Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy to diagnose osteoarthritis in equine serum.

BACKGROUND: Reliable and validated biomarkers for osteoarthritis (OA) are currently lacking. OBJECTIVES: To develop an accurate and minimally invasive method to assess OA-affected horses and provide potential spectral markers indicative of disease. STUDY DESIGN: Observational, cross-sectional study. METHODS: Our cohort consisted of 15 horses with OA and 48 without clinical signs of the disease, which were used as controls. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy was used to investigate serum samples (50 µL) collected from these horses. Spectral processing and multivariate analysis revealed differences and similarities, allowing for detection of spectral biomarkers that discriminated between the two cohorts. A supervised classification algorithm, namely principal component analysis coupled with quadratic discriminant analysis (PCA-QDA), was applied to evaluate the diagnostic accuracy. RESULTS: Segregation between the two different cohorts, OA-affected and controls, was achieved with 100% sensitivity and specificity. The six most discriminatory peaks were attributed to proteins and lipids. Four of the spectral peaks were elevated in OA horses, which could be potentially due to an increase in lipids, protein expression levels and collagen, all of which have been previously reported in OA. Two peaks were found decreased and were tentatively assigned to the reduction of proteoglycan content that is observed during OA. MAIN LIMITATIONS: The control group had a wide range of ages and breeds. Presymptomatic OA cases were not included. Therefore, it remains unknown whether this test could also be used as an early diagnostic tool. CONCLUSIONS: This spectrochemical approach could provide an accurate and cost-effective blood test, facilitating point-of-care diagnosis of equine OA.

Temperature, plant species and residence time effects on nitrogen removal in model treatment wetlands.

Total nitrogen (TN) removal in treatment wetlands (TWs) is challenging due to nitrogen cycle complexity and the variation of influent nitrogen species. Plant species, season, temperature and hydraulic loading most likely influence root zone oxygenation and appurtenant nitrogen removal, especially for ammonium-rich wastewater. Nitrogen data were collected from two experiments utilizing batch-loaded (3-, 6-, 9- and 20-day residence times), sub-surface TWs monitored for at least one year during which temperature was varied between 4 and 24 °C. Synthetic wastewater containing 17 mg/l N as NH4 and 27 mg/l amino-N, 450 mg/l chemical oxygen demand (COD), and 13 mg/l SO4-S was applied to four replicates of Carex utriculata, Schoenoplectus acutus and Typha latifolia and unplanted controls. Plant presence and species had a greater effect on TN removal than temperature or residence time. Planted columns achieved approximately twice the nitrogen removal of unplanted controls (40-95% versus 20-50% removal) regardless of season and temperature. TWs planted with Carex outperformed both Typha and Schoenoplectus and demonstrated less temperature dependency. TN removal with Carex was excellent at all temperatures and residence times; Schoenoplectus and Typha TN removal improved at longer residence times. Reductions in TN were not accompanied by increases in NO3, which was consistently below 1 mg/l N.

On fitting the k-C* first order model to batch loaded sub-surface treatment wetlands.

The k-C* first order model was fit to time-series COD data collected from batch-loaded model wetlands. Four replicates of four plant species treatments; Carex utriculata (sedge), Schoenoplectus acutus (bulrush), Typha latifolia (cattail) and unplanted controls were compared. Temperature was varied by 4 degrees C from 24 degrees C to 4 degrees C to 24 degrees C over a year-long period. One mathematical fit was made for each wetland replicate at each temperature setting (192 fits). Temperature effects on both parameters were subsequently estimated by fitting the Arrhenius relationship to the estimated coefficients. Inherent interactions between k and C* make values dependent on sample timing and statistical technique for either time series (batch load) or distance profile (plug flow) data. Coefficients calibrated using the Levenberg-Marquardt method are compared to values previously reported using a nonlinear mixed effect regression technique. Overall conclusions are similar across approaches: (a) the magnitude of the coefficients varies strongly by species; (b) the rate constant k decreases with increasing temperature; and (c) temperature and species variation in the residual concentration C* is greater than the variation in k, such that variation in k alone is a poor predictor of performance. However, the magnitudes of the coefficients, especially the rate parameter k, vary between the statistical techniques, highlighting the need to better document the statistical routines used to calibrate the k-C* model.

