Paralyzed flagella mutants of Chlamydomonas reinhardtii. Defective for axonemal doublet microtubule arms.
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Chlamydomonas↗
Biomedical subjects
B Huang
Publications and source records attributed to B Huang.
Modulation of protein synthesis during the growth cycle of a culture of scarlet rose.
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Adenosine Triphosphate↗
Two-dimensional analysis of flagellar proteins from wild-type and paralyzed mutants of Chlamydomonas reinhardtii.
Flagellar polypeptides of Chlamydomonas reinhardtii were analyzed in two-dimensions by isoelectric focusing and electrophoresis in the presence of sodium dodecyl sulfate. In addition to flagellar tubulin, over 130 polypeptides were resolved and 100 of these were identified as axonemal components in wild-type organisms. Flagella of two nonconditional paralyzed mutants, pf 14 and pf 1, were also analyzed and, at the same time, electron microscopic studies were carried out. pf 14 flagella, which completely lack radial spokes and associated spokeheads, are missing 12 polypeptides. Six of these polypeptides are also missing from pf 1 flagella in which spokes are clearly present but spoke heads appear to be absent.
Cell Movement↗
Flagellar mutants of Chlamydomonas: studies of radial spoke-defective strains by dikaryon and revertant analysis.
The motility mutant of Chlamydomonas reinhardtii pf14 lacks radial spoke structures in its flagellar axonemes, and 12 proteins present in wild type are missing from a two-dimensional map (isoelectrofocusing/sodium dodecyl sulfate electrophoresis) of its (35)S-labeled flagellar proteins. Six of these same proteins are missing in pf1, which lacks spoke-heads. To determine whether any of the missing proteins represent the mutant gene product two experimental approaches have been applied. The first makes use of the fact that gametes of either mutant strain when fused with wild-type gametes to form quadriflagellate dikaryons undergo recovery of flagellar function. Recovery at the molecular level was monitored by prelabeling the mutant proteins with (35)S and allowing recovery to occur in the absence of protein synthesis. It is to be expected that the mutant gene product would not be restored as a radioactive protein and that recovery would depend on the assembly of the wild-type counterpart that is not labeled. The second technique makes use of revertants induced by UV irradiation. Dikaryon rescue in the case of pf14 leads to restoration of 11 radioactive components; only protein 3 fails to appear as a radioactive spot. For pf1 only two radioactive proteins are restored; proteins 4, 6, 9, and 10 were not radioactive. Analysis of revertants of pf1 gave evidence (altered map positions) that protein 4 is the mutant gene product. In the case of pf14, analysis of 22 revertants has not provided similar positive evidence that protein 3 is the gene product.
Alleles↗
Temperature-sensitive mutations affecting flagellar assembly and function in Chlamydomonas reinhardtii.
A series of conditional mutants of the algal, biflagellate Chlamydomonas reinhardtii with temperature-sensitive defects in flagellar assembly and function were isolated. The genetics and phenotypes of 21 mutants displaying a rapid alteration in flagellar function upon shift from the permissive (20 degrees C) to the restrictive (32 degrees C) temperatures are described. These mutants designated as "drop-down" or dd-mutants have been placed in four categories on the basis of their defective phenotypes: (a) dd-assembly mutants - the preformed flagella are resorbed at 32 degrees C and reassembly of flagella is inhibited; (b) dd-fragile flagella mutants - the flagella are lost by detachment at 32 degrees C, but can be reassembled; (c) dd-motility mutants - the flagella are retained at 32 degrees C, but are functionally defective; (d) dd-lethal mutants - display combined defects in flagellar function and cell growth. Tetrad analysis of the mutants back-crossed to wild-type, recombination analysis of intermutant crosses, and complementation tests in the construction of heterozygous diploid strains indicate that at least 14 nuclear genetic loci are represented among 21 mutants. The availability of temperature-sensitive mutations affecting the assembly and function of the flagellum suggests that the morphogenesis of this complex eukaryotic organelle is amenable to genetic dissection.
Chlamydomonas↗
Microtubules and filaments in ciliate contractility.
The basis for cell body contractility in heterotrich ciliates resides in the interaction of two discrete contractile fiber systems, the myonemes and km fibers. The motive force for rapid cell contraction in these ciliates has been associated with a calcium-induced change in the macromolecular conformation of the contractile filaments of the myonemes. In Stentor coeruleus, changes in the contractile state of the myonemes are reflected in a reversible calcium-dependent transformation of thin, oriented filaments to shorter tubular forms. Dimensional changes in the km fibers in Stentor coeruleus are generated by the relative sliding of the component parallel microtubule arrays. Changes in the morphology of cross-bridges extending between the sliding components suggest that these structures function in the process of cell extension either in developing the sliding forces required for active displacement of adjacent microtubule ribbons or regulating the extent to which sliding occurs.
