Cartilage boundary lubrication of ovine synovial fluid following anterior cruciate ligament transection: a longitudinal study.

OBJECTIVE: To assess ovine synovial fluid (oSF) from different post-injury time points for (1) proteoglycan-4 (PRG4) and hyaluronan (HA) concentration, (2) HA molecular weight (MW) distribution, (3) cartilage boundary lubrication function, and (4) lubricant composition-function relationships. The association between cartilage boundary lubrication and gross cartilage changes after injury was also examined. METHODS: oSF was collected 2, 4, 10, and 20 weeks post anterior cruciate ligament (ACL) transection in five skeletally mature sheep. PRG4 and HA concentrations were measured using sandwich enzyme-linked immunosorbent assay, and HA MW distribution by agarose gel electrophoresis. Cartilage boundary lubrication of oSF was assessed using a cartilage-cartilage friction test. Gross damage to articular cartilage was also quantified at 20 weeks using modified Drez scoring protocol. RESULTS: Early (2-4 weeks) after ACL injury, PRG4 concentrations were significantly higher (P = 0.045, P = 0.037), and HA concentrations were substantially lower (P = 0.005, P = 0.005) compared to 20 weeks. The HA MW distribution also shifted towards lower ranges in the early post-injury stage. The kinetic friction coefficients were significantly higher 2-4 weeks post injury (P = 0.008 and P = 0.049) compared to 20 weeks. Poor cartilage boundary lubricating ability early after injury was associated with cartilage damage at 20 weeks. CONCLUSION: Altered composition and diminished boundary lubrication of oSF early after ACL transection may pre-dispose the articular cartilage to degenerative changes and initiate osteoarthritis (OA). These observations also provide potential motivation for biotherapeutic interventions at earlier time points post injury.

The effect of molecular weight on hyaluronan's cartilage boundary lubricating ability--alone and in combination with proteoglycan 4.

OBJECTIVES: (1) assess the molecular weight dependence of hyaluronan's (HA) cartilage boundary lubricating ability, alone and in combination with proteoglycan 4 (PRG4), at physiological concentrations; (2) determine if HA and PRG4 interact in solution via electrophoretic mobility shift assay (EMSA). METHODS: The cartilage boundary lubricating ability of a broad range of MW HA (20 kDa, 132 kDa, 780 kDa, 1.5 MDa, and 5 MDa) at 3.33 mg/ml, both alone and in combination with PRG4 at 450 µg/ml, was assessed using a previously described cartilage-on-cartilage friction test. Static, µ(static, Neq), and kinetic, <µ(kinetic, Neq)>, were calculated. An EMSA was conducted with PRG4 and monodisperse 150 kDa and 1,000 kDa HA. RESULTS: Friction coefficients were reduced by HA, in a MW-dependent manner. Values of <µ(kinetic, Neq)> in 20 kDa HA, 0.098 (0.089, 0.108), were significantly greater compared to both 780 kDa, 0.080 (0.072, 0.088), and 5 MDa, 0.079 (0.070, 0.089). Linear regression showed a significant correlation between both µ(static, Neq) and <µ(kinetic, Neq)>, and log HA MW. Friction coefficients were also reduced by PRG4, and with subsequent addition of HA; however the synergistic effect was not dependent on HA MW. Values of <µ(kinetic, Neq)> in PRG4, 0.080 (0.047, 0.113), were significantly greater than values of PRG4+various MW HA (similar in value, averaging 0.040 (0.033, 0.047)). EMSA indicated that migration of 150 kDa and 1,000 kDa HA was retarded when combined with PRG4 at high PRG4:HA ratios. CONCLUSIONS: These results suggest alterations in HA MW could significantly affect synovial fluid's cartilage boundary lubricating ability, yet this diminishment in function could be circumvented by physiological levels of PRG4 forming a complex, potentially in solution, with HA.

Relationship between development, metabolism, and mitochondrial organization in 2-cell hamster embryos in the presence of low levels of phosphate.

