberculosis among health care workers during three recent decades.

Some studies have found that health care workers have an increased risk of tuberculosis, whereas other studies have reported the opposite. This study examined data of Finnish health care workers (HCWs) for the incidence of tuberculosis disease. Cases of occupational tuberculosis in Finland were analysed over a period of 30 yrs (1966-1995). Control subjects were all other incident tuberculosis cases at working age. The material thus obtained included 658 people with notified occupational tuberculosis, and 56,146 control cases. The authors studied incidence, and age specific rate, as well as their trends. The incidence of tuberculosis among health care workers decreased from 57.9 to 6.1 per 100,000 and the corresponding figures among control subjects decreased from 156.8 to 9.1 per 100,000. The overall risk in health care workers was lower than in the general population throughout the study period. Analysis of age specific rates revealed no age group at increased risk. The reasons are multifactorial, among them the successful tuberculosis programme, and the lack of impact of such risk factors as immigration, human immunodeficiency virus infection and drug resistance; high coverage of bacille Calmette-Guérin vaccination may also be a factor, although it is difficult to assess.

Isolation and analysis of the genes for cytochrome c oxidase in Paracoccus denitrificans.

Synthetic oligonucleotide probes were used to clone two loci from the chromosomal DNA of Paracoccus denitrificans that contain the genes for cytochrome c oxidase (cytochrome aa(3)). One locus seems to contain four or five genes probably forming an operon. Two of these code for the oxidase subunits II and III. Three open reading frames are found between the COII and COIII genes. The other locus codes for the subunit I. A short open reading frame is found upstream of this gene. All three subunits of the Paracoccus enzyme show remarkable homology to the corresponding subunits of the mitochondrial cytochrome oxidase. Possible protein products of the open reading frames have not yet been identified.

Toothbrushing and the occurrence of salivary mutans streptococci children at day care centers.

Risk factors for the occurrence of salivary mutans streptococci (MS) were surveyed in 677 children aged 1-8 years (4.9 +/- 1.2; mean +/- SD) at 20 municipal day care centers in Oulu. As a part of an intervention program to reduce the spread of infectious diseases, toothbrushing was discontinued in 10 centers, the other 10 serving as controls. The test for MS was performed on 506 children before the 8-month intervention, on 358 at the end and on 345 on both occasions. Past caries experience was recorded from the children's dental health cards (dmf). Dental health habits were evaluated by means of questionnaires filled in by the parents. The occurrence of MS varied from 43.4 to 48.0%. Although those children who cleaned their teeth regularly at home carried less MS than those who did so irregularly (46.4 vs. 64.9%, p < 0.001), the withdrawal of toothbrushing at the day care center did not increase the incidence of MS. The occurrence of MS was increased by consumption of sweets containing sucrose (p < 0.01) and reduced by the regular use of fluoride tablets (p < 0.02). Logistic modeling showed a positive MS test to be associated significantly with older age (p < 0.01) and female sex (p < 0.05) in addition to toothbrushing at home (p < 0.001). The children with positive tests had significantly higher mean (+/- SD) dmf than those with negative ones (1.6 +/- 2.7 vs. 0.3 +/- 1.2, p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of single risk indicators in relation to caries increment in adolescents.

Base-line data on a series of risk indicators were related to 11-month caries increment in 181 subjects with a mean age of 13 years and 3 months. A caries increment equalling or exceeding one tooth surface was recorded in 21% of the subjects. The risk indicators consisted of past caries experience, white spot lesions, visible plaque and gingivitis, and six salivary tests: secretion rate, buffer effect, sucrase, mutans streptococci, lactobacilli, and Candida. Significant associations between caries increment and past caries experience (p = 0.002), white spot lesions (p = 0.01), lactobacilli (p = 0.02), Candida (p = 0.006), and sucrase (p = 0.02) were observed. The ensuing odds ratios were thus recorded: past caries experience, 3.6; white spot lesions, 2.9; salivary sucrase activity, 2.9; lactobacilli, 2.5; and Candida, 2.8.

Multifactorial modeling for prediction of caries increment in adolescents.

