The use of enamel matrix derivative in the treatment of periodontal osseous defects: a clinical decision tree based on biologic principles of regeneration.

An investigation of the periodontal literature reveals four critical factors necessary for a regenerative response in the treatment of intrabony defects. These include root preparation to remove toxins and altered cementum, space creation by the graft or membrane barrier for migration of progenitor cells, stabilization and flap coverage of the graft, and use of a membrane barrier or enamel matrix derivative (EMD). This article reviews the literature supporting this concept and presents a clinical decision tree to determine when to use EMD alone or with autogenous grafts and membrane barriers in the treatment of defects of varying morphologies. The clinical decision tree is designed to increase the predictability of a positive clinical response. Cases are shown to demonstrate the indication for each treatment.

Purification of native myosin filaments from muscle.

Analysis of the structure and function of native thick (myosin-containing) filaments of muscle has been hampered in the past by the difficulty of obtaining a pure preparation. We have developed a simple method for purifying native myosin filaments from muscle filament suspensions. The method involves severing thin (actin-containing) filaments into short segments using a Ca(2+)-insensitive fragment of gelsolin, followed by differential centrifugation to purify the thick filaments. By gel electrophoresis, the purified thick filaments show myosin heavy and light chains together with nonmyosin thick filament components. Contamination with actin is below 3.5%. Electron microscopy demonstrates intact thick filaments, with helical cross-bridge order preserved, and essentially complete removal of thin filaments. The method has been developed for striated muscles but can also be used in a modified form to remove contaminating thin filaments from native smooth muscle myofibrils. Such preparations should be useful for thick filament structural and biochemical studies.

Comparison of information processing technologies.

OBJECTIVE: To examine the type of information obtainable from scientific papers, using three different methods for the extraction, organization, and preparation of literature reviews. DESIGN: A set of three review papers was identified, and the ideas represented by the authors of those papers were extracted. The 161 articles referenced in those three reviews were then analyzed using 1) a formalized data extraction approach, which uses a protocol-driven manual process to extract the variables, values, and statistical significance of the stated relationships; and 2) a computerized approach known as "Idea Analysis," which uses the abstracts of the original articles and processes them through a computer software program that reads the abstracts and organizes the ideas presented by the authors. The results were then compared. The literature focused on the human papillomavirus and its relationship to cervical cancer. RESULTS: Idea Analysis was able to identify 68.9 percent of the ideas considered by the authors of the three review papers to be of importance in describing the association between human papillomavirus and cervical cancer. The formalized data extraction identified 27 percent of the authors' ideas. The combination of the two approaches identified 74.3 percent of the ideas considered important in the relationship between human papillomavirus and cervical cancer, as reported by the authors of the three review articles. CONCLUSION: This research demonstrated that both a technically derived and a computer derived collection, categorization, and summarization of original articles and abstracts could provide a reliable, valid, and reproducible source of ideas duplicating, to a major degree, the ideas presented by subject specialists in review articles. As such, these tools may be useful to experts preparing literature reviews by eliminating many of the clerical-mechanical features associated with present-day scientific text processing.

Excessive base excision repair of 5-hydroxymethyluracil from DNA induces apoptosis in Chinese hamster V79 cells containing mutant p53.

We have demonstrated previously that the toxicity of 5-hydroxymethyl-2'-deoxyuridine (hmdUrd) to Chinese hamster fibroblasts (V79 cells) results from enzymatic removal of large numbers of hydroxymethyluracil residues from the DNA backbone [Boorstein,R. et al. (1992) Mol. Cell. Biol., 12, 5536-5540]. Here we report that a significant portion of the hmdUrd-induced cell death that is dependent on DNA base excision repair in V79 cells is apoptosis. Incubation of V79 cells with pharmacologically relevant concentrations of hmdUrd resulted in the characteristic changes of apoptosis as measured by gel electrophoresis, flow cytometry and phase contrast microscopy. However, hmdUrd did not induce apoptosis in V79mut1 cells, which are deficient in DNA base excision repair of 5-hydroxymethyluracil (hmUra). Apoptosis was not prevented by addition of 3-aminobenzamide, which inhibits synthesis of poly(ADP-ribose) from NAD, indicating that apoptosis was not the direct consequence of NAD depletion. Pulsed field gel electrophoresis indicated that hmdUrd treatment resulted in high molecular weight (2.2-4.5 Mb) DNA double-strand breaks prior to formation of internucleosomal ladders in V79 cells. Simultaneous measurement of DNA strand breaks with bromodeoxyuridine/terminal deoxynucleotidyl transferase-fluorescein isothiocyanate labeling and of cell cycle distribution indicated that cells with DNA strand breaks accumulated in late S/G(2) and that hmdUrd-treated cells underwent apoptosis after arrest in late S/G(2) phase. Our results indicate that excessive DNA base excision repair results in the generation of high molecular weight DNA double-strand breaks and eventually leads to apoptosis in V79 cells. Thus, delayed apoptosis following DNA damage can be a consequence of excessive DNA repair activity. Immunochemical analysis showed that both V79 and V79mut1 cells contained mutant p53, indicating that apoptosis induced by DNA base excision repair can be independent of p53.

