Cell-Dependent Chemiluminescence. Modulation of the N-Formyl Chemotactic Peptide (Fnlpntl) Mediated Oxidative Burst in Human Polymorphonuclear Leukocytes (Pmnl) By Murine Monoclonal Antibody Nms-1

Binding of purified monoclonal antibody (moAB) IgM NMS-1 to suspended initially spherical living human PMNLs is not associated with the generation of chemiluminescence but was found to enhance the chemiluminescence response to the N-formyl chemotactic peptide FNLPNTL.

We investigated quantitatively the kinetics of oxygen metabolite generation by PMNLs stimulated with FNLPNTL ± moAB NMS-1 using luminol-dependent chemiluminescence as a very sensitive detection system. Chemiluminescence detection allowed the analysis of the time sequence of onset and development of reactive oxygen metabolites following stimulation of PMNLs by FNLPNTL in the presence of moAB NMS-1. The increase of response of PMNLs stimulated with FNLPNTL in the presence of moAB NMS-1 depended on the concentration of the antibody and the sequence of stimulus addition.

Stimulation of human PMNLs by 10nM FNLPNTL induced a rapid burst of chemiluminescence which peaked ∼5min after stimulus addition. The subsequent addition of moAB NMS-1 (≥2μg/ml DPBS(+)—0.1% HSA, 37°C) to FNLPNTL-stimulated PMNLs—after the FNLPNTL-mediated response had already decayed (16-18 min) - without delay induced a second burst of oxygen metabolite generation. The magnitude of this second peak of activation was dose-dependent.

Treatment of PMNLs with moAB NMS-1 (≥ 1μg/ml DPBS(+)—0.1% HSA, 3 min, 37°C)—prior to FNLPNTL (10nM) stimulation - increased rate and magnitude of the FNLPNTL-mediated response. This response is biphasic with the first peak at the FNLPNTL position and a second, higher peak ∼16 min after FNLPNTL addition. The magnitude of response was dose-dependent. The latency (lag time) of the respone was not changed compared to controls which received no moAB NMS-1 treatment.

The observed moAB NMS-1 dependent increase in FNLPNTL-mediated chemiluminescence is transient (5-60 min), persistent activation was not detected.     

Monoclonal antibody NMS-1 increases N-formyl chemotactic peptide-mediated oxidative burst generation in human neutrophils

Murine monoclonal antibody (mAb) NMS-1 was generated which binds to the surface of living human neutrophils. The antigens on neutrophil plasma membranes recognized by mAb NMS-1 were solubilized in Nonidet P-40 and immunopurified on matrix-bound mAb NMS-1. mAb NMS-1 binds to four periodate-sensitive structures of 70,000, 95,000, 140,000, and 170,000 Da on the plasma membrane surface of human neutrophils as was shown by Western blot analysis. Binding of mAb NMS-1 to human neutrophils induced a rapid transient rise in cytosolic free calcium (Quin 2 fluorescence) but no detectable generation of reactive oxygen metabolites. The oxidative burst of N-formyl peptide-treated neutrophils, however, increased in the presence of mAb NMS-1. The kinetics of N-formyl peptide (N-formyl-norleucyl-leucyl-phenylalanyl-norleucyl-tryrosyl-lysine; FNLPNTL)-mediated hydrogen peroxide formation (p-hydroxy phenyl acetate oxidation) in the presence of mAb NMS-1 was analyzed quantitatively. 1) When neutrophils were incubated with mAb NMS-1 before FNLPNTL addition, an increase in rate, magnitude, and duration of hydrogen peroxide formation was observed compared with controls which received no mAb NMS-1 treatment. After termination of the initial linear phase of response, a second transient linear phase of hydrogen peroxide formation was induced. This second phase of activation was not observed in neutrophils which received no mAb NMS-1 treatment. The onset of the response and latency before attainment of the initial linear rate of hydrogen peroxide formation was not changed by mAb NMS-1 pretreatment. 2) When neutrophils were stimulated with FNLPNTL, the addition of mAb NMS-1--after termination of the FNLPNTL-induced response--without delay induced a second transient burst of hydrogen peroxide formation. Persistent activation of hydrogen peroxide formation by mAb NMS-1 in FNLPNTL-stimulated neutrophils was not observed.     

