Surgical pearl: the cross-stitch.
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Humans↗
Biomedical subjects
T S Wang
Publications and source records attributed to T S Wang.
Role of fission yeast primase catalytic subunit in the replication checkpoint.
To investigate the cell cycle checkpoint response to aberrant S phase-initiation, we analyzed mutations of the two DNA primase subunit genes of Schizosaccharomyces pombe, spp1(+) and spp2(+) (S. pombe primase 1 and 2). spp1(+) encodes the catalytic subunit that synthesizes the RNA primer, which is then utilized by Polalpha to synthesize the initiation DNA. Here, we reported the isolation of the fission yeast spp1(+) gene and cDNA and the characterization of Spp1 protein and its cellular localization during the cell cycle. Spp1 is essential for cell viability, and thermosensitive mutants of spp1(+) exhibit an allele-specific abnormal mitotic phenotype. Mutations of spp1(+) reduce the steady-state cellular levels of Spp1 protein and compromised the formation of Polalpha-primase complex. The spp1 mutant displaying an aberrant mitotic phenotype also fails to properly activate the Chk1 checkpoint kinase, but not the Cds1 checkpoint kinase. Mutational analysis of Polalpha has previously shown that activation of the replication checkpoint requires the initiation of DNA synthesis by Polalpha. Together, these have led us to propose that suboptimal cellular levels of polalpha-primase complex due to the allele-specific mutations of Spp1 might not allow Polalpha to synthesize initiation DNA efficiently, resulting in failure to activate a checkpoint response. Thus, a functional Spp1 is required for the Chk1-mediated, but not the Cds1-mediated, checkpoint response after an aberrant initiation of DNA synthesis.
Amino Acid Sequence↗
Fission yeast Rad17 associates with chromatin in response to aberrant genomic structures.
Fission yeast checkpoint protein Rad17 is required for the DNA integrity checkpoint responses. A fraction of Rad17 is chromatin bound independent of the other checkpoint proteins throughout the cell cycle. Here we show that in response to DNA damage induced by either methyl methanesulfonate treatment or ionizing radiation, increased levels of Rad17 bind to chromatin. Following S-phase stall induced by hydroxyurea or a cdc22 mutation, the chromatin-bound Rad17 progressively dissociates from the chromatin. After S-phase arrest by hydroxyurea in cds1Delta or rad3Delta cells or by replication mutants, Rad17 remains chromatin bound. Rad17 is able to complex in vivo with an Rfc small subunit, Rfc2, but not with Rfc1. Furthermore, cells with rfc1Delta are checkpoint proficient, suggesting that Rfc1 does not have a role in checkpoint function. A checkpoint-defective mutant protein, Rad17(K118E), which has similar nuclear localization to that of the wild type, is unable to bind ATP and has reduced ability in chromatin binding. Mutant Rad17(K118E) protein also has reduced ability to complex with Rfc2, suggesting that Lys(118) of Rad17 plays a role in Rad17-Rfc small-subunit complex formation and chromatin association. However, in the rad17.K118E mutant cells, Cds1 can be activated by hydroxyurea. Together, these results suggest that Rad17 binds to chromatin in response to an aberrant genomic structure generated from DNA damage, replication mutant arrest, or hydroxyurea arrest in the absence of Cds1. Rad17 is not required to bind chromatin when genomic structures are protected by hydroxyurea-activated Cds1. The possible checkpoint events induced by chromatin-bound Rad17 are discussed.
Cell Cycle Proteins↗
Low triglyceride levels affect calculation of low-density lipoprotein cholesterol values.
The Friedewald formula for the calculation of low-density lipoprotein cholesterol (LDL-C) values is fairly accurate provided the triglyceride value is less than 400 mg/dL. It is not clear whether the estimation is also valid in the presence of low triglyceride and high cholesterol levels. We describe herein a patient with a low triglyceride value of approximately 50 mg/dL, a high cholesterol level, and a discrepant LDL-C level. The LDL-C level using the Friedewald calculation turned out to be much higher than the LDL-C level using direct measurement. We, therefore, suggest that in the presence of low triglyceride and high cholesterol levels, the LDL-C level should be measured directly instead of using the Friedewald calculation.
Cholesterol, LDL↗
The forehead flap for nasal reconstruction.
