Ultrastructural and histochemical study of anural development in the ascidian Molgula pacifica (Huntsman)

Summary Tadpole development is eliminated in the life cycle of the ascidian Molgula pacifica. The elimination of a tailed larva is termed anural development, in contrast to urodele development which is exhibited by most ascidian species. In the present study, transmission electron microscopy and histochemistry were used to gain a better understanding of anural development in M. pacifica. The fine structure of M. pacifica oocytes and fertilized eggs was similar to urodele oocytes and eggs, except that a perivitelline space and test cells were absent. M. pacifica embryos exhibited the typical cleavage pattern of urodele embryos. Gastrulation was initiated at the vegetal pole, as in urodeles, and occurred at the same time as in two urodele species (Molgula manhattensis and Pyura haustor). However, changes in cell shapes and cell movements of the vegetal pole cells that participate in gastrulation were highly modified compared to commonly studied ascidians. The changes in shapes and movements of the vegetal pole cells were minimal and resulted in embryos having a very small archenteron and blastopore. The presence of large, yolky cells in the interior of the embryo likely restricted vegetal cell movements. Two ultrastructurally distinct types of epidermal cells were evident at the gastrula stage. When gastrulae were manually dechorionated from their surrounding mucous-follicular envelope layers, the embryos were already surrounded by a thin tunic. When day 1 juveniles in the process of hatching were sectioned along the anterior-posterior axis, regional differences in cell types were evident. Differentiated muscle cells in the posterior region were not evident. Day 1 M. pacifica juveniles, anural-developing M. provisionalis juveniles and tadpoles from three urodele species were tested for their abilities to express AchE activity. The highest levels of AchE activity were detected in the larval tail muscle cells of urodeles, low levels of activity were detected in the posterior region of M. provisionalis juveniles, whereas M. pacifica juveniles did not exhibit AchE activity. The results are discussed in terms of evolutionary mechanisms responsible for anural development in ascidians. Offprint requests to: W.R. Bates     

A yeast artificial chromosome telomere clone spanning a possible location of the Huntington disease gene

The Huntington disease (HD) gene has been mapped to the most distal subband of chromosome 4p. Analysis of recombination events has not provided an unequivocal location of the HD gene, but it indicates a position very close to the telomere as one possibility. We have constructed a yeast artificial chromosome (YAC) vector (containing a rare-cutter polylinker) for the cloning of mammalian telomeres, used it to prepare a BssHII-telomere library with DNA from an individual homozygous for HD, and have identified a 115-kb clone containing the telomere of 4p. One probable recombinant would confine the telomeric candidate location for the gene to the region covered by the YAC, which makes it possible that the clone described here contains the HD locus in its mutant form.     

Nonlinear mixed effects models for repeated measures data

We propose a general, nonlinear mixed effects model for repeated measures data and define estimators for its parameters. The proposed estimators are a natural combination of least squares estimators for nonlinear fixed effects models and maximum likelihood (or restricted maximum likelihood) estimators for linear mixed effects models. We implement Newton-Raphson estimation using previously developed computational methods for nonlinear fixed effects models and for linear mixed effects models. Two examples are presented and the connections between this work and recent work on generalized linear mixed effects models are discussed.     

Chromosomal analysis of Nicotiana asymmetric somatic hybrids by dot blotting and in situ hybridization

Summary A species-specific, dispersed repetitive DNA sequence was cloned from Nicotiana plumbaginifolia and used in dot blots and in situ hybridizations to analyze asymmetric somatic hybrids of N. tabacum(+)kanamycin-resistant N. plumbaginifolia. Dot blot hybridization data, using the cloned, species-specific repetitive DNA as a probe, showed that some of the hybrids contain only 1%–5% N. plumbaginifolia DNA, whereas others contain 15%–25%. In situ hybridization of the probe to chromosome spreads showed that the extremely asymmetric hybrids retain a single N. plumbaginifolia chromosome; the hybrids with higher dot blot values were found to have 8 to 12 N. plumbaginifolia chromosomes and chromosome fragments. In situ hybridization also revealed translocations between N. plumbaginifolia and N. tabacum chromosomes in 3 of 8 hybrids studied. RFLP analysis using a 5S gene probe showed the presence of N. plumbaginifolia-specific 5S banding patterns in most hybrids examined, including those that retain only a single N. plumbaginifolia chromosome.     

Transforming growth factor-beta induces platelet-derived growth factor (PDGF) messenger RNA and PDGF secretion while inhibiting growth in normal human mammary epithelial cells