Effect of a new fluorinated quinolone (ofloxacin) on the chemiluminescence of phagocytosing human neutrophils

We measured the chemiluminescence (CL) of human neutrophils (PMNLs) exposed to different concentrations of ofloxacin (2, 4, and 6 μg/ml) readily achievable in therapy. CL reaction during zymosan phagocytosis by PMNLs obtained from human healthy volunteers was registered in a computer-linked LKB 1251 luminometer. Ofloxacin did not induce significant variations on the respiratory burst of PMNLs.     

Automation and computerization of chemiluminescence: A new methodological approach in the study of human phagocytes

Peripheral blood phagocytic cells (PMNLs) are activated by contact with opsonized particles. Metabolic activation of PMNLs is associated with a remarkable increase in the respiratory burst and generates high energy oxygen compounds which are responsible for the bactericidal activity of PMNLs and for their ability to produce luminol-dependent chemiluminescence (CL). The CL phenomenon is measured by an automated and computerized photoluminometer (Berthold LB950) in whole blood stimulated with opsonized zymosan. This whole blood method of CL measurement has been applied to the study of the phagocytic process and to the investigation of cellular and humoral abnormalities in several pathologies, indicating this assay as a simple, rapid and reliable test.     

Effect of Staphylococcus aureus serine proteinase on the respiratory burst in phagocytic cells in vitro

The in vitro effects of the Staphylococcus aureus serine proteinase (SASP) on the respiratory burst of human blood mononuclear phagocytes and rat lung macrophages were investigated. The generation of reactive oxygen species (ROS), determined by means of luminol-based chemiluminescence, was stimulated by treatment with SASP in both types of the defense cells. Cell activation depended on the concentration of the enzyme and the response of monocytes was an order of magnitude stronger relative to macrophages. The chemiluminescence emission kinetics were different for both cell types and the maximum signal was achieved in approximately 3 and 17 min, respectively. In experiments involving further cell activation by latex particles, macrophages pretreated with various SASP concentrations reacted with enhanced ROS generation whereas for monocytes, the latex-induced chemiluminescence was weakened by the enzyme. The results concerning the modification of the phagocytic host cell activity by SASP suggest that this enzyme might play an important role in pathomechanisms of staphylococcal infections in vivo.     

Studies on the susceptibility of different culture morphotypes of Listeria monocytogenes to uptake and survival in human polymorphonuclear leukocytes

This study demonstrated that atypical virulent filaments of Listeria monocytogenes (rough variant type II and designated FR for this study), isolated from clinical specimens or generated during exposure to pulsed-plasma gas discharge in liquids, were shown to be capable of survival when engulfed by human polymorphonuclear leukocytes (PMNLs). Factors shown to significantly influence the maximal respiratory burst response in PMNLs and survival of different internalized cell or filament forms of L. monocytogenes were bacterial strain, culture form, degree of opsonization (with and without the use of 10% serum) and composition of the bacterial growth media used before uptake by PMNLs. Opsonized regular-sized L. monocytogenes cells grown on blood agar (BA) elicited the greatest respiratory burst response and survived best in PMNLs. The filamentous (FR) and multiple cell chain (MCR) rough variants were significantly less susceptible to uptake and survival in PMNLs. Supplementation of tryptone soya agar with hemin resulted in significantly reduced chemiluminescence responses in phagocytosing PMNLs compared with the maximal levels observed from prior bacterial growth on BA or brain heart infusion agar that also contained a source of iron. The MCR variants secreting decreased levels of a peptidoglycan hydrolase CwhA protein exhibited the lowest percentage survival when internalized in PMNLs compared with wild-type smooth or FR culture variants as determined by the macrophage-killing assay.     

