Equine sperm-bound antisperm antibodies are associated with poor semen quality.

Antisperm antibodies (ASAs) have been associated with infertility in stallions. The objectives of this study were to investigate the frequency of ASA-positive semen samples in satisfactory and non-satisfactory breeder stallions, the association between ASA binding and semen quality, and factors that may affect the diagnosis. Breeding soundness examinations were performed in 21 stallions and the percentage of IgG- and IgA-bound spermatozoa was evaluated using flow cytometry. Median IgG and IgA binding did not differ between the first and second ejaculates. The percentage of IgA-bound spermatozoa was higher in non-satisfactory (n = 10) than satisfactory breeder stallions (n = 11). However, IgG binding or frequency of IgG-positive ejaculates did not differ with stallion classification. The IgG-positive stallions had significantly lower total sperm motility, concentration and total numbers than IgG-negative stallions in the first ejaculate, and lower sperm concentration in the second ejaculate. The IgA-positive stallions had lower total sperm motility, normal spermatozoa and total numbers than IgA-negative stallions in the first ejaculate, and lower total sperm motility, normal spermatozoa and total numbers in the second ejaculate. While IgG binding did not differ with season, IgA binding was higher in the non-breeding season (n = 6 stallions) than the breeding season (n = 15 stallions) in the first ejaculate. Stallion age did not differ with ASA classification. In conclusion, IgG binding was highly prevalent in both groups of stallions, while IgA binding was higher and more prevalent in non-satisfactory breeders. Both isotypes were associated with poor semen quality. Season and sexual rest had an effect on IgA but not IgG binding.

Sperm-bound antisperm antibodies prevent capacitation of bovine spermatozoa.

It was hypothesized here that sperm-bound antisperm antibodies (ASAs) impair the ability of bovine spermatozoa to undergo capacitation, bind to the zona pellucida, and complete the acrosome reaction. The effect of ASA binding on these functions was evaluated in frozen/thawed spermatozoa from four bulls before and after induction of ASAs. Ejaculates were divided into ASA negative (<10% immunoglobulin [Ig]G- and IgA-bound spermatozoa) or ASA positive (≥20% IgG and/or IgA-bound spermatozoa). The percentage of capacitated (Merocyanine 540 positive) live spermatozoa in response to heparin was lower in ASA-positive than ASA-negative ejaculates (P < 0.0001). Treatment with heparin resulted in a higher percentage of capacitated spermatozoa compared with control treatments in ASA-negative but not ASA-positive ejaculates. The percentage of capacitated spermatozoa after heparin treatment was negatively correlated with IgA (P = 0.02, R2 = -0.48) but not IgG binding. Sperm binding to the zona pellucida was lower in IgA-positive (six spermatozoa/oocyte; 3-10 spermatozoa/oocyte) than IgA-negative ejaculates (seven spermatozoa/oocyte; 4-13 spermatozoa/oocyte) (P = 0.019). Zona binding was negatively correlated with the percentage of IgA-bound spermatozoa (P = 0.04; R2 = -0.24) but not IgG-bound spermatozoa. The percentage of acrosome-reacted spermatozoa was higher in calcium ionophore A23187-treated than control aliquots in both ASA-negative and ASA-positive ejaculates (P < 0.0001). However, the percentage of acrosome-reacted spermatozoa did not differ between ASA-positive and ASA-negative samples, and no correlation was identified with IgG or IgA binding. It was concluded that sperm-bound IgA affected the ability of bovine spermatozoa to undergo capacitation. ASAs inhibited the changes in plasma membrane fluidity associated with capacitation and binding of spermatozoa to the zona pellucida.

The phonological neighbourhood effect on short-term memory for order.

