In Vitro Assessment of Dimensional Accuracy of Three Different Types of Interocclusal Recording Materials.

AIM: The aim of the current study was to evaluate the dimensional accuracy of three various interocclusal recording materials. MATERIALS AND METHODS: A total of 90 disc-shaped samples were prepared using polyether paste, wax, and polyvinyl siloxane material with the support of stainless steel die. For the purposes of this investigation, three frequently utilized interocclusal recording materials were chosen, and 30 samples from each material were prepared. Group I: Bite registration using polyether paste; Group II: Bite registration using wax; Group III: Bite registration using polyvinyl siloxane material. No samples were exposed to direct sunlight during storage and were kept at room temperature. Using a 10x universal measuring microscope, each sample disc was examined for the presence of horizontal and vertical lines inscripted in the die. For each of the samples, readings were taken at different time periods of 24, 48, and 72 hours. RESULTS: After 24 hours, the less dimensional changes were found in polyether paste group (0.11 ± 0.07) followed by polyvinyl siloxane material group (0.19 ± 0.04) and wax group (0.25 ± 0.12). After 48 hours, the less dimensional changes were found in polyether paste group (0.34 ± 0.02) followed by polyvinyl siloxane material group (0.42 ± 0.01) and wax group (0.94 ± 0.12). After 72 hours, the least dimensional changes were found in polyether paste group (0.46 ± 0.14) followed by polyvinyl siloxane material group (0.92 ± 0.03) and wax group (1.14 ± 0.09). CONCLUSION: The present study concluded that both the material and time factors had an impact on dimensional stability. The most dimensionally stable group was the polyether paste group, which was followed by the polyvinyl siloxane and wax material groups. CLINICAL SIGNIFICANCE: Interocclusal recording material records the occlusal connection between real or artificial teeth for occlusal rehabilitation planning and for creating removable and fixed dentures. The creation of a clinically acceptable prosthesis is dependent upon the accuracy of the patient's diagnostic or working casts and the interocclusal record. How to cite this article: Sonkesriya S, Subramanian D, Saha P, et al. In Vitro Assessment of Dimensional Accuracy of Three Different Types of Interocclusal Recording Materials. J Contemp Dent Pract 2023;24(12):936-939.

A randomized, double blind, placebo controlled, multicenter clinical trial to assess the efficacy and safety of Emblica officinalis extract in patients with dyslipidemia.

BACKGROUND: Dyslipidemia is one of the most frequently implicated risk factors for development of atherosclerosis. This study evaluated the efficacy of amla (Emblica officinalis) extract (composed of polyphenols, triterpenoids, oils etc. as found in the fresh wild amla fruit) in patients with dyslipidemia. METHODS: A total of 98 dyslipidemic patients were enrolled and divided into amla and placebo groups. Amla extract (500 mg) or a matching placebo capsule was administered twice daily for 12 weeks to the respective group of patients. The patients were followed up for 12 weeks and efficacy of study medication was assessed by analyzing lipid profile. Other parameters evaluated were apolipoprotein B (Apo B), apolipoprotein A1 (Apo A1), Coenzyme Q10 (CoQ10), high-sensitive C-reactive protein (hsCRP), fasting blood sugar (FBS), homocysteine and thyroid stimulating hormone (TSH). RESULTS: In 12 weeks, the major lipids such as total cholesterol (TC) (p = 0.0003), triglyceride (TG) (p = 0.0003), low density lipoprotein cholesterol (LDL-C) (p = 0.0064) and very low density lipoprotein cholesterol (VLDL-C) (p = 0.0001) were significantly lower in amla group as compared to placebo group. Additionally, a 39% reduction in atherogenic index of the plasma (AIP) (p = 0.0177) was also noted in amla group. The ratio of Apo B to Apo A1 was reduced more (p = 0.0866) in the amla group as compared to the placebo. There was no significant change in CoQ10 level of amla (p = 0.2942) or placebo groups (p = 0.6744). Although there was a general trend of FBS reduction, the numbers of participants who may be classified as pre-diabetes and diabetes groups (FBS > 100 mg/dl) in the amla group were only 8. These results show that the amla extract used in the study is potentially a hypoglycaemic as well. However, this needs reconfirmation in a larger study. CONCLUSIONS: The Amla extract has shown significant potential in reducing TC and TG levels as well as lipid ratios, AIP and apoB/apo A-I in dyslipidemic persons and thus has scope to treat general as well as diabetic dyslipidemia. A single agent to reduce cholesterol as well as TG is rare. Cholesterol reduction is achieved without concomitant reduction of Co Q10, in contrast to what is observed with statins. TRIAL REGISTRATION: Registered with Clinical Trials Registry- India at www.ctri.nic.in (Registration number: CTRI/2015/04/005682 ) on 8 April 2015 (retrospectively registered).

