Addition Of Sera Research Articles

Effect of oocyte collection techniques and maturation media on in vitro maturation of buffalo oocytes

The study was conducted to know the effect of oocyte collection techniques and various maturation media on in vitro maturation of buffalo oocytes obtained from slaughterhouse derived ovaries. Highest maturation rate was observed in TCM 199 + FBS + hCG media, followed by Ham’s F-10 + FBS + hCG, TCM 199 and Ham’s F-10 alone. The difference between them was statistically nonsignificant. Maturation rate was significantly higher in oocytes collected by aspiration than by slicing technique. Nonsignificant difference was observed in maturation rates in BCB+ and BCB– oocytes. In conclusion, addition of sera and hormones into basic maturation media, viz. TCM-199 and Ham’s F-10 resulted into better in vitro maturation rate.

Targeted in Vivo O-GlcNAc Sensors Reveal Discrete Compartment-specific Dynamics during Signal Transduction

β-O-N-acetyl-D-glucosamine (O-GlcNAc) is a post-translational modification involved in a plethora of biological systems ranging from cellular stress to insulin signaling. This modification shares many hallmarks with phosphorylation, including its dynamic cycling onto a host of proteins such as transcription factors, kinases, and phosphatases, and regulation of cellular functions, including cell signaling. Herein, we report the development of an improved genetically based O-GlcNAc FRET sensor and compartmentalized targeted variants for the characterization of the spatiotemporal dynamics of O-GlcNAc. During serum-stimulated signal transduction, rapid increases in O-GlcNAc activity were observed at both the plasma membrane and the nucleus, with a concomitant decrease detected in the cytoplasm. These findings suggest the existence of compartment specific dynamics for O-GlcNAc in response to signal-inducing stimuli, pointing to complex regulation of this modification. In addition, inhibition of the PI3K pathway by wortmannin abolished the O-GlcNAc response, suggesting that the activity observed is modulated downstream of the PI3K pathway. Taken together, our data argues that O-GlcNAc is a rapidly induced component of signaling and that the interplay between O-GlcNAc and kinase signaling may be more akin to the complex relationship between kinase pathways.

Effect of rabbit anti-asialo-GM1 (GA1) polyclonal antibodies on neuromuscular transmission and acetylcholine-induced action potentials: neurophysiological and immunohistochemical studies.

We produced anti-asialo-GM1 (GA1) polyclonal antibodies by sensitizing New Zealand rabbits with GA1 and investigated the epitopes and pathogenic role of anti-GA1 antibodies that appeared in serum. The serum blocked neuromuscular transmission, but not acetylcholine (ACh)-induced potentials, in muscle-spinal cord cocultured cells. The effect was complement independent. The antibodies inhibited voltage-gated Ca2+ channel (VGCC). The epitopes recognized by the antibodies were located in the outer membrane of Schwann cells and motor axons of Wistar rat ventral roots and on motor axons extended from spinal cord to muscle cells in muscle-spinal cocultured cells. The ACh-induced potential was not reduced by the addition of sera, suggesting the blockade is presynaptic. Thus, anti-GA1 antibodies may block neuromuscular transmission by suppressing VGCC on axonal terminals of motor nerves.

In vitro studies on viability and proliferation of Enteromyxum scophthalmi (Myxozoa), an enteric parasite of cultured turbot Scophthalmus maximus.

In vitro cultivation of the myxozoan Enteromyxum scophthalmi was attempted using different culture media and conditions. The progress of the cultures was monitored using dye-exclusion viability counts, tetrazolium-based cell-proliferation assays, measuring the incorporation of BrdU during DNA synthesis, and by morphological studies using light and electron microscopes. In preliminary experiments, the persistence of viable stages for a few days was ascertained in both medium 199 (M199) and in seawater. An apparent initial proliferation was noticed in the culture media, with many young stages observed by Day 7 post-inoculation (p.i.). In contrast, fast degeneration occurred in seawater, with but a few living stages persisting to Day 1 p.i and none to Day 5 p.i. Both tetrazolium-based cell-proliferation assays and dye-exclusion viability counts demonstrated a progressive degeneration of the cultures. Although M199 medium and neutral pH with the addition of sera appeared to provide the most favourable conditions during the first few hours, all cultures degenerated with time and no parasite proliferation or maintenance could be achieved in the long term in any of the conditions assayed, including attempts of co-cultivation with a turbot cell line. The ultrastructure of stages cultured for 15 d demonstrated complete degeneration of organelles and mitochondria, although the plasma membrane remained intact in many stages. Unknown factors related to the metabolism or the life cycle of this myxozoan are probably responsible for the inability to culture the parasite, which seems to be strictly dependent on the target host tissues for survival.

