Simplified analysis of pathogenic leptospiral serovars by random amplified polymorphic DNA fingerprinting

Recent studies have suggested that platelet activating factor (PAF) plays an important role in various reproductive functions, including ovulation, implantation and parturition, and that the local concentration of PAF is modulated by PAF-acetylhydrolase (PAF-AH), a potent PAF inactivator. In this study, we investigated the possible effects of various bioactive substances, which are present at high concentrations in the human pregnant uterus, on PAF-AH secretion from decidual macrophages using a monocyte-macrophage model system, human myelocytic leukaemia cells (HL-60). By treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), HL-60 cells were transformed to macrophage-like cells, which secreted PAF-AH into the culture medium time- and dose-dependently. After treatment with 10(-8) M TPA, the effects of various substances on the secretion of PAF-AH were examined. Among the substances examined, cortisol and TGF-beta suppressed PAF-AH secretion from TPA-stimulated HL-60 cells in a significant and dose-dependent way. Endothelin, epidermal growth factor, and brain natriuretic peptide had no significant effect on PAF-AH secretion from TPA-stimulated HL-60 cells. These results suggest that local PAF concentrations in the pregnant uterus might be regulated, at least partly, by cortisol and TGF-beta; thus these substances may play a role in the initiation of parturition via regulation of local PAF concentrations.     

The application of random amplified polymorphic DNA for sandfly species identification

A case of disseminated North American blastomycosis is described in which the full extent of the multifocal osteomyelitis was depicted by Tc-99m MDP scintigraphy. Skeletal involvement by this disease is not uncommon, yet the utility of bone scintigraphy has not been reported. The efficacy with which the whole body may be examined makes this technique especially appropriate for a hematogenously disseminated and often multifocal form of osteomyelitis where radiographically occult and asymptomatic lesions may be present.     

Diversity among clinical isolates of Proteus penneri detected by random amplified polymorphic DNA analysis

Previous findings indicate that wheel running can have either an aversive or an appetitive effect. That is, wheel running for 30 min induces conditioned taste aversion (CTA) in rats trained while hungry and thirsty but facilitates feeding in non-deprived rats. In Experiment 1, wheel running was also found to be effective in producing CTA in non-deprived rats. Therefore, Experiment 2 tested whether wheel running produces the aversive and appetitive effects simultaneously. During each of four training trials, two groups of non-deprived rats were given a flavored solution to drink for 10 min. Then those in the wheel group were put in running wheels for 30 min whereas those in the cage group spent 30 min in small cages. Finally, all rats were given a 60-min feeding test. After the first trial, the wheel group drank less flavored solution than the cage group during each of the remaining trials. The wheel group also ate more than the cage group on each feeding test. These results indicate that wheel running produces CTA and facilitates eating at the same time. A role for the mesolimbic dopamine reward system in these effects was considered.     

Segregation analysis of random amplified polymorphic DNA (RAPD) markers in Picea abies Karst

The reliability of arbitrarily primed amplification products was tested. The segregation analysis of 266 amplification products obtained using 17 different 10-mer oligonucleotides in 34 megagametophytes from a single tree of Picea abies was carried out. Fifty-four out of the 165 variable bands fit the 1:1 segregation ratio expected for Mendelian traits. The segregation ratio of a subset of six RAPD markers in five other individuals from the same population confirmed their genetic nature. Our results strengthen the evidence previously reported that RAPDs markers can be considered Mendelian traits useful in the detection of genetic variability among both different individuals and populations.     

