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PubMed1993

The use of polymorphic Alu insertions in human DNA fingerprinting.

We have characterized several Human Specific (HS) Alu insertions as either dimorphic (TPA25, PV92, APO), slightly dimorphic (C2N4 and C4N4) or monomorphic (C3N1, C4N6, C4N2, C4N5, C4N8) based on studies of Caucasian, Asian, American Black and African Black populations. Our approach is based upon: 1) PCR amplification using primers complementary to the unique DNA sequences that flank the site of insertion of the different Alu elements studied; 2) gel electrophoresis and scoring according to the presence or absence of an Alu insertion in one or both homologous chromosomes; 3) allele frequencies determined by gene counting and compared to Hardy-Weinberg expectations. Our DNA fingerprinting procedure using PCR amplification of diallelic polymorphic (dimorphic) Human Specific Alu insertions, may be used as a tool for genetic mapping, to characterize populations, study human migrational patterns, and track the inheritance of human genetic disorders.

DNA Fingerprinting↗
PubMed1990

An altered constitutive peptide in sym 5 mutants of Pisum sativum L.

Mutational analysis of Pisum sativum L. was used to search for constitutive proteins that might function in nodule formation. The sym 5 locus is a mutational hot spot, represented by seven independently derived mutant lines with decreased nodulation. Comparison of two-dimensional polyacrylamide gels of in vitro-translated root RNA showed a consistent difference in the migrational pattern of one peptide. In the nodulating parental cultivar 'Sparkle', a 66 kDa peptide had a pI of 5.9. In four of the five tested sym 5 mutants, the 66 kDa peptide had a more acidic pI of 5.8. This 66 kDa peptide is found in lateral root, tap root, and shoot. Its expression was independent of rhizobial inoculation, root temperature, or light.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1989

The cell-surface antigens of Bacteroides thetaiotaomicron.

Three strains of B. thetaiotaomicron of different origin were investigated. Lipopolysaccharides were extracted from the studied strains using a phenol-water method. The best purification of LPS was achieved by digestion with nuclease and subsequent ultracentrifugation. Capsular material (CPS) was obtained from the most heavily encapsulated strain. The preparation were analyzed chemically, and their serological activity was determined. All antigens were active with homologous antibacterial sera in immunodiffusion, crossed immunoelectrophoresis, and passive hemagglutination tests. In the CPS equal amounts of saccharides and proteins were detected. All antigens were analyzed by polyacrylamide gel electrophoresis with SDS. Capsular antigen slowly migrated in the gel in the form of a single band. Migration pattern of lipopolysaccharides of the studied B. thetaiotaomicron strain was characteristic for S-type LPS.

Antigens, Bacterial↗
PubMed1996

Identification of picobirnavirus from faeces of Italian children suffering from acute diarrhea.

Polyacrylamide gel electrophoresis of nucleic acid extracted from stool samples of diarrhoeic children revealed in 3 out of 690 (0.43%) specimens two electrophoretic bands with a migration pattern characteristic of picobirnavirus ds-RNA. In none of the 92 control children were similar bands detected. No other potential enteric pathogens were found in the patients with picobirnavirus infection.

Child↗
PubMed1995

Cytokeratins as a marker for epicardial formation in the quail embryo.

Several techniques have been used to visualize the migration pattern of the epicardial cells from the proepicardial organ over the myocardial surface. As the epicardial cells contain keratin tonofilament bundles, we have incubated 92 whole-mount quail hearts with an anti-keratin antibody. This immunohistochemical method showed that the complete epicardial covering of the embryonic heart is preceded by the formation of three epicardial rings. The epicardial rings are formed on the outer myocardial surface in the grooves that separate the cardiac segments from each other. We have also documented timing and patterning of isolated epicardial islands. They are not encountered at random over the myocardial surface, but only along the edge of the advancing epicardial front border and in two defined future epicardial ring areas on the ventral side of the outflow tract. The epicardial islands suggest that in the quail free-floating parts of epicardium can attach to the myocardium. Characteristics of the surface of the myocardium at the transitional zones between the cardiac segments, as well as the three-dimensional remodelling of the heart during cardiac morphogenesis seem to play a role in the pattern in which the epicardium eventually completely ensheaths the myocardial surface. Congenital heart defects are often related to malpositioned transitional zones that dictate the pattern of epicardial outgrowth. As the embryonic position of the epicardial rings is mirrored in the pattern of the main arterial stems, the coronary vascularization pattern might be altered in congenitally malformed hearts as well.

