The effect of perineural dexamethasone on duration of sciatic nerve blockade: a randomized, double-blind study.

BACKGROUND: Major hindfoot and ankle surgery is associated with severe postoperative pain, which is effectively alleviated by combined sciatic and saphenous nerve blockade. Local anaesthetics with added dexamethasone consistently prolongs the duration of pain relief compared to local anaesthetics alone. However, whether the extended duration of pain relief is due to an effect on duration of sensorimotor block per se vs. systemic absorption of the dexamethasone is still not fully elucidated. We aimed to investigate the postoperative duration of sensorimotor blockade with either dexamethasone or saline added to bupivacaine-epinephrine. METHODS: Fifty six patients scheduled for surgery were randomly assigned to a popliteal sciatic nerve block of 18 ml 0.5% bupivacaine-epinephrine with either 2 ml of 0.4% dexamethasone or 2 ml 0.9% normal saline added. Sensory and motor functions were tested every 30 min until normalized nerve functions. Primary outcome was time until complete return of sensorimotor functions. RESULTS: Mean (SD) time until return of normal sensory and motor functions was 26 (6) vs. 16 (4) hours, P < 0.001, postponing block remission by 10 (95% CI: 8-13) hours. Mean (SD) time until first opioid request was 34 (11) vs. 15 (7) hours, P < 0.001, extending first opioid request by 19 (95% CI: 13-25) hours. Total oral morphine equivalents administered 0-48 h differed significantly between the two groups by 39 (95% CI: 23-55) mg. CONCLUSIONS: Addition of 8 mg dexamethasone to 0.5% bupivacaine-epinephrine significantly prolongs the duration of sensorimotor popliteal sciatic nerve blockade, and reduces pain and opioid consumption in patients after major hind foot and ankle surgery.

A novel type of three band pattern at STR loci.

SE33 or ACTBP2 is the most polymorphic locus in many national DNA databases and in the commercial STR kits used to type both crime scene samples and reference samples to populate these databases. We describe the molecular reason for a three band pattern of SE33 seen in several samples. A SNP in the flanking SE33 region causes the binding of the unlabelled D3S1358 primer. As a result, a "chimeric" PCR product of the labelled SE33 primer and the D3S1358 primer is generated that is smaller than the regular SE33 amplicon. We call this "Type 3 three band pattern" as the genetic base differs from the Type 1 three band pattern caused by somatic mosaicism and the Type 2 that results from copy number variation.

A new method for the preparation of rat liver lysosomes. Separation of cell organelles of rat liver by carrier-free continuous electrophoresis.

A combination of differential centrifugation and carrier-free continuous electrophoresis is introduced as a new method for the isolation of animal cell organelles. Various buffers were systematically checked in order to find the system which preserves the organelles and gives as well a good separation in the free-flow electrophoresis apparatus. Triethanolamine-acetate buffer (10 mM), pH 7.4 was used. The isolated lysosomes were pure according to marker enzymes and electron micrographs. A heterogeneity of the lysosomes in electrophoretic mobility was demonstrated with respect to the marker enzymes arylsulfatase and beta-glucuronidase. The lysosomes with higher mobility showed a maximum enrichment of 240-fold with respect to arylsulfatase. The lysosomes with lower electrophoretic mobility showed a 65-fold enrichment with respect to beta-glucuronidase. The ratio of beta-glucuronidase to arylsulfatase varied from 2:1 to 1:2 in lysosomes of different mobility. The yield amounted to approximately 1 mg of lysosomal protein per gram of liver protein. 5-8 mg of lysosomes can be obtained in one experiment. The electrophoretic separation proves to be an effective tool in obtaining pure and well preserved lysosomes.

The surface charge of rat liver mitochondria and their membranes. Clarification of some controversies concerning mitochondrial structure.

The surface charge of intact mitochondria and submitochondrial particles was examined by the technique of preparative free flow electrophoresis. When submitochondrial preparations obtained by a swelling-contraction procedure were examined with this technique, two fractions were observed. One of these fractions exhibited the same electrophoretic properties as intact mitochondria, which indicated that it was derived from the outer limiting membrane of the mitochondrion. This fraction was found to contain the enzymes monoamine oxidase and rotenone-insensitive NADH-cytochrome c reductase which have been reported to be localized in the outer mitochondrial membrane. The other fraction exhibited an electrophoretic mobility which was different from that of intact mitochondria, and this fraction contained enzymes characteristic of the inner membrane-matrix fraction such as soluble and particulate enzymes of the Krebs cycle. Microsomes exhibited an electrophoretic mobility which was almost identical with that of the outer mitochondrial membrane. In addition to resolving the localization of enzymes in mitochondrial membranes, these data indicate that the outer limiting membrane of the mitochondrion is the sole determinant of the surface charge of mitochondria.

