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Natural killing and antibody-dependent cellular cytotoxicity in specific-pathogen-free miniature swine and germ-free piglets. II. Ontogenic development development of NK and ADCC.

The ontogenic development of natural killing (NK) and antibody-dependent cellular cytotoxicity (ADCC) in germ-free and specific-pathogen-fee (SPF) miniature swine were compared. Activities of NK and ADCC were tested by a short-term (2.5 to 4 h) 51Cr-labelled human myeloid cell line K562 and TNP-conjugated human B-cell line SB as target cells for NK and ADCC, respectively. Animals obtained by aseptic hysterectomy 3-5 days prior to term showed ADCC activities similar to adult levels but lacked NK activity. Hysterectomy-derived piglets which were colostrum-deprived and maintained in germ-free isolators developed NK activity at 3-4 weeks of age. In comparison, naturally-farrowed, colostrum-fed piglets maintained in our SPF facility developed NK activity at 2-3 weeks of age. Thereafter, there was no significant difference in the levels of either NK or ADCC between germ-free and SPF animals. This suggests that microbial flora and environment do not affect the development of effector cells for ADCC but do play some role in the maturation of NK cells during ontogeny. The difference in ontogeny of NK an ADCC further support our previous suggestion that the effector cells for NK and ADCC in swine are distinct sub-populations.

Animals↗

Miniaturized linear time-of-flight mass spectrometer with pulsed extraction.

Improved resolution for a miniaturized instrument is demonstrated at high masses using a pulsed extraction, 3(") linear time-of-flight (TOF) mass analyzer. This illustrates the utility of a small and simple mass spectrometer for biological/medical analyses. Current and future applications suggested by this instrument include rapid mass spectral reading of oligonucleotides that differ in one base (single nucleotide polymorphisms), distinction of biomarker signatures from different species of bacterial spores (biological weapons detection) and point-of-care instruments for proteomics-based diagnostics. We have incorporated a two-stage, pulsed-extraction design that places the focal plane of the ions at the detector channel plate surface. The ions are accelerated to a total energy of 12 keV to enable detection of high-mass proteins in a design that incorporates a floatable flight tube set at the voltage of the front channel plate of the detector. The resultant elimination of post-acceleration at the detector is intended to improve mass resolution by reducing the difference in arrival times between ions and their neutral products. Resolutions of one part in 1200 at m/z 4500 and one part in 600 at m/z 12 000 have been achieved. Proteins with molecular masses up to 66 000 Da, mixtures of oligonucleotides, and biological spores have all been successfully measured, results that increase the potential use of this TOF analyzer.

Adrenocorticotropic Hormone↗

Qualitative study of in vivo melphalan adduct formation in the rat by miniaturized column-switching liquid chromatography coupled with electrospray mass spectrometry.

In a general study of DNA adduct formation with melphalan, rats were intravenously injected with a single high dose (10 mg kg(-1)). Adduct formation was studied at the nucleoside level in the target organs liver, bone marrow, kidney and blood with the use of 2D liquid chromatography/tandem mass spectrometry (LC/MS/MS). Adducts of dGuo and dAdo were detected under selected reaction monitoring in liver and bone marrow 10 h after administration of melphalan. In the DNA hydrolysates from kidney and blood a Gua-melphalan adduct was found, although in very low abundance. These first results of the search for in vivo-formed melphalan adducts in the rat showed that our miniaturized LC/MS technique is useful for investigating this type of compound. More experiments will be performed in this area to gather more information about the pharmacokinetics and the quantity of adducts formed.

Animals↗

Thyroid hormones regulate the frequency of miniature end-plate currents in pre- and prometamorphic stages of the tadpole tail.