Platelet-derived growth factor (PDGF) is a potent mitogen in human serum which specifically stimulates the proliferation of mesenchymal cells. We have now examined normal human mammary epithelial cells (HMEC) derived from reduction mammaplasties and grown in a serum-free defined medium. Medium conditioned by HMEC contained a PDGF-like activity that competed with [125I]PDGF for binding to PDGF receptors in normal human fibroblasts. When conditioned media were incubated with antiserum specific for either PDGF-A or PDGF-B, only PDGF-A antiserum was capable of inhibiting binding of conditioned media to PDGF receptors. Using an RNase protection assay, mRNA from normal HMEC was probed for both the PDGF-A and PDGF-B chains. Little or no PDGF-B was found in HMEC strains, while a strong signal was seen with the PDGF-A probe. When HMEC were grown in the presence of transforming growth factor-beta (TGF beta) for 48 h, inhibition of growth was observed in association with a 20- to 40-fold stimulation of PDGF-B mRNA and a 2-fold stimulation of PDGF-A mRNA. This mRNA induction was extremely rapid (within 1 h), and secreted PDGF activity was induced 2- to 3-fold. Two other HMEC growth inhibitors and differentiating agents, sodium butyrate and phorbol ester 12-O-tetradecanoylphorbol-13-acetate, had no effect on PDGF mRNA regulation. The current study suggests that PDGF gene induction is an extremely rapid and specific indicator of TGF beta function regardless of whether TGF beta is acting in a growth stimulatory or inhibitory manner. Any role of PDGF-B in TGF beta modulation of differentiation of normal or malignant mammary gland remains to be determined.     

Use of synthetic oligoribonucleotides to probe RNA-protein interactions in the MS2 translational operator complex.

Synthetic oligoribonucleotides have been used to probe the interaction of MS2 coat protein with the translational operator of the MS2 replicase gene. We have investigated the possible formation of a transient covalent bond between the single-stranded uridine residue, at position -5, and a cysteine side-chain on the coat protein, by the incorporation of a chemically modified residue (5-BrU) at this position. This chemically synthesised operator variant has a binding constant of between 10 and 50 times greater than that of the wild type and is therefore comparable with the tight binding variant having a cytidine substituted at the -5 position. Dissociation kinetics show that the complex with the 5-BrU operator is more stable than the -5C variant; a result which is consistent with the formation of a Michael adduct at the -5 position. In addition, a number of other chemical variants of the operator have been analysed. These include operators incorporating deoxyadenine residues at each of the important single-stranded adenine sites. Recently the Michael adduct proposal has been challenged on the basis of mutagenesis of the coat protein cysteine residues. These results are discussed in the light of our data in support of Michael adduct formation.     

Slow interaction of 5'-adenylyl-beta,gamma-imidodiphosphate with Escherichia coli DNA gyrase. Evidence for cooperativity in nucleotide binding.

We have examined the kinetics of interaction between Escherichia coli DNA gyrase and the nonhydrolyzable ATP analog 5'-adenylyl-beta,gamma-imidodiphosphate (ADPNP) in the presence and absence of ATP. In the absence of ATP, [alpha-32P]ADPNP binds extremely slowly to gyrase, with an apparent second-order rate constant (k1) of 120 M-1 min-1. Similarly, the limited negative supercoiling of closed-circular DNA caused by ADPNP binding is slow, requiring at least 2 h to reach completion in the presence of 100 microM ADPNP. A very slow but detectable rate of dissociation of ADPNP from gyrase was measured, with a rate constant of 3.5 x 10(-4) min-1. The calculated dissociation constant for ADPNP is thus 2.9 microM. ADPNP is a potent competitive inhibitor of ATP-dependent DNA supercoiling. Inhibition is established much more rapidly than can be accounted for by the slow rate of ADPNP binding in the absence of ATP. We have found that ATP can accelerate the rate of [32P]ADPNP binding by more than 15-fold (k1 = 1,850 M-1 min-1). The ATP-promoted rate enhancement requires the presence of DNA; in the absence of DNA, ATP has no effect on the rate of binding. Relaxed closed-circular, nicked-circular, and linear pBR322 DNA are all equally effective cofactors for ATP-stimulated binding of ADPNP. After a short lag, the presence of ATP also greatly speeds up ADPNP dissociation from gyrase bound initially to closed-circular DNA, with the restoration of DNA supercoiling activity. This effect is not observed in the presence of nicked-circular or linear DNA, suggesting that ADPNP dissociates more rapidly from gyrase bound to supercoiled DNA. The results of ADPNP binding provide evidence for cooperative interactions between the nucleotide binding sites. To account for these data, a model is proposed for the interaction of nucleotides at the two ATP binding sites on DNA gyrase.     

Low-frequency respiratory system resistance in the normal dog during mechanical ventilation

The low-frequency resistances of the respiratory system, lung, and chest wall were investigated in four anesthetized paralyzed dogs mechanically ventilated at various frequencies between 0.08 and 0.83 Hz. The resistances were calculated by three different methods: 1) as the real part of the complex impedance obtained from regular ventilation data, 2) as the effective resistance of a two-compartment model fitted to the same data, and 3) as the resistance of a single-compartment model fitted to data obtained during sinusoidal ventilation at various frequencies. Alveolar pressures were measured by a closed-chest alveolar capsule technique and afforded a direct measure of airways resistance. All three resistance estimates were very similar and decreased markedly with frequency between 0 and 1 Hz. The real part of lung impedance at the higher frequencies investigated (around 5 Hz) closely approximated airways resistance, as predicted by the eight-parameter viscoelastic model of respiratory mechanics proposed by Bates et al. (J. Appl. Physiol. 67:2276-2285, 1989).