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Biomedical subjects

C Little

Publications and source records attributed to C Little.

At least 73 records · Page 4Linked to original sources
PubMed1987

Platelet storage lesion: formation of platelet fragments with platelet factor 3 activity.

Certain samples of stored platelet concentrates exhibited marked platelet factor 3 activity. This activity was associated almost completely with platelet fragments formed during storage. Platelet concentrates containing high levels of these fragments and hence high platelet factor 3 activity were characterized after storage by alkaline plasma pH values, high levels of extracellular lactate dehydrogenase activity and relatively low cell counts.

Blood Platelets↗
PubMed1987

An X-ray-crystallographic study of beta-lactamase II from Bacillus cereus at 0.35 nm resolution.

Crystals of beta-lactamase II (EC 3.5.2.6., 'penicillinase') from Bacillus cereus were grown with Cd(II) in place of the natural Zn(II) cofactor and stabilized by cross-linking with glutaraldehyde. Their space group is C2, the cell dimensions are a = 5.44 nm, b = 6.38 nm, c = 7.09 nm and beta = 93.6 degrees, and there is one molecule in the asymmetric unit. Diffraction data were collected from cross-linked crystals of the Cd(II)-enzyme, the apoenzyme and six heavy-atom derivatives. The electron-density map calculated at 0.35 nm resolution reveals the essential Cd(II) ion surrounded by three histidine residues and one cysteine residue. The position of a glutamic acid residue, modification of which destroys activity [Little, Emanuel, Gagnon & Waley (1986) Biochem. J. 233, 465-469], suggests the probable location of the active site of the enzyme. Two minor Cd(II) sites not essential for activity were also located. The structure of the apoenzyme at this resolution appears to differ from that of the Cd(II)-enzyme only in the orientation of two of the histidine residues and the cysteine residue that surround the metal ion.

Apoenzymes↗
PubMed1987

Binding of catecholamines to alpha-1 acid glycoprotein, albumin and lipoproteins in human serum.

The binding of catecholamines in human serum was determined by equilibrium dialysis at 37 degrees. For serum concentrations of 10-15 nM the bound fractions were 28.8 +/- 2.2%, 25.7 +/- 1.7% and 22.2 +/- 2.2% for (+/-)-isoproterenol (IPR), (+/-)-norepinephrine (NE) and (+/-)-epinephrine (EPI), respectively. At higher serum concentrations saturation occurred. Alpha-1 acid glycoprotein (AAG) possessed one high affinity binding site and approximately 10 low affinity sites. The catecholamines were bound to AAG with the same order of potency for both classes of binding sites: IPR (Kd1: 100 microM Kd2: 2.2 mM) greater than NE (Kd1: 120 microM, Kd2: 6.5 mM) greater than EPI (Kd1: 140 microM, Kd2: 14 mM). Human serum albumin (HSA) and lipoproteins (SLP) interacted with the catecholamines in a non-saturable manner. IPR showed the strongest and EPI the weakest association to both of these serum protein fractions. (-)-Propranolol was able to inhibit the binding of IPR in serum and to isolated AAG, but not to HSA or to SLP. The present results show that AAG is an important catecholamine-binding protein in human serum. AAG, but not HSA or SLP, possesses binding sites shared by adrenergic receptor stimulators and blockers.

Binding Sites↗
PubMed1987

Pelvic fractures in horses: 19 cases (1974-1984).

The clinical features of 19 horses with pelvic fractures were reviewed. The most common problem was a grade III or IV unilateral hind limb lameness. The prevalence of fractures was greater in females and horses less than 4 years old. All horses were treated with a combination of analgesic administration and stall rest for 2 to 6 months. Seven of 11 horses (64%) available for long-term follow-up evaluation recovered and 4 of 11 (36%) had no lameness when being ridden. Five of seven (71%) horses with fractures that involved the acetabulum recovered, and 4 (57%) had complete resolution of lameness. No correlation was found between the long-term outcome of the horses and the bone fractured, age of horse, or duration of injury before admission.

Animals↗
PubMed1987

Increased arachidonic acid metabolites from cells in culture after treatment with the phosphatidylcholine-hydrolyzing phospholipase C from Bacillus cereus.