Confirmation of an excess of the high enzyme activity COMT val allele in heroin addicts in a family-based haplotype relative risk study.

A previous case control study by Vandenbergh et al. [1997: Am J Med Genet 74:439-442] showed an association between the high activity catechol O-methyltransferase (COMT) polymorphism and polysubstance abuse in a group of North American subjects. In the current study we confirm these results by genotyping 38 Israeli heroin addicts and both parents using a robust family-based haplotype relative risk (HRR) strategy. There is an excess of the val COMT allele (likelihood ratio = 4.48, P = 0.03) and a trend for an excess of the val/val COMT genotype (likelihood ratio = 4.97, P = 0.08, 2 df) in the heroin addicts compared to the HRR control group. We also genotyped an additional 101 nonrelated heroin addicts and 126 control subjects using a case control design and found no significant difference in COMT val allele frequency (25.4% vs. 29.7%, likelihood ratio = 1.04, P = 0.31). A significant difference is observed in COMT allele frequency among the three principal Israeli ethnic groups (Ashkenazi Jewish, non-Ashkenazi Jewish, and Palestinian Arab) in a large group of control subjects we have so far examined (chi-square = 7.9, P = 0.019, df = 2, n = 1,422 alleles) suggesting that population stratification is responsible for our failure to observe an excess of the COMT val allele when using the case-control design.

Lymphoepithelioma-like carcinoma of the uterine cervix.

We describe two rare cervical tumors having morphologic features closely resembling those of the nasopharyngeal lymphoepithelioma. This entity has historically been classified as a subtype of squamous cell carcinoma, but after reviewing the literature and the two cases presented here, we propose that this tumor is a distinct carcinoma of the cervix that differs from squamous cell carcinoma in that it carries a more favorable prognosis, typically affects a younger population of women, is more prevalent in noncaucasian populations (especially those of Asian descent), and lacks a clearly defined association with infection due to human papilloma virus (HPV).

"The birthing from within holistic sphere": a conceptual model for childbirth education.

An expanded conceptual model of childbirth education is offered, proposing the benefits of balancing informative teaching processes with creative, experiential, introspective learning processes for parents. The application of these two teaching dimensions to exploring four different perspectives of birth (the mother's, the father's, the baby's, and the culture's) is discussed, along with examples from "Birthing From Within" classes. Implications for current practice and the evolving role of childbirth educator are noted.

Tropomyosin positions in regulated thin filaments revealed by cryoelectron microscopy.

Past attempts to detect tropomyosin in electron micrograph images of frozen-hydrated troponin-regulated thin filaments under relaxing conditions have not been successful. This raised the possibility that tropomyosin may be disordered on filaments in the off-state, a possibility at odds with the steric blocking model of muscle regulation. By using cryoelectron microscopy and helical image reconstruction we have now resolved the location of tropomyosin in both relaxing and activating conditions. In the off-state, tropomyosin adopts a position on the outer domain of actin with a binding site virtually identical to that determined previously by negative staining, although at a radius of 3.8 nm, slightly higher than found in stained filaments. Molecular fitting to the atomic model of F-actin shows that tropomyosin is localized over sites on actin subdomain 1 required for myosin binding. Restricting access to these sites would inhibit the myosin-cross-bridge cycle, and hence contraction. Under high Ca(2+) activating conditions, tropomyosin moved azimuthally, away from its blocking position to the same site on the inner domain of actin previously determined by negative staining, also at 3.8 nm radius. These results provide strong support for operation of the steric mechanism of muscle regulation under near-native solution conditions and also validate the use of negative staining in investigations of muscle thin filament structure.

Nucleosomes, linker DNA, and linker histone form a unique structural motif that directs the higher-order folding and compaction of chromatin.