The polarity of the proximal tubule cell in rat kidney. Different surface charges for the brush-border microvilli and plasma membranes from the basal infoldings.

Two different membrane fractions were obtained from a brush-border fraction of rat kidney cortex by using their different electrical surface charges in preparative free-flow electrophoresis. One membrane fraction contained only morphologically intact microvilli and was characterized by a high specific activity of alkaline phosphatase. The other fraction morphologically resembled classical plasma membranes by possessing junctional complexes and a high Na-K-ATPase activity The contamination of the isolated membrane fractions by other cell organelles was extremely low These two fractions represent the apical (luminal) and the basal (interstitial) area of the renal proximal tubule cell membrane and clearly demonstrate the polarity of this cell.

Rapid purification of clathrin-coated vesicles by free-flow electrophoresis.

Free-flow electrophoresis was successfully used as the final step in the purification of clathrin-coated vesicles from bovine brain. Based on biochemical analysis, the material obtained in this way was found to be of equal purity with respect to the protein composition and lipid content as that purified by the previously widely used methods of permeation chromatography on controlled pore glass or Sephacryl S-1000. However, as judged by electron microscopy, the electrophoretically purified coated vesicles contained less smooth membranes than the coated vesicle preparations that had been obtained by permeation chromatography. Free-flow electrophoresis offers considerable advantages in speed of purification, in the total amount of material processed and in flexibility of operation. Analysis of the electrophoretic mobility of purified coated vesicles showed that this is governed by the coat proteins rather than by the vesicle contained therein. A shift in electrophoretic mobility of purified coated vesicles was obtained by the binding of coat protein specific monoclonal antibodies. This raises the possibility of purifying subpopulations of coated vesicles with respect to coat protein composition.

Androgen-like and anti-androgen-like effects of antiprogestins in human mammary cancer cells.

In addition to their antiprogestational activity, the antiprogestins RU486, ZK98.299 and ZK98.734 possess varying antiglucocorticoid as well as androgen-like or antiandrogen-like properties in human mammary cancer cells. The human mammary cancer cell line MFM-223, which contains only androgen receptors, was used as a model to investigate androgen receptor mediated effects of these antiprogestins. Proliferation of MFM-223 cells is inhibited by androgens and does not respond to oestrogens, progestins and glucocorticoids. As shown in proliferation assays, ZK98.734 was a strong inhibitor of cell proliferation. This effect was antagonised by the antiandrogen hydroxyflutamide. ZK98.734 was found to displace [3H]R1881 from the androgen receptor in MFM-223 cells, substantiating the involvement of the androgen receptor. The antiprogestin ZK98.299 failed to influence the proliferation of MFM-223 cells. ZK98.299 did not bind to the androgen receptor and was devoid of androgenic or antiandrogenic activity. RU486 bound to the androgen receptor. It was a weak inhibitor of MFM-223 cell proliferation, but the inhibition of proliferation by RU486 was not antagonised by hydroxyflutamide. This effect was probably not mediated by the androgen receptor. RU486 had antiandrogenic activity in this cell line, as it antagonised the inhibitory effect of dihydrotestosterone at a 100-molar excess. These results were confirmed by transfection experiments with an MMTV-CAT construct in the same cell line, demonstrating the biological function of the ZK98.734-androgen receptor complex. ZK98.299 and RU486 were not able to induce CAT activity. The different androgenic or antiandrogenic properties of the antiprogestins investigated should be considered when selecting antiprogestational properties of the antiprogestins investigated should be considered when selecting antiprogestational compounds for clinical applications, as a partial androgenic activity may be of benefit in breast cancer but can have undesired side-effects in other diseases.

Countercurrent centrifugal elutriation in a table-top centrifuge.

