Biomedical subjects
Publications and source records attributed to R Strong.
We describe two patients awaiting orthoptic liver transplantation in whom diagnostic paracentesis for the diagnosis of spontaneous bacterial peritonitis led to major hemorrhage. Accordingly, we advise caution in patients such as ours, particularly if the prothrombin time or partial thromboplastin time is more than twice the control value.
Neurotransmission is impaired in age-related disorders, such as Alzheimer's and Parkinson's diseases, which has prompted many investigations into the neurochemistry of the aging human brain. Of all the neurotransmitter systems studied, age-related changes in parameters of the serotonergic, cholinergic, and dopaminergic systems are the most reliably measured. The association of these neurotransmitters, respectively, with mood, memory, and motor function has fueled interest in how changes in neurochemistry may contribute to age-associated behavioral changes and possibly predispose older persons to diseases of late life. The evidence suggests that impaired neurotransmission may be responsible for at least some of the behavioral abnormalities associated with aging. Moreover, age-related neurodegenerative diseases may evolve from the interaction between defects in specific neurochemical mechanisms and as-yet undefined pathophysiologic processes.
Chromatography and electrophoresis have become extremely valuable and important methods for the separation, purification, detection and analysis of biopolymers and HPLC/HPCE may become the premier, preferable approaches for both qualitative and quantitative analyses of most proteins, especially from recombinant materials. This includes smaller peptides, polypeptides, proteins, antibodies and all types of protein or antibody-conjugates (antibody-enzyme, protein-fluorescent probe, antibody-drug and so forth). This entire Topical Issue of Journal of Chromatography emphasizes the application of chromatography and electrophoresis to protein analysis. This particular review deals with approaches to the selective tagging or labeling of proteins at trace (minute) levels, again using either chromatography or electrophoresis, with the emphasis on modern HPLC/HPCE methods and approaches. We discuss here both pre- and post-column labeling methods and reagents, techniques for realizing selective labeling of proteins or antibodies, applicable approaches to protein preconcentration in both HPLC and HPCE areas and in general, methods for improving (lowering) detection limits for proteins utilizing chemical or physical derivatization and/or preconcentration techniques. There are really two major goals or emphases in that which follows: (1) methods for selective labeling of proteins prior to or after HPLC/HPCE and (2) labeling of proteins at trace levels for improved separation-detection and lowered detection limits. We discuss here a large number of specific references related to both pre- and post-column/capillary derivatizations for proteins, as well as methods for improved detectability in both HPLC and HPCE by, for example, analyte preconcentration on a solid-phase extractor or membrane support, capillary isotachophoresis and other methods. Selective reactions or derivatizations on proteins refers to the ability to tag the protein at specific (e.g. reactive amino sites) in a controlled manner, with the products having the same number of tags all at the very same site or sites. The products are all the same species, having the same number of tags at the same locations on the protein. Selective reactions can also refer to the idea of tagging all of the protein sample at only a single, same site or at all available sites, homogeneously.
We have developed HPCE methods for the analysis of sulfomycin (trivial name) and related compounds (code name, crude material = U82127 = I), which is an animal growth promoter derived from a fermentation beer. The crude material, I, isolated from the fermentation consisted of more than 40 components which were not completely separated by conventional HPLC. Thus, as a complementary analysis method, we have optimized HPCE conditions for I using various capillaries including uncoated, coated, and packed using various buffers. The optimized HPCE conditions were obtained with an uncoated fused-silica capillary and a buffer that consisted of 30 mM Tris-tricine, 10 mM SDS, 10 mM NaCl and 20% MeOH, pH 8.0. Using these HPCE conditions, we were able to separate the one main component collected from the HPLC effluent into two or three components. In order to identify the main components of the fermentation product, an off-line HPLC-HPCE-MS analysis for I was performed. From the MALDI-TOF-MS results, the separated components collected from HPCE had different molecular masses. Four lots of I samples having different characteristics were also analyzed by HPCE to investigate lot-to-lot differences in peak profiles. The four lots of I were found to have very similar peak profiles. In this paper, I refers to the crude fermentation product and sulfomycin to the purified, major HPLC component of I.