A stress-induced oxidative burst in Eucheuma platycladum (Rhodophyta)

A hurst of hydrogen peroxide has been found in the red macroalga Eucheuma platycladwn Schmitz as a response to mechanical stress. After exposure of pieces of thalli (2 cm) broken from the plant and stirred with a magnetic bar an oxidative burst was registered, as measured by luminol dependent chemiluminescence (LDC). The burst was totally inhibited by cataluse (EC 1.11.1.6). showing the generation of H_2:O_2; Ten g of seaweed in 300 ml sea water caused a maximal medium concentration of LDC corresponding to 7 u .M H₂O_2; The burst decayed after about 30 min. The decay is probably caused by increased catalase aciivity of the sea water. due to leakage of catalasc from the seaweed. Addition of NaN₃ caused a dramatic increase in LDC. probably due to inhibition of catalase. Similar bursts of active oxygen, involving active oxygen species such as O₂, H₂O₂ and OH. have been reported as pan of the hypcrsensitive reaction in some higher plants, e.g. tobacco. potato and soybean. Exposure of plants or cell suspension cultures to some pathogenic bacteria, fungi, inorganic elicitors or physical damage causes an oxidalive burst that is often followed by necrosis. The production ot active oxygen is thought to he a first defence against invading pathogens. We assume that the oxidative burst from E. platycladum is of a defensive nature, providing a protection against grazers and pathogenic organisms. To our knowledge this is the first repoil of an oxidalive burst from seaweeds.     

b-adrenergic modulation of FMLP- and zymosan-induced intracellular and extracellular oxidant production by polymorphonuclear leukocytes

Evaluation of catecholamine modulation of PMNL extracellular and intracellular oxidant production may reflect beneficial and harmful effects of b-adrenergic agonists in various disease states. We investigated the kinetics and potency of adrenaline-mediated inhibition of oxidant generation in FMLP- and zymosan-stimulated PMNLs. In FMLP-stimulated cells, the short-term burst of oxidant generation was inhibited by adrenaline in a dose-dependent fashion. Intra- and extracellular chemiluminescence and extracellular superoxide anion and hydrogen peroxide generation showed similar IC50 values for adrenaline (1.3-3.0 ï 10-8 M) indicating that both extracellular and intracellular events were inhibited with the same potency. In contrast, intracellular oxidant production evoked by the phagocytosis of zymosan was only minimally affected by 3 ï 10-5 - 3 ï 10-12 M adrenaline. Extracellular inhibition of oxidant production was also apparent in zymosan-stimulated cells. In conclusion, adrenaline's ability to depress extracellular generation of oxygen metabolites while retaining prolonged intracellular oxidant production for phagocytosis supports its beneficial role as selectively targeted physiological protector.     

Analysis of luminol-dependent chemiluminescence from granule depleted neutrophil cytoplasts reveals two different light-emitting mechanisms

When neutrophil cytoplasts (granule-free vesicles of cytoplasm enclosed by plasmalemma) were exposed to the chemotactic peptide formylmethionyl-leucyl-phenylalanine, no luminol-dependent chemiluminescence was detected, despite a pronounced production of superoxide anions and hydrogen peroxide. Addition of purified myeloperoxidase (MPO) or human serum albumin (HSA) to the cytoplasts before the stimulus resulted in a chemiluminescence response. In contrast to the bimodal response obtained from normal PMNL, only a single peak of chemiluminescence was obtained from the cytoplasts responding to the peptide. The time-course of the response obtained in the presence of albumin was more prolonged than the response obtained in the presence of MPO. Furthermore, the involvement of different oxidative metabolites in the MPO and the HSA systems, respectively, was demonstrated by the accumulated chemiluminescence effect obtained when MPO, but not HSA, was introduced in the measuring system after FMLP addition. From these results it can be concluded that there are at least two different light-generating mechanisms in FMLP-induced luminol-dependent chemiluminescence of neutrophil cytoplasts. One of these is dependent on myeloperoxidase and possibly related to the myeloperoxidase-hydrogen peroxide reaction, whereas the other one is hydrogen peroxide independent.     