An elutriation system has been developed which fits into a small table-top centrifuge. The new CS1 rotor functioned as accurately as the JE-6 rotor, commercially available from Beckman Instruments. However, fewer cells were required for the separation experiments. Leukocytes obtained from 25 ml whole blood by Ficoll-Hypaque gradient centrifugation were sufficient for separating lymphocytes from monocytes and for purifying granulocytes. Furthermore, when lymphocytes were harvested from cell cultures 40 ml of a suspension of 1.2 x 10(6) cells/ml were enough to yield an enriched preparation of antibody producing cells.

Antigen-specific electrophoretic cell separation (ASECS): isolation by human T and B lymphocyte subpopulations by free-flow electrophoresis after reaction with antibodies.

The electrophoretic mobility of human lymphocytes can be reduced by incubation with surface antigen specific antibodies under non-capping conditions. This renders subpopulations of human peripheral blood lymphocytes accessible to separation by free-flow electrophoresis. After reaction of lymphocyte preparations with anti-IgM antibody and a fluorescent second antibody, B lymphocytes showed a considerable shift in position in preparative cell electrophoresis and could be separated with high yield, purity and vitality. Similarly, a T cell subpopulation reactive with the monoclonal antibody T811 could be isolated, even though only small amounts of this antibody were bound, by using a double-sandwich method. Non-specific antibody uptake via Fc-receptors did not contribute to the observed shift of antibody-labelled cells to lower electrophoretic mobility. Flow cytometric analysis showed that cells were separated according to their antigen density. Thus cell electrophoresis can be used to separate antibody-labelled cells. With a flow rate of 100,000 cells/sec this method has a much higher separation capacity than fluorescence-activated cell sorting. The described method should be applicable to the separation of a wide range of cell populations for which specific antibodies are available.

Electrofused mammalian cells analyzed by free-flow electrophoresis.

Somatic cell fusion is a powerful and widely used technique. In recent years, electrofusion has become increasingly popular because it is a gentle process that can be optically controlled and carefully monitored using appropriate fusion chambers and because it permits the efficient fusion of smaller cell numbers. However, damage of the cell membrane and cell lysis occurs during application of the electrical field and is accompanied by changes in surface charge which can be detected by free-flow electrophoresis. In this study, we evaluated free-flow electrophoresis to detect changes in cell viability after application of electric-field conditions employed in mammalian cell electrofusion and to separate dead cells and cell debris from intact unfused or fused cells.

Cytofluorometric analysis of R-Thy-1. antigens in various rat lymphocytes with different electrophoretic mobility and organ distribution.

Small bone marrow lymphocytes, which had been previously enriched by velocity sedimentation, thymocytes, lymph node cells and spleen cells were electrophoretically separated, stained with fluorescein conjugated rabbit a-rat-Thy-1. globulin and their fluorescence intensities analyzed with a flow cytophotometer. Thy-1. antigens were found in 80% of the bone marrow small lymphocytes showing low electrophoretic mobility (EPM), in all thymocytes, about 80% of which show low and the rest medium to high EPM, and in a few lymph node cells of high EPM. Thy-1. positive cells were not observed in the spleen. All fluorescence intensity histograms obtained were modal and could be properly fitted with normal curves showing coefficients of variation (C.V.) in the range of 20% to 30%. It was observed that the thymocytes of low EPM had an antibody binding affinity significantly different from that of the other stained lymphocytes. Moreover the surface antigen density decreased in the sequence: thymocytes of low EPM, bone marrow lymphocytes of low EPM and thymocytes of high EPM. The fluorescence intensity of stained lymph node cells of high EPM appeared similar to that of thymocytes of high EPM but was not evaluated precisely. Thus the two dimensional cell analysis provided by a combination of EPM and surface fluorescence of Thy-1.+ cells, allows the characterization of different lymphocyte populations which cannot be clearly identified with normal one dimensional techniques. The biological significance of the results is discussed briefly.

Partial characterization of lymphokines that change the surface charge density of human polymorphonuclear leukocytes.

