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PubMed2006

Ultrasensitive native fluorescence detection of proteins with miniaturized polyacrylamide gel electrophoresis by laser side-entry excitation.

Direct detection of separated proteins inside polyacrylamide gels has many advantages compared to staining methods. Ultrasensitive native fluorescence detection of proteins with miniaturized 1-D and 2-D PAGE was achieved with laser side-entry excitation. The detection limit for R-phycoerythrin protein spots in 1-D SDS-PAGE with 532 nm excitation was as low as 15 fg, which corresponds to only 40,000 molecules. The average detection limit of six standard native proteins was 5 pg per band with 275 nm excitation. The dynamic range spanned more than three orders of magnitude. By using the same detection setup, approximately 150 protein spots from 30 ng of total Escherichia coli extraction were detected on a 0.8 cm x 1 cm gel in 2-D separation. The significant improvement in sensitivity for laser side-entry excitation comes from higher excitation power and lower background level compared with other excitation modes.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2002

Key issues involved with the use of miniature specimens in the characterization of the mechanical behavior of polymeric biomaterials--a review.

This article is arranged in three parts. In the first, a brief history of the use of miniature specimens for characterizing the mechanical behavior of materials in general is given. In the second part, several trends in literature reports of small punch and small shear punch tests of ultra-high-molecular-weight polyethylene and acrylic bone cement specimens are examined critically. In this exercise, special attention is paid to one test metric; namely, the work to failure [which is the area under the punch load (P) versus punch displacement (Delta) plot up to the failure point]. In the third part of the article, seven key issues in this field are discussed. In each case, the gaps in the current knowledge base are pointed out. Among those issues are determination of sensitivity of test results to test variables, development of methods for converting P-Delta results to bulk material properties, and constitutive modeling of the mechanical behavior of a polymeric biomaterial, under both small punch and small shear punch loading.

Arthroplasty↗

PubMed1991

Influence of surface characteristics on bone integration of titanium implants. A histomorphometric study in miniature pigs.

The purpose of the present study was to evaluate the influence of different surface characteristics on bone integration of titanium implants. Hollow-cylinder implants with six different surfaces were placed in the metaphyses of the tibia and femur in six miniature pigs. After 3 and 6 weeks, the implants with surrounding bone were removed and analyzed in undecalcified transverse sections. The histologic examination revealed direct bone-implant contact for all implants. However, the morphometric analyses demonstrated significant differences in the percentage of bone-implant contact, when measured in cancellous bone. Electropolished as well as the sandblasted and acid pickled (medium grit; HF/HNO3) implant surfaces had the lowest percentage of bone contact with mean values ranging between 20 and 25%. Sandblasted implants with a large grit and titanium plasma-sprayed implants demonstrated 30-40% mean bone contact. The highest extent of bone-implant interface was observed in sandblasted and acid attacked surfaces (large grit; HCl/H2SO4) with mean values of 50-60%, and hydroxylapatite (HA)-coated implants with 60-70%. However, the HA coating consistently revealed signs of resorption. It can be concluded that the extent of bone-implant interface is positively correlated with an increasing roughness of the implant surface.

Animals↗

PubMed1992

Collection of blood mononuclear cells by leukapheresis for transplantation in a Yucatan miniature swine model.

A large animal model is needed to evaluate new apheresis technologies. These technologies include novel methods of harvesting the blood mononuclear cell population which contains the hematopoietic stem cells needed to restore hematopoiesis in recipients of hematopoietically lethal therapy and the use of cytokines to provide a safe and predictable method of manipulating these circulating hematopoietic stem cells. We describe the methods used to collect mononuclear cells by leukapheresis from Yucatan miniature swine. These animals are of sufficient size to tolerate the procedures and have many physiologic and hematologic similarities to man. They are of good temperament and are easily trained. Long-term venous access was obtained using single lumen silicone rubber catheters placed in the inferior vena cava. The animals were apheresed while fully awake using a Haemonetics Model V50 machine and a modified lymphocyte collection protocol. The procedure was highly efficient for the collection of mononuclear cells and a 10 pass procedure yielded a product which contained 19.7 x 10(9) mononuclear cells, 10.7 x 10(9) granulocytes, and 17 ml of erythrocytes in a volume of approximately 100 ml. This product can be cryopreserved and used for subsequent transplantation. The content of four apheresis procedures provides hematopoietic reconstitution of lethally irradiated swine on a time scale equivalent to transplantation of optimal numbers of bone marrow cells.

