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Acetylcholinesterase (AChE) molecular isoforms from anglionic and adjacent unaffected (control) colonic tissue in patients with Hirschsprung's disease (HD) were analysed by density gradient centrifugation in order to determine the major AChE isoforms and the effect of a reported MgCl2 inactivation assay method upon them, with a view to improving the AChE assay used in the diagnosis of Hirschsprung's disease. The total AChE level was greater in the HD-affected colonic tissue than the control tissue (HD: 9.0 vs. control: 7.3 units/g tissue) and this was due to a consistently greater elevation of the globular tetrameric form, G4 (HD: 48.8% vs. control: 35.5% of total AChE). The inactivation of whole tissue homogenates by brief exposure to 4 mol/l MgCl2 followed the same pattern (HD: 48.4% vs. control: 28.7% inactivation). The detergent-extractable G4 is inhibited to a greater extent than the low salt-soluble G4 by exposure to 4 mol/l MgCl2 (83.8% vs. 51.1%). These results imply that the elevated AChE levels in HD are mainly due to increases of the hydrophobic globular tetrameric form of AChE of the same type that is found in differentiating nervous tissue before synapse formation. The monomeric globular isoform G1 is not inhibited but the asymmetric A4, A8 and A12 isoforms are completely inhibited by exposure to 4 mol/l MgCl2. All isoforms of Torpedo (electric ray) electroplax and human erythrocyte AChE, mainly amphiphilic G2, are almost completely inhibited. The inhibition by 4 mol/l MgCl2 of the main G4 isoform in HD-affected intestine accentuates the difference between aganglionic and unaffected intestine in fractionated samples, but does not provide a sufficiently specific G4 isoform assay. The use of 2-4 mol/l MgCl2 in histochemical AChE staining reduces the activity slightly but does not differentiate the tetrameric AChE isoform that is increased in Hirschsprung's disease but does reduce contaminating erythrocyte AChE levels that can obscure the result in blood-stained biopsies. A specific immunochemical stain for hydrophobic AChE tetramers or the associated 20 kDa membrane associated subunit is therefore needed to provide specificity.

Periodate modification of human serum transferrin produces a species that binds Fe(III) weakly at pH 7.4 contrary to previous reports that Fe(III)-binding activity is completely lost. Ternary complexes of periodate-modified transferrin and either Fe(III) with nitrilotriacetate (NTA), oxalate, citrate, or EDTA, or of Cu(II) with oxalate could be formed. Peak wavelength maxima of these spectral bands are identical to those reported for native transferrin in the absence of bicarbonate. No carbonate ternary complexes of periodate-modified transferrin with Fe(III), Al(III), Cu(II), or Zn(II) could be formed. Conditional (Fe(NTA)) binding constants (log K) for C- and N-terminal modified sites are 7.33 and 7.54, respectively. The respective extinction coefficients at 470 nm are decreased 45% compared with the native protein. The electron paramagnetic resonance spectrum of the complex closely resembles that of the Fe(III)-NTA ternary complex formed with native transferrin in the absence of bicarbonate. Anions, including bicarbonate, at high concentrations destabilize formation of this Fe(III)-NTA ternary complex, while Fe(III) chelators readily remove the bound Fe(III). Bicarbonate, sulfate, and pyrophosphate still bind to the modified binding sites in the absence of metal although with slightly lower affinity and with lower molar difference absorptivities. Results are interpreted as an inhibition of a crucial protein conformational change by an intramolecular cross-link, preventing formation of the particularly stable metal-carbonate ternary complex from the less stable metal-chelate ternary complex. The method can be used to produce monosited transferrins.

Every polymerase chain reaction (PCR) requires use of a temperature cycler for about 3 hours. Since there are many diagnostic tests using this technology, it is important that robust but inexpensive machinery is available. Such a stand-alone machine has been designed and used to analyse an interesting family in which a mother and her 2 children were diagnosed as having cystic fibrosis.

PAV protease is able to cleave between the crosslinked sites of the gamma-chain of fibrin and fibrin-derived D-dimer. The rate of digestion can be increased fourfold to tenfold by the addition of zinc ions. Although the PAV protease is a zinc-containing metalloenzyme, the enhanced rate of cleavage can be shown to be due to histidine-specific zinc binding by D-dimer. It is proposed that zinc ions cause a distortion of the inter-crosslink peptide chain, creating a novel protease-susceptible site. The physiologic relevance is uncertain due to the relatively low measured zinc binding constant Kd = 10(-3.88) M. Zinc binding could, nevertheless, create a useful fibrin-specific neoepitope for antibody recognition in vitro.

