D. Ross Boswell

Elevated levels of acute phase plasma proteins in major depression

Levels of acute phase and other plasma proteins were measured in 21 men with major depression, 28 men with alcohol dependence, and 12 men who acted as controls. The depressed men had significantly elevated levels of the acute phase proteins, haptoglobin and alpha-1-antichymotrypsin, and of immunoglobulin G. The elevations in haptoglobin and alpha-1-antichymotrypsin were highly correlated with each other, and were correlated with the severity of depression and negatively correlated with the thyroid stimulating hormone response to thyrotropin. The alcoholic men had elevated haptoglobin levels, but significantly decreased levels of immunoglobulin G. These findings provide further evidence for an inflammatory response during depression.

ASSESSMENT OF A COMPUTERIZED SYSTEM FOR THE DIAGNOSIS OF IRON DEFICIENCY

&NA; The aim of this study was to develop a computer expert system that could reproduce a pathologists diagnosis of iron deficiency from the data obtained from blood tests. 275 cases were collected for construction and testing of the expert system. The expert system used a combination of fuzzy set logic and cut‐off points from 14 parameters to arrive at one of 5 diagnostic categories graded from “iron deficient” to “no evidence of iron deficiency”. The agreement between pathologist and expert system was 0.91 (Spearman rank correlation coefficient) in the learning population; this dropped to 0.79 in the test population. Absolute agreement on diagnostic category was reached in 71% of cases. In no case was there disagreement by more than 3 grades.

Circulating proalbumin associated with a variant proteinase inhibitor

The unique finding of normal proalbumin in human plasma provides an insight into the mechanism of propeptide cleavage. Proalbumin, present as 1-5% of the total albumin, was found in a boy whose prime problem was the presence of a mutant proteinase inhibitor, alpha 1-antitrypsin Pittsburgh (358 Met----Arg) [2]. The inferred structure of human proalbumin was confirmed as Arg-Gly-Val-Phe-Arg-Arg-Alb. On incubation with various enzymes (trypsin, tryptase, thrombin, chymotrypsin, chymase and cathepsin B), only trypsin was capable of converting proalbumin to albumin. There was no conversion when proalbumin was incubated with whole blood, plasma or serum. However, intravenous injection of proalbumin into a rat resulted in complete conversion to albumin, the half-life of this process being 6 h. We conclude that propeptide cleavage is dependent on a serine proteinase which is inhibited intracellularly, by the mutant inhibitor, and that all the albumin in the boy was secreted as proalbumin, but was subjected to a separate cleavage process after export from the hepatocyte.

Active site of α1-antitrypsin: Homologous site in antithrombin-III

Abstract Examination of peptides resulting from reaction of bovine trypsin and human α 1 -antitrypsin in near-equimolar amounts showed anomalous cleavage of antitrypsin at a Met-Ser bond 37 residues from the C-terminus, giving evidence that this is the active site for trypsin inhibition. Alignment of the C-terminal 141 residues of α 1 -antitrypsin with the C-terminal 147 residues of human antithrombin-III showed homology with 30% identity and allowed the identification of a homologous active site in antithrombin.

Novel human proalbumin variant with intact dibasic sequence facilitates identification of its converting enzyme

We describe here the identification of a new genetic variant of human proalbumin with an N-terminal sequence of Arg-Gly-Val-Phe-Arg-Arg-Val-Ala-His-Lys-. Proalbumin Blenheim (10%) and mature albumin Blenheim (38%) with an initial sequence of Val-Ala-His-Lys-make up nearly half the serum albumin in affected individuals. Despite retaining an intact dibasic processing site, proalbumin Blenheim (1 Asp----Val) enters the circulation unprocessed. The observed ratio of proalbumin to albumin can be accounted for by proteolysis in the periphery. Employed as a potential substrate, proalbumin Blenheim provides a unique means of identifying the physiologically relevant proalbumin convertase. In vitro studies showed that the variant is readily cleaved by trypsin. However, it is not cleaved by the proposed proalbumin convertase, a membrane-bound Ca2+-dependent proteinase prepared from rat liver Golgi vesicles, which gives authentic cleavage of normal human proalbumin.

Reactive site of α1-antitrypsin is C-terminal, not N-terminal

Abstract α 1 -Antitrypsin recovered from trypsin- α 1 -antitrypsin complexes was shown to be a mixture of two peptides which remained associated in 6 M guanidine and in 1% acetic acid, but were separated by SDS-polyacrylamide gel electrophoresis. The larger peptide had an M r of 47 000 and gave low yields on end-group analysis; the smaller had an M r of 4000 and was the C-terminal 36-residue fragment of α 1 -antitrypsin. These results explain the consistent but erroneous finding of a reactive site near the N-terminus of α 1 -antitrypsin, and confirm that the reactive site is 36 residues from the C-terminus.

P1 variant antithrombins Glasgow (393 Arg to His) and Pescara (393 Arg to Pro) have increased heparin affinity and are resistant to catalytic cleavage by elastase Implications for the heparin activation mechanism

The heparin affinity of normal and two P1 variants of antithrombin‐III (AT) was studied by gradient elution with NaCl in Tris buffer on heparin‐Sepharose. At pH 7.4 normal AT eluted art [Na+] 0.78 mol/l and the variants both showed increased affinity with AT Pescara eluting at [Na+] 0.86 mol/l and AT Glasgow at [Na+] 0.92 mol/l. We have earlier proposed a model for heparin activation in which the native state of AT maintains a salt bridge involving the P1 Arg‐393 residue. Binding of heparin induces a higher heparin affinity conformation in which the salt bridge is disrupted to reveal the reactive centre for inhibition of thrombin. The Glasgow and Pescara variants, lacking a reactive centre P1 basic residue, would be unable to form this salt bridge, and we suggested that the high affinity conformation which they adopt as their native state would resemble the heparin induced conformation. To examine this model, we measured the heparin induced fluorescence of two P1 variants and tested the susceptibility of their reactive loops to catalytic cleavage. Both variants had fluorescence spectra indistinguishable from normal AT. In the absence of heparin, neither variant was more susceptible than normal to catalytic cleavage by human neutrophil elastase. These findings suggest that the conformation of these P1 variants is different to that of fully heparinized normal AT.

Haemoglobin Manukau β67[E11] Val→Gly : transfusion-dependent haemolytic anaemia ameliorated by coexisting alpha thalassaemia

Summary Haemoglobin Manukau (β67 Val→Gly) is a novel haemoglobin variant presenting in two brothers as non‐spherocytic haemolytic anaemia which became transfusion dependent by 6 months of age. The severity of clinical expression seems to be modulated by coexisting alpha thalassaemia: the severely affected children have a normal complement of alpha globin genes with an unusual genotype (‐α3,7/ααα3,7), while their father, who carries the abnormal gene with minimal symptoms, has homozygous α+ thalassaemia (‐α3,7/ ‐α3,7) Another unusual feature of this case is the association of the β67 Val→Gly mutation with modification of β141 Leu to a residue (believed to be hydroxyleucine) that is not detected by standard amino acid analysis. This finding offers an explanation for the previous report of an association of another mutation at this site (Hb Sydney β67 Val→Ala) with Hb Coventry (deletion of β141 Leu).