Effect of hypoxia on UDP-glucuronosyl transferase mRNA expression in human hepatocarcinoma functional liver celL4 cell line.

Although hypoxic conditions have been reported to affect the expression levels of various enzymes like cytochrome P450, the effect of hypoxia for UDP-glucuronosyl transferase (UGT) expression has been unclear. We evaluated the mRNA expression of UGTs (UGT1A1·1A6·1A9·2B7) in a functional liver cell-4 (FLC-4) cell line by three-dimensional culture under hypoxic conditions (37 °C, 1% O2, 5% CO2) fo 7 days. The mRNA expression of UGT1A1·1A6·1A9·2B7 decreased significantly after 3 days and that of UGT1A1·1A6·1A9 decreased significantly after 7 days. Hypoxic conditions affect the expression levels of UGT enzymes, thus the adjustment of dosage and interval should be considered in drug therapy that metabolized by UGT.

IC138 defines a subdomain at the base of the I1 dynein that regulates microtubule sliding and flagellar motility.

To understand the mechanisms that regulate the assembly and activity of flagellar dyneins, we focused on the I1 inner arm dynein (dynein f) and a null allele, bop5-2, defective in the gene encoding the IC138 phosphoprotein subunit. I1 dynein assembles in bop5-2 axonemes but lacks at least four subunits: IC138, IC97, LC7b, and flagellar-associated protein (FAP) 120--defining a new I1 subcomplex. Electron microscopy and image averaging revealed a defect at the base of the I1 dynein, in between radial spoke 1 and the outer dynein arms. Microtubule sliding velocities also are reduced. Transformation with wild-type IC138 restores assembly of the IC138 subcomplex and rescues microtubule sliding. These observations suggest that the IC138 subcomplex is required to coordinate I1 motor activity. To further test this hypothesis, we analyzed microtubule sliding in radial spoke and double mutant strains. The results reveal an essential role for the IC138 subcomplex in the regulation of I1 activity by the radial spoke/phosphorylation pathway.

Mutacin production in Streptococcus mutans genotypes isolated from caries-affected and caries-free individuals.

Relationships between genetic diversity and mutacin production in Streptococcus mutans were evaluated in 319 clinical isolates from eight caries-affected and eight caries-free individuals. The isolates were submitted to mutacin typing and AP-PCR (arbitrarily primed polymerase chain reaction) assay. The mutacin production was detected for 12 Streptococcus sp. indicator strains. Results showed significant variations in the mutacin production profiles and the inhibitory spectra of both groups. A possible association was seen between mutacin activity and the distinct patterns of Streptococcus sp. colonization in the two groups. Genotyping by AP-PCR using the primers OPA-02 and OPA-13 revealed 101 distinct genotypes against 48 phenotypes identified by mutacin typing. No correlation was observed between the inhibitory spectra of mutacin and genotypic similarities based on AP-PCR analyses. According to our results, strains of the same S. mutans genotype showed different mutacin profiles, suggesting a high degree of interstrain diversity. In conclusion, mutacin production seems to be of clinical importance in the colonization of S. mutans and is highly diversified in the S. mutans species.

Recovery of flagellar dynein function in a Chlamydomonas actin/dynein-deficient mutant upon introduction of muscle actin by electroporation.

Flagellar and ciliary inner-arm dyneins contain actin as a subunit; however, the function of this actin subunit remains unknown. As a first step toward experimental manipulation of actin in dynein, we developed a method for introducing exogenous actin into Chlamydomonas cells by electroporation. A non-motile mutant, ida5oda1, lacking inner-arm dyneins due to the absence of conventional actin, was electroporated in the presence of rabbit skeletal muscle actin. About 20% of the electroporated cells recovered motility under optimal conditions. In addition, by taking advantage of their phototactic behavior, the rescued cells could be concentrated. Motility was also recovered with fluorescently labeled actin; in this case, axonemes became fluorescent after electroporation, suggesting that actin was in fact incorporated as a dynein subunit. The feasibility of incorporating a substantial amount of macromolecules by electroporation will be useful not only for studying actin function, but also for a variety of studies using Chlamydomonas in which no efficient methods have been developed for expressing or introducing foreign proteins and other macromolecules.