There is a growing body of literature that suggests that long-term memory (LTM) and short-term memory (STM) structures that were once thought to be distinct are actually co-dependent, and that LTM can aid retrieval from STM. The mechanism behind this effect is commonly argued to act on item memory but not on order memory. The aim of the current study was to examine whether LTM could exert an influence on STM for order by examining an effect attributed to LTM, the phonological neighbourhood effect, in a task that reduced the requirement to retain item information. In Experiment 1, 18 participants completed a serial reconstruction task where neighbourhood density alternated within the lists. In Experiment 2, 22 participants completed a serial reconstruction task using pure lists of dense and sparse neighbourhood words. In Experiment 3, 22 participants completed a reconstruction task with both mixed and pure lists. There was a significant effect of neighbourhood density with better recall for dense than sparse neighbourhood words in pure lists but not in mixed lists. Results suggest that LTM exerts an influence prior to that proposed by many models of memory for order.

Effect of Bovine Sperm-Bound Antisperm Antibodies on Oviductal Binding Index.

This study was designed to test the hypothesis that sperm-bound IgG and IgA decrease binding of bull spermatozoa to oviductal epithelial cells in vitro. Three ejaculates were cryopreserved from each of four antisperm antibody (ASA)-negative satisfactory breeder bulls. Bulls were then immunized with autologous spermatozoa, and three ASA-positive ejaculates were cryopreserved from each bull post-immunization. First, microscopy methods were compared to select the most appropriate assay for evaluation of oviductal binding index (BI). The BI did not differ when the evaluation was performed under fluorescence microscopy (131.1 sperm/mm(2); 62.5-251.1 sperm/mm(2)), phase-contrast microscopy (160.5 sperm/mm(2); 56.8-397.4 mm(2)) or their combination (116.4 sperm/mm(2); 56.8-249.6 sperm/mm(2)) (Median; IQR). The combination of microscopy methods was selected as it allowed better visualization of cells. Then, BI was compared between ASA-negative and ASA-positive ejaculates, and the association between BI and ASA binding was evaluated. The BI was less in ASA-positive (114.9; 0 to 201.8 sperm/0.1 mm(2)) than ASA-negative samples (218.9; 24.7 to 276.8 sperm/0.1 mm(2)) (P = 0.0002). This reduction in BI was significant in three of the four bulls. Regression analysis identified a negative association between BI and the percentage of IgG-bound (p = 0.013) but not IgA-bound spermatozoa. In conclusion, sperm-bound IgG decreased the ability of bovine spermatozoa to bind to oviductal epithelial cells in vitro.

Effects of cows grazing toxic endophyte-infected tall fescue or novel endophyte-infected tall fescue in late gestation on cow performance, reproduction, and progeny growth performance and carcass characteristics.

Very little is known regarding the effects of cow exposure to toxic ergot alkaloids in late gestation and the subsequent, long-term effects on progeny. Therefore, the objectives were to determine the effects of grazing toxic endophyte-infected tall fescue () or novel endophyte-infected tall fescue during late gestation on cow BW, BCS, hair coat score (HCS), respiration rates, milk production, and reproduction and on growth performance and carcass characteristics of progeny. Eighty gestating, Angus × Simmental cows (age = 6.68 ± 0.32 yr; 588 ± 16 kg initial BW; initial BCS = 5.66 ± 0.28) were stratified by initial BW and allocated into 8 pasture groups (10 cows/group) with 4 groups per treatment. Each group was allotted to 1 of 2 grazing treatments: toxic endophyte-infected tall fescue ('Kentucky-31'; E+) or novel endophyte-infected tall fescue ('Jesup MaxQ'; NOV). Cows were placed on grazing treatments at 110 ± 6 d prepartum (May 28, 2014) and remained on treatment until the end of the calving period (October 8, 2014; 23 ± 6 d postpartum). Cow BW and BCS did not diverge ( ≥ 0.15) at any time point among grazing treatments. However, cows grazing E+ had increased ( < 0.05) respiration rates and HCS and reduced ( < 0.05) prolactin concentration compared with cows grazing NOV. Calf birth BW, Julian calving date, milk production, AI conception rate, and overall pregnancy rate did not differ ( ≥ 0.23) by grazing treatment. In a subsequent grazing period of the progeny, dam grazing treatment did not affect ( ≥ 0.14) respiration rates, HCS, or prolactin concentration of the progeny when all progeny grazed E+ pastures. However, progeny born to dams grazing NOV tended ( > 0.06 to ≤0.10) to have increased BW at 70 ± 6 d of age, 205 d (adjusted weaning BW), and throughout the postweaning calf grazing period. Despite a tendency for BW to differ throughout the postweaning calf grazing period, finishing phase performance and carcass characteristics of progeny did not differ ( ≥ 0.20) between dam grazing treatment. In conclusion, results indicate that by the time progeny enter the feedlot and are finished on a high-grain diet, any fetal programming effects are minimal.

