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L R Berry

Publications and source records attributed to L R Berry.

14 recordsLinked to original sources
PubMed2006

Covalent antithrombin-heparin complexes.

Unfractionated heparin (UFH) and low molecular weight heparin (LMWH) have been utilized as primary anticoagulants for thrombosis prophylaxis and treatment. However, a number of biophysical and safety limitations have led to development of new anticoagulants. Covalent antithrombin-heparin (ATH) complexes may address many of these issues. Early ATH products were prepared that had increased intravenous half-lives relative to UFH but lacked any improvement in anti-factor Xa activity or had no catalytic activity or reactivity against thrombin. However, a recent conjugate developed by Chan et al. has displayed a number of superior properties. Chan et al. ATH has an increased direct thrombin inhibition rate and can catalyze coagulant enzyme inhibition by exogenous antithrombin with very high specific activity. Unlike UFH, clot-bound thrombin is readily inhibited by ATH and, at similar antithrombotic efficacy, the ATH has improved bleeding profiles compared to heparins. Given the preclinical findings, Chan et al. ATH may warrant clinical trial testing for control of clot propagation.

Animals↗
PubMed2006

Effect of substrate and fibrin polymerization inhibitor on determination of plasma thrombin generation in vitro.

BACKGROUND: Thrombin generation potential, a critical haemostatic measure, can be determined by continuous detection of total thrombin or direct subsampling. However, differences between methods exist in area under the curve or peak thrombin calculated. Also, impact of anticoagulants on thrombin generation may vary depending on mode of analysis. OBJECTIVE: We studied the effect of components on thrombin generation in the presence or absence of anticoagulants. METHODS: The continuous method was conducted with plasma +/- fibrin(ogen) +/- fibrin polymerization inhibitor. Plasma contained slow-reacting TG5134 substrate at 37 degrees C and reaction was started with dilute thromboplastin in CaCl(2)/Tris buffer. Absorbance (405 nm) was recorded over time and free thrombin calculated from total thrombin activity. For the subsampling method, similar plasma mixtures +/- TG5134 were reacted and free thrombin measured directly as the difference in activity against S2238 substrate of timed subsamples taken into EDTA or EDTA + antithrombin + heparin. RESULTS: Slow-reacting substrate in the continuous method acted as a competitor for thrombin, giving delayed but greater free thrombin than direct subsampling. These differences persisted to varying extents with all anticoagulants tested. In either method, presence of polymerization inhibitor increased the amount of free thrombin. Continuous method detection of alpha(2)macroglobulin complexes was hampered by sensitivity limits leading to inordinate free thrombin calculations. Especially with hirudin, although free thrombin remained at the end of the subsampling method, continuous method calculations assumed no residual free thrombin. CONCLUSION: In vitro plasma thrombin generation is delayed and increased by slow-acting substrate and fibrin polymerization inhibitor.

Adult↗
PubMed1988

Fibrinogen has a rapid turnover in the healthy newborn lamb.

The half-lives for coagulation factors in the healthy newborn infant are not known and may be different than for the adult. We measured the half-life for fetal sheep fibrinogen and compared it to the half-life of adult sheep fibrinogen. Fibrinogen was purified from adult and fetal sheep plasma and radiolabeled with either 125I or 131I. The half-lives for these fibrinogens were determined in the adult sheep and newborn lamb. In addition, the fetal and adult sheep fibrinogens were compared by reptilase time, thrombin clotting time, sialic acid content, and the behavior of the N-glycans derived from these fibrinogens on the immobilized lectin, Sepharose-concanavalin A. Finally, the in vivo response of coinjected radiolabeled fibrinogens to increasing doses of infused thrombin was determined. The fetal sheep fibrinogen differed from the adult as indicated by a prolonged reptilase time and an increased sialic acid content (fetal: 10-11 residues/340 Kd versus adult: 8-9 residues/340 Kd). The latter was also reflected in differing chromatographic profiles for the N-glycans on Sepharose-concanavalin A. The half-lives for both the adult and fetal fibrinogen were significantly more rapid in the newborn lamb (fetal: 47 +/- 2.0 h; adult: 46 +/- 2.4 h, mean +/- SEM) than in the adult (fetal: 116 +/- 6.5 h; adult: 121 +/- 6.9 h). Finally, the adult and fetal sheep fibrinogen responded to thrombin in an identical fashion in the intact animal. In summary, both adult and fetal fibrinogen have faster half-lives in the lamb compared tot he adult, despite a higher sialic acid content for the fetal fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1987

A comparison of two procedures used for complexing Fe(III) with human apotransferrin. II. Uptake of Fe(III) by K-562 cells from 55Fe . transferrins and Fe . [3H]transferrins.