Association between actin and light chains in Chlamydomonas flagellar inner-arm dyneins.

Inner dynein arms in cilia and flagella contain actin as a subunit; however, the function of this actin is totally unknown. Here we performed chemical crosslinking experiments to examine the interaction of actin with other subunits. Six of the seven Chlamydomonas inner-arm dynein species separated by anion-exchange chromatography contain actin and either one of the two previously identified light chains, p28 and centrin, in a mutually exclusive manner. Western blotting of chemically crosslinked dyneins indicated that actin is directly associated with p28 and centrin but not with the dynein heavy chains (HCs). In contrast, p28 and centrin both appeared to interact directly with the N-terminal half of the HCs. Thus it is likely that actin is associated with the heavy chains through p28/centrin. These light chains may well function in the assembly or targeting of the inner arm to the correct axonemal location.

The sensitivity of chlamydomonas photoreceptor is optimized for the frequency of cell body rotation.

For phototactic migration, Chlamydomonas scans the surrounding light environment by rotating the cell body with an eyespot located on the equator. The intensity of the light signal received by the eyespot should therefore change cyclically at the frequency of the cell body rotation. In this study, the response of the photoreceptor to cyclically changing light stimuli was analyzed using immotile mutant cells. To simulate the light intensity change perceived by a rotating cell, light stimuli were applied that consisted of a light phase with the intensity changing similar to a half cycle of a sine wave and a dark phase of the same length. The fluence rate at the peak of the sine wave was of the order of 10(19) photons m(-2) s(-1), i.e. high intensity at which phototaxis is saturated. A photoreceptor current (PRC) was produced at the onset of each light phase. Interestingly, its amplitude varied depending on the frequency and was largest at 1-5 Hz, a frequency range similar to the frequency of cell body rotation. Experiments on the kinetics of the PRC indicate that the response was small at low frequency because of the inactivation of the PRC before full activation. In contrast, at high frequency the PRC was suppressed by adaptation to the repetitive stimuli. These characteristic kinetics of the PRC should be important for Chlamydomonas cells to extract information from the signals generated by the cell body rotation.

Transport and arrangement of the outer-dynein-arm docking complex in the flagella of Chlamydomonas mutants that lack outer dynein arms.

The outer dynein arms of Chlamydomonas flagella are attached to a precise site on the outer doublet microtubules and repeat at a regular interval of 24 nm. This binding is mediated by the outer dynein arm docking complex (ODA-DC), which is composed of three protein subunits. In this study, antibodies against the 83- and 62-kD subunits (DC83 and DC62) of the ODA-DC were used to analyze its state of association with outer arm components within the cytoplasm, and its localization in the axonemes of oda mutants. Immunoprecipitation indicates that DC83 and DC62 are preassembled within the cytoplasm, but that they are not associated with outer arm dynein. Both proteins are lost or greatly diminished in oda1 and oda3, mutants in the structural genes of DC62 and DC83, respectively, demonstrating that their association is necessary for their stable presence in the cytoplasm. Immunoelectron microscopy indicates that DC83 repeats at 24-nm intervals along the length of the doublet microtubules of oda6, which lacks outer arms; thus, outer arm periodicity may be determined by the ODA-DC. Flagellar regeneration and temporary dikaryon experiments indicate that the ODA-DC can be rapidly transported into the flagellum and assembled on the doublet microtubules independently of the outer arms and independently of flagellar growth. Unexpectedly, the intensity of ODA-DC labeling decreased toward the distal ends of axonemes of oda6 but not wild-type cells, suggesting that the outer arms reciprocally contribute to the assembly/stability of the ODA-DC.

Vigorous beating of Chlamydomonas axonemes lacking central pair/radial spoke structures in the presence of salts and organic compounds.