Does batch operation enhance oxidation in subsurface constructed wetlands?

Two side-by-side experimental sub-surface flow systems allowed direct comparison of wetland performance under batch and continuous-flow operation. One system consisted of microcosm "columns" operated in 20-day batch mode while the second consisted of continuous-flow "cells" operated at a five-day residence time. Both systems treated identical synthetic domestic wastewater for two years and then treated identical synthetic mine-impacted water for one year. Each system had replicates planted with Typha latifolia, Scirpus acutus and unplanted controls. Temperature was cycled annually between 4 to 24 degrees C. Results indicated that plant species, season, and mode of operation interacted strongly in controlling dynamics of COD, nitrogen species, phosphate, sulfate, and redox potentials. In batch-loaded columns, between-species differences in oxidation and COD removal were large in winter, during plant dormancy, but absent in summer; COD removal, sulfate concentration, and redox potentials were closely correlated, suggesting that variation in root-zone oxygenation due to seasonal plant growth patterns and temperature-dependent plant and microbial respiration may explain observed differences. In the continuous-flow cells, species and seasonal differences were minimal or non-existent, indicating that under continuous-flow operation plants either did not influence root zone oxidation or that this influence had no effect on wetland performance for COD and nutrient removal or sulfate reduction.

Changes in myosin structure and function in response to glycation.

Nonenzymatic glycosylation (glycation) is recognized as an important post-translational modification underlying alterations of structure and function of extracellular proteins. The effect of glycation on intracellular proteins is, on the other hand, less well known despite the vital importance of intracellular proteins for cell, tissue, and organ function. The aim of this study was to explore the effects of glycation on the structure and function of skeletal muscle myosin. Myosin was incubated for up to 30 min with glucose and subsequently tested for structural and functional modifications by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and a single-fiber in vitro motility assay, respectively. MALDI spectra revealed glycation-related structural alterations as evidenced by the disappearance of specific Lys-C proteolysis products and the appearance of higher mass peaks that are attributed to cross-linking by glucose. This change was paralleled by a significant reduction in the in vitro motility speed, suggesting a structure-related decline in myosin mechanics in response to glucose exposure. Further evidence that early glycation products form in the regulatory regions of the myosin molecule is derived from the fact that there is complete reversal of motility speed after reaction with the Schiff base-cleaving agent hydroxylamine hydrochloride. Thus, glycation of skeletal muscle myosin has a significant effect on both the structural and functional properties of the protein, a finding that is important in understanding the mechanisms underlying the impairment in muscle function associated with aging and diabetes.

Effects of aging on actin sliding speed on myosin from single skeletal muscle cells of mice, rats, and humans.

The effects of aging on the mechanical properties of myosin were measured in 87 fibers from muscles of humans (n = 40), rats (n = 21), and mice (n = 26) using a single fiber in vitro motility assay. Irrespective of species, an 18-25% aging-related slowing in the speed of actin filaments was observed from 62 single fibers expressing the slow (type I) beta-myosin heavy chain isoform. The mechanisms underlying the aging-related slowing of motility speed remain unknown, but it is suggested that posttranslational modifications of myosin by oxidative stress, glycation, or nitration play an important role. The aging-related slowing in the speed of actin filaments propelled by the type I myosin was confirmed in three mammalian species with an approximately 3,400-fold difference in body size. Motility speed from human myosin was 3-fold slower than from myosin of the approximately 3,400-fold smaller mouse and approximately twofold slower when compared with the approximately 130-fold smaller rat, irrespective of age. A strong correlation was observed between the log values of actin sliding speed and body mass, suggesting that the effects of scaling is, at least in part, due to altered functional properties of the motor protein itself.

Effects of aging on regulation of muscle contraction at the motor unit, muscle cell, and molecular levels.