The purpose of the study was to develop a multifactorial model for the prediction of 11-month caries increment in adolescents. The mean age of the subjects (n = 181) at the base-line examination was 13 years and 3 months. The risk indicators consisted of past caries experience, white spot lesions, visible plaque, gingivitis, salivary secretion rate, buffer effect, sucrase, mutans streptococci, lactobacilli, and Candida. The multifactorial modeling included all the above risk indicators, age, and gender and resulted in different models in boys and girls, indicating the difficulty of caries prediction in adolescents. When boys and girls were combined, the final model included past caries experience, Candida, and salivary sucrase. Although the accuracy of the model was slightly below the 80% level recommended for screening purposes, the results provide clinically valuable information. The risk of caries increases with an increasing number of positive tests within the model.

Wegener's granulomatosis--treatment under revision?

A 44-year-old man with nasal and respiratory symptoms combined with positive serum antibodies to neutrophil cytoplasmic antigens (ANCA) suggestive of Wegener's granulomatosis was treated with antibacterial agents. Complete clinical response was achieved with co-trimoxazole, and the titer of ANCA declined. After a 12-month treatment period, the patient contracted fever and respiratory symptoms and fatigue again, and he had proteinuria and hematuria. After the institution of conventional treatment with oral prednisolone and cyclophosphamide, a favorable response was achieved. Wegener's-like granulomatosis is difficult to diagnose at its early stage, but the presence of ANCA may be helpful. We suggest that co-trimoxazole should be considered as a first-line treatment, under careful supervision, for young patients whose disease is limited to the respiratory organ.

A system for specific, high-throughput genotyping by allele-specific primer extension on microarrays.

This study describes a practical system that allows high-throughput genotyping of single nucleotide polymorphisms (SNPs) and detection of mutations by allele-specific extension on primer arrays. The method relies on the sequence-specific extension of two immobilized allele-specific primers that differ at their 3'-nucleotide defining the alleles, by a reverse transcriptase (RT) enzyme at optimized reaction conditions. We show the potential of this simple one-step procedure performed on spotted primer arrays of low redundancy by generating over 8000 genotypes for 40 mutations or SNPs. The genotypes formed three easily identifiable clusters and all known genotypes were assigned correctly. Higher degrees of multiplexing will be possible with this system as the power of discrimination between genotypes remained unaltered in the presence of over 100 amplicons in a single reaction. The enzyme-assisted reaction provides highly specific allele distinction, evidenced by its ability to detect minority sequence variants present in 5% of a sample at multiple sites. The assay format based on miniaturized reaction chambers at standard 384-well spacing on microscope slides carrying arrays with two primers per SNP for 80 samples results in low consumption of reagents and makes parallel analysis of a large number of samples convenient. In the assay one or two fluorescent nucleotide analogs are used as labels, and thus the genotyping results can be interpreted with presently available array scanners and software. The general accessibility, simple set-up, and the robust procedure of the array-based genotyping system described here will offer an easy way to increase the throughput of SNP typing in any molecular biology laboratory.

Deletion of the gene for subunit III leads to defective assembly of bacterial cytochrome oxidase.

COIII is one of the major subunits in the mitochondrial and a bacterial cytochrome c oxidase, cytochrome aa3. It does not contain any of the enzyme's redox-active metal centres and can be removed from the enzyme without major changes in its established functions. We have deleted the COIII gene from Paracoccus denitrificans. The mutant still expresses spectroscopically detectable enzyme almost as the wild-type, but its cytochrome c oxidase activity is much lower. From 50 to 80% of cytochrome a is reduced and its absorption maximum is 2-3 nm blue-shifted. The EPR signal of ferric cytochrome a is heterogeneous indicating the presence of multiple cytochrome a species. Proteolysis of the membrane-bound oxidase shows new cleavage sites both in COI and COII. DEAE-chromatography of solubilized enzyme yields fractions that contain a COI + COII complex and in addition haem-binding, free COI as well as free COII. The mutant phenotype can be complemented by introducing the COIII gene back to cells in a plasmid vector. We conclude that cytochrome oxidase assembles inefficiently in the absence of COIII and that this subunit may facilitate a late step in the assembly. The different oxidase species in the mutant represent either accumulating intermediates of the assembly pathway or dissociation products of a labile COI + COII complex and its conformational variants.