1. After simultaneous intravenous injection as a mixture, 99mTc-labelled diethylenetriaminepentaacetic acid (99mTc-DTPA; molecular mass 492 Da) and inulin (approximately 6000 Da) gave arterial plasma clearance curves consisting of three exponentials, the time courses of which were not significantly different between the two solutes. 2. The ratio of 99mTc-DTPA to inulin concentration in antecubital venous plasma (normalized to the ratio in arterial plasma at 30 s) was 0.6, significantly less than unity, within 2 min after intravenous injection, but increased to reach unity by 60 min. The minimum concentration ratio of 99mTc-DTPA to inulin in arterial plasma was 0.75 at 4 min, also rising to just above unity at 60 min. 3. The extraction fraction from plasma to interstitial space was higher for 99mTc-DTPA (approximately 0.5) than for inulin (approximately 0.2). For both solutes, the net extraction fraction decreased with time, becoming negative at about 25 min after injection. Thereafter, the net extraction fractions remained negative, between -0.05 and -0.1, and not significantly different between the two solutes. 4. 99mTc-DTPA time-activity curves recorded over the limbs with scintillation probes were biphasic, with an initial phase corresponding closely in time with the first exponential of the arterial 99mTc-DTPA plasma clearance curve. The second phase corresponded in time to the intermediate exponential of the arterial 99mTc-DTPA plasma clearance curve. 5. The time course of net 99mTcm-DTPA extraction fraction across the forearm vascular bed was bi-exponential, with phases corresponding in time with the two phases of the limb uptake curves. 6. Deconvolution analysis of the limb time-activity curves, using the arterial time-concentration curve as the input function, gave bi-exponential 99mTc-DTPA impulse response curves in which the time courses of the exponentials corresponded with the first and intermediate exponentials of the arterial 99mTc-DTPA clearance curve. 7. The bi-exponential nature of the equilibrium of 99mTc-DTPA between vascular and interstitial compartments suggests the presence of two separate functional volumes within the interstitial space. Although 99mTc-DTPA and inulin clearly diffuse at different rates across the endothelium, as would be expected from their disparate sizes, the similarity in the time courses of their initial exponentials and simultaneous equalization of transfer rates (i.e. when net extraction fraction was zero) is consistent with the hypothesis that inulin moves initially into a smaller functional interstitial fluid volume than 99mTc-DTPA. The total distribution volumes, however, are not significantly different between the two solutes.
During reperfusion after ischemia, deleterious biochemical processes can be triggered that may antagonize the beneficial effects of reperfusion. Research into the understanding and treatment of reperfusion injury (RI) is an important objective in the new era of reperfusion therapy for stroke. To investigate RI, permanent and reversible unilateral middle cerebral artery/common carotid artery (MCA/CCA) occlusion (monitored by laser Doppler) of variable duration in Long-Evans (LE) and spontaneously hypertensive (SH) rats and unilateral MCA and bilateral CCA occlusion in selected LE rats was induced. In LE rats, infarct volume after 24 hours of permanent unilateral MCA/CCA occlusion was 31.1 +/- 34.6 mm3 and was only 28% of the infarct volume after 120 to 300 minutes of reversible occlusion plus 24 hours of reperfusion, indicating that 72% of the damage of ischemia/reperfusion is produced by RI. When reversible ischemia was prolonged to 480 and 1080 minutes, infarct volume was 39.6 mm3 and 16.6 mm3, respectively, being indistinguishable from the damage produced by permanent ischemia and significantly smaller than damage after 120 to 300 minutes of ischemia. Reperfusion injury was not seen in SH rats or with bilateral CCA occlusion in LE rats, in which perfusion is reduced more profoundly. Reperfusion injury was ameliorated by the protein synthesis inhibitor cycloheximide or spin-trap agent N-tert-butyl-alpha-phenylnitrone pretreatment.