Phage typing combined with pulsed-field gel electrophoresis and random amplified polymorphic DNA increases discrimination in the epidemiological analysis of Salmonella enteritidis strains

In adult rabbits, mid-diaphyseal segments of the radius or ulna were excised to produce defects greater than the critical size for spontaneous bone repair. The defects were enveloped in sleeves composed of nonbiodegradable expanded polyfluoroethylene (ePTFE), pore size 30, 60, 90 microns, and compared with sleeves of three biodegradable materials. Bone morphogenetic protein and associated noncollagenous bone matrix protein (BMP/NCP) or recombinant human morphogenetic protein (rhBMP-2) were implanted inside the sleeves. Albumin was implanted for a control system. Without intracompartmental BMP, only about 10%-15% of the defect was repaired by bone growth extending from the bone ends into the sleeves composed of ePTFE, pore size 30 microns. With sleeves with pore size 60 or 90 microns and intracompartmental BMP/NCP, 54%-96% regeneration occurred within 8 weeks after the operation. Sleeves of biodegradable nonimmunogenic materials such as polyorthoester (POE) and polylactic-polyglycollic acids (PLA/PGA) permitted 86%-98% restoration of bone continuity, but only when BMP was present in the lumen. With puncture holes (0.5 mm in diameter), implants of BMP/NCP in the 30-micron PTFE sleeve produced transmembrane external callus formation and bone regeneration to 147%. Sleeves composed of aorta first calcified, then induced complete intracompartmental bone regeneration. Atelocollagen sleeves incited a low-grade inflammatory cell reaction and did not promote complete regeneration. Under conditions presently undisclosed segments of the ulna bridged with ePTFE, were incompletely paired, even with intracompartmental BMP/NCP. Puncture holes of 0.5 mm admitted ingrowth of capillaries and introduced local conditions favorable for the response to BMP/NCP. BMP/NCP may promote proliferation of nutrient vessels and differentiation of bone marrow stroma cells between the open bone ends. For further investigation, the hypothesis to be examined is that the optimum response to BMP/NCP and rhBMP-2 would emerge in compartments containing first a high concentration gradient and second proliferating perivascular cells.     

Random amplified polymorphic DNA (RAPD) interpretation requires a sensitive method for the detection of amplified DNA

The random amplified polymorphic DNA technique (RAPD) has found wide use in molecular genetics because of its speed and ease of use. For various reasons, with this method the amplified DNA fragments are produced at different concentrations between genotypes and even between polymerase chain reaction (PCR) runs. Since the detection of the multiple amplified fragments is performed routinely by agarose gel, and seldom by acrylamide gel electrophoresis, we have found that by capillary zone electrophoresis (CZE), which is more sensitive and accurate than gel electrophoresis, it is possible to unequivocally detect amplified fragments even at low concentration, avoiding polymorphism misinterpretation. CZE is also useful to make more potentially polymorphic fragments evident per random primer used, with obvious economical benefits.     

A pseudoautosomal random amplified polymorphic DNA marker for the sex chromosomes of Silene dioica

The segregation pattern of an 810-bp random amplified polymorphic DNA (RAPD) band in the F1 and backcross generations of a Silene dioica (L.) Clairv. family provides evidence that this molecular marker is located in the pseudoautosomal region (PAR) of the X and Y chromosomes. The marker was found through a combination of bulked segregant analysis (BSA) and RAPD techniques. Recombination rates between this pseudoautosomal marker and the differentiating portion of the Y chromosome are 15% in both generations. Alternative explanations involving nondisjunction or autosomal inheritance are presented and discussed. Chromosome counts provide evidence against the nondisjunction hypothesis, and probability calculations argue against the possibility of autosomal inheritance. This constitutes the first report of a pseudoautosomal DNA marker for plant sex chromosomes.     

Characterization of clinical isolates of Streptococcus agalactiae by random amplified polymorphic DNA using degenerate oligonucleotides

Epidemiological studies of Streptococcus agalactiae strains have been limited by the lack of sensitive and discriminatory methods for comparing clinical isolates. Serotyping, albeit a widely used methodology, has been shown to possess low capability to distinguish between epidemiologically related and unrelated isolates. We have employed here a random amplification of polymorphic DNA (RAPD) assay, using degenerate oligonucleotides as primers, to characterize S. agalactiae isolates from related or unrelated clinical samples. Epidemiologically-related isolates (mother-infant pairs) showed identical profiles by this methodology. On the contrary, 12 epidemiologically-unrelated isolates (classified into 5 different serotypes) resulted in 11 distinct RAPD patterns. This suggests that the proposed modified RAPD assay provides a highly discriminatory tool for the analysis of genomic diversity among isolates from pathogenic organisms.     