Prevalence of bovine sperm-bound antisperm antibodies and their association with semen quality.

The objectives of this study were to determine reference intervals (RIs) for sperm-bound immunoglobulins G and A (IgG and IgA), prevalence of antisperm antibodies (ASAs) in satisfactory and nonsatisfactory breeders, and association between ASAs and semen quality in beef bulls. It was hypothesized that ASA binding differed with breeding soundness classification and semen quality. The percentage of IgG- (IgGperc) and IgA-bound (IgAperc) spermatozoa was evaluated in satisfactory (n = 134) and nonsatisfactory (n = 71) breeder beef bulls using flow cytometry. The RI for IgGperc was 0% to 13.5%. The RIs for IgAperc were 0% to 25.8% in yearling Aberdeen Angus bulls and 0% to 12% in all other bulls. The prevalence of IgA-positive samples was higher in nonsatisfactory (14.1%) than that in satisfactory (1.5%) breeders (P = 0.0003). However, the prevalence of IgG-positive samples did not differ. Similarly, IgA binding was higher in nonsatisfactory (median; interquartile range; 2.18; 0.77%-8.57%) than that in satisfactory breeders (median; interquartile range; 1.11; 0.32%-3.16%; P = 0.0035), but IgG binding did not differ. Among ASA-positive bulls, median IgA and IgG binding was 39.7% (range, 18.8%-96.2%) and 24.8% (range, 14.2%-33.1%), respectively. Immunoglobulin A binding correlated with the percentage of total (P < 0.0001; r(2) = -0.345) and progressively motile spermatozoa (P < 0.0001; r(2) = -0.329), morphologically normal spermatozoa (P = 0.0004; r(2) = -0.256), sperm head abnormalities (P = 0.0416; r(2) = 0.149), proximal droplets (P = 0.0227; r(2) = 0.167), and coiled tails (P = 0.0338; r(2) = 0.156). Immunoglobulin G binding correlated with the percentage of total (P < 0.0001; r(2) = -0.373) and progressively motile spermatozoa (P < 0.0001; r(2) = -0.455) and sperm concentration (P = 0.0332; r(2) = -0.195). Reference intervals were established for determination of cutoffs for clinically significant sperm-bound IgA and IgG with flow cytometry. Immunoglobulin A binding was both higher and more prevalent in nonsatisfactory breeder bulls. Although IgG binding did not differ with breeding soundness classification, detection of surface-bound IgG and IgA was associated with changes in semen quality.

Evidence of lipid peroxidation and protein phosphorylation in cells upon oxidative stress photo-generated by fullerols.

An oxidative stress (OS) state is characterized by the generation of Reactive Oxygen Species (ROS) in a biological system above its capacity to counterbalance them [1]. Exposure to OS induces the accumulation of intracellular ROS, which in turn causes cell damage in the form of protein, lipid, and/or DNA oxidations. Such conditions are believed to be linked to numerous diseases or simply to the ageing of tissues. However, the controlled generation of ROS via photosensitizing drugs or photosensitizers (PS) is now widely used to treat various tumors and other infections [2,3]. Here we present a method to track the chemical changes in a cell after exposure to oxidative stress. OS is induced via fullerols, a custom made water soluble derivative of fullerene (C(60)), under visible light illumination. Synchrotron-based Fourier Transform InfraRed Microspectroscopy (S-FTIRM) was used to assess the chemical makeup of single cells after OS exposure. Consequently, a chemical fingerprint of oxidative stress was probed in this study through an increase in the bands linked with lipid peroxidation (carbonyl ester group at 1740 cm(-1)) and protein phosphorylation (asymmetric phosphate stretching at 1240 cm(-1)).

