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.

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.

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.

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.

Meningoencefalitis por cryptococcus: reporte de un caso clínico y revisión de la literatura / Cryptococcal meningoencefalitis: a clinical case report and literature review

La infección por cryptococcus en el niño es muy infrecuente especialmente si es inmunocompetente. Se presenta un preescolar inmunocompetente con meningoencefalitis grave con líquido cefalorraquídeo claro y signos neurológicos de focalización, tratado como meningoencefalitis herpética. Evolucionó inicialmente con leve mejoría, reingresando por aumento del compromiso neurológico. Se amplió el estudio etiológico y se detectó cryptococcus en el LCR. Se comenta el cuadro clínico de la meningoencefalitis cryptococócica su diagnóstico y revisión de la literatura. Nos parece aconsejable incluir el test de tinta china en toda meningoencefalitis a líquido claro de etiología no precisada

Colitis hemorrágica por escherichia coli enterohemorrágica O157 H:7: comentario en relación a dos casos clínicos / Haemorrhagic colitis caused by escherichia coli H7-O157: a review of 2 clinical cases

Escherichia coli enterohemorrágica O157 H:7 es una infección emergente en nuestro país. Dos casos graves en nuestra unidad permiten actualizar aspectos poco conocidos. A diferencia de países desarrollados (edad media 15 años), en nuestro país afecta a niños pequeños (media 1,5 años) indicando probablemente transmisión doméstica desde el adulto, que no enferma por inmunidad. La colitis puede ser grave y requerir colectomía cuyas indicaciones no son precisas. No está comprobado que los antibióticos aumenten la incidencia de SHU pero se sugiere evitar su uso temprano. No todos los laboratorios clínicos buscan este germen aun cuando la técnica es fácil. El tratamiento debe incluir una observación cuidadosa buscando las complicaciones como síndrome hemolítico urémico y un manejo adecuado de contactos. La prevención se basa en buen lavado de utensilios de cocina en contacto con carne cruda, de las manos antes de tomar o alimentar niños, y la ingestión de carne de vacuno y cerdo bien cocida, en especial si es molida. Proposiciones: el médico debe buscar la etiología, y las autoridades difundir medidas de prevención e instaurar notificación obligatoria

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.

Production of mitogen-contamination free alginates with variable ratios of mannuronic acid to guluronic acid by free flow electrophoresis.

Commercial alginates consisting of variable homopolymeric regions of beta-D-mannuronic acid and alpha-L-guluronic acid, interspaced with regions of alternating blocks, are potent stimulators of macrophages and lymphocytes. Therefore, inflammatory reactions and fibrotic overgrowth of the beads result if Langerhans islets are encapsulated in raw alginate hydrogel beads (cross-linked with divalent cations). The result is random failure of the islets some time after transplantation. Analysis of raw alginates by using free flow electrophoresis demonstrated that commercial alginates contained at least 10-20 fractions (characterized by different electrophoretic mobilities) which showed mitogenic activity. These fractions could be quantitatively separated from the alginic acids by free flow electrophoresis on a preparative scale. The purified alginates cross-linked with Ca2+ ions exhibited no mitogenic reactions as proved by an in vitro assay. In addition, examination of purified Ba2+ alginate beads implanted intraperitoneally in rats or mice for three weeks showed no fibrotic overgrowth in contrast to implants made from unpurified alginate.

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.

Free-flow electrophoresis under microgravity: evidence for enhanced resolution of cell separation.

A mixture of fixed rabbit, guinea pig and rat erythrocytes, suspended in a relatively conductive solution, was separated by means of continuous free flow electrophoresis (CFFE) under 1 g- and microgram- conditions using a specially designed electrophoretic module. Short duration microgram conditions were realized on board a sounding rocket. Due to the energy input and the associated thermal convection a separation of the three differently charged cell types in distinct peaks was not possible under 1 g-conditions as shown by reference experiments on the ground before launch. In contrast to the poor resolution under 1 g-conditions, clear separation of the cell mixture could be recorded after lift-off of the rocket under microgram-conditions. Repeated measurements demonstrated that the separation profile was completely stable during the entire microgram-phase of about 6 min. Since the CFFE experiment in space was an exact replica of the ground reference experiments, the results demonstrated unambiguously the potential of CFFE for cell separation under microgram-conditions in media of high ionic strength.

Antigen-specific electrophoretic cell separation for immunological investigations.

