Leukocyte filtration and miniature perfusion during arrested heart CABG on a Jehovah's Witness patient.

Bloodless surgery and a reduction in the use of allogeneic blood products has long been the standard of care in medicine. Many individuals in our communities have demanded this form of surgical treatment for personal and religious reasons. On 6 December 2002, a 72-year-old male patient was admitted to our institution as a critical air flight transfer. The patient's height was 190.5 cm and weight was 59.3 kg (body surface area 1.83 m2). His preliminary diagnosis was chest pain with myocardial infarction as evidenced by elevated blood cardiac isoenzymes. His principle diagnosis was subendocardial infarction with paroxysmal ventricular tachycardia. Cardiac catheterization was performed and demonstrated severe triple vessel disease with an ejection fraction of 30%. He was evaluated and accepted as a candidate for coronary artery bypass grafting. Multidisciplinary consultation concluded that a safe and effective method of perioperative treatment would involve the use of arrested heart support with cold blood cardioplegia using a low prime miniature perfusion circuit as no blood products would be considered for use. Additionally, the combined modalities of perfusion interventions to minimize hemodilution consisted of intraoperative autologous blood collection totaling 500 mL and rapid autologous priming of the miniature perfusion circuit. The miniature perfusion system was a low prime Cardiovention (Santa Clara, CA) CORx device which includes a hollow-fiber oxygenator and integral centrifugal pump with a surface area of 1.2 m2. This system also incorporates an air sensing solenoid which triggers rapid air evacuation in a bolus range of 1 mL or greater. Kinetic venous drainage is another feature of this device as the centrifugal pump is integrated into the oxygenator. We believed that a miniature extracorporeal circuit would enhance the desired clinical outcome as opposed to the risk of: (1) off-pump coronary artery bypass (OPCAB) approach and the concern of emergent transition to an on-pump procedure and (2) use of larger surface area with conventional systems that impose a greater hemodilutional effect. Leukocyte filtration was employed as the patient had a significant past medical history of chronic obstructive pulmonary disease. We herein report our clinical experience with this method of treatment on a patient who refused the use of blood products in his surgical treatment. It is our belief that the multiple modalities utilized in combination during this procedure resulted in positive clinical outcomes as demonstrated by an intubation time of 8 hours 35 min with a discharge on the fifth postoperative day.

Homeopathically prepared gibberellic acid and barley seed germination.

The potentisation process by which homeopathic preparations are produced raises the concern that these medicines have placebo effects only, since they theoretically no longer contain active molecules of the diluted substance. Plant models offer a method of examining the efficacy of homeopathically prepared solutions. This study examined the effects of homeopathically prepared gibberellic acid (HGA3) on the germination performance of barley (Hordeum vulgare L.) seeds. The effect of HGA3 (4-200 cH) on seed germination rate and seedling development was compared to that of the most commonly used form of gibberellic acid (GA3), 0.5 g l(-1), and control (distilled water). The extent and type of response was dependent on the vigour level of the seedlot. Treating seeds from three vigour groups in HGA3 consistently resulted in larger seedlings. High-vigour seeds treated with HGA3 4, 30 and 200 cH germinated faster, and roots of medium-vigour seedlots treated in HGA3 15 cH were longer. Biphasic effects of HGA3 were also demonstrated. As a plant model, germinating barley seeds successfully demonstrated the ability of HGA3 to produce a biological response.

Indications for off pump coronary surgery.

The learning curve in off pump surgery must be followed due to the fact that beating heart coronary surgery is a completely different operation. Beating heart coronary surgery is truly a team approach. Both the surgeon and the anesthesiologist must work in concert to attain a smooth, safe and efficient operation. A sternotomy is performed. All conduits are harvested as for traditional coronary artery bypass grafting (CABG). The pericardium is opened using a "hockey stick" incision. Another incision is made to complete the reflection of the pericardium from the pulmonary artery to the aorta. The heart is then repositioned with the surgeon's hand and exposure device is placed at the apex of the heart. Additional pericardial sutures may place for positioning as needed for exposure to complete the other anastomosis using stabilizers. The left internal mammary artery (LIMA) to left anterior descending artery (LAD) graft is performed first, after the postero-lateral wall or the right side of the heart can be revascularized. After each anastomosis is performed, measurement of the flow through the conduit is recommended. The chest is closed in the standard fashion. Off pump coronary artery grafting has been established as a safe and effective procedure. It involves a totally different mind-set for the surgeon. Indication for off pump coronary surgery depends on the experience and comfort level of the surgeon. Currently, there are a multitude of devices available for both exposure and stabilization to efficiently perform this operation. Therefore, most patients should be considered candidates for off pump coronary revascularization.