Flagella of Chlamydomonas mutants lacking the central pair of microtubules or radial spokes do not beat; however, axonemes isolated from these mutants were found to display vigorous bending movements in the presence of ATP and various salts, sugars, alcohols, and other organic compounds. For example, about 15% of the total axonemes isolated from pf18, a mutant lacking the central pair, displayed beating in the presence of 10 mM MgSO(4) and 0.2 mM ATP at about 22 Hz, while none beat with the same concentration of ATP and < or = 5 mM or > or = 25 mM MgSO(4). The beat frequency and waveform of beating pf18 axonemes were similar to those of wild type axonemes beating under the same conditions. Similarly, 10-50% of the axonemes beat in the presence of 0.5 M sucrose, 2.0 M glycerol, or 1.7 M[10% (v/v)] ethanol. The appearance of motility did not correlate with the change in axonemal ATPase; however, these substances at those concentrations commonly increased the amplitude of nanometer-scale oscillation (hyper-oscillation) in pf18 axonemes, as well as the extent of ATP-induced sliding disintegration of protease-treated axonemes. Axonemes of double mutants lacking both the central pair and various subspecies of inner-arm dynein also beat at increased MgSO(4) concentrations, but axonemes lacking outer-arm dynein in addition to the central pair did not beat. These and other observations suggest that small molecules perturb the regulation of microtubule sliding through some change in water activity or osmotic stress. Axonemes must have an intrinsic ability to beat without the central pair/radial spokes under a variety of non-physiological solution conditions, as long as the outer dynein arms are present. Apparently, the major function of the central pair/radial spoke structures is to restore this activity under physiological conditions.

Analysis of cell vibration for assessing axonemal motility in Chlamydomonas.

Flagellar mutants of Chlamydomonas have greatly contributed to our understanding of the function of axonemes and axonemal dyneins. An important step in studying mutants is to correlate the molecular and structural defects in the axoneme with motility. This is not always easy, however, partly because it is often necessary to quantify axonemal motility by measuring the cell's swimming velocity, the flagellar beat frequency, or flagellar waveform in a number of cells or axonemes. To skip this time-consuming step, a quick method for measuring the average flagellar beat frequency in a population of cells is developed based on fast Fourier transform (FFT) analysis of the vibration of cell bodies. This method yields the average beat frequency within 10-60 s and has been used as a powerful tool for identifying mutants lacking various dynein species. It is also particularly useful for studies analyzing detergent-extracted cell models under various reactivation conditions.

Direct measurement of inter-doublet elasticity in flagellar axonemes.

The outer doublet microtubules in ciliary and flagellar axonemes are presumed to be connected with each other by elastic links called the inter-doublet links or the nexin links, but it is not known whether there actually are such elastic links. In this study, to detect the elasticity of the putative inter-doublet links, shear force was applied to Chlamydomonas axonemes with a fine glass needle and the longitudinal elasticity was determined from the deflection of the needle. Wild-type axonemes underwent a high-frequency, nanometer-scale vibration in the presence of ATP. When longitudinal shear force was applied, the average position of the needle tip attached to the axoneme moved linearly with the force applied, yielding an estimate of spring constant of 2.0 (S.D.: 0.8) pN/nm for 1 microm of axoneme. This value did not change in the presence of vanadate, i.e., when dynein does not form strong cross bridges. In contrast, it was at least five times larger when ATP was absent, i.e., when dynein forms strong cross bridges. The measured elasticity did not significantly differ in various mutant axonemes lacking the central-pair microtubules, a subset of inner-arm dynein, outer-arm dynein, or the radial spokes, although it was somewhat smaller in the latter two mutants. It was also observed that the shear displacement in an axoneme in the presence of ATP often took place in a stepwise manner. This suggests that the inter-doublet links can reversibly detach from and reattach to the outer doublets in a cooperative manner. This study thus provides the first direct measure of the elasticity of inter-doublet links and also demonstrates its dynamic nature.

Isolation and characterization of novel Chlamydomonas mutants that display phototaxis but not photophobic response.