Adenosine Triphosphate↗
The contractile process in the ciliate, Stentor coeruleus. I. The role of microtubules and filaments.
The structural basis for the function of microtubules and filaments in cell body contractility in the ciliate Stentor coeruleus was investigated. Cells in the extended state were obtained for ultrastructural analysis by treatment before fixation with a solution containing 10 mM EGTA, 50-80 mM Tris, 3 mM MgSO(4), 7.5 mM NH(4)Cl, 10 mM phosphate buffer (pH 7.1). The response of Stentor to changes in the divalent cation concentrations in this solution suggests that Ca(+2) and Mg(+2) are physiologically important in the regulation of ciliate contractility. The generation of motive force for changes in cell length in Stentor resides in two distinct longitudinal cortical fiber systems, the km fibers and myonemes. Cyclic changes in cell length are associated with (a) the relative sliding of parallel, overlapping microtubule ribbons in the km fibers, and (b) a distinct alteration in the structure of the contractile filaments constituting the myonemes. The microtubule and filament systems are distinguished functionally as antagonistic contractile elements. The development of motive force for cell extension is accomplished by active microtubule-to-microtubule sliding generated by specific intertubule bridges. Evidence is presented which suggests that active shortening of contractile filaments, reflected in a reversible structural transformation of dense 4-nm filaments to tubular 10-12-nm filaments, provides the basis for rapid cell contraction.
Animals↗
Chemical heterogeneity of protofilaments forming the outer doublets from sea urchin flagella.
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Alkylation↗
Single intravenous dose and steady-state oral dose pharmacokinetics of nicardipine in healthy subjects.
Nicardipine HCl oral doses (10-40 mg) were administered sequentially to six healthy subjects. For each regimen the capsule dose was administered every 8 hours (q 8 h) for 3 days and the plasma profiles of nicardipine and its pyridine analogue (M5) were determined following the last dose on day 4. Steady-state plasma concentrations of nicardipine for each subject were fitted very well by the Michaelis-Menten equation. An intravenous tracer dose (0.885 mg nicardipine HCl) was administered simultaneously with the final oral dose on the fourth day of the 30 mg q 8 h regimen. The steady-state bioavailability of nicardipine was shown to be dose-dependent and averaged 19 per cent (10 mg), 22 per cent (20 mg), 28 per cent (30 mg), and 38 per cent (40 mg). Nicardipine undergoes linear first-pass metabolism to M5. Other metabolic pathways are responsible for the saturable first-pass metabolism observed for nicardipine.
Administration, Oral↗
Socioeconomic status, depression, and health service utilization among adolescent women.
This article examines relationships among socioeconomic status (SES), depression, and health services utilization among 5,735 adolescent women. In cross-sectional analyses, effects of SES on having obtained a routine physical examination and use of psychological/emotional counseling in the past year are examined. Then, longitudinal analyses determine the effects of health service utilization on depression at 1 year follow-up (T2) controlling for baseline depression and SES. SES was associated with medical but not mental health service use. SES and health service use independently predicted T2 depression and an income x baseline depression interaction was noted. The findings and their implications are discussed.
Adolescent↗
GABA and GABA(A) receptor antagonists alter developing cone photoreceptor development in neonatal rabbit retina.
