[Clinical pathway for total knee arthroplasty. I: Pathway conception and effect on functional quality of results]. / Klinischer Behandlungspfad zur Implantation einer Oberflächenknieendoprothese (EGON). I: Pfadkonzeption und Effekt auf funktionelle Ergebnisqualität.

PURPOSE: The aim of the current study was to evaluate patient-centred and economic outcomes after introduction of a clinical pathway for total knee arthroplasty. METHODS: In a prospective trial two sequential cohorts of patients undergoing total knee arthroplasty were recruited. Baseline treatment was surveyed in cohort I and the clinical pathway was developed and evaluated in cohort II. Data from WOMAC, EQ-5D as well as partial cost data were collected. The study design was ratified by the local Independent Ethics Committee. RESULTS: There was an increase in WOMAC score of 39% for cohort I and 35% for cohort II in 3 months follow-up. Similar results were found for ED-5D with an increase of 30% for cohort I and 25% for cohort II. Partial cost rates could be lowered from 4303 EUR to 419 EUR. Despite this significant cost saving we were not able to improve the ratio of improvement in quality of life to costs. CONCLUSION: With the aid of a clinical pathway the process for implementation of a total knee arthroplasty was improved and treatment quality assured.

High-affinity sodium-vitamin C co-transporters (SVCT) expression in embryonic mouse neurons.

The sodium-vitamin C co-transporters SVCT1 and SVCT2 transport the reduced form of vitamin C, ascorbic acid. High expression of the SVCT2 has been demonstrated in adult neurons and choroid plexus cells by in situ hybridization. Additionally, embryonic mesencephalic dopaminergic neurons express the SVCT2 transporter. However, there have not been molecular and kinetic analyses addressing the expression of SVCTs in cortical embryonic neurons. In this work, we confirmed the expression of a SVCT2-like transporter in different regions of the fetal mouse brain and in primary cultures of neurons by RT-PCR. Kinetic analysis of the ascorbic acid uptake demonstrated the presence of two affinity constants, 103 microM and 8 microM. A K(m) of 103 microM corresponds to a similar affinity constant reported for SVCT2, while the K(m) of 8 microM might suggest the expression of a very high affinity transporter for ascorbic acid. Our uptake analyses also suggest that neurons take up dehydroascorbic acid, the oxidized form of vitamin C, through the glucose transporters. We consider that the early expression of SVCTs transporters in neurons is important in the uptake of vitamin C, an essential molecule for the fetal brain physiology. Vitamin C that is found at high concentration in fetal brain may function in preventing oxidative free radical damage, because antioxidant radical enzymes mature only late in the developing brain.

Elevated expression of glucose transporter-1 in hypothalamic ependymal cells not involved in the formation of the brain-cerebrospinal fluid barrier.

Glucose transporters play an essential role in the acquisition of glucose by the brain. Elevated expression of glucose transporter-1 has been detected in endothelial cells of the blood-brain barrier and in choroid plexus cells of the blood-cerebrospinal fluid barrier. On the other hand, there is a paucity of information on the expression of glucose transporters in the ependymal cells that line the walls of the cerebral ventricles. The tanycytes are specialized ependymal cells localized in circumventricular organs such as the median eminence that can be segregated into at least three types, alpha, beta1 and beta2. The beta2 tanycytes form tight junctions and participate in the formation of the cerebrospinal fluid-median eminence barrier. Using immunocytochemistry and in situ hybridization, we analyzed the expression of hexose transporters in rat and mouse hypothalamic tanycytes. In both species, immunocytochemical analysis revealed elevated expression of glucose transporter-1 in alpha and beta1 tanycytes. Intense anti-glucose transporter-1 staining was observed in cell processes located throughout the arcuate nucleus, in the end-feet reaching the lateral sulcus of the infundibular region, and in cell processes contacting the hypothalamic capillaries. On the other hand, there was very low expression of glucose transporter-1 in beta2 tanycytes involved in barrier function. In contrast with the results of the cytochemical analysis, in situ hybridization revealed that tanycytes alpha, beta1, and beta2 express similar levels of glucose transporter-1 mRNA. Further analysis using anti-glial fibrillary acidic protein antibodies to identify areas rich in astrocytes revealed that astrocytes were absent from areas containing alpha and beta1 tanycytes, but were abundant in regions containing the barrier-forming beta2 tanycytes. Overall, our data reveal a lack of correlation between participation in barrier function and expression of glucose transporter-1 in hypothalamic tanycytes. Given the virtual absence of astrocytes in areas rich in alpha and beta1 tanycytes, we speculate whether the tanycytes might have astrocyte-like functions and participate in the metabolic coupling between glia and neurons in the hypothalamic area.

