Non-invasive early prenatal molecular diagnosis using retrieved transcervical trophoblast cells

Fetal DNA was recovered from 17 of 39 (44%) transcervical cell (TCC) samples obtained between 7 and 9 weeks of gestation by endocervical canal flushing. Trophoblast retrieval was adequate for polymerase chain reaction (PCR) amplification of Y chromosome-specific DNA sequences and detection of paternal-specific microsatellite alleles. The fetal sex predicted by PCR in TCCs was confirmed in all cases by karyotype analysis of chorionic villi at 10 weeks of gestation. The absence of the disease-associated paternal alleles in TCC samples from two pregnancies at risk for spinal muscular atrophy and myotonic dystrophy predicted unaffected fetuses in agreement with subsequent results on chorionic villi and newborns' leukocytes. A trisomy 21 fetus was diagnosed in TCCs using fluorescent in situ hybridization (FISH) and semiquantitative PCR analysis of superoxide dismutase-I (SOD 1). Present experience indicates that TCC sampling is a promising technique for early prenatal monitoring of Mendelian disorders and chromosome aneuploidy.     

Scorbutic and fasted guinea pig sera contain an insulin-like growth factor I-reversible inhibitor of proteoglycan and collagen synthesis in chick embryo chondrocytes and adult human skin fibroblasts

Chick embryo chondrocytes cultured in sera from scorbutic and fasted guinea pigs exhibited decreases in collagen and proteoglycan production to about 30-50% of control values (I. Oyamada et al., 1988, Biochem. Biophys. Res. Commun. 152, 1490-1496). Here we show by pulse-chase labeling experiments that in the chondrocyte system, as in the cartilage of scorbutic and fasted guinea pigs, decreased incorporation of precursor into collagen was due to decreased synthesis rather than to increased degradation. There was a concomitant decrease in type II procollagen mRNA to about 32% of the control level. As in scorbutic cartilage, proteoglycan synthesis by chondrocytes in scorbutic serum was blocked at the stage of glycosaminoglycan chain initiation. Scorbutic and fasted guinea pig sera also caused a 50-60% decrease in the rates of collagen and proteoglycan synthesis in adult human skin fibroblasts, which synthesize mainly type I collagen. Decreased matrix synthesis in both cell types resulted from the presence of an inhibitor in scorbutic and fasted sera. Elevated cortisol levels in these sera were not responsible for inhibition, as determined by the addition of dexamethasone to chondrocytes cultured in normal serum. Insulin-like growth factor I (IGF-I, 300-350 ng/ml) reversed the inhibition of extracellular matrix synthesis by scorbutic and fasted guinea pig sera in both cell types and prevented the decrease in type II procollagen mRNA in chondrocytes. Therefore, in addition to its established role in proteoglycan metabolism, IGF-I also regulates the synthesis of several collagen types. An increase in the circulating inhibitor of IGF-I action thus could lead to the negative regulation of collagen and cartilage proteoglycan synthesis that occurs in ascorbate-deficient and fasted guinea pigs.     

Structural analysis of human neutrophil migration: Centriole, microtubule, and microfilament orientation and function during chemotaxis

Orientation of nucleus, centriole, microtubules, and microfilaments within human neutrophils in a gradient of chemoattractant (5 percent Escherichia coli endotoxin-activated serum) was evaluated by electron microscopy. Purified neutropils (hypaque-Ficoll) were placed in the upper compartment of chemotactic chambers. Use of small pore (0.45 μm) micropore filters permitted pseudopod penetration, but impeded migration. Under conditions of chemotaxis with activated serum beneath the filter, the neutrophil population oriented at the filter surface with nuclei located away from the stimulus, centrioles and associated radial array of microtubules beneath the nuclei, and microfilament-rich pseudopods penetrating the filter pores. Reversal of the direction of the gradient of the stimulus (activated serum above cells) resulted in a reorientation of internal structure which preceded pseudopod formation toward the activated serum and migration off the filter. Coordinated orientation of the entire neutrophil population did not occur in buffer (random migration) or in a uniform concentration of activated serum (activated random migration). Conditions of activated random migration resulted in increased numbers of cells with locomotory morphology, i.e. cellular asymmetry with linear alignment of nucleus, centriole, microtubule array, and pseudopods. Thus, activated serum increased the number of neutrophils exhibiting locomotory morphology, and a gradient of activated serum induced the alignment of neutrophils such that this locomotory morphology was uniform in the observed neutrophil populayion. In related studies, cytochalasin B and colchicines were used to explore the role of microfilaments and microtubules in the neutrophil orientation and migration response to activated serum. Cytochalasin B (3.0 μg/ml) prevented migration and decreased the microfilaments seen, but allowed normal orientation of neutrophil structures. In an activated serum gradient, colchicines, but not lumicolchicine, decreased the orientation of nuclei and centrioles, and caused a decrease in centriole-associated microtubules in concentrations as low as 10(-8) to 10(-7) M. These colchicines effects were associated with the rounding of cells and impairment of pseudopod formation. The impaired pseudopod formation was characterized by an inability to form pseudopods in the absence of a solid substrate, a formation of narrow pseudopods within a substrate, and a defect in pseudopod orientation in an activated serum gradient. Functional studies of migration showed that colchicines, but not lumicolchicine, minimally decreased activated random migration and markedly inhibited directed migration, but had not effect on random migration. These studies show that, although functioning microfilaments are probably necessary for neutrophil migration, intact microtubules are essential for normal pseudopod formation and orientation, and maximal unidirectional migration during chemotaxis.     