Animals↗

PubMed1992

The mechanism of suckling in two species of infant mammal: miniature pigs and long-tailed macaques.

Suckling is the form of feeding unique to infant mammals. The mechanism used by infant mammals to withdraw liquid from the nipple is the subject of considerable debate. Suckling has been examined in two species of infant mammals: miniature pigs and long-tailed macaques. In both species radio-opaque markers were inserted into the tongue and jaws; the movements of the jaw and tongue (and also of specific regions within the tongue) plus the movement of milk containing barium were studied by high-speed cineradiography (100 and 150 frames/sec). In the case of macaques, simultaneous pressure transducer recordings were also made. In both species, liquid moved out of the nipple as the intraoral space was expanded by a combination of tongue movement (negative pressure pumping) coupled with jaw opening. There was no evidence for expression (positive pressure on the nipple) in either species, strongly supporting the view that a suction mechanism is responsible for acquisition of milk from the nipple. Subsequent intraoral transport was different in the two species. The pigs used a second pump mechanism at the base of the tongue to transport liquid through the pillars of the fauces into the valleculae. The monkeys used a "squeeze-back" mechanism similar to the transport mechanism documented for adult macaques. Further work with other species can test our tentative hypothesis that all mammals use a negative pressure suction for acquisition, but, as is true for adult mammals, infants may use different transport mechanisms to form and move the bolus.

Animals↗

PubMed2003

A miniaturized matrix-assisted laser desorption/ionization time of flight mass spectrometer with mass-correlated acceleration focusing.

Mass correlated acceleration (MCA) has now been integrated into a 4 in (10.2 cm) matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer to achieve high resolving power across a broader mass range, without sacrificing detection of higher mass ions. The goal was to combine MCA with a custom-built miniaturized instrument such as those that might be used for field-portable applications. Unlike other pulsed extraction methods, MCA is not mass dependent and mass spectra can be achieved with a single tuning of instrument parameters. Additionally, the multi-channel recording advantage is better realized because ions of all masses can be brought into focus simultaneously. The MCA dual-stage ion source compensates dynamically for the mass dependence by incorporating an extraction pulse region followed by an acceleration region that contains a time-dependent waveform correlated with mass. The technique was validated with applications in peptide mixtures and protein digestions. Diagnostic studies for the instrument include m/z range and limits of detection.

Acceleration↗

PubMed2005

Evaluation of the sensitivity of miniaturized liquid chromatography-electrospray ionization-mass spectrometry for pharmaceutical analysis.

LC-ESI-MS is applied frequently in pharmaceutical analysis. The sample amount is generally not restricted, however with LC-ESI-MS, a lack of sensitivity may still be observed with standard-bore LC columns in isocratic mode. Therefore, it was investigated whether increased sensitivity could be achieved by using miniaturized LC-ESI-MS. Seven columns ranging from 0.1 to 4.6 mm ID were tested using several instrument setups. For proper comparison, a sensitivity gain factor (SGF) was introduced. The SGF expresses the extra sensitivity that may be obtained on top of the normal increase of peak concentration, which can be expected when the column ID is reduced. Desogestrel, mirtazapine, and sugammadex sodium were used as test compounds. For desogestrel and sugammadex sodium, the SGF increased up to a factor of 5-13 when the column ID was reduced, indicating enhanced ionization efficiencies at lower flow rates. Optimum sensitivity was found for the 0.3 mm column coupled in combination with a microinjection valve and a dedicated low flow rate interface. For mirtazapine, no increase of SGF was observed when the column ID was decreased. Apparently, the ionization efficiency of this compound is not affected by the flow rate and the spray quality.

Chromatography, High Pressure Liquid↗

PubMed2005

Miniature self-contained intravascular magnetic resonance (IVMI) probe for clinical applications.

A miniature (1.73 mm in diameter) NMR probe, which contains a magnet and a radiofrequency (RF) coil, is presented. This probe is integrated at the tip of a standard catheter and can be inserted into the human coronary arteries, creating local magnetic fields needed to obtain the NMR signal from the blood vessel walls, without the need for external magnet or RF coils. The basic theory governing the probe performance in terms of signal-to-noise-ratio and contrast parameters is presented, along with measured results from test samples. The NMR signal can be analyzed to obtain tissue contrast parameters such as T1, T2 and the diffusion coefficient, which may be used to detect lipid-rich vulnerable plaques in the coronary arteries.