BACKGROUND: The hypothesis being tested in this paper is that endotoxin levels in donors and in recipients during liver transplantation influences postoperative outcome. METHODS: Endotoxin levels in systemic venous and portal venous blood were measured using in 46 adult donors and 44 adult recipients (47 liver transplants) during the period 1992-95. Endotoxin was measured using a modification of the Limulus amoebocyte lysate (LAL) assay. RESULTS: In the donor, systemic endotoxin levels were above normal levels at 10.0 +/- 1.3 pg/mL from the start and rose to 15.8 +/- 2.9 pg/mL after dissection of the hilar structures, but fell to 10.6 +/- 0.8 pg/mL just prior to the removal of the liver (control = 7.8 pg/mL). The mean portal venous endotoxin levels were 18.2 +/- 3.4 pg/mL after dissection of the hilar structures and 12.6 +/- 0.9 pg/mL after cannulation of the portal vein. In the recipients, the highest level in the portal venous blood occurred at the end of the anhepatic phase (46.5 +/- 6.7 pg/mL). The systemic venous samples in the recipients were elevated to start with, but fell rapidly to 19.3 +/- 1.5 pg/mL 24 h postoperatively, and to 13.2 +/- 1.0 pg/mL by day 7. The endotoxin concentrations were higher in recipients who developed complications. CONCLUSIONS: Endotoxin is elevated throughout the recipient transplantation procedure and up to 7 days postoperatively. High levels of endotoxin at induction, the anhepatic phase and at certain time points correlated with patients who developed postoperative complications.
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The presence of auto-antibodies and hypergammaglobulinaemia in patients with primary sclerosing cholangitis (PSC) suggest an overactive humoral immune system. Serum cytokines, measured using in-house double monoclonal sandwich ELISA, were used to assess the state of cellular and humoral immunity in this condition by comparison with sex and age matched normal controls and patients with alcoholic cirrhosis (AC). Soluble CD23 (sCD23) as a marker of humoral immunity was significantly elevated in PSC (N = 31) relative to patients with AC (N = 12) and the control group (N = 20) (P < 0.0001 and P < 0.001 respectively). Serum interleukin (IL) 10, as an anti-inflammatory cytokine and IL8, as a marker of neutrophil activation were significantly elevated in patients with PSC relative to those with AC and the controls (P < 0.001 and P < 0.05 respectively). Interferon gamma, as a marker of cellular immunity, and granulocyte-macrophage colony stimulating factor, a marker of monocyte/macrophage function were similar in all the groups. Cytokines and sCD23 were no different between patients with AC and the control group. While more than two thirds of the patients with PSC were positive for ANCA, there was no correlation between the presence of ANCA or ANCA titre and serum levels of either IL8, IL10 and sCD23. These results suggest exaggerated humoral immunity in PSC. The raised levels of IL10 and IL8 in PSC are discussed in the context of inflammatory bowel disease and liver dysfunction.
We have developed high-performance immunoaffinity chromatography (HPIAC) methods for the detection and quantitation of bovine growth hormone releasing factor (GHRF), which could also be applicable to its metabolites in biofluids. These approaches have involved a combination of IAC using immobilized antibody (Ab) to GHRF, together with reversed-phase high-performance liquid chromatography (RP-HPLC) separations of initially isolated and concentrated protein, followed by selective detection, involving on-line immunodetection (ID) schemes. ID methods involved HPIAC supports of the Ab, together with synthesized Ab-fluorescein isothiocyanate conjugates. We have demonstrated optimization methods for each step of the entire hyphenated technique (IAC-HPLC-ID), and then actually quantitated GHRF using this overall system. The minimum detectable concentration was about 1 ng/5 ml (200 ppt) with fluorescence detection (excitation wavelength, 490 nm; emission wavelength, 510-650 nm). We have also tested a single blind, spiked biological sample (bovine plasma), spiked with a known level of GHRF. Accuracy (7.4%) and precision (S.D. = +/- 22%) were quite acceptable for a double immunoassay method.
