Evolution of α2-macroglobulin. The structure of a protein homologous with human α2-macroglobulin from plaice (Pleuronectes platessa L.) plasma

The plaice (Pleuronectes platessa L.) papain-binding protein previously demonstrated to be homologous with human alpha(2)-macroglobulin, and designated plaice alpha(2)-macroglobulin homologue or alphaMh, was shown to be a glycoprotein of s(20,w) 11.86S. In polyacrylamide-gel pore-limit electrophoresis under non-denaturing conditions plaice alphaMh migrated to the same position as half-molecules of human alpha(2)-macroglobulin, and treatment with methylamine or a proteinase caused no change in its electrophoretic properties. Either denaturation in urea (4m) or mild reduction by dithiothreitol (1mm) partially dissociated plaice alphaMh into half-molecules. Denaturation with reduction further dissociated the protein into quarter-subunits. In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reducing conditions plaice alphaMh dissociated into subunits of M(r) 105000 (I) and 90000 (II). Approximately equal amounts of each subunit were formed, and peptide ;mapping' showed subunits I and II to be distinct polypeptide chains. Under alkaline denaturing conditions, a proportion of the I chains of alphaMh were cleaved into fragments of M(r) about 60000 and 40000. This cleavage was favoured by reducing conditions and prevented by prior inactivation of the alphaMh with methylamine. [(14)C]Methylamine allowed to react with alphaMh became covalently linked to subunit I. These properties suggested the existence of an autolytic site on subunit I analogous to the autolytic site of human alpha(2)-macroglobulin. Reaction of alphaMh with a proteinase resulted in cleavage of a fragment of M(r) 10000-15000 from subunit I. A proportion of the proteinase molecules trapped by alphaMh became covalently linked to the inhibitor. A scheme is proposed for the evolution of human alpha(2)-macroglobulin and plaice alphaMh from a common ancestral protein, which may also have been an ancestor of complement components C3 and C4.     

Evolution of alpha 2-macroglobulin. The purification and characterization of a protein homologous with human alpha 2-macroglobulin from plaice (Pleuronectes platessa L.) plasma.

A papain-binding protein (PB-protein) was purified to homogeneity from the plasma of plaice (Pleuronectes platessa L.). PB-protein inhibited the activity of trypsin and pancreatic elastase (serine proteinases), thermolysin (a metalloproteinase) and papain (a cysteine proteinase). Presaturation of PB-protein with trypsin prevented the subsequent inhibition of thermolysin, and vice versa. Only catalytically active endopeptidases were bound by PB-protein. The catalytic activity of trypsin bound by PB-protein was inhibited by 95% against an insoluble protein substrate, but only by 38% against a low-molecular-weight synthetic substrate. The remaining activity of the bound trypsin was partially protected against further inhibition by soya-bean trypsin inhibitor. Trypsin bound by PB-protein showed a decrease of 67% in its reactivity with antibodies. The inhibitory activity of PB-protein was inactivated at pH 8.0 by methylamine (0.2M) or dithiothreitol (1 mM). The inhibition of proteinases by plaice PB-protein shows the distinctive characteristics of inhibition by human alpha 2-macroglobulin, and it is concluded that the plaice protein is a homologue of the human macroglobulin.     

Changes in serum amylase and its isoenzymes after whole body irradiation.

A study was carried out to assess the effect of total body irradiation on pancreatic and parotid isoenzymes of amylase in patients about to undergo bone-marrow transplantation who had received high-dose cyclophosphamide. Twelve patients were studied, enzyme activity being measured before and at various times after total body irradiation. Serum total amylase activity rose rapidly within 12 hours of irradiation to a maximum at 36 hours, returning to normal by six days; most of the increase was derived from salivary damage, with a much smaller pancreatic component. These results confirm that radiation produces acute changes in amylase activity, which may be of use in assessing radiation-induced damage.     

Cyclosporin A as prophylaxis against graft-versus-host disease in 36 patients.

Oral cyclosporin A was used as prophylaxis against graft-versus-host disease in (a) 31 patients with acute leukaemia or aplastic anaemia given transplants of HLA-matched bone marrow and (b) five patients with inborn errors of metabolism given transplants of haplotype-identical (parental) bone marrow. Twenty-six patients survived longer than two months after the operation. Despite the cyclosporin A, 31 patients (86%) suffered an acute form of graft-versus-host disease and 22 (61%) a chronic form. Nevertheless, the disease was usually treatable with immunosuppressive agents and caused the death of only one patient. Cyclosporin A caused renal toxicity in all cases; occasionally this was associated with a "capillary leak" syndrome, fatal in two patients. In children hypertension, fits, and fluid retention were common side effects. Blood concentrations of cyclosporin A correlated with blood urea values and blood pressure but did not predict the occurrence of graft-versus-host disease. Four different dose schedules were used to find the optimum way to administer this drug. Oral cyclosporin A is extremely effective at reducing the severity of graft-versus-host disease, but prevention of the disease is limited by toxicity of the drug and variable absorption. Better results might be achieved with parenteral administration or by using the drug in combination with other methods.     

