Evaluation of a new fiber-reinforced resin composite.

Efficacy of the usage of an experimental fiber-reinforced composite (FRC) on mechanical properties of an indirect composite was investigated by means of three-point bending and Charpy impact tests. Bond strength between the FRC and the indirect composite was also evaluated by tensile testing. The FRC consisted of a matrix resin with 25% silanized milled glass fiber (11-microm diameter, 150-microm length) and 5% colloidal silica. The values of strain of proportional limit, total strain, and fracture energy of the FRC during the bending test (1.2%, 10.4%, and 41.6 x 10(-3) J) were significantly higher than those of the indirect composite (0.1%, 2.5%, and 11.9 x 10(-3) J). The impact strengths of the 1-mm specimens with FRC ranged from 15.2 to 15.9 kJ/m(2), and were significantly higher than that of the control (3.1 kJ/m(2)). The 2-mm specimens showed significant difference from the control when the FRC thickness was equal or greater than 0.5 mm. The bond strength after the thermocycling was 15.2 MPa, and all of the specimens exhibited cohesive fracture inside the indirect composite. Based upon the results, it was concluded that the FRC tested in this study improved toughness and impact resistance of the indirect composite. The interfacial bonding between the FRC and the indirect composite was strong enough to prevent delamination.

Effects of filler composition on flexibility of microfilled resin composite.

The effects of the filler composition on physical and mechanical properties of microfilled composites was investigated by measuring water absorption, solubility, compressive, flexural, and impact strength. A series of experimental composites, consisting of UDMA/TEGDMA comonomer matrix and prepolymerized fillers, was fabricated. The prepolymerized fillers were composed of hydrophobic colloidal silica and two monomers in varying ratios, trimethylolpropanetrimethacrylate (TMPT), and polyesterdiacrylate (PEDA). TMPT/PEDA ratios were 100:0, 64:36, 46:54, 18:82, and 0:100%. There were no significant differences in water sorption and solubility, regardless of the amount of PEDA monomer. Young's modulus and modulus of resilience increased with decreasing PEDA ratio. Fracture energy exhibited drastic changes (30.1 x 10(-5) J to 93.4 x 10(-5) J). The highest value of flexural strength (96.0 +/- 3.5 MPa) was obtained when the TMPT-PEDA filler was 46:54. The impact strengths of composites fabricated with TMPT-PEDA filler of 46:54 (11.2 +/- 1.4 kJ/m(2)), 18:82 (10.6 +/- 3.2 kJ/m(2)), and 0:100 (13.1 +/- 3.8 kJ/m(2)) were significantly higher than those with 100:0 (6.0 +/- 1.8 kJ/m(2)) or 64:36 (7.1 +/- 2.4 kJ/m(2)). Based upon the results, it was concluded that the mechanical properties of microfilled composites were improved by the modification of prepolymerized filler composition.

K(+)-channel transgenes reduce K(+) currents in Paramecium, probably by a post-translational mechanism.

PAK11 is 1 of more than 15 members in a gene family that encodes K(+)-channel pore-forming subunits in Paramecium tetraurelia. Microinjection of PAK11 DNA into macronuclei of wild-type cells results in clonal transformants that exhibit hyperexcitable swimming behaviors reminiscent of certain loss-of-K(+)-current mutants. PAK2, a distant homolog of PAK11, does not have the same effect. But PAK1, a close homolog of PAK11, induces the same hyperexcitability. Cutting the PAK11 open reading frame (ORF) with restriction enzymes before injection removes this effect entirely. Microinjection of PAK11 ORF flanked by the calmodulin 5' and 3' UTRs also induces the same hyperexcitable phenotype. Direct examination of transformed cells under voltage clamp reveals that two different Ca(2+)-activated K(+)-specific currents are reduced in amplitude. This reduction does not correlate with a deficit of PAK11 message, since RNA is clearly produced from the injected transgenes. Insertion of a single nucleotide at the start of the PAK11 ORF does not affect the RNA level but completely abolishes the phenotypic transformation. Thus, the reduction of K(+) currents by the expression of the K(+)-channel transgenes reported here is likely to be the consequence of a post-translational event. The complexity of behavioral changes, possible mechanisms, and implications in Paramecium biology are discussed.