Rodent motor units, muscle fibers, and motor proteins undergo significant aging-related changes. Such changes include spatial organization and physiological properties of fast- and slow-twitch single motor units, regulation of contractile speed and force generation capacity at the muscle fiber level, and altered functional properties of the motor protein myosin. In addition to specific changes, there also appears to be a "disorganization" of the coordinated expression of contractile, sarcoplasmic reticular, and mitochondrial protein isoforms in aging skeletal muscle. This is suggested to have a strong impact on aging-related impairments in muscle function in addition to the changes in specific muscle proteins.

Actomyosin interactions in a novel single muscle fiber in vitro motility assay.

A novel in vitro motility assay has been developed to study the actomyosin interaction, in which the molecular motor protein myosin has been extracted and immobilized directly from 2-4 mm single rat skeletal muscle fiber segments. This method study was carried out to investigate: (1) the amount of myofibrillar proteins extracted from the fiber segment; (2) the effects of temperature on the speed at which actin is propelled by fast and slow myosin; and (3) the effects of myosin isoform expression on motility speed. Approximately 80% of the myosin and myosin-associated proteins were extracted from the fiber segments. while no significant extraction was shown of the thin filament proteins. Fluorescently labeled actin filaments moved with constant speed in a bi-directional motion over the high-density myosin region in the experimental chamber, and motility speed was highly dependent on the myosin heavy chain (MyHC) isoform extracted. At 25 degrees C, significant (P < 0.001) differences in motility speed were obtained between type I (1.31 +/- 0.23 microm/s, n = 11) and IIxb (5.81 +/- 0.35 microm/s, n = 6), or llb (6.07 +/- 0.33 microm/s, n = 8) MyHC isoforms. The motility speed and maximum velocity of unloaded shortening (V0) in single fibers were well correlated, indicating that filament speed is a good molecular analogue to contractile speed at the fiber level. The effects of temperature on filament motility speed were analyzed from 10 to 35 degrees C. The Q10 values, calculated in the 10-25 degrees C temperature range, differed between slow (4.20) and fast (2.38) myosin. In conclusion, this in vitro motility assay offers a unique possibility to compare the regulatory and modulatory influence of myosin isoforms and thin filament proteins on shortening velocity, at the cellular and molecular level in the same muscle fiber.

Teachers' skill ratings of children with learning disabilities: a comparison of the United States and Japan.

This study compared U.S. and Japanese grade school teachers' perceptions of the strengths and weaknesses of children in their classrooms identified as fitting commonly used criteria for a learning disability. U.S. teachers identified 4.0 percent of their children as meeting the criteria and Japanese teachers identified 1.5 percent. The teachers then rated these children's abilities in the areas of listening, speaking, reading/writing, reasoning, mathematics, social, and study skills. Overall, U.S. and Japanese teachers' rating patterns were similar on 70 percent of the skills. In most areas where significant differences were found-listening, speaking, reading/writing and study skills-U.S. teachers rated higher percentages of their children as "weaker" than Japanese teachers. A noteworthy exception was the area of social skills where Japanese children received higher percentages of "weak" ratings. U.S. and Japanese teachers also differed in their perceptions of causative factors leading to their children's learning difficulties. We discuss the findings in terms of U.S.-Japanese differences in writing systems and cultural expectations.

In vitro motility speed of slow myosin extracted from single soleus fibres from young and old rats.

1. Isolated soleus muscle fibres from aged rats contract more slowly than those from young rats. To determine whether this effect is due to a difference between the myosin molecules, we measured the rate at which actin filaments are driven over a myosin coated surface in the presence of ATP by using a novel in vitro motility assay where myosin is extracted from single muscle fibre segments. 2. Motility was dependent on the myosin density on the coverslip. In regions of high myosin density, actin motility was orientated parallel and anti-parallel to the direction of flow during myosin adhesion to the coverslip. In contrast, in regions of lower myosin density, actin motility was more random. The speed was about 20 % higher in the high density regions (P < 0.001). Further, the speed of filaments in the high density region, moving away or towards the fibre was less variable (P < 0.05) than that of more randomly moving filaments in the low density region. 3. The speed with myosin from slow soleus fibres of young adult rats (3-6 months old; v = 1.43 +/- 0.23 microm s-1; mean +/- s.d.) was faster (P < 0.001) than with myosin from aged rats (20-24 months old; v = 1.27 +/- 0.23 microm s-1). 4. No difference in myosin isoforms between young adult and aged fibres could be detected using electrophoretic and immunocytochemical techniques. Fibres of both ages expressed the beta/slow myosin heavy chain (MyHC) isoform and slow isoforms of essential and regulatory myosin light chains (MyLCs). 5. It is concluded that an age-related alteration in myosin contributes to the slowing of the maximum shortening velocity (V0) observed in soleus muscle fibres expressing the beta/slow MyHC isoform.