Comparison of expectation and satisfaction among new and existing denture wearers and correlation of duration of previous denture-wearing experience to satisfaction in completely edentulous patients: A pilot study.

AIM: The aim of the study was to evaluate the patients' expectation and satisfaction with complete dentures before and after the treatment concerning retention, mastication, phonetics, esthetics, and comfort among the first-time denture wearers and already denture wearers and to find the correlation between duration of denture-wearing experience and satisfaction. SETTINGS AND DESIGN: Cross sectional survey . MATERIALS AND METHODS: A questionnaire was given to the patient before the onset of the treatment, and ratings were given by the patient for expectation regarding retention, mastication, phonetics, esthetics, and comfort. Patient-related variables regarding previous denture experience and duration of wearing were also recorded. After the treatment was completed, the patient was asked to complete the same questionnaire to assess the satisfaction. STATISTICAL ANALYSIS USED: Descriptive statistics, frequency and percentage analysis, Wilcoxon signed- rank test, Mann-Whitney U-test. RESULTS: Expectation and satisfaction were met for all the variables except mastication (P = 0.004) for first-time wearers and except mastication (P = 0.001) and comfort (P = 0.007) among existing denture wearers. However, no significant correlation was elicited between patient expectation and satisfaction in both these groups as the overall mean satisfaction percentage was similar. A clinical significance of P = 0.037 was seen with respect to the duration of denture-wearing experience among existing wearers. Regression model analysis showed a decreased satisfaction with increased duration of wearing (r = 0.396). CONCLUSIONS: Satisfaction and expectation were found to be almost the same irrespective of whether they are first-time wearers or existing wearers. With the increase in the duration of denture-wearing experience, the satisfaction of the patients decreased.

Interplay between TAp73 Protein and Selected Activator Protein-1 (AP-1) Family Members Promotes AP-1 Target Gene Activation and Cellular Growth.

Unlike p53, which is mutated at a high rate in human cancers, its homologue p73 is not mutated but is often overexpressed, suggesting a possible context-dependent role in growth promotion. Previously, we have shown that co-expression of TAp73 with the proto-oncogene c-Jun can augment cellular growth and potentiate transactivation of activator protein (AP)-1 target genes such as cyclin D1. Here, we provide further mechanistic insights into the cooperative activity between these two transcription factors. Our data show that TAp73-mediated AP-1 target gene transactivation relies on c-Jun dimerization and requires the canonical AP-1 sites on target gene promoters. Interestingly, only selected members of the Fos family of proteins such as c-Fos and Fra1 were found to cooperate with TAp73 in a c-Jun-dependent manner to transactivate AP-1 target promoters. Inducible expression of TAp73 led to the recruitment of these Fos family members to the AP-1 target promoters on which TAp73 was found to be bound near the AP-1 site. Consistent with the binding of TAp73 and AP-1 members on the target promoters in a c-Jun-dependent manner, TAp73 was observed to physically interact with c-Jun specifically at the chromatin via its carboxyl-terminal region. Furthermore, co-expression of c-Fos or Fra1 was able to cooperate with TAp73 in potentiating cellular growth, similarly to c-Jun. These data together suggest that TAp73 plays a vital role in activation of AP-1 target genes via direct binding to c-Jun at the target promoters, leading to enhanced loading of other AP-1 family members, thereby leading to cellular growth.