Treatment of rat liver cells (the C-9 cell line), porcine aorta endothelial cells, bovine aorta smooth muscle cells, bovine aorta endothelial cells, mouse fibroblasts and rat keratinocytes with highly purified, crystallized Bacillus cereus phospholipase C, which hydrolyzes phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine but has little or no effect on phosphatidylinositol, phosphatidylglycerol, cardiolipin, sphingomyelin, lysophosphatidylcholine or lysophosphatidylethanolamine, increased metabolism of arachidonic acid. Hydrolysis of phosphatidylcholine (and/or phosphatidylethanolamine) by a phosphatidylcholine (or phosphatidylethanolamine)-hydrolyzing phospholipase C appears to contribute to liberation of substrate for arachidonic acid metabolism.

Animals↗
PubMed1986

Carboxy groups as essential residues in beta-lactamases.

Beta-lactamases are divided into classes A, B and C on the basis of their amino acid sequences. Beta-Lactamases were incubated at pH 4.0 with the carboxy-group reagent 1-(3-dimethylaminopropyl)-3-ethylcarbodi-imide plus a coloured nucleophile and the extents of inactivation and nucleophile incorporation were monitored. Two class A enzymes (from Bacillus cereus and Bacillus licheniformis) and two class C enzymes (from Enterobacter cloacae P99 and Pseudomonas aeruginosa) were examined. All four enzymes were inactivated, with total inactivation corresponding to the incorporation of approx. 2-3 mol of nucleophile/mol of enzyme. In the case of beta-lactamase I from Bacillus cereus, some 53% of the incorporated nucleophile was located on glutamic acid-168 in the amino acid sequence.

Amino Acid Sequence↗
PubMed1986

A spectral study of cobalt(II)-substituted Bacillus cereus phospholipase C.

The coordination sphere of both the structural and catalytic zinc ions of Bacillus cereus phospholipase C has been probed by substitution of cobalt(II) for zinc and investigation of the resultant derivatives by a variety of spectroscopic techniques. The electronic absorption, circular dichroic, magnetic circular dichroic, and electron paramagnetic resonance spectra were found to be strikingly similar when cobalt(II) was substituted into either site and are consistent with a distorted octahedral environment for the metal ion in both sites. Octahedral coordination appears comparatively rare in zinc metalloenzymes but has been suggested for glyoxalase I [Sellin, S., Eriksson, L. E. G., Aronsson, A.-C., & Mannervik, B. (1983) J. Biol. Chem. 258, 2091-2093; Garcia-Iniguez, L., Powers, L., Chance, B., Sellin, S., Mannervik, B., & Mildvan, A. S. (1984) Biochemistry 23, 685-689], transcarboxylase [Fung, C.-H., Mildvan, A. S., & Leigh, J. S. (1974) Biochemistry 13, 1160-1169], and the regulatory binding site of Aeromonas aminopeptidase [Prescott, J. M., Wagner, F. W., Holmquist, B., & Vallee, B. L. (1985) Biochemistry 24, 5350-5356]. Phospholipase C is so far unique in having two such sites.

Bacillus cereus↗
PubMed1986

Molecular forms of myeloperoxidase in human plasma.

A radioimmunoassay for myeloperoxidase was established with the use of affinity-purified anti-(human myeloperoxidase) immunoglobulins. By the use of ion-exchange followed by immunoaffinity chromatography a preparation of immunoreactive, catalytically active myeloperoxidase was obtained from fresh human plasma. In non-denaturing gel electrophoresis, the plasma preparation showed about four catalytically active components of mobility very similar to that of the granulocyte enzyme. SDS/polyacrylamide-gel electrophoresis combined with protein blotting showed that the two polypeptides of strongest antigenicity in the plasma preparation corresponded in Mr to the large and the small subunits of the granulocyte enzyme. In addition, the plasma preparation contained a higher-Mr immunoreactive polypeptide, possibly a precursor form of the enzyme, together with another of Mr similar to that of the large subunit of eosinophil peroxidase.

Chromatography, Affinity↗
PubMed1986

Identification of an essential glutamic acid residue in beta-lactamase II from Bacillus cereus.

