Applicability of SSCP analysis for MHC genotyping: fingerprinting of Ovar-DRB1 exon 2 alleles from Finnish and Russian breeds

Recent studies have implicated leptin in the modulation of bone mass during skeletal development. Whether leptin also exerts an influence on bone after growth has stopped is unknown at present. In this cross-sectional study on 94 women (60 premenopausal, 34 postmenopausal) aged 40-60 years, we analyzed the relationship between serum leptin and bone density and bone cortex geometry and bone metabolism. Total and trabecular bone density as well as total and cortical bone area were determined by quantitative computed tomography (QCT) at the distal radius. Bone metabolism was assessed by measuring bone-specific alkaline phosphatase, osteocalcin, procollagen type I C-terminal propeptide (PICP) and collagen type I C-terminal telopeptide in serum, and deoxypyridinoline in urine samples. None of the indices of bone density or geometry was significantly related to leptin serum concentrations (P > 0.05) before or after adjustment for body mass index (BMI). PICP was associated with serum leptin in the postmenopausal group only (r = -0.40 after adjustment for BMI; P = 0.009). Yet, as none of the other markers of bone metabolism exhibited a significant correlation with serum leptin in any of the menopausal groups, this association is likely to be due to the influence of extraskeletal factors on PICP serum levels. Thus, it appears that leptin has less influence on the mature than on the growing skeleton.     

Application of DNA fingerprinting to obstetrics and gynecology

Restriction fragment length polymorphisms (RFLP) are variations in the size of restriction fragments of genomic DNA that hybridize to specific probes. They are the consequence of changes in primary DNA sequences, most of which result from small-scale changes in DNA. Recently minisatellite DNA probes that detect many regions of great variability within the human genome have been described. Minisatellite probes consist of multiple repeated copies of a common 10-15 base pair core sequence. On hybridization to restriction enzyme digests of human DNA, they simultaneously detect many highly polymorphic minisatellites at different loci in the genome, and produce band patterns that are individual specific. The band patterns are called "DNA fingerprints" or "DNA barcode" which can be used for individual identification on forensic and legal medicine. In addition to forensic and legal medicine, DNA fingerprinting can be used in both basic research and clinical examination of obstetrics and gynecology. The RFLP bands in DNA fingerprinting are inherited as single Mendelian co-dominants and we can use such minisatellite DNA probe for the determination of zygosity in multiple pregnancy. This probe can be used for the determination of androgenesis as a cause of complete hydatidiform mole. Each polymorphic band in molar tissues could be identified as being of paternal but not maternal origin. Some polymorphic bands of paternal origin were not observed in molar tissues, indicating that endoreduplication of a normal haploid sperm or fertilization by dispermy to an anuclear oocyte with no effective genome could be the cause of complete hydatidiform mole (androgenesis).(ABSTRACT TRUNCATED AT 250 WORDS)     

Echinococcus granulosus: cloning of a thioredoxin peroxidase

Disseminated histoplasmosis has been recognized as a serious opportunistic infection in patients with acquired immunodeficiency syndrome (AIDS). However, cases reported in the literature have been predominantly in adult patients. Here we report an infant with AIDS who presented with fever, cough, rhinorrhea, hepatosplenomegaly, pancytopenia and coagulopathy, and died of respiratory failure. Autopsy revealed disseminated histoplasmosis involving multiple organs including lungs, intestines, liver, spleen, bone marrow, lymph nodes, kidneys, and meninges. The diagnosis was established based on histomorphology and confirmed by blood culture.     

Predicting ligand binding to proteins by affinity fingerprinting

BACKGROUND: There are many ways to represent a molecule's properties, including atomic-connectivity drawings, NMR spectra, and molecular orbital models. Prior methods for predicting the biological activity of compounds have largely depended on these physical representations. Measuring a compound's binding potency against a small reference panel of diverse proteins defines a very different representation of the molecule, which we call an affinity fingerprint. Statistical analysis of such fingerprints provides new insights into aspects of binding interactions that are shared among a wide variety of proteins. These analyses facilitate prediction of the binding properties of these compounds assayed against new proteins. RESULTS: Affinity fingerprints are reported for 122 structurally-diverse compounds using a reference panel of eight proteins that collectively are able to generate unique fingerprints for about 75% of the small organic compounds tested. Application of multivariate regression techniques to this database enables the creation of computational surrogates to represent new proteins that are surprisingly effective at predicting binding potencies. We illustrate this for two enzymes with no previously recognizable similarity to each other or to any of the reference proteins. Fitting of analogous computational surrogates to four other proteins confirms the generality of the method; when applied to a fingerprinted library of 5000 compounds, several sub-micromolar hits were correctly predicted. CONCLUSIONS: An affinity fingerprint database, which provides a rich source of data defining operational similarities among proteins, can be used to test theories of cryptic homology unexpected from current understanding of protein structure. Practical applications to drug design include efficient pre-screening of large numbers of compounds against target proteins using fingerprint similarities, supplemented by a small number of empirical measurements, to select promising compounds for further study.     

