Multiplex ligation-dependent probe amplification using a completely synthetic probe set

The recent development of multiplex ligation-dependent probe amplification (MLPA) has provided an efficient and reliable assay for dosage screening of multiple loci in a single reaction. However, a drawback to this method is the time-consuming process of generating a probe set by cloning in single-stranded bacteriophage vectors. We have developed a synthetic probe set to screen for deletions in a region spanning 18.5 Mb within chromosome 3q. In a pilot study, we tested 15 synthetic probes on 4 control samples and on 2 patients previously found to possess a heterozygous deletion in the region 3q26-q28. These synthetic probes detected deletions at all previously known deleted loci. Furthermore, using synthetic probes, the variability of results within samples was similar to that reported for commercially available M13-derived probes. Our results demonstrate that this novel approach to MLPA provides a generic solution to the difficulties of probe development by cloning; such synthetically generated probes may be used to screen a large number of loci in a single reaction. We conclude that the use of synthetic probes for MLPA is a rapid, robust, and efficient alternative for research (and potentially diagnostic) deletion and duplication screening of multiple genomic loci.     

Lung Function in Patients Receiving Busulphan

An attempt was made to achieve earlier detection of busulphan lung (fibrosing alveolitis) and to determine its incidence by means of serial studies during life, including measurement of the gas transfer factor. Twenty-three patients were investigated over an average period of nearly two years of busulphan treatment. One case of busulphan lung was detected and subsequently confirmed at necropsy, but in the remainder there was no clinical, radiological, or physiological evidence of fibrosing alveolitis. It is concluded that the development of fibrosing alveolitis may be related to individual genetic or immunological factors rather than to busulphan dosage.     

Developmental and environmental factors that enhance binding of Bordetella pertussis to human epithelial cells in relation to sudden infant death syndrome (SIDS)

Abstract Asymptomatic infection due to Bordetella pertussis has been suggested to be one cause of sudden infant death syndrome (SIDS). We examined developmental and environmental factors previously found to affect binding of another toxigenic species, Staphylococcus aureus , to human epithelial cells: expression of the Lewis^a antigen; infection with respiratory syncytial virus (RSV); exposure to cigarette smoke; and the inhibitory effect of breast milk on bacterial binding. Binding of two strains of B. pertussis (8002 and 250825) to buccal epithelial cells was significantly reduced by treating the cells with monoclonal antibodies to Lewis^a ( P < 0.05) and Lewis^x ( P < 0.01) antigens. Both strains bound in significantly greater numbers to cells from smokers compared with cells from non-smokers ( P < 0.05). HEp-2 cells infected with RSV subtypes A or B had higher binding indices for both 8002 ( P < 0.001) and 250825 ( P < 0.01). On RSV-infected cells, there was significantly enhanced binding of monoclonal antibodies to Lewis^x ( P < 0.05), CD14 ( P < 0.001) and CD18 ( P < 0.01); and pre-treatment of cells with anti-CD14 or CD18 also significantly reduced binding of both strains of B. pertussis . Pre-treatment of the bacteria with human milk significantly reduced their binding to epithelial cells. The results are discussed in relation to our three-year survey of bacterial carriage among 253 healthy infants, their mothers and local SIDS cases between 1993–1995 and in relation to the change to an earlier immunisation schedule for infants and the recent decline in SIDS in Britain.     