The effect of low concentrations of inorganic phosphate (P(i)) on development, metabolic activity, and mitochondrial organization in the same cohorts of cultured hamster embryos was evaluated. Two-cell embryos were collected from eCG-stimulated golden hamsters and cultured in HECM-10 with 0.0 (control), 1.25, 2.5, or 5.0 microM KH(2)PO(4). Glucose utilization through the Embden-Meyerhof pathway (EMP) and tricarboxylic acid (TCA)-cycle activity were determined following 5 h of culture. Mitochondrial organization in living embryos was evaluated using multiphoton microscopy at 6 h of culture. Development was assessed at 27 h (on-time 8-cell stage) and 51 h (on-time blastocyst stage) of culture. Total cell numbers, as well as cell allocation to the trophectoderm and inner cell mass were determined for morula- and blastocyst-stage embryos. Culture with P(i) did not alter TCA-cycle activity. However, culture with > or 2.5 microM P(i) significantly increased (P < 0.01) EMP activity compared to control. Mitochondrial organization was significantly (P < 0.01) disrupted by P(i) in a dose-dependent manner. Development to the 8-cell, morula/blastocyst, and blastocyst stages was significantly reduced (P < 0.05) in the presence of > or =2.5 microM P(i) compared to both control and 1.25 microM P(i). This study clearly demonstrates that, for hamster embryos, inclusion of even exceptionally low concentrations of P(i) in culture medium dramatically alters embryo physiology. Additionally, although 2-cell embryos can tolerate some structural disruption without concomitant, detrimental effects on development or metabolic activity, metabolic disturbance is associated with decreased developmental competence.

Granting forgiveness or harboring grudges: implications for emotion, physiology, and health.

Interpersonal offenses frequently mar relationships. Theorists have argued that the responses victims adopt toward their offenders have ramifications not only for their cognition, but also for their emotion, physiology, and health. This study examined the immediate emotional and physiological effects that occurred when participants (35 females, 36 males) rehearsed hurtful memories and nursed grudges (i.e., were unforgiving) compared with when they cultivated empathic perspective taking and imagined granting forgiveness (i.e., were forgiving) toward real-life offenders. Unforgiving thoughts prompted more aversive emotion, and significantly higher corrugator (brow) electromyogram (EMG), skin conductance, heart rate, and blood pressure changes from baseline. The EMG, skin conductance, and heart rate effects persisted after imagery into the recovery periods. Forgiving thoughts prompted greater perceived control and comparatively lower physiological stress responses. The results dovetail with the psychophysiology literature and suggest possible mechanisms through which chronic unforgiving responses may erode health whereas forgiving responses may enhance it.

Autocrine/paracrine action of oxytocin in pig endometrium.

Luminal epithelial cells of porcine endometrium are unresponsive to oxytocin (OT) in vitro although they express the greatest quantity of OT and receptors for OT in vivo. Therefore, the objective of this study was to determine if oxytocin acted in an autocrine manner on luminal epithelial cells to stimulate prostaglandin (PG)F(2alpha) secretion. Treatment of endometrial explants or enriched luminal epithelial cells with OT antagonist L-366,948 decreased (P < 0.05) basal secretion of PGF(2alpha). Oxytocin increased (P < 0.01) PGF(2alpha) secretion from luminal epithelial cells that were pretreated with 1:5000 or 1:500 OT antiserum for 3 h to immunoneutralize endogenously secreted OT. However, OT only increased (P < 0.05) PGF(2alpha) secretion from glandular epithelial cells when pretreated with 1:500 OT antiserum. Pretreatment with OT antiserum did not alter the ability of OT to induce PGF(2alpha) secretion from stromal cells. Medium conditioned by culture of luminal epithelial cells stimulated (P < 0.05) phospholipase C activity in stromal cells, indicative of the presence of bioactive OT. Oxytocin was secreted by luminal epithelial cells and 33% was released from the apical surface. These results indicate that luminal epithelial cells secrete OT that acts in an autocrine and/or paracrine manner in pig endometrium to stimulate PGF(2alpha) secretion.

Differential effect of hexoses on hamster embryo development in culture.