Detection of Echinococcus multilocularis in the definitive host: coprodiagnosis by PCR as an alternative to necropsy

Recently, extensions of the range of Echinococcus multilocularis in Europe and North America and drastic increases in fox populations in Europe put an increasing proportion of the human population at risk of alveolar echinococcosis. To obtain data on the local infection pressure, studies of the prevalence of the parasite in the animals that transmit the parasite, foxes, dogs, and cats, are urgently required. Such investigations, however, have been hampered by the need for necropsy of the host animal to specifically diagnose infection with the parasite. In this study, a nested PCR and an improved method for DNA extraction were developed to allow the sensitive and specific diagnosis of E. multilocularis infections directly from diluted fecal samples from foxes. The target sequence for amplification is part of the E. multilocularis mitochondrial 12S rRNA gene. The specificity of the method was 100% when it was tested against 18 isolates (metacestodes and adult worms) of 11 cestode species, including E. granulosus. The sensitivity of the method was evaluated by adding egg suspensions and individual eggs to samples of diluted feces from uninfected foxes. The presence of one egg was sufficient to give a specific signal. To confirm the PCR results, an internal probe which hybridized only with E. multilocularis amplification products but not with the DNA of other cestodes was constructed. In order to investigate the applicability of this method for epidemiological studies, 250 wild foxes from a area in southern Germany where echinococcosis is highly endemic were examined by both necropsy and PCR of rectal contents. The sensitivity correlated with the parasites' number and stage of maturity. It ranged from 100% (>1,000 gravid worms) to 70% (<10 nongravid worms). On the basis of positive PCR results for 165 foxes, the sensitivity of the traditional and widely used necropsy method was found to be not higher than 76%. We therefore present this PCR system as an alternative method for the routine diagnosis of E. multilocularis in carnivores.     

A rare cause of respiratory failure: Echinococcus of the pulmonary artery

To combine the advantages of the standard technique and the bicaval technique of orthotopic heart transplantation, we use a muscular flap of recipient heart right atrium for connecting the superior vena cava with the donor heart right atrium. The results in respect to the maintenance of atrioventricular valve competence as well as atrial conduction are promising.     

cDNA fingerprinting of osteoprogenitor cells to isolate differentiation stage-specific genes

A cDNA fingerprinting strategy was developed to identify genes based on their differential expression pattern during osteoblast development. Preliminary biological and molecular staging of cDNA pools prepared by global amplification PCR allowed discrim-inating choices to be made in selection of expressed sequence tags (ESTs) to be isolated. Sequencing of selected ESTs confirmed that both known and novel genes can be isolated from any developmental stage of interest, e.g. from primitive progenitors, intermediate precursors or mature osteoblasts. EST expression provides insight into possible interrelated physiological functions and putative interacting molecules during differentiation. This method offers a functional genomics approach to isolate differentiation stage-specific genes in samples as small as a single cell.     

Application of DNA fingerprinting to enforcement of hunting regulations in Ontario

DNA fingerprinting has been used in investigations of 40 cases of infractions of hunting regulations involving white-tailed deer (Odocoileus virginianus) and moose (Alces alces) in Ontario. In most of these cases, individual-specific DNA fingerprints obtained with the Jeffrey's 33.15 multilocus probe were used to link the animal remains found at the illegal kill site to blood and tissue samples of the dead animal associated with a suspect. DNA fingerprints from 27 white-tailed deer and 19 moose were obtained in order to establish the level of band-sharing in DNA fingerprints among unrelated individuals in each species. We also determined the levels of band-sharing among animals from the same region and calculated the probability of two individuals sharing the same DNA fingerprint. Details are presented from cases in which the evidence was presented and accepted by Ontario courts.     

Forensic applications of DNA fingerprinting

Although previous research has shown that childhood adversity has long-term effects on adult depression, little is known about the causal pathways involved in these effects. In this report data from a two-wave longitudinal survey of the U.S. household population are used to study these pathways as they affect the association between childhood family violence and adult recurrence of depression. We focus on recurrence of depression because most episodes of clinically significant depression in adulthood occur to persons with a history of depression. We find that chronic interpersonal stress in adulthood mediates the effect of childhood family violence on recurrence of depression, and that childhood family violence modifies the effect of chronic adult interpersonal stress on recurrence of depression. Furthermore, in the absence of chronic adult interpersonal stress there is no association between childhood family violence and adult recurrence of depression.     