Expression of the hexose transporters GLUT1 and GLUT2 during the early development of the human brain.

We used immunohistochemistry with anti-glucose transporter antibodies to document the presence of facilitative hexose transporters in the fetal human brain. GLUT1 is expressed in all regions of the fetal brain from ages 10 to 21 weeks. GLUT1 was present in the endothelial cells of the brain capillaries, the epithelial cells of the choroid plexus and neurons. High expression of GLUT2 was observed in the granular layer of the cerebellum in brains 21 weeks old, but GLUT2 immunoreactivity was absent at earlier stages. GLUT3 and GLUT4 immunoreactivities were absent at all stages studied. GLUT5 immunoreactivity was evident only in the cerebellar region of 21-week old fetal brains. We conclude that GLUT1 plays a fundamental role in early human brain development. The data also suggest that the cerebellum of the developing brain has the capacity to transport fructose, a substrate that has not been previously identified as a source of metabolic energy in the adult human brain.

Oxytocin inhibits the uptake of serotonin into uterine mast cells.

The uptake of serotonin (5HT) into mouse uterine horns, the localization of sites at which this amine could be stored and the effect of oxytocin on 5HT uptake were studied. To analyze the characteristics of the 5HT uptake process, the tissue was incubated with [3H]serotonin. The uptake of [3H]5HT was Na+ dependent and saturable (Kmapp: 166 +/- 15 nM, Vmax: 404 +/- 25 fmol/mg tissue, 30 min (diestrous); and Km: 165 +/- 39 nM, Vmax: 276 +/- 43 fmol/mg tissue, 30 min (estrous), n = 6), and was inhibited by imipramine, fluoxetine and 6-nitroquipazine (IC50: 2; 0.09 and 0.5 nM, respectively). In the myometrium the main 5HT uptake process was localized in uterine mast cells. This was determined by treating the uterine horns with 6-hydroxydopamine, by using an immunocytochemical approach and by studying the outflow of 3H under the action of stimuli directed to either mast cells (compound 48/80: 10 microgram/ml) or sympathetic nerves (high K+: 100 mM and veratridine: 20 microM) in uterine preparations. Oxytocin inhibited [3H]5HT uptake into uterine mast cells during estrus, but not in ovarectomized mice treated with progesterone. Maximal inhibition was attained at 0.03 nM, with a significant reduction in both Kmapp and Vmax (87 +/- 15 nM and 184 +/- 36 fmol/mg tissue/30 min, n = 3, respectively). This effect was reversed by the addition of OVT16, an oxytocin antagonist, at a concentration of 4 nM (Kmapp 158 +/- 35 nM, Vmax: 278 +/- 24 fmol/mg tissue, 30 min, n = 3). These findings support a new potential role of oxytocin and mast cells as a local regulators of serotonin bioavailability in myometrium. Because serotonin is recognized as an important endogenous uterotonic compound, this effect could be considered as an indirect action of oxytocin that may contribute to its potency as a labor inducer after genomic effects of estrogens are expressed in uterine tissue.

Ethodin: pharmacological evidence of the interaction between smooth muscle and mast cells in the myometrium.

Ethodin has been used to induce labor through a mechanism that does not involve the estrogen-preparatory process being postulated as necessary for ensuring the events in a normal labor. The cellular mechanisms involved in that process are unknown. We used an isolated organ bath preparation for mouse uterine horns and a primary culture of mouse myometrial smooth muscle cells to analyze the cellular mechanisms involved in the contractile action of this drug in the myometrium. Ethodin at a concentration of 10 microM and Compound 48/80 (1 microg/ml) evoked contractions of uterine horns in an isolated organ bath preparation. Uterine contractile responses showed a transient increase in contractile tension that lasted 2 to 3 min. Tachyphylaxis was observed after four or five successive stimuli, which consisted in additions and washings of the drug at an interval of 10 min. The primary smooth muscle mouse myometrium cells contained a high proportion of relaxed cells that varied widely in length (5-160 microm). Cell lengths decreased in response to the application of serotonin (10 microM) and oxytocin (0.1 microM) but were not affected after the addition of ethodin (10 microM). However, the cells contracted after a purified fraction of mast cells that had been degranulated by the action of the drug ethodin, which was added to the culture medium. These results provide some evidence related to the mechanism of myometrial contractile action of ethodin and support the hypothesis that mast cells may be involved in the regulation of myometrium contractility.

