Biomedical subjects

L Cook

Publications and source records attributed to L Cook.

At least 109 records · Page 6Linked to original sources

PubMed1987

Discriminative stimulus properties of a sigma receptor agonist in the rat: role of monoamine systems.

The role of the dopaminergic, noradrenergic and serotonergic systems in the production of the discriminative stimulus (DS) properties of the sigma receptor agonist (+)-N-allylnormetazocine [(+)-NANM/(+)-SKF 10,047] was measured in rats. The partial generalization of the noradrenergic agonist clonidine with (+)-NANM and partial antagonism of (+)-NANM by yohimbine suggest that the noradrenergic system may play a role in (+)-NANM DS. Activation of the dopaminergic system by apomorphine did not produce (+)-NANM DS however, haloperidol, which binds to the sigma receptor with high affinity, antagonized (+)-NANM DS. These data suggest that the haloperidol antagonism of (+)-NANM DS is a function of antagonism at the sigma receptor rather than antagonism at the dopamine receptor. Since yohimbine does not bind to the sigma receptor, yohimbine antagonizes (+)-NANM by a mechanism different from that of haloperidol. There was no evidence to suggest serotonergic mediation of (+)-NANM DS.

Animals↗

PubMed1987

Rabbit secretory components: identification of a third allotype, t63.

A third allotype of rabbit secretory component has been identified. The allotype previously referred to as t62 by our laboratory can now be subdivided into two allotypes, t62 and t63, with alloantisera capable of discriminating between the two. Results of family studies are consistent with a three allele system (t61, t62 and t63) at the t-locus. By SDS PAGE, electrophoretic mobilities of the multiple SC bands for each of the three allotypes are characteristic of the allotype; the apparent molecular sizes of the bands of the t62 allotype are 2 to 3 kDa lower than those for the t61 allotype. The banding patterns of the t61 and t63, although similar, are not identical to each other. Results of serologic cross-reaction studies and of tryptic peptide mapping studies suggest multiple structural differences between the allotypes as well as a closer relationship between t62 and t63 than between either of these allotypes and t61.

Alleles↗

PubMed1987

Biochemical and immunological identity of the hepatic peroxisomal and microsomal trans-2-enoyl CoA hydratase bifunctional protein.

In the present study, the hepatic microsomal and peroxisomal bifunctional trans-2-enoyl CoA hydratases were isolated and purified from rats treated with 2% di-(2-ethylhexyl)phthalate for 8 days. These two enzymes (microsomal and peroxisomal) were purified with the identical purification procedures and had identical molecular masses of 76 kDa. A single band was observed on an electrophoretic gel of an equimixture of the two proteins. Both preparations had identical pI's of 8.6 and pH optima of 6.0 for the dehydrogenase (reductase) and 7.5 for the hydratase activity. Two-dimensional gel analysis of an equimixture of the two preparations showed only one band. Ouchterlony double-diffusion analysis showed that an antibody raised against the purified microsomal enzyme interacted at a point with the peroxisomal enzyme, indicating immunologic identity. Western blot analysis demonstrated that the antibody formed a single band with total microsomal and peroxisomal fractions. The antibody inhibited the enzymatic activities of both preparations in a similar manner. Interestingly, the antibody had a markedly greater inhibitory effect on the reductase activity of the two enzyme preparations, and a much less inhibitory effect on the hydratase activity, suggesting that the antigenic determinants reside at or near the catalytic site of the reductase portion of the protein. These results suggest that the microsomal and peroxisomal bifunctional proteins are identical.

Animals↗

PubMed1987

Source of the hepatic microsomal trans-2-enoyl CoA hydratase bifunctional protein: endoplasmic reticulum or peroxisomes.