Beta-Lactamase II from Bacillus cereus was readily inactivated by incubation at pH 4.75 with a water-soluble carbodiimide plus a suitable nucleophile. In the early stages of the reaction, 1 equivalent of nucleophile was incorporated/equivalent of enzyme, whereas during the later stages a second equivalent of nucleophile was also incorporated. This latter process correlated with the blocking of the enzyme's single thiol group. Enzyme inactivated in the presence of the coloured nucleophile N-(2,4-dinitrophenyl)ethylenediamine was fragmented by pepsin digestion, and coloured peptides were isolated by gel filtration and h.p.l.c. Two major peptides, representing 52% of the incorporated label, were isolated and sequenced. Both peptides contained the incorporated label on glutamic acid-37, and it is concluded that this latter residue represents a catalytically essential carboxylic residue in beta-lactamase II.

Bacillus cereus↗
PubMed1986

Current status of treatment of acute leukemia in adults: an overview of the Memorial experience and review of literature.

The results of treatment of 629 previously untreated adults with acute leukemia at Memorial Hospital are reviewed. During the past 14 years, 135 adults (greater than 15 years) with acute lymphoblastic leukemia (ALL) have been treated with one of three successive multidrug-intensive treatment protocols (L2, L10/10M, and L17/17M), each calling for 2.5 to 3 years of systemic chemotherapy and prophylactic intrathecal methotrexate without cranial irradiation. The complete remission (CR) rates were L2 (n = 22) = 77%; L10/10M (n = 69) = 86%; L17/17M (n = 44) = 77%. The median durations of survival and remission were, respectively, L2 = 33 and 30 months; L10/10M = 62 months and not reached; and L17/17M = not reached. Almost all relapses occurred within the first 3 years while still continuing treatment, and there were only rate late relapses after stopping treatment. It appears that approximately half of the patients may have been cured with the latest two protocols. During the last 17 years, 494 adults aged 15 to greater than 70 with acute nonlymphoblastic leukemia (ANLL) were treated with one of five successive multiple drug treatment protocols of varying intensity (arabinosylcytosine + 6-thioguanine [n = 36]; L6 [n = 101]; L12 [n = 104]; L14/14M [n = 121]; and L16/16M [n = 132]). Patients with myelodysplastic syndromes generally were not treated until they developed acute leukemia, but were then entered and included in the results. Secondary leukemias following treatment of other neoplastic diseases were not included. The complete remission rates were fairly constant between 47 and 64% and the median durations of remissions were between 9 and 21 months. The intensive treatment L14 and L16 protocols were associated with more early deaths and did not result in a significantly improved remission incidence or duration or survival. With all protocols, the majority of relapses occurred within the first 2 years, but relapses continued to occur at a decreasing rate for 4 years and occasionally even later. Whereas a small fraction (approximately 10 to 15%) of adults with ANLL are now apparently being cured with combination chemotherapy, despite intensive efforts there has been little improvement during the last decade and more selective and effective forms of treatment are urgently needed.

Acute Disease↗
PubMed1986

Morphological changes associated with pH changes during storage of platelet concentrates in first-generation 3-day container.

The platelet injury and loss of viability that has been shown to occur with storage of platelet concentrates (PC) under conditions with increasing or falling pH were examined using scanning and transmission electron microscopy. After storage, samples were taken for measurement of pH value, platelet count and size distribution, release of lactate dehydrogenase (LDH) into plasma, and for SEM and TEM. Increased levels of LDH were observed in PC with pH above 7.3 and below 6.1. In PC with pH above 7.3 this was related to an increased number (23%) of platelets that were lysed or had a swollen disintegrated internal structure (balloons) as seen with TEM. SEM and Coulter counter studies also showed that platelet fragmentation and formation of microvesicles were prominent in PC with pH above 7.3. The electron microscopic pictures confirmed previous suggestions that platelet disc-to-sphere transformation and cytoplasmic swelling occur when pH falls below 6.7-6.8 during storage. SEM studies showed that concomitant with this change, folds and bulky projections appeared on the platelet surface. In PC with pH below 6.1 the morphological change was irreversible with the appearance of more than 90% lysed and balloon platelets. In conclusion, these studies suggest that the loss of viability observed with PC with pH above 7.3 or below 6.1 after storage is related to an increased percentage of lysed and balloon platelets.

Blood Platelets↗
PubMed1986

Morphological changes associated with pH changes during storage of platelet concentrates.