Blood Vessel Prosthesis↗

PubMed1997

A high-density screening format for encoded combinatorial libraries: assay miniaturization and its application to enzymatic reactions.

A novel, miniaturized high-throughput screening format is described for assay of combinatorial libraries generated on beads. This approach, which is ideally suited to encoded libraries synthesized on beads, utilizes the photolytic cleavage of individual compounds into a high-density well array (>6500 wells within a standard 96-well microtiter plate footprint) with well volumes as low as 0.37 microl. As a model study, an encoded dipeptide library (324 members) acylated with isobutyl succinate was assayed using this format to search for potential inhibitors of matrilysin, a member of the matrix metalloproteinase superfamily. In situ release of compounds from solid support was accomplished by photochemical cleavage after beads and enzyme were distributed to the wells. After the addition of a fluorogenic substrate to the array, the extent of enzyme inhibition and identification of active compounds was quantitated by imaging of the fluorescence emission upon uv irradiation. The structure-activity relationship data generated from the identified inhibitors in this study corroborate previous findings, thus validating the utility of this approach as a means of high-throughput screening of bead-based libraries.

Acylation↗

PubMed1993

Trichinella spiralis: genetic basis and kinetics of the anti-encysted muscle larval response in miniature swine.

Primary-infected swine lymphocyte antigen (SLA) inbred miniature swine, NIH minipigs, of the SLAa/a haplotype (aa) display markedly reduced Trichinella spiralis-encysted muscle larval (ML) burdens 6 weeks after homologous challenge and are therefore referred to as responders against encysted T. spiralis ML. To investigate the kinetics and genetics of the anti-encysted ML response, and in an attempt to ascertain the mechanisms underlying this phenomenon, NIH minipigs of multiple SLA haplotypes, designated either as ax (aa, ac, ad, af, ag, dh and hh) or non-ax (cc, cd, and dd), received a primary inoculation of 300 T. spiralis ML followed by a challenge inoculation of 10,000 ML 6 or 17 weeks later. Pigs were examined at necropsy and at Weeks 1, 2, 3.5, and 6 after challenge. Statistical cluster analyses, based on final ML burdens, performed on 129 pigs in 13 trials, revealed that 47% of primary-plus-challenged ax pigs exhibited responder phenotype as early as 1-2 weeks after T. spiralis challenge whereas this response was manifested in only 8% of non-ax pigs. No correlation was observed between responder phenotype and T. spiralis-specific blastogenesis, cell expression of class I or class II SLA molecules, lymphoid cell subsets, adult worm numbers, or fecundity. Although some ax responder pigs exhibited a localized inflammatory response with a prominent eosinophilic infiltrate surrounding degenerating encysted ML, no correlation was observed between responder phenotype and either peripheral blood eosinophilia or the ability of eosinophil-enriched cell populations from responder pigs to adhere to or destroy encysted ML in vitro.

Animals↗

PubMed1982

Evaluation of a miniature CdTe detector for monitoring left ventricular function.

A miniature CdTe probe interfaced to a microcomputer was used to measure left ventricular ejection fraction (LVEF) in 25 patients. LVEF obtained with the CdTe module, in the beat-to-beat mode, or the integrated gated mode agreed well with LVEF obtained with a gamma camera (r = 0.80; r = 0.82 respectively). Similarly, LVEF by CdTe probe agreed with LVEF obtained by gated equilibrium studies performed with a computerized NaI probe. The CdTe probe can provide comparable measurement of LVEF at a fraction of the cost of a camera-computer system and, being small and lightweight, the CdTe probe is adaptable for monitoring patients in intensive care facilities.

Adult↗

PubMed1988

Class II genes of miniature swine. I. Class II gene characterization by RFLP and by isolation from a genomic library.