STUDIES ON NITRIC OXIDE FREE RADICALS GENERATED FROM POLYMORPHONUCLEAR LEUKOCYTES (PMN) STIMULATED BY PHORBOL MYRISTATE ACETATE (PMA)

The interaction of NO and O^?₂free radicals generated from PMA (phorbol myristate acetate)-stimulated PMN (polymorphonuclear leukocytes) was studied by a nitroxide spin trap, DMPO (5,5-dimethyl-1-pyrroline-1-oxide). It was found that addition of L-arginine to the system would significantly decrease the trapped O^?₂by DMPO and addition of N^G-monomethyl-arginine (NGMA) would significantly increase the trapped O^?₂by DMPO. It was proved that the formation of ONOO^?by the reaction of NO and O^?₂was the main reason for the decrease of trapped O^?₂in the experiment with xanthine/xanthine oxidase and irradiation of riboflavin systems. The yield of NO during this process was calculated. The generation dynamic of NO was studied by a luminol-dependent chemiluminescence technique and it was found that after stimulation of PMN by PMA, there would be an immediate, significant chemi-luminescence, which came mainly from the active oxygen free radicals generated by PMN. If L-arginine was added to this system, the chemiluminescence would increase about 100-fold, but NGMA inhibited the increase of the chemiluminescence. Ten minutes after addition of L-arginine, this increase did not change, the chemiluminescence peak decreased gradually, but the half life increased. The ESR and chemiluminescence properties of NO and ONOO^?synthesized were also studied in model systems.     

Flow-cytometric characterization of stimulation, free radical formation, peroxidase activity and phagocytosis of human granulocytes with 2,7-dichlorofluorescein (DCF)

A standardized four-step assay for the flow cytometric determination of the oxidative activity of human polymorphonuclear leukocytes (PMNL) from normal human individuals and from septic patients was developed, using 2,7-dichlorofluorescin-diacetate (DCFH-DA) as indicator for the intracellular formation of H2O2 and free radicals. Spontaneous H2O2 and free radical formation was measured by preincubation of buffy coat PMNLs from fresh peripheral venous blood at 37 degrees C and pH 7.4 with 10 microM DCFH-DA. Intracellular peroxidase activity was determined by addition of 1 mM external H2O2 to this assay. A maximum of granulocyte oxidative burst activity was elicited by the addition of 150 nM phorbol-myristate-acetate (PMA). A physiological burst was generated by incubating buffy coat PMNLs together with E. coli bacteria. The DNA of dead cells was in all instances simultaneously counterstained with propidium iodide (PI). Quiescent or H2O2 or bacteria treated granulocytes moved as a single cell cluster to higher fluorescences. Stimulation with PMA, in contrast, generated always a bimodal distribution of granulocyte fluorescence with the high activity cell cluster being approximately sevenfold more active than the low activity cell cluster. Roughly half of the granulocytes in normal individuals had high fluorescence. An increase of the high activity granulocytes was observed in septic patients. Model experiments with the nonfluorescent DCFH-DA cleavage product DCFH (2,7-dichlorofluorescin) showed that DCFH was quickly photo-oxidized to fluorescent DCF (2,7-dichlorofluorescein) by UV-light and to a lower degree by daylight. DCFH even slowly autooxidized in the dark.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacology of MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)- indol-2-yl]-2,2-dimethyl propanoic acid), a potent, orally active leukotriene biosynthesis inhibitor.

MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)- indol-2-yl]-2,2-dimethyl propanoic acid, previously L-686,708) is a potent inhibitor of leukotriene (LT) biosynthesis in intact human and elicited rat polymorphonuclear leukocytes (PMNLs) (IC50 values 3.1 and 6.1 nM, respectively) and in human, squirrel monkey, and rat whole blood (IC50 values 510, 69, and 9 nM, respectively). MK-0591 had no effect on rat 5-lipoxygenase. MK-0591 has a high affinity for 5-lipoxygenase activating protein (FLAP) as evidenced by an IC50 value of 1.6 nM in a FLAP binding assay and inhibition of the photoaffinity labelling of FLAP by two different photoaffinity ligands. Inhibition of activation of 5-lipoxygenase was shown through inhibition of the translocation of the enzyme from the cytosol to the membrane in human PMNLs. MK-0591 was a potent inhibitor of LT biosynthesis in vivo, first, following ex vivo challenge of blood obtained from treated rats and squirrel monkeys, second, in a rat pleurisy model, and, third, as monitored by inhibition of the urinary excretion of LTE4 in antigen-challenged allergic sheep. Inhibition of antigen-induced bronchoconstriction by MK-0591 was observed in inbred rats pretreated with methysergide, Ascaris-challenged squirrel monkeys, and Ascaris-challenged sheep (early and late phase response). These results indicate that MK-0591 is a potent inhibitor of LT biosynthesis both in vitro and in vivo indicating that the compound will be suitable for assessing the role of leukotrienes in pathological situations.     

