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Biomedical subjects

K A Harris

Publications and source records attributed to K A Harris.

At least 55 records · Page 3Linked to original sources
PubMed1995

In vivo structural analysis of spliced leader RNAs in Trypanosoma brucei and Leptomonas collosoma: a flexible structure that is independent of cap4 methylations.

The formation of the mRNA 5' end in trypanosomatid protozoa is carried out by trans-splicing, which transfers a spliced leader (SL) sequence and its hypermethylated cap (cap4) from the SL RNA to the pre-mRNA. Previous in vitro studies with synthetic uncapped RNAs have shown that the SL sequence of Leptomonas collosoma can assume two alternate conformations, Form 1 and Form 2, with Form 1 being the dominant one. To gain information about the structure of the SL RNA in vivo, in its protein-rich environment, we have used permeable Trypanosoma brucei and L. collosoma cells for chemical modification experiments. We introduce the use in vivo of the water-soluble reagents CMCT and kethoxal. In contrast to the in vitro results, the Form 2 secondary structure predominates. However, there are chemically accessible regions that suggest conformational flexibility in SL RNPs and a chemically inaccessible region suggestive of protection by protein or involvement in tertiary interactions. Using complementary 2'-O-methyl RNA oligonucleotides, we show that T. brucei SL RNA can be induced to switch conformation in vivo. SL RNA stripped of proteins and probed in vitro does not display the same Form 2 bias, indicating that SL RNA structure is determined, at least in part, by its RNP context. Finally, the methyl groups of the cap4 do not seem to affect the secondary structure of T. brucei SL RNA, as shown by chemical modification of undermethylated SL RNA probed in vivo.

Aldehydes↗
PubMed1995

Tau phosphorylation in brain slices: pharmacological evidence for convergent effects of protein phosphatases on tau and mitogen-activated protein kinase.

Tau is a neuron-specific, microtubule-associated protein that forms paired helical filaments (PHFs) of Alzheimer's disease when aberrantly phosphorylated. We have attempted to elucidate the protein kinases and phosphatases that regulate tau phosphorylation. Incubation of rat, human, and rhesus monkey temporal neocortex slices with the phosphatase inhibitor okadaic acid induced epitopes of tau similar to those found in PHFs. Okadaic acid (1-20 microM) induced variant forms of tau at 60-68 kDa, which were recognized by the monoclonal antibodies Alz-50 (in humans only) and 5E2 and two polyclonal antipeptide antisera, OK-1 and OK-2. The phosphorylation-sensitive monoclonal antibody Tau-1 failed to recognize the slowest mobility forms of tau after okadaic acid treatment. FK-520 (1-10 microM), a potent inhibitor of calcineurin activity, was tested in brain slices and found not to alter tau mobility. However, combinations of FK-520 (5 microM) and okadaic acid (100 nM) caused tau mobility shifts similar to those seen after 10 microM okadaic acid treatment; similar results were seen using the calcineurin-selective inhibitor cypermethrin. Treatment of human slices with 10 microM okadaic acid decreased both protein phosphatase 2A and calcineurin activity; FK-520 inhibited only protein phosphatase 2B activity. A proposed tau-directed kinase, 42-kDa mitogen-activated protein kinase (p42mapk), was activated by okadaic acid (> 100 nM) but not FK-520 (5 microM). Nerve growth factor (100 ng/ml) activated p42mapk, particularly when used in combination with 100 nM okadaic acid; changes in tau mobility were seen when this kinase was activated. Forskolin (2 microM) antagonized the effects of nerve growth factor on both p42mapk activity and tau phosphorylation; forskolin alone had little effect on PHF-like tau formation induced by phosphatase inhibitors. These results outline complex interactions between tau-directed protein kinases and protein phosphatases and suggest potential sites for therapeutic intervention.

Amino Acid Sequence↗
PubMed1995

Skeletal muscle injury induced by ischemia-reperfusion.