Gamma aminobutyric acid (GABA) has been established as an important developmental signal in a number of regions of the central nervous system (CNS), including retina. Our previous studies have shown that GABAergic horizontal cells act as the initial synaptic target for developing cone photoreceptors in neonatal rabbit retina. Since intraocular injections of the GABA(A) receptor antagonists, picrotoxin or bicuculline, disrupt cone synaptogenesis in vivo, GABA released from horizontal cells may provide a necessary signal for cone axon growth and/or synapse formation. In the current report, we have used cultured retinal explants to examine the effects of GABA(A) receptor antagonists on other aspects of developing cones. These include the distribution pattern of cone cell bodies across the outer surface of the retina and the expression of GABA(A) receptors within both cone cell bodies and axonal processes. Peanut agglutinin (PNA), a plant lectin that specifically labels cone plasma membrane and extracellular matrix, was used to monitor cone development, and a GABA(A) receptor antibody against the beta2/3 subunits of the protein was used to label GABA(A) receptors. Results showed that cones maintained in the explant culture express GABA(A) receptors in a temporal and spatial pattern similar to that observed in vivo, namely a low expression of receptors on cone cell bodies at postnatal day 1 (P1), peaking around P3 and diminishing by P7. Neonatal retinal explants exposed to the GABA(A) receptor antagonists, bicuculline (10 microM) or SR95531 (5 microM), for 24 h in culture showed disruption of the normal distribution of cone cell bodies. When GABA (100 microM) was added along with either antagonist, cone cell bodies appeared normal. Neither bicuculline nor SR95531 alone had any effect on the general morphology of other retinal layers, suggesting that these GABA(A) receptor antagonists at the concentrations used were not acting as nonspecific disruption agents. The effects of GABA antagonists were confined to the first week after birth with no disruption seen in P9 or adult explant cultures. These data provide a direct demonstration of the necessity for GABAergic input to cones during active synaptogenesis. As we have previously shown, GABA(A) receptor activation causes a substantial increase in intracellular calcium concentrations in cones and thereby could provide a mechanism by which GABA regulates cone maturation.
Animals↗
GABA(A) receptor immunoreactivity is transiently expressed in the developing outer retina.
Extensive evidence has suggested a trophic role of gamma-aminobutyric acid (GABA) on developing cone photoreceptors in postnatal retina. In a previous study, we showed that GABA raises intracellular calcium levels in the developing cones via activation of GABA(A) receptors. Using confocal microscopy in conjunction with immunocytochemistry, we have now demonstrated that (1) GABA(A) receptor subunits are localized on cone cell bodies as well as on cone pedicles, indicating that GABA has a direct, rather than indirect, effect on cones and (2) the temporal expression of GABA(A) receptor subunits coincides with the developmental effects of GABA on cone synaptogenesis. An antibody against the beta 2/3 subunits of the GABA(A) receptor and a specific cone marker peanut-agglutinin lectin (PNA) were used to double-label wholemount neonatal retinal preparations. Results show that GABA(A) receptors are transiently expressed on cone photoreceptors in the early stages of postnatal retinal development. GABA (A)receptor immunoreactivity is clearly present on cone cell bodies and their processes and on other--as yet unidentified--elements (horizontal cells?) in the outer plexiform layer. Immunoreactivity decreases within cone photoreceptor somata after postnatal day 5, but persists in the processes of the outer plexiform layer until day 7. Our results provide support for the hypothesis that GABA acts as an important developmental regulator of cone photoreceptor maturation.
Animals↗
NMR structure and mutagenesis of the Fas (APO-1/CD95) death domain.
Programmed cell death (apoptosis) mediated by the cytokine receptor Fas is critical for the removal of autoreactive T cells, the mechanism of immune privilege, and for maintenance of immune-system homeostasis. Signalling of programmed cell death involves the self-association of a conserved cytoplasmic region of Fas called the death domain and interaction with another death-domain-containing protein, FADD (also known as MORT1). Although death domains are found in several proteins, their three-dimensional structure and the manner in which they interact is unknown. Here we describe the solution structure of the Fas death domain, as determined by NMR spectroscopy. The structure consists of six antiparallel, amphipathic alpha-helices arranged in a novel fold. From the structure and from site-directed mutagenesis, we have identified the region of the death domain involved in self-association and binding to the downstream signalling partner FADD.
Amino Acid Sequence↗
Spermatogenesis in early and chronic phases of experimental spinal cord injury in the rodent model.
A rodent model was used to study the degree and dynamics of potential spermatogenic alterations during both acute and chronic phases after experimental spinal cord injury (SCI). Sexually mature Sprague-Dawley rats underwent controlled impact SCI by exposure of the thoracic spine, T-10 laminectomy, and intraoperative somatosensory-evoked potential latency and amplitude. A 50 gm-cm SCI was produced in 35 experimental subjects. Sham surgery was performed on 16 control subjects through exposure of the dura without weight drop. SCI was verified by obliteration of the somatosensory-evoked potential following injury and subsequent neurologic assessment (modified hindlimb Tarlov scale) 4 weeks after injury. Flow cytometry with acridine orange as the DNA probe was used to measure potential spermatogenic alterations in testicular cell development and integrity of epididymal sperm chromatin structure between 2 and 20 weeks following SCI. Flow cytometric analysis revealed that nine of the 35 SCI animals demonstrated altered spermatogenesis; it is not clear whether these effects are specific or nonspecific stress related. These responder animals contributed to dramatic differences in relative percent testicular haploid cells (spermatids) and concurrent differences in percent diploid cells at 2, 4, 8, 12, and 16 weeks. Percentages within the three spermatid populations (round, elongating, and elongated) also differed at these time points. The sperm chromatin structure assay (SCSA) revealed significant epididymal sperm nuclear structure differences at 2, 4, and 12 weeks (P < 0.001). These findings are in concordance with our clinical observations of spermatogenesis in spinal cord injured men and suggest that significant spermatogenic deficit may occur, even in the early phase of injury.
