Affinity chromatography of thymidine kinase from a rat colon adenocarcinoma

Thymidine kinase from a transplantable colon adenocarcinoma, induced by 1,2-dimethylhydrazine and maintained in CDF rats, was purified by affinity chromatography using thymidine-3'(4-aminophenylphosphate) coupled to carboxyhexyl-Sepharose. Most of the contaminating protein passed through the column; non-specifically adsorbed protein was washed from the column by 0.1 M KC1 in 0.01 M Tris-HC1, 7.5. Thymidine kinase was eluted with 0.1 mM thymidine, 0.1 M KC1 in 0.01 M Tris-HC1, pH 7.5. The purified enzyme accounted for about 26% of the applied activity; the specific activity of the purified material (peak fraction) was 3,500 moles TMP formed per mg protein per 10 min., a 1,800-fold purification of the applied extract. The preparation is free of nucleoside phosphotransferase, but contains other protein impurities. Purification was completed in less than 1 hour, making this a useful procedure for isolation of this unstable enzyme.     

A rapid method for hemoglobin chain recombination

A rapid method for hemoglobin chain recombination which gives a homogeneous product was developed. The method utilizes a small carboxymethylcellulose column as a medium for chain recombination and concentration of the hemoglobin. Equimolar amounts of p-hydroxymercuribenzoate derivatives of alpha- and beta-chains were mixed with 300 x molar excess of beta-mercaptoethanol over the p-hydroxymercuribenzoate groups. After 10 min of incubation in an ice bath, the mixture was adjusted to pH 5.85, and was loaded on a carboxymethylcellulose column. The column was washed with 10 mM phosphate buffer-1 mM Na2EDTA-47 mM beta-mercaptoethanol, pH 5.85 and then with 10 mM phosphate buffer, pH 5.85. The hemoglobin was eluted from the column by use of 15 mM K2HPO4. The hemoglobin was homogeneous on polyacrylamide gel electrophoresis and had a visible spectrum, electrophoretic mobility, and number of -SH groups comparable to those shown by control hemoglobin.     

Characterization and representative structures of N-oligosaccharides bound to apolipoprotein H

We studied the structure of N-linked carbohydrates bound to apolipoprotein H by a combination of two methods which make use of lectins. Digoxigenin-labelled lectins are used for the structural characterization of carbohydrate chains of glycoproteins. Concanavalin A lectin affinity chromatography was used to analyse apolipoprotein H according to the characteristics of its carbohydrate chain inner to sialic acid residues. Our results from digoxigenin-labelled lectins analysis showed that apolipoprotein H gave positive bands to SNA, DSA, GNA, PNA and AAA lectins. Apolipoprotein H gave a negative band when reacted with MAA lectin. When we applied apolipoprotein H onto the Concanavalin A lectin column no detectable amounts of protein were eluted with Concanavalin A buffer. After adding a buffer with low sugar concentration (10 mM glucoside) a large amount of apolipoprotein H was recovered. These molecules of apolipoprotein H weakly bound to the lectin. When a higher sugar concentration (500 mM mannoside) was added most of the sample applied was eluted. These molecules of apolipoprotein H firmly bound to the column having high affinity for the lectin. These results combined with those coming from the digoxigen-labeled lectins method enable us to understand the inner structure of carbohydrate chains with their outer branches. Molecules of apolipoprotein H which weakly bind to Concanavalin A could bear complex N-glycans organized in biantennary or truncated hybrid structures. Firmly bound apolipoprotein H referred to molecules rich in N-glycan hybrid structures. They have an outer branch belonging to the high mannose carbohydrate chains which explain the ability to bind to the column and an other main branch bearing the sequence galactose beta-(1-4)-N-acetylglucosamine beta-(1-2) mannose. Galactose could be the terminal sugar or, alternatively, be masked with sialic acid alpha-(2-6) terminally linked.     