The human T-cell leukemia virus type-1 Tax protein regulates the activity of the IkappaB kinase complex

Two cytokine-inducible kinases, IKKalpha and IKKbeta, are components of a 700-kDa kinase complex that specifically phosphorylates IkappaB. Phosphorylation of IkappaB by IKK leads to its ubiquitination and subsequent degradation, resulting in the nuclear translocation of NF-kappaB. The oncogenic protein Tax, encoded by human T-cell leukemia virus type-1 (HTLV-1), stimulates IKK activity to result in constitutive nuclear levels of NF-kappaB. In an attempt to gain insights into the mechanism by which Tax mediates constitutive activation of the NF-kappaB pathway, we analyzed the chromatographic distribution of IKK proteins using cellular extracts prepared from three T lymphocytes either lacking or containing Tax. IKK kinase activity and the distribution of proteins in the IKK complex were characterized. In extracts prepared from cells containing Tax, the activity of both IKKalpha and IKKbeta present in the 700-kDa IKK complex were increased. Surprisingly, cell lines expressing Tax also contained an additional peak of IKKbeta, but not IKKalpha activity, that migrated at 300 kDa rather than at 700 kDa. We noted that extracts containing Tax had extremely low levels of IkappaBbeta, but not IkappaBalpha, and contained predominantly a truncated form of the MAP3K MEKK1. These results suggest that Tax may target several components of the NF-kappaB pathway leading to constitutive activation of this important regulator of cellular gene expression.     

Projection of sensory neurons from a homeotic mutant appendage,Antennapedia, in Drosophila melanogaster

Central projections of sensory neurons from homeotic mutant appendages (Antennapedia) of Drosophila melanogaster were compared with those of wild-type antennae and wild-type legs by means of degeneration and cobalt backfilling methods. Sensory axons originating from wild-type thoracic legs terminate within the ventral ipsilateral half of the corresponding neuropile segment and do not project to the brain. Sensory fibers from the third antennal segment (AIII) of wild-type animals project into the ipsilateral antennal glomerulus (AG) and to a lesser extent into the contralateral AG, whereas those from the second antennal segment terminate principally within the ipsilateral posterior antennal center. The sensory terminals of femur, tibia, and tarsi of the homeotic leg show a distribution very similar to that of the homologous wild-type antennal segment AIII, differing to a minor degree only in the size and precise localization of terminals within the antennal glomeruli. No degenerating axons were evident in ultrastructural examination of neck connectives after removal of homeotic legs. It is thus very improbable that any sensory fibers of the homeotic leg project to normal leg projection areas in the thoracico-abdominal ganglion. Several alternative explanations are offered for the apparent retention of antennal specificity by axons from the transformed appendage.     

Adaptive line transect sampling

Adaptive line transect sampling offers the potential of improved population density estimation efficiency over conventional line transect sampling when populations are spatially clustered. In adaptive sampling, survey effort is increased when areas of high animal density are located, thereby increasing the number of observations. Its disadvantage is that the survey effort required is not known in advance. We develop an adaptive line transect methodology that, by varying the degree of adaptation, allows total effort to be fixed at the design stage. Relative to conventional line transect surveys, it also provides better survey coverage in the event of disruption in survey effort, e.g., due to poor weather. In analysis, sightings from the adaptive sections are downweighted in proportion to the increase in effort. We evaluate the methodology by simulation and report on surveys of harbor porpoise in the Gulf of Maine, in which the approach was compared with conventional line transect sampling.