Pulsed-field gel electrophoresis is more efficient than ribotyping and random amplified polymorphic DNA analysis in discrimination of Pasteurella haemolytica strains

OBJECTIVE: Factors such as size of hyphema, intraocular pressure, initial visual acuity, and use of steroids or antifibrinolytic drugs may be associated with the likelihood of rebleeding in traumatic hyphema. The association of the visual outcome with secondary hemorrhage has been questioned. DESIGN: Randomized, placebo-controlled, clinical trial. PARTICIPANTS: Two hundred and thirty-eight patients who had hyphema develop after blunt trauma. INTERVENTION: Eighty patients received oral tranexamic acid, 80 patients received placebo, and 78 patients received oral prednisolone. MAIN OUTCOME MEASURES: Secondary hemorrhage and vision at the time of discharge from the hospital were measured. RESULTS: Rebleeding occurred in 43 (18%) of the patients and was prevented significantly by oral tranexamic acid compared with the placebo (odds ratios [OR] = 0.39; 95% confidence interval [CI], 0.17, 0.89). Occurrence of secondary hemorrhage had weak associations with initial high intraocular pressure (OR = 2.7; 95% CI, 0.99, 7.3) and initial visual acuity of 6/60 or less (OR = 1.8; 95% CI, 0.9, 3.7). Secondary hemorrhage had no statistical association with age, gender, oral prednisolone, size of hyphema, and retinal damage. Visual acuity of 6/60 or less at the time of discharge was significantly associated with rebleeding (OR = 10.5; 95% CI, 3.7, 29.2), initial visual acuity of 6/60 or less (OR = 9.9; 95% CI, 2.8, 38.0), retinal damage (OR = 14.6; 95% CI, 3.8, 55.8), and male gender (OR = 6.5; 95% CI, 1.4, 31.9). Final visual acuity had no significant statistical association with age, use of oral prednisolone or tranexamic acid, and size of hyphema. CONCLUSIONS: High intraocular pressure and low vision at the time of first examination may be associated with increased chance of rebleeding. Retinal damage, secondary hemorrhage, male gender, and initial poor vision are associated with a worse visual outcome in patients with traumatic hyphema.     

Persistence in bovine mastitis of Staphylococcus aureus clones as assessed by random amplified polymorphic DNA analysis, ribotyping and biotyping

The physiological properties of joint capsule mechanoreceptors in the ankle joint of the chicken were studied in the 3-h period immediately after intra-articular injection of microcrystal sodium urate. The electrical activity was recorded from single C- and A-delta sensory fibres dissected from the parafibular nerve. C-fibres showed high levels of spontaneous activity and receptive fields that varied from single spots 1 mm in diameter up to 4 x 4 mm. Thresholds to mechanical stimulation ranged from 0.1 to 8 g and 80% of the units responded to movement of the joint. A-delta fibres showed little spontaneous activity and receptive fields that varied from 1 mm to 9 x 1 mm. Thresholds to mechanical stimulation ranged from 0.1 to 16 g and 17% responded to joint movement. A comparison of the physiological properties of the C- and A-delta fibres in sodium urate arthritis with similar fibres in normal and monoarthritic animals indicated an increased sensitivity in the C-fibres but not in the A-delta fibres. Sensitisation was observed in the significantly increased receptive field size, decreased response thresholds, increased response to joint movement and the high level of spontaneous activity. These changes in the sensitivity of the joint capsule C-fibre receptors provides peripheral neural evidence for the pain experienced in acute gouty arthritis.     

