Serum matrix metalloproteinase-3 concentration is influenced by MMP-3 -1612 5A/6A promoter genotype and associated with myocardial infarction.

OBJECTIVES: Matrix metalloproteinase-3 (MMP-3) is implicated in the formation of atherosclerotic plaques, and the MMP-3 -1612 5A/6A polymorphism is associated with myocardial infarction (MI) and stable coronary artery disease (CAD). The present study examined whether the -1612 5A/6A polymorphism in the promoter region of the MMP-3 gene influences serum concentrations of MMP-3 and whether serum concentrations of MMP-3 are related to extent of coronary atherosclerosis and risk of MI. DESIGN AND SUBJECTS: This case-control study was conducted in three hospitals in the northern part of Stockholm. A total of 755 MI patients aged below 60 were screened, 433 entered and 387 completed the study. Three hundred and eighty-seven sex- and age-matched control subjects were recruited from the general population of the same county. METHODS: The MMP-3 genotype was determined by Pyrosequencing(TM) and the serum MMP-3 concentration was quantified with an immunoassay. Severity and extension of CAD was assessed by quantitative coronary angiography in a subgroup of patients (n=243). RESULTS: Patients had lower serum MMP-3 concentration than controls. There was a strong association between MMP-3 -1612 5A/6A genotype and serum concentrations of MMP-3. The presence of one or two copies of the 6A-allele was associated with a graded increase in serum MMP-3. In female patients there was an inverse correlation (r=-0.39, P<0.05) between serum MMP-3 concentration and plaque area. Conclusion. In conclusion, the serum concentration of MMP-3 is influenced by MMP-3 -1612 5A/6A genotype and associated with MI.

Role of genetic resistance in invasive pneumococcal infection: identification and study of susceptibility and resistance in inbred mouse strains.

From a panel of nine inbred mice strains intranasally infected with Streptococcus pneumoniae type 2 strain, BALB/c mice were resistant and CBA/Ca and SJL mice were susceptible to infection. Further investigation revealed that BALB/c mice were able to prevent proliferation of pneumococci in the lungs and blood, whereas CBA/Ca mice showed no bacterial clearance. Rapidly increasing numbers of bacteria in the blood was a feature of CBA/Ca but not BALB/c mice. In the lungs, BALB/c mice recruited significantly more neutrophils than CBA/Ca mice at 12 and 24 h postinfection. Inflammatory lesions in BALB/c mice were visible much earlier than in CBA/Ca mice, and there was a greater cellular infiltration into the lung tissue of BALB/c mice at the earlier time points. Our data suggest that resistance or susceptibility to intranasal pneumococci may have an association with recruitment and/or function of neutrophils.

A recQ family DNA helicase gene from Aspergillus nidulans.

We have identified an Aspergillus nidulans gene encoding a RecQ family helicase which we have therefore named recQ. The A. nidulans recQ protein is most closely related in sequence to human recQ helicase 5. Like the latter polypeptide, A. nidulans recQ consists of little more than the conserved helicase domain, lacking the long amino- and carboxy-terminal extensions seen in other recQ family members such as BLM and WRN and in the sole RecQ family helicase of the yeast Saccharomyces cerevisiae (Sgs1p). By analogy with other eukaryotic RecQ helicases, A. nidulans recQ helicase is likely to play an important role in the maintenance of genomic integrity.

A novel 'two-component' protein containing histidine kinase and response regulator domains required for sporulation in Aspergillus nidulans.

We have characterised a-novel Aspergillus nidulans gene encoding a 'two-component' signalling protein (tcsA). tcsA encodes both a histidine kinase domain and a response regulator domain similar to those found in bacterial, lower eukaryotic and plant members of the two-component family of proteins, while two PAS domains in the amino-terminal region of the predicted tcsA product may monitor the signal which regulates a tcsA histidine kinase-response regulator phosphorelay. While tcsA is nonessential for vegetative growth, cells lacking the gene are unable to produce conidia on standard Aspergillus growth media. However, tcsA is not absolutely required for production since this defect is suppressed by growth on 1 M sorbitol.

Characterisation of Candida albicans infections of haematogenous and mucosal origin in mice lacking the interferon gamma receptor protein.