Animals↗
PubMed1991

Fatal hyperammonemia resulting from a C-to-T mutation at a MspI site of the ornithine transcarbamylase gene.

Ornithine transcarbamylase (OTC) deficiency is the most common inborn error of the urea cycle in humans and is responsible for lethal neonatal hyperammonemia in males. Partial OTC deficiency also occurs in females and can be responsible for life-threatening hyperammonemic comas in heterozygotes. The cosegregation of the trait with a 5.8-kb abnormal MspI fragment in an affected family led us to hypothesize that this unexpected migration pattern was related to the mutation event in this particular family. Using polymerase chain reaction amplification of the specific mRNA derived from a post-mortem biopsy of the liver, we found that the MspI site located in the seventh exon of the gene was abolished and we finally identified a C-to-T transition at codon 225 of the cDNA, changing a proline to a leucine in the protein. Subsequent digestion of amplified exon 7 using the restriction enzyme MspI allowed direct screening for the mutant genotype during the next pregnancy. The present study supports the view that direct detection of the mutant genotype using either Southern blotting or digestion of amplified exons of the gene can contribute to genetic counselling in noninformative families. Finally, since MspI digestions are routinely performed for restriction fragment length polymorphism-based family studies in OTC deficiency, we suggest that the possible presence of the 5.8-kb abnormal fragment should be investigated on Southern blots of affected individuals.

Amino Acid Metabolism, Inborn Errors↗
PubMed1994

Factor IX gene mutations causing haemophilia B: comparison of SSC screening versus systematic DNA sequencing and diagnostic applications.

The search for mutations of the factor IX gene responsible for haemophilia B should nowadays be used routinely for the molecular diagnosis of this inherited disorder, i.e. carrier detection and prenatal diagnosis. A number of methodologies have been proposed, most of them being delicate or expensive. We have used a simple strategy based on a preliminary screening of eight factor IX gene fragments by single-strand conformation analysis (SSCA), followed by direct sequencing of fragments displaying an abnormal migration pattern. Carrier testing is then performed by use of an enzyme restriction site altered by the mutation or by the SSCA itself. By using this strategy we were able readily to identify the factor IX molecular defect of nine unrelated haemophilia B patients from southern France. We validated the efficiency and reliability of the SSC-based detection of mutations by sequencing all the polymerase chain reaction (PCR) fragments studied in the haemophilic patients. No other sequence alteration could be found except the one detected by SSC analysis. We conclude that this method can be advantageously used for diagnosis purposes in a routine laboratory involved in haemophilia B diagnosis and report nine previously undescribed haemophilia B families with their factor IX mutation.

DNA Mutational Analysis↗
PubMed1994

Population screening of lactate dehydrogenase deficiencies in Fukuoka Prefecture in Japan and molecular characterization of three independent mutations in the lactate dehydrogenase-B(H) gene.

Screening for lactate dehydrogenase (LDH) subunit deficiencies was performed on 2880 blood samples from healthy individuals in the Fukuoka Prefecture in Japan by means of electrophoresis. The frequencies of heterozygotes with either LDH-A or LDH-B deficiency were found to be 0.104% at each locus. These estimated frequencies of either LDH-A or LDH-B deficiencies were slightly lower than, but not significantly different from, those found previously in Shizuoka Prefecture. The genetic mutations in individuals heterozygous for LDH-B deficiency were analyzed by the polymerase chain reaction and DNA conformation polymorphism. Abnormal migration patterns were observed in individuals heterozygous for LDH-B deficiency. Subsequent sequence determination of the mutant alleles revealed three novel mutations: an eight-base duplication in exon 3, a four-base duplication in exon 4, and a one-base deletion in exon 7 of the LDH-B gene. These three mutations result in frame-shift translation and premature termination. In addition, the mutations resulting in the duplication of eight or four nucleotides appear to cause a decrease in the levels of LDH-B mRNA.