OBJECTIVE: To study the temporal progression of injury in skeletal muscle after ischemia-reperfusion insult by means of intravital videomicroscopy and nuclear fluorescent dyes. DESIGN: A controlled study in an animal model. SETTING: A vascular research laboratory at a university-affiliated hospital. SUBJECTS: Eight male Wistar rats, for each of which the extensor digitorum longus muscle of the hind limb was exposed and prepared. INTERVENTIONS: Two hours of complete no-flow ischemia followed by 90 minutes of reperfusion in five of the rats; the other three rats acted as controls and underwent the same surgical procedure but not ischemia. During the reperfusion period the fluorescent vital dyes bisbenzimide, which permeates all cells, and ethidium bromide, which permeates cells with damaged membranes, were applied. Recordings to videotape were made with the intravital microscope very 15 minutes during the reperfusion period. MAIN OUTCOME MEASURES: The number of perfused capillaries crossing three straight lines on the video monitor were counted as a measure of microvascular dysfunction. An index of tissue injury was calculated as the ratio of the number of nuclei stained by ethidium bromide to the number stained by bisbenzimide (E/B). The number of stuck and rolling leukocytes and the velocity of the rolling leukocytes were determined in postcapillary venules. RESULTS: The mean number of perfused capillaries (and standard error of the mean) fell from 20.71 (1.64)/mm before ischemia to 11.69 (1.18)/mm during reperfusion in the experimental group but remained constant in the control group. In the experimental group E/B progressed from 0.43 (0.05) at the onset of reperfusion to 0.87 (0.03) at the end of reperfusion, the number of rolling leukocytes increased from a preischemia mean of 4.00 (1.90) to 14.80 (1.30)/1000 microns2, and the number of stuck leukocytes increased from 1.42 (0.20) to 9.20 (0.70)/1000 microns2. The velocity of the rolling leukocytes did not differ between the control and the experimental groups. CONCLUSIONS: Although microvascular perfusion decreased quickly to a constant level after 2 hours of noflow ischemia plus reperfusion, a progressive increase in tissue injury occurred, which may correlate with the number of stuck leukocytes.

Animals↗
PubMed1994

Postarteriographic femoral pseudoaneurysms: treatment with ultrasound-guided compression.

Pseudoaneurysms following femoral arterial catheterization are increasingly common and are related to factors such as catheter size, periprocedural anticoagulation, hypertension, and improper technique. Ultrasound-guided compression as a noninvasive technique for repair of these lesions was prospectively evaluated in 16 patients whose mean age was 61 years. Nine patients were on anticoagulants and six had hypertension. All patients presented with an enlarging groin hematoma, a pulsatile mass, and/or a bruit following femoral catheterization. Seventeen femoral artery pseudoaneurysms, including one with an associated arteriovenous fistula, were detected using color-flow Doppler imaging. Three pseudoaneurysms thrombosed spontaneously. The remaining 14 were managed with compressive therapy lasting from 20 to 100 minutes. No complications were encountered during the compressions and 10 false aneurysms (71%) responded completely (mean time to thrombosis of 38 minutes). Two lesions responded partially to compression and there were two failures, the latter associated with excessive anticoagulation in one patient and a well-established pseudoaneurysm in the second patient. Only one pseudoaneurysm (6%) in the series required surgical correction. Ultrasound-guided compression of acute pseudoaneurysms in the groin is a safe, inexpensive, and effective method of treatment.

Adult↗
PubMed1994

Tau phosphorylation in human, primate, and rat brain: evidence that a pool of tau is highly phosphorylated in vivo and is rapidly dephosphorylated in vitro.

The extent of tau phosphorylation is thought to regulate the binding of tau to microtubules: Highly phosphorylated tau does not bind to tubules, whereas dephosphorylated tau can bind to microtubules. It is interesting that the extent of tau phosphorylation in vivo has not been accurately determined. Tau was rapidly isolated from human temporal neocortex and hippocampus, rhesus monkey temporal neocortex, and rat temporal neocortex and hippocampus under conditions that minimized dephosphorylation. In brain slices, we observed that tau isolated under such conditions largely existed in several phosphorylated states, including a pool that was highly phosphorylated; this was determined using epitope-specific monoclonal and polyclonal antibodies. This highly phosphorylated tau was dephosphorylated during a 120-min time course in vitro, presumably as a result of neuronal phosphatase activity. The slow-mobility forms of tau were shifted to faster-mobility forms following in vitro incubation with alkaline phosphatase. Laser densitometry was used to estimate the percent of tau in slow-mobility, highly phosphorylated forms. Approximately 25% of immunoreactive tau was present as slow-mobility (66- and 68-kDa) forms of tau. The percentage of immunoreactive tau in faster-mobility pools (42-54 kDa) increased in proportion to the decrease in content of 66-68-kDa tau as a function of neuronal phosphatases or alkaline phosphatase treatment. These data suggest that the turnover of phosphorylated sites on tau is rapid and depends on neuronal phosphatases. Furthermore, tau is highly phosphorylated in normal-appearing human, primate, and rodent brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1994

Peripheral aneurysms.

Aneurysms of the peripheral arteries usually involve the femoral or popliteal regions. Although not as common as abdominal aortic aneurysms, they carry a significant morbidity if left untreated. Rupture is a rare complication, but embolization or thrombosis may frequently threaten the viability of the limb. Elective repair of peripheral aneurysms will prevent complications of these lesions. This paper outlines the history, diagnosis, and treatment of peripheral aneurysms with emphasis on nursing care.

Aneurysm↗
PubMed1994

Intraoperative thrombolytic therapy as a treatment of small vessel thrombosis: an experimental study.

