Prenatal diagnosis of the fragile X syndrome: loss of mutation owing to a double recombinant or gene conversion event at the FMR1 locus.

The fragile X syndrome, an X linked mental retardation syndrome, is caused by an expanded CGG repeat in the first exon of the FMR1 gene. In patients with an expanded repeat the FMR1 promoter is methylated and, consequently, the gene is silenced and no FMR1 protein (FMRP) is produced, thus leading to the clinical phenotype. Here we describe a prenatal diagnosis performed in a female from a fragile X family carrying a large premutation. In chorionic villus DNA of the male fetus the normal maternal CGG allele and a normal pattern on Southern blot analysis were found in combination with the FRAXAC2 and DXS297 allele of the maternal at risk haplotype. A second chorionic villus sampling was performed giving identical results on DNA analysis and, in addition, expression of FMRP was shown by immunohistochemistry. We concluded that the male fetus was not affected with the fragile X syndrome. Subsequent detailed haplotype analysis showed a complex recombination pattern resembling either gene conversion or a double crossover within a 20 kb genomic region.     

Effect of reduced temperature on glycoprotein (Ig, HLA) processing and transport in lymphoid cells

Secretion of Igs and surface expression of HLA antigens was examined in lymphoid cells as a function of temp. Upon reducing the temp from 37 to 20 degrees C a progressive decrease in the secretion of Ig and surface expression of HLA antigens was noted. When the status of the oligosaccharides present on these glycoproteins was examined, conversion of high-mannose [endo-beta-N-acetylglucosaminidase-(Endo H) sensitive] to complex-type (Endo H resistant) oligosaccharides diminished with decreasing temp. At no time was an accumulation of Endo H resistant glycoproteins seen intracellularly. These results show that the phenomenon observed for synthesis and intracellular transport of viral glycoproteins in epithelial cells at reduced temp, namely intracellular accumulation of viral glycoproteins carrying complex sugar moieties, does not necessarily apply to glycoprotein transport in lymphoid cells. A difference in subcellular organization of epithelial and lymphoid cells may be responsible for this discrepancy.     

Quantitative flow cytometric analysis of opsonophagocytosis and killing of nonencapsulated Haemophilus influenzae by human polymorphonuclear leukocytes.

Since nonencapsulated Haemophilus influenzae persists in the lower respiratory tracts of patients with chronic bronchitis despite the presence of specific antibodies, complement, and polymorphonuclear leukocytes (PMNs), opsonophagocytosis of H. influenzae was analyzed. Nonencapsulated H. influenzae isolated from the sputa of chronic bronchitis patients was labeled with fluorescein isothiocyanate and incubated with human PMNs in the presence of complement and antibodies for 30 min at 37 degrees C. Candida albicans was added to each sample as an internal standard, and the reduction of the number of bacteria was determined by flow cytometry. Fluorescence quenching with ethidium bromide was used to discriminate between intracellular and extracellular bacteria. Opsonophagocytosis of viable H. influenzae d1 was 17% +/- 29% in the presence of complement and human pooled sera containing high titers of strain-specific antibodies. Opsonophagocytosis of six other H. influenzae strains was also poor. Under the same conditions, opsonophagocytosis of Staphylococcus aureus was 90% +/- 5%, and opsonophagocytosis of C. albicans was 55% +/- 23%. About half of the number of H. influenzae bacteria associated with PMNs was internalized. Opsonophagocytosis of heat-killed H. influenzae d1 (41% +/- 20%) was higher than that of viable bacteria of the same strain (P < 0.05). This result suggests that the accessibility of epitopes on H. influenzae for opsonizing antibodies is better on killed than on viable bacteria. We conclude that viable nonencapsulated H. influenzae is poorly opsonophagocytized in the presence of strain-specific antibodies and complement.     