Samples of human apotransferrin (apo . HTr) were saturated with Fe(III) by two different techniques, a method employing excess trisodium citrate to chelate Fe(III) and a nonchelating approach which involves the ferroxidase activity of ceruloplasmin to convert Fe(II)----Fe(III). The samples were radiolabelled with either 55Fe or 3H. Using an initial molar Fe/apo . HTr ratio of 2.0-2.1, preparations of human transferrin with bound Fe (Fe . HTr) using the citrate method invariably contained 2.2-2.4 atoms Fe/molecule, whereas Fe . HTr (ceruloplasmin method) contained 2.0 atoms/molecule as shown by spectrophotometric and radioactivity measurements. Uptake of Fe from these Fe . HTr preparations by K-562 cells grown in a serum-free medium was marginally, but consistently, more rapid from 55Fe . HTr (citrate) than from 55Fe . HTr (ceruloplasmin). Taking account of the different Fe contents of the Fe . HTr preparations, the rate measured over a 2-h period amounted to approximately 12,700 and 16,100 Fe atoms/(cell . min) for Fe . HTr (ceruloplasmin) and Fe . HTr (citrate), respectively. However, cell binding by the two Fe . [3H]HTr preparations did not differ significantly over the 8-h incubation period. Furthermore, from the 3H distribution, the quantities of Fe . HTr bound reversibly at the cell surface and contained within the cell were similar for the two Fe . HTr preparations. The results indicate that apo . HTr may bind Fe in different ways depending on the method of Fe presentation and that the Fe . HTr product can donate Fe to K-562 cells at a rate which may reflect the method used for Fe-complex formation.

Apoproteins↗
PubMed1987

Maternal low-carbohydrate high-protein diet affects mandibular growth in diabetic newborn rats.

Dams with 7 pups each were randomly assigned to two different diets. Twelve dams were fed a normal (20%) protein diet and were divided into two groups of 4 and 8 animals. Pups from group 1 (n = 28) were injected with citrate buffer as a control. Pups from group 2 (n = 56) were injected with streptozotocin. Twelve additional dams were fed a 40% protein diet. They were also divided into two groups of 4 and 8 animals. Pups from group 3 (n = 28) were injected with citrate buffer as a control. Pups from group 4 (n = 56) were injected with streptozotocin. Forty-eight hours later, diabetic status was determined using Dextrostix. On Day 15, pups were injected with [14C]proline to determine collagen synthesis and 45Ca to study mineralization. After the pups were killed, blood glucose levels were determined. Then mandibles were removed. Milk from each dam was also collected after injection of oxytocin. At the time of killing, blood glucose levels in diabetic pups were less than earlier levels, though still higher than those of controls on either diet. The weights of body and mandible, collagen contents, and the total calcium contents in the diabetic group were in general less than those of the nondiabetic group on the 20 and 40% protein diets. 45Ca uptake in the diabetic group was significantly increased compared with those of the nondiabetic rats on both diets. The percentage reduction in the mandibles of diabetic rats from those of nondiabetic rats on the 40% protein diets was consistently less than that of animals on the 20% protein diets. The higher protein contents of the maternal milk in the 40% protein group may partly be responsible for the smaller impairment of mandibular development in the diabetic over nondiabetic animals. It is concluded that maternal low-carbohydrate high-protein diets will play indirectly a beneficial role in the development of the mandibles of diabetic newborns.

Animals↗
PubMed1986

A comparison of two procedures used for complexing Fe(III) with human apotransferrin: I. Physicochemical properties of the Fe(III) . transferrin products.