Cutting edge: FAS (CD95) mediates noncanonical IL-1ß and IL-18 maturation via caspase-8 in an RIP3-independent manner.

Fas, a TNF family receptor, is activated by the membrane protein Fas ligand expressed on various immune cells. Fas signaling triggers apoptosis and induces inflammatory cytokine production. Among the Fas-induced cytokines, the IL-1ß family cytokines require proteolysis to gain biological activity. Inflammasomes, which respond to pathogens and danger signals, cleave IL-1ß cytokines via caspase-1. However, the mechanisms by which Fas regulates IL-1ß activation remain unresolved. In this article, we demonstrate that macrophages exposed to TLR ligands upregulate Fas, which renders them responsive to receptor engagement by Fas ligand. Fas signaling activates caspase-8 in macrophages and dendritic cells, leading to the maturation of IL-1ß and IL-18 independently of inflammasomes or RIP3. Hence, Fas controls a novel noncanonical IL-1ß activation pathway in myeloid cells, which could play an essential role in inflammatory processes, tumor surveillance, and control of infectious diseases.

Evaluation of the Impact of Different Surface Treatments on Implant Surface: An In Vitro Study.

Aim: The current study was conducted to assess the effects of various surface treatments on the implant surface. Materials and Methods: A total of 45 dental implants measuring 16 mm in length and 5 mm in diameter were used, along with the appropriate abutments. Depending on the surface treatment used, the samples were randomly split into three groups, with 15 implants in each group: Group 1: Control, Group 2: UV light-treated, Group 3: Sandblasted and acid-etched (SLA)-treated. After surface treatment, a scanning electron microscope (SEM) was used to assess the test samples' surface properties. All sample images were captured using a 3000× magnification. After all three groups' surfaces were treated, the surface roughness was measured using a digital optical profilometer with a stylus speed of 0.5 mm/s that was connected to computer software. Results: The maximum surface roughness was found in the group treated with SLA (0.714 ± 0.12), followed by the group treated with UV light (0.692 ± 0.09) and the control group (0.516 ± 0.12). There was a significant difference found between different surface treatment methods. Conclusion: The present study concluded that the group that received the SLA treatment had the highest surface roughness when compared to the UV light and control groups.

Singapore Undiagnosed Disease Program: Genomic Analysis aids Diagnosis and Clinical Management.

OBJECTIVE: Use next-generation sequencing (NGS) technology to improve our diagnostic yield in patients with suspected genetic disorders in the Asian setting. DESIGN: A diagnostic study conducted between 2014 and 2019 (and ongoing) under the Singapore Undiagnosed Disease Program. Date of last analysis was 1 July 2019. SETTING: Inpatient and outpatient genetics service at two large academic centres in Singapore. PATIENTS: Inclusion criteria: patients suspected of genetic disorders, based on abnormal antenatal ultrasound, multiple congenital anomalies and developmental delay. EXCLUSION CRITERIA: patients with known genetic disorders, either after clinical assessment or investigations (such as karyotype or chromosomal microarray). INTERVENTIONS: Use of NGS technology-whole exome sequencing (WES) or whole genome sequencing (WGS). MAIN OUTCOME MEASURES: (1) Diagnostic yield by sequencing type, (2) diagnostic yield by phenotypical categories, (3) reduction in time to diagnosis and (4) change in clinical outcomes and management. RESULTS: We demonstrate a 37.8% diagnostic yield for WES (n=172) and a 33.3% yield for WGS (n=24). The yield was higher when sequencing was conducted on trios (40.2%), as well as for certain phenotypes (neuromuscular, 54%, and skeletal dysplasia, 50%). In addition to aiding genetic counselling in 100% of the families, a positive result led to a change in treatment in 27% of patients. CONCLUSION: Genomic sequencing is an effective method for diagnosing rare disease or previous 'undiagnosed' disease. The clinical utility of WES/WGS is seen in the shortened time to diagnosis and the discovery of novel variants. Additionally, reaching a diagnosis significantly impacts families and leads to alteration in management of these patients.