Post-operative effects on insulin resistance and specific tension of single human skeletal muscle fibres.

Surgery and accidental trauma are associated with a transient period of insulin resistance, substrate catabolism and muscle weakness. In the present study, we evaluated the changes in the force-generating capacity of chemically skinned single muscle fibres following abdominal surgery. Biopsies of the m. vastus lateralis were obtained in three patients 1 day before and 3 or 6 days after surgery. Part of the biopsy was frozen for histochemical analysis of the fibre cross-sectional area (FCSA) and myofibrillar protein content, and another part was used for single-fibre contractile measurements. All patients developed insulin resistance following surgery. The maximum velocity of unloaded shortening of single muscle fibres did not change following surgery. The FCSA did not decrease after surgery, as determined either from histochemical sections or from single fibres measured at a fixed sarcomere length of 2.76+/-0.09 microm (mean+/-S.D.). Further, the force-generating capacity of the single fibres, measured as maximal Ca(2+)-activated force (P(0)) or as P(0) normalized to FCSA (specific tension), remained unchanged, as did the myofibrillar protein content of the muscle. In conclusion, the muscle weakness associated with post-operative insulin resistance is not related to a decreased specific tension or a loss of myofibrillar proteins. Other potential cellular mechanisms underlying post-operative weakness are discussed.

Regulation of human muscle contraction at the cellular and molecular levels.

The rat is the most extensively characterized species with regard to regulation of muscle contraction and myofibrillar protein isoform expression, but there is reason to question whether results from small mammals, such as the rat, can be extrapolated directly to larger mammals, such as man. Studies of human muscle contraction have primarily used different in vivo muscle function measurements, i.e. measurements of force at different speeds of movement during electrical stimulation or voluntary activation. These measurements give important information on overall muscle function, but they are of limited value for our understanding of regulation of muscle contraction. In basic science, cellular- and molecular-physiological methods have been used for many years, but these techniques have so far only rarely been used in studies of human muscle contraction. Detailed studies of human muscle contraction can be performed in the short muscle fibre segments obtained by the percutaneous muscle biopsy technique both at the cellular and molecular level. The skinned fibre preparation in combination with a novel in vitro motility assay offers a unique possibility to investigate regulation of human muscle contraction at the cellular and molecular levels in the same muscle cell segment in both health and disease, i.e. in muscle cells characterized according to the type and amount of expressed myofibrillar protein isoforms.

The effect of age on in vitro motility speed of slow myosin extracted from single rat soleus fibres.

The effect of age on the motor protein myosin was examined in a novel in vitro motility assay. Myosin was extracted from soleus fibres of young (3-6 month) and old (20-24 month) rats. All fibres expressed the type I myosin heavy chain (MyHC) and the slow isoforms of the myosin light chains (MyLCs). In vitro motility speed was significantly (P < 0.001) faster in the young adult (1.43 +/- 0.23 microm s-1) than in the aged group (1.27 +/- 0.23 microm s-1). The result indicates that the age-related decrease in contractile speed observed in slow fibres may be the effect of a change in the properties of myosin with age.

An overview of carbohydrate-protein interactions with specific reference to myosin and ageing.

Non-enzymatic glycosylation (glycation), a post-translational modification of proteins, results from the reaction of proteins with reducing sugars. Glycation is implicated in various pathologies like diabetes, Alzheimer's disease and it has been suggested to play an important role in the ageing process. Research on protein glycation has primarily studied extracellular proteins such as albumin, haemoglobin and collagen. However, there is increasing evidence that intracellular proteins may also be affected by glycation, and glycation of myosin is reported to decrease myosin ATPase activity. Glycated adducts are detected by various techniques such as chromatography, electrophoresis, fluorescence and immunochemistry. Inhibition or removal of these adducts has been achieved by chemical compounds such as aminoguanidine (amG), beta-mercaptoethanol (bME) and N-phenacylthiazolium bromide (PTB). In the present pilot study, using a novel in vitro motility assay, we have observed an attenuation in the motility speed of actin (approximately 13%) on myosin extracted from single muscle fibre segments after 15-min glucose incubation. Addition of bME to the incubation medium maintained actin motility speed.