CONTEXT: Reconstruction of extensive nasal defects often represents a significant challenge owing to several unique qualities of the nose, such as complex topography, mobile free margins, and multiple nasal subunits. Furthermore, loss of internal nasal lining and/or structural skeletal support may be present following removal of extensive skin cancers. OBJECTIVE: To describe our experience with the use of forehead flap reconstruction for extensive nasal defects. DESIGN: Retrospective case series. SETTING: Academic health care hospital system. PATIENTS/INTERVENTION: One hundred forty-seven patients with extensive nasal defects repaired with a forehead flap. MAIN OUTCOME MEASURES: The functional and aesthetic results were assessed. The characteristics of defects repaired with the forehead flap and the need for lining and/or cartilage were examined. RESULTS: The forehead flap was used to repair 147 nasal defects after Mohs excision of nonmelanoma skin cancer. Full-thickness skin was lost in all cases, structural skeletal support in 68 cases (46%), and internal mucosal lining in 45 cases (31%). Our experience and surgical technique using the forehead flap are described. CONCLUSIONS: The forehead flap represents one of the best methods for repair of extensive nasal defects. Near-normal functional and cosmetic results can be achieved.
Adult↗
Inhibition of nitric oxide release by an aqueous extract of Tinospora tuberculata.
An aqueous extract of Tinospora tuberculata stems was found to scavenge nitric oxide (NO) in vitro in both cell and cell-free systems. When the aqueous extract was added to lipopolysaccharide-stimulated murine macrophages, it inhibited NO release dose-dependently, and similar activity was found in a cell-free system using sodium nitroprusside as a NO donor. These findings may help to explain, in part, certain pharmacological activities of Tinospora tuberculata.
Animals↗
The preclinical pharmacologic study of dopamine transporter imaging agent [99mTc]TRODAT-1.
The purpose of this study was to investigate the pharmacologic characteristics of TRODAT-1 (2beta-((N,N'-bis(2-mercaptoethyl)ethylene diamino)methyl), 3beta-(4-chlorophenyl)tropane) labeled with [99mTc] as an imaging agent for dopamine transporter (DAT). Radiochemical purity of [99mTc]TRODAT-1 was over 90%. The partition coefficients in octanol and buffer were 2.12 and 2.19 at pH 7.0 and 7.4, respectively. Animal studies have been performed in rats, rabbits, and normal and hemi-Parkinsonian model monkeys. Biodistribution displayed moderate uptake in rat brain (0.28 %ID/organ at 2 min) and the striatal uptake was 0.193, 0.142, and 0.136 %ID/g at 2, 60, and 120 min, respectively. The ratios of striatal/cerebellar (ST/CB) uptake were 2.4, 4.45, and 2.45 %ID/g at 60, 120, and 240 min, respectively. The major radioactivity was excreted by the hepatobiliary system. Blood clearance kinetics was performed in rabbits, and the initial half-life of 1.18 min and late half-life of 367.8 min were obtained. Brain single photon emission computed tomography imaging studies in normal monkeys showed the ratios of ST/CB uptake were 1.56-2.0 %ID/g and indicated that both uptake and retention in the striatal area were associated with the DAT. The imaging of hemi-Parkinsonian model monkeys also displayed the expected selectivity, the highest uptake being observed in the basal ganglia area of the normal side. Thereby, it is suggested that [99mTc]TRODAT-1 is a safe and useful imaging agent for localization of the presynaptic DAT in the brain.
Animals↗
Nasal reconstruction utilizing a muscle hinge flap with overlying full-thickness skin graft.
BACKGROUND: Deep nasal defects of the dorsum, sidewall, and ala can be challenging to repair. OBJECTIVE: The article describes our experience with a muscle hinge transposition flap with overlying local full-thickness skin grafting for repair of deep nasal defects in a single-stage procedure. METHODS: A muscle hinge transposition flap with overlying local full-thickness skin grafting was used immediately after Mohs micrographic surgery to repair 12 deep nasal defects of the dorsum, sidewall, alar lobule, and supratip. RESULTS: No cases of infection, flap, or graft necrosis occurred in our series. Cosmetic and functional outcomes were judged from good to excellent by patient and surgeon. To enhance the cosmetic outcome, 5 patients underwent spot dermabrasion within 2 months of repair. CONCLUSION: For properly selected small to medium-sized deep nasal defects (1-2 cm) that lack a sufficiently loose adjacent tissue reservoir for a single-stage local flap, a muscle hinge transposition flap with local full-thickness skin grafting can provide consistently satisfying aesthetic and functional results.
Basal Cell Carcinoma↗
Analysis of fission yeast primase defines the checkpoint responses to aberrant S phase initiation.