We report here on the lactate dehydrogenase (LD) of a patient who presented with an additional LD isoenzyme band between LD 3 and LD4 found during routine examination. The abnormal LD was found to be of increased molecular size, which was not due to complexing with immunoglobulins. The abnormal LD was purified by affinity chromatography, labelled and electrophoresed on a SDS-polyacrylamide gel. Autoradiography suggested abnormal aggregation as the cause of increased molecular size.

Ther serum concentration of alpha-fetoprotein (AFP) was measured by radio-immunoassay in 98 patients with liver disease including hepatoma, chronic active hepatitis, alcoholic cirrhosis, and acute virus B hepatitis. Raised AFP levels, above 30 ng/ml, were found in 87% of patients with acute viral hepatitis, in 82% of patients with primary liver cell carcinoma, in 58% with chronic active hepatitis and in 14% of patients with alcoholic cirrhosis. However, levels above 1 000 ng/ml were found only in patients with hepatoma and in acute viral hepatitis.

Eight patients with diffuse plasma cell infiltration of the small bowel who had the clinical features of immunoproliferative small intestinal disease (IPSID), but whose serum was negative for free alpha-heavy chains, were investigated for evidence of a non-secretory form of alpha-chain disease (alpha-CD). Molecular sieving and immunoblotting of serum, immunoperoxidase staining of biopsy specimens, and in vitro protein synthesis studies utilising an immunoprecipitation technique and polyacrylamide gel electrophoresis, failed to detect any new cases of alpha-CD. Four of the eight cases were found to have diffuse intestinal lymphoma. The remaining four patients, who were unsuccessfully investigated for evidence of a significant abnormality in cellular immunity, have not developed detectable alpha-CD protein or lymphoma over a mean of 143 months. Despite continuing exposure to possible environmental stimuli, it is concluded that not all cases of IPSID elaborate detectable alpha-CD protein or evolve to lymphoma.

The pattern of cancer in African gold miners over the 8-year period 1964-71, comprising 2,926,461 man-years of employment was studied. Of the 1344 cancers found, primary liver cancer accounted for 52-8%, oesophageal cancer 12-1%, cancer of the respiratory system 5-4% and cancer of the bladder 4-8%. Analysis of the spatial distribution of these four cancers, both on subcontinental and local scale, showed distinct gradients of occurrence between areas of significantly higher and lower incidence than expected. In the case of primary liver cancer in Mozambique and oesophageal cancer in the Transkei, the spatial distribution reflects closely that found in the general resident population of each territory. The crude incidence rate of primary liver cancer in gold miners from Mozambique dropped sharply over the period of the survey.

Rat fibrinogen plasmic fragment E was found to contain one oligosaccharide chain per gamma-chain attached by a glycosylamine linkage. The oligosaccharide was composed of 1 sialic acid, 1 galactose, 2 mannose and 2 glucosamine residues. The probable sequence from the nonreducing end was sialic acid leads to galactose beta leads to mannose alpha leads to mannose alpha leads to glucosamine leads to glucosamine. No difference in the rate of clearance from the rat circulation could be detected between native and desialated fragment E. A non-denaturing method for the purification of fragment E is described.

Histone octamers have been reconstituted from acid-extracted chicken erythrocyte histones. By the criteria of molecular size on exclusion chromatography as well as sedimentation velocity and conformational properties established by circular dichroism, fluorescence spectroscopy and imido-ester cross-linking, the reconstituted octamers have a structure identical to that of salt-extracted octamers.

Simple mixing of acid purified histones H3 and H4 in equimolar quantities at low ionic strength near pH 7 does not yield the tetramer but rather a high Mr aggregate. Dialysis of acid extracted total or core histones into 2 M NaCl 150 mM phosphate (pH 7.4) followed by fractionation of the histone complexes at lower ionic strength (150 mM NaCl) results in an H3-H4 tetramer of a structure identical to that derived from salt-extracted histones. Dialysis of acid extracted total or core histones directly into the lower ionic strength buffer with subsequent fractionation, results in H3-H4 tetramer of closely similar structure.

Previous studies are in conflict over the effect of infusing mixed fibrinogen-fibrin degradation products on fibrinogen synthesis, as determined by changes in fibrinogen concentration or by incorporation of labeled amino acids into fibrinogen. We have injected purified homologous fragments D1 and E into rats and measured their fibrinogen and albumin synthetic rates by the [14C]carbonate technique, a method that provides quantitative estimates of hepatic secretory protein synthesis. Fibrinogen fractional synthetic rates were increased 2.5 times in animals injected with fragment D1, compared with saline-injected controls. No increase were observed in fragment E-injected animals. Neither fragment produced changes in albumin synthesis. Fragment D increased plasma fibrinogen concentration, but did not raise plasma haptoglobin levels. These results suggest that fragment D is a regulator of fibrinogen synthesis.