DR4 subtypes and their molecular properties in a population-based study of Swedish childhood diabetes.

The aim of this study was to determine the association between childhood insulin-dependent diabetes mellitus (IDDM) and HLA-DR4 subtypes and to test in a population-based investigation whether the DR4 association has an effect independent to that of DQ. First, HLA genotyping identified DR4 in 337/425 (79%) patients and 148/367 (40%) controls (Odds Ratio 5.67; p < 0.01). Second, a total of 14 DR4 subtypes were detected by PCR and sequence specific oligo probes. Only two DR4 subtypes, DRB1*0401 (62% patients and 25% controls; OR 4.95, p < 0.01) and *0404 (16% patients and 10% controls; OR 1.67, p < 0.05) were however positively associated with the disease. These two subtypes were positively associated only when linked to DQB1*0302-DQA1*0301 (DQ8) (56% patients and 14% controls; OR 7.69, p < 0.01; 15% patients and 10% controls; OR 1.55, p < 0.05, respectively). When DRB1*0401 was linked to DQB1*0301-DQA1*0301 (DQ7) (6% patients and 11% controls; OR 0.52, p < 0.05), this DR4 subtypes was negatively associated with IDDM. Third, tests of strongest association allowed the following ranking of alleles or haplotypes DQB1*0302-DQA1*0301 (DQ8) > DQB1*0302 > DRB1*0401 > DRB1*0404 and the association of DRB1*0401 has a significant effect in DQ8 positive IDDM patients. We conclude that the DR4 association with IDDM is secondary to DQ by linkage disequilibrium, which support the role of HLA-DQ as a primary genetic risk factor for IDDM.

Angiofollicular lymph node hyperplasia of the parotid.

Angiofollicular lymph node hyperplasia (AFLNH) is a condition of uncertain aetiology usually presenting with mediastinal lymphadenopathy. Occasionally other lymph node groups are affected and there may be associated wide ranging systemic manifestations. A case of AFLNH affecting parotid lymph nodes is presented. The histological features of AFLNH and its variants are discussed, and current ideas on its pathogenesis are summarised.

Cloning of the amino terminal nucleotides of the antigen I/II of Streptococcus sobrinus and the immune responses to the corresponding synthetic peptides.

A portion of the antigen I/II (spaA, B, P1) gene of Streptococcus sobrinus 6715, containing the coding sequence for the amino terminal 684 amino acids of the protein, was cloned in bacteriophage lambda GT10. Selection was by immunological detection using a polyclonal antiserum to the antigen I/II from Strep. mutans. From the amino acid sequence, peptides were synthesized, 15 amino acids in length, that covered the entire sequence. In total, 260 synthetic peptides were synthesized and evaluated for their immunogenicity in Balb/C mice. Thirty-nine peptides were immunogenic, without carrier, and the antisera generated were tested for their ability to bind cells of Strep. mutans and Strep. sobrinus in a solid-phase assay. Antisera corresponding to peptides from five regions on the I/II molecule bound cells of both bacterial species. These peptides were then evaluated for their ability to stimulate in vitro murine lymphocyte proliferation, after in vivo immunization with Strep. sobrinus cells. Two of the peptides were capable of stimulating proliferation, as determined by incorporation of [3H]-thymidine into murine lymph node cells. The sequences of these 5 peptides were then compared to sequences found in the antigen I/II from Strep. mutans (Kelly et al., 1989). As expected, there was considerable homology between the cross-reactive peptides synthesized and the analogous region from Strep. mutans. This homology was not usually contiguous and suggests that the antibodies bind a face of antigen I/II that is in an alpha-helical conformation.(ABSTRACT TRUNCATED AT 250 WORDS)