Effect of calcium and magnesium on binding of beta, gamma-methylene ATP to sarcoplasmic reticulum

Isolated sarcotubular membranes (SR) from skeletal muscle bound 3.7 nmol of beta, gamma-methylene [8-3H]ATP (AMP-PCP) per mg of membrane protein. Only one class of binding site was identified and the dissociation constant (K) for this site was 1.5 X 10(-5) M. Addition of 0.05% Triton X-100 increased the number of binding sites to 5.7 nmol/mg. ATP and ADP competitively inhibited AMP-PCP binding. The dissociation constants for ATP and ADP were 3.5 X 10(-5) M and 3.3 X 10(-6) M, respectively. Since this data was obtained in the presence of 5 mM EDTA, it was established that the sarcoplasmic reticulum has a high affinity for the metal free forms of ATP, ADP, and AMP-PCP. Magnesium concentrations in excess of 1 X 10(-4) M inhibited AMP-PCP binding. Lower concentrations of magnesium had little effect on AMP-PCP binding. The effect of calcium on AMP-PCP binding was biphasic. Calcium concentration between 1 X 10(-6) and 1 X 10(-4) M inhibited AMP-PCP binding. Inhibition was maximal at 1 X 10(-5) M. Calcium concentration above 1 X 10(-4) M facilitated analogue binding. Possible sites of magnesium and calcium actions are discussed.     

Cyclin D1 protein expression is related to clinical progression in laryngeal squamous cell carcinomas

The expression of cyclin D1 gene was investigated in 74 laryngeal squamous cell carcinomas (LSCCs) in order to determine its clinical and prognostic value. Overexpression of cyclin D1 was detected immunohistochemically using DCS6 monoclonal antibody on formalin-fixed, paraffin-embedded tissue sections. Cyclin D1 expression was detected in 22 of the 74 cases investigated (30 per cent), thirteen of which presented nodal metastases (59 per cent); of the patients without any detectable cyclin D1 protein expression, six presented nodal metastases (12 per cent). Cyclin D1 protein expression was found in five per cent of the specimens of normal mucosa, eight per cent of those with low-grade dysplasia and 20 per cent of those with high-grade dysplasia. A statistically significant association was found between cyclin D1 expression and the supraglottic site (p < 0.05), tumour extension (p < 0.001), the presence of lymph node metastases (p < 0.001), and advanced clinical stage (p < 0.001). Cyclin D1 expression analysis is an important tool in the selection of LSCC patients with an aggressive clinical course.     

Cytogenetic evaluation of butylated hydroxytoluene

Ischaemic injury was produced in the dog heart by occluding the left anterior descending coronary artery just below the second diagonal branch for the duration of 3 h. A prostacyclin analogue, ZK 36374 (Iloprost) was administered (10 micrograms/kg) just before the coronary artery occlusion. During 3 h occlusion ATP levels in the ischaemic area declined from 65.4 +/- 7.1 mM/g protein in sham-operated group to 31.5 +/- 5.2 microM/g protein in no-drug group and to 44.1 +/- 5.0 microM/g protein in Iloprost group. Creatine phosphate decreased from 112 +/- 14 microM/g protein, to 81 +/- 10 microM/g protein in no-drug group and to 95 +/- 12 microM/g protein in Iloprost group. The energy charge (ATP + 0.5 ADP/ATP + ADP + AMP) decreased slightly but not significantly in no-drug and Iloprost group. Three hours of LAD occlusion produced a significant fall in SOD activity in the ischaemic heart in comparison to the non-ischaemic and Iloprost-treated hearts.     

Preparation of phosphopeptides derived from alpha s-casein and beta-casein using immobilized glutamic acid-specific endopeptidase and characterization of their calcium binding