OBJECTIVE: This paper studies the effect of three doses of Streptokinase infused intra-operatively into an animal model of small vessel thrombosis. EXPERIMENTAL DESIGN: This is a controlled trial of intraoperative infusion of thrombolytic agent into a thrombosed arterial segment compared to no infusion into the contralateral limb. EXPERIMENTAL SUBJECTS: 19 New Zealand White rabbits were stratified into 3 groups. INTERVENTIONS: Thrombosis was achieved by infusing a mixture of topical thrombin and autologous blood into individual iliac arteries of the New Zealand white rabbit. A randomly selected hind limb had an infusion of one of three doses of streptokinase (A = 2,500u; B = 5,000u; C = 10,000u) in saline over 20 minute period. Preinfusion and post infusion angiography was performed. MEASUREMENTS: Angiograms were ranked by a radiologist blinded to the side of infusion and clotting parameters were assessed. RESULTS: All limbs at all doses of streptokinase infusion (SK) showed significant clot lysis when compared to the non-infused limb(C). The percentage of improved segments is as follows: iliac: SK = 100%, C = 79%; femoral SK = 79%, C = 32%; tibial SK = 52%, C = 5%. Although there was an elevation in clotting time and a reduction in fibrinogen, levels remained within normal limits. CONCLUSIONS: Streptokinase infused directly into thrombus in arteries even in low doses significantly enhances thrombolysis of vessels which are too small to be cleared by mechanical means. No significant systemic complications were encountered.

Animals↗
PubMed1993

Okadaic acid induces hyperphosphorylated forms of tau protein in human brain slices.

Hyperphosphorylated forms of the microtubule-associated protein tau are components of the paired helical filaments (PHFs) seen in patients with Alzheimer's disease. Slices of human lateral temporal cortex were obtained from tissues removed incidental to resections for intractable hippocampal epilepsy. Tau phosphorylation in temporal lobe slices was determined using mobility shifts after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodetection with the monoclonal antibodies Alz-50, 5E2, and Tau-1. The results indicate that tau phosphorylation was altered in a dose-dependent manner by the phosphatase inhibitor okadaic acid, but not by N-methyl-D-aspartate, quisqualate, or kainate. The slowest mobility forms of tau, termed "PHF-like tau," produced by okadaic acid treatment were dephosphorylated by purified protein phosphatase 2B (calcineurin). Formation of PHF-like tau peptides was blocked by KN-62, 1[N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazi ne, an inhibitor of Ca2+/calmodulin-dependent protein kinase II. The protein kinase inhibitor staurosporine also prevented formation of PHF-like tau. These data suggest that phosphorylation of tau is regulated by Ca(2+)-dependent protein kinases and okadaic acid-sensitive protein phosphatases, alterations of which may be implicated in the pathogenesis of Alzheimer's disease.

Adult↗
PubMed1993

Spatial proximity and sequence localization of the reactive sulfhydryls of porphobilinogen synthase.

The zinc metalloenzyme porphobilinogen synthase (PBGS) contains several functionally important, but previously unidentified, reactive sulfhydryl groups. The enzyme has been modified with the reversible sulfhydryl-specific nitroxide spin label derivative of methyl methanethiosulfonate (MMTS), (1-oxyl-2,2,5,5-tetramethyl-delta 3-pyrroline-3-methyl)methanethiosulfonate (SL-MMTS) (Berliner, L. J., Grunwald, J., Hankovszky, H. O., & Hideg, K., 1982, Anal. Biochem. 119, 450-455). EPR spectra show that SL-MMTS labels three groups per PBGS subunit (24 per octamer), as does MMTS. EPR signals reflecting nitroxides of different mobilities are observed. Two of the three modified cysteines have been identified as Cys-119 and Cys-223 by sequencing peptides produced by an Asp-N protease digest of the modified protein. Because MMTS-reactive thiols have been implicated as ligands to the required Zn(II), EPR spectroscopy has been used to determine the spatial proximity of the modified cysteine residues. A forbidden (delta m = 2) EPR transition is observed indicating a through-space dipolar interaction between at least two of the nitroxides. The relative intensity of the forbidden and allowed transitions show that at least two of the unpaired electrons are within at most 7.6 A of each other. SL-MMTS-modified PBGS loses all Zn(II) and cannot catalyze product formation. The modified enzyme retains the ability to bind one of the two substrates at each active site. Binding of this substrate has no influence on the EPR spectral properties of the spin-labeled enzyme, or on the rate of release of the nitroxides when 2-mercaptoethanol is added.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Ancrod versus heparin for anticoagulation during vascular surgical procedures.