Thyroid hormones (THs), primarily 3,3',5-triiode-(L)-thyronine (T(3)), have been clearly established as natural inducers of apoptosis during metamorphosis of anuran embryos. We decided to use this phenomenon to test the hypothesis that, prior to genomic activation, T(3) has acute actions in the neuromuscular junction (NMJ) of the tail of amphibian embryos. We detected a dramatic increase in the production of miniature end-plate currents (MEPCs) 2-5 min after continuous application of T(3) (250 nM) using focal recordings under voltage clamp. Furthermore, this increase in the spontaneous release of neurotransmitter, evaluated by the MEPC frequency, was maintained for several hours. Reverse-T(3), the "inhibitory" form of THs, prevented this increase in MEPC frequency, suggesting that this is probably a highly specific action of T(3). In addition, the elevation in MEPC frequency induced by T(3) was unchanged in the presence or absence of extracellular calcium. The T(3)-mediated increase in MEPC frequency was blocked by niflumic acid, a nonsteroidal antinflammatory fenamate used to prevent the apoptotic volume decrease observed in many systems. The present study demonstrated that T(3) induces a remarkable nongenomic action in the NMJ of the tadpole tail at pre- and promatamorphic stages.

Acetylcholine↗

Miniature endplate potentials induced by ammonium chloride, hypertonic shock, and botulinum toxin.

Intracellular recordings were made at the neuromuscular junction (NMJ) of the mouse diaphragm to study alteration of miniature endplate potential (MEPP) amplitude and rise time after different treatments. Following either hyperosmotic shock or 3 to 5 min of incubation in 10 to 50 mM ammonium chloride (NH4Cl) (replacing NaCl, a treatment which is known to raise intracellular pH) MEPP frequencies increased and the amplitudes of MEPPs decreased. These treatments as well as type A botulinum toxin (BoTx) gradually prolonged the rising phase of some MEPPs, which increased their time-to-peak (slow-MEPPs; Vautrin and Kriebel: Neuroscience 41:71-88, 1991) and increased eventually their amplitude. Fasciculation after hyperosmotic shock or during NH4Cl challenge was blocked by D-tubocurarine and was due to large slow-MEPPs that reached threshold for the muscle fiber action potential. The development of fasciculation provided the time course for the development of giant-MEPPs. Increased frequency of giant MEPP is accompanied by a block of the nerve-evoked muscle contraction. Effects of BoTx on spontaneous release were functionally antagonized either by NH4Cl or hyperosmotic shock. NH4Cl delayed BoTx blockage of bell-MEPPs. Data suggest that BoTx alters the formation of transmitter packets gradually but similarly to other treatments which increase incidence of skew-MEPPs.

Action Potentials↗

Evaluation of the teratogenicity of morphine sulfate administered via a miniature implantable pump.

A technique has been developed for the implantation of miniature infusion pumps in pregnant mice with minimal teratogenic and toxic side effects. In 7- to 10-day pregnant CF-1 mice receiving constant low doses of morphine sulfate via the infusion pump, the results, including fetal weight reduction and various skeletal and soft tissue abnormalities, were similar to those reported in previous investigations using single injections.

Animals↗

A myasthenia gravis plasma immunoglobulin reduces miniature endplate potentials at human endplates in vitro.

A particular myasthenia gravis (MG) plasma Ig has previously been shown to block a single alpha-bungarotoxin (alpha-BuTx) binding site on embryonic rat muscle acetylcholine receptor (AChR). We have investigated its effect on embryonic/denervated and adult human AChR both in extracts and in situ. Plasma Ig blocked 125I-alpha-BuTx binding by greater than 85% to the AChR extracted from denervated muscle, but only by 55% to AChR extracted from normal human muscle. Incubation of intact human muscle fibers with the plasma Ig reduced 125I-alpha-BuTx binding to the endplate AChRs by 63%, and substantially decreased the amplitude of miniature endplate potentials. We conclude that anti-alpha-BuTx site antibodies, when present, can be important in the pathophysiology of the disease.

Biopsy↗

Effects of octopamine on miniature excitatory junction potentials from developing and adult moth muscle.

Intracellular recordings of excitatory junction potentials (EJPs) and miniature EJPs (MEJPs) were made from the dorsal longitudinal muscle of Manduca sexta to determine the sites of action of octopamine. MEJPs increased in amplitude and frequency as the moth developed during the 3 days before eclosion. DL-Octopamine (5 X 10(-6) M) increased the amplitude of excitatory junction potentials in both immature moths (one day before eclosion) and adults. Octopamine (10(-5) M) also increased the amplitude and frequency of MEJPs from immature animals (one and two days before eclosion) but had the opposite effect on adults and pharate adults ready to eclose. Treatment with octopamine (10(-5) M) resulted in a decrease in input resistance and a hyperpolarization in both immature and adult muscle fibers. The results suggest that octopamine acts both presynaptically and postsynaptically but that the increase in the amplitude of the evoked response is due primarily to influences on presynaptic processes.