The murine tub (rd5) mutation is not associated with a primary axonemal defect

Some genetic syndromes causing loss of hearing and vision, such as some forms of Usher’s syndrome, also cause reduced sperm cell motility, bronchiectasis, and other pathologies involving cilia- and flagella-bearing cells. In some Usher’s patients, ultrastructural defects of axonemes within photoreceptor ciliary bridges, nasal cilia, and sperm cell flagella have been found, indicating a primary defect of axonemal conformation. Mice homozygous for the tub (rd5) mutation exhibit progressive retinal degeneration, sensorineural hearing loss, reduced fertility, and obesity, and presently represent the only animal model with neuroepithelial degeneration of both cochlea and retina without other neurological abnormalities. They provide a good phenotypic match to human genetic sensory syndromes, particularly human sensory/obesity syndromes, such as Alstrom’s and Bardet/Biedl, although no human candidate genes have been identified. Because of their unique phenotype, tubby mice are an appropriate model in which to look for a primary axonemal defect. We studied the axonemal ultrastructure of photoreceptors and sperm cells and performed functional testing of sperm in tub/tub mice before and after the onset of obesity. Approximately 15% of photoreceptor axonemes appeared abnormal in tub/tub animals, compared to 0% in controls. Both tub homozygotes and controls exhibited approximately 10% abnormal sperm cell axonemes, and no differences in sperm cell motile function were found at any age. The modest occurrence of axonemal defects in photoreceptors of tub/tub animals is likely to be a secondary effect of retinal degeneration. We conclude that the tubby phenotype is not associated with a generalized defect of cilia- and flagella-bearing cells and that the tub mutation does not primarily affect axonemal structure.     

Lumbar anomalies in the Shanidar 3 Neandertal

Recent examination of the Shanidar 3 remains revealed the presence of anomalous bilateral arthroses in the lumbar region. This paper describes this developmental anomaly, as well as several degenerative changes and offers potential etiologies.The Shanidar 3 remains represent an adult male Neandertal, approximately 35–50 years of age, dating to the Last Glacial. Although the partial skeleton is fragmentary, preserved elements include an almost complete set of ribs, portions of all thoracic vertebrae, all lumbar vertebrae, and the sacrum. Vertebral articulations from S1–T1 can be confidently assigned. The vertebra designated L1 is well preserved but lacks transverse processes. Instead, well defined bilateral articular surfaces, rather than transverse processes, are located on the pedicles. The skeletal elements associated with the anomalous L1 articulations were not recovered.The most likely interpretation is that the arthroses in question represent the facets for a 13th pair of ribs, a rare condition in modern hominid populations. Such lumbar developmental anomalies are an infrequent expression of a larger complex of cranial-caudal border shifting seen in the vertebral column. These shifts result in a change in the usual boundaries between the distinctive vertebral regions and are responsible for the majority of variability present in the vertebral column.     

Behavioural thermoregulation and cold adaptation in two strains of white mice

Survival and degree of cold adaptation were determined for Swiss Webster and Connaught strain white mice exposed to 2°C and caged as follows: (I) a single animal in an empty plastic cage; (II) a single animal in a plastic cage containing bedding material; (III) 2 animals in an empty wire-mesh cage, and (IV) 3 animals in a plastic cage containing bedding material. After 10 days of exposure under these conditions, survival was 54, 62, 87 and 94% in Swiss Webster, and 87, 100, 92 and 92% in Connaught mice. When the degree of cold adaptation was estimated on the basis of the average decrease in core temperature during a 45-minute exposure to -10°C, values of 4.4 (I), 5.1 (II), 4.2 (III), 6.8 (IV) and 11.0°C (control) were determined for Swiss Webster mice, and 1.7 (I, II and III), 3.2 (IV) and 9.0°C (control) for Connaught mice. The study revealed that there are significant intrastrain differences in the ability of small mammals to both tolerate and adapt to a cold stress.

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Zusammenfassung Die Überlebensrate und das Ausmass der Kälte-adaptation von Mäusen des Swiss-Webster und Connaught Stammes wurden während 2°C-Kälteexponierung bei folgender Haltung bestimmt: (1) einzeln in leeren Kunststoffkäfigen; (2) einzeln in Kunststoffkäfigen mit Einstreu; (3) 2 Tiere in Drahtkäfig und (4) 3 Tiere in Kunststoffkäfig mit Einstreu. Nach 10 Tagen überlebten unter diesen Bedingungen von den Swiss-Webster Mäusen 54, 62, 87 und 94% und von den Connaught-Mäusen 87, 100, 92 und 92%. Die Abschätzung der Kälteadaptation aus dem mittleren Abfall der Kerntemperatur nach 45 Min bei -10°C ergab bei den Swiss-Webster-Mäusen 4,4; 5,1; 4,2 und 6,5°C und 11, 0°C bei den Kontrollen während bei den Connaught-Mäusen die Senkung nur 1,7°C (1, 2, 3), 3,2° (4) und 9,0°C bei den Kontrollen betrug. Danach bestehen signifikante Unterschiede in der Kältetoleranz und der Anpassungsfähigkeit zwischen den beiden Mäusestämmen.