Increased affinity of histamine H1 binding to membranes of human myometrium at the end of pregnancy.

1. The characterization of H1 binding sites in membrane preparations of human myometrium obtained from pregnant and non-pregnant women was performed by using 3H-mepyramine as the radioactive ligand. 2. Saturation curve analysis revealed that 3H-mepyramine is bound to a single class of binding sites. Changes in the H1 site binding parameters were observed at the end of pregnancy, resulting in an increased affinity relative to non-pregnant tissue (Kd: 131.0 +/- 8.8 (non-pregnant) and 72.5 +/- 7.5 (pregnant) nM, n = 6, P < 0.01). 3. A reduction in receptor concentration at the end of pregnancy was also observed, [Bmax: 565.2 +/- 43.7 (non-pregnant) and 309.6 +/- 25.9 (pregnant) fmol/mg prot, n = 6, P < 0.01]. It is possible that this reduction in Bmax could be attributed to a dilution factor due to the increase in membraneous proteins that occurs during gestation.

Distribution of mast cells and the effect of their mediators on contractility in human myometrium.

OBJECTIVE: To examine the distribution of mast cells in human uterine tissue and to study the interactions between mast cell mediators (histamine and serotonin) and PGF2 alpha in human myometrium contractile activity. DESIGN: Distribution of mast cells were analysed in cryostat sections of myometrium samples stained with Toluidine blue. Contractile activity was evaluated in an isolated organ bath preparation on myometrial strips obtained from women whose pregnancies ended in elective caesarean section. SETTING: Biological Science Laboratories at the University of Concepción. SUBJECTS: Twenty women undergoing elective caesarean and 10 women undergoing hysterectomy at the G. Grant Hospital. MAIN OUTCOME MEASUREMENTS: Cumulative concentration-response curves for histamine and serotonin before and after the addition of subumbral concentrations of the PGF2 alpha or serotonin were performed. RESULTS: Serotonin was more active than histamine to evoke contractions (EC50:0.20 (SE 0.02) mumol/l vs 1.5 (SE 0.2) mumol/l, respectively). Furthermore, threshold concentrations of serotonin (0.05 mumol/l) potentiated the contractile effect of histamine (EC50:0.3 (SE 0.06) mumol/l and 50% increase in E(max)). PGF2 alpha had a poor contractile effect, but threshold concentrations (0.05 and 0.10 mumol/l) enhanced the contractile effect of both serotonin and histamine. A population of mast cells was found in close apposition to smooth muscle fibres. CONCLUSIONS: It is postulated that the simultaneous release of mast cell mediators (histamine and serotonin) in myometrium could be an important stimulus for evoking strong contractions in the human uterus. PGF2 alpha may have indirect effects in myometrium by amplifying the effects of histamine and serotonin.

Characterization and distribution of cholinesterase activity in mouse uterine horns: changes in estrous cycle.

1. Both butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) are present in the mouse uterus, BChE being more abundant. 2. Their molecular forms were sequentially solubilized by different extraction media obtaining three ChE fractions whose specific activity was different, depending on the stage of the estrous cycle: hydrosoluble (estrous: 75.5 +/- 6.6 and diestrous: 47.9 +/- 8.7 mU/mg prot); detergent-soluble or amphiphilic (estrous 26.6 +/- 2.4 and diestrous 14.7 +/- 3.3 mU/mg prot.), and high ionic strength-soluble (estrous: 18.7 +/- 4.2 and diestrous 12.8 +/- 1.2 mU/mg prot.). 3. Histochemical procedures demonstrated a different distribution for both ChE activities. AChE was found in nerves next to smooth muscle cells of the circular layer and blood vessels, while BChE was concentrated in the longitudinal stratum surrounding the smooth muscle cells. Under the predominance of progesterone, BChE was also found in the endometrial glands. 4. Maximal contractions evoked by the addition of ACh to the isolated organ bath were concentration dependent and greater in estrous than in diestrous. Nevertheless the difference at the two stages of the estrous cycle disappeared when contractions were normalized to smooth muscle cross-sectional area. 5. BChE but not AChE inhibition augmented maximal contractions elicited by ACh in longitudinal but not in circular smooth muscle. 6. The effect of BChE inhibition on the contractile force developed was greater at lower concentrations of ACh and did not depend on the stage of the estrous cycle.