Samples of human Fe.transferrin (Fe.HTr) were prepared from a single batch of apotransferrin (apo.HTr) by either the Fe(III)-citrate or the Fe(II)-ceruloplasmin (ferroxidase) method. By using 55Fe, 55Fe.HTr prepared by the citrate method and 55Fe.HTr prepared by the ceruloplasmin method contained 2.2-2.3 and 2.0 Fe/mol, respectively. For both 55Fe.HTr preparations, the isotope was shown to be associated with the protein from the measurement of absorbance at 465 nm and dialysis studies. However, passage of the 55Fe.HTr (ceruloplasmin) reaction mixture through DEAE-cellulose caused 55-60% of 55Fe to be lost from the protein, although no decrease in absorbance at 465 nm was observed. Ion-exchange chromatography of 55Fe.HTr (citrate) did not induce loss of 55Fe. Absorbance measurements showed significant differences between the two Fe.HTr preparations with respect to the ratios A212/A278 and A463/A278. Using an excitation wavelength of 275 nm, the fluorescence intensity ratios relative to apo.HTr were 0.275 and 0.309 for Fe.HTr (citrate) and Fe.HTr (ceruloplasmin), respectively. Electron spin resonance (ESR) measurements confirmed that Fe.HTr (citrate) and Fe.HTr (ceruloplasmin) were saturated with Fe. Hyperfine coupling constants and other features of the resonance profile revealed distinct differences between the two Fe.HTr preparations. Dialysis against H2O caused Fe.HTr (citrate), but not Fe.HTr (ceruloplasmin), to lose absorbance at 465 nm. The ESR profile of Fe.HTr (citrate), after dialysis against H2O, was reduced to multiple splittings and a lack of resolution of the central hyperfine structure. Addition of Na2CO3 restored the absorbance (465 nm) and the ESR pattern of Fe.HTr (citrate). In contrast, these properties of Fe.HTr (ceruloplasmin) were little affected by dialysis against H2O. However, the addition of trisodium citrate to Fe.HTr (ceruloplasmin) caused a reduction in absorbance at 465 nm and a change in ESR profile to resemble that of Fe.HTr (citrate) after dialysis in H2O; these changes, caused by citrate binding to Fe.HTr (ceruloplasmin), were restored to normal by the addition of Na2CO3. The data indicate that different protein conformations result from complexing Fe(III) with apo.HTr by these two different procedures. The two Fe.HTr products may differ, conceivably, in their abilities to transfer Fe to cells.

Apoproteins↗
PubMed1985

On the interaction between 5-hydroxytryptamine and N-acetylneuraminic acid under aqueous conditions.

A complex designated 5-HT-NeuAc was formed between 5-hydroxytryptamine (5-HT) and N-acetylneuraminic acid (NeuAc) under aqueous conditions. Complex formation was encouraged by exposure to light (3000-3800 A; 1 A = 0.1 nm) and freeze-drying and the freeze-dried complex was isolated by gel filtration chromatography. Although stable to rechromatography on Bio-Gel P-2 if H2O was the eluent, 5-HT-NeuAc dissociated into the free components when placed in 0.1 M NaCl. Chemical analyses of the isolated complex showed that an equimolar amount of 5-HT and NeuAc was present and that all group functions were intact; these data suggested that the association between 5-HT and NeuAc was noncovalent. Spectrophotometric measurements demonstrated a small increase (approximately 12%) in extinction coefficient (275 nm) and a large increase (340- to 440-fold) in fluorescence emission (340 nm) compared with 5-HT alone. Data obtained from 1H nuclear magnetic resonance spectroscopy (250 MHz) of 5-HT and NeuAc standards compared closely to published reports. In comparison, measurements made with 5-HT-NeuAc showed that all 5-HT protons were slightly deshielded; of the NeuAc protons, slight deshielding of H8 and significant shielding of H3eq, H3ax, and H6 was observed. From these observations, a model describing the association between 5-HT and NeuAc is proposed.

Kinetics↗
PubMed1983

The role of proteolytic enzymes derived from crude bacterial collagenase in the liberation of hepatocytes from rat liver. Identification of two cell-liberating mechanisms.