To investigate the checkpoint response to aberrant initiation, we analyzed the cell cycle checkpoint response induced by mutations of Schizosaccharomyces pombe DNA primase. DNA primase has two subunits, Spp1 and Spp2 (S. pombe primases 1 and 2). Spp1 is the catalytic subunit that synthesizes the RNA primer, which is then extended by DNA polymerase alpha (Polalpha) to synthesize an initiation DNA structure, and this catalytic function of Polalpha is a prerequisite for generating the S-M phase checkpoint. Here we show that Spp2 is required for coupling the function of Spp1 to Polalpha. Thermosensitive mutations of spp2(+) destabilize the Polalpha-primase complex, resulting in an allele-specific S phase checkpoint defect. The mutant exhibiting a more severe checkpoint defect also has a higher extent of Polalpha-primase complex instability and deficiency in the hydroxyurea-induced Cds1-mediated intra-S phase checkpoint response. However, this mutant is able to activate the Cds1 response to S phase arrest induced by temperature. These findings suggest that the Cds1 response to the S-phase arrest signal(s) induced by a initiation mutant is different from that induced by hydroxyurea. Interestingly, a polalphats mutant with a defective S-M phase checkpoint and an spp2 mutant with an intact checkpoint have a similar Polalpha-primase complex stability, and the Cds1 response induced by hydroxyurea or by the mutant arrests at the restrictive temperature. Thus, the Cds1-mediated intra-S phase checkpoint response induced by hydroxyurea can also be distinguished from the S-M phase checkpoint response that requires the initiation DNA synthesis by Polalpha.
Alleles↗
A novel mutant allele of the chromatin-bound fission yeast checkpoint protein Rad17 separates the DNA structure checkpoints.
To further dissect the genetic differences between the checkpoint pathway following S-phase cdc arrest versus DNA damage, a genetic screen was performed for checkpoint mutants that were unable to arrest mitosis following cell-cycle arrest with a temperature-sensitive DNA polymerase delta mutant, cdc20-M10. One such checkpoint mutant, rad17-d14, was found to display the cut phenotype following S-phase arrest by cdc20-M10, but not by the DNA synthesis inhibitor hydroxyurea, reminiscent of the chk1 mutant. Unlike chk1 , rad17-d14 was not sensitive to UV irradiation. Interestingly, the ionising radiation sensitivity of rad17-d14 was only at higher doses, and cells were found to be defective in properly arresting cell division following irradiation in S phase, but not G(2) phase. Biochemical analysis attributes the checkpoint defects of rad17-d14 to the failure to phosphorylate the checkpoint effector Chk1p. To investigate if Rad17p monitors the genome for abnormal DNA structures specifically during DNA synthesis, chromatin association of Rad17p was analysed. Rad17p was found to be chromatin associated throughout the cell cycle, not just during S phase. This interaction occurred irrespective of the arrest with cdc20-M10 and, surprisingly, was also independent of the other checkpoint Rad proteins, and the cell-cycle effectors Chk1p and Cds1p.
Alleles↗
Analysis of relative binding affinity of E7-pRB of human papillomavirus 16 clinical variants using the yeast two-hybrid system.
A number of genotypes of the human papillomaviruses (HPV) are associated with malignancies of the uterine cervix. Sequencing work has revealed the existence of intratype HPV variants with minor differences in the nucleotide sequence. More recent data suggest the possibility that some of the variants may have different modes of clinical manifestation. In this study, sequences of the E6 and E7 oncogenes of 17 HPV16 isolates derived from PAP smear samples of Taiwanese patients were analyzed. A number of E6 and E7 novel variants were found. Particularly, a prevalent (64.7%) E6 polymorphic site A442C with an E113D amino acid substitution seems specific to Taiwanese patients. In E7, two novel but silent polymorphic sites G663A (41.2%) and T846C (88.2%) were also prevalent in the samples analyzed. The yeast two-hybrid system was adopted for rapid assessment of relative E7-pRb binding affinity in the variants. The relative binding affinities of the E7 proteins of different HPV types to pRB were in close agreement with previous biochemical data. A T663G/C24W polymorphic change in E7 correlated with a decrease in E7-pRb relative binding affinity the significance of which remains to be clarified. This semi-quantitative biochemical and genetic approach may be useful as a first step in the development of clinical protocols for the screening and identification of important HPV variants for clinical interpretation and for further functional analysis by transfection or other bioassays.