The effects of glucose, fructose, and galactose on hamster embryo development in the absence of phosphate were studied in culture. One- and two-cell embryos were cultured to the blastocyst stage in HECM-9 medium without hexose or in medium with increasing concentrations of hexoses. Embryo development, cell number, and cell allocation were assessed in blastocysts. Blastocyst viability was determined by transfer to pseudopregnant recipients. Although 0.25 mM fructose increased mean cell number, low glucose concentrations had no stimulatory effect on development to blastocyst. Both galactose and 5.0 mM glucose were detrimental to embryos. Addition of 0.5 mM glucose increased implantation and fetal viability as compared with controls. Compared with 0.5 mM glucose, treatment with 0.25 mM fructose gave similar implantation and fetal viability, whereas 5.0 mM glucose tended to decrease implantation and significantly decreased fetal development. These data demonstrate that morphology is a poor indicator of embryo viability and that exposure of preimplantation embryos to glucose or fructose is important for embryo viability post-transfer. Although no difference in blastocyst viability was detected between embryos cultured with 0.25 mM fructose and those cultured with 0.5 mM glucose, increased cell numbers obtained with fructose suggest that fructose may be more appropriate than glucose for inclusion in culture medium.

Phosphate induced developmental arrest of hamster two-cell embryos is associated with disrupted ionic homeostasis.

Culture of hamster embryos with 0.35 mM inorganic phosphate results in developmental arrest at the 2-cell stage. These arrested 2-cell embryos were found to have significantly elevated levels of both intracellular pH and intracellular free calcium. Culture of 2-cell embryos with both glucose and phosphate did not further alter intracellular ionic homeostasis. Developmental arrest of 2-cell embryos was dependent on the concentration of phosphate used. Culture with 1.25 microM phosphate did not alter development, while concentrations of 2.5 microM and 5.0 microM resulted in a percentage of embryos arresting development at the 2-cell stage. Analysis of intracellular levels of pH and calcium after culture with different phosphate concentrations revealed a significant negative correlation between intracellular calcium levels and development beyond the 2-cell stage. There was no correlation between the increase in intracellular pH and embryo development in the presence of phosphate. The increase in intracellular calcium levels after culture with phosphate appears to be derived from intracellular pools, as preventing the influx of extracellular calcium did not alter development beyond the 2-cell stage. Therefore, it is apparent that a disruption in ionic homeostasis is associated with developmental arrest of hamster embryos cultured with phosphate.

Effect of oxytocin on concentration of PGF2 alpha in the uterine lumen and subsequent endometrial responsiveness to oxytocin in pigs.

The aim of this study was to determine the effect of oxytocin on PGF2 alpha secretion into the uterine lumen of pigs and subsequent endometrial responsiveness to oxytocin in vitro. Cyclic, pregnant and oestradiol-induced pseudopregnant gilts were injected i.v. with vehicle or 20 iu oxytocin 10 min before hysterectomy on day 16 after oestrus. Concentrations of PGF2 alpha and 13,14-dihydro-15-keto PGF2 alpha (PGFM) were significantly increased in uterine flushings collected at hysterectomy (P < 0.05) in pregnant oxytocin-injected gilts. Concentrations of PGF2 alpha and PGFM were greater (P < 0.001) in pregnant than in pseudopregnant and cyclic gilts, and greater (P < 0.01) in pseudopregnant than in cyclic gilts. The ratio of PGFM:PGF2 alpha tended to be greater in cyclic (P < 0.06) and pseudopregnant gilts (P < 0.1) than in pregnant gilts. At 85 +/- 5 min after oxytocin injection, endometrium from each gilt was incubated for 3 h for determination of phosphoinositide hydrolysis and PGF2 alpha secretion in response to treatment with 0 or 100 nmol oxytocin l-1. Endometrial phosphoinositide hydrolysis in response to 100 nmol oxytocin l-1 in vitro was greater (P < 0.05) in cyclic oxytocin-injected gilts than in cyclic vehicle-injected gilts. Treatment with oxytocin in vitro did not stimulate phosphoinositide hydrolysis significantly in vehicle- or oxytocin-injected pregnant gilts or pseudopregnant gilts. Endometrial PGF2 alpha secretion increased after treatment with 100 nmol oxytocin l-1 in vitro in cyclic vehicle-injected (P < 0.01), cyclic oxytocin-injected (P < 0.01), pregnant vehicle-injected (P = 0.06), pseudopregnant vehicle-injected (P < 0.05) and pseudopregnant oxytocin-injected (P < 0.05) gilts, but not in pregnant oxytocin-injected gilts. The increase in PGF2 alpha in pseudopregnant oxytocin-injected gilts was less (P < 0.05) than that in cyclic oxytocin-injected gilts. These results indicate that oxytocin increases the concentration of PGF2 alpha and PGFM in the uterine lumen during pregnancy and may upregulate endometrial responsiveness to oxytocin during late dioestrus in pigs, but does not have the latter effect during early pregnancy or oestradiol-induced pseudopregnancy.