Comparison between mutagenesis in normal and transformed syrian hamster fibroblasts: Difference in the temporal order of HPRT gene replication

A highly tumorigenic subdiploid cell line, BP6T, derived in our laboratory from Syrian hamster embryo (SHE) cells, is amenable to studies of somatic mutation in vitro. Cellular and biochemical characterization of clonally derived BP6T cells resistant to 6-thioguanine (TG^r) or ouabain (Oua^r) demonstrated these mutants to be similar qualitatively to mutants of SHE cells characterized previously (Barrett et al., 1978). BP6T TG^r mutants resistant to 6-thioguanine are cross-resistant to 8-azaguanine, lack HPRT activity, exhibit a low frequency of reversion and arise spontaneously at a rate of 5 × 10⁻⁷ mutants per cell per generation. BP6T Oua^r mutants were shown to be highly resistant to ouabain-mediated inhibition of ⁸⁶Rb influx, indicating an alteration in the Na⁺/K⁺ ATPase. These studies on the BP6T cell line provide the experimental basis for a comparative study of the mutagenic responses of normal, diploid SHE cells versus those of related, but transformed aneuploid cells. Highly synchronized cultures of these 2 cells were mutagenized by pulse treatment with BrdU during different periods of S phase, followed immediately by near-UV irradiation. The induced mutation frequencies so obtained provided information about the temporal order of replication of genes encoding HPRT and Na⁺/K⁺ ATPase in both SHE and BP6T cells. The temporal pattern of replication of Na⁺/K⁺ ATPase gene loci is similar in both cell types, but the temporal order of replication of the HPRT gene is significantly different between SHE and BP6T cells (mid-late S phase, versus early S phase, resp.). This observed difference emphasizes the caution required in the study of mutagenesis and DNA replication using transformed, aneuploid cells under the assumption that the underlying mechanisms are the same for normal, diploid cells.     

Quantification of surfactant phospholipids in the dog lung

We quantified total phospholipid (PL), total and disaturated phosphatidylcholine (PC and DSPC), phosphatidylglycerol (PG), and total protein in alveolar washings and lung tissue in 22 dog lungs. Quantitative recovery of alveolar material and assessment of its possible contamination by blood lipids were important determinants of methodology. To remove blood, the vessels of half the lungs were perfused with a fluorocarbon emulsion before lavage. The volume of blood removed by perfusion and the quantity and fatty acid patterns of its whole blood and plasma PL and PC were determined. Washings of unperfused lungs contained means of 21% more PL and 24% more PC than those of perfused lungs. Although this excess could be accounted for by the PL and PC in pulmonary blood, the hemoglobin and total protein content of washings and their PC fatty acid patterns indicated that blood lipids were not a major source of the excess lipid in washings of unperfused lungs. Using more recent morphometric estimates rather than the indirect ones previously used by others, the quantity of alveolar DSPC (1 mg/g lung) is calculated to be 1.8 times the amount necessary to form a packed monolayer on the internal surface of the lung at functional residual capacity.     

Calcium-dependent slow potassium conductance in rat skeletal myotubes

A prolonged hyperpolarizing afterpotential (amplitude 5–20 mV, half decay time about 400 msec at 25°C) follows the action potential in myotubes and myosacs cultured from rat skeletal muscle. This slow hyperpolarizing afterpotential (hap) is mediated by an increase in membrane K conductance, because its reversal potential follows the Nernst potential for K and is not affected by other ions. The conductance increase measured during the hap (up to four times the resting input conductance) correctly predicts the time course of the slow hap. The slow hap is Ca dependent. Its amplitude decreases when bath [Ca] is lowered, and both amplitude and duration increase when bath [Ca] is raised. The slow hap is blocked by intracellular injection of the calcium chelator, EGTA. It is inhibited by solutions containing 2–4 mM manganese or 1–5 mM barium, but is not blocked by 5–20 mM tetraethylammonium. Myotubes bathed in zero [Na], high [Ca] solutions show calcium action potentials, which are inhibited by 2–10 mM manganese, nickel or cobalt. Myotubes bathed in isotonic Ca salts (or in 2 mM Ca plus 5 mM caffeine) show long-lasting (up to 10 sec) spontaneous hyperpolarizations accompanied by prolonged contractions. These hyperpolarizations are associated with a large increase in input conductance, and they reverse in sign near the K equilibrium potential. They appear to reflect activation of the Ca-sensitive K conductance by Ca released from intracellular stores. The observation that spontaneous hyperpolarizations usually occur with no prior depolarization argues that at least a portion of the slow, Ca-sensitive K conductance system can be activated by internal Ca alone, with no requirement for plasma membrane depolarization. Cultured myotubes also have a faster K conductance system, which is inhibited by 5–20 mM tetraethylammonium or 1–5 mM barium, and is not dependent on Ca for its activation.     