Female↗
The C-terminal region of Schizosaccaromyces pombe proliferating cell nuclear antigen is essential for DNA polymerase activity.
Proliferating cell nuclear antigen (PCNA), the processivity factor (sliding clamp) of DNA polymerases (Pols), plays essential roles in DNA metabolism. In this report, we examined the functional role of the C-terminal region of Schizosaccaromyces pombe PCNA both in vitro and in vivo. The deletion or Ala substitution of the last 9 aa (252-260A), as well as Ala replacement of only 4 aa (252-255A) at the C terminus, failed to substitute for the wild-type PCNA protein for cell growth in S. pombe. Two other PCNA mutant proteins, A251V and K253E, exhibited cold-sensitive phenotypes. Several yeast strains harboring mutations, including those at the acidic C-terminal region, showed elevated sensitivity to DNA damage. The ability of the mutant PCNA proteins to stimulate DNA synthesis by Poldelta and Polepsilon also was studied in vitro. The mutant proteins that did not support cell growth and a mutant protein containing a single amino acid substitution at position 252, where Pro is replaced by Ala, stimulated Poldelta and Polepsilon activities poorly. All mutant PCNA proteins, however, were assembled around DNA by the clamp loader, replication factor C, efficiently. Thus, the C-terminal region of PCNA is important for interactions with both Poldelta and Polepsilon and for cell survival after DNA damage. The C terminus of sliding clamps from other organisms has been shown to be important for clamp loading as well as polymerase interactions. The relationship between the conserved sequence in this region in different organisms is discussed.
Amino Acid Sequence↗
Intrathymic immunomodulation and the "infectious" tolerance pathway in allograft recipients.
BACKGROUND: We have previously shown that >75% of LEW cardiac allografts survive indefinitely in BUF rats pretreated at Day -21 intrathymically (IT) with donor alloantigen in conjunction with a single intravenous dose of ALS. Spleen cells can adoptively transfer the tolerant state to new cohorts of test recipients. This study was designed to analyze cellular and humoral events contributing to the "infectious" tolerance pathway in this model. METHODS: Spleen cells (25 x 10(6)) harvested from BUF recipients bearing long-term cardiac allografts were injected intravenously into lightly irradiated (450 R) secondary BUF rats, followed 24 h later by transplantation of LEW of ACl hearts. Cardiac allografts were then analyzed serially by reverse transcription polymerase chain reaction for Th1 and Th2 cytokine gene expression. Donor-specific IgM and IgG alloantibody responses in host serum were screened by flow cytometry. RESULTS: Transfer of regulatory spleen cells harvested between Days 80 and 140 from tolerant hosts induced tolerance to heart grafts in a donor-specific manner. In the early posttransplant period, selective sparing of Th2 cytokines was noted. Adoptively transferred hosts showed overall depression of IgM, but a vigorous IgG1 and IgG2a alloantibody response. CONCLUSION: IT + ALS-induced tolerance can be transferred in a donor-specific "infectious" manner to new cohorts of engrafted recipients. The development of tolerance is nontemporally bound, and associates with an early Th2-type immune deviation at the graft site. The elevated levels of T cell-dependent IgG1 and IgG2 may interfere with the antigen reactivity and alloresponsive effector functions, contributing to graft acceptance in the "infectious" tolerance pathway.
Animals↗
Fanconi anemia C protein acts at a switch between apoptosis and necrosis in mitomycin C-induced cell death.
Deregulation of apoptosis seems to be a hallmark of the Fanconi anemia (FA) syndrome. In order to further define the role of the FA protein from complementation group C (FAC) in apoptosis, we characterized parameters modified during the mitomycin-C (MMC)-induced apoptotic program. It is shown that despite a higher level of cell death for FA compared to normal lymphoblasts after MMC treatment, FA cells do not display a marked DNA fragmentation. Furthermore, while playing a central role in MMC apoptosis of normal lymphoblasts, the activity of caspase-3-like proteases is altered in FA cells. Interestingly, the disruption of the mitochondrial transmembrane potential (Deltapsi), an early event that can lead to apoptotic or to necrotic death, is accomplished similarly in FA and in normal cells. Finally, it is shown that the overexpressed FAC protein inhibited the apoptotic steps, with the exception of the decrease of the Deltapsi. Altogether, our results indicate that the FAC protein acts at a step preceding the activation of the caspases and after the modification of the Deltapsi, a decision point at which cells can be pushed toward either apoptosis or necrosis and which, consequently, regulates the balance between the two modes of cell death.