Class II genes of miniature swine have been characterized by restriction fragment length polymorphism (RFLP) analysis and by analysis of a series of clones isolated from a lymphocyte genomic library. For RFLP analysis, DNA samples from three independent major histocompatibility complex homozygous lines and three intra-MHC recombinant lines were digested with a variety of restriction enzymes and analyzed in Southern blots using human cDNA probes for DP, DQ, DR, and DZ alpha genes, and DP, DQ, DR, and DO beta genes. One, or at most two, unique fragments were detected by hybridization with each of the human alpha probes tested. In contrast, multiple bands (five to six for most enzymes examined) were detected by each of the human beta probes tested, the majority of which were found to cross-react with at least three of these probes under conditions of moderate stringency. Genomic DNA from the SLAc haplotype was cloned into an EMBL-3 bacteriophage vector, and the corresponding genomic library was screened with each of these human cDNA probes. The class II genes thereby isolated from this library showed characteristics consistent with those anticipated from the RFLP analysis. Thus, unique alpha genes were obtained which showed no evidence of cross-hybridization, while beta genes showed extensive cross-hybridization and were frequently detected in the library by more than one human beta gene probe. These data are consistent with early evolutionary divergence of alpha genes, prior to mammalian speciation, and with continuing evolution of beta genes, with possible shared usage of these genes by different alpha loci. The data also imply that alpha genes can readily be assigned to loci homologous to their human counterparts, but that beta genes will require further mapping and/or sequence analysis to confirm assignments.

Animals↗

PubMed1993

A glucose monitoring system for on line estimation in man of blood glucose concentration using a miniaturized glucose sensor implanted in the subcutaneous tissue and a wearable control unit.

We have developed a miniaturized glucose sensor which has been shown previously to function adequately when implanted in the subcutaneous tissue of rats and dogs. Following a glucose load, the sensor output increases, making it possible to calculate a sensitivity coefficient to glucose in vivo, and an extrapolated background current in the absence of glucose. These parameters are used for estimating at any time the apparent subcutaneous glucose concentration from the current. In the previous studies, this calibration was performed a posteriori, on the basis of the retrospective analysis of the changes in blood glucose and in the current generated by the sensor. However, for clinical application of the system, an on line estimation of glucose concentration would be necessary. Thus, this study was undertaken in order to assess the possibility of calibrating the sensor in real time, using a novel calibration procedure and a monitoring unit which was specifically designed for this purpose. This electronic device is able to measure, to filter and to store the current. During an oral glucose challenge, when a stable current is reached, it is possible to feed the unit with two different values of blood glucose and their corresponding times. The unit calculates the in vivo parameters, transforms every single value of current into an estimation of the glucose concentration, and then displays this estimation. In this study, 11 sensors were investigated of which two did not respond to glucose. In the other nine trials, the volunteers were asked to record every 30 s what appeared on the display during the secondary decrease in blood glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1980

A new miniature plethysmograph to measure volume changes in small circumscribed tissue areas.

With an ultrasonic method tissue layer thickness was measured in man in circumscribed superficial tissue areas where the underlaying bone provided good backwall echos. In a 5 mm tissue layer changes of +/- 0.2% could be reliably detected. Knowing the height of the tissue cylinder between the surface of the skin and the bone allowed to calculate the tissue volume. The ultrasonic probes could therefore serve as miniature plethysmograph. Several probes were attached in the frontal region, sternum, along the vertebral column and along the tibia simultaneously. Changes of the volume content of the superficial shell tissues were induced by orthostasis, water immersion and heat exposure. It was possible to assess quantitatively the volume shifts into or out of the superficial tissues. During orthostasis 166 ml of fluid left the superficial tissues of the cephalad parts of the body and 164 ml could be traced in the dependent parts. Heat exposure was followed by a pooling 140 ml in the tissues studied. The most pronounced tissue volume changes were observed in the forehead region during heat exposure.

Electronics↗

PubMed1993

Effects of a new calcium antagonist, CD-832, on experimental coronary artery spasm in miniature pigs.

The effects of a new calcium antagonist, CD-832, on experimental coronary artery spasms were studied in Göttingen miniature pigs. Pigs underwent endothelial denudation at the left anterior descending coronary artery using a balloon catheter. Changes in the diameter of the denuded and nondenuded site in response to an intracoronary administration of serotonin (10 micrograms/kg) or histamine (10 micrograms/kg) were assessed quantitatively by selective coronary arteriography 1 week after endothelial denudation. Percent reductions of the coronary artery diameter induced by serotonin or histamine in the denuded site were significantly greater than those in the nondenuded site (p < 0.01). Coronary artery spasm induced by serotonin or histamine in the denuded site was attenuated in a dose-dependent manner by intravenous infusion of CD-832 (10 and 30 micrograms/kg/min) or nifedipine (1 and 3 micrograms/kg/min). The degrees of inhibition of coronary artery spasm by CD-832 were similar to those produced by nifedipine. CD-832 and nifedipine at the high dose caused comparable increases in the basal coronary artery diameter. These results suggest that CD-832 may be a useful drug for the treatment of coronary artery spasm.