Interactions between p53, hMSH2-hMSH6 and HMG I(Y) on Holliday junctions and bulged bases.

The ability of the tumor suppressor protein, p53, to recognize certain types of DNA lesions may represent one of the mechanisms by which this protein modulates cellular response to DNA damage. p53 DNA binding properties are regulated by several factors, such as post-translational modifications including phosphorylation and acetylation, regulation by its own C-terminal domain and interactions with other cellular proteins. Substrates resembling Holliday junctions and extra base bulges were used to study the effect of three nuclear proteins, HMG-1, HMG I(Y) and hMSH2-hMSH6, on the lesion binding properties of p53. Gel retardation assays revealed that the three proteins had varying effects on p53 binding to these substrates. HMG-1 did not influence p53 binding to Holliday junctions or 3-cytosine bulges. HMG I(Y) rapidly dissociated p53 complexes with Holliday junctions but not 3-cytosine bulges. Finally, the mismatch repair protein complex, hMSH2-hMSH6, enhanced p53 binding to both substrates by 3-4-fold. Together, these results demonstrate that p53 DNA binding activity is highly influenced by the presence of other proteins, some having a dominant effect while others have a negative effect.

Pharmacokinetic study of amaranth extract in healthy humans: A randomized trial.

OBJECTIVE: Nitric oxide (NO) is one of the most important signaling molecules produced within the body. Continuous generation of NO is essential for the integrity of the cardiovascular system. The aim of this study was to assess whether oral intake of a nitrate (NO3-)-rich dietary supplement (amaranth extract) is able to increase NO3- and nitrite (NO2-) levels in blood plasma and saliva of healthy adults. METHODS: In the present study, bioavailability and pharmacokinetics of NO3- and NO2- from amaranth extract (2 g as single dose) was studied in 16 healthy individuals and compared with placebo in a crossover design. The NO3- and NO2- levels in plasma as well as saliva were measured up to 24 h. RESULTS: After administration of amaranth extract, the NO3- levels in plasma as well as saliva were found to be significantly (P < 0.001) higher than in the placebo group. The NO2- level in plasma was slightly higher (P < 0.05) in the amaranth group (test group) compared with that in the placebo group, whereas the saliva NO2- level was significantly high (P < 0.001) in the amaranth extract-treated group than the placebo group. CONCLUSIONS: These results clearly indicate that a single oral dose of amaranth extract is able to increase the NO3- and NO2- levels in the body for at least 8 h. The increase in NO3- and NO2- levels can help to improve the overall performance of people involved in vigorous physical activities or sports.

Cell proliferation and apoptosis: dual-signal hypothesis tested in tuberculous pleuritis using mycobacterial antigens.

Antigens and mitogens have the innate ability to trigger cell proliferation and apoptosis thus exhibiting a dual-signal phenomenon. This dual-signal hypothesis was tested with mycobacterial antigens (PPD and heat killed Mycobacterium tuberculosis - MTB) in tuberculous pleuritis patients where the immune response is protective and compartmentalized. We compared and correlated the cell-cycle analysis and antigen-induced apoptosis in normal and patients' peripheral blood mononuclear cells (PBMCs) and patients' pleural fluid mononuclear cells (PFMCs). In cell-cycle analysis, PFMCs showed good mitotic response with PPD and MTB antigens where 10% and 7% of resting cells entered the S and G2/M phases of cell cycle, respectively. This antigen-induced proliferation of PFMCs correlated well with the lymphocyte transformation test (LTT) results. On the other hand, PFMCs also showed 21% of spontaneous apoptosis, which further increased to 43%, by induction with known apoptotic agent like Dexamethasone (DEX) and the mycobacterial antigens PPD and MTB. Further we demonstrated by anti-CD3 induction experiments that prior activation of cells is prerequisite for them to undergo apoptosis. Our results showed that PPD and MTB antigens induced both cell proliferation and apoptosis in PFMCs, which were pre-sensitized to mycobacterial antigens in vivo. Thus the dual-signal phenomenon was operative against these antigens in tuberculous pleuritis. We also demonstrated that the activated cells are more predisposed to apoptosis.

