Attitudes to dental care, Sweden 2003-2013, and clinical correlates of oral health-related quality of life in 2013.

OBJECTIVE: To investigate attitudes to dental care, and to assess possible associations with socio-economic and clinical variables over a period of ten years, and to investigate the association between OHRQoL assessed by oral impact on daily performance (OIDP), and socio-economic, dental care habits, smoking and oral status. MATERIALS AND METHODS: Cross-sectional studies performed in the county of Dalarna, Sweden, in 2003, 2008 and 2013. Random samples of 1,107-1,115 dentate individuals, aged 30-85 years, who answered a questionnaire and who were radiographically and clinically examined were included. RESULTS: The importance of preventive treatment, regular recalls and meeting the same caregiver as on previous visits became less important. In individuals with alveolar bone loss, meeting the same caregiver as on previous visits was important (P<.05). In individuals with manifest caries, information on treatment cost was important, while prevention became less important (P<.05). OIDP was reported by 31% of the individuals in the study, and frequent impact was reported by 10%. Individuals with manifest caries lesions, less than 20 remaining teeth, and temporomandibular disorders (TMD) reported OIDP to a significantly higher degree, compared to orally healthy individuals. CONCLUSION: Attitudes important in maintaining and improving good oral health, such as preventive care and regular recalls to dentistry, became less important during this period of 10 years. Oral impact was found to be associated with irregular dental visits and limited economy for dental care, individuals with less than 20 remaining teeth, TMD and manifest caries.

Trends over 30 years in the prevalence and severity of alveolar bone loss and the influence of smoking and socio-economic factors--based on epidemiological surveys in Sweden 1983-2013.

OBJECTIVE: Epidemiological studies of the prevalence of periodontitis over an extended time using the same methodology to investigate and classify periodontitis are sparse in the literature. Smoking and socio-economic factors have been proven to increase the risk for periodontal disease. The objective of this study was to investigate 30-year time trends, using the same methodology to classify the prevalence and severity in alveolar bone loss (ABL) and to investigate the influence of tobacco and socio-economic factors. METHODS: Four cross-sectional epidemiological studies in an adult population were performed in the county of Dalarna, Sweden, in 1983, 2003, 2008 and 2013. Random samples of 787-1133 individuals aged 35-85 who answered a questionnaire about tobacco use and socio-economic factors were radiographically and clinically examined. A number of teeth, ABL and calculus visible on radiographs were registered. The severity of ABL as detected on radiographs was classified into no bone loss, moderate or severe. RESULTS: The prevalence of moderate ABL decreased from 45% in 1983 to 16% in 2008, but increased to 33% in 2013 (P < 0.05). The prevalence of severe ABL remained the same from 1983 (7%) to 2013 (6%). Calculus visible on radiographs increased from 22% in 2008 to 32% in 2013 (P < 0.05). Socio-economic factors had limited impact on the severity of ABL. CONCLUSION: Moderate ABL and calculus visible on radiographs significantly increased between 2008 and 2013. Smoking was the strongest factor associated with ABL overall.

A two-nucleotide deletion renders the mannose-binding lectin 2 (MBL2) gene nonfunctional in Danish Landrace and Duroc pigs.

The mannose-binding lectins (MBLs) are central components of innate immunity, facilitating phagocytosis and inducing the lectin activation pathway of the complement system. Previously, it has been found that certain single-nucleotide polymorphisms (SNPs) in porcine MBL1 and MBL2 (pMBL1, pMBL2) affect mRNA expression, serum concentration, and susceptibility to disease, but the combinatory effect of pMBL1 and pMBL2 genotypes needs further elucidation. In the present study, pMBL1 and pMBL2 alleles, combined pMBL haplotypes, and MBL-A concentration in serum were analyzed in purebred Landrace (N = 30) and Duroc (N = 10) pigs. Furthermore, the combined pMBL haplotypes of 89 Piètrain × (Large White × Landrace) crossbred pigs were studied, and the genotypes of 67 crossbreds challenged with Escherichia coli were compared to their individual disease records. In the purebred animals, three non-synonymous SNPs and a two-nucleotide deletion were detected in the coding sequence of pMBL2. The two-nucleotide deletion was present at a frequency of 0.88 in the Landrace pigs and 0.90 in the Duroc pigs, respectively. In the crossbreds, the T allele of the SNP G949T in pMBL1-previously shown to have profound effect on MBL-A concentration even in the heterozygote condition-was detected in 47 % of the animals. Finally, an association was found between low-producing MBL genotypes and low body weight on the day of weaning in the same animals.