Age Factors↗

Protein chip based miniaturized assay for the simultaneous quantitative monitoring of cancer biomarkers in tissue extracts.

A multiplexed fluorescence immunoassay using a novel planar waveguide technology-based microarray system, ZeptoMARK (Zeptosens), was developed to detect simultaneously urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and vascular endothelial growth factor (VEGF) in extracts of breast cancer tissues. The three analytes assay was cross-validated with single-analyte ELISA/chemiluminescence immunosorbent assay tests, revealing good correlations and enhanced assay sensitivities (LODs) of 1 pg/mL for uPA, 33 pg/mL for PAI-1, and 1 pg/mL for VEGF. Values were well within the 80-120% limits for assay recovery and within the +/-20% limits for assay precision. The uPA, PAI-1, and VEGF results obtained from 50 breast cancer cytosols using the protein array system demonstrated that the microarray-based multiplexed assay is a sensitive and robust tool to be used for the simultaneous quantification of cancer markers in small breast cancer tissue samples (core biopsies). The miniaturized, multiplexed assay format has a potential to be used for the quantitative analysis of a larger set of validated markers with significance in disease management.

Biomarkers, Tumor↗

Accelerated high-sensitivity microsequencing of proteins and peptides using a miniature reaction cartridge.

Greatly reduced automated protein sequencing degradation times of less than 25 min with concurrent on-line phenylthiohydantoin (PTH) derivative analysis times of less than 16 min have been achieved by using a miniaturized reaction cartridge with optimized chemical and analytical cycles. Using this method a wide range of standard and novel peptides and proteins have been sequenced with reproducibly high initial and repetitive cycle yields. In these accelerated analyses the recovery of the more labile PTH derivatives was markedly improved by using elevated pressure during cleavage steps and temperature programming throughout the Edman cycle.

Amino Acid Sequence↗

Direct analysis of volatile organic compounds in human breath using a miniaturized cylindrical ion trap mass spectrometer with a membrane inlet.

Membrane introduction mass spectrometry (MIMS) coupled to a miniature mass spectrometer equipped with a cylindrical ion trap (CIT) analyzer was used to monitor the flavor components, 3-phenyl-2-propenal and methyl salicylate, found in cinnamon and wintergreen candies, respectively, directly from human breath. The poly(dimethylsiloxane) (PDMS) membrane was operated in a trap-and-release mode, where the temperature of the membrane was cycled during the experiments, which permitted temporal resolution of the two compounds of interest, facilitating their observation in the complex sample. Under these thermally driven conditions, the 10-90% rise times for both compounds are similar (15 s for methyl salicylate, 17 s for 3-phenyl-2-propenal), but the difference in diffusivity means that the signal for 3-phenyl-2-propenal is delayed and the 10% point occurs 6 s later than that for wintergreen. Additional specificity needed for complex samples was gained by using tandem mass spectrometry.

Acrolein↗

A liquid-Ga-filled carbon nanotube: a miniaturized temperature sensor and electrical switch.

Temperature control on the nanometer scale is a challenging task in many physical, chemical, and material science applications where small experimental volumes with high temperature gradients are used. The crucial difficulty is reducing the size of temperature sensors while keeping their sensitivity, working temperature range, and, most importantly, their simplicity and accuracy of temperature reading. In this work, we demonstrate the ultimate miniaturization of the classic thermometer using an expanding column of liquid gallium inside a multi-walled C nanotube for precise temperature measurements. We report that electrical conductivity through unfilled nanotube regions is diffusive with a resistance per unit length of approximately 10 kOmega microm(-1), whereas Ga-filled segments of the nanotube show metallic behavior with a low resistance of approximately 100 Omega microm(-1). No noticeable Schottky barrier exists between the nanotube carbon shell and the inner Ga filling. Based on these findings, an individual carbon nanotube partially filled with liquid Ga is used as a temperature sensor and/or switch. The nanotube's electrical resistance decreases linearly with increasing temperature as the metallic Ga column expands inside the tube channel. In addition, the tube resistance drops sharply when two encapsulated Ga columns approaching each other meet inside the nanotube, producing a switching action that can occur at any predetermined temperature, as the Ga column position inside the nanotube can be effectively pre-adjusted by nanoindentation using an atomic force microscope.