Mesoscale phenomena in ternary solutions of tertiary butyl alcohol, water, and propylene oxide.

The phase behavior and mesoscopic inhomogeneities in the ternary system of tertiary butyl alcohol (TBA), water, and propylene oxide (PO) have been studied by static and dynamic light scattering, gas chromatography, mass spectrometry, and molecular dynamics simulations. Mesoscale inhomogeneities are observed in this system in a broad range of PO concentrations, from 0.02 to about 65 mass %, and at certain TBA/water mass ratios varying from about 3/97 to about 30/70. This TBA/water composition domain corresponds to a region where short-lived micelle-like molecular clustering and thermodynamic anomalies are observed in the TBA-water binary system. At dilute PO concentrations (0.02 to about 1 mass %) the mesoscale inhomogeneities are Brownian diffusive droplets, with a size of the order of a hundred nanometers. We hypothesize that these droplets have a hydrophobic core enriched by oily impurities and oligomerized PO molecules. A hydrogen-bonded layer of TBA, water, and PO molecules surrounds this hydrophobic core. At high PO concentrations (beyond 50 mass %), the interfacial curvature of the mesoscopic inhomogeneities changes its sign and the exteriors of these inhomogeneities become hydrophobic. The inversion of the internal curvature may result in the formation of a spongelike bicontinuous mesoscale structure at intermediate PO concentrations. This mesostructure appears to be at a nonequilibrium state, although extremely long-lived.

Mesoscale inhomogeneities in aqueous solutions of small amphiphilic molecules.

Small amphiphilic molecules, also known as hydrotropes, are too small to form micelles in aqueous solutions. However, aqueous solutions of nonionic hydrotropes show the presence of a dynamic, loose, non-covalent clustering in the water-rich region, This clustering can be viewed as "micelle-like structural fluctuations". Although these fluctuations are short ranged (approximately 1 nm) and short lived (10 ps-50 ps), they may lead to thermodynamic anomalies. In addition, many experiments on aqueous solutions of hydrotropes show the occasional presence of mesoscale (approximately 100 nm) inhomogeneities. We have combined results obtained from molecular dynamics simulations, small-angle neutron scattering, and dynamic light-scattering experiments carried out on tertiary butyl alcohol (hydrotrope)-water solutions and on tertiary butyl alcohol-water-cyclohexane (hydrophobe) solutions to elucidate the nature and structure of these inhomogeneities. We have shown that stable mesoscale inhomogeneities occur in aqueous solutions of nonionic hydrotropes only when the solution contains a third, more hydrophobic, component. Moreover, these inhomogeneities exist in ternary systems only in the concentration range where structural fluctuations and thermodynamic anomalies are observed in the binary water-hydrotrope solutions. Addition of a hydrophobe seems to stabilize the water-hydrotrope structural fluctuations, and leads to the formation of larger (mesoscopic) droplets. The structure of these mesoscopic droplets is such that they have a hydrophobe-rich core, surrounded by a hydrogen-bonded shell of water and hydrotrope molecules. These droplets can be extremely long-lived, being stable for over a year. We refer to the phenomenon of formation of mesoscopic droplets in aqueous solutions of nonionic hydrotropes containing hydrophobes, as mesoscale solubilization. This phenomenon may represent a ubiquitous feature of nonionic hydrotropes that exhibit clustering in water, and may have important practical applications in areas, such as drug delivery, where the replacement of traditional surfactants may be necessary.

Resolving the mystery of aqueous solutions of tertiary butyl alcohol.

We have resolved a long-standing issue in the discussion on the origin of the mesoscale inhomogeneities observed in aqueous solutions of tertiary butyl alcohol (TBA). We have shown that the formation of stable mesoscale particles (of about 100 nm in size) can be triggered by the addition of trace amounts of propylene oxide (an impurity expected to be present in all commercial samples of TBA) to a solution, which was previously filtered at a low temperature to remove these inhomogeneities. We hypothesize that these particles are aggregates of mixed clathrate-hydrates that are formed through the stabilization of fluctuations of the intrinsic structure in TBA aqueous solutions by the clathrate-forming ability of propylene oxide.