Improved replication efficiency of echovirus 5 after transfection of colon cancer cells using an authentic 5' RNA genome end methodology.

Oncolytic virotherapy is a promising novel form of cancer treatment, but the therapeutic efficiency needs improvement. A potential strategy to enhance the therapeutic effect of oncolytic viruses is to use infectious nucleic acid as therapeutic agent to initiate an oncolytic infection, without administrating infectious viral particles. Here we demonstrate improved viral replication activation efficiency when transfecting cells with 5' end authentic in vitro transcribed enterovirus RNA as compared to genomic RNA with additional non-genomic 5' nucleotides generated by conventional cloning methods. We used echovirus 5 (E5) as an oncolytoc model virus due to its ability to replicate in and completely destroy five out of six colon cancer cell lines and kill artificial colon cancer tumors (HT29 spheroids), as shown here. An E5 infectious cDNA clone including a hammerhead ribozyme sequence was used to generate in vitro transcripts with native 5' genome ends. In HT29 cells, activation of virus replication is approximately 20-fold more efficient for virus genome transcripts with native 5' genome ends compared to E5 transcripts generated from a standard cDNA clone. This replication advantage remains when viral progeny release starts by cellular lysis 22 h post transfection. Hence, a native 5' genomic end improves infection activation efficacy of infectious nucleic acid, potentially enhancing its therapeutic effect when used for cancer treatment. The clone design with a hammerhead ribozyme is likely to be applicable to a variety of oncolytic positive sense RNA viruses for the purpose of improving the efficacy of oncolytic virotherapy.

Residual force enhancement after stretch in striated muscle. A consequence of increased myofilament overlap?

When skeletal muscle is stretched above optimal sarcomere length during tetanic activity there is an increase in force that stays above the isometric force level throughout the activity period. This long-lasting increase in contractile force, generally referred to as 'residual force enhancement after stretch' (FE(resid)), has been studied in great detail in various muscle preparations over more than half a century. Substantial evidence has been presented to show that non-uniform sarcomere behaviour plays a major part in the development of FE(resid). However, in a great number of recent studies the role of sarcomere non-uniformity has been challenged and alternative mechanisms have instead been proposed to explain the increase in force such as enhancement of cross-bridge function and/or strengthening of parallel elastic elements along the muscle fibres. This article presents a short review of the salient features of FE(resid) and provides evidence that non-uniform sarcomere behaviour is indeed likely to play a major role in the development of FE(resid). Electron microscopical studies of fibres rapidly fixed after active stretch demonstrate that, dispersed in the preparation, there are assymetrical length changes within the two halves of myofibrillar sarcomeres resulting in greater filament overlap in one half of the sarcomere than in the opposite sarcomere half. Sarcomere halves with increased filament overlap will consequently be in a situation where they are able to produce a greater force than that recorded in the isometric control. Weaker regions in series will be able to keep the enhanced force by recruitment of elastic elements.

Extensive polymorphism in the porcine Toll-like receptor 10 gene.