A TRP homolog in Saccharomyces cerevisiae forms an intracellular Ca(2+)-permeable channel in the yeast vacuolar membrane.

The molecular identification of ion channels in internal membranes has made scant progress compared with the study of plasma membrane ion channels. We investigated a prominent voltage-dependent, cation-selective, and calcium-activated vacuolar ion conductance of 320 pS (yeast vacuolar conductance, YVC1) in Saccharomyces cerevisiae. Here we report on a gene, the deduced product of which possesses significant homology to the ion channel of the transient receptor potential (TRP) family. By using a combination of gene deletion and re-expression with direct patch clamping of the yeast vacuolar membrane, we show that this yeast TRP-like gene is necessary for the YVC1 conductance. In physiological conditions, tens of micromolar cytoplasmic Ca(2+) activates the YVC1 current carried by cations including Ca(2+) across the vacuolar membrane. Immunodetection of a tagged YVC1 gene product indicates that YVC1 is primarily localized in the vacuole and not other intracellular membranes. Thus we have identified the YVC1 vacuolar/lysosomal cation-channel gene. This report has implications for the function of TRP channels in other organisms and the possible molecular identification of vacuolar/lysosomal ion channels in other eukaryotes.

The cloning and molecular analysis of pawn-B in Paramecium tetraurelia.

Pawn mutants of Paramecium tetraurelia lack a depolarization-activated Ca(2+) current and do not swim backward. Using the method of microinjection and sorting a genomic library, we have cloned a DNA fragment that complements pawn-B (pwB/pwB). The minimal complementing fragment is a 798-bp open reading frame (ORF) that restores the Ca(2+) current and the backward swimming when expressed. This ORF contains a 29-bp intron and is transcribed and translated. The translated product has two putative transmembrane domains but no clear matches in current databases. Mutations in the available pwB alleles were found within this ORF. The d4-95 and d4-96 alleles are single base substitutions, while d4-662 (previously pawn-D) harbors a 44-bp insertion that matches an internal eliminated sequence (IES) found in the wild-type germline DNA except for a single C-to-T transition. Northern hybridizations and RT-PCR indicate that d4-662 transcripts are rapidly degraded or not produced. A second 155-bp IES in the wild-type germline ORF excises at two alternative sites spanning three asparagine codons. The pwB ORF appears to be separated from a 5' neighboring ORF by only 36 bp. The close proximity of the two ORFs and the location of the pwB protein as indicated by GFP-fusion constructs are discussed.

Recent advances in the molecular genetics of Paramecium.

Paramecium continues to be used to study motility, behavior, exocytosis, and the relationship between the germ and the somatic nuclei. Recent progress in molecular genetics is described. Toward cloning genes that correspond to mutant phenotypes, a method combining complementation with microinjected DNA and library sorting has been used successfully in cloning several novel genes crucial in membrane excitation and in trichocyst discharge. Paramecium transformation en masse has now been shown by using electroporation or bioballistics. Gene silencing has also been discovered in Paramecium, recently. Some 200 Paramecium genes, full length or partial, have already been cloned largely by homology. Generalizing the use of gene silencing and related reverse-genetic techniques would allow us to correlate these genes with their function in vivo.

An unusual complementation in non-excitable mutants in Paramecium.

A non-excitable behavioural mutant, d4-662, was previously characterized as the fourth pawn locus mutant pwD in Paramecium tetraurelia. We now provide data demonstrating that d4-662 is in fact controlled by a pwB allele that has the unusual feature of complementing other pwB alleles in heterozygous F1 progeny. Neither the cytoplasm nor the nucleoplasm of d4-662 cured the mutational defects of pwB and in the reverse combination of d4-662 and pwB, the result was the same. On the other hand, pwA, another non-excitable mutant, was cured upon cross-injection with d4-662 and mutants carrying trichocyst non-discharge marker genes were also cured. This evidence suggests that d4-662 is a new mutant belonging to pwB, and would be better designated as pwB662. Extensive crossbreeding analyses, however, showed an unusual genetic relationship between d4-662 and pwB (pwB95 or pwB96). When d4-662 was crossed with pwB mutants, many progeny expressing wild-type phenotype or mixed clones of wild-type and pawn cells were obtained in the F1. Less than 12.5% expressed the pawn phenotype. The appearance of wild-type progeny in this F1 strongly suggests that an inter-allelic interaction between pwB662 and other pwB alleles may occur during development of the macronucleus.