Amino Acid Sequence↗
PubMed1985

Genetic control of immune response to staphylococcal nuclease. XII: Analysis of nuclease antigenic determinants using anti-nuclease monoclonal antibodies.

SJL mice, which are high responders to Staphylococcal nuclease (nuclease), were immunized and used to produce hybridoma cell lines secreting anti-nuclease monoclonal antibodies (mAb). Ten stable clones were derived from a single fusion. Seven of these produced antibodies of the IgG1, kappa isotype and were more precisely characterized for antigenic specificity. Only one hybridoma cell line (54-10-4) produced anti-nuclease antibodies capable of inhibiting enzymatic activity of nuclease. Binding inhibition analyses strongly suggest that the other monoclonal antibodies, which failed to inhibit nuclease activity detect two different antigenic regions, or epitopes, of the molecule: epitope cluster 1 domain is defined by hybridomas 54-2-7, 54-5-2, 54-9-8, and 54-10-8; epitope cluster 2 by 54-5-1 and 54-1-9. Because of its capacity to inhibit nuclease enzymatic activity mAb 54-10-4 was considered specific for a third epitope of the nuclease molecule called epitope 3. Binding studies of these monoclonal antibodies were extended to peptide fragments of the nuclease molecule in order to examine possible cross-reactions with such fragments, as has previously been reported for antibodies purified from polyclonal antisera. Monoclonal antibodies specific for epitope cluster 1 on the native molecule also bound to the fragments 1-126 and 49-149 but failed to bind to fragment 99-149, suggesting that the corresponding epitope(s) is determined by amino acids localized between residues 49 and 99. The epitope clusters 2 and 3 appeared to be expressed only on the native molecule. Monoclonal antibodies of different clusters exhibited very different migration patterns on isoelectric focusing while monoclonal antibodies of the same cluster were indistinguishable, which suggests that they may have originated from the same B cell precursor. Taken together these data suggest that this panel of monoclonal antibodies detects at least three distinct epitopes of the nuclease molecule, one of which could be involved in the determination of the enzymatic site.

Animals↗
PubMed1993

Detection and characterization of new genetic mutations in individuals heterozygous for lactate dehydrogenase-B(H) deficiency using DNA conformation polymorphism analysis and silver staining.

Human lactate dehydrogenase (LDH)--B(H) mutant genes were analyzed by polymerase chain reaction (PCR) and DNA conformation polymorphism. We used polyacrylamide gradient gel and silver staining procedures for DCP analysis, and observed abnormal migration patterns in individuals heterozygous for the LDH-B deficiency. Subsequent sequence determination of the mutant alleles consistently resulted in detection of three single base substitutions (transversions), viz., a C to A at residue "35" (GCG, Ala-->GAG, Glu), a T to G at residue "172" (TTT, Phe-->GTT, Val), and an A to T at residue "176" (ATG, Met-->TTG, Leu). Furthermore, mismatched PCR or amplification refractory mutation system was developed for the rapid screening and confirmation of these mutations. These amino acid replacements may cause conformational changes in neighboring residues; this probably affects the active site arrangement and results in the loss of enzyme activity.

Amino Acid Sequence↗
PubMed1987

Statement on the nomenclature of dog C4 allotypes.

As a collaborative work of three laboratories the polymorphism of the canine fourth complement component (C4) was studied in a total of 131 unrelated dogs from different breeds and mongrels. Using high voltage electrophoresis followed by an immunoblotting technique, we detected eight distinct variants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of canine C4 showed an additional heterogeneity of the alpha and gamma chains which resulted in a total of 11 variants in the population studied. So that more precise information concerning the respective C4 allotypes will be available, a nomenclature is proposed designating not only the migration pattern of the C4 variants in agarose gels but also the heterogeneity of the C4 chains observed in SDS-PAGE.

Alleles↗
PubMed1996

Identification of single and dual infections with distinct subtypes of human immunodeficiency virus type 1 by using restriction fragment length polymorphism analysis.

