Expression of the heparan sulfate proteoglycan, perlecan, during mouse embryogenesis and perlecan chondrogenic activity in vitro.

Expression of the basement membrane heparan sulfate proteoglycan (HSPG), perlecan (Pln), mRNA, and protein has been examined during murine development. Both Pln mRNA and protein are highly expressed in cartilaginous regions of developing mouse embryos, but not in areas of membranous bone formation. Initially detected at low levels in precartilaginous areas of d 12.5 embryos, Pln protein accumulates in these regions through d 15.5 at which time high levels are detected in the cartilage primordia. Laminin and collagen type IV, other basal lamina proteins commonly found colocalized with Pln, are absent from the cartilage primordia. Accumulation of Pln mRNA, detected by in situ hybridization, was increased in d 14.5 embryos. Cartilage primordia expression decreased to levels similar to that of the surrounding tissue at d 15.5. Pln accumulation in developing cartilage is preceded by that of collagen type II. To gain insight into Pln function in chondrogenesis, an assay was developed to assess the potential inductive activity of Pln using multipotential 10T1/2 murine embryonic fibroblast cells. Culture on Pln, but not on a variety of other matrices, stimulated extensive formation of dense nodules reminiscent of embryonic cartilaginous condensations. These nodules stained intensely with Alcian blue and collagen type II antibodies. mRNA encoding chondrocyte markers including collagen type II, aggrecan, and Pln was elevated in 10T1/2 cells cultured on Pln. Human chondrocytes that otherwise rapidly dedifferentiate during in vitro culture also formed nodules and expressed high levels of chondrocytic marker proteins when cultured on Pln. Collectively, these studies demonstrate that Pln is not only a marker of chondrogenesis, but also strongly potentiates chondrogenic differentiation in vitro.     

Fourier transform IR studies of the self-association of N-tert-butoxycarbonyl-α-amino acids

The self-association of several N-urethanyl-L-amino acids (N-t-Boc-glycine, N-t-Boc-L-alanine, N-t-Boc-L-methionine, and N-t-Boc-O-Bz-L-tyrosine has been investigated in carbon tetrachloride by Fourier transform (FTIR) spectrometry. The fractions of nonbonded OH and NH groups have been determined from the intensities of the free OH and NH-stretching vibrations. The FTIR spectra have been examined in the carbonyl stretching regions by using Fourier self-deconvolution techniques. The results show that intermolecular hydrogen bonds are formed between the carboxylic acid group of one molecule and the urethane group of another molecule, suggesting that the leading factor for the self-association of N-t-Boc amino acids is the acidity of the OH groups. In N-t-Boc glycine, more heterodimers are formed than in the other amino acids. In N-t-Boc-O-Bz-L-tyrosine where the steric hindrance of the substituent implanted on the C^α atom is higher, the concentration of homodimers and heterodimers is lower than in N-t-Boc-L-alanine or N-t-Boc-L methionine. © 1997 John Wiley & Sons, Inc.     

POPULATION DYNAMICS OF THE SPORE-FORMING DIATOM LEPTOCYLINDRUS DANICUS IN NARRAGANSETT BAY. RHODE ISLAND1

A field study to assess resting spore importance in long-and short-term population dynamics of Leptocylindrus danicus ( Ehr.) Gran was carried out, based on the observation that germinating spores produce only vegelative cells of maximum diameter. From May through November 1978 . L. danicus was present in lower Narragansett Bay, but resting spores were not found. May and June bioassays suggest that Bay water could not support sexuality (hence spore formation) in L. danicus during this period. Mean valve diameter decreased abruptly in early June. as did cell number. When mean valve diameter increased abruptly in August, with no resting spores seen, 70 additional clonal isolates showed that one clone had the "normal" sexual and spore-forming life cycle while 69 clones had an "alternate" life cycle lacking sperm and spore formation. Valvar fine structure of the two types differed with respect to the presence or absence of a subcentral valvar pore, and a new subspecific taxon is here distinguished: variety apora. Examination of the early June data indicates the replacement of one population by another. Populations of L. danicus may be sporadically introduced into Narragansett Bay from Gulf Stream rings .     