Upregulation of Respiratory Burst of Polymorphonuclear Leukocytes By A Bleomycin Derivative, Peplomycin

The influence of peplomycin (PLM) on the respiratory burst of peripheral blood polymorphonuclear leukocytes (PMN) was investigated. Short-term (5 min) treatment of human PMN with 0.1μg/ml to 100μg/ml of PLM increased phorbol myristate acetate (PMA)-and formyl-methionyl-leucyl-phenylalanine (FMLP)-induced luminol-dependent chemiluminescence. PMN, as well as alveolar macrophages from rabbits treated with 0.5 to 1.0 mg/kg of peplomycin per day for 5 days, generated more superoxide (O₂^-) than the cells from untreated rabbits. In both PLM-treated and untreated PMN, chemiluminescence induced by FMLP and PMA was decreased to less than 50% of the control by staurosporine, superoxide dismutase (SOD) and catalase. However, the peak intensity in PLM-untrcated PMN was decreased to about 30% of the control by genislein, while this agent induced a slight decrease in peak intensity in the PLM-treated PMN. Inositol triphosphate and diacyl glycerol levels were not clearly increased by PLM, but an increase of intracellular Ca and a shift of protein kinase C (PKC) to the membrane occurred in PMN within 1 min after PLM treatment. Western blotting revealed that the tyrosine phosphorylation of a 115 kDa protein was upregulated by 5 to 50μg/ml of PLM. While, PLM suppressed SOD activity in alveolar macrophages and PMN. These results seem to indicate that PLM increases the respiratory burst of PMN and macrophages both by way of direct PKC activation and by the upregulation of protein tyrosine phosphorylation. This increased reactive oxygen generation, together with the suppression of SOD activity seems to be tissue-impairing.     

Comparative study of β-glucan induced respiratory burst measured by nitroblue tetrazolium assay and real-time luminol-enhanced chemiluminescence assay in common carp (Cyprinus carpio L.)

The respiratory burst is an important feature of the immune system. The increase in cellular oxygen uptake that marks the initiation of the respiratory burst is followed by the production of reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide which plays a role in the clearance of pathogens and tissue regeneration processes. Therefore, the respiratory burst and associated ROS constitute important indicators of fish health status. This paper compares two methods for quantitation of ROS produced during the respiratory burst in common carp: the widely used, single-point measurement based on the intracellular reduction of nitroblue tetrazolium (NBT) and a real-time luminol-enhanced assay based on the detection of native chemiluminescence. Both assays allowed for detection of dose-dependent changes in magnitude of the respiratory burst response induced by β-glucans in head kidney cells of carp. However, whereas the NBT assay was shown to detect the production of only superoxide anions, the real-time luminol-enhanced assay could detect the production of both superoxide anions and hydrogen peroxide. Only the chemiluminescence assay could reliably record the production of ROS on a real-time scale at frequent and continual time intervals for time course experiments, providing more detailed information on the respiratory burst response. The real-time chemiluminescence assay was used to measure respiratory burst activity in macrophage and neutrophilic granulocyte-enriched head kidney cell fractions and total head kidney cell suspensions and proved to be a fast, reliable, automated multiwell microplate assay to quantitate fish health status modulated by β-glucans.     