The compaction level of arrays of nucleosomes may be understood in terms of the balance between the self-repulsion of DNA (principally linker DNA) and countering factors including the ionic strength and composition of the medium, the highly basic N termini of the core histones, and linker histones. However, the structural principles that come into play during the transition from a loose chain of nucleosomes to a compact 30-nm chromatin fiber have been difficult to establish, and the arrangement of nucleosomes and linker DNA in condensed chromatin fibers has never been fully resolved. Based on images of the solution conformation of native chromatin and fully defined chromatin arrays obtained by electron cryomicroscopy, we report a linker histone-dependent architectural motif beyond the level of the nucleosome core particle that takes the form of a stem-like organization of the entering and exiting linker DNA segments. DNA completes approximately 1.7 turns on the histone octamer in the presence and absence of linker histone. When linker histone is present, the two linker DNA segments become juxtaposed approximately 8 nm from the nucleosome center and remain apposed for 3-5 nm before diverging. We propose that this stem motif directs the arrangement of nucleosomes and linker DNA within the chromatin fiber, establishing a unique three-dimensional zigzag folding pattern that is conserved during compaction. Such an arrangement with peripherally arranged nucleosomes and internal linker DNA segments is fully consistent with observations in intact nuclei and also allows dramatic changes in compaction level to occur without a concomitant change in topology.

Diabetes mellitus after renal transplantation: as deleterious as non-transplant-associated diabetes?

BACKGROUND: Despite use of lower doses of corticosteroid hormones after renal allotransplantation in the era of cyclosporine and tacrolimus, posttransplant diabetes mellitus remains a common clinical problem. METHODS: We prospectively investigated the effect of posttransplant diabetes on long-term (mean follow-up, 9.3+/-1.5 years) graft and patient survival in the 11.8% of our renal transplant population (n = 40) who developed diabetes after kidney transplantation, and we compared outcome in 38 randomly chosen nondiabetic control patients who had received transplants concurrently. RESULTS: Twelve-year graft survival in diabetic patients was 48%, compared with 70% in control patients (P = 0.04), and Cox's regression analysis revealed diabetes to be a significant predictor of graft loss (P = 0.04, relative risk = 3.72) independent of age, sex, and race. Renal function at 5 years as assessed by serum creatinine level was inferior in diabetic patients compared to control patients (2.9+/-2.6 vs. 2.0+/-0.07 mg/dl, P = 0.05). Two diabetic patient who experienced graft loss had a clinical course and histological features consistent with diabetic nephropathy; other diabetes-related morbidity in patients with posttransplant diabetes included ketoacidosis, hyperosmolar coma or precoma, and sensorimotor peripheral neuropathy. Patient survival at 12 years was similar in diabetic and control patients (71% vs. 74%). CONCLUSIONS: Posttransplant diabetes mellitus is associated with impaired long-term renal allograft survival and function, complications similar to those in non-transplant-associated diabetes may occur in posttransplant diabetes, and, hence, as in non-transplant-associated diabetes, tight glycemic control may also be warranted in patients with posttransplant diabetes.

Evaluation of a surgical debridement bur for use in periodontal surgery.

The Evian Debridement Bur facilitates two fundamental techniques in periodontal surgery--soft-tissue debridement and root planing. The bur debrides granulation and other soft tissues attached to the root or bone and removes hard deposits from root surfaces. It can also be used as an adjunct while performing crown-lengthening procedures and endodontic surgery. The bur is made with blunted flutes and, if used with a relatively light touch, will not cut into the alveolus and will not damage the tooth surface. This article reviews the use and advantages of this bur and shows its effect on the root surface at the ultrastructural level.

Effects of perturbations of pools of deoxyribonucleoside triphosphates on expression of ribonucleotide reductase, a G1/S transition state enzyme, in p53-mutated cells.

Effects of drug treatment with antimetabolites on a human colon cancer cell line, SW480, were studied. Cells were treated with 10 microM of 5-fluorouracil (5FU), an inhibitor of pyrimidine synthesis, or 1000 microM of hydroxyurea (HU), an inhibitor of both purine and pyrimidine syntheses, or the combination. Recombinant alpha-2a-interferon (IFN), 500 U/mL, also was employed, as this augments the effects of both antimetabolites in vitro and in vivo. The predominant effect of this combination was to block cells in early S phase as measured by 5-bromo-2'-deoxyuridine (BrdUrd) incorporation. By 24 hr, 86% of the cells had accumulated in S phase, but failed to progress to G2/M. This was accompanied by an early, rapid decline in all four deoxyribonucleoside triphosphates (dNTPs) by 38-86% at 4-24 hr. Despite these effects, expression of the G1/S transition state enzyme, ribonucleotide reductase (RR), increased at 24 hr as measured by a 3 to 5-fold increase in mRNA levels for the M2 subunit, in the absence of a measurable effect on protein levels. The rise in levels of RR mRNA and the continued progression of cells into S phase were associated with a synergistic inhibition of cell cycle proliferation resulting from treatment with the three-drug combination. This suggests that in the presence of antimetabolite-induced depletion of dNTPs, SW480 cells, which lack a normal p53 gene, will proceed into S phase, and that this is associated with a rise in expression of the G1/S transition state enzyme, RR. Cells arrested in S phase by a p53-independent mechanism will undergo a synergistic enhancement of cell death.