Flow Perfusion Culture of Marrow Stromal Cells Seeded on Porous Biphasic Calcium Phosphate Ceramics

Calcium phosphate ceramics have been widely used for filling bone defects to aid in the regeneration of new bone tissue. Addition of osteogenic cells to porous ceramic scaffolds may accelerate the bone repair process. This study demonstrates the feasibility of culturing marrow stromal cells (MSCs) on porous biphasic calcium phosphate ceramic scaffolds in a flow perfusion bioreactor. The flow of medium through the scaffold porosity benefits cell differentiation by enhancing nutrient transport to the scaffold interior and by providing mechanical stimulation to cells in the form of fluid shear. Primary rat MSCs were seeded onto porous ceramic (60% hydroxyapatite, 40% β-tricalcium phosphate) scaffolds, cultured for up to 16 days in static or flow perfusion conditions, and assessed for osteoblastic differentiation. Cells were distributed throughout the entire scaffold by 16 days of flow perfusion culture whereas they were located only along the scaffold perimeter in static culture. At all culture times, flow perfused constructs demonstrated greater osteoblastic differentiation than statically cultured constructs as evidenced by alkaline phosphatase activity, osteopontin secretion into the culture medium, and histological evaluation. These results demonstrate the feasibility and benefit of culturing cell/ceramic constructs in a flow perfusion bioreactor for bone tissue engineering applications.     

Multiplex PCR assay for immediate identification of the infecting species in patients with mycobacterial disease.

Rapid identification of infecting mycobacterial species enables appropriate medical care decisions to be made. Our aim was to demonstrate the clinical usefulness of the multiplex PCR assay, a test based on PCR, which permits direct identification of 12 mycobacterial species in clinical specimens. A total of 259 specimens from 177 patients who had clinical symptoms of mycobacterial disease but for whom there were difficulties in diagnosis were tested. Specimens were analyzed within 48 h of receipt of the sample. Mycobacteria were identified in 102 specimens; 66 specimens contained nontuberculous mycobacteria, and 36 specimens contained Mycobacterium tuberculosis complex mycobacteria. The PCR assay identified the mycobacterial species in 43 (97.7%) of 44 microscopy- and culture-positive specimens and in 15 (93.8%) of 16 culture-positive, microscopy-negative specimens. It also permitted species identification in infections caused by more than one mycobacterial species. For 56 (96.5%) of the 58 specimens from patients with infections caused by opportunistic mycobacteria, the organisms were identified with the PCR assay. The test was useful also for the identification of fastidious mycobacteria, e.g., M. genavense, and those that cannot be cultured, e.g., M. leprae. After resolution of discrepant results, the sensitivity of the PCR assay was 97.9%, the specificity was 96.9%, the positive predictive value was 95.0%, and the negative predictive value was 98.7%. For culture these values were 60.8, 100, 100, and 81.0%, respectively. Thus, the multiplex PCR assay enables prompt diagnosis when rapid identification of infecting mycobacteria is necessary.     

The inferior alveolar artery in its bony course

Based on the dissection of 30 hemi-mandibles, the authors report a study of the inferior alveolar artery in its intraosseous course. On morphologic considerations they propose a classification of the collaterals into two groups: the principal collaterals destined for the teeth and the bony alveolar tissue and the secondary collaterals destined for the sheath and the nerve as well as the bony tissue around the canal. Loss of the teeth and absorption of the alveolar bone modify the caliber of the inferior alveolar arterial axis, the distribution of its collaterals and possibly its mode of termination. These facts suggest a consideration of the vascularization of the mandible in terms of four sectors. They arrive at practical conclusions that may be drawn from this study in stomatology.     

Recombinant 25-kDa CD23 and interleukin 1 alpha promote the survival of germinal center B cells: evidence for bifurcation in the development of centrocytes rescued from apoptosis.