Histamine H1 receptor binding sites in mouse uterine horns.

1. The specific binding of both [3H]histamine and [3H]pyrilamine to crude membrane preparations obtained from mouse uterine horns was studied with the aid of radioligand binding techniques. 2. Saturation isotherms and binding of 300 nM of [3H]histamine in the presence of increasing concentrations of unlabelled histamine suggested the presence of two populations of [3H]histamine binding sites (IC50: 56 +/- 5.6 nM and 100 +/- 9.8 microM, respectively). 3. Pyrilamine displaced the high affinity binding site (IC50: 100 +/- 10 nM), while cimetidine displaced the low affinity one (IC50: 98 +/- 10 microM). 4. Saturation curve analysis by using [3H]pyrilamine as radioligand, revealed that H1 binding site varied according to the predominance of sexual hormones (Kd: 29 nM; 102 nM and 130 nM in uterine membranes from ovariectomized, diestrous and estrous, respectively). At the end of pregnancy Kd was found to be 71.1 nM.

Mast cells mediators evoke contractility and potentiate each other in mouse uterine horns.

1. The ability of prostaglandin F2 alpha (PGF2 alpha), histamine and serotonin to induce and/or potentiate contractions in mouse uterine horns was studied in an attempt to analyze whether uterine mast cells degranulation could favor contractions during labor. 2. PGF2 alpha was the most potent of the three compounds (EC50 = 0.7 microM), being followed by serotonin (EC50 = 1.2 microM) and histamine. Between 10 and 100 microM histamine only evoked weak contractions, not higher than 50% of maximal amplitude of contractions. 3. Serotonin (0.1 microM) potentiated the contractile effect of both histamine and PGF2 alpha when added simultaneously with the corresponding compound to the isolated organ bath. Also, histamine at threshold contractile concentration (3 microM) was able to potentiate the contractile effect of both serotonin and PGF2 alpha. 4. The potentiating effect of PGF2 alpha on both histamine and serotonin evoked contractions was recorded after treating the preparation with 10 microM of the compound for at least 10 min before the corresponding concentration-effect curves were performed. 5. It is postulated that the simultaneous presence of these mast cells mediators in myometrium could be an important stimulus for both to trigger and/or to maintain contractions during labor.

Uptake of L-leucine and L-phenylalanine across the basolateral cell surface in isolated oxyntic glands.

The time course, kinetic, specificity and sodium-dependence of L-leucine and L-phenylalanine uptake by rabbit isolated oxyntic glands were studied in order to identify the systems involved in the transport of branched-chain and aromatic neutral amino acids through the basolateral cell membrane. The uptake was measured directly in the disrupted cells after incubation of the glands with the 3H-labelled amino acid both in a sodium-containing and a sodium-free medium. The uptake of L-leucine was largely carrier-mediated whilst L-phenylalanine was taken up by either carrier-mediated and nonsaturable processes. Both amino acids were taken up by a Na(+)-independent process. The kinetic parameters of L-leucine and L-phenylalanine carrier-mediated influx were, respectively: Kt = 2.71 mM and Jmax = 1390 nmol mg-1 s-1, Kt = 1.03 mM and Jmax = 176 nmol mg-1 s-1. From cross-inhibition studies it can be inferred that L-leucine is primarily transported by a Na(+)-independent system which shows specificity for bulky side chains dipolar amino acids. The system displays similar affinities for L-phenylalanine (Ki = 2.81 mM) and L-isoleucine (Ki = 2.62 mM). Similar results were obtained from self-inhibition experiments: the Ki of the carrier-mediated uptake of L-leucine and L-phenylalanine were 2.12 and 2.40 mM (from a Hanes plot) or 3.2 and 0.8 mM (from a Dixon plot), respectively. It is concluded that a sodium-independent transport system, like Christensen's 'L' type, is shared by branched-chain and aromatic dipolar amino acids, which only shows slight differences in their affinities for the carrier.