Oxytocin-stimulated phosphoinositide hydrolysis and prostaglandin F secretion by luminal epithelial, glandular epithelial, and stromal cells from pig endometrium. I. Response of cyclic pigs on day 16 postestrus.

Oxytocin (OT) is the physiological stimulus for pulsatile release of endometrial prostaglandin (PG) F2alpha during luteolysis in domestic ungulates, and the cellular mechanism for this appears to involve phosphoinositide (PI) hydrolysis. To determine which endometrial cell type(s) was responsive to OT during luteolysis in swine, luminal epithelial (LEC), glandular epithelial (GEC), and stromal cells (SC) were isolated from endometrium by differential enzymatic digestion and sieve filtration on Day 16 postestrus and cultured. For PI hydrolysis in experiment 1, SC were most responsive to 100 nM OT (p < 0.001), whereas LEC were least responsive and GEC had an intermediate response (p < 0.001). For PGF secretion in experiment 2, the response to OT was greatest for SC, least for LEC, and intermediate for GEC. In experiment 3, 100 nM OT increased PI hydrolysis in SC within 30 min (p < 0.05) and in GEC within 60 min (p < 0.05) but did not increase PI hydrolysis in LEC. In experiment 4, PI hydrolysis in SC was increased (p < 0.05) by 33-333 nM OT but was not increased by

Endometrial responsiveness to oxytocin during diestrus and early pregnancy in pigs is not controlled solely by changes in oxytocin receptor population density.

These studies were performed to test the hypotheses that: 1) endometrial responsiveness to oxytocin (OT) in pig endometrium is associated with changes in OT receptor (OTr) population density resulting from corresponding regulation of OTr gene transcription, 2) endometrial responsiveness to OT is controlled solely through a mechanism involving changes in OTr population density, and 3) OTr population density and endometrial responsiveness to OT differ between diestrus and early pregnancy in pigs. In experiment 1, OTr population density and dissociation constant (Kd) in cyclic pigs were constant on Days 10-16 but increased (p < 0.05) between Days 10 and 12 of pregnancy before decreasing (p < 0.05) through Day 16. OT induced phosphoinositide (PI) hydrolysis and prostaglandin (PG) F2 alpha secretion in cyclic pigs only on Day 16 (p < 0.05), and during pregnancy only on Day 12 (p < 0.05). Activation of G protein by aluminum fluoride (AIF4-) treatment maximally stimulated (p < 0.05) PI hydrolysis and PGF2 alpha secretion in cyclic pigs on all days, indicating that downstream from the OTr, the PGF2 alpha secretory pathway was fully functional. During pregnancy, PI hydrolysis and PGF2 alpha secretion in response to AIF4- decreased (p < 0.01) on Days 14 compared to Days 10 and 12, and AIF4- did not stimulate PGF2 alpha release on Day 16. In experiment 2, abundance of OTr mRNA in cyclic pigs decreased between Days 0 and 5 before increasing between Days 5 and 12 (p < 0.05), but it was higher (p < 0.05) on Days 10-15 of pregnancy than on equivalent days in cyclic gilts. These results indicate that control of PGF2 alpha secretion in cyclic pigs appeared to occur primarily at the level of OTr coupling to G protein because changes in OTr number were not associated with increased sensitivity to OT or G-protein activation by AIF4-. During pregnancy, control was exerted at multiple levels, which included the OTr, G protein, phospholipase C, and subsequent aspects of the secretory pathway. The present study also indicated that endometrium was responsive to OT during luteolysis in cyclic pigs but not during corpus luteum maintenance in pregnant pigs.