Phosphopeptides that were derived from alpha s-CN or beta-CN were prepared with immobilized glutamic acid-specific endopeptidase, and their Ca2+ binding was characterized. alpha s-Casein or beta-CN was hydrolyzed in a fluidized bed bioreactor containing 2 ml of immobilized glutamic acid-specific endopeptidase by recirculating 20 ml of alpha s-CN or beta-CN solution (10 mg/ml in 50 mM Tris.HCl and 0.02% NaN3, pH 8.0) for 3 h at 20 degrees C. The molecular masses of casein peptides were monitored by SDS-PAGE. Each hydrolysate was applied to an anion-exchange column using stepwise elution with various concentrations of KCl to separate peptides. The casein phosphopeptide content of the elution profile was monitored by analysis of protein and P concentrations. Calcium binding in phosphopeptide-enriched fractions was determined by CaCl2 titration and measurement of free Ca2+ with a Ca-selective electrode. The electrophoresis patterns showed four major peptides having molecular masses of 10.8, 9.0, 6.6, and 3.6 kDa in the alpha s-CN hydrolysate and 9.3, 8.2, and 6.2 kDa in the beta-CN hydrolysate. The highest concentrations of P were detected in the fractions that eluted with 0.4 and 0.5 M KCl for the alpha s-CN hydrolysate and with 0.4 M KCl for the beta-CN hydrolysate. The calcium-binding ability was found only in the fraction that was eluted with 0.4 M KCl; the maximum Ca2+ binding and the apparent binding constant were 0.24 mmol/mg of protein and 75 M-1, and 0.14 mmol/mg of protein and 148 M-1, respectively. alpha s-Casein phosphopeptides had different patterns for Ca2+ binding than did beta-CN phosphopeptides as the total Ca concentration was increased. Calcium binding to these casein phosphopeptides differed from that previously characterized for the tryptic peptides.     

High-performance liquid chromatography of penimocycline (author's transl)

Penimocycline is an antibiotic obtained by Mannich reaction between tetracycline and ampicilline. Separation of penimocycline from tetracyclines and other impurities has been studied by high-performance liquid chromatography. The most effective method is liquid-liquid partition on a Micropak CH column (non-polar hydrocarbon bonded on porous silica microparticles) and gradient elution with water-methanol, 0.02 M phosphate buffer (pH 7.6) and 1 mM EDTA. Some results on hydrolysis of penimocycline are given.     

Gas chromatographic assay for free and total plasma levels of thiopental

A rapid gas chromatographic assay for the determination of free and total plasma thiopental is described. Free thiopental was obtained by ultrafiltration through Amicon Centroflo membrane cones. Gas chromatographic assay utilized secobarbital as an internal standard and employed on-column methylation of the barbiturates to improve peak resolution. In 73 blood samples from 22 patients total thiopental concentrations ranged from 4.2 to 134 mug/ml plasma, with a mean of 28 mug/ml. Free thiopental values ranged from 8.6 to 22.7 per cent of total, with a mean of 13.7 per cent free thiopental and a standard deviation of 3.2 per cent. At a total thiopental level of 10 mug/ml, unbound thiopental averaged 10.7 per cent with ultrafiltration, compared with 11.5 per cent with equilibrium dialysis. Assays of thiopental by gas chromatography and 14C scintillation counting gave similar results. There were progressive increases in the percentages of thiopental that were unbound when thiopental was added to plasma, purified crystalline albumin (4.5 g/l), and normal serum albumin (5 g/l), and a solution of purified protein fractions (5 g/l). Differences in protein binding determined by this method and previously reported methods are discussed.     

Purification and partial characterization of a highly immunogenic human leukocyte protein, the L1 antigen

L1 is a major and highly immunogenic protein component of human granulocytes. It was purified from random samples of blood-donor leukocytes. An antiserum to L1 was initially raised in rabbits by immunization with protein fractions from preparative agarose gel electrophoresis. The main problem in purifying L1 was its poor stability when carried through multiple steps. Preparative isoelectric focussing was therefore adopted as an efficient one-step method. The isoelectric focussing pattern of L1 was greatly influence by the presence of EDTA or calcium ions in the sample buffer. With low EDTA concentrations or calcium excess, L1 focussed as seven protein bands in two regions, pH 6.1-6.5 and pH 7.6-8.4. Conversely, L1 was found only in the pH-6.1-6.5 region when excess EDTA was added to the sample. Irrespective of conditions, the bulk of L1 focussed at pH 6.3 and pH 6.5. Also the electrophoretic mobility of L1 was strongly influenced by calcium ions, suggesting uptake of calcium by the protein. In the presence of calcium, L1 adhered to glass surfaces and filters used for concentration of protein solutions. The latter problem could be prevented by addition of EDTA. The molecular mass of L1 was determined to be about 36.5 kDa. The molecule was shown to consist of three non-covalently linked 12.5-kDa subunits.     

