THE WIDELY SPREAD PHENOMENON OF IN VIVO AND IN VITRO INDUCTION OF ROD/RING STRUCTURES AND ITS INFLUENCE ON THE GENERATION OF AUTOANTIBODIES AGAINST IMPDH2

Abstract Methyldopa-treated gamma globulin can be demonstrated serologically on either the red cell surface or on latex beads by the indirect antiglobulin reaction. The development of a positive antiglobulin reaction was related to methyldopa concentration and the length and temperature of incubation of methyldopa with protein and could be partially inhibited by the addition of albumin to the incubation mixtures. After more prolonged incubation, antiglobulin positivity also developed with plasma-treated with methyldopa. 14C-methyldopa was covalently bound to gamma globulin. Aggregation of gamma globulin following treatment with methyldopa could be demonstrated by both sedimentation velocity and molecular weight determinations employing low-speed equilibrium centrifugation. Protein aggregation was a function of time, temperature, and methyldopa concentration. Detectability by the antiglobulin reaction, the darkening noted in solutions to which methyldopa or hydroquinone had been added, as well as the aggregation of protein was inhibited by a reducing agent which prevented formation of a quinone from the hydroquinone. Some of the immunologically atypical features of the sensitization of red cells by methyldopa or its structural analogues are explicable by the adherence, in vivo, of chemically modified, nonantibody gamma globulin which renders the red cell directly antiglobulin positive.

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Mammalian Dynamin-like Protein DLP1 Tubulates Membranes

Molecular Biology of the Cell ◽

10.1091/mbc.12.9.2894 ◽

2001 ◽

Vol 12 (9) ◽

pp. 2894-2905 ◽

Cited By ~ 216

Author(s):

Yisang Yoon ◽

Kelly R. Pitts ◽

Mark A. McNiven

Keyword(s):

Cultured Cells ◽

Immunogold Labeling ◽

Wild Type ◽

Tubule Formation ◽

Defective Mutant ◽

Ring Structures ◽

Membrane Tubules ◽

Large Gtpases

Dynamins are large GTPases with mechanochemical properties that are known to constrict and tubulate membranes. A recently identified mammalian dynamin-like protein (DLP1) is essential for the proper cellular distribution of mitochondria and the endoplasmic reticulum in cultured cells. In this study, we investigated the ability of DLP1 to remodel membranes similar to conventional dynamin. We found that the expression of a GTPase-defective mutant, DLP1-K38A, in cultured cells led to the formation of large cytoplasmic aggregates. Electron microscopy (EM) of cells expressing DLP1-K38A revealed that these aggregates were comprised of membrane tubules of a consistent diameter. High-magnification EM revealed the presence of many regular striations along individual membrane tubules, and immunogold labeling confirmed the association of DLP1 with these structures. Biochemical experiments with the use of recombinant DLP1 and labeled GTP demonstrated that DLP1-K38A binds but does not hydrolyze or release GTP. Furthermore, the affinity of DLP1-K38A for membrane is increased compared with wild-type DLP1. To test whether DLP1 could tubulate membrane in vitro, recombinant DLP1 was combined with synthetic liposomes and nucleotides. We found that DLP1 protein alone assembled into sedimentable macromolecular structures in the presence of guanosine-5′-O-(3-thio)triphosphate (GTPγS) but not GTP. EM of the GTPγS-treated DLP1 revealed clusters of stacked helical ring structures. When liposomes were included with DLP1, formation of long membrane tubules similar in size to those formed in vivo was observed. Addition of GTPγS greatly enhanced membrane tubule formation, suggesting the GTP-bound form of DLP1 deforms liposomes into tubules as the DLP1-K38A does in vivo. These results provide the first evidence that the dynamin family member, DLP1, is able to tubulate membranes both in living cells and in vitro. Furthermore, these findings also indicate that despite the limited homology to conventional dynamins (35%) these proteins remodel membranes in a similar manner.

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301 THE EFFECT OF EQUINE FOLICULLAR FLUID ON IN VITRO INDUCTION OF THE ACROSOME REACTION IN FROZEN - THAWED STALLION SPERMATOZOA

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab301 ◽

2010 ◽

Vol 22 (1) ◽

pp. 307

Author(s):

D. S. Silva ◽

P. Rodriguez ◽

N. S. Arruda ◽

R. Rodrigues ◽

J. L. Rodrigues

Keyword(s):

Contact Area ◽

Follicular Fluid ◽

Acrosome Reaction ◽

Reproductive Tract ◽

Female Reproductive Tract ◽

Heparin Group ◽

Fluorescent Stain ◽

In Vitro Induction

The capacitation process occurs in vivo upon exposure of the spermatozoa through the female reproductive tract, but can be induced in vitro in the presence of several compounds. This study was conducted to assess the effect of heparin or equine follicular fluid on hyperactivated motility and in vitro induction acrosome reaction swim-up method with frozen-thawed stallion semen. Two hundred microliters of frozen-thawed equine semen was placed in a tube (45°C) to increase contact area and incubated at 37°C for 1 h. After incubation 800 μL of the supernatant was collected by centrifugation (500 × g, 10 min) to collect spermatozoa. The resulting pellet was resuspended in capacitation medium Fert-TALP supplemented with 5.0 μg mL-1 heparin or 100% follicular fluid and incubated for different times (1, 2, 3, 4, and 5 h) at 37°C. After incubation the hyperactivated motility and acrosome-reacted spermatozoa were evaluated. Hoechst stain was used to differentiate live and dead spermatozoa, and chlortetracycline (CTC) fluorescent stain was used to assess the capacitation response of sperm; data were analyzed by ANOVA. The effect of equine follicular fluid resulted in improved percentage of spermatozoa with acrosome reaction at all times of incubation (60, 63, 57, 52, and 58%) but immediately after 3 h of incubation, the hyperactivated motility decreased in heparin group and follicular fluid (42 and 30%, respectively).

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Induction of acetylcholine receptor-specific suppression. An in vitro model of antigen-specific immunosuppression in myasthenia gravis.

Journal of Experimental Medicine ◽

10.1084/jem.159.1.292 ◽

1984 ◽

Vol 159 (1) ◽

pp. 292-304 ◽

Cited By ~ 11

Author(s):

S Bogen ◽

E Mozes ◽

S Fuchs

Keyword(s):

T Cell ◽

Myasthenia Gravis ◽

Clinical Importance ◽

Lymphocyte Transformation ◽

Specific Suppression ◽

Target Autoantigen ◽

High Concentrations ◽

In Vitro Induction

This report describes the in vivo and in vitro induction of murine (AChR)-specific suppressor T cells (Ts) and T cell factors (TsF), and the development of an appropriate assay system for their measurement. The assay described is based on the in vitro Mishell-Dutton culture system. Using this assay, it was shown that the AChR-specific helper cell is an Lyt-2- radiosensitive T cell. Moreover, the proliferating cell measured in the lymphocyte transformation assay was shown to provide AChR-specific T cell help. In vivo induction of Ts cells is achieved by injection of soluble AChR; potent AChR-specific suppression is found in the spleen 1 wk later. In vitro induction of Ts cells involves the primary education of naive splenocytes by culturing them with high concentrations of AChR. Both the in vivo- and in vitro-induced Ts cells were shown to secrete AChR-specific factors that mediate their suppressive effects. The possibility of specifically suppressing the AChR-immune response may be of a particular clinical importance since the AChR is the target autoantigen in the neuromuscular autoimmune disease myasthenia gravis.

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