Control of endometrial phosphoinositide hydrolysis and prostaglandin F2alpha secretion in pigs.

This study examined the control of endometrial phosphoinositide (PI) hydrolysis and prostaglandin (PG) F2alpha release by oxytocin (OT) and vasopressin in cyclic pigs on Day 15 post oestrus. In Expt 1, OT and arginine-vasopressin (AVP) increased endometrial PI hydrolysis (P<0.01), but only OT stimulated (P<0.01) PGF2alpha secretion. In Expt 2, OT and lysine-vasopressin (LVP) increased PI hydrolysis (P<0.01). No difference was detected between the 100 nM and 200 nM concentrations of OT or between the 100 nM and 200 nM concentrations of LVP. The increase in PI hydrolysis was greater (P<0.05) for 100 nM OT plus 100 nM LVP than for the 100 or 200 nM concentrations of OT or LVP alone. In Expt 3, OT (P<0.01) and LVP increased (P<0.01) PI hydrolysis. An OT antagonist abolished (P<0.01) the OT-induced increase in PI hydrolysis, but did not significantly alter the LVP-induced increase. A type 2 VP antagonist completely inhibited (P<0.01) the LVP-induced increase in PI hydrolysis, but only partially antagonized the stimulatory effect of OT (P<0.01). These results are consistent with the proposal that OT acts through specific receptors to promote endometrial PGF2alpha secretion in pigs.

Maximizing the bilateral field advantage on verbal and nonverbal matching tasks.

Research has established that response latencies are generally shorter on visual matching tasks when one target is projected to each hemifield (bilateral presentation) than when both targets are projected to the same hemifield (unilateral presentation). This effect, called the bilateral field advantage (BFA), has recently shown promise as a predictor of callosal dysfunction. As a step toward developing a reliable BFA index, the present study examined two factors that appear to influence the extent of BFA in normal subjects. Twenty-seven right-handed college students performed a verbal matching task (using letter pairs drawn from the set AaBb) and a nonverbal matching task (using dot pattern pairs constructed with 4 dots in a 3 x 3 matrix). Order of task varied across subjects (dots followed by letters, or letters followed by dots, or letter trials and dot trials interleaved at random). The targets were presented either unilaterally or bilaterally. Results revealed a robust BFA for the letter-matching task in all three task order conditions, suggesting that the letter task may be suitable for inclusion in a battery of tasks for clinical assessment. The dot-matching task did not yield a significant BFA when administered as the first task. The dot task BFA increased when the letter task preceded it, and became comparable to the letter task BFA in the interleaved condition.

A proposed role for oxytocin in regulation of endometrial prostaglandin F2 alpha secretion during luteolysis in swine.

Pulsatile secretion of endometrial prostaglandin (PG)F2 alpha is stimulated by oxytocin (OT) during late diestrus in domestic ruminants (i.e., cattle, sheep and goats) and results in corpus luteum (CL) regression leading to the onset of a new estrous cycle. Pulsatile PGF2 alpha release is also responsible for CL regression in swine, but the stimulus for its secretion from the uterine endometrium is not known. We propose that OT binds to specific OT receptors (OTR) on the endometrium to stimulate phosphoinositide (PI) hydrolysis, thereby activating the inositol trisphosphate (IP3)-diacylglycerol (DAG) second-messenger system to promote pulsatile PGF2 alpha secretion. Exogenous OT administered to cyclic gilts during late diestrus (days 10-16) decreased interestrous interval in three of four experiments. However, OT did not promote CL regression in hysterectomized gilts indicating that the effect of OT was uterine-dependent. Circulating concentrations of 13,14-dihydro-15-keto PGF2 alpha (the major stable metabolite of PGF2 alpha) were increased (p < 0.01) 10 min after i.v. injection of OT on days 14 and 16 in cyclic gilts and on days 10-16 in pregnant gilts, but the magnitude of the response to OT on all days in pregnant gilts was markedly reduced compared to the response in cyclic gilts on days 14 and 16. Mean density and Kd of OTR detected on endometrium of cyclic pigs 15 days post-estrus were 29.2 +/- 5.5 fmol/mg protein and 1.59 +/- 0.23 nM, respectively. Density of OTR was correlated with OT-stimulated PI hydrolysis (r = 0.83, p < 0.05) and PGF2 alpha secretion (r = 0.87, p < 0.10). Endometrial IP3 was increased within 30 seconds after OT treatment and preceded the increase in PGF2 alpha release stimulated by OT. Endometrial PI hydrolysis and PGF2 alpha secretion were similarly increased by AIF4-(phospholipase C activator), but not by cholera toxin (adenylyl cyclase activator). Although OT binding to OTR could be displaced by lysine-vasopressin and lysine-vasopressin stimulated PI hydrolysis, lysine-vasopressin did not stimulate PGF2 alpha release. Distinct receptors for OT and lysine-vasopressin on pig endometrium were confirmed by treatment with 100 nM OT + 100 nM lysine-vasopressin which stimulated PI hydrolysis more than 100-200 nM OT or lysine-vasopressin alone. These results support the hypothesis that OT stimulates phospholipase C to hydrolyze PI, yielding IP3 and DAG second-messengers which promote endometrial PGF2 alpha release during CL regression in pigs.