Computer interpreted fetal electroencephalogram. II. Patterns in infants who were neurologically abnormal at 1 year of age

A computer program for pattern recognition of fetal electroencephalogram has been used to analyze the records of nine fetuses, known to be neurologically abnormal at 1 year of age. In 4,913 10 second epochs of adequate FEEG, Low Voltage Irregular (LVI) accounted for 17.8 per cent, Mixed activity (MIX) for 30.5 per cent, High Voltage Slow (HVS) for 18.1 per cent, and Trace Alternant (T/A) for 33.2 per cent of the epochs. The numbers of observed FEEG patterns in these abnormal cases appear to be significantly different from those in 11 normal cases (p less than 0.001)3. Specifically, the relative frequency of LVI was found to be increased in the abnormal cases (p congruent to 0.05). Moreover, LVI was significantly associated with visually interpreted prolonged voltage suppression (p less than 0.025) and lowered one-minute Apgar score (p congruent to 0.025). Using discriminant function analysis for LVI, MIX, HVS, and T/A patterns from FEEG recorded during labor, 10 of 11 infants were correctly classified as being neurologically normal at one year of age and 6 of 9 infants were correctly classified as being neurologically abnormal at 1 year of age. These studies confirm previous associations based on visual analysis of FEEG and suggest that the relative frequencies of FEEG patterns may be useful in the prediction of neurologic outcome 1 year later.     

Preliminary X-ray crystallographic analysis of biotin carboxylase isolated from Escherichia coli

Acetyl CoA carboxylase catalyzes the first committed step in the biosynthesis of long chain fatty acids. In Escherichia coli, the enzyme consists of three subunits that are isolated separately and display distinct functional properties. Here we report the crystallization and preliminary X-ray analysis of one of these components, namely biotin carboxylase. The crystals are grown by microdialysis against 10 mM potassium phosphate (pH 7.0), 1 mM EDTA, 2 mM DTT and 1 mM NaN3 at 4 degrees C. They belong to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 61.9 A, b = 96.1 A and c = 180.6 A and contain one dimer per asymmetric unit. The crystals diffract to a nominal resolution of 2.2 A. From a mechanistic standpoint, biotin carboxylase is especially interesting in that it is the smallest protein within its class and is one of only two carboxylases that can utilize free biotin as a substrate.     

Thermal injury functionally alters bone marrow-derived macrophages: a study of monocyte-hepatocyte interactions