Homeopathically prepared gibberellic acid and barley seed germination

The potentisation process by which homeopathic preparations are produced raises the concern that these medicines have placebo effects only, since they theoretically no longer contain active... (AU)

Prospective analysis of endoscopic vein harvesting.

BACKGROUND: Utilization of bridging vein harvesting (BVH) of saphenous vein grafts (SVG) for coronary artery bypass grafting (CABG) results in large wounds with great potential for pain and infection. Endoscopic vein harvesting (EVH) may significantly reduce the morbidity associated with SVG harvesting. METHODS: A prospective database of 200 matched patients receiving EVH and BVH was compared. The patients all underwent CABG done over a period of 4 months (April to August 2000). Patients were excluded if they had prior vein harvesting. RESULTS: The EVH and BVH group included 100 patients each with similar demographics. The patients in the EVH group had significantly fewer wound complications, mean days to ambulation, and total length of stay (P <0.05). There was no difference in harvest time or vein injuries. CONCLUSION: Endoscopic vein harvesting results in significantly fewer wound complications, decrease in days to ambulation, and the total length of stay. EVH is superior to BVH in patients undergoing CABG.

Miniaturized FRET assays and microfluidics: key components for ultra-high-throughput screening.

Assay miniaturization applicable across a wide range of target classes, along with automation and process integration, are well-recognized goals for ultra-high-throughput screening on an industrial scale. This report summarizes the implementation of fluorescence resonance energy transfer (FRET)-based biochemical and cell-based assays in 3456-well NanoWelltrade mark assay plates using key components of Aurora's ultra-high-throughput screening system.

Identification of a membrane-spanning domain of the thiol-activated pore-forming toxin Clostridium perfringens perfringolysin O: an alpha-helical to beta-sheet transition identified by fluorescence spectroscopy.

Clostridium perfringens perfringolysin O (PFO or theta-toxin) is a cytolytic toxin that binds to cholesterol-containing membranes and then self-associates to spontaneously form aqueous pores of varying size in the bilayer. In this study, a membrane-spanning domain has been identified in PFO by a combination of fluorescence spectroscopic methods using the fluorescent dye N, N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1, 3-diazolyl)ethylenediamine (NBD) whose emission properties are sensitive to water. PFO was substituted with a single cysteine at most of the residues between amino acids K189 and N218, and then each cysteine was modified with NBD. Each purified NBD-labeled PFO was then bound to membranes, and the probe's environment was ascertained by measuring its fluorescence lifetime, emission intensity, and collisional quenching with either aqueous (iodide ions) or nonaqueous (nitroxide-labeled phospholipids) quenchers. Lifetime and intensity measurements revealed that the amino acid side chains in this region of the membrane-bound PFO polypeptide alternated between being in an aqueous or a nonaqueous environment. This pattern indicates that this portion of the membrane-bound PFO spans the membrane in an antiparallel beta-sheet conformation. The alternating exposure of these residues to the hydrophobic interior of the bilayer was demonstrated by their susceptibility to quenching by nitroxide moieties attached to phospholipid acyl chains. Residues K189-N218 therefore form a two-stranded, amphipathic beta-sheet in the membrane-bound PFO that creates a stable interface between the pore and the membrane. This same region packs as three short alpha-helices in the soluble, monomeric form of PFO, and therefore, the cholesterol-dependent conversion of PFO to a membrane-bound oligomer involves a major structural transition in which three alpha-helices unfold to form a membrane-spanning amphipathic beta-sheet.

BiP maintains the permeability barrier of the ER membrane by sealing the lumenal end of the translocon pore before and early in translocation.

