High serum total cholesterol is a long-term cause of osteoporotic fracture.

SUMMARY: Risk factors for osteoporotic fractures were evaluated in 1,396 men and women for a period of 20 years. Serum total cholesterol was found to be an independent osteoporotic fracture risk factor whose predictive power improves with time. INTRODUCTION: The purpose of this study was to evaluate long-term risk factors for osteoporotic fracture. METHODS: A population random sample of men and women aged 25-64 years (the Gothenburg WHO MONICA project, N = 1,396, 53% women) was studied prospectively. The 1985 baseline examination recorded physical activity at work and during leisure time, psychological stress, smoking habits, coffee consumption, BMI, waist/hip ratio, blood pressure, total, HDL and LDL cholesterol, triglycerides, and fibrinogen. Osteoporotic fractures over a period of 20 years were retrieved from the Gothenburg hospital registers. Poisson regression was used to analyze the predictive power for osteoporotic fracture of each risk factor. RESULTS: A total number of 258 osteoporotic fractures occurred in 143 participants (10.2%). As expected, we found that previous fracture, smoking, coffee consumption, and lower BMI each increase the risk for osteoporotic fracture independently of age and sex. More unexpectedly, we found that the gradient of risk of serum total cholesterol to predict osteoporotic fracture significantly increases over time (p = 0.0377). CONCLUSIONS: Serum total cholesterol is an independent osteoporotic fracture risk factor whose predictive power improves with time. High serum total cholesterol is a long-term cause of osteoporotic fracture.

Reducing uncertainty in health-care resource allocation.

A key task for health policymakers is to optimise the outcome of health care interventions. The pricing of a new generation of cancer drugs, in combination with limited health care resources, has highlighted the need for improved methodology to estimate outcomes of different treatment options. Here we introduce new general methodology, which for the first time employs continuous hazard functions for analysis of survival data. Access to continuous hazard functions allows more precise estimations of survival outcomes for different treatment options. We illustrate the methodology by calculating outcomes for adjuvant treatment of gastrointestinal stromal tumours with imatinib mesylate, which selectively inhibits the activity of a cancer-causing enzyme and is a hallmark representative for the new generation of cancer drugs. The calculations reveal that optimal drug pricing can generate all win situations that improve drug availability to patients, make the most of public expenditure on drugs and increase pharmaceutical company gross profits. The use of continuous hazard functions for analysis of survival data may reduce uncertainty in health care resource allocation, and the methodology can be used for drug price negotiations and to investigate health care intervention thresholds. Health policy makers, pharmaceutical industry, reimbursement authorities and insurance companies, as well as clinicians and patient organisations, should find the methodology useful.

G-quadruplex DNA structures--variations on a theme.

To be functional, nucleic acids need to adopt particular three-dimensional structures. For a long time DNA was regarded as a rigid and passive molecule with the sole purpose to store genetic information, but experimental data has now accumulated that indicates the full dynamic repertoire of this macromolecule. During the last decade, four-stranded DNA structures known as G-quadruplexes, or DNA tetraplexes, have emerged as a three-dimensional structure of special interest. Motifs for the formation of G-quadruplex DNA structures are widely dispersed in eukaryotic genomes, and are abundant in regions of biological significance, for example, at telomeres, in the promoters of many important genes, and at recombination hotspots, to name but a few in man. Here I explore the plethora of G-quadruplex DNA structures, and discuss their possible biological functions as well as the proteins that interact with them.

The human TINF2 gene organisation and chromosomal localization.

Telomeres have emerged as possible targets for cancer therapy. In order to effectively pursuit them, we need to understand how the genes that control telomere length are regulated. TIN2, the protein product of the human TINF2 gene, was recently identified as a negative regulator of telomere length. Here I report the organisation and chromosomal localization of the TINF2 gene. It contains six exons spanning over 1912 bp of genomic DNA. Consistent with the finding that a wide variety of human tissues expresses low endogenous levels of TINF2, its proximal promoter region has the typical features of a housekeeping gene. It lacks a canonical TATA box but exhibits several GC boxes, which are potential binding sites for ubiquitous transcription factors like NF kappa B and Sp1.