Process parameters within a 750,000 litre anaerobic digester during a year of disturbed fermenter performance.

A 750,000litre fermenter was studied throughout one entire year by investigating the concentrations of volatile fatty acids (acetic, butyric, i-butyric, propionic, valeric and i-valeric acids), pH, concentrations of total C, N, S and NH(4)(+)-N, amounts of chemical and biological oxygen demand, and abundance of acetogenic microorganisms. Additionally several process parameters such as temperature, retention time, dry weight and input of substrate and liquids, and the concentrations and amounts of CH(4), H(2), CO(2) and H(2)S within the biogas were monitored continuously. Various volatile fatty acids and the ratio of acetic to propionic acid were shown to allow a rough indication on the fermentation but were not sufficiently precise to describe the fermenter performance. Nutrient compounds and special fractions, such as easily extractable carbohydrates or the concentration of total fats were more strongly correlated to the gas production of the fermenter. Results of an MPN-method for the determination of acetogenic microorganisms point to an important role of these microorganisms during the phase of restoration of the fermenter performance.

Strontium ranelate does not stimulate bone formation in ovariectomized rats.

INTRODUCTION: Strontium ranelate (SrR) is suggested to function as a dual-acting agent in the treatment of postmenopausal osteoporosis with anti-resorptive and anabolic skeletal benefits. We evaluated the effects of SrR on the skeleton in ovariectomized (OVX) rats and evaluated the influence of dietary calcium. METHODS: Three-month old virgin female rats underwent ovariectomy (OVX, n = 50) or SHAM surgery (SHAM, n = 10). Four weeks post-surgery, rats were treated daily by oral gavage with distilled water (10 ml/kg/day) or SrR (25 or 150 mg/kg/day) for 90 days. Separate groups of animals for each dose of SrR were fed a low (0.1%) or normal (1.19%) calcium (Ca) diet. Static and dynamic histomorphometry, DXA, mu-CT, mechanical testing, and serum and skeletal concentrations of strontium were assessed. RESULTS: SrR at doses of 25 and 150 mg/kg/day did not increase bone formation on trabecular or periosteal bone surfaces, and failed to inhibit bone resorption of trabecular bone regardless of Ca intake. There were no improvements in bone mass, volume or strength with either dose of SrR given normal Ca. CONCLUSION: These findings demonstrate that SrR at dosages of 25 and 150 mg/kg/day did not stimulate an anabolic bone response, and failed to improve the bone biomechanical properties of OVX rats.

The effect of the microscopic and nanoscale structure on bone fragility.

Bone mineral density is the gold-standard for assessing bone quantity and diagnosing osteoporosis. Although bone mineral density measurements assess the quantity of bone, the quality of the tissue is an important predictor of fragility. Understanding the macro- and nanoscale properties of bone is critical to understanding bone fragility in osteoporosis. Osteoporosis is a disease that affects more than 75 million people worldwide. The gold standard for osteoporosis prognosis, bone mineral density, primarily measures the quantity of bone in the skeleton, overlooking more subtle aspects of bone's properties. Bone quality, a measure of bone's architecture, geometry and material properties, is evaluated via mechanical, structural and chemical testing. Although decreased BMD indicates tissue fragility at the clinical level, changes in the substructure of bone can help indicate how bone quality is altered in osteoporosis. Additionally, mechanical properties which can quantify fragility, or bone's inability to resist fracture, can be changed due to alterations in bone architecture and composition. Recent studies have focused on examination of bone on the nanoscale, suggesting the importance of understanding the interactions of the mineral crystals and collagen fibrils and how they can alter bone quality. It is therefore important to understand alterations in bone that occur at the macro-, micro- and nanoscopic levels to determine what parameters contribute to decreased bone quality in diseased tissue.

Two thalamic pathways to primary auditory cortex.