The electrophoretically ''slow'' and ''fast'' forms of the alpha 2-macroglobulin molecule.

alpha 2-Macroglobulin (alpha 2M) was isolated from human plasma by a four-step procedure: poly(ethylene glyco) fractionation, gel chromatography, euglobulin precipitation and immunoadsorption. No contaminants were detected in the final preparations by electrophoresis or immunoprecipitation. The protein ran as a single slow band in gel electrophoresis, and was designated 'S-alpha 2M'. S-alpha 2M bound about 2 mol of trypsin/mol. Treatment of S-alpha 2M with a proteinase or ammonium salts produced a form of the molecule more mobile in electrophoresis, and lacking proteinase-binding activity (F-alpha 2M). The electrophoretic mobility of the F-alpha 2M resulting from reaction with NH4+ salts was identical with that of proteinase complexes. We attribute the change in electrophoretic mobility of the alpha 2M to a conformation change, but there was no evidence of a change in pI or Strokes radius. Electrophoresis of S-alpha 2M in the presence of sodium dodecylsulphate gave results consistent with the view that the alpha 2M molecule is a tetramer of identical subunits, assembled as a non-covalent pair of disulphide-linked dimers. Some of the subunits seemed to be 'nicked' into two-thires-length and one-third-length chains, however. This was not apparent with F-alpha 2M produced by ammonium salts. F-alpha 2M produced by trypsin showed two new bands attributable to cleavage of the subunit polypeptide chain near the middle. Immunoassays of F-alpha 2M gave 'rockets' 12-29% lower than those with S-alpha 2M. The nature of the interactions between subunits in S-alpha 2M and F-alpha 2M was investigated by treating each form with glutaraldehyde before electrophoresis in the presence of sodium dodecyl sulphate. A much greater degree of cross-linking was observed with the F-alpha 2M, indicating that the subunits interact most closely in this form of the molecule. Exposure of S-alpha 2M to 3 M-urea or pH3 resulted in dissociation to the disulphide-bonded half-molecules; these did not show the proteinase-binding activity characteristic of the intact alpha 2M. F-alpha 2M was less easily dissociated than was S-alpha 2M. S-alpha 2M was readily dissociated to the quarter-subunits by mild reduction, with the formation of 3-4 new thiol groups per subunit. Inact reactive alpha 2M could then be regenerated in high yield by reoxidation of the subunits. F-alpha 2M formed by reaction with a proteinase or ammonium salts was not dissociated under the same conditions, although the interchain disulphide bonds were reduced. If the thiol groups of the quarter-subunits of S-alpha 2M were blocked by carboxymethylation, oxidative reassociation did not occur. Nevertheless treatment of these subunits with methylammonium salts or a proteinase caused the reassembly of half-molecules and intact (F-) tetramers. It is emphasized that F-alpha 2M does not have the properties of a denatured form of the protein...     

Long-term culture of human aortas

Summary Segments of human thoracic aorta were maintained in long-term explant culture for 18 weeks in serum-supplemented medium. The aortas were grossly normal in appearance, and random samples fixed for light microscopy prior to culture revealed a normal morphology. The intima contained no more than five layers of smooth muscle cells. After 7 days in culture, the intima was noticeably thicker than the uncultured segments. The increased thickness was due to proliferating smooth muscle cells and production of extracellular material. After several months in culture, extracellular material consisting of collagen and flocculent material was present in areas resembling atherosclerotic fibrous plaques. A peripheral growth, which formed around the explant, was composed of fibroblastlike cells and added to the overall thickness of the intima. However, aortic segment maintained for up to 2 months in serum-free culture medium showed no cellular proliferation. This study demonstrates that changes resembling early stages of atherosclerosis occur in human aortas maintained in explant culture using routine culture procedures. Supported in part by the Pangborn Fund and the Graduate School of the University of Maryland. This is publication 443 from the Cellular Pathobiology Laboratory.     

Report of a trisomy 8p infant with carrier father.

This report describes an infant with fatal congenital heart disease, cleft palate, brain malformations, and trisomy 8p resultant from the paternal balanced reciprocal translocation, rcp(8;15) (p11;p11). Review of six previously reported trisomy 8p patients (resultant from parental balanced translocation in each instance) revealed severe mental retardation in five, short stature in all, and a variety of brain, skeletal, and cardiac defects. The features of the seven trisomy 8p patients reviewed here are not sufficiently similar to suggest a distinct dysmorphic syndrome. In addition the features differ from those in the trisomy 8 mosaicism syndrome, in which the mental retardation and malformations are generally less severe.