The great importance of the Toll-like receptors (TLRs) in innate immunity is well established, but one family member--TLR10--remains elusive. TLR10 is expressed in various tissues in several species, but its ligand is not known and its function is still poorly understood. The open reading frame of TLR10 was sequenced in 15 wild boars, representing three populations, and in 15 unrelated domestic pigs of Hampshire, Landrace and Large White origin. Amino acid positions corresponding to detected nonsynonymous single nucleotide polymorphisms (SNPs) were analysed in the crystal structures determined for the human TLR1-TLR2-lipopeptide complex and the human TLR10 Toll/Interleukin 1 receptor (TIR) dimer. SNP occurrence in wild boars and domestic pigs was compared, and haplotypes for the TLR10 gene and the TLR6-1-10 gene cluster were reconstructed. Despite the limited number of animals sequenced in the present study (N = 30), a larger number of SNPs were found in TLR10 than recently reported for TLR1, TLR6 and TLR2. Thirty-three SNPs were detected, of which 20 were nonsynonymous. The relative frequency of nonsynonymous (d(N) ) and synonymous (d(S) ) SNPs between wild boars and domestic pigs was higher in TLR10 than recently reported for TLR1, TLR6 and TLR2. However, the polymorphism reported in the present study seems to leave the function of the TLR10 molecule unaffected. Furthermore, no nonsynonymous SNPs were detected in the part of the gene corresponding to the hinge region of the receptor, probably reflecting rigorously acting functional constraint. The total number of SNPs and the number of nonsynonymous SNPs were significantly lower (P < 0.05) in the wild boars than in the domestic pigs, and fewer TLR10 haplotypes were present in the wild boars. The majority of the TLR6-1-10 haplotypes were specific for either wild boars or domestic pigs, probably reflecting differences in microbial environment and population history.

Contractile performance of striated muscle.

The single muscle fiber preparation provides an excellent tool for studying the mechanical behaviour of the contractile system at sarcomere level. The present article gives an overview of studies based on intact single fibers from frog and mouse skeletal muscle. The following aspects of muscle function are treated: (1) The length-tension relationship. (2) The biphasic force-velocity relationship. (3) The maximum speed of shortening, its independence of sarcomere length and degree of activation. (4) Force enhancement during stretch, its relation to sarcomere length and myofilament lattice width. (5) Residual force enhancement after stretch. (6) Force reduction after loaded shortening. (7) Deactivation by active shortening. (8) Differences in kinetic properties along individual muscle fibers.

Dynamic allosteric communication pathway directing differential activation of the glucocorticoid receptor.

Allosteric communication within proteins is a hallmark of biochemical signaling, but the dynamic transmission pathways remain poorly characterized. We combined NMR spectroscopy and surface plasmon resonance to reveal these pathways and quantify their energetics in the glucocorticoid receptor, a transcriptional regulator controlling development, metabolism, and immune response. Our results delineate a dynamic communication network of residues linking the ligand-binding pocket to the activation function-2 interface, where helix 12, a switch for transcriptional activation, exhibits ligand- and coregulator-dependent dynamics coupled to graded activation. The allosteric free energy responds to variations in ligand structure: subtle changes gradually tune allostery while preserving the transmission pathway, whereas substitution of the entire pharmacophore leads to divergent allosteric control by apparently rewiring the communication network. Our results provide key insights that should aid in the design of mechanistically differentiated ligands.

Laser diffraction studies on single skeletal muscle fibers.

Sarcomere movements during isometric tetanic contractions were resolved to 50 angstroms by diffraction techniques. After a latent period that followed the first stimulus, all sarcomeres shortened simultaneously and uniformly. Oscillations in length and tension in synchrony with the stimuli occurred during an incomplete tetanus. However, no oscillations in length were detected during the plateau of a tetanus.

Release of calcium into the myofibrillar space in response to active shortening of striated muscle.

AIM: The study was undertaken to explore whether shortening of striated muscle during activity is associated with release of bound Ca2+ into the myofibrillar space as has previously been proposed in order to explain the depressant effect of active shortening. METHODS: The experiments were carried out on single muscle fibres isolated from the anterior tibialis muscle of Rana temporaria. The fibres were loaded with the calcium sensitive indicator Fluo-3. The fibres, stimulated to produce a partially fused isometric tetanus, were subjected to a shortening ramp or, alternatively, to a stretch ramp during activity while force, fibre length, sarcomere length and the Fluo-3 signal were recorded. RESULTS: A shortening ramp performed during a partially fused tetanus caused an increase in the myofibrillar free calcium concentration and produced, simultaneously, a decrease in active force. The isometric force recovered gradually after the shortening ramp, while the intracellular Ca2+ concentration stayed above the control level during the remainder of the stimulation period. A stretch ramp applied during a partially fused tetanus caused a considerably smaller change in the myofibrillar Ca2+ concentration. CONCLUSION: The results provide evidence that the myosin cross-bridges interact with the calcium binding sites on the thin filaments during active shortening, causing sustained release of calcium and reduced contractile strength.