Preincubation of human blood lymphocytes with cell surface antigen specific antibodies under non-capping conditions reduces the electrophoretic mobility of the corresponding lymphocyte subpopulation. Antigen-positive and antigen-negative cells can be separated by free flow electrophoresis with high yield, purity and viability. The use of fluorescence-labelled second antibodies augments the induced decrease in net surface charge density, and allows rapid detection of antigen-positive cells in the fractions of electrophoresis. Carrier-free cell electrophoresis of human peripheral blood lymphocytes after reaction with anti-IgM-antibody or the monoclonal antibodies OKT4 or OKT8, and sandwich staining with tetrarhodamine isothiocyanate-labelled anti-IgG resulted in the large-scale separation of high pure human B and T lymphocyte subpopulations. Their functional integrity was shown in assays of lymphocyte transformation and of antigen-specific induction and regulation of antibody synthesis in vitro. These separate lymphocyte subpopulations are useful tools for immunological investigations. While, for instance, the effects of drugs on human lymphocytes are obscured by coincident changes in cell composition of the peripheral blood tested that do not by themselves reflect whole body immunocompetence, the cell separation and in vitro assays at a defined cell number and cell composition allow the recording of quantitative changes in the function of different cell subpopulations. We studied the influence of the anesthetic thiopental on separated human lymphocyte subsets. In both polyclonal lectin stimulation and in vitro antibody production, thiopental exhibited a noncytotoxic suppression of lymphocyte functions. B-Cells, T-helper and T-suppressor cells were equally affected and showed the same dose response.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Cell electrophoresis of group B streptococci: separation of types Ia, Ib/c, II, III and IV before and after neuraminidase treatment.

Group B streptococcal strains of types Ia, Ib/c, II, III and IV could be characterized by distinct electrophoretic mobilities before and after neuraminidase treatment. By this method of cell electrophoresis in the type I a strain, two subpopulations could be detected; whereas in all strains the electrophoretic mobility is markedly reduced after enzymatic removal of neuraminic acid, type II remained unaffected. The method of "bacteriopheresis" offers a new approach to classification of bacteria with respect to the primary and secondary surface structures.

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.

The negative surface charge density is a maturation marker of human B lymphocytes.

Small resting B lymphocytes were highly enriched and completely depleted of all preactivated large B lymphocytes using countercurrent centrifugal elutriation and free flow electrophoresis. They required T lymphocytes, monocytes, and a mitogen to produce antibodies after 5 days of preincubation. Large activated B lymphocytes were obtained in cell fractions which were free of resting ones. They produced antibodies even in the absence of a mitogen. Two groups were distinguished, differing in their stage of differentiation and their negative surface charge density. The cells of one group had an electrophoretic mobility (EM) like resting B lymphocytes ranging from 0.85 to 0.99 X 10(-4) (cm2 V-1 s-1). They took 2 to 3 days of preincubation before they started to secrete antibodies. Interleukin 2 and pokeweed mitogen enhanced their antibody production capability. The cells of the other group had an EM between 0.99 and 1.13 X 10(-4) (cm2 V-1 s-1). They secreted antibodies even during the first day of incubation. The quantity of the antibodies which they produced depended only on the blood donor. It could not be influenced by a mitogen or by interleukin 2. The study shows that large B lymphocytes with high negative surface charge density are in a later maturation stage than those with lower negative surface charge density.

Studies on the activation mechanism of human mononuclear leukocytes isolated by physical methods.

Responder lymphocytes, accessory lymphocytes and monocytes were isolated using countercurrent centrifugal elutriation and free flow electrophoresis. The Concanavalin A (Con A) binding behaviour of the isolated cell populations was studied. Responder lymphocytes and accessory cells bound Con A by different mechanisms. Cells in all the isolated populations were able to interact directly with Con A. Con A binding to responder lymphocytes was inhibited by alpha-methylmannoside (alpha MM) and by a distinct plasma protein. Accessory lymphocytes and monocytes bound Con A even in the presence of 10% human plasma or 50 mM alpha MM. The plasma protein which inhibited interaction of responder lymphocytes with Con A also reduced lymphocyte proliferation, when resting monocytes were used as accessory cells. If, however, monocytes were used after activation by lipopolysaccharide no inhibition effect was observed. From the results we conclude that there is a distinct plasma protein which protects the organism against lymphocyte stimulation not controlled by accessory cells. This inhibitory plasma protein appears to be a euglobulin.

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.