The present study was designed to investigate the hepatic localization of the microsomal bifunctional trans-2-enoyl CoA hydratase. Despite the low activity (less than 10%) of peroxisomal marker enzymes in isolated hepatic microsomes (acyl CoA oxidase (this study), catalase, and urate oxidase (L. Cook, M. N. Nagi, J. Piscatelli, T. Joseph, M. R. Prasad, D. Ghesquier, and D. L. Cinti, 1986, Arch. Biochem. Biophys. 245, 24-26), additional evidence in this study suggests that the microsomal enzyme is derived from peroxisomes. For example, the microsomal hydratase activity was associated with the ribosomal fractions but not with the smooth endoplasmic reticulum. In addition, when an extract of the peroxisomal enzyme was incubated with either free ribosomes or membrane-bound ribosomes, marked binding was observed with each of the fractions. Furthermore, the ease of release of the bifunctional enzyme from both free ribosomes and membrane-bound ribosomes by only KCl suggests that the bound enzyme is not a nascent protein. Labeling of liver tissue from DEHP-treated rats with rabbit immune IgG made to the purified microsomal hydratase followed by gold conjugated goat anti-rabbit IgG suggested a single subcellular site for the bifunctional hydratase--the peroxisomal organelle.

Adsorption↗

PubMed1987

Spontaneous antithrombin in a patient with benign paraprotein.

A 66-year-old man with peptic ulcer disease developed a paraprotein that resulted in a spontaneously prolonged prothrombin time, activated partial thromboplastin time, and thrombin clotting time. Although the reptilase time was normal, the thrombin clotting time failed to correct with the addition of normal plasma, calcium, or protamine sulfate. The patient's purified fibrinogen was normal, but his serum contained an IgG that inhibited the clotting of normal plasma and purified fibrinogen in the presence of thrombin. In contrast to previously described paraproteins, this patient's IgG appeared to inhibit the activity of thrombin per se rather than to interfere with fibrinogen cleavage or fibrin polymerization. Although immunoprecipitation between thrombin and the paraprotein could not be demonstrated, the patient's purified IgG, in the presence of thrombin, decreased the thrombin activity on a chromogenic substrate. Further, increasing concentrations of thrombin overcame the inhibitory effect of the patient's paraprotein. Thus, the patient's paraprotein appeared to possess antithrombin activity.

Aged↗

PubMed1987

Discriminative stimulus properties of (+)-N-allylnormetazocine in the rat: correlations with (+)-N-allylnormetazocine and phencyclidine receptor binding.

Recent studies have identified a stereospecific (+)-NANM binding site that binds psychotomimetic opioids and phencyclidine (PCP) but has a distribution in brain different from the PCP binding site. Since (+)-NANM has no opioid activity and (-)-NANM has opioid activity, rats were trained to discriminate (+)-NANM from saline in order to develop an ability to distinguish the (+)-NANM cues from other opioid agonist and antagonist activities. Cyclazocine, PCP, and ketamine all produced (+)-NANM-like stimuli in a dose-dependent manner. Behaviorally, cyclazocine and PCP are equipotent to (+)-NANM whereas ketamine is 6.7 times less potent than (+)-NANM. Pentazocine had the highest affinity for the (+)-[3H]NANM binding site, yet did not produce (+)-NANM-like discriminative stimuli. By contrast, ketamine had the lowest binding affinity for the (+)-[3H]NANM binding site and did produce (+)-NANM-like discriminative stimuli. Drug discrimination potencies relative to (+)-NANM were not predictive of relative binding affinities at (+)-NANM or PCP binding sites, although there was a trend toward a stronger correlation with the PCP binding site. Therefore, the discriminative stimulus properties of (+)-NANM cannot be explained by pharmacologic actions at either (+)-NANM or PCP binding sites alone, and may involve concurrent actions at both sites.

Animals↗

PubMed1987

Myo-osseous intercostal pedicle flaps for tracheal reconstruction in puppies.

We evaluated a myo-osseous intercostal pedicle flap for distal tracheal reconstruction. Mongrel puppies, 6 to 10 weeks old, underwent tracheal repair, 12 for primary defects and seven for stenotic lesions created in the distal trachea. A composite flap was constructed from the anterior portion of the fourth rib with the overlying pleura and periosteum and a posteriorly based intercostal muscle/neurovascular pedicle. The animals did well following tracheal reconstruction without evidence of airway obstruction. Bronchoscopy documented normal tracheal diameter and the repair site could not be discerned from the surrounding mucosa. Histologically, there was complete respiratory epithelial ingrowth with no inflammatory changes. The rib graft had active hematopoietic marrow. This work suggests that the myo-osseous intercostal pedicle flap provides a surface for normal epithelial ingrowth of tracheal mucosa without stimulating granulation tissue, interposes a stent capable of growth, therefore minimizing anastomotic stricture, and is an effective alternative in the management of distal tracheal stenosis.