Germinal centers contain a proliferating pool of centroblasts which give rise to non-dividing centrocyte. Centrocytes are programmed to die by apoptosis unless they receive a positive signal for rescue. Rescue, in vivo, is likely to be dependent, initially, on interaction with antigen held on follicular dendritic cells (FDC). A subset of FDC located in that part of the germinal center furthest from centroblasts is particularly rich in CD23. Supernatants containing high levels of soluble CD23 were found not only to encourage the survival of germinal center B cells but also to promote their differentiation toward a plasmacytoid morphology; these activities were diminished following removal of CD23 from the supernatants. Recombinant 25-kDa CD23 was initially found to be incapable of providing the signal for germinal center cell development but on the addition of interleukin 1 alpha which, by itself, was inactive, rescue and differentiation of germinal center B cells were now achieved. Apoptosis in germinal center cells could also be prevented by the ligation of surface CD40 with monoclonal antibody: however, rescue via this pathway was not accompanied by plasmacytoid differentiation. These findings provide a functional rationale to the high level expression of CD23 found within a discrete subset of FDC and indicate a bifurcation in the development of germinal center B cells following their rescue from apoptosis.     

Chloroplast DNA from lettuce and Barnadesia (Asteraceae): structure,gene localization,and characterization of a large inversion

Summary We have cloned into plasmids 17 of 18 lettuce chloroplast DNA SacI fragments covering 96% of the genome. The cloned fragments were used to construct cleavage maps for 10 restriction enzymes for the chloroplast genomes of lettuce (Lactuca sativa) and Barnadesia caryophylla, two distantly related species in the sunflower family (Asteraceae). Both genomes are approximately 151 kb in size and contain a 25 kb inverted repeat. We also mapped the position and orientation of 37 chloroplast DNA genes. The mapping studies reveal that chloroplast DNAs of lettuce and Barnadesia differ by a 22 kb inversion in the large single copy region. Barnadesia has retained the primitive land plant genome arrangement, while the inversion has occurred in a lettuce lineage. The endpoints of the derived lettuce inversion were located by comparison to the well-characterized spinach and tobacco genomes. Both endpoints are located in intergenic spacers within tRNA gene clusters; one cluster being located downstream from the atpA gene and the other upstream from the psbD gene. The endpoint near the atpA gene is very close to one endpoint of a 20 kb inversion in wheat (Howe et al. 1983; Quigley and Weil 1985). Comparison of the restriction site maps gives an estimated sequence divergence of 3.7% for the lettuce and Barnadesia genomes. This value is relatively low compared to previous estimates for other angiosperm groups, suggesting a high degree of sequence conservation in the Asteraceae.     

High expression of MDR1, MRP1, and MRP3 in the hepatic progenitor cell compartment and hepatocytes in severe human liver disease

An increase in bile ductular structures is observed in diverse human liver diseases. These structures harbour the progenitor cell compartment of the liver. Since ATP-binding cassette (ABC) transporters may have a cytoprotective role in liver disease, an immunohistochemical study was performed on human liver specimens from patients with primary biliary cirrhosis (PBC), chronic hepatitis C virus (HCV) infection, submassive cell necrosis, and normal liver. The expression of MDR1, MDR3, BSEP, MRP1, MRP2, and MRP3 was determined using specific antibodies. Dilution series were constructed to determine the critical staining level in order to estimate the factor of up-regulation. In normal liver, hepatocytes showed canalicular staining for MDR3, BSEP, and MRP2. MDR1 stained the canalicular membrane of hepatocytes as well as that of cholangiocytes. MRP3 showed low immunoreactivity of bile duct epithelial cells and centrilobular hepatocytes only. Normal liver showed no immunoreactivity for MRP1. In diseased liver, the expression of MDR3, BSEP, and MRP2 was relatively stable. In PBC, HCV, and submassive necrosis, the expression levels of MDR1, MRP1, and MRP3 were increased. The strongest immunoreactivity was seen after submassive necrosis, where remaining islands of hepatocytes showed strong canalicular staining for MDR1 and MRP3. Regenerating bile ductules at the interface of portal tracts and necrotic areas stained intensely for MDR1, MRP1, and MRP3. In conclusion, MDR1, MRP1, and MRP3 are up-regulated in hepatocytes in severe human liver disease. Strong MDR1, MRP1, and MRP3 reactivity is seen in regenerating human bile ductules.