Mice harbouring a null deletion mutation in the IFNgamma receptor gene were used to study the role of IFNgamma responsiveness during experimental systemic candidiasis of mucosal or haematogenous origin. After intravenous (i.v.) or intranasal (i.n.) challenge with Candida albicans the progression of infection and concomitant cellular and antibody anti-C. albicans immune responses were analysed. During the week following i.v. challenge, the rate of C. albicans multiplication in kidneys, liver and spleen was faster in IFNgammaR (-/-) than IFNgammaR (+/+) mice. As a result, IFNgammaR (-/-) mice perished earlier than IFNgammaR (+/+) mice when challenged with equal numbers of live yeast cells. However, the overall susceptibility of the two mouse strains, in terms of survival against different C. albicans challenge doses over a 60-day period, was similar. No differences were found in the cellular anti-C. albicans response generated by i.v. challenge in both mouse strains. In contrast the kinetics and strength of the serum anti-C. albicans antibody responses were markedly different. Significantly stronger, predominantly IgG2a antibody responses accompanied the eventual control of C. albicans infection in IFNgammaR (-/-) mice. Following intranasal challenge, there was no difference in the rate of C. albicans clearance from the lungs of IFNgammaR (-/-) and IFNgammaR (+/+) mice. However, 48 h after challenge, large, conspicuous abscesses appeared in the lungs, liver, kidneys and spleen of IFNgammaR (-/-) mice. These abscesses were characterised by the presence of C. albicans and abundant neutrophilic infiltrates, but very few macrophages. No such abscesses developed in i.n. challenged IFNgammaR (+/+) mice. In both mouse strains, i.n. challenge induced strong systemic anti-C. albicans cellular responses, but relatively low titre systemic antibody responses. Mucosal anti-C. albicans antibody responses were detected in IFNgammaR (+/+), but not IFNgammaR (-/-) mice. Splenic adherent macrophages obtained from IFNgammaR (-/-) mice exhibited a significantly lower candidacidal activity than those of IFNgammaR (+/+) mice, and as expected, were not responsive to IFNgamma. In summary, these data suggest that IFNgamma has a role in limiting C. albicans multiplication during the early stages of infection, as well as in preventing the development of C. albicans-associated abscesses. Activation of macrophages by IFNgamma might be pivotal in mediating this role.

Cloning and expression in murine erythroleukemia cells: the soluble forms of the type I and type II tumor necrosis factor receptors fused to an immunogenic affinity tag.

We have cloned, expressed, and purified the extracellular domains of types I and II human tumor necrosis factor receptors. Both proteins were expressed in and secreted by murine erythroleukemia cells under the control of the human beta-globin promoter placed down-stream from the human globin locus control region. Secretion of both proteins was directed by the respective tumor necrosis factor receptor signal sequence. Each tumor necrosis factor receptor extracellular domain was expressed as a chimeric protein, fused to a carboxy terminal flexible peptide linker and an antigenic affinity tag. Secretion of both proteins into the growth medium in a hollow fiber bioreactor was achieved. A monoclonal antibody generated against the affinity tag allowed the purification of both proteins. These were isolated as biologically active products in that they bound human tumor necrosis factor-alpha in a 125I-radioiodinated ligand binding assay. The two proteins also bound tumor necrosis factor-alpha at approximately equimolar ratios as demonstrated by BIAcore sensorgram analysis.

Pharmacological characterization of cloned human NK-2 (neurokinin A) receptor expressed in a baculovirus/Sf-21 insect cell system.

Using the novel ligand [4,5-3H-Leu9]neurokinin A ([4,5-3H-Leu9] NKA) in a receptor binding assay, we characterized the pharmacology of a cloned neurokinin NK-2 receptor from human lung (hNK-2R), expressed in baculovirus-infected Sf-21 insect cells. Functional hNK-2R cDNA clones were isolated from human lung using a polymerase chain reaction-based methodology. hNK-2R was cloned into pAcYM1, a vector designed to couple expression to the polyhedrin promoter, and the recombinant baculovirus was isolated and used to infect Sf-21 insect cells. hNK-2R expression levels were monitored by Northern blots and 125-I-NKA binding assays. Isolates demonstrating the highest specific binding of 125-I-NKA were grown and membrane preparations from high-speed centrifugations were prepared from both hNK-2R-expressing and wild-type virus-infected cells. [3H]NKA bound in a protein-dependent, saturable (Bmax = 820 +/- 167 fmol/mg of protein), and highly specific (88 +/- 5%) manner to hNK-2R, but not to membranes from cells infected with wild-type virus (14 +/- 8%, 7 +/- 10 fmol/mg of protein). [3H]NKA binding was rapid (k1 = 0.085 nM-1 x min-1) and reversible (t1/2 = 4-5 min). Equilibrium binding experiments demonstrated binding to a mixture of receptors in high and low affinity states (Kd1 = 2.28 +/- 0.26 nM and Kd2 = 266 +/- 91 nM). Binding to hNK-2R was greatly enhanced (400%-600%) by Ca2+ and Mg2+ (EC50 values of 30 microM and 140 microM, respectively), whereas guanosine-5'-O-(3'-thio)triphosphate and guanosine-5'-(beta, gamma-imido)diphosphate were inhibitory. Competition experiments with agonists also demonstrated binding to high and low affinity states, with the following order of potency: NKA > [Nle10]NKA(4-10) > [beta-Ala8]NKA(4-10) >> substance P; Senktide and the NK-1 antagonist CP96,345 (10 microM) did not inhibit binding. Inhibition of binding by selective NK-2 antagonists was consistent with a single affinity state and demonstrated the following order of affinity: SR48,968 >> MEN10,376 > L659,877 > R396. These data suggest that infection of Sf-21 cells with baculovirus expression vector harboring the cDNA of hNK-2R resulted in expression of high affinity, G protein-coupled hNK-2R, with pharmacological selectivity compatible with the NK-2A receptor subtype.