Analysis of genetic markers by random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR)

RAPD-PCR is a new technique that, starting from genomic DNA allows, with the use of a single primer of "random" base composition to amplify a variable number of sequences that can give important informations if analyzed for linkage studies, gene mapping or phylogenetic purposes. In order to detect the possible application of this simple way of DNA-fingerprinting in individual identification and in cell lineages characterization we analyzed human and non-human Primates DNA. Six different single primers of variable length were used and resulted in individual or specific electrophoretic patterns. As already reported we found a better resolution using "short" primers. The individual electrophoretic patterns obtained by RAPD-PCR can be a simple and reliable approach to DNA analysis.     

Comparison of PCR, nested PCR, and random amplified polymorphic DNA PCR for detection and typing of Ureaplasma urealyticum in specimens from pregnant women

A PCR assay, using three primer pairs, was developed for the detection of Ureaplasma urealyticum, parvo biovar, mba types 1, 3, and 6, in cultured clinical specimens. The primer pairs were designed by using the polymorphic base positions within a 310- to 311-bp fragment of the 5' end and upstream control region of the mba gene. The specificity of the assay was confirmed with reference serovars 1, 3, 6, and 14 and by the amplified-fragment sizes (81 bp for mba 1, 262 bp for mba 3, and 193 bp for mba 6). A more sensitive nested PCR was also developed. This involved a first-step PCR, using the primers UMS-125 and UMA226, followed by the nested mba-type PCR described above. This nested PCR enabled the detection and typing of small numbers of U. urealyticum cells, including mixtures, directly in original clinical specimens. By using random amplified polymorphic DNA (RAPD) PCR with seven arbitrary primers, we were also able to differentiate the two biovars of U. urealyticum and to identify 13 RAPD-PCR subtypes. By applying these subtyping techniques to clinical samples collected from pregnant women, we established that (i) U. urealyticum is often a persistent colonizer of the lower genital tract from early midtrimester until the third trimester of pregnancy, (ii) mba type 6 was isolated significantly more often (P = 0.048) from women who delivered preterm than from women who delivered at term, (iii) no particular ureaplasma subtype(s) was associated with placental infections and/or adverse pregnancy outcomes, and (iv) the ureaplasma subtypes most frequently isolated from women were the same subtypes most often isolated from infected placentas.     

Geographic discrimination of Paracoccidioides brasiliensis strains by randomly amplified polymorphic DNA analysis

The structured clinical interview for diagnosis (axis 1) according to the Diagnostic and Statistical Manual for Mental Disorders (DSM-III-R) was used to assess psychiatric morbidity in 110 infertile patients. They were divided into two groups according to whether referral to the service of psychosomatic medicine was deemed advisable by the physician in charge. Psychiatric disorders were diagnosed in 39 of 56 (69.6%) patients in the referred group and in 13 of 54 (24.1%) in the non-referred group. Psychiatric morbidity was found in 61.1% of females and 21% of males. Adjustment disorders were found in 59.6% (31/52) of all patients, in 59% (24/39) of patients among the referred group and in 61.5% (8/13) of patients among the non-referred group. Fourteen (67%) of 21 women in the referred group with adjustment disorders suffered from anxiety. In addition, 33.3% of patients in the non-referred group showed important psychological dysfunction, although DSM-III-R criteria were not met. Psychiatric morbidity was significantly associated with the number of treatment cycles and female gender in the whole study population, as well as with the type and length of infertility in the non-referred group. Psychological services in an infertility clinic help to identify at an early stage those individuals who are more likely to be vulnerable. This would enable psychological interventions to be targeted towards those in greater need.     