Rapid binding of guanosine 5'-O-(3-thiotriphosphate) to an apparent complex of beta-adrenergic receptor and the GTP-binding regulatory protein Gs

When reconstituted phospholipid vesicles that contain purified beta-adrenergic receptors and the GTP-binding regulatory protein Gs were preincubated with agonist before the addition of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), the typical receptor-stimulated GTP gamma S binding reaction was preceded by an even more rapid burst of GTP gamma S binding. This burst was studied in detail at 0 degree C. The rate of the burst was second order in nucleotide and Gs [k assoc approximately 2 X 10(7) (M.min)-1], consistent with diffusion-controlled binding. The magnitude of the burst was always less than the number of receptors present and was roughly linear with receptor number when similarly prepared vesicles were compared. There was no obvious quantitative correlation between the burst and the amount of Gs. The species that gave rise to the burst formed with t1/2 approximately 15 min at 0 degree C in the presence of agonist and decayed by approximately 3 min upon addition of antagonist or detergent. Formation and decay of this species was much faster at at 30 degrees C. The data suggest that a complex of agonist, receptor, and Gs that is primed for the rapid binding of guanine nucleotide can form and be analyzed in reconstituted vesicles.     

Chemiluminescence response of phagocytes in E. coli induced experimental ascending pyelonephritis.

The mechanism of tissue injury at the cellular level by following the chemiluminescence response of various phagocytes in E. coli induced experimental pyelonephritis in mice was investigated. There was a marked increase in the capacity of various phagocytic cells viz; renal neutrophils and macrophages peritoneal macrophages, blood monocytes and neutrophils to produce reactive oxygens species through the respiratory burst activity as monitored by the chemiluminescence response. The chemiluminescence response was observed to be increased significantly (p less than 0.001) with increasing days post infection in all phagocytic cells. However, the quantity of total reactive oxygen species produced per million cells was much more in the renal and peritoneal macrophages as compared to blood monocytes and neutrophils. The peak chemiluminescence response time was observed to be decreased from 4 to 2 minutes with the progression of the diseases. The implications of these findings have been discussed.     

Three-peaked chemilluminescent response of human peripheral blood leukocytes following stimulation with phytohaemagglutinin (PHA)

Luminol-enhanced chemiluminescence (CL) was used to examine the response of various leukocyte populations following stimulation with a crude extract of Phaseolus vulgaris, namely phytohaemagglutinin (PHA-C). Populations stimulated included a human peripheral mixed leukocyte preparation (MLP), and purified preparations of lymphocytes, monocytes and polymorphonuclear leukocytes (PMNL). Mouse peritoneal exudate cells and the lymphocytic cells lines Molt #4 and Daudi were also stimulated. Following stimulation, a characteristic three-peaked chemiluminescent response was obtained from the MLP population. Little or no response was obtained from the purified lymphocytes. Monocytes produced a sharp peak corresponding to the second peak of the MLP response and PMNL produced a broad peak corresponding to the third peak of the MLP response. Mouse peritoneal exudate cells containing lymphocytes and monocytes/macrophages showed a two-peaked stimulation which corresponded to the first two peaks of the MLP response. Molt #4 and Daudi showed no chemiluminescence if stimulated individually, but if added to a MLP substantial enhancement of the first and second peaks was observed. These results indicate some form of lymphocyte/monocyte interaction leading to enhanced CL following PHA-C stimulation.     