Crude bacterial collagenase was chromatographed on DEAE-cellulose to yield three peaks with proteolytic activity: an arginine esterase (DEAE-1), gelatinase (DEAE-2) and a caseinolytic activity (DEAE-3). The arginine esterase and gelatinase activity fractions were slightly contaminated with each other but neither possessed caseinolytic activity; the caseinolytic fraction was devoid of arginine esterase and gelatinase activities. In addition, crude collagenase was fractionated by ZnII-affinity chromatography to produce a gelatinase peak (ZnII peak 1), which was free from arginine esterase and caseinolytic activities. The four fractions were compared to crude collagenase in their ability to liberate rat hepatocytes by using either liver slices or a standard perfusion technique. Compared to crude collagenase (0.05-0.1% w/v), which produced 70-80% liver digestion with approximately 80% cell viability, digestion with equivalent quantities of the isolated enzymic activities was relatively poor. Gelatinase activity (ZnII peak 1) was wholly ineffective and DEAE-1 and DEAE-2 each possessed only slight digestive properties. Hepatocyte liberation by the caseinolytic activity, DEAE-3, was partially successful (30-40% digestion, 25-30% viability) but only a portion of liver tissue was digested regardless of the quantity of DEAE-3 used. However, by mixing certain fractions before perfusion two gelatinase-dependent, cell-releasing mechanisms were identified: (a) DEAE-3 with ZnII peak 1 and (b) DEAE-1 mixed with either DEAE-2 or ZnII peak 1. Each system compared creditably with the digestive properties of an equivalent activity of crude collagenase. At present we are attempting to determine any differences between hepatocytes produced by the two enzymic mechanisms.

Animals↗
PubMed1982

The structural heterogeneity of the carbohydrate moiety of desialylated human transferrin.

Human transferrin consists of a single chain polypeptide which supports two N-glycosidically linked glycans at sequons a and b. Glycopeptides were released from human transferrin by proteolytic digestion, desialylated by mild acid hydrolysis, and then isolated by chromatographic methods. The structures of the glycans located on each sequon were determined by a combination of analytical techniques including Smith degradation, permethylation, and enzymic degradation. Approximately 79% of the total glycan from sequon a was of the biantennary type as previously described by Dorland and his colleagues (FEBS Lett. 77, 15-20 (1977)). The remaining 21% consisted of a mixture of triantennary and tetraantennary glycans, each amounting to approximately 10% of the total glycan for this sequon. The triantennary structure resembled that described for the N-glycosidic triantennary glycans of bovine fetuin by Nilsson and his colleagues (J. Biol. Chem. 254, 4545-4553 (1979)). Of the tetraantennary glycan, approximately half of the structures were incomplete, i.e., one antenna terminated by N-acetylglucosamine. On sequon b, 81% of the glycan was biantennary, identical to those biantennary glycans of sequon a, and the reminder was triantennary, also of the fetuin type. The glycan structures and their locations on the polypeptide are related to the known subpopulations of human transferrin.

Amino Acids↗
PubMed1979

Bi-and tri-antennary human transferrin glycopeptides and their affinities for the hepatic lectin specific for asialo-glycoproteins.

Glycopeptides were isolated from a proteolytic digest of human transferrin. After mild acid hydrolysis the desialylated glycopeptides were labelled by the galactose oxidase/NaB(3)H(4) procedure and then fractionated by Sephadex-gel filtration or by anion-exchange chromatography. Either technique allowed separation of the two heterosaccharide chains (designated glycan I and glycan II) previously described for this protein by Spik, Vandersyppe, Fournet, Bayard, Charet, Bouquelet, Strecker & Montreuil (1974) (in Actes du Colloque Internationale No. 221 vol. 1, pp. 483-499). Subsequent chromatography on Sepharose-concanavalin A separated fractions containing different quantities of carbohydrates for each glycan, as indicated by analyses. The isolated glycan fractions were then tested for their abilities to bind to the immobilized rabbit hepatic lectin. Our studies suggest that either glycan can have a bi- or tri-antennary structure. Desialylated biantennary glycans I and II did not bind to the hepatic lectin. Desialylated triantennary glycan I was slightly retarded by the hepatic lectin, whereas the triantennary glycan II consisted of equal quantities of a retarded and a bound type. Desialylated triantennary glycan II was totally displaced from the hepatic lectin by using a buffer containing 0.05m-EDTA. The results suggest that greater structural heterogeneity exists in the carbohydrate moiety of human transferrin than was previously envisaged. Such heterogeneity could be reflected in several molecular forms of human transferrin, which, after desialylation, differ significantly in their affinities for the hepatic lectin.

Amino Acids↗