Effect of thermocycling on the flexural strength of porcelain laminate veneers.

AIM: The aim of this study was to examine the impact of thermocycling on the flexural strength and development of surface flaws on the glazed surface of porcelain laminate veneer restorations with and without resin luting cement. MATERIALS AND METHODS: 80 Vitadur alpha dentin porcelain discs (10 mm diameter, 0.9 mm thickness) were glazed on one side and divided into two groups: A (porcelain laminate veneer only without resin luting cement) and B (porcelain laminate veneer luted with resin cement), each containing 40 discs. The discs in groups A and B were then thermocycled at different temperatures and were subjected to SEM analysis to evaluate the effect of thermocycling on crack propagation. Mean flexural strength was determined by using the ball-on-ring test. Student's t -test was used to find out the difference between strength values of the thermocycled porcelain discs and discs luted with resin cement. RESULTS: SEM analysis revealed crack propagation in the subgroups subjected to extremes of temperature, i.e., 4 +/- 1 degrees C, 37 +/- 1 degrees C and 4 +/- 1 degrees C, 65 +/- 1 degrees C in the porcelain laminate veneers luted with resin cement. Flexural strength analysis revealed superior flexural strength for porcelain laminate veneers: 88.58 +/- 6.94 MPa when compared to porcelain laminate veneers luted with resin cement: 8.42 +/- 2.60 MPa. Results were tabulated and statistically analyzed using Student's t -test. CONCLUSION: Laminate veneer specimens exhibited greater flexural strength than those which were luted with resin cements. Laminate veneer specimens luted with resin cement and subjected to extremes of temperature, 4 +/- 1 degrees C and 37 +/- 1 degrees C and 4 +/- 1 degrees C and 65 +/- 1 degrees C, showed a marked decrease in flexural strength. After thermocycling at extremes of temperature, laminate veneer specimens luted with resin cement showed crack propagation. Fit of laminate veneers cannot / should not be compensated by the thickness of luting agent.

p53 Monitors replication fork regression by binding to "chickenfoot" intermediates.

The tumor suppressor protein, p53, utilizes multiple mechanisms to ensure faithful transmission of the genome including regulation of DNA replication, repair, and recombination. Monitoring these pathways may involve direct binding of p53 to the DNA intermediates of these processes. In this study, we generated templates resembling stalled replication forks and utilized electron microscopy to examine p53 interactions with these substrates. Our results show that p53 bound with high affinity to the junction of stalled forks, whereas two cancer-derived p53 mutants showed weak binding. Additionally, some of the templates were rearranged to form "chickenfoot" structures in the presence of p53. These were mostly formed due to p53 trapping intermediates of spontaneous fork regression; however, in a small population, the protein appeared to be promoting their formation. Collectively, these results demonstrate the importance of sequence-independent binding in p53-mediated maintenance of genomic integrity.

Modulation of p53 binding to Holliday junctions and 3-cytosine bulges by phosphorylation events.

Recognition of certain types of DNA lesions by the tumor suppressor protein, p53, represents one of the several downstream functions of this protein in response to DNA damage. This binding property is regulated by several factors including posttranslational modifications and interactions with other proteins. Phosphorylation by several stress-response kinases activates p53 by increasing protein stability as well as transactivation properties. Here we examined the effect of phosphorylation events on the sequence-independent binding properties of p53 using two DNA substrates: One resembling Holliday junctions and the other containing extra base bulges. Gel retardation assays showed that dephosphorylation of serine 392 in the C-terminal domain of p53 greatly reduces Holliday junction and lesion recognition. In contrast, sequence-specific binding is disrupted by the removal of some N-terminal phosphates but not serine 392. Rephosphorylation of p53 by certain kinases can restore p53 recognition of Holliday junctions and 3-cytosine bulges. In all cases, phosphorylation of serine 392 occurs; however, reactivation also involves other residues. Together, the results show that p53 DNA binding activity is strongly regulated by the phosphorylation state of the protein.

p53 binds telomeric single strand overhangs and t-loop junctions in vitro.