Vitrification of buffalo (Bubalus bubalis) oocytes

The objective of the present study was to develop a method for the cryopreservation of buffalo oocytes by vitrification. Cumulus-oocyte complexes (COCs) were obtained from slaughterhouse ovaries. Prior to vitrification of COCs in the vitrification solution (VS) consisting of 4.5 M ethylene glycol, 3.4 M dimethyl sulfoxide, 5.56 mM glucose, 0.33 mM sodium pyruvate and 0.4% w/v bovine serum albumin in Dulbecco's phosphate buffered saline (DPBS), the COCs were exposed to the equilibration solution (50% VS v/v in DPBS) for 1 or 3 min at room temperature (25 to 30 degrees C). The COCs were then placed in 15-microL of VS and immediately loaded into 0.25-mL French straws, each containing 150 microL of 0.5 M sucrose in DPBS. The straws were placed in liquid nitrogen (LN2) vapor for 2 min, plunged and stored in LN2 for at least 7 d. The straws were thawed in warm water at 28 degrees C for 20 sec. For dilution, the COCs were equilibrated in 0.5 M sucrose in DPBS for 5 min and then washed 4 to 5 times in the washing medium (TCM-199+10% estrus buffalo serum). The proportion of oocytes recovered in a morphologically normal form was significantly higher (98 and 88%, respectively; P<0.05), and the proportion of oocytes recovered in a damaged form was significantly lower (2 and 12%, respectively; P<0.05) for the 3-min equilibration than for 1 min. For examining the in vitro developmental potential of vitrified-warmed oocytes, the oocytes were placed in 50-microL droplets (10 to 15 oocytes per droplet) of maturation medium (TCM-199+15% FBS+5 microg/mL FSH-P), covered with paraffin oil in a 35-mm Petri dish and cultured for 26 h in a CO2 incubator (5% CO2 in air) at 38.5 degrees C. Although the nuclear maturation rate did not differ between the 1- and 3-min equilibration periods (21.5+/-10.7 and 31.5+/-1.5%, respectively), the between-trial variation was very high for the 1-min period. This method of vitrification is simple and rapid, and can be useful for cryopreservation of buffalo oocytes.     

Immunomodulating action of low intensity millimeter waves on primed neutrophils

Comparative investigation of the susceptibility of intact and primed neutrophils of the NMRI strain mice to low intensity millimeter wave (mm wave) irradiation (41.95 GHz) was performed. The specific absorption rate was 0.45 W/kg. Isolated neutrophils were primed by a chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) at a subthreshold concentration of 10 nM for 20 min, and then the cells were activated by 1 microM fMLP. Production of the reactive oxygen species (ROS) was estimated by the luminol dependent chemiluminescence technique. It was found that the preliminary mm wave irradiation of the resting cells at 20 degrees C did not act on the ROS production induced by the chemotactic peptide. The exposure of the primed cells results in a subsequent increase in the fMLP response. Therefore, the primed neutrophils are susceptible to the mm waves. Specific inhibitors of the protein kinases abolished the mm wave effect on the primed cells. The data indicate that protein kinases actively participate in transduction of the mm wave signal to effector molecules involved in neutrophil respiratory burst.     

Investigation of burst generation by the electrically coupled cyberchron network in the snail Helisoma using a single-electrode voltage clamp

This paper describes the results of investigating burst generation by the cyberchron network in the snail Helisoma. The cyberchron network is composed of approximately 20 electrically coupled neurons and controls the feeding behavior of the snail. The electrical coupling between network members has made it particularly difficult to distinguish between the importance and involvement of single-cell and network properties in burst generation by this system. The present investigations utilized the new single-electrode voltage clamp to examine the membrane properties and network interactions of the cyberchron neurons: (1) A slow outward current is activated by moderately large depolarizing commands (?40 to 0 mV) and does not undergo inactivation decay (i.e., decline in magnitude) during a command potential step maintained for 10 sec or more. The lack of inactivation of the outward current in cyberchron neurons appears to be due to the dominating role of a Ca-dependent K current. (2) There are two functionally distinct classes of cyberchrons—current generator cyberchrons and follower cyberchrons. (3) Primary current generator cyberchrons have membrane properties similar to endogenous bursting neurons (e.g., persistent inward Ca current and negative resistance region in I–V plot) and appear to provide the main driving and timing current for the rest of the network. (4) The vast majority of cyberchrons are secondary current generator cyberchrons with membrane properties which exhibit inward-going rectification and appear to burst as a result of regenerative excitation with one another and the primary current generator cyberchrons. (5) The second class of cyberchrons are driven by the electrical synaptic input from the current generator cyberchrons, do not exhibit inward-going rectification, and are called follower cyberchrons. (6) Burst termination is due to activation of a slow outward tail current in most cyberchrons during the burst (probably Ca-activated K current) which causes a hyperpolarization in individual cyberchrons, terminating the burst. (7) Decay of the outward tail current causes the cyberchrons to depolarize, which activates the persistent inward Ca current in the primary current generator cyberchrons, starting the burst cycle anew.