The bilateral field advantage on a letter-matching task.

A series of studies have reported that responding is faster when letter pairs to be matched are projected to two hemispheres rather than one. Four experiments described here tested this bilateral field advantage and identified factors that influence its extent. Subjects were shown letter pairs drawn from the ensemble "AaBb", and classified the letter pairs as "Match" if the letters had the same name (regardless of case) and "No Match" if they did not. In the first two experiments the letter pairs were presented unilaterally (both letters in one visual field), bilaterally (one letter in each visual field), or centrally (both letters on the vertical midline, above and below fixation), in order to investigate how the bilateral field advantage is influenced by screen location. The third experiment added a bilateral-diagonal position (to check for artefacts related to horizontal scanning strategies), and the fourth experiment added distractor digits (to equate initial processing demands in the bilateral and unilateral conditions). Results indicate that the bilateral field advantage is a robust phenomenon, although several manipulations reduced its magnitude. Implications of these findings for models of hemispheric collaboration and interhemispheric processing are discussed.

Source density analysis of scalp potentials during linguistic and non-linguistic processing of visual stimuli.

Event-related potentials (ERPs) were recorded from 40 locations, covering most of the scalp, during repeated tasks in which the observer (O) had to judge either the tense of a printed verb (V) or the symmetry of a spatial pattern (S). Stimuli were drawn at random from large ensembles. A simplified method of Laplacean analysis (MacKay 1983, 1984) allowed the corresponding source densities to be mapped at up to 28 locations, relatively free of artefacts due to eye movements or tongue movements. O signalled his judgement in each case by pressing one of two buttons on a given cue. The decision time allowed was kept short (about 1 s) but long enough for the task to be handled successfully. When stimuli 'V' and 'S' were drawn from geometrically different ensembles, the source-density distributions for the two tasks differed significantly at a number of locations. When 'V' and 'S' were drawn from a common ensemble, however, and O was instructed on each trial (in random order) to assess each stimulus as a word or as a geometrical pattern, the similarities in the source-density maps were more striking than the differences. It would seem that during sufficiently rapid verbal and spatial judgments, little sign of hemispheric specialization or task-specific differences may appear in the spatiotemporal profile of ERP source densities. More salient differences, some lateralized, appeared during the preparation interval prior to verbal and spatial tasks; but their pattern varied widely from subject to subject.

Age differences in mental synthesis.

In a series of three experiments, a mental synthesis task was administered to groups of college students and older adults. Simple symmetrical or asymmetrical straightline patterns were divided into two parts; the two parts were presented either sequentially or simultaneously. Participants were instructed to mentally combine the two parts and either reproduce or recognize the complete pattern. The college students made more correct responses than older adults on both the reproduction and recognition measures. Both groups performed better when urged to visualize the complete pattern than when instructed to memorize the individual component parts. Overall, the results suggest that age differences in synthesis performance are related to, but perhaps not limited to, differences in memory ability.