The lung is the target organ most frequently involved in the early phase of multiple organ failure. Microembolisation of the pulmonary vasculature by bacterial and non-bacterial particles and debris with failure of the clearance mechanism of the reticuloendothelial system (RES) and depletion of plasma fibronectin have been implicated in the pathogenesis. The present study examined the concurrent changes in plasma fibronectin, RES phagocytic function, organ localisation of bacterial and non-bacterial particles and the levels of circulating endotoxin and fibrin degradation products in a clinically relevant murine model of severe intra-abdominal infection. Progressive sepsis was associated with deteriorating RES phagocytic function to 45% of control values within 48 h of sepsis induction. There was decreased hepatosplenic uptake and increased pulmonary localisation of bacterial and lipid emulsion particles. Plasma fibronectin increased in septic animals within 48 h suggesting increased fibronectin production. These changes would support the hypothesis that altered RES function may facilitate pulmonary microembolisation in the pathogenesis of septic multiple organ failure.

An established and validated method using loops of intestine in vivo in rats was used to study the effects of cytoskeletal inhibitors on iron absorption. Radioactive iron instilled into the loop of intestine pretreated with test substance was monitored in the blood and, after death, ferritin loading with radioactive iron was measured on density gradients of mucosal cell homogenates and absorbed iron in the carcass was determined. Colchicine, vincristine and cytochalasin B all caused dose- and time-dependent inhibition of iron absorption, and the effects of cytochalasin B were reversible within 1 h. It is not known which cellular component is the vehicle for the transcellular movement of iron from the intestinal lumen onto plasma transferrin; however, this study showed that the uptake of iron by ferritin in an iron-absorbing loop of intestine paralleled the actual absorption of iron into the carcass. This phenomenon did not occur in non-iron-absorbing intestinal and was inhibited by the action of the cytoskeletal inhibitors in the iron-absorbing region. Previously we had shown that iron uptake into cells and onto cellular transferrin was virtually the same throughout the small intestine, irrespective of the iron-absorbing capacity of the region. The results of this study therefore suggest that iron absorption depends on an intact cytoskeletal system and that ferritin in the iron-absorbing cell is able to load from the pool of iron committed to transcellular movement onto plasma transferrin.

The transferrin that is isolated from washed intestinal mucosal cell preparations consists partly of a fraction that has properties distinguishing it from serum transferrin. The serum transferrin contaminating mucosal preparations, even when fully saturated with iron and in the presence of proteinase inhibitors, also acquires the properties of the mucosal transferrin when the mucosa is homogenised. The mucosal transferrin is modified by a single cleavage of the polypeptide chain yielding a disulphide-linked peptide of 6550 daltons linked to the parent protein by a disulphide bridge. The amino-terminal sequence of the first 11 residues of this peptide could be aligned with both the known rat and human transferrin carboxy-terminal sequences. In both cases the sequence is preceded by a phenylalanine residue (residue 622 of human transferrin). This suggested that a mucosal chymotryptic enzyme was responsible even though rat transferrin is not susceptible to alpha-chymotrypsin if fully iron-saturated. Since transferrin mRNA is not found in the intestinal mucosa it must be imported from the serum. It remains uncertain whether the modified transferrin is present naturally and plays a role in iron absorption but these findings do indicate the eventual fate of any transferrin imported into an intestinal cell.

We have compared serum viscosities in 2 patients with the IgA type of multiple myeloma. One patient had the rare condition of IgA-2 myeloma. We confirm that hyperviscosity is associated with higher polymers. It would appear that there is no barrier to polymer formation with the IgA-2 subclass.

We have examined the plasmic digestion products of fibrin formed in the presence of dansylcadaverine, the fluorescent D dimer, to determine whether they are held together not only by the cross-link region on the gamma chain but also by other interactions on the D domain. Antibodies to the D dimer reacted 8X more strongly with sites on the D dimer (purified or in the presence of E) than with sites on fibrinogen or plasmin-digested fibrinogen. The reactivity of this surface site was lost when the gamma chain was cleaved by plasmin after the molecule had been destabilized by the removal of calcium ions, thus breaking the covalent linkage of the homodimer. The noncovalent D dimer retained its dimeric structure by the criteria of molar volume, measured by fluorescence polarization, and molecular sieving. The noncovalently attached, cross-link-containing peptide bound tightly to the parent molecules at higher temperatures but rotated more freely below 15 degrees C, and could be lost from the parent molecules without destroying the dimeric structure. We therefore propose that the forces maintaining the dimeric structure of the noncovalently joined molecule are not solely located at the gamma-chain cross-link region. These other sites on the D domain are therefore candidates for the initial fibrinogen polymerization site and may also have a role in fibrinogen half-molecule assembly.