K(+)-dependent composite gating of the yeast K(+) channel, Tok1.

TOK1 encodes an outwardly rectifying K(+) channel in the plasma membrane of the budding yeast Saccharomyces cerevisiae. It is capable of dwelling in two kinetically distinct impermeable states, a near-instantaneously activating R state and a set of related delayed activating C states (formerly called C(2) and C(1), respectively). Dwell in the R state is dependent on membrane potential and both internal and external K(+) in a manner consistent with the K(+) electrochemical potential being its determinant, where dwell in the C states is dependent on voltage and only external K(+). Whereas activation from the C states showed high temperature dependencies, typical of gating transitions in other Shaker-like channels, activation from the R state had a temperature dependence nearly as low as that of simple ionic diffusion. These findings lead us to conclude that although the C states reflect the activity of an internally oriented channel gate, the R state results from an intrinsic gating property of the channel filter region.

A new multigene family encoding calcium-dependent calmodulin-binding membrane proteins of Paramecium tetraurelia.

Ca2+/calmodulin (CaM) regulates various physiological processes in a wide variety of organisms, metazoa and protists alike. To better understand Ca2+/CaM-dependent processes, particularly those with membrane-associated components, we studied Ca2+/CaM-binding membrane proteins in Paramecium tetraurelia, a unicellular model system. A CaM-binding protein, PCM1 (Paramecium CaM-binding membrane-bound protein), from a detergent-solubilized ciliary membrane fraction was identified and purified through Ca2+-dependent CaM-affinity chromatography. PCM1 has an apparent molecular mass of approx. 65kDa. It binds radiolabeled CaM in blot overlay assays and binds to CaM-affinity columns, both only in the presence of 10 microM or higher Ca2+. Three peptide sequences from PCM1 were obtained, and polymerase chain reaction (PCR) and Southern hybridization experiments were designed accordingly, leading to a partial cDNA clone for PCM1 and the discovery of three homologs: PCM2, PCM3 and PCM4. Amino acid sequences predicted by the full-length coding sequence for PCM3 and partial genes for PCM1, PCM2 and PCM4 are very similar (approx. 85% amino-acid identities). Their sequences indicate that they are hitherto novel proteins with beta/gamma-crystallin domains, cysteine-rich regions and potential CaM-binding domains. These protein motifs are suggested to mediate protein-protein interaction important for Ca2+/CaM signal transduction event(s) through the PCM family of proteins.

Transformation of Paramecium tetraurelia by electroporation or particle bombardment.

Methods for mass transformation of Paramecium tetraurelia were established using plasmids bearing neomycin-resistance or calmodulin gene fragments. Phenotypic and molecular analyses showed that, although variable, up to 5% transformation can be achieved by electroporation. Concentrations of divalent cations Ca2+ and Mg2+ in the electroporation medium were crucial for efficient transformation. Strong neomycin-resistance transformation using bioballistic particle bombardment with gold particles was observed. For both methods, hybridization to transformant DNA revealed plasmid signals consistent with macronuclear transformation and correlated with transformed phenotypes. Complementation of a known calmodulin gene mutation was also achieved by mass transformation. Possible sources of variation and the general utility of these methods are discussed.

A comparison of internal eliminated sequences in the genes that encode two K(+)-channel isoforms in Paramecium tetraurelia.

We examined both the somatic (macro-) and the germinal (micronuclear) DNAs that encode two K(+)-channel isoforms, PAK1 and PAK11, in Paramecium tetraurelia. The coding regions of these two isoforms are 88% identical in nucleotides and 95% identical in amino acids. Their introns are also highly conserved. Even some of the internal eliminated sequences in PAK1 and PAK11 are clearly related. PAK1 has five IESs; PAK11 has four. The first (5'-most) IESs of the two genes are located at the same site in the coding sequence but differ in size. The 2nd IES in PAK1 (206-bp), the largest among the nine IESs, has no PAK11 counterpart. The 3rd, 4th and 5th IESs in PAK1 have a counterpart in PAK11 that is similar in size and in sequence, and identical in its position in the coding sequence. In addition, the first IES of PAK11 bears some resemblance to the 4th one of PAK1. The similarities and differences between the two sets of IESs are discussed with respect to the origin and divergence of the two K(+)-channel isoforms.