A nuclease hypersensitive element in the human c-myc promoter adopts several distinct i-tetraplex structures.

Nucleic acid structure-function correlations are pivotal to major biological events like transcription, replication, and recombination. Depending on intracellular conditions in vivo and buffer composition in vitro, DNA appears capable of inexhaustible structure variation. At moderately acidic, or even neutral pH, DNA strands that are rich in cytosine bases can associate both inter- and intramolecularly to form i-tetraplexes. The hemiprotonated cytosine(+)-cytosine base pair constitutes the building block for the formation of i-tetraplexes, and motifs for their formation are frequent in vertebrate genomes. A major control element upstream of the human c-myc gene, which has been shown to interact sequence specifically with several transcription factors, becomes hypersensitive to nucleases upon c-myc expression. The control element is asymmetric inasmuch as that one strand is uncommonly rich in cytosines and exhibits multiple motifs for the formation of i-tetraplexes. To investigate the propensity for their formation we employ circular dichroism (CD) in combination with ultra violet (UV) spectroscopy and native gel electrophoresis. Our results demonstrate the cooperative formation of well-defined i-tetraplex structures. We conclude that i-tetraplex formation occurs in the promoter region of the human c-myc gene in vitro, and discuss implications of possible biological roles for i-tetraplex structures in vivo. Hypothetical formation of intramolecular fold-back i-tetraplexes is important to c-myc transcription, whereas chromosomal translocation events might involve the formation of bimolecular i-tetraplex structures.

DNA tetraplex formation studied with fluorescence resonance energy transfer.

It is emerging that DNA tetraplexes are pivotal for many major cellular processes, and techniques that assess their structure and nature to the point are under development. Here we show how the structural conversion of largely unstructured single-stranded DNA molecules into compact intrastrand DNA tetraplexes can be monitored by fluorescence resonance energy transfer. We recently reported that intrastrand tetraplex formation takes place in a nuclease hypersensitive element upstream of the human c-myc proto-oncogene. Despite the highly repetitive guanine-rich sequence of the hypersensitive element, fluorescence resonance energy transfer measurements indicate that only one well defined tetraplex structure forms therein. The proposed structure, which is specifically stabilized by potassium ions in vitro, has a core of three stacked guanine tetrads that is capped by two intrastrand A-T base pairs.

TGGA repeats impair nucleosome formation.

Nucleosomes, the building blocks of chromatin, are responsible for DNA packaging in eukaryotic cell nuclei. They play a structural role in genome condensation, and influence transcription and replication. Properties of the DNA sequence, such as curvature and flexibility, direct the location of nucleosomes. DNA sequences that position nucleosomes have been identified and rules that govern their properties have been formulated. However, DNA sequences that are refractory to nucleosome formation have been less well characterised and it is possible that they may perturb or alter chromatin structure. Here we identify such sequences by selecting those that refrain from nucleosome formation from a large pool of synthetic DNA fragments with a central region of 146 random base-pairs fitted with adapters for PCR amplification. These were used for in vitro salt-induced reconstitution of nucleosomes under thermodynamic equilibrium conditions. Fragments that did not form nucleosomes were purified, amplified by PCR, and the reconstitution was repeated. After 17 rounds of negative selection, the material was highly enriched in sequences reluctant to form nucleosomes. Cloning and sequencing revealed that 35% of the molecules had long repeats of TGGA, and their affinity for histone octamers was about half that of average DNA.

DNA tetraplex formation in the control region of c-myc.

The c-myc oncogene is one of the most commonly malfunctioning genes in human cancers, and is an attractive target for anti-gene therapy. Although synthetic oligonucleotides designed to silence c-myc expression via one of its major control elements function well in vitro, their mode of action has been indefinite. Here we show that the targeted control element adopts an intrastrand fold-back DNA tetraplex, which requires potassium ions for stability in vitro. We believe formation of the tetraplex is important for c-myc activation in vivo, and propose a transcription initiation mechanism that explains how anti-gene therapy silence c-myc at the molecular level.