We have investigated the effects of stimulated human lymphocyte supernatants on the surface charge density of human polymorphonuclear leukocytes using analytical free-flow electrophoresis. Unfractionated ConA-induced human lymphocyte supernatants were found to decrease the electrophoretic mobility of PMN leukocytes by 10-16%. Optimal conditions for the production of this lymphokine as well as for the PMN leukocyte-lymphokine interaction are described. In order to obtain more information about the factor responsible for the effect, active and control supernatants were fractionated by Sephacryl S-200 column chromatography, Sephadex G-50, and Amicon membrane filtration. The fractions obtained were dialysed, concentrated, and tested for activity on PMN leukocytes in free-flow electrophoresis. Fractions obtained in parallel were tested for Leukocyte Inhibition Factor (LIF) activity using the Clausen assay. Decrease in electrophoretic mobility of human PMN leukocytes was found to be due to two distinct lymphokines with molecular weights of about 10,000-20,000 and 50,000-60,000 d, respectively. These fractions showed no activity in the Clausen assay. Fraction V (60,000-80,000 d) revealed strong activity in the Clausen assay, but had no effect on the surface charge density of human PMN leukocytes. The ConA-induced lymphokines that changed the surface charge density of PMN leukocytes retained their activity after heating to 56 degrees C for 30 min. They were not synthesized in the presence of puromycin, but mitomycin C treatment of the lymphocytes had no effect on the production of the lymphokines. Moreover, pretreatment of human PMN leukocytes with puromycin for 30 min blocked the reaction, indicating an active role of the PMN leukocytes following the reaction with the lymphokines. Finally these lymphokines preferentially influenced the surface charge density of human PMN leukocytes but not that of rat or guinea-pig peritoneal exudate cells.

Physical therapy for spinal accessory nerve injury complicated by adhesive capsulitis.

BACKGROUND AND PURPOSE: The authors found no literature describing adhesive capsulitis as a consequence of spinal accessory nerve injury and no exercise program or protocol for patients with spinal accessory nerve injury. The purpose of this case report is to describe the management of a patient with adhesive capsulitis and spinal accessory nerve injury following a carotid endarterectomy. CASE DESCRIPTION: The patient was a 67-year-old woman referred for physical therapy following manipulation of the left shoulder and a diagnosis of adhesive capsulitis by her orthopedist. Spinal accessory nerve injury was identified during the initial physical therapy examination, and a program of neuromuscular electrical stimulation was initiated. OUTCOMES: The patient had almost full restoration of the involved muscle function after 5 months of physical therapy. DISCUSSION: This case report illustrates the importance of accurate diagnosis and suggests physical therapy intervention to manage adhesive capsulitis as a consequence of spinal accessory nerve injury.

Free flow electrophoresis in space shuttle program (Biotex).

In the space shuttle program free flow electrophoresis will be applied for separation of proteins, biopolymers and cells. Proteins are to be separated according to the "Feldsprung-Gradienten" procedure by Prof. H. Wagner, University of Saarbruecken, biopolymers are to be separated by the isotachophoresis technique by Prof. Schmitz, University of Muenster and we intend to separate cells in order to increase the efficiency of recovery of hybrid cells after electrofusion performed under microgravity in collaboration with Prof. U. Zimmermann, University of Wuerzburg. There are supposed two ways for reaching this goal: 1) Enrichment of cells before electrofusion may enhance the probability that the cells of interest are immortalized. 2) Separation of cells after electrofusion may help to clone the hybrid cells of interest. Under microgravity, the combination of improved electrophoresis with higher electrofusion rates may provide new possibilities for immortalization of cells. This may be a new way to obtain cellular products, which are physiologically glycosylated.

Losing uniqueness - shifts in carabid species composition during dry grassland and heathland succession.

Dry sand ecosystems, such as dry grasslands and heathlands, have suffered habitat loss and degradation due to land-use changes and are today among the most endangered habitats in Central Europe. To evaluate the impact of degradation processes on habitat quality, we investigated how succession from sparse vegetated sand ecosystems to grass-invaded and tree-dominated ecosystems and the environmental parameters associated with it influences carabid assemblages. We also determined to what extent typical xerophilic species assemblages still exist. Pitfall trapping at 28 study sites in northwestern Germany yielded 111 carabid species that were grouped using Kendall's W coefficient of concordance. Ordination revealed that the differences between the four species groups resulted from vegetation cover and soil humidity, indicating that carabid distribution clearly reflects degradation processes. Our results suggest that areas in which succession proceeds were unsuitable for assemblages typical of dry grasslands and heathlands. In all, 35 species are lost due to succession from dry grassland and heathland to grass-invaded and tree-dominated sites. We discuss implications for habitat management and restoration, since dry sand ecosystems comprise a very high number of specialized and endangered species.