Identification of Bordetella pertussis in nasopharyngeal swabs by PCR amplification of a region of the adenylate cyclase gene.

The polymerase chain reaction (PCR) was used to amplify a 522-bp region of the adenylate cyclase toxin (cyaA) gene of Bordetella pertussis. As few as 100 cfu from a suspension of B. pertussis could be detected by this procedure when the amplified PCR product was detected by ethidium bromide staining of agarose gels. However, simulated clinical specimens, prepared from swabs impregnated with known numbers of B. pertussis cells, only yielded a positive reaction with > or = 10(4) cfu. Hybridisation of a Southern blot of the PCR products from the swab samples with a cya-specific probe gave a positive reaction with as few as 8 cfu, but the hybridisation signal was uniformly weak with fewer than 10(4) cfu. Nevertheless, three of 13 nasopharyngeal swabs, taken from suspected clinically defined cases of whooping cough and stored frozen for up to 18 months, gave a positive PCR reaction.

The mouse peroxisome proliferator activated receptor recognizes a response element in the 5' flanking sequence of the rat acyl CoA oxidase gene.

Peroxisome proliferators are a diverse group of chemicals, including several hypolipidaemic drugs, that activate a nuclear hormone receptor termed the peroxisome proliferator activated receptor (PPAR). The peroxisomal enzyme acyl CoA oxidase (ACO) is the most widely used marker of peroxisome proliferator action. We have examined the 5' flanking region of the rat ACO gene for sequences that mediate the transcriptional effect of peroxisome proliferators and have identified an element located 570 bp upstream of the ACO gene that confers responsiveness to the hypolipidaemic peroxisome proliferator Wy-14,643. This peroxisome proliferator response element (PPRE) contains a direct repeat of the sequence motifs TGACCT and TGTCCT and binds PPAR. These data therefore indicate an important role of PPAR in mediating the action of peroxisome proliferators including the induction of ACO.

Amplification of DNA by the polymerase chain reaction for the efficient diagnosis of pertussis.

The standard diagnostic methods for pertussis have several shortcomings. With the increased knowledge of the Bordetella pertussis genome a specific and conserved DNA sequence, present in about 70-80 copies in each genome, was selected for amplification with the polymerase chain reaction (PCR) technique in order to evaluate its diagnostic potential in children with suspected pertussis. The 400 basepair DNA sequence chosen was present and amplified in all 112 B. pertussis strains and in no other bacterial species examined. The specificity of the amplified material was documented by restriction enzyme cleavage. In nasopharyngeal aspirates a B. pertussis specific PCR product was visualized in 19/25 culture positive and in 5/50 culture negative children. In conclusion the present PCR assay for B. pertussis can be clinically useful and permit a specific diagnosis within 1 day after sampling. Further studies are requested to document its sensitivity, specificity and predictive value for positive and negative results.

Unknown sequence amplification: application to in vitro genome walking in Chlamydia trachomatis L2.

A recently described technique, 'Chemical Genetics' unknown sequence amplification method, which requires only one specific oligonucleotide, has broadened the applicability of the polymerase chain reaction to DNA of unknown sequence. We have adapted this technique to the study of the genome of Chlamydia trachomatis, an obligate intracellular bacterium, and describe modifications that significantly improve the utility of this approach. These techniques allow for rapid genomic analysis entirely in vitro, using DNA of limited quantity or purity.

Analysis of the chromosomal location of two copies of a Bordetella pertussis insertion sequence.