Secretory proteins are cotranslationally translocated across the mammalian ER membrane through an aqueous pore in the translocon while the permeability barrier is maintained by a tight ribosome-membrane junction. The lumenal end of the pore is also blocked early in translocation. Extraction of soluble lumenal proteins from microsomes and reconstitution with purified proteins demonstrate, by fluorescence collisional quenching, that BiP seals the lumenal end of this pore. BiP also seals translocons that are assembled but are not engaged in translocation. These ribosome-free translocons have smaller pores (9-15 A diameter versus 40-60 A in functioning translocons) and are generated when ribosomes dissociate from functioning translocons with large pores. BiP therefore maintains the permeability barrier by sealing both nontranslocating and newly targeted translocons.

The aqueous pore through the translocon has a diameter of 40-60 A during cotranslational protein translocation at the ER membrane.

Eukaryotic secretory proteins are cotranslationally translocated through the endoplasmic reticulum (ER) membrane via aqueous pores that span the lipid bilayer. Fluorescent probes were incorporated into nascent secretory proteins using modified Lys-tRNAs, and the resulting nascent chains were sealed off from the cytosol in fully assembled translocation intermediates. Fluorescence quenching agents of different sizes were then introduced into the ER lumen in order to determine which were small enough to enter the pore and to quench the fluorescence of probes inside the ribosome and/or the pore. These accessibility studies showed that the aqueous pore in a functioning translocon is 40-60 A in diameter, making it the largest hole observed to date in a membrane that must maintain a permeability barrier.

Rotational and conformational dynamics of Escherichia coli ribosomal protein L7/L12.

Fluorescence methods were utilized to study dynamic aspects of the 24 kDa dimeric Escherichia coli ribosomal protein L7/L12. Oligonucleotide site-directed mutagenesis was used to introduce cysteine residues at specific locations along the peptide chain, in both the C-terminal and N-terminal domains, and various sulfhydryl reactive fluorescence probes (iodoacetamido) fluorescein, IAEDANS, pyrenemethyl iodoacetate) were attached to these residues. In addition to the full-length proteins, a hinge-deleted variant and variants corresponding to the C-terminal fragment and the N-terminal fragment were also studied. Both steady-state and time-resolved fluorescence measurements were carried out, and the results demonstrated that L7/L12 is not a rigid molecule. Specifically, the two C-terminal domains move freely with respect to one another and with respect to the dimeric N-terminal domain. Removal of the hinge region, however, significantly reduces the mobility of the C-terminal domains. The data also show that the rotational relaxation time monitored by the fluorescent probe-depends upon the probe's excited state lifetime. This observation is interpreted to indicate that a hierarchy of motions exists in the L7/L12 molecule including facile motions of the C-terminal domains and dimeric N-terminal domain, in addition to the overall tumbling of the protein. Probes attached to the N-terminal domain exhibit global rotational relaxation times consistent with the molecular mass of the dimeric N-terminal fragment. Upon reconstitution of labeled L7/L12 with ribosomal cores, however, the motion associated with the dimeric N-terminal domain is greatly diminished while the facile motion of the C-terminal domains is almost unchanged.

Dimer/monomer equilibrium and domain separations of Escherichia coli ribosomal protein L7/L12.

The dimer to monomer equilibrium and interdomain separations of cysteine variants of L7/L12 have been investigated using fluorescence spectroscopy. Steady-state polarization measurements on cysteine containing variants of L7/L12, labeled with 5-(iodoacetamido)fluorescein, demonstrated dimer to monomer dissociation constants near 30 nM for variants labeled at position 33, in the N-terminal domain, and positions 63 and 89, in the C-terminal domain. A dissociation constant near 300 nM was determined for a variant labeled at position 12, in the N-terminal domain. The polarization of a labeled C-terminal fragment did not change over the range of 200 microM to 1 nM, indicating that this construct remains monomeric at these concentrations, whereas a dimer to monomer dissociation constant near 300 nM was observed for an FITC labeled N-terminal fragment. Intersubunit fluorescence resonance energy self-transfer was observed when appropriate probes were attached to cysteines at residues 12 or 33, located in the N-terminal domain. Probes attached to cysteines at positions 63 or 89 in the C-terminal domain, however, did not exhibit intersubunit self-transfer. These results indicate that these residues in the C-terminal domains are, on average, separated by greater than 85 A. Intersubunit self-transfer does occur in a C-89 double mutation variant lacking 11 residues in the putative hinge region, indicating that the loss of the hinge region brings the two C-terminal domains closer together. Rapid subunit exchange between unlabeled wild-type L7/L12 and L7/L12 variants labeled in the N-terminal domain was also demonstrated by the loss of self-transfer upon mixing of the two proteins.