PURPOSE OF THE STUDY: The purpose of this study was to evaluate the results of the anterior cruciate ligament (A.C.L.) reconstruction according to two scoring systems (Arpege and IKDC), to analyse the influence of different factors on the results, to study the effect of a lateral extra-articular tenodesis, the morbidity of patellar tendon graft harvesting, and the advantage of arthroscopically assisted reconstruction. MATERIAL: Seventy nine patients, 17 to 39 years old (average 27 years), underwent an anterior cruciate ligament reconstruction for chronic instability, using a free bone-patellar tendon-bone graft. In 43 cases, a lateral extra-articular plasty was added (Lemaire's procedure). The reconstruction was arthroscopically assisted in 17 cases. Interval between initial injury and surgery was 24 months (2 months to 9 years and 7 months). The average follow-up was 2.5 years (range 1 to 18). METHOD: All patients were reviewed for evaluation with two scoring systems (Arpege and IKDC). Roentgenograms of both knees, including antero-posterior weight-bearing and lateral view, patellar view, dynamic radiographs, allowed evaluation of post-operative arthrosis and residual anterior laxity in extension. Fischer's test and chi square test were used for statistical evaluation. RESULTS: Using the Arpege CLAS system, functional results were excellent or good in 75.9 per cent of cases (excellent in 44.3 per cent, good in 31.6 per cent), fair in 15.2 per cent, poor in 8.9 per cent; according to the IKDC system, 65.8 per cent were excellent or good. 84.8 per cent of the patients were satisfied in Arpege system and 91.2 per cent in IKDC system. The pivot-shift test was negative in 86 per cent, equivocal in 7.6 per cent and positive in 6.4 per cent. The radiological Lachman's test (difference between control and affected knee) was 0-2 mm in 53.2 per cent, 3-5 mm in 39.2 per cent, 6-10 mm in 7.6 per cent, never greater than 10 mm. Antero-posterior weight-bearing radiographs were normal in 83.5 per cent, showed joint remodeling in 10.1 per cent pre-arthrosis in 6.3 per cent but no arthrosis. Functional results were not correlated with age at time of surgery, interval between initial injury and surgery, nor clinical Lachman's test. Competitive sportsmen had a better result (p = 0.001). Residual laxity in extension was correlated with lesions of medial meniscus (p = 0.035). Degenerative changes in femoro-tibial joint were correlated with residual laxity in extension (p = 0.019). There was no significative difference between A.C.L. reconstruction isolated or associated with lateral extra-articular tenodesis. Time to return to work was shorter for patients with arthroscopically assisted procedure (p = 0.067). DISCUSSION AND CONCLUSION: Functional results after A.C.L. reconstruction using a free bone-patellar tendon-bone graft are satisfactory and confirm the reliability of this procedure. Arpege CLAS and IKDC systems give comparable functional results, but IKDC evaluate anatomical results, residual laxity and degenerative changes of the joint, that constitute essential long-term pronostic factors. Morbidity of patellar tendon harvesting appears to be of short duration and largely reversible. Added lateral extra-articular tenodesis doesn't improve the results. Arthroscopically assisted procedure seems to allow a faster rehabilitation.     

Tissue fibronectin is an endogenous ligand for galectin-1

A 14K beta-galactoside-binding lectin (galectin-1) is present in many animal tissues. In a search for endogenous ligands, we surveyed galectin-1-binding proteins in human placenta. Extract of human placenta with 2 M urea was applied to a Sepharose 4B column conjugated with galectin-1 purified from frog (Rana catesbeiana) eggs. Two major proteins eluted with 100 mM lactose from the column-bound fraction showed apparent molecular masses of 220 and 180 kDa on SDS-PAGE under reducing conditions. Western blotting analysis using monoclonal antibodies indicated that these proteins were fibronectin and laminin, respectively. Most placental and amniotic fibronectins bound strongly to the column, whereas almost all plasma fibronectin passed through the column. The galectin-1, fibronectin and laminin were immunohistochemically shown to be co-localized in the extracellular matrix of placental tissue. In a cell attachment assay, rhabdosarcoma cells adhered to a plate coated with placental fibronectin, even in the presence of GRGDS peptide, if galectin-1 were also present. This adhesive effect of galectin-1 was inhibited by lactose. These results indicate that tissue fibronectin, as well as laminin, serve as endogenous ligands for galectin-1, suggesting that galectin-1 may play a role in assembly of the extracellular matrix, or in the control of cell adhesion based on lectin-extracellular matrix interaction.     

Estimates of the chromium(VI) reducing capacity in human body compartments as a mechanism for attenuating its potential toxicity and carcinogenicity

Cathepsin L was purified from carp hepatopancreas by a method involving ammonium sulfate precipitation and a series of column chromatographies, in which the enzyme had an affinity toward Concanavalin A and Cibacron Blue F3GA. Its homogeneity was established by Native-PAGE, but two protein bands corresponding to molecular masses of 30,000 (single chain) and 24,000 (heavy chain) migrated on SDS-PAGE. The enzyme exhibited a maximum activity for carbobenzoxy-L-phenylalanyl-L-arginyl-4-methylcoumaryl-7-amide (Z-Phe-Arg-MCA) at pH 5.5-6.0 and 50 degrees C and the remarkable stability at pH 5.0-6.5 and below 40 degrees C. All tested cysteine protease inhibitors and TLCK and chymostatin markedly inhibited its activity, whereas the other serine protease inhibitors and a metalloprotease inhibitor negligibly affected it. In addition, several metal compounds reduced either its activity or stability to differing extents. Although EDTA alone caused an only marginal activation of the enzyme, its maximum activation required both 2 mM cysteine and 1 mM EDTA. The enzyme had an ability to hydrolyze three peptidyl-MCA substrates including Z-Phe-Arg-MCA, but all kinetic constants indicate that Z-Phe-Arg-MCA is the optical substrate to the enzyme.     