The unicellular green alga Chlamydomonas displays two distinct kinds of behavioral response to light: phototaxis, in which cells swim toward or away from the light source under constant illumination; and photophobic responses (also called stop responses or photoshock responses), in which cells transiently convert their flagellar waveform and swim backward upon sudden increase in light intensity. It has been suggested that the two responses partly share a common signal transduction pathway, but exactly how the different responses are produced has not been established. In this study, to help understand the molecular and cellular mechanisms that bring about the photophobic response, we isolated novel mutants (ppr1, ppr2, ppr3, and ppr4) that do not show the photophobic response. Importantly, these mutants retain the ability to display phototaxis, with almost the same sensitivities as in the wild type cell. Demembranated and reactivated flagellar axonemes of the ppr mutants were found to convert the bending patterns depending on the Ca2+ concentration, indicating that the axonemal mechanism for waveform conversion required for the photophobic response was unaffected by the mutations. In addition, measurements of electric currents in cell suspensions showed that these mutants generate normal photoreceptor currents (PRC) upon photostimulation, suggesting that they retain the normal activity of photoreception and the ionic channels that produce PRCs. However, the all-or-none flagellar current (FC), a Ca2+ current generated by PRC-induced depolarization of flagellar membrane, was absent or seriously impaired in the mutants. These findings clearly indicate that the all-or-none FC is necessary for the photophobic response but not for phototaxis. The isolation of the four genetically independent ppr mutants suggests that the generation of the FC is based on multiple components that are not used in the mechanism for phototaxis, and implies that the Chlamydomonas flagellar membrane possesses a voltage-dependent Ca2+-channel specifically used for generation of photophobic responses.

Highly divergent actin expressed in a Chlamydomonas mutant lacking the conventional actin gene.

The Chlamydomonas mutant ida5 is deficient in the conventional actin gene and its axoneme lacks a subset of inner dynein arms that contain actin as a subunit. However, this mutant retains some other inner dynein arms because a novel protein (NAP) is expressed as a substitute for actin. In this study, we show by sequence analysis that NAP is identical to a putative actin-related protein, the cDNA sequence of which has recently been reported and shown to have 64% amino acid identity with conventional actin. A polyclonal antibody raised against a synthetic polypeptide corresponding to the NH2-terminal sequence of this protein specifically reacted with the spot corresponding to NAP in two-dimensional electrophoresis patterns. NAP apparently can substitute for conventional actin in some, but not all, cellular functions, and therefore can be regarded as a highly divergent actin. This unconventional actin appears to be expressed only when conventional actin is absent.

Real-time observation of Ca2+-induced basal body reorientation in Chlamydomonas.

The two basal bodies of Chlamydomonas are connected by a bridge, the distal fiber, that contains a Ca2+-binding protein, centrin. Although various fibrous structures in many organisms containing centrin or similar proteins have been shown to contract at Ca2+ concentrations >10(-7)-10(-6) M, the contractility of the distal fiber in Chlamydomonas has not been demonstrated. To determine whether it undergoes Ca2+-dependent contraction, we isolated the flagella-basal body complex from the paralyzed-flagella mutant pf18 and measured the angle between the two axonemes at different Ca2+ concentrations. Use of a double mutant with the mutant fa1, deficient in the mechanism for Ca2+-dependent flagellar amputation, enabled the measurement at Ca2+ concentrations > or = 10(-4) M. The angle, 80-120 degrees at 10(-9) M Ca(2-), was found to decrease by about 20 degrees when the Ca2+ concentration was raised above 10(-6) M. The angle increased again when the Ca2+ concentration was lowered below 10(-7) M. The flagellar apparatuses isolated from the double mutant between pf18 and the mutant vfl2 deficient in the structural gene of centrin had an angle of 90-130 degrees at 10(-9) M Ca2+, but the angle did not change when the Ca2+ concentration was increased. Thus centrin must be involved in the basal body reorientation. In detergent-extracted cell models of the pf18fa1 mutant, the angle between the two axonemes was found to decrease transiently by about 15 degrees upon iontophoretic application of Ca2+. Hence, the Ca2+-induced basal body reorientation can take place even when the basal body is contained in the cell body covered by the cell wall. It may function as part of the mechanism for phobic responses wherein Chlamydomonas cells swim backward transiently upon reception of strong light or mechanical stimuli.

A dynein light chain of sea urchin sperm flagella is a homolog of mouse Tctex 1, which is encoded by a gene of the t complex sterility locus.