Neurons in the center of cat primary auditory cortex (AI) respond to a narrow range of sound frequencies and the preferred frequencies in local neuron clusters are closely aligned in this central narrow bandwidth region (cNB). Response preferences to other input parameters, such as sound intensity and binaural interaction, vary within cNB; however, the source of this variability is unknown. Here we examined whether input to the cNB could arise from multiple, anatomically independent subregions in the ventral nucleus of the medial geniculate body (MGBv). Retrograde tracers injected into cNB labeled discontinuous clusters of neurons in the superior (sMGBv) and inferior (iMGBv) halves of the MGBv. Most labeled neurons were in the sMGBv and their density was greater, iMGBv somata were significantly larger. These findings suggest that cNB projection neurons in superior and iMGBv have distinct anatomic and possibly physiologic organization.

Rapid establishment of chemical and mechanical properties during lamellar bone formation.

The development of prophylaxes and treatments of bone diseases that can effectively increase the strength of bone as a structure necessitates a better understanding of the time course by which chemical properties define the stiffness of the material during primary and secondary mineralization. It was hypothesized that these processes would be relatively slow in the actively growing skeleton. Seven-week-old Sprague-Dawley female rats (n = 8) were injected with multiple fluorochrome labels over a time span of 3 weeks and killed. Chemical and mechanical properties of the tibial mid-diaphysis were spatially characterized between the endocortical and periosteal surface by in situ infrared microspectroscopy and nanoindentation. The phosphate-to-protein ratio of bone 2-6 days old was 20% smaller at the periosteal surface and 22% smaller at the endocortical surface (P < 0.05 each) compared to older intracortical regions. The ratios of carbonate to protein, crystallinity, type A/type B carbonate, collagen cross-linking, and bone elastic modulus did not differ significantly between bone 2-6, 10-14, and 8-22 days old and intracortical regions. Intracortical properties of 10-week-old rats, except for the carbonate-to-protein ratio which was 23% smaller (P < 0.01), were not significantly different from intracortical matrix properties of young adult rats (5 months, n = 4). Spatially, the phosphate-to-protein ratio (R(2) = 0.33) and the phosphate-to-carbonate ratio (R(2) = 0.55) were significantly correlated with bone material stiffness, while the combination of all chemical parameters raised the R(2) value to 0.83. These data indicate that lamellar bone has the ability to quickly establish its mechanical and chemical tissue properties during primary and secondary mineralization even when the skeleton experiences rapid growth.

Liver histopathology and liver and serum alanine aminotransferase and alkaline phosphatase activities in epileptic dogs receiving phenobarbital.

Phenobarbital (PB) therapy is frequently associated with elevated serum alanine aminotransferase (ALT) and alkaline phosphatase (AP) activities in dogs without clinical signs of liver disease. The goal of this study was to determine if increased serum ALT and AP activities in clinically healthy PB-treated epileptic dogs are due to hepatic enzyme induction or to subclinical liver injury. Liver biopsies were obtained from 12 PB-treated dogs without clinical signs of liver disease but with elevated serum ALT and/or AP activities or both. Liver biopsies were obtained from eight healthy control dogs not receiving PB. Biopsies were evaluated histopathologically (all dogs) and liver homogenates were assayed for ALT (all dogs) and AP (six treated dogs, all controls) activities. As a positive control, liver cytochrome P4502B, an enzyme known to be induced by PB, was measured by benzyloxyresorufin-O-dealkylase activity and immunoblotting (five treated dogs, all controls). Serum AP isoenzyme analyses were performed. Results showed that ALT and AP activities in liver homogenates were not increased in treated dogs compared with controls, whereas the positive control for induction, CYP2B, was dramatically increased in treated dogs. Histopathological examination of liver biopsies revealed more severe and frequent abnormalities in treated dogs compared to controls, but similar types of abnormalities were found in both groups. Serum AP isoenzyme analyses in treated dogs demonstrated increased corticosteroid-induced and liver isoenzyme activities compared to controls. Results do not support induction of ALT or AP in the liver as the cause of elevated serum activities of these enzymes due to PB.