Characterization of Scedosporium prolificans clinical isolates by randomly amplified polymorphic DNA analysis

Fingerprinting by randomly amplified polymorphic DNA (RAPD) analysis was used to differentiate Scedosporium prolificans isolates. A total of 59 arbitrary primers were screened with six unrelated S. prolificans isolates, and a panel of 12 primers was selected. The 12 primers were then used to detect DNA polymorphisms among 17 S. prolificans isolates from 11 patients with systemic S. prolificans infections diagnosed in three hospitals located in geographically different areas of Spain. Eight patients were diagnosed with S. prolificans infection in a single institution over a 6-year period, and two other patients were diagnosed with S. prolificans infection in a different hospital over a 1-year period. No single primer allowed for the discrimination of all the isolates from different patients, but this was possible by combining the RAPD patterns from three primers (UBC 701, AB1.08, and AB1.11 or UBC 701, AB1.08, and UBC 707). However, multiple isolates from the same patient were identical. In this study, we also compared a visual method and a computerized method for the analysis of the RAPD patterns. Both methods were satisfactory and gave few discordances, but given the advantages and disadvantages of each method, both systems should be used together. RAPD analysis provided a fast and economical means of typing S. prolificans isolates, with a high level of discrimination among unrelated isolates. Typing by RAPD analysis confirmed that the S. prolificans infections were epidemiologically unrelated.     

Validation of random amplified polymorphic DNA assays by ribotyping as tools for epidemiological surveys of Pasteurella from animals

Two collections of strains of Pasteurella were studied for epidemiological purposes by ribotyping and random amplified polymorphic DNA (RAPD) assays. These strains were isolated through two different structures of animal productions: cattle and rabbit. Forty strains of P. haemolytica from cattle reared in independent breeding-herds belonged to only 3 ribotypes after digestion with HindIII and PvuII. No further discrimination of these strains was obtained by RAPD assays. All these 40 strains showed more than 90% of similarity. This result was consistent with the hypothesis of a clonal dissemination of these strains in bovine herds, possible favoured by the large use of antibiotics. Forty-one strains of P. multocida were isolated in rabbits flocks belonging to 16 breeders. Six of these were linked by commercial relationships. Twenty-eight out of the 29 strains isolated through this commercial network belonged to only three ribotypes whereas the 12 strains from independant breeders belonged to 9 ribotypes. Results of RAPD assays were in accordance with those of ribotyping and validate the use of RAPD assays for epidemiological studies of Pasteurella strains.     

Differentiation of Arthroderma spp. by random amplification of polymorphic DNA (RAPD) and Southern hybridization

To develop the molecular differentiation analysis of dermatophytes, we carried out RAPD and Southern hybridization analyses using genomic DNAs of six Arthroderma species, including A. fulvum, A. grubyi, A. gypseum, A. incurvatum, A. otae and A. racemosum. The RAPD analysis gave different band patterns specific to each of the six Arthroderma fungi. However, minor differences in the banding patterns were observed between the strains of plus (+) and minus (-) mating types of A. gypseum, A. fulvum and A. incurvatum. Southern blot analysis using a probe (1S) obtained from A. grubyi DNA gave specific bands only in the DNA samples of A. grubyi and A. incurvatum. On the other hand, Southern blot analysis using a probe (C3) obtained from A. otae DNA gave specific bands in all six Arthroderma species examined, and the size of the bands were specific to each species. These findings indicate that RAPD and Southern hybridization analyses are useful in the differentiation of these Arthroderma species.     

Restriction fragment length polymorphism analysis and random amplified polymorphic DNA analysis of Campylobacter jejuni strains isolated from patients with Guillain-Barré syndrome

Campylobacter jejuni serotype O19 strains associated with the Guillain-Barré syndrome (GBS) and other strains were examined by restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction products of the flaA genes and by random amplified polymorphic DNA (RAPD) analysis. RFLP analysis showed that regardless of LIO serotype, geographic origins, or association with GBS, the O19 isolates shared an identical digestion pattern by each of four restriction endonucleases, DdeI, MboI, MseI, and AluI. In contrast, among C. jejuni O1 or O2 strains, RFLP patterns were different even among strains of the same LIO serotype. The results of the RAPD analysis were consistent with the flaA RFLP data. These data indicate that all of the O19 strains that were tested were closely related to one another whether they were or were not associated with GBS.     