Additional evidence for an association between the dopamine D4 receptor (D4DR) exon III seven-repeat allele and substance abuse in opioid dependent subjects: relationship of treatment retention to genotype and personality.

The long form of the dopamine D4 receptor (D4DR) exon III repeat polymorphism has been linked in some but not all studies to impulsive, extravagant and novelty-seeking personality traits that are prominent in affiliated behaviours such as attention deficit disorder and substance abuse. In particular, we have reported previously an increased frequency of the long seven-repeat D4DR exon III allele in a group of 141 opioiddependent subjects compared to 110 control subjects. In order to further substantiate the role of D4DR in contributing to heroin addiction we have genotyped an additional, smaller cohort of opioid-dependent subjects. In this new group of 57 opioid-dependent subjects compared to an expanded group of 143 control subjects a significant difference was observed in overall genotype frequency (p=0.04). An excess of the seven-repeat allele of the D4DR receptor gene was also observed compared to control subjects (p=0.06). The frequency of the seven-repeat allele is 15.8% in the heroin addict population vs. 8.1% in the control group, conferring a relative risk of 2.07 (95% CI: 0.98-4.38). An association between two polymorphisms considered together (D4DR and dopamine D3 receptor) and treatment retention was observed (p=0.02). In a subgroup of 38 opioid-dependent subjects, who were successfully administered the TPQ, higher Harm Avoidance (p< 0.001) and Novelty Seeking (NS3; extravagant vs. reserved, p< 0.001) scores were found. In contrast to some previous reports, no relationship was apparent between TPQ scores and treatment retention in this small group of opioid-dependent subjects.

Measurement of deoxyuridine triphosphate and thymidine triphosphate in the extracts of thymidylate synthase-inhibited cells using a modified DNA polymerase assay.

New inhibitors of the enzyme thymidylate synthase (TS) are now reaching clinical application. Alteration of the dUTP: dTTP ratio may be critical to TS inhibition-induced tumor cell death. The DNA polymerase assay with modification was used to rapidly and sensitively measure dUTP, dTTP, and dUTP:dTTP ratios in cell extracts of HT29 human colon carcinoma cells treated with the specific TS inhibitor ZD1694 [N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl)-L-glutamic acid]. These results revealed an increase in the dUTP:dTTP ratio at 2 hr after a 2-hr exposure to ZD1694 at concentrations of 0.05 to 0.2 microM with significant normalization at 16 hr after a 2-hr exposure despite evidence of continued TS inhibition. This assay is highly sensitive and reproducible for levels of dUTP and is less labor intensive than traditional assays.

Combination therapy with 5-fluorouracil and IFN-alpha2a induces a nonrandom increase in DNA fragments of less than 3 megabases in HT29 colon carcinoma cells.

We have used pulsed-field gel electrophoresis to examine 5-fluorouracil (5FU)-induced DNA double-strand breaks (DSBs), both with and without modulation by IFN-alpha2a (IFNalpha), in HT29 human colon adenocarcinoma cells. Although 24-h treatment with either 10 microM 5FU or 500 units/ml IFNalpha did not result in significant DNA fragmentation, the combination of 5FU + IFNalpha resulted in a significant increase in DNA DSBs versus either drug alone (P < 0.05). The pattern of fragmentation induced by treatment with 5FU + IFNalpha was compared to that induced by gamma-radiation, which generates lesions at random sites, digestion with NotI restriction endonuclease, which cleaves at the specific sequence 5' ellipsis GCGGCCGCellipsis 3', and HhaI restriction endonuclease, which cleaves at the specific sequence 5'ellipsis GCGCellipsis 3'. 5FU + IFNalpha resulted in a specific pattern characterized by the accumulation of fragments of <3 Mb in the absence of fragments of >3 Mb, which differed from that of gamma-radiation and restriction endonuclease digestion. Because neither morphological nor DNA fragmentation characteristic of apoptosis was observed after 5FU + IFNalpha treatment, the nonrandom pattern of DSBs that was observed did not appear to be the result of the initiation of programmed cell death within these cells.