Lower fitness of hatchery and hybrid rainbow trout compared to naturalized populations in Lake Superior tributaries.

We have documented an early life survival advantage by naturalized populations of anadromous rainbow trout Oncorhynchus mykiss over a more recently introduced hatchery population and outbreeding depression resulting from interbreeding between the two strains. We tested the hypothesis that offspring of naturalized and hatchery trout, and reciprocal hybrid crosses, survive equally from fry to age 1+ in isolated reaches of Lake Superior tributary streams in Minnesota. Over the first summer, offspring of naturalized females had significantly greater survival than offspring of hatchery females in three of four comparisons (two streams and 2 years of stocking). Having an entire naturalized genome, not just a naturalized mother, was important for survival over the first winter. Naturalized offspring outperformed all others in survival to age 1+ and hybrids had reduced, but intermediate, survival relative to the two pure crosses. Averaging over years and streams, survival relative to naturalized offspring was 0.59 for hybrids with naturalized females, 0.37 for the reciprocal hybrids, and 0.21 for hatchery offspring. Our results indicate that naturalized rainbow trout are better adapted to the conditions of Minnesota's tributaries to Lake Superior so that they outperform the hatchery-propagated strain in the same manner that many native populations of salmonids outperform hatchery or transplanted fish. Continued stocking of the hatchery fish may conflict with a management goal of sustaining the naturalized populations.

Scrapie-infected cells, isolated prions, and recombinant prion protein: a comparative study.

Fourier -transform infrared microscopic spectra of scrapie-infected nervous tissue measured at high spatial resolution (approximately 6 microm) were compared with those obtained from the purified, partly proteinase K digested scrapie isoform of the prion protein isolated from nervous tissue of hamsters infected with the same scrapie strain (263K) to elucidate similarities/dissimilarities between prion structure investigated in situ and ex vivo. A further comparison is drawn to the recombinant Syrian hamster prion protein SHaPrP(90-232) after in vitro conformational transition from the predominantly alpha-helical isoform to beta-sheet-rich structures. It is shown that prion protein structure can be investigated within tissue and that detectability of regions with elevated beta-sheet content as observed in microspectra of prion-infected tissue strongly depends on spatial resolution of the experiment.

Imaging capabilities of synchrotron infrared microspectroscopy.

It has become increasingly clear that synchrotron infrared micro-spectroscopy is an extremely valuable analysis tool when determining the chemical composition of biological and biomedical samples, at the diffraction-limited spatial resolution. Highly resolved infrared micro-spectroscopy, together with the high signal-to-noise level of the recorded spectra, is essential in generating chemical and statistical (multivariate) images. This is illustrated in the case of individual cell and hair section studies. Unprecedented chemical images of lipid distribution and secondary structure relative concentration have been achieved using the synchrotron source. A comparison with a Focal plane Array imaging system, on the same hair section, shows that, despite the fast imaging processing and improved quality achieved with the focal plane array detectors, spectral quality is markedly superior in the case of the synchrotron source. It is clear that the two approaches could be very complementary if combined on the same sample area, in a synchrotron facility.

Synchrotron-based Biological Microspectroscopy: From the Mid-Infrared through the Far-Infrared Regimes.

Infrared radiation from synchrotron storagerings serves as a high-brightness source fordiffraction-limited microspectroscopy inboth the mid- and far-infrared spectralranges. Mid-infrared absorption, due to localvibrational modes within complex molecules,is shown to be sensitive to small chemicalchanges associated with certain diseases.Farinfrared modes are believed to result from thefolding or twisting of larger, morecomplex molecules. The ability for thesynchrotron source to perform microscopy ata frequency of 1 THz is demonstrated.

Center for Synchrotron Biosciences' U2B beamline: an international resource for biological infrared spectroscopy.