Dihydropyridine and ryanodine binding in ventricles from rat, trout, dogfish and hagfish.

In the adult mammalian heart, the majority of Ca2+ required for contraction is released from the sarcoplasmic reticulum (SR) via the Ca2+-release channel or ryanodine receptor (RyR). Such release is dependent upon a relatively small influx of Ca2+ entering the cell across the sarcolemma (SL) by means of the L-type Ca2+ channel or the dihydropyridine receptor (DHPR). In lower vertebrates, there is indirect evidence suggesting that Ca2+ influx across the SL may be sufficient to support contraction in the absence of Ca2+ release from the SR. This apparent difference in myocardial excitation-contraction (E-C) coupling was investigated further by determining DHPR and RyR densities in ventricular homogenate preparations from rat, trout, dogfish and hagfish. DHPR Bmax values (means +/- S.E.M.) were highest in rat (0.30 +/- 0.01 pmol mg-1), lower in trout (0.16 +/- 0.01 pmol mg-1) and dogfish (0.27 +/- 0.03 pmol mg-1), and slightly above the level of detection in hagfish (0.03 +/- 0.01 pmol mg-1). The DHPR dissociation constants (Kd) of 40-70 pmoll-1 in these three species were of similar magnitude. RyR binding revealed both high- and low-affinity sites in all species. RyR Bmax for the high-affinity site was greatest in the rat (0.68 pmol mg-1), lower in trout (0.19 pmol mg-1) and dogfish (0.07 pmol mg-1) and lowest in hagfish (0.01 pmol mg-1). The RyR Kd1 values for the high-affinity sites were comparable in all preparations (range 12-87 nmoll-1). The quantitative expression of RyRs in these species is consistent with the relative amount of SR present as indicated in physiological experiments and electron micrographs. Taking into consideration myocyte morphology of teleost and elasmobranch species, the data are consistent with a greater reliance on Ca2+ influx across the SL during E-C coupling in lower vertebrates, although a functional role for Ca2+ release from the SR in the more active species await further investigation.

Tetramethylrhodamine dimer formation as a spectroscopic probe of the conformation of Escherichia coli ribosomal protein L7/L12 dimers.

The fluorescent probe tetramethylrhodamine iodoacetamide was attached to cysteine residues substituted at various specific locations in full-length and deletion variants of the homodimeric Escherichia coli ribosomal protein L7/L12. Ground-state tetramethylrhodamine dimers form between the two subunits of L7/L12 depending upon the location of the probe. The formation of tetramethylrhodamine dimers caused the appearance of a new absorption band at 518 nm that was used to estimate the extent of interaction of the probes in the different protein variants. Intersubunit tetramethylrhodamine dimers form when tetramethylrhodamine acetamide is attached to two different sites in the N-terminal domain of the L7/L12 dimer (residues 12 or 33), but not when attached to sites in the C-terminal domain (residues 63, 89, or 99). The tetramethylrhodamine dimers do form at sites in the C-terminal domain in L7/L12 variants that contain deletions of 11 or 18 residues within the putative flexible hinge that separates the N- and C-terminal domains. The tetramethylrhodamine dimers disappear rapidly (within 5 s) upon addition of excess unlabeled wild-type L7/L12. It appears that singly labeled L7/L12 dimers are formed by exchange with wild-type dimers. Binding of L7/L12:tetramethylrhodamine cysteine 33 or cysteine 12 dimers either to L7/L12-depleted ribosomal core particles, or to ribosomal protein L10 alone, results in disappearance of the 518-nm absorption band. This result implies a conformational change in the N-terminal domain of L7/L12 upon its binding to the ribosome, or to L10.

Location and domain structure of Escherichia coli ribosomal protein L7/L12: site specific cysteine crosslinking and attachment of fluorescent probes.