Animals↗

PubMed1987

Inhibition of human sputum elastase by substituted 2-pyrones.

Nineteen 4-hydroxy- and 4-methoxy-2-pyrones related to elasnin (I) have been assayed for in vitro inhibition of human sputum elastase (HSE), porcine pancreatic elastase, alpha-chymotrypsin, and trypsin. Inhibition is reported as Ki and Ki'; percentage inhibition was dependent on [S] in a number of cases, making it unsuitable as a measure of relative inhibition. The 3-(1-oxoalkyl)-4-hydroxy-6-alkyl-2-pyrones were found to be most effective, the octyl homologue 11 being the most potent inhibitor (Ki = 4.6 microM, 30 times better than the lead compound). A further reduction in inhibition was observed when the hitherto hydrophobic 6-substituent was substituted by a branched functionality of hydrophilic nature. Conversely, methylation of the 4-hydroxy group of the 6-alkyl-2-pyrones increased inhibitory activity. The mechanism of inhibition varied from pure noncompetitive to mixed type to uncompetitive and was found to be dependent on the pattern of substitution. We believe that the 4-hydroxy-2-pyrone binds to the S4 subsite, with the 6-substituent extending across the S4-S1 subsites and the 3-substituent occupying the S5 subsite. The length of the inhibitor binding region was calculated to be approximately 24 A. None of the hydrophobic compounds were found to have any appreciable inhibition (less than 10%) with porcine pancreatic elastase, bovine alpha-chymotrypsin, and bovine trypsin when tested at the limit of their solubility. The hydrophilic compounds were nonspecific, inhibiting all four enzymes. Dialysis was used to show that the interaction is fully reversible.

Humans↗

PubMed1987

Evaluation of latex-based heterophile antibody assay for diagnosis of acute infectious mononucleosis.

The diagnosis of an acute episode of infectious mononucleosis (IM) is made on the basis of clinical symptoms and serological evidence for development of heterophile antibodies. In an effort to obtain a very sensitive and specific assay for diagnosis of IM, we have developed MERISTAR:IM. The MERISTAR assay uses latex beads coated with purified bovine heterophile antigen. We compared the sensitivity and specificity and performance characteristics of the latex-based test to those of the commercially available erythrocyte-based Monospot test with serum samples from 363 suspected-IM patients and controls. A total of 28 samples from patients with confirmed IM were positive with the MERISTAR assay, while only 23 of the samples were positive with the Monospot assay. The two assays had equal specificity, as each test gave two false-positive results. In the technical performance of the assay, the MERISTAR assay was simpler and faster to perform than the Monospot assay.

Acute Disease↗

PubMed1987

Total anomalous pulmonary venous return: report of a case diagnosed on ECMO.

After repeated echocardiographic assessment and cardiology consultation, a 2,750-g, 36 weeks' gestation neonate with esophageal atresia, tracheo-esophageal fistula, and pneumonia thought to be complicated by persistent pulmonary hypertension was placed on extracorporeal membrane oxygenation (ECMO). While on ECMO, pre-oxygenator and umbilical artery blood gas patterns suggested the diagnosis of total anomalous pulmonary venous return which was subsequently confirmed by cardiac catheterization.

Diagnosis, Differential↗

PubMed1986

(+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [(+)-3-PPP] but not (-)-3-PPP produces (+)-N-allylnormetazocine-like (SKF 10,047) discriminative stimuli.

In rats trained to discriminate the prototypic sigma receptor agonist, (+)-N-Allylnormetazocine [(+)-N-Allylnormetazocine [(+)-NANM/SKF 10,047], from saline, the (+)- but not the (-)-isomer of 3-(3-hydroxyphenyl)-N-(1-propyl)piperidine (3-PPP) produced (+)-NANM-like discriminative stimuli. (+)-3-PPP binds stereo selectively to the (+)-NANM binding site, but not to the phencyclidine binding site. Additionally, phencyclidine was found to produce (+)-NANM-like discriminative stimuli. Although the 3-PPP isomers were shown to produce changes in central dopaminergic activity (Hjorth et al. Life Sci 37, 673, 1985), the discriminative stimulus properties of (+)-3-PPP are apparently not mediated via the dopaminergic system. This hypothesis is supported by the fact that apomorphine did not produce (+)-NANM-like discriminative stimuli. These stimuli are thus non-dopaminergic and may be due to the (+)-3-PPP actions at the sigma binding site. However, it is possible that (+)-NANM, PCP, and (+)-3-PPP may have common non-sigma pharmacologic properties that account for the similar discriminative stimulus properties of these compounds.