The simultaneous presence of multiple HIV-1 subtypes has become common in communities with the growth of the pandemic. As a consequence, the potentiality for an increased frequency of HIV-1 mixed infections caused by viruses of distinct subtypes could be expected. Thus, there is a need to estimate the prevalence and geographic distribution of infections caused by viruses of a singular subtype as well as coinfections caused by two or more HIV-1 strains of distinct subtypes. To address this need, we have developed a genetic method based on restriction fragment length polymorphism (RFLP) to screen for these two types of infections within infected populations. In this assay, restriction enzymes may be used to predict the phylogroup of HIV-1 infected samples. A 297 bp pol fragment spanning the entire viral protease gene and a 311 bp fragment of the p24 gag region are used for this analysis. The viral regions are amplified by nested PCR using DNA templates from uncultured peripheral blood mononuclear cells (PBMC) or virus culture. Classification of HIV-1 strains to well defined subtypes B, D, F, and A/C is done by sequential endonuclease restriction analysis of a PCR amplified-protease gene followed by analysis of the p24 gag region. The electrophoretic migration patterns visualized by ethidium bromide staining or by radiolabeled probes are then determined on a 10% polyacrylamide gel. In infections caused by viruses of a singular subtype, a single restriction pattern is detected, whereas in multiple infections caused by two or more viral strains of different subtypes, the combination of different digestion patterns are observed in infected individuals. Using this methodology we have screened for genetic variations in HIV-1 proviral DNA from thirty-three Brazilian samples. Our RFLP procedure classified thirty-two samples as single infections caused by viruses of subtypes B (31) and F (1), and one sample as dual infection caused by distinct viral strains. Subsequent sequence and phylogenetic analysis of the viral protease gene in lymphocytes of all these patients confirmed our RFLP findings in single infections, and demonstrated the existence of two distinct HIV-1 strains of subtypes F and D in a patient which lymphocytes showed the simultaneous presence of two different digestion patterns. As up to now, single infections caused by subtype D variants were not identified in Brazil, our data provide the first evidence of subtype D HIV-1 in this country. Because sequencing of HIV proviral DNA is not particularly practical for large-scale molecular epidemiological studies, the protease/gag-based RFLP screening method will be useful to predict the phylogroup of HIV-1, and to identify multiple infections caused by HIV-1 strains of distinct subtypes. We believe that this information is crucial for both evaluation of the HIV-1/AIDS pandemic and intervention strategies.

Adult↗
PubMed1991

Sequence of a rotavirus gene 4 associated with unique biologic properties.

The genome segment 4 of the simian rotavirus variant SA 11-4F was sequenced. This gene is of probable bovine origin, and it contains a few amino acid differences when compared with other SA 11 variants (4 fm and fem) that were isolated independently and that have fast migration patterns of their gene 4 segments. Hypotheses for the role of sequence changes are made relative to the unique properties of the SA 11-4F variant.

Amino Acid Sequence↗
PubMed1989

Detection of a large number of subgroup 1 human rotaviruses with a "long" RNA electropherotype.

The long or short electrophoretic migration patterns of group A human rotaviruses are linked to their subgroup antigenic specificities. Long pattern isolates usually belong to subgroup 2 (SG2) and short pattern to subgroup 1 (SG1). To date detection of only 4 isolates which do not follow this linkage, have been reported. In the present communication we report the detection of unusually large number (39 isolates) of long pattern human isolates with SG1 specificities.

Antibodies, Monoclonal↗
PubMed1978

Cross-linking of Newcastle disease virus (NDV) proteins.

The proxomity and spatial relationships of the structural proteins of Newcastle disease virus (NDV) were studied by chemical cross-linking with a series of imidoesters. When the virions were reacted by the cross-linker with a distance 6.1A or longer between the functional groups and analyzed by polyacrylamide gel electrophoresis, remarkable changes were observed in the migration patterns of the viral proteins. The most striking one was the extensive decrease in the intensity of the M protein band, and although not so strikingly, glycoprotein and nucleocapsid protein bands were reduced significantly. Instead, several protein complexes appeared at and near the top of the gels. The protein complexes formed by a reversible cross-linker, dimethyl-3,3'-dithiobispropionimidate (DTBP), were analyzed by two dimensional electrophoresis; the complexes on the first-dimension cylindrical gels were cleaved by reduction with 2-mercaptoethanol and electrophoresed laterally on the second-dimension slab gels. The results indicated that homodimers of glycoprotein, nucleocapsid protein and M protein were generated under the condition of the most gentle cross-linking employed. At the same time, however, trimer and higher homopolymers of M protein were already detectable. Under the more extensive conditions, the bulk of M protein was cross-linked to form a large protein complex with very high molecular weight. Further, small but significant amounts of glycoprotein and nucleocapsid protein were always detected in this complex. These results suggest that M protein may be present in the virion in close enough proximity to interact with each other and may further have some interactions with glycoprotein and nucleocapsid protein. On the basis of these findings possible roles of M protein in virus assembly were discussed.