Five different variants of L7/L12 containing single cysteine substitutions, two in the N-terminal (NTD) and three in the C-terminal domain (CTD), were produced, modified with [125I]N-[4-(p-azidosalicylamido)butyl]-3-(2'-pyridyldithio) propionamide ([125I]APDP), a sulfhydryl-specific, heterobifunctional, cleavable photo-cross-linking reagent, and reconstituted into ribosomes. These were irradiated, the total proteins were extracted and reductively cleaved, and the cross-linked proteins were identified. The effect of zero-length disulfide cross-linking on binding and activity was also determined. The same sites in L7/L12 were used to attach a rhodamine dye. The formation of ground-state rhodamine dimers caused the appearance of a new absorption band at 518 nm that was used to estimate the extent of interaction of the probes in the free protein and in complexes with L10. The three sites in the CTD, but not the N-terminal sites, cross-linked to L2 and L5 and to 30S proteins S2, S3, S7, S14, and S18 in a manner influenced by elongation factors. Binding to the ribosome and, therefore, function were blocked by zero-length cross-linking within the NTD, but not the CTD. Binding also disrupted rhodamine dimers in the NTD. No rhodamine dimers formed in the CTD.

Inhibition of the creatine kinase reaction decreases the contractile reserve of isolated rat hearts.

To define the relation between phosphoryl transfer via creatine kinase (CK) and the ability of the intact beating heart to do work, we chemically inhibited CK activity and then measured cardiac performance under physiological and acute stress conditions. Isolated perfused rat hearts were exposed to iodoacetamide (IA) and subjected to one of three cardiac stresses: hypercalcemic (Ca2+ = 3 mM) buffer perfusion (n = 7), norepinephrine (2 mumol/min) infusion (n = 6), or hypoxic buffer perfusion (n = 5). IA decreased CK activity to near zero, measured in intact hearts by 31P magnetization transfer, and to 2% of control CK activity, measured in myocardial homogenates. The CK isoenzyme profile was unchanged, suggesting nonselective IA inhibition of all isoenzymes. Mitochondria isolated from IA-treated hearts had normal ADP:O ratios, state 3 respiratory rates, and unchanged acceptor and respiratory control ratios. Neither actomyosin adenosinetriphosphatase nor adenylate kinase activities were changed. After IA exposure, end-diastolic pressure, left ventricular developed pressure, and heart rate were unchanged for at least 30 min at physiological perfusion pressures, but large changes were observed during stress conditions. The increase in left ventricular developed pressure induced by hypercalcemic perfusion and by norepinephrine infusion decreased by 39 and 54%, respectively. During hypoxia, the rate of phosphocreatine depletion was decreased by 57%, left ventricular developed pressure declined, and end-diastolic pressure increased faster than in controls. These results show that inhibition of CK to < 2% of control activity by IA reduced contractile reserve by approximately 50%. We conclude that CK activity is essential for the expression of the full dynamic range of myocardial performance.

Temperature dependence of unitary properties of an ATP-dependent potassium channel in cardiac myocytes.

The temperature dependence of the properties of unitary currents in cultured rat ventricular myocytes has been studied. Currents flowing through an ATP-dependent K+ channel were recorded from inside-out patches with the bath temperature varied from 10 degrees to 30 degrees C. The channel conductance was 56 pS at room temperature (22 degrees C), and the amplitudes of unitary currents and the channel conductance exhibited a relatively weak (Q10 from 1.4 to 1.6) dependence on temperature. The temperature dependence of channel mean open times was biphasic with the low temperature (10-20 degrees C) range showing a relatively stronger temperature dependence (Q10 of 2.3) than the high temperature (20-30 degrees C) range (Q10 of 1.6). The activation energies for the two regions were determined from an Arrhenius plot with the activation energy, corresponding to the lower temperature range, near 16 kcal/mol. Thermodynamic analysis, using transition rate theory, indicated that the formation of a transition state prior to channel closure to be associated with a positive entropy component for the high Q10 region.

Injuries resulting from motorized personal watercraft.

To determine if there is a widespread problem with personal watercraft (jet ski) injuries throughout the United States, we reviewed the hospital records of patients who were treated at this institution for injuries incurred while they were operating a motorized personal watercraft or jet ski. All of the patients were under the age of 19 and suffered severe fractures or lacerations. To assess the extent of the problem with these injuries regionally, we collected data from 8 midwestern states for 1989. Sixty-four personal watercraft accidents involving 90 victims were reported in the survey. Fifty-three of 90 patients sustained fractures, lacerations, or head injuries. Seventy-nine of 90 were under age 25, and 24 patients were under 16 years of age. The need for supervision and the potential for serious injury while operating personal watercraft is supported by these findings.