Animals↗

PubMed1986

Do rat hepatic microsomes contain multiple NADPH-supported fatty acid chain elongation pathways or a single pathway?

[2-14C]-trans-2-hexadecenoyl CoA (16:1) and [2-14C]-trans-2-cis-8,11,14-eicosatetraenoyl CoA (20:4) were chemically synthesized and employed as competitive substrates for the liver microsomal trans-2-enoyl CoA reductase component of the fatty acid chain elongation system. Both 7.5 microM and 15 microM 20:4 competitively inhibited the reduction of 16:1 CoA to palmitoyl CoA. In addition, the reduction of both substrates was identically inhibited to the same extent by the acetylenic derivative, dec-2-ynoyl CoA. Furthermore, trypsin, chymotrypsin and subtilisin inhibited trans-2-enoyl CoA reductase activity when three different substrates were employed--16:1, 20:4 and trans-2-cis-11-octadecadienoyl CoA (18:2). These results are consistent with the hypothesis of multiple condensing enzymes connected to a single elongation pathway.

Acyl-CoA Dehydrogenases↗

PubMed1986

Site of inhibition of rat liver microsomal fatty acid chain elongation system by dec-2-ynoyl coenzyme A. Possible mechanism of inhibition.

The present study examines the effect of the acetylenic thioester dec-2-ynoyl-CoA (delta 2 10 identical to 1-CoA) on the microsomal fatty acid chain elongation pathway in rat liver. When the individual reactions of the elongation system were measured in the presence of delta 2 10 identical to 1-CoA, the trans-2-enoyl-CoA reductase activity was markedly inhibited (Ki = 2.5 microM), whereas the activities of the condensing enzyme, the beta-ketoacyl-CoA reductase, and the beta-hydroxyacyl-CoA dehydrase were not affected. The absence of inhibition of total microsomal fatty acid elongation was attributed to the significant accumulation of the intermediates, beta-hydroxyacyl-CoA and trans-2-enoyl-CoA, without formation of the saturated elongated product, indicating that the trans-2-enoyl-CoA reductase-catalyzed reaction was the only site affected by the inhibitor. The nature of the inhibition was noncompetitive. In contrast to the delta 2 10 identical to 1-CoA, delta 3 10 identical to 1-CoA did not inhibit trans-2-enoyl-CoA reductase activity, suggesting that the mode of inhibition was not via formation of the 2,3-allene derivative. Based on the observation (a) that p-chloromercuribenzoate markedly inhibits reductase activity, (b) that dithiothreitol protects the enzyme against inactivation by delta 2 10 identical to 1-CoA, (c) of the spectral manifestation of the interaction between thiol reagents and delta 2 10 identical to 1-CoA depicting an absorbance peak similar to that of the beta-ketoacyl thioester-Mg2+ enolate complex, (d) of a similar absorbance spectrum formed by the interaction between delta 2 10 identical to 1-CoA and liver microsomes, and (e) of the absence of formation of a similar spectrum by delta 3 10 identical to 1-CoA, trans-2-10:1-CoA, or delta 2 10 identical to 1 free acid with liver microsomes, we propose that delta 2 10 identical to 1-CoA inactivates trans-2-enoyl-CoA reductase by covalently binding to a critical sulfhydryl group at or in close proximity to the active site of the enzyme.

Acyl Coenzyme A↗

PubMed1986

Evidence for multiple condensing enzymes in rat hepatic microsomes catalyzing the condensation of saturated, monounsaturated, and polyunsaturated acyl coenzyme A.