Critical sarcomere extension required to recruit a decaying component of extra force during stretch in tetanic contractions of frog skeletal muscle fibers.

29 single frog skeletal muscle fibers were stretched during fused tetanic contractions. The force increase during stretch exhibited a breakpoint at a critical length change (average: 16.6 nm per one-half sarcomere) that was independent of velocity of stretch and of sarcomere length between 1.8 and 2.8 microns. After stretch there was an early decaying force component with a force-extension curve similar to that during stretch, which disappeared over approximately 2 s. This component was removed by a small, quick release, leaving a longer-lasting component. The critical amplitude of release required to produce this result was found by clamping the fiber to a load at which there was zero velocity of shortening. This amplitude increased with time up to the angle in the force record during stretch, was constant for the remainder of the stretch, and decreased with time after the end of stretch; it was consistently less than the critical amplitude of stretch required to reach the breakpoint of force enhancement during stretch but was also independent of sarcomere length. The force drop accompanying the critical release showed a small increase up to an optimum magnitude at 2.4--2.7 microns sarcomere length, with a decrease at longer lengths.

Residual force enhancement after stretch of contracting frog single muscle fibers.

Single fibers from the tibialis anterior muscle of Rana temporaria at 0.8-3.8 degrees C were subjected to long tetani lasting up to 8 s. Stretch of the fiber early in the tetanus caused an enhancement of force above the isometric control level which decayed only slowly and stayed higher throughout the contraction. This residual enhancement was uninfluenced by velocity of stretch and occurred only on the descending limb of the length-tension curve. The absolute magnitude of the effect increased with sarcomere length to a maximum at approximately 2.9 micrometers and then declined. The phenomenon was further characterized by its dependence on the amplitude of stretch. The final force level reached after stretch was usually higher than the isometric force level corresponding to the starting length of the stretch. The possibility that the phenomenon was caused by nonuniformity of sarcomere length along the fiber was examined by (a) laser diffraction studies that showed sarcomere stretch at all locations and (b) studies of 9-10 segments of approximately 0.6-0.7 mm along the entire fiber, which all elongated during stretch. Length-clamped segments showed residual force enhancement after stretch when compared with the tetanus produced by the same segment held at the short length as well as at the long length. It is concluded that residual force enhancement after stretch is a property shown by all individual segments along the fiber.

Differential uptake of estramustine phosphate metabolites and its correlation with the levels of estramustine binding protein in prostate tumor tissue.

Estracyt (EMP) has been used for the treatment of hormone refractory prostate cancer for many years. Recently, new data from combination studies have given rise to new interest in this old drug. Explanations for the synergy found in the clinic are many, but one major factor may be the previous indication that the drug accumulates in the prostate tumor. We have, therefore, examined the level of the four metabolites, estromustine (EoM), estramustine (EaM), estrone, and estradiol in the tumor and serum of 14 patients with T2 and T3 prostate cancer receiving a single i.v. dose of 600 mg of EMP, about 12 h before radical prostatectomy. Because it has been suggested that the uptake into the prostate tumor is due to binding to the estramustine binding protein (EMBP), we have in addition measured the level of EMBP in the prostate tumor tissue. The main serum and tissue metabolite in all patients was EoM followed by EaM, estrone, and estradiol. The levels for EoM ranged from 63.8-162.8 ng/ml in the serum and from 64.8-1209 ng/ml in the prostate tumor, resulting in a mean ratio for serum to tumor of 1:5. The levels for EaM ranged from 8.3-51.4 ng/ml in the serum and 73.9-563.4 ng/ml in the tumor, giving a mean ratio for serum to tumor of 1:13. The levels of EMBP were higher in T3 tumors than in T2 tumors, 54.1 and 40.7 ng/g tissue, respectively. A significant correlation was found between the levels of EaM (r = 0.60) and the levels of EMBP in the tumor. These data demonstrate that 12 h after a single i.v. dose of 600 mg of EMP the levels of the cytotoxic metabolites EoM and EaM are substantially higher in the tumor than in the serum of the same patient and that a correlation exists between the levels of EaM in the tumor and the levels of EMBP. Thus, this supports the hypothesis that the EMBP is responsible for the retention of EoM and EaM in the prostate tumor.

