Physiochemical and biological properties of the major basic protein from guinea pig eosinophil granules.

Guinea pig eosinophil granules are characterized by the presence of a basic protein of low molecular weight which accounts for greater than 50% of granule protein. This protein, termed the major basic protein (MBP), readily aggregates and becomes insoluble, and the formation of aggregates is dependent on the establishment of disulfide bonds. Analysis of concentrated preparations of MBP often revealed a series of bands which were multiples of a monomeric unit with a mol wt of approximately 11,000. Analysis of reduced and alkylated MBP on a 10% agarose column equilibrated with 6 M guanidinium chloride revealed a single polypeptide chain with a mol wt of 10,800. Amino acid analysis of the protein revealed the presence of 13% arginine, consistent with the basic character of the molecule. Four residues of tryptophan, were present, indicating that MBP is not a histone. The MBP did not increase vascular permeability when injected into the skin of guinea pigs, nor did it antagonize the effect of histamine and bradykinin in the skin. MBP also did not contract the isolated guinea pig ileum and when mixed with histamine or bradykinin did not inhibit their activity on the gut. MBP had only weak, if any, antihistaminic activity. MBP possessed weak bactericidal activity when compared to histone and then only with one strain of E. coli. MBP precipitated DNA, neutralized heparin, and activated papain. On a molar basis MBP was more active than cysteine in activating papain. These results do not point to any unique biological activity associated with MBP other than those expected of a protein as basic as it is and one which possesses reactive sulfhydryl groups. Possible functions of eosinophils based on the properties of the MBP are discussed.

Trypsin inhibition by mouse serum: sexual dimorphism controlled by testosterone.

The trypsin inhibiting activity in the serum of male mice is substantially greater than that in females. In five strains of mice and two large groups of interstrain hybrids this difference ranged from 14 (in ICR mice) to 55 percent (in DBA mice). Castration of males significantly decreased the serum trypsin inhibiting activity, whereas the administration of testosterone restored the activity to its original level. Administration of testosterone to female mice increased the activity to a level similar to that in males of the same strain. Because almost all the change in inhibiting activity occurred in the electrophoretic alpha-1 region, alpha-1 region, alpha-1-antitrypsin is probably responsible for this effect.

Inherited variations of human serum alpha-1-antitrypsin.

The normal serum alpha(1)-antitrypsin migrates as a three banded patternwhen separated electrophoretically in starch gel with a sodium acetateethylenediaminetetraacetic acid buffer of pH 4.95. The results obtained when certain inherited variants of the serum alpha(1)-antitrypsin are separated electrophoretically suggest that the previously described variations in the region preceding the albumin band represent inherited variations of the serum alpha(1)-antitrypsin.

Obstructive lung disease and alpha-1-antitrypsin deficiency gene heterozygosity.

The phenotypes of serum alpha(1)-antitrypsin were determined by antigenantibody crossed electrophoresis. There were five homozygotes and 25 heterozygotes for the deficiency gene found in a group of 103 patients with obstructive lung disease. The frequency of heterozygotes was 14 and 9 percent in two control groups with different mean ages of 36 and 80. There was only one heterozygote among 39 healthy males over 70 years of age. Heterozygosity may be a predisposing factor in chronic obstructive lung disease, especially in the male population.

Regulation of factor XIa activity by platelets and alpha 1-protease inhibitor.

We have studied the complex interrelationships between platelets, Factor XIa, alpha 1-protease inhibitor and Factor IX activation. Platelets were shown to secrete an inhibitor of Factor XIa, and to protect Factor XIa from inactivation in the presence of alpha 1-protease inhibitor and the secreted platelet inhibitor. This protection of Factor XIa did not arise from the binding of Factor XIa to platelets, the presence of high molecular weight kininogen, or the inactivation of alpha 1-protease inhibitor by platelets. The formation of a complex between alpha 1-protease inhibitor and the active-site-containing light chain of Factor XIa was inhibited by activated platelets and by platelet releasates, but not by high molecular weight kininogen. These results support the hypothesis that platelets can regulate Factor XIa-catalyzed Factor IX activation by secreting an inhibitor of Factor XIa that may act primarily outside the platelet microenvironment and by protecting Factor XIa from inhibition, thereby localizing Factor IX activation to the platelet plug.

Alpha-1-antitrypsin-Pittsburgh. A potent inhibitor of human plasma factor XIa, kallikrein, and factor XIIf.