Biosensing Techniques↗

Calcium-insensitive miniature endplate potentials at the neuromuscular junction.

Several different types of acetylcholine secretion have been shown to coexist at the neuromuscular junction along with the Ca2+-dependent quantal release producing miniature endplate potentials (mepps) and endplate potentials. One of these, the Ca2+-insensitive, slow-rising mepps (slow mepps), is present in normal untreated muscles but is most prominent in many conditions where the Ca2+-dependent quantal release mechanism is not functioning properly. Slow mepps occur at a frequency of less than 0.1 Hz in normal muscles, with large variability between fibres and muscles, and can reach frequencies of 1-2 Hz in several pathological conditions. The potentials are also highly variable in size and shape, being generally of high amplitude (0.1-15 mV) and prolonged time course (1-15 ms rise time). Most importantly, slow mepps are not affected by procedures which increase the intraterminal Ca2+ concentration, including nerve stimulation, thus being unable to contribute to the function of synaptic transmission. The cellular source of the Ca2+-insensitive mepps has been determined to be the nerve terminal and not the Schwann cells or nerve sprouts. The release process producing slow mepps is generally insensitive to many drugs, ions, and procedures, stimulation being observed with vinblastine, cytochalasin B, and caffeine. Depression of this secretion is effected by uncouplers of oxidative phosphorylation and by a drug (AH5183) which inhibits the vesicular active acetylcholine transport system. It is concluded that the slow mepps are due to an exocytic fusion of unique synaptic vesicles with the plasma membrane near the active zones, in a process insensitive to many intracellular ions and regulators. Since slow mepps are prominent in many pathological conditions of nerve and muscle, it is speculated that they play some role in the recovery or development of synaptic function.

Acetylcholine↗

Teratogenic potential of blighted potato concentrate in rabbits, hamsters, and miniature swine.

Freeze-dried preparations of potatoes infected with phytophthora infestans or Alternaria solani were fed to pregnant hamsters, rabbits, and miniature swine. No evidence was observed that these diets or the extracts of respective blights were capable of producing teratologic effects in hamsters. Lesions of neurulation and anencephaly were noticed in rabbits and swine, respectively. The need for further pharmacological and toxicological evaluation in sensitive species is emphasized.

Alternaria↗

Immunocompetence of bivalve hemocytes as evaluated by a miniaturized phagocytosis assay.

Immune function in bivalves can be adversely affected by long-term exposure to environmental contaminants. Investigating alterations in immunity can therefore yield relevant information about the relationship between exposure to environmental contaminants and susceptibility to infectious diseases. We have developed a rapid, cost-effective, and miniaturized immunocompetence assay to evaluate the phagocytic activity, viability, and concentration of hemocytes in freshwater and marine bivalves. Preliminary experiments were performed to optimize various aspects of the assay including 1) the time required for adherence of hemocytes to polystyrene microplate wells, 2) the time required for internalization of fluorescent bacteria, 3) the ratio of hemocytes to fluorescent bacteria in relation to phagocytosis, 4) hemolymph plasma requirements, and 5) the elimination of fluorescence from (noninternalized) bacteria adhering to the external surface of hemocytes. The results of these experiments showed the optimal adherence time for hemocytes in microplate wells to be 1 h, that phagocytosis required at least 2 h of contact with fluorescently labeled E. coli cells, that the number of fluorescent E. coli cells had a positive effect on phagocytic activity, that at least 2.5 million cells/mL were required to measure a significant intake, and that a linear increase in uptake of bacteria (R = 0.91; p < 0.01) could be obtained with concentrations of up to 1.3 x 10(6) hemocytes/mL. Afterward, the assay was used in two field studies to identify sites having the potential to affect the immunocompetence of bivalves. The first study was conducted on Mya arenaria clams collected at selected contaminated sites in the Saguenay River (Quebec, Canada), and the second examined Elliptio complanata freshwater bivalves that had been exposed to a municipal effluent plume in the St. Lawrence River (Quebec, Canada). In the Saguenay River field study a significant increase in phagocytosis was observed at sites closest to polluted areas. Phagocytotic activity varied over time and was highest during the warmest months (June, July, and August), closely paralleling the spawning period of Mya arenaria clams. In contrast, a drop in phagocytic activity was observed in Elliptio complanata mussels exposed to surface water 4 km downstream of a major municipal effluent plume, with a concomitant increase in the number of hemocytes in the hemolymph. It appears that both immunosuppressive and immunostimulative effects are likely to occur in the field and that responses will be influenced by the type and intensity of contaminants at play.