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Resume On a examiné le taux de survie et d'adaptation au froid de souris blanches des races Swiss-Webster en Connaught placées à 2°C dans les conditions suivantes: (1) bêtes isolées dans des cages de plastique vides; (2) bêtes isolées dans des cages de plastique pourvues d'une litière; (3) ensemble de deux bêtes dans des cages grillagées vides et enfin (4) ensemble de trois bêtes dans des cages de plastique pourvues d'une litiére. Après 10 jours, le taux de survie fut de 54, 62, 87 et 94% chez les souris Swiss-Webster et de 87, 100, 92 et 92% chez les souris Connaught. Le degré d'adaptation au froid a été estimé par l'abaissement de la température centrale après exposition de 45 minutes à -10°C. Les résultats furent: Swiss-Webster 4,4, 5,1, 4,2 et 6,5°C (11,0°C pour des bêtes de contrôle); Connaught 1,7, 1,7, 1,7 et 3,2°C (9,0°C pour le contrôle). Il en résulte que la tolérance au froid et les facultés d'adaptation sont différentes pour ces deux races et cela de façon significative.

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This paper is based on research conducted at Defence Research Establishment Toronto, Downsview, Ontario (DRET Research Paper No. 680).     

Conformational properties of branched RNA fragments in aqueous solution

The conformational properties of branched trinucleoside diphosphates ACC, ACG, AGC, AGG, AUU, AGU, AUG, ATT, GUU, and aAUU [XYZ = X(2'p5'Y)3'p5'Z] have been studied in aqueous solution by nuclear magnetic resonance (1H, 13C), ultraviolet absorption, and circular dichroism. It is concluded from these studies that the purine ring of the central residue (X; e.g., adenosine) forms a base-base stack exclusively with the purine or pyrimidine ring of the 2'-nucleotidyl unit (Y; 2'-residue). The residue attached to the central nucleoside via the 3'-5'-linkage (Z; 3'-residue) is "free" from the influence of the other two heterocyclic rings. The ribose rings of the central nucleoside and the 2'- and 3'-residues exist as equilibrium mixtures of C2'-endo (2E)-C3'-endo (3E) conformers. The furanose ring of the central nucleoside (e.g., A) when linked to a pyrimidine nucleoside via the 2'-5'-linkage shows a higher preference for the 2E pucker conformation (e.g., AUG, AUU, ACG, ca. 80%) than those linked to a guanosine nucleoside through the same type of bond (AGU, AGG, AGC, ca. 70%). This indicates some correlation between nucleotide sequence and ribose conformational equilibrium. The 2E-3E equilibrium of 2'-pyrimidines (Y) shows significant, sometimes exclusive, preference (70-100%) for the 3E conformation; 3'-pyrimidines and 2'-guanosines have nearly equal 2E and 3E rotamer populations; and the ribose conformational equilibrium of 3'-guanosines shows a preference (60-65%) for the 2E pucker. Conformational properties were quantitatively evaluated for most of the bonds (C4'-C5', C5'-O5', C2'-O2', and C3'-O3') in the branched "trinucleotides" AUU and AGG by analysis of 1H-1H, 1H-31P, and 13C-31P coupling constants. The C4'-C5' bond of the adenosine units shows a significant preference for the gamma + conformation. The dominant conformation about C4'-C5' and C5'-O5' for the 2'-and 3'-nucleotidyl units is gamma + and beta t, respectively, with larger gamma + and beta t rotamer populations for the 2'-unit. The increased conformational purity in the 2'-residue, compared to the 3'-residue, is ascribed to the presence of an ordered (adenine----2'-residue) stacked state. The favored rotamers about C3'-O3' and C2'-O2' are epsilon- and epsilon'-, respectively. The conformational features of AUU and AGG were compared to those of their constitutive dimers A3'p5'G, A2'p5'G, A3'p5'U, and A2'p5'U and monomers 5'pG and 5'pU.