Acute Disease↗
[Isolation and expression profiling of the Pto-like gene SsPto from Solanum surattense].
A novel Pto-like gene (designated as SsPto) is cloned from yellow-fruit nightshade (Solanum surattense). The full-length cDNA of SsPto is 1331 bp long with an open reading frame of 960 bp encoding a polypeptide of 320 amino acid residues. The deduced SsPto protein has a calculated molecular weight of 36.21 kDa with an isoelectric point of 6.18. Multiple sequence alignment shows that SsPto protein shares 71.4% and 71.6% identities to Pto proteins from Lycopersicon pimpinellifolium and L. hirsutum respectively. Genomic Southern blot analysis indicates the presence of a small family of SsPto in the S. surattense genome. SsPto is found to be constitutively expressed in the S. surattense plant with the highest expression in stems. However, under induction by TMV for 6 days, SsPto expresses the highest in roots. Further expression analysis reveals that the signaling components of defense/stress pathways, such as methyl jasmonate (MeJA), salicylic acid (SA), gibberellic acid (GA3) and hydrogen peroxide (H2O2), up-regulate the SsPto transcript levels over the control. Cold treatment, nevertheless, has no significant effect on SsPto expression whereas SsPto expression is down-regulated by dark treatment. Our findings suggest that this novel stress- and pathogen-inducible SsPto from S. surattense may participate not only in the defense/stress responsive pathways, but also in diverse processes of plant's growth and development.
Amino Acid Sequence↗
Niemann-Pick disease type C presenting as neonatal hepatitis: report of one case.
A three-month old Chinese male infant was a victim of neonatal hepatitis presenting with prolonged jaundice, poor body weight gain, progressive hepatosplenomegaly and extremely elevated serum alpha-fetoprotein level. Niemann-Pick disease (NPD) type C was confirmed by autopsy, which revealed sphingolmyelin deposition in multiple visceral organs, and normal sphingomyelinase activity in liver. This is the first case of NPD type C in Taiwan. In idiopathic neonatal hepatitis with hepatosplenomegaly here, NPD type C must be taken into consideration.
Hepatitis↗
Hemolytic disease of the newborn due to anti-Di(a): report of one case.
In the Diego blood group, the frequency of the Di(a + b +) or Di(a + b -) phenotype among Chinese in Taiwan is estimated to be 3.2%. Here we report a case of severe hemolytic disease caused by anti-Di(a). The baby's total bilirubin elevated to 23 mg/dl at the age of 72 hours. A blood exchange transfusion and phototherapy were performed. We suggest to include Diego positive cell panels in testing antibody specificities that are likely to be encountered in this population.
Blood Group Antigens↗
A system linking occupation history questionnaire data and magnetic field monitoring data.
A method is presented which links on-site electromagnetic field monitoring data with pre-existing work history data. The linkage is used to estimate cumulative and average annualized magnetic field exposure for a case-control study. On-site electromagnetic field monitoring data for 1,966 volunteer utility employees, at 59 sites in the United States and three other countries, were obtained from a large project (the EMDEX project) designed to collect, analyze, and document 60-Hz electric and magnetic field exposures for a diverse population. These data represent 9 primary work environments, and 16 job classification categories, amounting to 144 unique job categories which were consolidated using the job-exposure matrix presented into 282 three-digit Dictionary of Occupational Title (DOT) codes. The DOT code categories were then linked to lifetime occupational histories from a case-control study of leukemia. The method may be extended to link additional job titles with monitoring information. Job titles linked with electromagnetic field monitoring information provide more specific estimates of exposure intensity than previous ordinal estimates of exposure. Therefore, estimates of cumulative electromagnetic field exposure are achievable, as well as high and low level exposure estimates.
Case-Control Studies↗