The outer-arm dynein of sea urchin sperm flagella contains six light chains with molecular masses of 23.2, 20.8, 12.3, 11.5, 10.4 and 9. 3kDa. We have cloned a cDNA for the 12.3kDa polypeptide (light chain 3) and found that this protein is highly homologous to mouse Tctex1, a protein encoded by a member of the multigene family in the t complex region that is involved in male sterility and the development of the germ cells. Tctex1 has recently been shown to be homologous to a light chain of cytoplasmic dynein. Therefore, the cytoplasmic dynein light chain has been implicated in the mechanism for the transmission ratio distortion (meiotic drive) that is characteristic of t haplotypes in mice. Our present finding, however, indicates that axonemal light chain 3 must be considered equally important.

Recovery of flagellar inner-arm dynein and the fertilization tubule in Chlamydomonas ida5 mutant by transformation with actin genes.

The ida5 mutant of Chlamydomonas, first isolated as a mutant lacking a subset of axonemal inner-arm dyneins, has recently been shown to lack conventional actin owing to a serious mutation in its gene. It lacks inner-arm dyneins probably because actin is an essential subunit for their assembly. In addition, male gametes of ida5 are unable to produce the fertilization tubule, a structure that contains a core of actin filament bundles. To establish that those observed deficiencies are solely attributable to the loss of actin, and to provide a basis for future studies on the actin function in this organism, we examined in this study whether transformation of this mutant with cloned actin genes can rescue the mutant phenotypes. Cotransformation of the double mutant ida5arg2 with the wild-type actin gene and arginino-succinate lyase gene that suppresses the arg2 mutation yielded several transformants that displayed increased motility. All of them were found to have acquired the introduced actin gene in the genome and the product actin in the flagella, and regained the missing inner-arm dyneins and wild-type motility. In addition, most transformants also became able to grow the fertilization tubule when mating reaction was induced. In addition to the wild-type actin gene, we also used a chimeric actin gene in which the N-terminal 12 amino-acid sequence of Chlamydomonas actin was replaced by that of the greatly divergent Tetrahymena actin. Transformants with this gene also resulted in recovery of inner-arm dynein and 70-80% of the wild-type level of motility. These results established that the lack of inner-arm dynein and the fertilization tubule in ida5 are consequences of its loss of conventional actin. Furthermore, they demonstrate that Chlamydomonas offers an excellent experimental system with which to study the structure-function relationship of actin by means of mutant analysis.

Chlamydomonas inner-arm dynein mutant, ida5, has a mutation in an actin-encoding gene.

Chlamydomonas flagellar inner-arm dynein consists of seven subspecies (a-g), of which all but f contain actin as subunits. The mutant ida5 and a new strain, ida5-t, lack four subspecies (a, c, d, and e). These mutants were found to have mutations in the conventional actin gene, such that its product is totally lost; ida5 has a single-base deletion that results in a stop codon at a position about two-thirds from the 5' end of the coding region, and ida5-t lacks a large portion of the entire actin gene. Two-dimensional gel electrophoresis patterns of the axonemes and inner-arm subspecies b and g of ida5 lacked the spot of actin (isoelectric point [pI] = approximately 5.3) but had two novel spots with pIs of approximately 5.6 and approximately 5.7 instead. Western blot with different kinds of anti-actin antibodies suggested that the proteins responsible for the two novel spots and conventional actin are different but share some antigenicity. Since Chlamydomonas has been shown to have only a single copy of the conventional actin gene, it is likely that the novel spots in ida5 and ida5-t originated from another gene(s) that codes for a novel actin-like protein(s) (NAP), which has hitherto been undetected in wild-type cells. These mutants retain the two inner-arm subspecies b and g, in addition to f, possibly because NAP can functionally substitute for the actin in these subspecies while they cannot in other subspecies. The net growth rate of ida5 and ida5-t cells did not differ from that of wild type, but the mating efficiency was greatly reduced. This defect was apparently caused by deficient growth of the fertilization tubule. These results suggest that NAP can carry out some, but not all, functions performed by conventional actin in the cytoplasm and raise the possibility that Chlamydomonas can live without ordinary actin.

Induction of temporary beating in paralyzed flagella of Chlamydomonas mutants by application of external force.