The cloning by complementation of the pawn-A gene in Paramecium.

The genetic dissection of a simple avoidance reaction behavior in Paramecium tetraurelia has shown that ion channels are a critical molecular element in signal transduction. Pawn mutants, for example, were originally selected for their inability to swim backward, a trait that has since been shown to result from the loss of a voltage-dependent calcium current. The several genes defined by this phenotype were anticipated to be difficult to clone since the 800-ploid somatic macronucleus of P. tetraurelia is a formidable obstacle to cloning by complementation. Nonetheless, when the macronucleus of a pawn mutant (pwA/pwA) was injected with total wild-type DNA or a fractional library of DNA, its clonal descendants all responded to stimuli like the wild type. By sorting a fractional library, we cloned and sequenced a 2.3-kb fragment that restores the Ca2+ current and excitability missing in pawn-A. Data from RNase protection assays, followed by the sequencing of mutant alleles and cDNA clones, established an open reading frame. The conceptually translated product suggests a novel protein that may be glycophosphatidylinositol anchored. We also discuss the general usefulness of this method in cloning other unknown DNA sequences from Paramecium that are functionally responsible for various mutant phenotypes.

Random mutagenesis reveals a region important for gating of the yeast K+ channel Ykc1.

YKC1 (TOK1, DUK1, YORK) encodes the outwardly rectifying K+ channel of the yeast plasma membrane. Non-targeted mutations of YKC1 were isolated by their ability to completely block proliferation when expressed in yeast. All such mutations examined occurred near the cytoplasmic ends of the transmembrane segments following either of the duplicated P loops, which we termed the 'post-P loop' (PP) regions. These PP mutations specifically caused marked defects in the 'C1' states, a set of interrelated closed states that Ykc1 enters and exits at rates of tens to hundreds of milliseconds. These results indicate that the Ykc1 PP region plays a role in determining closed state conformations and that non-targeted mutagenesis and microbial selection can be a valuable tool for probing structure-function relationships of ion channels.

Toward cloning genes by complementation in Paramecium.

Conventional methods of gene cloning by complementing mutant defects is made difficult by the 800 ploidy of the Paramecium macronucleus. However, this nucleus is some 30 microns in diameter and readily propagates exogenous DNA fragments as cells divide. These attributes allow for massive injection of engineered DNA fragments and their maintenance in the transformed descendant. If a genomic DNA fraction injected into a mutant macronucleus effects complementation, it should be possible to sort a fractional library to isolate the complementing gene. Here, we investigated four aspects of establishing this method for general use. First, using the cloned CAM gene as a test case, we further investigated transformation by macronuclear injection and showed that phenotypic reversion is directly correlated with the copy number of the transgene, even when it is of a recessive allele, cam2, which has a missense mutation but produces a partially functional protein. Second, we examined the copy number of the transgene established in cells of older clonal age and discussed the likely dilution of the transgene in younger descendants of the injected cell. Third, we showed that the degree of phenotypic reversion is correlated with the transgene product, the cam2 calmodulin protein in the cell. Fourth, we extended the investigation to very recessive mutants whose genes are to be cloned. We showed that size fractions of wild-type genomic DNA digests effect strong phenotypic reversions in several pawn mutants, setting the stage for cloning these Ca(2+)-channel related genes. The general usefulness of this method in cloning genes that complement recessive alleles and current limitations of this method in dealing with dominant alleles are assessed and discussed.

Bonding to intact dentin.