The platelet injury and loss of viability that has been shown to occur with storage of platelet concentrates (PC) under conditions with increasing or falling pH were examined using scanning and transmission electron microscopy. After storage of PC in first (PL-146) and second generation containers (PL-1240 and PL-732), samples were taken for measurement of pH value, platelet count and size distribution, release of lactate dehydrogenase (LDH) into plasma, and for SEM and TEM. After storage of 83 PC in first generation containers for 4 days increased levels of LDH were observed in PC with pH above 7.3 (n = 2) and below 6.1 (n = 4). The electron microscopic pictures confirmed previous suggestions that platelet disc-to-sphere transformation and cytoplasmic swelling occur when pH falls below 6.7-6.8 during storage. In PC with pH below 6.1 the morphological change was irreversible with the appearance of more than 90% lysed and balloon platelets. In PC with pH above 7.3 because of low platelet concentration this was related to an increased number of platelets that were lysed of had a swollen disintegrated internal structure (balloons) as seen with TEM. SEM and Coulter counter studies also showed that platelet fragmentation and formation of microvesicles were prominent in PC with pH above 7.3. Second generation containers maintained the pH during 5-day-storage at higher levels but had enhanced risk of pH increase. A strong tendency for the platelets to become alkaline upon subsequent storage could be demonstrated as a result of excessive forces or prolonged centrifugation during the second spin of the preparation procedure. Whereas the platelet injury caused by low pH is easy to recognize because of the absence of swirling the morphological changes associated with high pH cannot be detected directly.

Acid-Base Equilibrium↗
PubMed1985

Further characterization of human eosinophil peroxidase.

The large and the small subunits (Mr 50 000 and 10 500 respectively) of human eosinophil peroxidase were isolated by gel filtration under reducing conditions. The subunits were very strongly associated but not apparently cross-linked by disulphide bridges. During storage, the large subunit tended to form aggregates, which required reduction to dissociate them. Amino acid analysis of the performic acid-treated large subunit showed the presence of 19 cysteic acid residues. The small subunit of eosinophil peroxidase had the same Mr value as the small subunit of myeloperoxidase. However, although these subunits have very similar amino acid compositions, they showed different patterns of peptide fragmentation after CNBr treatment. The carbohydrate of eosinophil peroxidase seemed associated exclusively with the large subunit and comprised mannose (4.5%, w/w) and N-acetylglucosamine (0.8%, w/w). The far-u.v.c.d. spectrum of the enzyme indicated the presence of relatively little ordered secondary structure.

Amino Acids↗
PubMed1984

Studies on the subunits of human myeloperoxidase.

The subunit composition of human myeloperoxidase was studied with the use of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration. The subunit pattern observed depended on the manner in which the enzyme was treated before analysis. Reduction before heat treatment in detergent led to two main protein species (Mr 57 000 and 10 500), whereas reduction during or after heat treatment yielded an additional species of Mr 39 000. Heating without any reductive pretreatment yielded the 39 000-Mr form as the major electrophoretic species. Carbohydrate staining showed large amounts of sugar on the 57 000-Mr species and little on the 10 500-Mr form. Significant amounts of haem were associated with this latter subunit. Haem also seemed to be associated with the 57 000-Mr form but not with the 39 000-Mr one. These three subunit forms were isolated and their amino acid composition analysed. The 57 000-Mr and 39 000-Mr forms had very similar amino acid composition and yielded an apparently identical collection of fragments on incubation with CNBr. Once separated, the subunits could not be interconverted. Generally, minor amounts of other molecular-mass forms were observed. The nature of the various molecular-mass forms originating from myeloperoxidase is discussed.

Amino Acids↗
PubMed1984

Effect of phospholipase C (Bacillus cereus) on freshly isolated and 4-day-stored human platelets.

Phospholipase C (from Bacillus cereus) was used to study fresh and stored human platelets. Provided that the enzyme was inactivated before lipid extraction, no significant degradation of phospholipid in fresh cells was noted, even when platelets were activated or induced to change shape by ADP, collagen or thrombin. With platelets isolated from concentrates stored for transfusion for 4 days at 22 degrees C, membrane phospholipids were degraded by the enzyme to an extent depending on the pH in the platelet concentrate at day 4 of storage. The extent of phospholipid hydrolysis in platelets correlated well with the extent of release of lactate dehydrogenase during storage, with both being minimal for platelets from concentrates of final pH 6.5-6.9. Under non-lytic conditions, phosphatidylcholine was the phospholipid most degraded (40%), with no significant degradation of phosphatidylserine being detected. Storage does not seem to alter the distribution of phospholipids at the external leaflet of the plasma membrane.

Bacillus cereus↗