A synchrotron infrared (IR) beamline, U2B, dedicated to the biomedical and biological sciences has been constructed and is in operation at the National Synchrotron Light Source (NSLS) of Brookhaven National Laboratory. The facility is operated by the Center for Synchrotron Biosciences of the Albert Einstein College of Medicine in cooperation with the NSLS. Owing to the broadband nature of the synchrotron beam with brightness 1000 times that of conventional sources, Fourier transform IR spectroscopy experiments are feasible on diffraction-limited sample areas at high signal-to-noise ratios and with relatively short data-acquisition times. A number of synchrotron IR microscopy experiments that have been performed in the mid-IR spectral range (500-5000 cm(-1)) are summarized, including time-resolved protein-folding studies in the microsecond time regime, IR imaging of neurons, bone and other biological tissues, as well as imaging of samples of interest in the chemical and environmental sciences. Owing to the high flux output of this beamline in the far-IR region (50-500 cm(-1)), investigations of hydrogen bonding and dynamic molecular motions of biomolecules have been carried out from 10 to 300 K using a custom-made cryostat and an evacuated box. This facility is intended as an international resource for biological IR spectroscopy fully available to outside users based on competitive proposal.

Characterization of bone mineral composition in the proximal tibia of cynomolgus monkeys: effect of ovariectomy and nandrolone decanoate treatment.

Life postmenopausal women, ovariectomized cynomolgus monkeys (Macaca fascicularis) experience accelerated loss of bone mass. Treatment of ovariectomized monkeys with nandrolone decanoate results in an increase in bone mass to levels comparable to those of intact animals. The changes in bone composition that occur with these treatments, however, are less well characterized. In the present study, we used synchrotron Fourier-transform infrared microspectroscopy (FT-IRM) and curve-fitting methods to monitor specific changes at cortical, subchondral, and trabecular bone regions in the proximal tibia. Four groups were studied: (1) sham-operated (sham); (2) ovariectomized and treated with placebo for 2 years (ovx); (3) ovx + nandrolone decanoate for 2 years (NAN); and (4) ovx + nandrolone decanoate beginning 1 year after ovx (dNAN). The results demonstrate that ovariectomy and nandrolone treatment did not affect the degree of mineralization as defined by the phosphate/protein ratio, but acid phosphate content (HPO(4)(2-)) in cortical and subchondral bone was increased by ovariectomy, suggesting this bone to be less mature due to increased remodeling that occurs after ovariectomy. In the subchondral and cortical bone regions, ovariectomized monkeys showed a lower total carbonate content (CO(3)(2-)/matrix ratio) than sham controls, specifically due to the decrease in labile carbonate content. In the trabecular region, no change of carbonate content was observed. Treatment with nandrolone decanoate was found to restore the loss in carbonate, where the resulting mineral had a larger quantity of type B carbonate. Finally, we correlated carbonate content with dual-energy X-ray absorptiometry measurements, and found a positive correlation between bone mineral density and type A carbonate in bone, which is stoichiometrically related to the amount of calcium in bone. Therefore, the results presented herein identify significant differences in bone chemistry after ovariectomy and nandrolone treatment, which may help explain previous findings that, although nandrolone decanoate treatment increased bone mass, it could not reverse the decrease in bone strength due to ovariectomy.

Informatics and multiplexing of intact protein identification in bacteria and the archaea.

Although direct fragmentation of protein ions in a mass spectrometer is far more efficient than exhaustive mapping of 1-3 kDa peptides for complete characterization of primary structures predicted from sequenced genomes, the development of this approach is still in its infancy. Here we describe a statistical model (good to within approximately 5%) that shows that the database search specificity of this method requires only three of four fragment ions to match (at +/-0.1 Da) for a 99.8% probability of being correct in a database of 5,000 protein forms. Software developed for automated processing of protein ion fragmentation data and for probability-based retrieval of whole proteins is illustrated by identification of 18 archaeal and bacterial proteins with simultaneous mass-spectrometric (MS) mapping of their entire primary structures. Dissociation of two or three proteins at once for such identifications in parallel is also demonstrated, along with retention and exact localization of a phosphorylated serine residue through the fragmentation process. These conceptual and technical advances should assist future processing of whole proteins in a higher throughput format for more robust detection of co- and post-translational modifications.