Identification and characterization of genomic nucleosome-positioning sequences.

Positioned nucleosomes are believed to play important roles in transcriptional regulation and for the organization of chromatin in cell nuclei. Here, we have isolated the DNA segments in the mouse genome that form the most stable nucleosomes yet characterized. In separate molecules we find phased runs of three to four adenine nucleotides, extensive CA repeats, and in a few cases phased TATA tetranucleotides. The latter forms the most stable nucleosome yet characterized. One sequence with CAG repeats was also found. By fluorescence in situ hydridization the selected sequences are shown to be localized at the centromeric regions of mouse metaphase chromosomes.

Effect of delmopinol on the cohesion of glucan-containing plaque formed by Streptococcus mutans in a flow cell system.

Glucan-containing plaque was formed by Streptococcus mutans adhering to saliva-coated glass slides in flow cells thermostated at 37 degrees C. The substrate was Brain Heart Infusion broth containing 1% sucrose and 10% sterile saliva. During the build-up of the plaque, which lasted for 29 h, the plaque was subjected to three two-minute exposures to either 0.1 mol/L sodium acetate buffer, pH 6.0, or the same buffer containing 6.4 mmol/L (0.2%) of the surface-active anti-plaque substance delmopinol hydro-chloride. The glass slides carrying the plaque were weighed, and plaques subjected to delmopinol treatment weighed only seven percent of the control plaques. The glass slides were then mounted in a beaker containing buffer, subjected to ultrasonication, and re-weighed. The delmopinol-treated plaques lost 59% of their wet weight upon sonication, while the controls lost only 19%. Control plaques having the same weight as delmopinol-treated plaques were not different from the control plaques grown for 29 h with regard to reduction of plaque weight after sonication. Transmission electron micrographs (TEM) showed a plaque dominated by globular or fibrillar matrix components in controls, while the delmopinol-treated plaque showed empty or unordered matrix areas between more densely packed cells. The TEM results were confirmed by scanning electron micrographs, which showed amorphous material associated with the bacterial cells in the control but not in the delmopinol-treated plaque. In conclusion, delmopinol reduced surface-associated glucan synthesis and lowered the cohesion of the plaque, indicating that glucan-containing plaque formed during repeated rinsings with delmopinol may be easier to remove by mechanical means than a non-treated plaque of this type.

Influence of delmopinol on bacterial zeta-potentials and on the colloidal stability of bacterial suspensions.

Delmopinol is a low molecular weight surface-active compound that has been shown to be effective against dental plaque both in vitro and in vivo and against gingivitis in vivo. To study the mode of action of delmopinol, the influence of the compound on the stability of bacterial suspensions, both with and without saliva, and on the zeta-potentials of oral streptococci was studied. The results showed that delmopinol reduced the magnitude of the zeta-potentials, but, in contrast, the colloidal stability of the bacterial suspensions without saliva was increased. The explanation of these observations could be that non-DLVO interaction components, such as repulsive hydration/steric forces, have come into effect at very close distances between two approaching bacterial cells. To judge from the present results, it is possible that delmopinol forms films on bacterial cells in a plaque, thereby facilitating mechanical removal.

The effect of ionic surfactants on salivary proteins adsorbed on silica surfaces.

The adsorption onto silicon oxide surfaces from water and 0.1 M acetate buffer containing 10% parotid saliva at 25 degrees C and 35 degrees C and at pH 6 was monitored in situ using ellipsometry. The silicon oxide surface was used as a model for dental enamel. The adsorption kinetics and the reversibility on rinsing were determined, and the desorbable fraction was found not to change after either 30 or 120 min of adsorption. Addition of sodium dodecyl sulfate after 30 or 120 min of saliva adsorption caused strong desorption. Rinsing 30 min after surfactant addition caused some redeposition if saliva was present, whereas continued desorption occurred in the absence of saliva. Cetyltrimethylammonium bromide caused either an increase or a slight decrease in the amount adsorbed when added after 30 min and 120 min, respectively. For both times, rinsing caused desorption, left the same amount adsorbed, and was not affected by the presence or absence of saliva in solution. No major effect from temperature and ionic strength was found.