Typing of rhizobia by PCR DNA fingerprinting and PCR-restriction fragment length polymorphism analysis of chromosomal and symbiotic gene regions: application to Rhizobium leguminosarum and its different biovars

Characterization of 43 strains of Rhizobium leguminosarum biovars viciae, trifolii, and phaseoli was performed by two methodologies based on PCR amplification, i.e., PCR DNA fingerprinting of interrepeat sequences and restriction fragment length polymorphism (RFLP) analysis of PCR -amplified chromosomal and symbiotic gene regions. Groupings generated by PCR DNA fingerprinting with either extragenic palindromic repetitive primers or two different single random primers were correlated with similar levels of resolution. Although less discriminating, PCR-RFLP analysis of intergenic spacer between genes coding for 16S and 23S rRNA (16S and 23S rDNA) yielded intraspecific polymorphisms. The classification of strains was independent of the biovar status and was in agreement with those obtained by PCR DNA fingerprinting. Intrabiovar variation within symbiotic gene regions was detected by PCR-RFLP analysis of nifDK and nodD gene regions, but the strains were grouped according to the biovar. The rDNA intergenic spacer and nif primers were verified to be universal for rhizobial species by testing of various reference strains, whereas the nod primers designed in this study were biovar or species specific for R. leguminosarum and Rhizobium etli. Classifications of R. leguminosarum strains by the PCR-based methods were correlated with those previously obtained by conventional total DNA restriction profile comparisons and RFLP analysis using chromosomal and symbiotic gene probes. Ranges of discriminating powers were also equivalent between the two approaches. However, the PCR-based methods are much less time-consuming and are therefore more convenient.     

Application of cycle dideoxy fingerprinting to screening heterogeneous populations of the equine infectious anemia virus

Nucleotide sequence heterogeneity in a population of the equine infectious anemia virus (EIAV) was investigated using a modification of the dideoxy fingerprinting (ddF) technique. PCR-amplified regions of the gag gene from EIAV isolates were ligated into plasmid vectors and used to transform bacteria. The single dideoxynucleotide sequencing step was performed using plasmid DNA prepared from individual bacterial colonies using an 35S end-labeled primer and Taq DNA polymerase. Analysis of the products of this reaction was conducted using non-denaturing polyacrylamide gel electrophoresis. Polymorphism within this gene was suggested by the presence of several distinct electrophoretic profiles. Significantly, each profile could be correlated with variations in nucleotide sequence, which demonstrates that cycle ddF (CddF) offers a rapid and sensitive approach to identify polymorphism in PCR-amplified products.     

Resistance of the the coraco-clavicular ligaments to traction: clinical application

One method for treating chronic incapacitating acromioclavicular dislocation is to resect the external extremity of the clavicle and to stabilise the stump by ligamentoplastic procedures, using the coracoclavicular ligament. The purpose of this work was to evaluate the mechanical quality of the ligamentoplastic approach. Twelve fresh cadavers, average age 80 years, were studied. The samples taken were 24 coracoclavicular ligaments, 24 coraco-acromial ligaments, 9 tendons from the palmaris longus muscle and 9 iliotibial tracts. The ligaments removed were tested in a Instron traction machine at a speed of 10 cm/mn. The mechanical properties of the coracoclavicular and coraco-acromial ligaments were studied. For comparison, those of the tendon of the palmaris longus muscle and the iliotibial tract were also studied. The results show the pre-rupture resistance of the coraco-acromial ligament to be 50% lower than that of the trapezoid and conoid parts of the coracoclavicular ligaments taken together. These results suggest the validity of ligamentoplastic treatment using the coraco-acromial ligament, but that reinforcement, using a tendon from the palmaris longus muscle or a piece of the iliotibial tract, may also be necessary, especially for subjects taking part in sports or with well-developed musculature.     

Immunological assessment of exposure to Echinococcus granulosus in a rural dog population in Uruguay

An ELISA was used to screen a dog population in Uruguay (Sarandi Del Yi, Durazno District) for the prevalence of specific serum antibodies (IgG, IgA and IgE) to Echinococcus granulosus. The sensitivity (61%) and specificity (97%) of the ELISA were determined using well-defined serum groups. A total of 408 dogs from Sarandi del Yi and environs were screened serologically, and 29.7% (8.6-13.8% for each antibody class) of dogs had positive levels of antibody to E. granulosus. This antibody prevalence (exposure) was significantly higher than the percentage of dogs found to be positive for E. granulosus worms by arecoline purgation (7.6%). This level of exposure to E. granulosus determined by ELISA is considered unacceptable from a public health perspective. Measures will now focus on obtaining data on the true prevalence of current infection in this dog population and on determining the transmission patterns of the disease in this endemic region.     

DNA vaccines: application to tuberculosis

Since the first observations in the early 1990s, the scientific literature on DNA vaccines has been growing exponentially. This article reviews the history and general principle, summarizes current knowledge on immune mechanisms, discusses safety considerations and highlights possible advantages of this technique as compared to the classic vaccines. Special emphasis is placed on the potential of DNA vaccines with respect to tuberculosis.     

Peptides from pigeon cerebrum: two-dimensional fingerprinting and amino acid analysis

The number and heterogeneity of endogenous peptides from pigeon cerebrum were studied by two-dimensional peptide fingerprinting. A highly consistent pattern of 8-9 peptides in the 1000-5000 molecular weight range was obtained from each cerebral sample. Amino acid analysis of the peptide material indicated that 30% of the identified residues were dicarboxylic amino acids and 30% were alphatic amino acids. There was no evidence for the presence of arginine or half-cystine in the peptide fraction.