Cytotoxic effect and uptake of estramustine in a rat glioma model.

The cytotoxic effect of estramustine-phosphate (EMP) and the uptake in tumor tissue were investigated in a rat glioma model in vitro and in vivo. EMP, a combination of nornitrogen mustard and 17beta-estradiol, is a cytotoxic drug which main target is assumed to be the microtubule system. EMP and its metabolite estramustine (EaM) have a demonstrated anti-tumorous effect on human glioma cells in vitro. The drug uptake in tumor tissue and subsequently also the cytotoxic effect, is believed to depend, at least partially, on a specific estramustine-binding protein (EMBP) which is present in human glioma tissue. In this study we have examined the effects and pharmacokinetics of EaM in the nitrosourea induced BT4C rat glioma model. The tumor was characterized by infiltrative growth with a histopathological picture resembling gliosarcoma. The presence of EMBP was demonstrated by immunohistology. In vitro EMP caused a dose-related inhibition of BT4C-cell growth. In vivo, in the rat model, a significant inhibition of tumor growth was obtained after administration of EaM 20 mg/kg/d i.p. The pharmacokinetics of EaM resembled that found in the human clinical situation with EaM as the main metabolite accumulating in tumor tissue. The mean concentration ratio of EaM was 15.6 in tumor versus serum, and 1.8 in tumor versus normal brain of 1.8. The cytotoxic effect demonstrated in the rat glioma model justifies further evaluation of EMP/EaM in the treatment of malignant gliomas.

Distribution of estramustine in the BT4C rat glioma model.

Estramustine (EaM), a carbamate ester of 17beta-estradiol and nor-nitrogen mustard, is a cytotoxic compound with antitumoral effect in malignant glioma in vitro and in vivo . However, knowledge of the pharmacokinetics of EaM in experimental glioma is limited. The objective of this study was therefore to investigate further the distribution of EaM in the BT4C rat glioma model. Assessment of EaM uptake and distribution was performed by quantitative whole-body autoradiography. In addition, the uptake of EaM and its metabolites estromustine (EoM), estradiol, and estrone were analyzed by gas chromatography. EaM was taken up from the circulation and was found to be the main product in glioma tissue. Whole-body autoradiography after [14C]-EaM administration revealed a strong 14C label simultaneously in tumor and normal brain tissue at 0.5 h after drug administration. In tumor tissue, sustained high levels of 14C label were detected at 12 h after drug administration. In contrast to the tumor, radioactivity in normal brain tissue rapidly leveled off, indicating a retention of radioactivity in the tumor. The tumor/brain radioactivity ratio reached a peak of 4.5 at 12 h after drug administration. High levels of 14C label were also found in pulmonary tissue. By gas chromatography, EoM was found to be the main metabolite in plasma. However, EaM reached higher levels in tumor tissue, with the mean tumor/plasma ratio being 11.7 as compared with 2.0 for EoM. Only low plasma levels of the estrogen metabolites were detected. In conclusion, EaM is taken up in the BT4C rat glioma tissue and is retained in the tumor as compared with normal brain tissue and plasma. EaM showed a greater selectivity for tumor tissue, exhibiting a high tumor/plasma ratio as compared with EoM. The distribution pattern after administration of EaM, as evaluated by both whole-body autoradiography and gas chromatography, supports the earlier suggestion that the uptake is related to a protein with EaM-binding characteristics.

Spectroscopic characterization of bacteriorhodopsin's L-intermediate in 3D crystals cooled to 170 K.