Alpha-1-antitrypsin-Pittsburgh is a human variant that resulted from a point mutation in the plasma protease inhibitor, alpha 1-antitrypsin (358 Met----Arg). This defect in the alpha 1-antitrypsin molecule causes it to have greatly diminished anti-elastase activity but markedly increased antithrombin activity. In this report, we demonstrate that this variant protein also has greatly increased inhibitory activity towards the arginine-specific enzymes of the contact system of plasma proteolysis (Factor XIa, kallikrein, and Factor XIIf), in contrast to normal alpha 1-antitrypsin, which has modest to no inhibitory activity towards these enzymes. We determined the second-order-inactivation rate constant (k'') of purified, human Factor XIa by purified alpha 1-antitrypsin-Pittsburgh and found it to be 5.1 X 10(5) M-1 s-1 (23 degrees C), which is a 7,700-fold increase over the k'' for Factor XIa by its major inhibitor, normal purified alpha 1-antitrypsin (i.e., 6.6 X 10(1) M-1 s-1). Human plasma kallikrein, which is poorly inhibited by alpha 1-antitrypsin (k'' = 4.2 M-1 s-1), exhibited a k'' for alpha 1-antitrypsin-Pittsburgh of 8.9 X 10(4) M-1 s-1 (a 21,000-fold increase), making it a more efficient inhibitor than either of the naturally occurring major inhibitors of kallikrein (C-1-inhibitor and alpha 2-macroglobulin). Factor XIIf, which is not inhibited by normal alpha 1-antitrypsin, displayed a k'' for alpha 1-antitrypsin-Pittsburgh of 2.5 X 10(4) M-1 s-1. This enhanced inhibitory activity is similar to the effect of alpha 1-antitrypsin-Pittsburgh that has been reported for thrombin. In addition to its potential as an anticoagulant, this recently cloned protein may prove to be clinically valuable in the management of septic shock, hereditary angioedema, or other syndromes involving activation of the surface-mediated plasma proteolytic system.

Enzymatic trimethylation of residue-72 lysine in cytochrome c. Effect on the total structure.

A highly purified protein methylase III from Neurospora crassa or Saccharomyces cerevisiae specifically methylates a single lysine residue of position 72 of horse heart cytochrome c. The enzymatically methylated cytochrome c has been separated from the unmethylated counterpart species by isoelectric focusing. Simultaneously, the pI values of these two species were found to be 9.49 and 10.03, respectively. Since methyl substitution increases the basicity associated with the epsilon-amino group of lysine residues, the observed decrease in pI value is in opposition to the predicted increase. Space-filling models revealed the possibility of a hydrogen bond between the oxygen of amide of residue-70 asparagine and the epsilon-amino nitrogen of residue-72 lysine in unmethylated horse heart cytochrome C. the enzymatic methylation of residue-72 lysine tends to dissociate this hydrogen bond, thereby possibly inducing the shift of 'effective charge' of the protein molecule. This paper also deals with the pI values of cytochromes c from 13 different sources, determined by the isoelectric focusing technique.

Genetic heterogeneity of rabbit alpha-1-antitrypsin.

Sixteen inbred or partially inbred strains of rabbits were investigated for electrophoretic and quantitative variations of alpha-1-antitrypsin (A-1-AT). We found interindividual differences in the electrophoretic A-1-AT patterns as well as quantitative differences in the concentrations of A-1-AT and the serum trypsin-inhibiting activity. Three electrophoretic phenotypes were distinguished: M, P and MP. M was characterized by a predominant anodal A-1-AT band, and P had a major cathodal component. The MP pattern can be explained by the occurrence of the M and P components in the same serum due to heterozygosity. The P pattern was associated with an A-1-AT concentration of approximately 56% of that in sera with the M phenotype. The levels of A-1-AT in sera with the MP phenotype were intermediate between those in M and P types. In addition to the type-specific quantitative variation, we found a quantitative sexual dimorphism of a moderate degree: Female rabbits had A-1-AT concentrations 16% less than males.

A rapid and quantitative method for the determination of interleukin-1 alpha and -beta mRNA expression in human monocytes and macrophages.

We have used IL-1 alpha and IL-1 beta specific RNA standards in an RNA dot blot assay to rapidly and quantitatively assess the differential expression of the two human interleukin-1 mRNAs. This approach enabled us to minimize inaccuracies due to differences in specific activities of the probes, transcript size, and transfer and/or hybridization efficiencies of the two transcripts. We were thus able to accurately determine the steady state levels and molar ratio of the two species of IL-1 mRNA in human peripheral blood monocytes (PBM) and alveolar macrophages (AM). PBM, upon stimulation by lipopolysaccharide (LPS), expressed approximately ten-fold more IL-1 beta mRNA than IL-1 alpha. AM, however, expressed only two-fold more IL-1 beta then IL-1 alpha. PBM aged in vitro for 7 days prior to LPS stimulation, expressed at least 20-fold more IL-1 beta than IL-1 alpha RNA and at levels significantly lower than either PBM or AM.