Effect of delmopinol on in vitro dental plaque formation, bacterial acid production and the number of microorganisms in human saliva.

This study investigated the effect of a surface active compound, delmopinol, on plaque formation and established plaque in vitro, on the bacterial acid production from glucose and on the total viable bacterial counts in saliva. The antimicrobial effect was compared with that of chlorhexidine. The plaque-inhibiting effect was evaluated in an artificial mouth system, and the effect on bacterial acid production was registered as a decrease of pH in bacterial suspensions with various concentrations of delmopinol. It was shown that delmopinol is able to prevent plaque formation, to dissolve established plaque in vitro, and that it has 5-125 times higher minimum inhibitory concentrations than chlorhexidine. Saliva samples collected 1 min after rinsing with delmopinol showed on differences in the total number of bacteria in saliva as compared with controls. It was also shown that the bacterial acid production from glucose was reduced successively with increasing concentrations of delmopinol. The results indicate that delmopinol might be as effective as chlorhexidine against plaque formation and that delmopinol is capable of penetrating established plaque, thus promoting a more effective mechanical cleansing.

Aspects of dental plaque formation with special reference to colloid-chemical phenomena.

In order to investigate factors of importance for plaque formation and the rate of plaque formation 133 randomly selected individuals went through a 3-day period of plaque accumulation. From these individuals one group of "heavy" and one group of "light" plaque formers were selected for further studies. These subjects were investigated with reference to clinical, biochemical, biophysical and microbiological variables, that in the literature have been suggested to influence plaque formation. The collected data were analysed statistically both by comparisons between the two groups and by multiple regression. In the comparative analyses there were only minor differences between the groups, and no single studied variable was considered as the only explanation to the great difference in the amount of plaque formed after 3 days between "heavy" and "light" plaque formers. The multiple regression showed that the initial bacterial colonization of tooth surfaces was dependent on the clinical wettability of tooth surfaces, the saliva-induced aggregation of oral bacteria and finally the relative salivary flow conditions around the tooth surfaces where the bacterial samples were collected. On basis of these results, it was suggested that saliva and oral bacteria could at least partly be looked upon as being a biological colloid system system. Further studies were designed to investigate whether oral bacteria could be regarded as colloid particles or not, and to study the properties of saliva as a colloid suspending medium. These studies showed that bacteria suspended in different salt solutions were dependent on pH, ionic concentration and the valency of the cations in the solutions to be able to aggregate. The aggregating capacity further seemed to be specific for different bacterial species and strains. Addition of saliva from "heavy" and "light" plaque formers influenced the colloid stability of bacteria suspended in water in different ways. Saliva from "light" plaque formers gave a lower colloid stability in suspensions of a plaque-forming bacterium (Streptococcus sanguis) as compared to the addition of saliva from "heavy" plaque formers. No such differences were observed in suspensions of S. salivarius, which is normally not a plaque-forming bacterium. The results indicate that colloid-chemical processes might be involved in bacterial attachment to tooth surfaces, and that initial bacterial colonization of tooth surfaces at least partly follow general biophysical laws.

Influence of saliva from 'heavy' and 'light' plaque formers on the colloidal stability of bacterial suspensions.

The influence of parotid saliva on the colloid stability of suspensions of Streptococcus sanguis and S. salivarius was studied in groups of previously identified 'heavy' and 'light' plaque formers. For S. sanguis it was observed that addition of parotid saliva from light plaque formers had more pronounced negative influence on the colloid stability than addition of such saliva from heavy plaque formers. No differences were observed for S. salivarius. The results indicate that saliva and bacteria might be regarded as a biological colloidal system and that the individual rate of plaque formation can perhaps be partially related to the colloid-chemical properties of bacteria and saliva.