Spectra are presented from a single 3D microcrystal of bacteriorhodopsin (bR) cooled to 170 K under various illumination conditions. This set is necessary and sufficient to assign the relevant crystal reference spectra. A spectral decomposition of the difference spectrum obtained following the trapping protocol of Royant et al. (2000) (Nature 406, 645-648) is given, confirming that the low temperature L-intermediate was the species that dominated the structural rearrangements previously reported. Smaller contributions from the K and M spectral intermediates are also quantified. Mechanistic insights derived from the X-ray structures of the early bR intermediates are discussed.

Mechanism underlying double-hyperbolic force-velocity relation in vertebrate skeletal muscle.

The force-velocity relation of frog striated muscle exhibits two distinct curvatures located on either side of a breakpoint that occurs near 80% of maximum isometric force (Po) where the shortening velocity is approximately 1/10 of Vmax. The present experiments have been performed to further elucidate the high-force deviation of the force-velocity curve in frog single muscle fibres. The biphasic shape of the force-velocity curve appears at the same relative values of Po and Vmax also after depressing the isometric force to 80% of the control value by dantrolene, a substance known to reduce the release of activator calcium from the sarcoplasmic reticulum. This finding suggests that the breakpoint of the force-velocity curve is not related to the force level per se but rather to the speed of shortening of the contractile system. Thus as the speed of shortening goes below 1/10 of Vmax, the performance of the myofilament system is changed such that less force and less motion are produced than expected from the main part of the force-velocity curve. In a series of experiments active force and fibre stiffness were simultaneously recorded while the fibre shortened at various speeds during tetanus. Stiffness was measured as the change in force that occurred in response to a 4 kHz sinusoidal length oscillation of the fibre. A plotting of stiffness against force recorded under these conditions provides a biphasic relationship with a distinct transition between the two phases near 80% of Po, i.e. at the same relative force at which the breakpoint occurs in the force-velocity curve. Above 0.8 Po stiffness increases more steeply with force than below this point. This means that while more crossbridges than expected attach to the thin filaments when the load is raised above 0.8 Po, the force output and the speed of shortening become lower than predicted from measurements at low and intermediate loads. The results suggest that the kinetics of crossbridge function is changed as the speed of filament sliding is reduced below a critical level, 1/10 of Vmax. Beyond this point a greater portion of myosin crossbridges would seem to accumulate in a state where less force is being produced. Data are also presented to further elucidate the force-velocity relation at negative loads. In these experiments the passive tension at long sarcomere lengths has been utilized to produce a longitudinal compressive force on the sarcomeres during unloaded shortening (force-clamp recording).(ABSTRACT TRUNCATED AT 400 WORDS)

Myofibrillar fatigue versus failure of activation.

Two principal mechanisms underlying fatigue of isolated muscle fibers are described: failure of activation of the contractile system and reduced performance of the myofibrils due to altered kinetics of crossbridge function. The relative importance of these two mechanisms during development of fatigue is discussed.

Double-hyperbolic nature of the force-velocity relation in frog skeletal muscle.

Load-clamp recordings were performed on single fibres of the anterior tibialis muscle of Rana temporaria to further characterize the relationship between force and velocity of shortening. With the techniques used the speed of shortening could be recorded at different loads both from the fibre as a whole and from individual segments (ca. 0.5 mm in length) along the intact fibre. In accordance with previous observations the force-velocity relation was found to have a break point near 78% of the measured isometric force, P0. The results furthermore demonstrate that the force-velocity relation is composed of two hyperbolic functions, above and below the break point, respectively. The hyperbola in the high-force region has a more pronounced curvature than that observed at low and intermediate loads. An increase in sarcomere length from 2.10 to 2.60 microns makes both portions of the force-velocity relation less curved and reduces, to a great extent, the difference in curvature between the two hyperbolas. Similar effects are produced by raising the tonicity of the extracellular medium. Addition of caffeine (0.5 mM), or changing the temperature within the range 1-11 degrees C, does not affect the shape of any portion of the force-velocity relation. The fact that the above features of the force-velocity relation appear in very short segments as well as in the whole fibre suggests that the double-hyperbolic shape of the force-velocity curve does represent the contractile behaviour at sarcomere level. The system behaves as if a fraction of myosin cross-bridges were neutralized, or abolished (leading to a relative decrease in force), as the speed of filament sliding is reduced below approximately 10% of the maximum.