Animals↗

PubMed1990

Diffusion of O2 in normal and ischemic retinas of anesthetized miniature pigs in normoxia and hyperoxia.

Transretinal PO2 profiles were recorded with O2-sensitive microelectrodes in the normal retina and in ischemic retinal foci induced by the occlusion of a retinal branch vein with argon laser photocoagulation in anesthetized miniature pigs. In the normal retina there are two PO2 gradients: one from the inner retina and the other from the choroid, both directed toward the middle of the retina. Both PO2 gradients persisted during hyperoxia. Thus, even in hyperoxia, the choroid does not supply the whole thickness of the normal retina with O2. Preretinal and transretinal PO2 measurements in ischemic inner retinal foci showed the existence of two PO2 gradients in steady-state systemic normoxia, as did those in the normal retina. This finding indicates that even in ischemia the choroid does not supply O2 to the inner retina; as a result, tissue hypoxia is maintained. During systemic hyperoxia, the intraretinal PO2 measurements in the ischemic foci showed only one gradient going from the choroid toward the inner retina. This gradient indicates that under these conditions, the choroid can supply O2 to the entire thickness of the ischemic retina. Extending a previously formulated hypothesis, we propose that in the ischemic retina as opposed to the normal retina, hyperoxia does not induce an increase in the O2 consumption of the outer retina. This suggestion could explain the rise in PO2 in the inner ischemic retina during hyperoxia.

Animals↗

PubMed1985

Phospholipids of normal and experimentally injured spinal cord of the miniature pig.

Experimental spinal cord trauma was produced in 3-month-old SS-1 miniature pigs by dropping a 25 g weight from a height of 20 cm upon the exposed spinal cord. The histological lesion consisted of edema and hemorrhage. Phospholipid concentration and composition, cholesterol concentration and phospholipid fatty acid composition were determined in whole spinal cord 3 hours after injury, and in spinal cord myelin 5 hours after injury. Three hours after injury phospholipid and cholesterol concentration were decreased by about 14% in the whole spinal cord. Trauma had no effect on the phospholipid composition of whole spinal cord and myelin. Fatty acid composition of myelin also did not change after injury, and changed very slightly in the whole spinal cord. It is concluded that edema following spinal cord trauma is much more extensive than previously assumed. Furthermore, peroxidation of membrane lipid fatty acids does not appear to be a significant factor in spinal cord pathology 3 hours after injury.

Animals↗

PubMed1995

Mechanisms of coronary hyperconstriction in response to serotonin induced by X-irradiation in miniature pigs: increased constrictive response of medial smooth muscle.

Experimental and clinical studies suggest that X-irradiation to the coronary artery may enhance vasoconstrictive response. This study aimed to clarify the effect of X-irradiation on the vasomotor response of porcine coronary artery. X-ray (15 Gy) was selectively irradiated to the left anterior descending coronary artery (LAD) in 14 Göttingen miniature pigs. The coronary vasomotor responses to serotonin at the irradiated site (LAD) and the control site (the left circumflex artery; LCX) were assessed by quantitative arteriography before and 1 h and 1, 2, and 4 weeks after X-irradiation. At 2 weeks, endothelium-dependent vasodilation in response to substance P was also evaluated arteriographically in vivo. At 2 weeks, isometric tension studies were performed to evaluate the constrictive responses of medial muscle strips and endothelium-dependent relaxation. Coronary vasoconstriction in response to serotonin was enhanced 1 week after, and further augmentation was noted 2 and 4 weeks after X-irradiation. Endothelium-dependent vasodilation in response to substance P in vivo was preserved 2 weeks after X-irradiation. Coronary arteriography showed no organic stenosis at the irradiated site. In vitro studies demonstrated that medial muscle strips of the irradiated site showed hypercontraction in response to serotonin and that endothelium-dependent relaxation in response to serotonin and substance P was preserved at the irradiated site. A histological study revealed no appreciable changes of the endothelial cells or intimal thickening.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