PURPOSE: The purpose of this study was to compare the conduct and early results of infrainguinal vascular reconstructions with use of ancrod or heparin for anticoagulation. METHODS: To test the hypothesis that ancrod was an effective alternative to heparin, 28 patients requiring infrainguinal bypass surgery were randomized to receive heparin during operation or ancrod before operation over a period of 12 hours to deplete circulating fibrinogen (0.2 to 0.5 gm/L). RESULTS: No clotting of blood within the grafts or native vessels was noted during the conduct of the surgical procedures in either group. No excessive bleeding was detected during operation in either group. The operative procedure, complication rate, and hospital course were also indistinguishable; patency at 1 month was also equal. CONCLUSION: Fibrinogen depletion with ancrod provides anticoagulation for the conduct of infrainguinal vascular reconstructions that is as effective as heparin. When heparin is contraindicated ancrod is an effective and safe alternative.

Aged↗
PubMed1993

Myocardial and circulatory performance during the ischemic phase of superior mesenteric artery occlusion.

OBJECTIVE: To investigate myocardial and circulatory parameters during the acute ischemic phase of mesenteric artery occlusion. DESIGN: A prospective, randomized, control trial. SUBJECTS: Twelve, adult, mongrel dogs. INTERVENTIONS: In seven dogs the superior mesenteric artery was occluded with two silk ligatures (experimental group). In five dogs the ligatures were not tied (control group). Measurements were made during 7 hours of occlusion. MAIN OUTCOME MEASURES: Myocardial performance and circulatory performance. RESULTS: There were significant (p < 0.05) reductions in arterial blood pressure, mean pulmonary artery pressure, diastolic pulmonary artery pressure and cardiac index in dogs exposed to intestinal ischemia compared with the control dogs. No differences were identified in ventricular performance, stroke volume index or peripheral vascular resistance index. CONCLUSIONS: Early cardiac and central circulatory changes in massive intestinal ischemia are due to intravascular hypovolemia. Sepsis and myocardial depressant factors were not found to be a cause of death.

Animals↗
PubMed1993

The management of chronic venous disease.

Chronic venous disease represents a spectrum of problems ranging from the merely cosmetic to disabling venous ulcers. Chronic venous insufficiency can result in disfigurement, disability, and a financial burden on the patient and society. Delivery of optimal care of patients with venous disease requires a thorough understanding of this complex system and of current methods of diagnosis and treatment. Successful outcome of treatment depends on appropriate patient selection, meticulous surgery, outstanding nursing care, and patient education.

Chronic Disease↗
PubMed1992

5-Chlorolevulinate modification of porphobilinogen synthase identifies a potential role for the catalytic zinc.

Porphobilinogen synthase (PBGS) is a Zn(II) metalloenzyme which catalyzes the asymmetric condensation of two molecules of 5-aminolevulinate (ALA). The nitrogen of the first substrate ends up in the pyrrole ring of product (P-side ALA); by contrast, the nitrogen of the second substrate molecule remains an amino group (A-side ALA). A reactive mimic of the substrate molecules, 5-chlorolevulinate (5-CLA), has been prepared and used as an active site directed irreversible inhibitor of PBGS. Native octameric PBGS binds eight substrate molecules and eight Zn(II) ions, with two types of sites for each ligand. As originally demonstrated by Seehra and Jordan [(1981) Eur. J. Biochem. 113, 435-446], 5-CLA inactivates the enzyme at the site where one of the two substrate molecules binds, and modification at four sites per octamer (one per active site) affords near-total inactivation. Here we report that 5-CLA-modified PBGS (5-CLA-PBGS) can bind up to four substrate molecules and four Zn(II) ions. Contrary to the conclusion of Seehra and Jordan, we find that the preferential site of 5-CLA inactivation is the A-side ALA binding site. On the basis of the dissociation constants, the metal ion binding sites lost upon 5-CLA modification are assigned to the four catalytic Zn(II) sites. 5-CLA-PBGS is shown to be modified at cysteine-223 on half of the subunits. We conclude that cysteine-223 is near the amino group of A-side ALA and propose that this cysteine is a ligand to the catalytic Zn(II). The vacant substrate binding site on 5-CLA-PBGS is that of P-side ALA. We have used 13C and 15N NMR to view [4-13C]ALA and [15N]ALA bound to 5-CLA-PBGS. The NMR results are nearly identical to those obtained previously for the enzyme-bound P-side Schiff base intermediate [Jaffe et al. (1990) Biochemistry 29, 8345-8350]. It appears that, in the absence of the catalytic Zn(II), 5-CLA-PBGS does not catalyze the condensation of the amino group of the P-side Schiff base intermediate with the C4 carbonyl derived from 5-CLA. On this basis we propose that Zn(II) plays an essential role in formation of the first bond between the two substrate molecules.

Amino Acid Sequence↗