Autoantibodies against CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) that impair glucose-induced insulin secretion in noninsulin- dependent diabetes patients.

Cyclic ADP-ribose (cADPR) has been shown to be a mediator for intracellular Ca2+ mobilization for insulin secretion by glucose in pancreatic beta cells, and CD38 shows both ADP-ribosyl cyclase to synthesize cADPR from NAD+ and cADPR hydrolase to hydrolyze cADPR to ADP-ribose. We show here that 13.8% of Japanese non-insulin-dependent diabetes (NIDDM) patients examined have autoantibodies against CD38 and that the sera containing anti-CD38 autoantibodies inhibit the ADP-ribosyl cyclase activity of CD38 (P </= 0.05). Insulin secretion from pancreatic islets by glucose is significantly inhibited by the addition of the NIDDM sera with anti-CD38 antibodies (P </= 0.04-0.0001), and the inhibition of insulin secretion is abolished by the addition of recombinant CD38 (P </= 0.02). The increase of cADPR levels in pancreatic islets by glucose was also inhibited by the addition of the sera (P </= 0.05). These results strongly suggest that the presence of anti-CD38 autoantibodies in NIDDM patients can be one of the major causes of impaired glucose-induced insulin secretion in NIDDM.

Effect of serum components on syncytium formation and virus production by cells infected with human immunodeficiency virus in vitro.

Previously it has been reported that cocultivation of human immunodeficiency virus type 1 (HIV-1)-infected cells with uninfected cells results in formation of multinuclear giant cells, generated via an interaction of gp120 on the surface of infected cells with CD4 on the uninfected cells. Formation of multinuclear giant cells as occurring in the presence of normal fetal calf serum was not observed when HIV-infected MOLT-4 or MOLT-3 cells (chronically infected with HTLV-IIIB) and uninfected cells were cocultured in both serum-free medium and fibrinogen-depleted serum. Addition of sera (human and rabbit) as well as of fibrinogen (human and bovine), fibronectin (human), and alpha-globulin (human), but not of albumin, transferrin or gamma-globulin to serum-free medium caused formation of multinuclear giant cells. In contrast, HIV production from MOLT-3 cells proceeds also in the absence of serum. In control experiments it was established that the cells maintained at reduced serum concentration, or in serum-free medium without or with fibrinogen are viable even though displaying a lower metabolic rate (ATP formation and DNA synthesis). From these findings we conclude that serum components (e.g., fibrinogen, fibronectin, and alpha-globulin) are absolutely required for syncytium formation but are not essential for virus release.

A Reliable Two-Hour Staphylococcus Plate Assay of Rifampicin, Erythromycin, Lincomycin, Chloramphenicol and Amikacin in Human Sera

A simple and precise agar diffusion microassay was developed for rifampicin (RIF) in serum. The method involved the addition of sera containing RIF (0.125-4.0 micrograms/ml) to wells cut in an agar medium (pH 6.6) surface seeded with an adjusted inoculum of Staphylococcus aureus ATCC 6538 P and incubated for 2h at 40 degrees C. Repetitive assays of sera containing known concentrations of RIF revealed an average mean recovery of 93.6% with a coefficient of variation of 5.7%. This rapid method allowed accurate determination of RIF in the presence of erythromycin, chloramphenicol, lincomycin and amikacin. These four antibiotics were also separately measured in sera by the rapid assay at different pH values.

Nerve Growth Factor‐like Activity Detected in Equine Peripheral Blood after Running Exercise

Addition of sera, collected from Thoroughbred horses after sprint exercise, induced significant neurite outgrowth from chick embryo dorsal root ganglia after a 24-hour culture. The nerve growth factor (NGF)-like activity was detected in sera collected immediately, or 1 hour or more, after the exercise. These findings suggest a possible role of serum NGF-like activity under stress conditions (running exercise) of horses.

PHA-induced cellular cytotoxicity. Inhibition by a nonimmunoglobulin factor present in sera from patients with active systemic lupus erythematosus.

Phytohemagglutinin-induced cellular cytotoxicity by normal mononuclear cells (MNC) is inhibited by serum of patients with active untreated systemic lupus erythematosus (SLE) but not by that of patients with untreated inactive SLE or rheumatoid arthritis. Three-hour preincubation of normal MNC with SLE sera suffices to inhibit throughout the assay. Preincubation with phytohemagglutinin (PHA) before addition of sera does not prevent the inhibition. DEAE-cellulose chromatography, Sephadex G-200 fractionation and immunoelectrophoretic analysis have shown the nonimmunoglobulin nature of this factor. It is thermostable and not affected by deoxyribonuclease or ribonuclease treatment. The mitogen-induced cellular cytotoxicity-inhibiting factor seems to be independent of other serum factors capable of blocking PHA-induced blastogenic transformation.