Impacts of a woody invader vary in different vegetation communities

The impact of an exotic species in natural systems may be dependent not only on invader attributes but also on characteristics of the invaded community. We examined impacts of the invader bitou bush, Chrysanthemoides monilifera ssp. rotundata , in fore and hind dune communities of coastal New South Wales, Australia. We compared invader impacts on vegetation structure, richness of both native and exotic growth forms and community variability in fore and hind dunes. We found that impacts of bitou invasion were context specific: in fore dune shrublands, functionally distinct graminoid, herb and climber rather than shrub growth forms had significantly reduced species richness following bitou invasion. However, in forested hind dunes, the functionally similar native shrub growth form had significantly reduced species richness following bitou invasion. Density of vegetation structure increased at the shrub level in both fore and hind dune invaded communities compared with non-invaded communities. Fore dune ground-level vegetation density declined at invaded sites compared with non-invaded sites, reflecting significant reductions in herb and graminoid species richness. Hind dune canopy-level vegetation density was reduced at invaded compared with non-invaded sites. Bitou bush invasion also affected fore dune community variability with significant increases in variability of species abundances observed in invaded compared with non-invaded sites. In contrast, variability among all hind dune sites was similar. The results suggest that effects of bitou bush invasion are mediated by the vegetation community. When bitou bush becomes abundant, community structure and functioning may be compromised.     

Dissecting lidocaine action: diethylamide and phenol mimic separate modes of lidocaine block of sodium channels from heart and skeletal muscle.

We have investigated block of sodium channels by diethylamide and phenol, which resemble the hydrophilic tertiary amine head and the hydrophobic aromatic tail of the lidocaine molecule, respectively. Diethylamide and phenol separately mimicked the fast and slow modes of block caused by lidocaine. Experiments were performed using single batrachotoxin-activated bovine cardiac and rat skeletal muscle sodium channels incorporated into neutral planar lipid bilayers. Diethylamide, only from the intracellular side, caused a voltage-dependent reduction in apparent single channel amplitude ('fast' block). Block was similar for cardiac and skeletal muscle channels, and increased in potency when extracellular sodium was replaced by N-methylglucamine, consistent with an intrapore blocking site. Thus, although occurring at 15-fold higher concentrations, block by diethylamide closely resembles the fast mode of block by lidocaine (Zamponi, G. W., D. D. Doyle, and R. J. French. 1993. Biophys. J. 65:80-90). For cardiac sodium channels, phenol bound to a closed state causing the appearance of long blocked events whose duration increased with phenol concentration. This slow block depended neither on voltage nor on the side of application, and disappeared upon treatment of the channel with trypsin. For skeletal muscle channels, slow phenol block occurred with only very low probability. Thus, phenol block resembles the slow mode of block observed for lidocaine (Zamponi, G. W., D. D. Doyle, and R. J. French. 1993. Biophys. J. 65:91-100). Our data suggest that there are separate sites for fast lidocaine block of the open channel and slow block of the "inactivated" channel. Fast block by diethylamide inhibited the long, spontaneous, trypsin-sensitive (inactivation-like) closures of cardiac channels, and hence secondarily antagonized slow block by phenol or lidocaine. This antagonism would potentiate shifts in the balance between the two modes of action of a tertiary amine drug caused by changes in the relative concentrations of the charged (fast blocking) and neutral (slow blocking) forms of the drug.     

Fast lidocaine block of cardiac and skeletal muscle sodium channels: one site with two routes of access.

We have studied the block by lidocaine and its quaternary derivative, QX-314, of single, batrachotoxin (BTX)-activated cardiac and skeletal muscle sodium channels incorporated into planar lipid bilayers. Lidocaine and QX-314, applied to the intracellular side, appear to induce incompletely resolved, rapid transitions between the open and the blocked state of BTX-activated sodium channels from both heart and skeletal muscle. We used amplitude distribution analysis (Yellen, G. 1984. J. Gen. Physiol. 84:157-186.) to estimate the rate constants for block and unblock. Block by lidocaine and QX-314 from the cytoplasmic side exhibits rate constants with similar voltage dependence. The blocking rate increases with depolarization, and the unblocking rate increases with hyperpolarization. Fast lidocaine block was virtually identical for sodium channels from skeletal (rat, sheep) and cardiac (beef, sheep) muscle. Lidocaine block from the extracellular side occurred at similar concentrations. However, for externally applied lidocaine, the blocking rate was voltage-independent, and was proportional to concentration of the uncharged, rather than the charged, form of the drug. In contrast, unblocking rates for internally and externally applied lidocaine were identical in magnitude and voltage dependence. Our kinetic data suggest that lidocaine, coming from the acqueous phase on the cytoplasmic side in the charged form, associates and dissociates freely with the fast block effector site, whereas external lidocaine, in the uncharged form, approaches the same site via a direct, hydrophobic path.