The interaction of p53 with a human model telomere in vitro was examined by electron microscopy. p53 demonstrated a sequence-independent affinity for telomeric DNA in vitro, localizing to the 3' single strand overhang and the t-loop junction both in the presence and absence of associated TRF2. Binding was not observed above background along the duplex telomeric repeats. However, the efficiency of TRF2-catalyzed t-loop formation on the model DNA was increased 2-fold in the presence of p53 although a variety of single strand or Holliday junction-binding proteins did not facilitate t-loop formation. These results suggest that p53 has an active role in telomere maintenance and structure through association with the t-loop junction.

The Epstein-Barr virus polymerase accessory factor BMRF1 adopts a ring-shaped structure as visualized by electron microscopy.

Epstein-Barr virus (EBV) encodes a set of core replication factors used during lytic infection in human cells that parallels the factors used in many other systems. These include a DNA polymerase and its accessory factor, a helicase/primase, and a single strand binding protein. The EBV polymerase accessory factor has been identified as the product of the BMRF1 gene and has been shown by functional assays to increase the activity and processivity of the polymerase. Unlike other members of this class of factors, BMRF1 is also a transcription factor regulating certain EBV genes. Although several polymerase accessory factors, including eukaryotic proliferating cell nuclear antigen, Escherichia coli beta protein, and T4 gene 45 protein have been shown to form oligomeric rings termed sliding clamps, nothing is known about the oligomeric state of BMRF1 or whether it forms a ring. In this work, BMRF1 was purified directly from human cells infected with an adenovirus vector expressing the BMRF1 gene product. The protein was purified to near homogeneity, and examination by negative staining electron microscopy revealed large, flat, ring-shaped molecules with a diameter of 15.5 +/- 0.8 nm and a distinct 5.3-nm diameter hole in the center. The size of these rings is consistent with an oligomer of 6 monomers, nearly twice as large as the trimeric proliferating cell nuclear antigen ring. Unlike the herpes simplex virus UL42 homologue, BMRF1 was found to self-associate in solution. These findings extend the theme of polymerase accessory factors adopting ring-shaped structures and provide an example in which the ring is significantly larger than any previously described sliding clamp.

Telomere looping in P. sativum (common garden pea).

Telomeres vary greatly in size among plants and, in most higher plants, consist of a long array of 5'-TTTAGGG-3'/3'-AAATCCC-5' (TTTAGGG) repeats. Recently, telomeric DNA in human, mouse, oxytricha, and trypanosome chromosomes have been found arranged into loops (t-loops), proposed to sequester the telomere from unwanted repair events and prevent activation of DNA damage checkpoints. We have asked whether t-loops exist in the higher order plant Pisum sativum (garden pea). DNA was isolated from the shoots and root tips of germinating seeds. Analysis of the telomeric restriction fragments showed that DNA hybridizing to a (TTTAGGG)n probe migrated as a smear centering around 25 kb, and direct sequencing verified the repeat to be (TTTAGGG)n. Total DNA in isolated nuclei was photo-cross-linked, and the telomeric restriction fragments were purified by gel filtration. Electron microscopic (EM) analysis revealed DNA molecules arranged as t-loops with a size distribution consistent with that seen by gel electrophoresis. Some molecules had loops as large as 75 kb. These results show that the arrangement of telomeric DNA into loops occurs in higher plants.

Human claspin is a ring-shaped DNA-binding protein with high affinity to branched DNA structures.

Claspin is an essential protein for the ATR-dependent activation of the DNA replication checkpoint response in Xenopus and human cells. Here we describe the purification and characterization of human Claspin. The protein has a ring-like structure and binds with high affinity to branched DNA molecules. These findings suggest that Claspin may be a component of the replication ensemble and plays a role in the replication checkpoint by directly associating with replication forks and with the various branched DNA structures likely to form at stalled replication forks because of DNA damage.