The diagnosis of graft-versus-host disease following liver transplantation may be delayed because the clinical and pathological features are nonspecific. We report the use of microsatellites to support a diagnosis of GVHD in a patient who developed fever and a skin rash 28 days after liver transplantation. The pattern of microsatellite alleles amplified from the peripheral blood on day 51 posttransplant indicated that recipient and donor DNA were present in approximately equal proportions. Microsatellite typing is a simple and rapid method to identify high levels of circulating donor DNA to support a diagnosis of GVHD following liver transplantation.
Lubeluzole is a neuroprotective compound in the final stages of clinical evaluation. We evaluated the effects of intravenous followed by intraperitoneal doses of lubeluzole on histological outcome after reversible tandem middle cerebral/common carotid artery occlusion in Long-Evans rats, with particular emphasis on the time window of efficacy. Lubeluzole, started 15 min after the onset of ischemia, had no adverse physiological or behavioral effects and reduce maximal infarct volume produced by 120 min or more of arterial occlusion by approximately 50%, from 143.2 +/- 11.8 mm3 (p < 0.05). Lubeluzole did not prolong the duration of middle cerebral artery occlusion which could be tolerated before histological damage occurred. Lubeluzole was still effective if started 30 min after the onset of ischemia (34% reduction of maximal infarct volume; p < 0.05), but not after delays of 60 or 120 min. we conclude that lubeluzole has promise as a neuroprotective drug, particularly for more severe strokes, but must be started very rapidly after the onset of ischemia to be effective.
Between January 1985 and December 1994, 164 liver transplantations were performed on 141 children. There were 100 reduced-size and 64 whole-liver grafts. Primary closure of the abdominal wound was not possible in 21 patients because of liver size, bowel edema, and distension. Temporary SILASTIC patch closure of the abdominal wound was used. For 16 of the 21 patients, removal of the SILASTIC patch and abdominal wall closure were completed by the seventh postoperative day; for the others, these were accomplished by the end of 2 weeks. The method is recommended when primary wound closure is not possible.
We evaluated the effect of chronic administration of CDP-choline, an intermediate of phospholipid synthesis, on outcome from middle cerebral artery occlusion, ranging from 30 to 120 min in duration in spontaneously hypertensive rats. Rats were randomly assigned to either CDP-choline 500 mg kg-1 or saline. CDP-choline treatment was initiated by intraperitoneal injection 15 min after the onset of ischemia and continued once a day for 14 days. Morphologic damage and behavioral dysfunction (motor and sensorimotor performance) were evaluated, and the maximal morphologic damage (Volmax), maximal behavioral dysfunction (BDmax) as well as the duration of ischemia producing half-maximal morphologic damage (T50) or behavioral dysfunction (BD50) were calculated using a curve-fitting program (ALLFIT). Ischemia in control animals produced a Volmax of 103.3 +/- 13.6 mm3. CDP-choline did not affect this value (Volmax of 101.6 +/- 11.4 mm3). However, CDP-choline significantly extended the T50 from 38.3 +/- 5.9 to 60.5 +/- 4.3 min (p < 0.05). Similar to the morphologic outcome, CDP-choline had no effect on BDmax but significantly extended BD50 from 41.9 +/- 4.6 to 72.9 +/- 24.5 min (p < 0.05). Our results suggest that the effectiveness of CDP-choline is greater in animals demonstrating submaximal ischemic injury which in this model is produced by 30-75 min of ischemia (effect on T50 and BD50), than in animals suffering maximal ischemic injury produced by ischemia longer than 75 min (no effect on Volmax and BDmax). These results may reflect a threshold of biological membrane damage within which CDP-choline is able to restore phospholipid content/arrangement and retain membrane integrity.