Allocation of random amplified polymorphic DNA markers and enzyme activities to Aspergillus nidulans and Aspergillus tetrazonus chromosomes

Chromosome-substituted haploid segregants of an A. nidulans x A. tetrazonus somatic hybrid were used to allocate several random amplified polymorphic DNA and isoenzyme markers to parental chromosomes. Twenty-six amplified DNA fragments, and nine isoenzyme activities, including lactate dehydrogenase, superoxide dismutase, and arylesterase isoenzymes were assigned to chromosomes. Chromosomes-specific markers were found for each A. nidulans and A. tetrazonus chromosome. These markers could be used to saturate the genetic map of A. nidulans. The formation of two secondary metabolites was also assigned to chromosomes III and VIII. Attempts were made to allocate extracellular enzyme activities to parental chromosomes, mostly without success, possibly because multiple enzyme forms located on different chromosomes could be responsible for the production of an enzyme activity.     

Identification and genetic mapping of random amplified polymorphic DNA (RAPD) markers to the sheep genome

The random amplified polymorphic DNA (RAPD) assay utilizes the polymerase chain reaction (PCR) and short primers of arbitrary nucleotide sequence to amplify DNA. In this study, the RAPD assay was used to identify and map polymorphic markers in the AgResearch International Mapping Flock (IMF) sheep pedigrees. Sires and dams of eight of the full-sib IMF pedigrees were screened with 131 different 10-mer oligonucleotide primers. An average of 85 RAPD polymorphisms was identified between each parental pair, and 53 markers were contributed to the AgResearch IMF collaboration. Forty-five of the RAPD markers were mapped in the AgResearch IMF genetic linkage map, and at least one marker was located on 17 of the 26 autosomes and both sex chromosomes. Three lines of evidence were used to check for the homology of scored polymorphisms in different pedigrees, pedigree evaluation, segregation analysis, and Southern blot analysis. These results demonstrate that the RAPD assay is a powerful approach for identifying polymorphisms that can be used as markers for constructing a sheep genetic linkage map.     

Random amplified polymorphic DNA analysis and variation of essential oil components of Atractylodes plants

Recently, air-abrasion technology has been examined for potential applications within dentistry, including the field of orthodontics. The purpose of this study was to compare the traditional acid-etch technique with an air-abrasion surface preparation technique, with two different sizes of abrading particles. The following parameters were evaluated: (a) shear bond strength, (b) bond failure location, and (c) enamel surface preparation, as viewed through a scanning electron microscope. Sixty extracted human third molars were pumiced and divided into three groups of 20. The first group was etched with a 37% phosphoric acid gel for 30 seconds, rinsed for 30 seconds, and dried for 20 seconds. The second and third groups were air-abraded with (a) a 50 microm particle and (b) a 90 microm particle of aluminum oxide, with the Micro-etcher microabrasion machine (Danville Engineering Inc.). All three groups had molar stainless steel orthodontic brackets bonded to the buccal surface of each tooth with Transbond XT bonding system (3M Unitek). A Zwick Universal Testing Machine (Calitek Corp.) was used to determine shear bond strengths. The analysis of variance was used to compare the three groups. The Adhesive Remnant Index (ARI) was used to evaluate the residual adhesive on the enamel after bracket removal. The chi square test was used to evaluate differences in the ARI scores among the groups. The significance for all tests was predetermined at p < or = 0.05. The results indicated that there was a significant difference in shear bond strength among the three groups (p = 0.0001). The Duncan Multiple Range test showed a significant decrease in shear bond strength in the air-abraded groups. The chi square test revealed significant differences among the ARI scores of the acid-etched group and the air-abraded groups (chi(2) = 0.0001), indicating no adhesive remained on the enamel surface after debonding when air-abrasion was used. In conclusion, the current findings indicate that enamel surface preparation using air-abrasion results in a significant lower bond strength and should not be advocated for routine clinical use as an enamel conditioner at this time.