Effect of progesterone on norepinephrine release from mouse adrenergic terminals in vitro.

1. The in vitro effect of progesterone on norepinephrine (NE) release and contractile activity was analyzed in uterine horns from estrogen-primed and progesterone-primed mice. 2. Progesterone (6-10 nmol/ml) evoked the release of [3H]NE above basal levels from uterine horns in both experimental conditions, the effect of progesterone on estrogen-primed being more important than on progesterone-primed mice uterus. 3. Progesterone also increased electrically evoked [3H]NE release in estrogen-primed uterine tissue, nevertheless no effect was observed in progesterone-primed ones. 4. Progesterone (0.6-10 nmol/ml) inhibited uterine horn isometric contractions only in estradiol-primed mice. This effect was partially blocked in uterine horns from reserpine-treated mice and when propanolol (1 microM) was added to the preparation of estradiol-primed mice uterus.

Histamine content and mast cells distribution in mouse uterus: the effect of sexual hormones, gestation and labor.

The histamine content of uteri from mice was analyzed in terms of both concentration and total amount per uterine horn a) at two stages of the estrous cycle (estrous and diestrous), b) under sex hormone treatment, c) during pregnancy and after delivery. Histamine concentration and mast cell density were greater during diestrous and in mice treated with progesterone (p less than 0.001). This effect was attributed to a reduction in uterine mass weight, since the amount of histamine per uterine horn remained constant throughout the estrous cycle. During pregnancy, both concentration and amount of histamine per uterine horn were increased, values were significantly higher than in estrous (p less than 0.001) from day 14-17 until day 21 when labor occurred. After six to eight hours post-partum an abrupt reduction on histamine content was observed. Mast cells were more abundant in myometrium than in endometrium, their density followed the same pattern as histamine concentration throughout the estrous cycle.

Efecto de fármacos tocolíticos sobre la actividad contractil evocada por prostaglandinas en el utero gestante / Tocolytic drugs effect in contractil activity evoked by prostaglandines in the pregnant uterus

Se estudió el efecto in vitro de ritodrine (28,7 ng/ml), fenoterol (30,0 ng/ml), verapamil (43 ng/ml), nifedipino (34,6 ng/ml) y sulfato de magnesio (6 meq/lt) sobre la actividad contráctil espontánea y evocada por prostaglandina PGF 2* en cuernos uterinos de ratones con 15 días de gestación. El rango de concentraciones usadas fue cercana a los niveles plasmáticos efectivos alcanzados para cada tocolítico respectivamente. A las dosis señaladas, la actividad contráctil espontánea fue completamente abolida por los tocolíticos investigados. En cambio, las contracciones evocadas por PGF 2* fueron inhibidas solamente por ritodrine, fenoterol y nifedipino. Se discuten posibles mecanismos involucrados en estas interacciones

Purification and properties of a new beta-lactamase from Streptomyces UCSM-104.

Extracellular beta-lactamase was produced in larger amounts by Streptomyces UCSM-104 than by Streptomyces albus G or strains R-39 and K-11. The anionic character displayed by the enzyme from Streptomyces USCM-104 on DEAE-cellulose chromatography was consistent with its mobility in polyacrylamide gel electrophoresis. This beta-lactamase is a low molecular weight (14,800) globular protein having approximately 124 amino acid residues. The enzyme behaved as a penicillinase toward several substrates studied, being most active on benzylpenicillin and ampicillin. On the other hand, it was inhibited by methicillin and cloxacillin, which behaved as competitive inhibitors for the hydrolysis of benzylpenicillin and cephaloridine.

Kinetic study on beta-lactamase from Streptomyces UCSM-104.

The beta-lactamase of Streptomyces UCSM-104 behaves as a penicillinase with the different substrates studied, being more active with benzylpenicillin (KM 2.6 mM) and ampicillin (KM 1.5 mM). On the other hand, it was competitively inhibited by methicillin (Ki 0.035 mM) and cloxacillin (Ki 0.35 mM) using benzylpenicillin as substrate. According to the criterion of Jack and Richmond in relation to the degree of inhibition obtained, the enzyme was found to be resistant to cloxacillin and sensible to methicillin.