IS481v1 and IS481v2 are two copies of a Bordetella pertussis insertion sequence element. We have shown that IS481v1 is located within 3 kbp of the start of the adenylate cyclase gene whilst IS481v2 is immediately adjacent to the end of the agglutinogen 2 gene and provides the stop codon for that gene. In addition, IS481v1 and IS481v2 were present at these two specific sites in nine strains of B. pertussis, including two Phase IV strains which expressed neither adenylate cyclase nor agglutinogen 2 and three Phase I strains which did not express agglutinogen 2. The loss of expression in these strains is not the result of DNA rearrangements at the sites of IS481v1 or IS481v2.

Analysis of separate isolates of Bordetella pertussis repeated DNA sequences.

Two independent isolates of a Bordetella pertussis repeated DNA unit were sequenced and shown to be an insertion sequence element with five nucleotide differences between the two copies. The sequences were 1053 bp in length with near-perfect terminal inverted repeats of 28 bp, had three open reading frames, and were each flanked by short direct repeats. The two insertion sequences showed considerable homology to two other B. pertussis repeated DNA sequences reported recently: IS481 and a 530 bp repeated DNA unit. The B. pertussis insertion sequence would appear to comprise a group of closely related sequences differing mainly in flanking direct repeats and the terminal inverted repeats. The two isolates reported here, which were from the adenylate cyclase and agglutinogen 2 regions of the genome, were numbered IS48lvl and IS48lv2 respectively.

Isolation of a repeated DNA sequence from Bordetella pertussis.

A repeated DNA sequence in the genome of Bordetella pertussis has been demonstrated. At least 20 copies of this sequence could be observed in either BamHI or EcoRI restriction enzyme digests of chromosomal DNA; fragments carrying the repeated DNA sequence ranged in size from about 1.5 to 20 kbp. The repeated DNA sequence was cloned from two separate regions of the B. pertussis genome, as shown by restriction enzyme site maps of the two clones and by hybridization studies. A small number of differences in the pattern of hybridization of the repeated DNA sequence to chromosomal DNA from several strains of B. pertussis was observed. No repeated DNA sequences were observed in one strain each of B. parapertussis and B. bronchiseptica, and there was no hybridization of B. pertussis DNA to Escherichia coli chromosomal DNA. The repeated DNA sequence was subcloned on a 2.54 kbp BamHI fragment from one of the two original clones. Restriction enzyme digests and hybridization studies showed that the repeated DNA sequence was about 1 kbp in size and had a single, internal ClaI site.

Preliminary evidence for a pyridine nucleotide cycle in Bordetella pertussis.

Preliminary evidence that Bordetella pertussis has a functional pyridine nucleotide cycle was the observation that [14C]-nicotinic acid was rapidly metabolized during its uptake by the bacteria to pyridine nucleotides and nicotinamide. Nicotinamide deamidase activity, necessary for the completion of the cycle by conversion of nicotinamide to nicotinic acid, was found in a soluble extract (20 000 X g supernatant) of B. pertussis cell lysates.

Modulation of Bordetella pertussis by nicotinic acid.

Growth of Bordetella pertussis in a high concentration of nicotinic acid (NA) had a modulating effect on several properties and activities of the bacteria. Compared with normally grown cells, those grown in a high concentration of NA had reduced capacity for taking up both NA and nicotinamide (ND); they had reduced adenylate cyclase activity and showed loss of agglutinogen factors 2 and 3, but an increase in factor 1. By contrast, cells grown in a high concentration of ND showed only a slightly decreased capacity for uptake of ND and none of the other changes. Modulation of B. pertussis by NA varied with the strain and culture conditions and appeared to be distinct from the antigenic modulation induced by high Mg2+ in the culture medium. Evidence is presented for the association of a small proportion of the extracytoplasmic adenylate cyclase with the outer membrane of B. pertussis.

Inhibition of nicotinic acid and nicotinamide uptake into Bordetella pertussis by structural analogues.

3-Pyridine-carboxaldehyde and 3-pyridine-aldoxime were effective and specific inhibitors of the uptake of both nicotinic acid (NA) and nicotinamide (ND) by Bordetella pertussis, although neither compound inhibited the growth of the bacteria in liquid medium or the oxidation of glutamate by washed suspensions. In contrast, the following pyridine derivatives did not inhibit uptake of NA or ND: iso-NA, iso-ND, isoniazid, 6-amino-NA and 6-amino-ND, 3-acetyl-pyridine, 3-pyridyl-acetic acid, N,N-diethyl-ND and 3-pyridine-sulphonic acid. 3- Pyridyl-carbinol was inhibitory, but less so than the first listed compounds.