Animals↗

Miniaturized amperometric biosensor based on xanthine oxidase for monitoring hypoxanthine in cell culture media.

Fabrication and characterization of miniaturized amperometric hypoxanthine biosensors are described and demonstrated for monitoring hypoxanthine in myocardial cell culture media. The sensors are based on xanthine oxidase (XO) immobilized on carbon fiber microelectrodes (CFMEs) using a composite film of Nafion and electropolymerized phenol (PPh). Nafion was used for XO immobilization because of its film hydrophobicity, enzyme-favored environment, and electrostatic interaction with XO, which was dispersed in Nafion film by immersing the Nafion-coated CFMEs in XO solution for 5 h. PPh film was formed as an overlay on Nafion and XO-modified CFMEs via electropolymerization. Hypoxanthine was measured with the sensor by the oxidation of enzymatic reaction products, hydrogen peroxide (H(2)O(2)), and uric acid (UA) at +0.60 V (vs Ag/AgCl). The use of Nafion and PPh as a matrix for XO immobilization yields enhanced specificity, sensitivity, and linearity toward hypoxanthine. A dynamic linear range of 5.0 microM to 1.8 mM was achieved with a calculated detection limit of 1.5 microM (S/N = 3) and a sensitivity of 3.144 nA/mM. In addition, the measurement was virtually interference-free from easily oxidizable species such as UA, ascorbic acid, physiological levels of neurotransmitters, and their principal metabolites. The biosensor was used to monitor hypoxanthine accumulation in myocardial cell culture media, in which the level of extracellular hypoxanthine was found to increase with ischemic tolerance.

Animals↗

Induction of giant miniature end-plate potentials during blockade of neuromuscular transmission by textilotoxin.

The present study investigated the action of textilotoxin, isolated from the venom of the Australian common brown snake Pseudonaja textilis, on neuromuscular transmission in isolated toad nerve-muscle preparations. Initial muscle twitch tension experiments revealed a triphasic pattern of changes in muscle tension and a irreversible binding action of textilotoxin (10 micrograms/ml) similar to other snake beta-neurotoxins. This was characterised by an initial depression of twitch tension, followed by a period of enhanced tension, eventually leading to a reduction in tension to complete neuromuscular blockade. These actions on muscle tension were investigated further by assessing the action of textilotoxin on end-plate potential amplitude (EPP). This revealed a similar triphasic alteration of the nerve-evoked release of acetylcholine from the motor nerve terminal. These actions on acetylcholine release were confirmed to be of a presynaptic origin since the modal amplitude of miniature end-plate potentials (MEPPs) was not reduced and in twitch tension experiments the muscle still contracted in response to direct muscle stimulation when nerve-evoked release was completely blocked. Interestingly dramatic effects were observed on the spontaneous release of acetylcholine, including an marked increase in MEPP frequency, a skewing of the MEPP amplitude frequency histogram to the right, and a resultant increase in the number of 'giant' MEPPs. These results indicate that textilotoxin causes a presynaptic blockade of neuromuscular transmission involving a disruption of the regulatory mechanism that controls acetylcholine release.

Animals↗

Miniaturization, genome size and the origin of functional constraints in the visual system of salamanders.

During their evolution, many species of lungless salamanders (fam. Plethodontidae) have experienced a great increase in genome size and consequently in the size of their cells, including sensory receptors and neurons. In addition, some have become extremely miniaturized. The consequences of these events and the morphological compensatory processes are studied in the visual system of juvenile and adult salamanders.

Animals↗