Calcium/calmodulin-dependent protein kinase II (CaM-kinase) is a central enzyme in regulating neuronal processes. Imbalances in the activity and distribution of this enzyme have been reported following in vivo ischemia, and sustained decreases in activity correlate with subsequent neuronal death. In this report, mice that had been rendered deficient in the alpha subunit of CaM-kinase using gene knock-out technology were utilized to determine whether this enzyme is causally related to ischemic damage. Using a focal model of cerebral ischemia, we showed that homozygous knock-out mice lacking the alpha subunit exhibited an infarct volume almost twice that of wild-type litter mates. Heterozygous mice exhibited slightly less damage following ischemia than did homozygous mice, but infarct volumes remained significantly larger than those of wild-type litter mates. We conclude that reduced amounts of the alpha subunit of CaM-kinase predisposes neurons to increased damage following ischemia and that any perturbation that decreases the amount or activity of the enzyme will produce enhanced susceptibility to neuronal damage.
We tested the hypothesis that increasing durations of focal ischemia that have been shown to result in enlargement of cortical infarct will be associated with progression of behavioral dysfunction that can be measured by a battery of tests sufficiently sensitive and reproducible to detect a positive effect of pharmacotherapy. Untreated or N-methyl-D-aspartate receptor antagonist (CNS-1102)-treated spontaneously hypertensive rats underwent 45, 60, 90, or 120 min of tandem middle cerebral and common carotid artery occlusion followed by reperfusion. We then evaluated the extent of damage and its recovery for up to 21 days using nine behavioral tests aimed at analyzing strength, coordination, and bilateral asymmetry. Also using a graded bioassay that employs a curve-fitting computer program (ALLFIT) to correlate duration of ischemia with degree of behavioral dysfunction, we calculated the average of maximal behavioral dysfunction and duration of ischemia required to produce half-maximal behavioral dysfunction and compared these values in untreated controls with analogous values obtained from animals treated with CNS-1102. Three behavioral tests, forearm flex, tape (somatosensory neutralization), and foot-fault placing, were each separately and combined able to distinguish between the degrees of damage produced by increasing durations of ischemia. The behavioral abnormalities assessed using the tape test were reversible within a week, whereas those using forearm flex or foot-fault tests persisted for at least 21 days. CNS-1102 significantly reduced behavioral dysfunction measured by all three tests. This analysis of behavioral dysfunction represents a useful experimental model to grade efficacy of therapies aimed at protecting the brain from damage produced by acute stroke and might also be used to assess recovery from preexisting ischemic damage.
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BACKGROUND AND PURPOSE: In search of a better treatment for acute ischemic stroke, we evaluated the use of lubeluzole and hemodilution with diaspirin cross-linked hemoglobin (DCLHb) therapy to test whether treatment with two complementary acting compounds provides more potent protection than either treatment alone. METHODS: We used unilateral reversible middle cerebral artery (MCA) and common carotid artery (CCA) occlusion of various durations in Long-Evans rats to produce ischemic cortical lesions. We calculated the average maximal lesion volume (Volmax) and the time required to produce half maximal lesion size (T50) in control animals (n = 31) and evaluated the effects on cerebral perfusion and infarct size of treatment with lubeluzole (n = 23), hemodilution (to 30% hematocrit) with albumin (n = 17) or DCLHb (n = 23), and combined lubeluzole + DCLHb therapy initiated 15 minutes after MCA/CCA occlusion. RESULTS: The Volmax produced by MCA/CCA occlusion in control animals was 138.5 +/- 7.7 mm3, and T50 was 98.5 +/- 10.2 minutes. Lubeluzole alone reduced Volmax by 53% with no significant effect on T50. In contrast to lubeluzole, DCLHb hemodilution prolonged T50 by 68% with no significant effect on Volmax. Prolongation of T50 by DCLHb was not due to hemodilution itself, since a similar degree of hemodilution with albumin had no effect. Finally, combined lubeluzole+DCLHb rescued 72% of the tissue and augmented the effect of lubeluzole alone by 40% (Volmax, 66.3 +/- 13.0 versus 39.4 +/- 12.2 mm3) while prolonging T50 by 31%. CONCLUSIONS: Combination therapy for acute stroke using compounds with complementary action can result in more complete attenuation of neuronal damage and demonstrates the possible clinical utility of combined neuroprotective and reperfusion therapies.