The condensation of palmitoyl-CoA with malonyl-CoA by rat hepatic microsomes was competitively inhibited by myristoyl-CoA, whereas it was noncompetitively inhibited by palmitoleoyl and gamma-linolenoyl-CoA. Furthermore, the condensation of palmitoleoyl-CoA with malonyl-CoA was also noncompetitively inhibited by gamma-linolenoyl-CoA. Replacement of normal diet by a fat-free high carbohydrate diet resulted in 8-, 2.5-, and 2.3-fold increases in the condensation rates of both palmitoyl- and myristoyl-CoA, palmitoleoyl-CoA, and gamma-linolenoyl-CoA, respectively. On the other hand, administration of di-(2-ethylhexyl)phthalate (DEHP) resulted in a 2-fold stimulation of the condensation activities with myristoyl- and palmitoyl-CoA, while those with palmitoleoyl- and gamma-linolenoyl-CoA decreased to about 83 and 63%, respectively. Similar results following dietary changes or DEHP administration were obtained for total elongation activities. Finally condensation activities of 16:0, 16:1, and gamma-18:3 CoA were differently affected by the proteolytic enzyme, chymotrypsin. The competitive substrate studies, those of dietary and DEHP administration, and the differential action of chymotrypsin strongly suggest the existence of at least three discrete condensing enzymes catalyzing the condensation of saturated, monounsaturated, and polyunsaturated acyl-CoAs. These studies also indicate that the condensation reaction is the regulating and rate-limiting step of the fatty acid chain elongation system.

Acyl Coenzyme A↗

PubMed1986

Hepatic subcellular distribution of short-chain beta-ketoacyl coenzyme A reductase and trans-2-enoyl coenzyme A hydratase: 25- to 50-fold stimulation of microsomal activities by the peroxisome proliferator, di-(2-ethylhexyl)phthalate.

The present study demonstrates unequivocally the existence of short-chain trans-2-enoyl coenzyme A (CoA) hydratase and beta-ketoacyl CoA reductase activities in the endoplasmic reticulum of rat liver. Subcellular fractionation indicated that all four fractions, namely, mitochondrial, peroxisomal, microsomal, and cytosolic contained significant hydratase activity when crotonyl CoA was employed as the substrate. In the untreated rat, based on marker enzymes and heat treatment, the hydratase activity, expressed as mumol/min/g liver, wet weight, in each fraction was: mitochondria, 684; peroxisomes, 108; microsomes, 36; and cytosol, 60. Following di-(2-ethylhexyl)phthalate (DEHP) treatment (2% (v/w) for 8 days), there was only a 20% increase in mitochondrial activity; in contrast, peroxisomal hydratase activity was stimulated 33-fold, while microsomal and cytosolic activities were enhanced 58- and 14-fold respectively. A portion of the cytosolic hydratase activity can be attributed to the component of the fatty acid synthase complex. Although more than 70% of the total hydratase activity was associated with the mitochondrial fraction in the untreated rat, DEHP treatment markedly altered this pattern; only 11% of the total hydratase activity was present in the mitochondrial fraction, while 49 and 29% resided in the peroxisomal and microsomal fractions, respectively. In addition, all four subcellular fractions contained the short-chain NADH-specific beta-ketoacyl CoA (acetoacetyl CoA) reductase activity. Again, in the untreated animal, reductase activity was predominant in the mitochondrial fraction; following DEHP treatment, there was marked stimulation in the peroxisomal, microsomal, and cytosolic fractions, while the activity in the mitochondrial fraction increased by only 39%. Hence, it can be concluded that both reductase and hydratase activities exist in the endoplasmic reticulum in addition to mitochondria, peroxisomes, and soluble cytoplasm.

Alcohol Oxidoreductases↗

PubMed1986

The effects of orthognathic surgery on speech production.

Speech characteristics of 20 patients who underwent orthognathic surgery for correction of various skeletal defects were studied before surgical treatment and at periodic intervals postoperatively. Patients completed questionnaires that focused on their perceptions of appearance and oral function after orthognathic surgery. Approximately 60% of the patients demonstrated preoperative articulation errors. These were classified as mild because they did not interfere with the ability to be understood. Periodic testing indicated positive changes in articulation for a majority of those who had exhibited preoperative errors. None of the subjects experienced postoperative deterioration in their articulation. The subjects' perceptions of their individual operations were positive on all variables assessed.