Apoptosis↗
Human papillomavirus DNA replication. Interactions between the viral E1 protein and two subunits of human dna polymerase alpha/primase.
Papovaviruses are valuable models for the study of DNA replication in higher eukaryotic organisms, as they depend on host factors for replication of their DNA. In this study we investigate the interactions between the human papillomavirus type 11 (HPV-11) origin recognition and initiator protein E1 and human polymerase alpha/primase (pol alpha/primase) subunits. By using a variety of physical assays, we show that both 180- (p180) and 70-kDa (p70) subunits of pol alpha/primase interact with HPV-11 E1. The interactions of E1 with p180 and p70 are functionally different in cell-free replication of an HPV-11 origin-containing plasmid. Exogenously added p180 inhibits both E2-dependent and E2-independent cell-free replication of HPV-11, whereas p70 inhibits E2-dependent but stimulates E2-independent replication. Our experiments indicate that p70 does not physically interact with E2 and suggest that it may compete with E2 for binding to E1. A model of how E2 and p70 sequentially interact with E1 during initiation of viral DNA replication is proposed.
Animals↗
Schizosaccharomyces pombe replication and repair proteins: proliferating cell nuclear antigen (PCNA).
Schizosaccharomyces pombe has a cell cycle progression with distinctive phases that serves as a perfect model system for investigating DNA replication and repair of eukaryotic cells. Here, we use proliferating cell nuclear antigen (PCNA) of S. pombe to demonstrate how the function of this protein in both DNA replication and repair can be assessed by genetic and biochemical approaches. We describe a method of introducing site-specific mutations into the fission yeast PCNA gene pcn1(+). The in vivo effects of these pcn1 mutants in a strain with a null pcn1 background are described and their in vitro biochemical properties are characterized. Mutants described here are those that are defective in enhancing processivity of DNA polymerase delta, show temperature-sensitive growth, and have increased sensitivity to hydroxyurea (HU), UV and gamma irradiation, and methyl methanesulfonate (MMS). Three mutants that show reduced growth rate in vivo and decreased capacity to enhance polymerase delta DNA synthetic activity and processivity in vitro-pcn1-1, pcn1-5, and pcn1-26-are described as examples of using a genetic approach to identify the biochemical function of replication proteins. One cold-sensitive growth allele, pcn1-3, that has a recessive cold-sensitive cdc phenotype and shows sensitivity to HU and UV and gamma irradiation is used as an example of using the genetic approach to reveal the function of replication proteins in repair. The power of combining both biochemical and genetic disciplines is emphasized. Methods for site-directed mutagenesis, in vitro analysis of mutant proteins, and in vivo characterization of mutants in response to UV or gamma irradiation, MMS, HU, and temperature, as well as genetic epistasis are described. Locations of functionally significant residues on the PCNA tertiary structure are summarized.
Cell Cycle↗
The birhombic transposition flap for soft tissue reconstruction.
BACKGROUND: Soft tissue reconstruction requires a thorough understanding of both anatomy and tissue movement. Flaps and grafts should be considered when simpler closure methods result in excessive tension or distortion of surrounding structures. OBJECTIVE: We describe our experience with the birhombic transposition flap. METHODS: The birhombic flap was used to repair 19 surgical defects after excision of skin cancer. RESULTS: The average defect size was 3.0 x 2.4 cm with the 19 wounds located on the nose (6), forearm (5), hand (4), forehead/temple (3), and preauricular cheek (1). No cases of excessive tension, infection, or flap necrosis occurred. CONCLUSION: Conceptualization of a larger defect into multiple smaller defects may be useful to facilitate closure of each smaller defect in an easier fashion. Our series demonstrates the usefulness of this concept with the birhombic transposition flap repair.
Humans↗
Isolated extragenital bowenoid papulosis of the neck.
We report a case of extragenital bowenoid papulosis (BP) in a healthy immunocompetent 42-year-old man. The lesions occurred on the anterolateral aspects of the neck and were not associated with genital, oral, or periungual lesions. Lesional skin tested positive with the Digene hybrid capture system cocktail assay that identifies infection with a mixture of high to intermediate oncogenic human papillomavirus (HPV) types, including types 16, 18, 31, 33, 35, 45, 51, 52, and 56. This cocktail assay identifies infection with HPV types typically associated with high-grade squamous intraepithelial lesions and invasive carcinoma. This case represents the sixth case of isolated cutaneous BP occurring a significant distance from the genital region.
Adult↗