Influence of pH on the suspension stability of some oral bacteria.

The purpose of the present study was to investigate the suspension stability of some oral bacteria at some clinically relevant pH levels. Through determinations of the electrophoretic mobility and sedimentation behavior at pH 5, 6, and 7, it was shown that within this pH range estimations of both the zeta-potentials and colloidal stability were pH-dependent for the bacteria and bacterial suspensions tested. The observations are in line with the DLVO theory for lyophobic sols and indicate that bacteria behave like colloidal particles when suspended in certain media. The colloidal behavior further seemed to vary in relation to bacterial species and strains.

Effects of cations on the colloidal stability of some oral bacteria.

The colloidal stability of seven strains of oral bacteria was studied at various concentrations of mono-, di-, and multi-valent metal counterions in the suspending media. Alterations in the concentration and the valency of the counterions were shown to influence the colloidal stability of the bacterial suspensions, as determined by their rate and time of initial sedimentation. The observations were in general in agreement with the accepted theories for colloidal stability. The results support previously made suggestions that under certain experimental conditions oral bacteria behave like colloidal particles.

Rate of plaque formation--some clinical and biochemical characteristics of "heavy" and "light" plaque formers.

The purpose of the present study was to give a clinical and biochemical characterization of two groups of individuals with different rates of plaque formation. From 133 individuals, 9 "heavy" and 10 "light" plaque formers were selected. The mean plaque index after 3 days of plaque accumulation, on buccal surfaces of premolars and first molars, was 2.6 for the "heavy" and 0.6 for the "light" plaque formers. The following variables were determined: periodontal status, DFS, dietary habits, salivary secretion rate and buffer effect, S. mutans and lactobacillus counts in saliva, salivary content of IgA, lactoferrin, lactoperoxidase and lysozyme, saliva-induced aggregation of certain oral streptococci, gel electrophoresis of saliva, amino acid composition of saliva and the acquired pellicle and retention depth of the dentogingival area. Comparing the two groups of plaque formers, statistically significant differences were found for the following three variables: parotid saliva-induced aggregation of a strain of S. sanguis, content of glutamic acid in the acquired pellicle and retention depth of the dentogingival area for maxillary premolars. Large variations for all studied variables were found, both within and between the groups. Several factors may be involved in plaque formation and none of the studied variables alone could explain the large difference in the amount of plaque formed after 3 days between the "heavy" and "light" plaque formers.

Biophysical and microbiologic studies of "heavy" and "light" plaque formers.

In order to trace factors possibly influencing initial bacterial colonization of tooth surfaces, a multiple linear regression analysis was performed on the present results as well as on those from certain clinical and biochemical studies previously performed in the same subjects. No statistically significant differences were observed between the two groups of plaque formers for the individual parameters. The multiple regression analysis showed, however, that the calculated work of adhesion for two polar and one non polar test liquid, the rate of unstimulated salivary secretion, morphologic differences in the dento-gingival areas of maxillary premolars and saliva-induced aggregation of S. mitior were crucial for about 90% of the variation in the number of CFU colonizing tooth surfaces after both 2 and 6 h. Both electrostatic and van der Waal's forces, as well as salivary flow conditions could thus have influenced the initial attachment of bacteria to the test surfaces. It is concluded that saliva including its suspended microorganisms could be looked upon as being a colloidal biological system, and if so, the initial bacterial colonization of tooth surfaces should follow known, general biophysical laws.

Evaluation of a simplified diagnostic aid (Oricult-N) for detection of oral candidoses.

The validity of a simplified diagnostic aid, Oricult-N, for detection of oral candidoses was compared with smears stained according to the periodic acid-Schiff's method. Samples were taken from 80 locations in 36 patients with lesions suspected for candidal infection. There was a statistically significant correlation between the two methods (P less than 0.001).