Adolescent↗

PubMed1986

Induction of rat liver mitochondrial fatty acid elongation by the administration of peroxisome proliferator di-(2-ethylhexyl)phthalate: absence of elongation activity in peroxisomes.

The administration of di-(2-ethylhexyl)phthalate (DEHP)3 to male Sprague-Dawley rats resulted in more than a threefold increase in activity of acetyl CoA-dependent hepatic mitochondrial fatty acid elongation. Peroxisomes obtained either from control or DEHP-treated rats were not capable of elongating any of the fatty acyl CoAs tested. Furthermore, the peroxisomes possessed no trans-2-enoyl CoA reductase activity. Therefore, the elongation activity in the 7500g fraction from both control and DEHP-fed animals can be attributed totally to the mitochondria. Maximal incorporation of acetyl CoA occurred in the presence of both NADH and NADPH, and octanoyl CoA (8:0) and decanoyl CoA (10:0) were found to be optimal primers for fatty acid elongation in both control and DEHP-treated animals. The apparent Km for 8:0 CoA was 17 microM in both animal groups while the Vmax was increased from 4.5 to 12.5 nmol/min/mg following treatment. The apparent Km for 10:0 CoA was 10 microM in both control and DEHP-treated groups while the apparent Vmax increased from 2.5 to 10 nmol/min/mg; palmitoyl-CoA (16:0) was a very poor primer for chain elongation. Although the acetyl CoA-dependent fatty acid elongation was stimulated by DEHP treatment, the mitochondrial trans-2-enoyl CoA reductase activity was unaffected. The mitochondrial total elongation activity following DEHP-treatment using 8:0 CoA as primer was about two times higher than enoyl CoA reductase activity using trans-2-decenoyl CoA (10:1). This was the result of accumulation of intermediates, which were identified as trans-2-10:1 (35%), beta-hydroxy 10:0 (25%), unidentified (15%), and elongated saturated product 10:0 (24%). Elongation by one acetate unit was found in both the control and DEHP-treated animals. The results are discussed in terms of physiological significance.

Acetyl Coenzyme A↗

PubMed1986

Isolation of rat liver microsomal short-chain beta-ketoacyl-coenzyme A reductase and trans-2-enoyl-coenzyme A hydratase: evidence for more than one hydratase.

An enzyme preparation (IIIB) isolated from liver microsomes of untreated male rats was found to contain two activities--short-chain trans-2-enoyl-CoA hydratase and beta-ketoacyl-CoA reductase. The hydratase was purified more than 1000-fold, while the reductase activity was purified over 600-fold. Employing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, a single band with a molecular weight of 76,000 was observed. Although attempts to separate these two activities have failed, it remains to be established whether the final preparation contains a single enzyme with two activities or two separate enzymes. The hydratase was most active toward crotonyl-CoA, followed by trans-2-hexenoyl-CoA (6:1) and -octenoyl-CoA (8:1); the enzyme was essentially inactive toward substrates containing more than eight carbon atoms. The Vmax for crotonyl-CoA was 2117 mumol/min/mg protein, while the Km was 59 microM. Using acetoacetyl-CoA as substrate, the Vmax for the beta-ketoacyl-CoA reductase was over 60 mumol/min/mg protein and the Km was 37 microM; the Vmax for beta-ketopalmitoyl-CoA was only 15% of that observed with acetoacetyl-CoA, although the Km was 6 microM. During the course of purification, a second short-chain hydratase was discovered (fraction IVA); unlike IIIB, this fraction catalyzed the hydration of 4:1, 6:1, and 8:1 at similar rates. The partially purified preparation yielded maximal activity with 8:1 CoA (apparent Vmax 35 mumol/min/mg), followed by 6:1 CoA, 4:1 CoA, and 10:1 CoA; longer chain CoA's were relatively poor substrates, with trans-2-hexadecenoyl CoA about 0.1 as active as 8:1 CoA. On SDS-gels, fraction IVA contained four bands, all of which were below 60,000 Mr. Proteases, such as trypsin, chymotrypsin, and subtilisin, were found to completely inactivate both enzyme fractions.

Alcohol Oxidoreductases↗