On-line sample preparation of paraquat in human serum samples using high-performance liquid chromatography with column switching

A new ion-pair high-performance liquid chromatographic method with column-switching has been developed for the determination of paraquat in human serum samples. The diluted serum sample was injected onto a precolumn packed with LiChroprep RP-8 (25-40 microm) and polar serum components were washed out by 3% acetonitrile in 0.05 M phosphate buffer (pH 2.0) containing 5 mM sodium octanesulfonate. After valve switching to inject position, concentrated compounds were eluted in the back-flush mode and separated on an Inertsil ODS-2 column with 17% acetonitrile in 0.05 M phosphate buffer (pH 2.0) containing 10 mM sodium octanesulfonate. The total analysis time per sample was about 30 min and mean recovery was 98.5+/-2.8% with a linear range of 0.1-100 microg/ml. This method has been successfully applied to serum samples from incidents by paraquat poisoning.     

Vitamin B12, folic acid, ferritin and haemoglobin status of rural women in child-bearing age in northeast Thailand

Serum vitamin B12, folic acid, ferritin and haematological variables were investigated in six hundred and seven women from 12 villages of 3 districts namely Chumpae, Srichumpu and Pupaman of Khon Kaen province, Northeast Thailand within the age range of 15-45 years. The cut-off point of haemoglobin concentration < 12 g/dl was applied for defining the normal and anaemic group (17.3%). The result showed that the concentration of ferritin, folic acid and vitamin B12 in the anaemic group were less than that of the normal group. Forty six out of 607 women or 7.6 per cent were found to be undernourished and 27.2 per cent of females were overnourished. The prevalence of deficiencies of vitamin B12, folic acid and ferritin were 6.3 per cent, 4.3 per cent and 12.5 per cent respectively.     

Isotachophoretic determination of ethylenediaminetetraacetate in salad dressing, mayonnaise, and margarine

An isotachophoretic method was developed for the determination of EDTA in foods imported by Japan. Skimmed samples of dressings, mayonnaise, or margarine were chromatographed on an anion exchange column, and interfering organic acids were eluted with water and 0.01N HCl. EDTA was eluted with 0.2N HCl and reacted with ferric chloride to form a stable EDTA--Fe complex. Electrophoresis was carried out with dilute HCl containing 0.05% Triton and beta-alanine (pH 3.5) as the leading electrolyte and 0.01M caproic acid as terminating electrolyte. Since uncoupled EDTA showed more than one zone, it was reacted with ferric chloride to form EDTA-Fe complex which showed only a single zone on an isotachopherogram. More than 90% of EDTA spiked at 100 or 1000 ppm level as disodium salt was recovered from the above mentioned three types of food. Detection limit was 10 ppm as disodium EDTA.     

Correlation between p53 mutations and antibody staining in breast carcinoma

Abnormalities of the p53 gene and protein were examined in 81 primary breast carcinoma samples. Using a polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) analysis, mutations in p53 exons 5-8 were identified in 13 of 81 tumours (16 per cent) and confirmed by DNA sequencing. Positive staining for p53 protein was detected in ten of 77 (13 per cent) of these tumours using polyclonal CM1 antibody on formalin-fixed tissue. Mutations detected by PCR-SSCP analysis were more common in grade III tumours (P = 0.015), but no correlation was found with tumour size, node status or level of epidermal growth factor receptor expression. A p53 mutation was associated with positive antibody staining in only two patients. Positive immunohistochemical staining using a p53 antibody may detect p53 protein expression, but this may not correlate directly with an underlying mutation in the hot spot region examined.