Dimethyl Adipimidate↗
PubMed1992

Comparison of herpesviruses isolated from reindeer, goats, and cattle by restriction endonuclease analysis.

A genomic comparison of bovine herpesvirus 1 (BHV-1), caprine herpesvirus (CHV-2) and reindeer herpesvirus (RHV), was performed using 5 restriction endonucleases. Cross neutralization of these three herpesviruses showed that BHV-1 and CHV-2 had a relatively low degree of cross reaction with heterologous viruses. RHV showed a higher degree of such cross reactivity. The restriction endonuclease analyses showed that the migration patterns of the DNA segments were different for the three groups of herpesviruses. The enteric caprine strain could be differentiated from genital strains using BstE II and Hpa I. The genome size of reindeer herpesvirus was estimated to be approximately 86.8 x 10(6) Da (131.8 kbp), and indications of isomerization of this genome were found. It is concluded that reindeer herpesvirus is a distinct species within the family Herpesviridae.

Animals↗
PubMed1989

Effect of duodenal ulcerogens cysteamine, mepirizole, and MPTP on duodenal myoelectric activity in rats.

Increased gastric acid secretion, enhanced acid delivery to the duodenum, and reduced alkaline secretion in the proximal duodenum are relatively well-established pathophysiologic abnormalities in duodenal ulcer. Impaired duodenal motility, however, may also contribute to duodenal ulceration by altering the distribution of acid and alkaline secretions along the upper digestive tract. We tested the hypothesis that the duodenal ulcerogens cysteamine, MPTP, and mepirizole modify duodenal motility in the rat and that motility changes might be a common and early alteration in experimental duodenal ulceration. All three duodenal ulcerogens rapidly produced extensive changes in duodenal myoelectric activity and reduced the frequency of myoelectric slow waves. Cysteamine induced marked hypermotility for at least 6 hr; MPTP rapidly decreased motility and fragmented the myoelectric migrating pattern. Mepirizole induced biphasic changes: an early hypermotility phase of about 30 min was followed by profound hypomotility. These results indicate that marked alterations of duodenal motility are common during experimental duodenal ulceration. In light of the differential effect of the ulcerogens on duodenal motility, it remains to be determined how these changes influence acid neutralization in the proximal duodenum. Nevertheless, our results suggest that all three duodenal ulcerogens, which are different in structure, alter duodenal motility.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMedDate not supplied

T cell clones and their products: experimental clues for the immunoprophylaxis and immunotherapy of intracellular bacterial infections?

In this paper experimental models utilizing murine T cell clones specific for the intracellular bacterium Listeria monocytogenes are described and some matters of possible relevance to the development of novel immunological strategies against intracellular bacterial infections discussed. Improved vaccines against intracellular bacteria should be selected for their expression of a maximum amount of immunoprotective epitopes and their lack of immunosuppressive epitopes. Analysis of these epitopes may be accomplished with T cell clones of known biological activities. Alternatively, active vaccination in the absence of the etiological agent using idiotypic or clonotypic antibodies may be considered. Improved vaccines against intracellular bacteria must also have the capacity to induce strong T cell responses. Intensified efforts should therefore be undertaken to overcome the current lack of medically acceptable adjuvants for the stimulation of cellular immunity. Once adjuvants as well as defined antigens have become available, the construction of a novel generation of highly effective vaccines should become feasible. In experimental models, T cell clones which confer antibacterial protection have been established. However, these T cell clones expressed low in vivo activity, most probably due to the acquisition of an aberrant migration pattern. Thus, the application of T cell clones for adoptive vaccination against intracellular bacteria in clinical medicine appears to be premature. The observation that lymphokines could protect mice against listeriosis indicates that immunotherapy of intracellular bacterial infections with lymphokines may be a realistic goal in the near future.

Antibodies, Bacterial↗