The distribution and quantitation of the iron-binding proteins of rat small intestinal mucosa was studied, in iron-deficient and replete animals, to explore their role in the absorption of iron. Adsorption (mucosal uptake) of iron in in situ ligated loops of small intestinal mucosa was found to be uniform throughout the length of the small intestine whereas absorption (carcass uptake) showed a steep decreasing gradient from the duodenum to the ileum. The disrupted, in vivo labeled mucosal cells were fractionated by isopycnic centrifugation and transferrin and ferritin were quantitated by radioimmunoassay. Transferrin derived from mucosal cells was shown to have a higher affinity for the antibody than transferrin in serum. Of the transferrin present in the mucosal extract, only a portion could be accounted for by contamination from the serum; the proteolysis resistant and intrinsic transferrin may be mucosal cell specific. Transferrin was found in similar amounts in all regions of the small intestine, was not affected by iron loading but doubled in response to iron deficiency. Mucosal ferritin was found in greater amounts in the iron-absorbing areas of the intestine, increased in the duodenum of iron-loaded animals, and decreased in iron-deficient animals. The incorporation of newly absorbed radioiron into ferritin was only found in iron absorbing regions and was completely inhibited by colchicine and cytochalasin-B, suggesting that ferritin was loaded with iron at the point of iron absorption and that the process is associated with vesicle movement and not simple diffusion. Transferrin and ferritin-specific immunoabsorption and also gel filtration established that no other soluble iron binding proteins were involved in absorption.

A technique for identifying and quantitating fibrin- and fibrinogen-related antigens (FRAs) in serum is described. Panspecific antifibrinogen IgG is bound covalently to diazotized aminophenylthio-ether cellulose paper discs. Under conditions of antibody excess, a disc probe extracts 95% of all fibrinogen-related antigen from appropriately diluted serum samples. Bound antigen is then eluted from the washed probe and electrophoresed on 4-11% gradient SDS-polyacrylamide gels. Derivatives of cross-linked fibrin, non-cross-linked fibrin and fibrinogen may thus be identified by their molecular weights, and their relative concentrations determined by densitometric analysis. The total amount of FRA is estimated by a radio-immunoassay which uses labelled D-dimer as the competitive antigen. The probes can extract as little as 0.20 and 0.15 microgram/ml of D-dimer and D-monomer, respectively. Specific FRA binding was confirmed with Western blotting. Potential interfering substances do not influence antigen binding to the discs and, since complete elution of all antigens is achieved, discs may be re-used. This procedure provides estimates of the serum levels of fragments YY, DY, X, DD, Y and D. The procedure is simple to perform and, as a research tool, may help to delineate some of the features of clotting disturbances in clinical states.

The mean serum fibronectin level of normal subjects, measured by radio-immunoassay, was 363 micrograms/ml (+/- 72 micrograms/ml; 1 SD). The levels were lower in younger subjects and in females using oral contraceptives. In most patients admitted to a respiratory intensive care unit fibronectin levels were low initially, and only remained low in those cases in which severe complications developed. There were no rapid fluctuations of fibronectin levels measured on a daily basis, suggesting therefore that a consumptive reduction of fibronectin is not a feature. The fibronectin molecule itself has abnormal properties in pathological cases and is poorly precipitable by heparin. These studies do not support the proposal that administration of plasma or cryoprecipitates would be beneficial chiefly because of the fibronectin content.

Histones can be extracted from chicken erythrocyte chromatin with 2 M CaCl2 10 mM Tris (pH 7.4). The core histones so extracted exist as H3-H4 tetramers and H2A-H2B dimers since calcium at greater than 0.5 M result in dissociation of the histone octamer. Reconstitution of octamers occurs on removal of the Ca2+ by dialysis. Although less than 0.5 M calcium do not result in octamer dissociation, perturbations in the structure can be detected by CD and tyrosine fluorescence spectroscopy.

Serum alpha-fetoprotein levels were measured serially by radioimmunoassay in 32 patients with acute viral hepatitis. With one exception, the levels were increased during the course of the illness. Raised concentrations of the protein occurred almost equally in patients with and without the hepatitis-B antigen. In the majority of patients the rise was modest but in seven levels were reached at which alpha-fetoprotein could be detected by immunodiffusion. Very high levels were more likely to occur in children. In eight patients the concentration increased as the serum glutamic pyruvic transaminase (SGPT) level, used as an index of hepatocyte damage, was returning to normal, a pattern which favoured increased synthesis of the protein during hepatocellular regeneration. In the remainder, the alpha-fetoprotein level paralleled that of SGPT, suggesting rather an acutephase reaction to liver injury.