To help understand the mechanism by which the sliding movement of outer-doublet microtubules in cilia and flagella is converted into bending waves, we examined the effect of mechanical force imposed on the flagella of Chlamydomonas mutants lacking the central pair or multiple dyneins. These mutants were almost completely nonmotile under normal conditions. A bend was produced in a flagellum either by holding a cell with a micropipette and quickly moving it with a piezoelectric actuator; or by pushing a flagellum with a microneedle. After removal of the external force, mutants lacking the central pair (pf18 and pf19) displayed beating at irregular intervals of > 1 second for one to several cycles. Similarly, a double mutant (ida2ida4) lacking four species of inner-arm dynein displayed beating at intervals of > 0.1 second for up to 80 cycles. However, paralyzed flagella of double mutants that lack the outer dynein arm in addition to the central pair or the inner dynein arm did not show cyclical movements upon application of external force. These results indicate that the central pair and the inner dynein arm are important for both stable bend formation at the base and efficient bend propagation along the flagellar length. They also suggest that the outer dynein arm, and not the inner dynein arm, enables the flagellar axoneme to propagate bends independently of the central pair. We propose that the axoneme is equipped with two independent motor systems for oscillatory movements: an outer-arm system controlled by the axonemal mechanical state independently of the central pair/radial spoke system, and an inner-arm system controlled by both the axonemal mechanical state and the central pair/radial spokes.

Ca2+-dependent waveform conversion in the flagellar axoneme of Chlamydomonas mutants lacking the central-pair/radial spoke system.

Chlamydomonas flagella undergo a striking waveform conversion from an asymmetrical ciliary type to a symmetrical flagellar type when the cell is stimulated by intense light and the Ca2+ concentration within the flagellum is increased above approximately 10(-6) M. To see whether the central-pair/radial spoke system is needed for this conversion as suggested by previous studies, we examined the effect of Ca2+ on the reactivated axonemes of the mutants lacking the central pair (pf18) or the radial spokes (pf14). Although the flagella of these mutants are paralyzed in vivo, demembranated axonemes can be reactivated to beat under certain nucleotide conditions such as in the presence of low concentrations (< 100 microM) of ATP. We examined the waveform of the axonemes reactivated at 20 microM ATP in the presence of 10(-8)-10(-4) M Ca2+ and found that these axonemes, as well as the wild-type axonemes, undergo a waveform conversion over a Ca2+ concentration range of 10(-7)-10(-5) M: a highly asymmetrical waveform at <10(-6) M Ca2+ and a symmetrical waveform at >=10(-5) M Ca2+. Although the waveform is different between the mutants and the wild type, the Ca2+ concentration at which the waveform conversion occurred was similar. These results indicate that the central pair/radial spoke system is not essential for the waveform conversion.

Beat frequency difference between the two flagella of Chlamydomonas depends on the attachment site of outer dynein arms on the outer-doublet microtubules.

The two flagella of Chlamydomonas, although similar to each other at first glance, differ in functional properties. A clear difference exists in the beat frequency: the trans-flagellum (the one farthest from the eyespot) beats with 30-40% higher frequency than the cis-flagellum (the one nearest to the eyespot) in demembranated and reactivated cell models. This difference is considered to be influenced by outer arm dynein, because the two flagella beat at almost the same frequency in cell models of oda mutants lacking the outer dynein arm. When a sample of outer arm dynein extracted and purified from the wild-type axoneme was mixed with the cell models of an oda mutant, oda1, an almost normal number of outer dynein arms became attached to the axonemes, and the wild-type level of beat frequency was recovered on reactivation with ATP addition. The frequency imbalance, however, was not restored. Unexpectedly, when a similar experiment was performed with the cell model of another oda mutant, oda6, the addition of outer arm dynein restored the cis-trans frequency imbalance in addition to the normal number of outer arms and the higher level of reactivated motility. Among other oda mutants, oda3 yielded results similar to those with oda1, whereas oda2, oda4, and oda5 yielded results similar to those with oda6. Because the only structural difference between the two groups of oda mutants is that the oda1 and oda3 axonemes lack the outer arm attachment site on the outer doublet A-tubule while the axonemes of the other mutants retain it, these findings suggest that the attachment site for the outer dynein arm is important in determining the flagellar beat frequency. This suggests that the basal portion of the outer arm dynein is important in regulating the flagellar activity and therefore the behavior of the cell.