It has been reported that the presence of a smear layer on dentinal substrates can compromise bonding. Typically, smear layers are removed by acidic agents that selectively extract calcium salts from dentin surfaces to leave a collagen-rich substrate. Acid-conditioned dentin (i.e., demineralized) is then primed and an adhesive agent applied. In the present study, we removed smear layers by "polishing" dentin specimens with a hydroxyapatite paste and ultrasonication. Bonding procedures were carried out by means of an aqueous solution of 20% 2-methacryloyloxyethyl phenyl phosphoric acid (phenyl-P) and 30% 2-hydroxyethyl methacrylate, referred to as 2OP-30H, a "self-etching primer". The 20P-30H solution was applied to "intact" dentin (i.e., non-demineralized) for either 30 or 60 s. Control samples received no application (O s) of the self-etching primer. Mean tensile bond strengths (10 MPa) were similar in both the 30-second- and 60-second-primed groups. The widths of formed hybrid layers varied from 0.3 +/- 0.2 micron at O s application (control) to 2.1 +/- 0.3 micron for the 30-second group and 4.1 +/- 0.2 micron for the 60-second group. SEM and TEM observations revealed that the 20P-30H self-etching primer created diffusion channels into "intact" calcium-rich dentin which permitted monomer to infiltrate dentin substrates. Hybrid layers identified under microscopic examination demonstrated resistance to both HCI and NaOCI treatments, suggesting that the hybrid layer was not defective, and that bonding was stable.

YKC1 encodes the depolarization-activated K+ channel in the plasma membrane of yeast.

Our previous patch-clamp studies showed that depolarization activates a K(+)-specific current in the plasma membrane of the budding yeast, Saccharomyces cerevisiae [Gustin et al. (1986) Science 233, 1195-1197]. The Yeast Genome Sequencing Project has now uncovered on the left arm of chromosome X an open reading frame (ORF) that predicts a 77-kDa protein reminiscent of a shaker-like alpha subunit with 6 membrane spans followed by a subunit with 2 spans. We found that deleting this ORF removes the yeast K+ current. Furnishing the ORF from plasmids restores or even greatly amplifies this current. These manipulations have no effects on the 40-pS mechanosensitive conductance also native to this membrane. Thus, this ORF, named YKC1 here, likely encodes a structure for the K(+)-specific channel of the yeast plasma membrane. This and other K+ channel subunits are compared and the possible uses of this gene in research are discussed. YKC1 has recently been shown by others to induce in frog oocytes a K+ current. Its activation is coupled to EK+ and its outward rectification depends on external divalent cations. We found the YKC1 channel in its native membrane activates at low voltages largely independent of EK+ and it remains so despite removal of divalents by chelation.

Functional reconstitution of ion channels from Paramecium cortex into artificial liposomes.

Toward isolating channel proteins from Paramecium, we have explored the possibility of functionally reconstituting ion channels in an artificial system. Proteins from Paramecium cortex reconstituted with soybean azolectin retained several channels whose activities were readily registered under patch clamp. The most commonly encountered activities were three: (i) a 71-pS cation channel that opens at all voltages unless di- or trivalent cations were added to close them, (ii) a 40 pS monovalent cation channel, and (iii) a large-conductance channel that prefers anions and exhibits many subconductance states. These channels survived mild detergent treatments without observable functional alterations. The possible origin of these channels from internal membranes, the possible role of 71-pS channel in internal Ca2+ release, and the prospects of their purification are discussed.

Paramecium Na+ channels activated by Ca(2+)-calmodulin: calmodulin is the Ca2+ sensor in the channel gating mechanism.

Paramecium Na+ channels, which were Ca(2+)-calmodulin activated, were studied in the inside-out mode of patch clamp. After excision of the membrane patch, they were active in the presence of 10(-5) to 10(-3) M Ca+ in the bath. They became much less active in the presence of 10(-6) M Ca2+, and their activity subsided completely at 10(-8) M Ca2+. A Hill plot showed a dissociation constant of 6 microM for Ca2+ binding. This dissociation constant shifted to a submicromolar range in the presence of 1 mM Mg2+. The channels also exhibited a mild voltage dependence. When exposed to 10(-8) M Ca2+ for an extended period of 2-4 min, channels were further inactivated even after bath Ca2+ was restored to 10(-4) M. Whereas neither high voltage (+100 mV) nor high Ca2+ (10(-3) M) was effective in reactivation of the inactive channels, addition of Paramecium wild-type calmodulin together with high Ca2+ to the bath restored channel activity without a requirement of additional Mg2+ and metabolites such as ATP. The channels reactivated by calmodulin had the same ion conductance, ion selectivity and Ca2+ sensitivity as those prior to inactivation. These inactivation and reactivation of the channels could be repeated, indicating that the direct calmodulin effect on the Na+ channel was reversible. Thus, calmodulin is a physiological factor critically required for Na+ channel activation, and is the Ca2+ sensor of the Na(+)-channel gating machinery.