Alpha1-antitrypsin phenotypes in chronic active liver disease and primary biliary cirrhosis.

Alpha1-antitrypsin concentrations and phenotypes were determined in groups of patients with chronic active liver disease and primary biliary cirrhosis. The concentrations of alpha1-antitrypsin were above normal values in both groups; the patients with primary biliary cirrhosis had higher concentrations than those with chronic active liver disease. The prevalence of common phenotypes in these two groups did not differ from that in a sample of healthy blood donors from this institution of from a large Norwegian sample. We interpret our data as disputing the view that alpha1-antitrypsin phenotypes, other than Z. significantly predispose adults to hepatic cirrhosis.

Host factors in chronic obstructive pulmonary disease in an upper Midwest rural community. Design, case selection, and clinical characteristics in a matched-pair study.

A series of 111 index subjects with chronic obstructive pulmonary disease (COPD) who had forced expiratory volume in 1 second (FEV1) of 70% or less of that predicted were matched on the basis of age, sex, occupation, and smoking history with control subjects who had an FEV1 of 85% or more of that predicted. Index and control subjects with seasonal or reversible airway disease were excluded. Men outnumbered women by a ratio of 4.5 to 1. Thirty-five percent of the women and 2% of the men were nonsmokers (0 pack-years). There were three PiZ phenotypes in the index group (two nonsmokers) and none in the controls. PiMZ phenotypes in the index group outnumbered those in the controls by 8 to 5. Host factors that might be important in these closely matched pairs were sought by history, physical examination, and a large battery of laboratory tests. A standard respiratory questionnaire revealed the anticipated significantly higher frequency of cough, phlegm, noisy respiration, and all grades of dyspnea in index subjects. Previous lower respiratory tract infections also were more frequent in index subjects than in controls. There were no detectable differences between groups in the frequency of upper airway infections, nasal polyps, sinus surgery, or reported allergy to any substance. If the British Medical Research Council's definition of chronic bronchitis were applied to our study, about two-thirds of our index subjects and almost one-third of our controls would be considered to have chronic bronchitis. Pack-years of smoking were not significantly associated with the amount and duration of cough and expectoration in male or female index subjects or controls. Significant differences between index and control groups on physical examination included the audible forced expiratory flow time over the trachea, the estimated maximal midexpiratory flow, breath sounds, rales, and total excursion of the hemidiaphragms. An endocrine questionnaire and measurement of blood sex hormones did not give any clues as to the propensity of males to develop COPD. Women with airway obstruction similar to that of men had histories of significantly fewer pack-years than did the men, and there was a much larger proportion of women who never smoked. Further studies, specifically on genetic and immunologic characteristics, are under way to identify potential host factors.

Alpha1-antichymotrypsin globules within hepatocytes in patients with chronic hepatitis C and cirrhosis.

Alpha1-antichymotrypsin (A1AC) is an acute phase serine protease inhibitor, similar to alpha1-antitrypsin (A1AT) in amino acid sequence. A1AT deficiency is known to be associated with emphysema and cirrhosis; deficiency of serum A1AC has been reported to be associated with emphysema, childhood asthma, and cryptogenic cirrhosis. The hepatocyte globules associated with A1AT deficiency have been well described; A1AC deficiency also has been reported to be associated with hepatocyte globules. The aim of this study was to describe the globules of A1AC and to compare them with A1AT globules. Immunohistochemistry for A1AC and A1AT was performed on liver biopsy specimens from 15 hepatitis C virus (HCV)-positive cirrhotic patients, 14 non-HCV cirrhotic patients, and 12 other patients with chronic hepatitis C but no cirrhosis, all of whom had known serum levels of A1AC; most had known serum levels of A1AT. Five of 15 HCV-positive cirrhotic patients, 1 of 14 non-HCV cirrhotic patients, and 1 of 12 noncirrhotic chronic hepatitis C patients had A1AC globules. Two of 15 HCV-positive cirrhotic patients and 2 of 14 non-HCV cirrhotic patients had A1AT globules. Histologically, the globules of A1AC were similar to those of A1AT but were smaller and fewer; the PAS/D stain was not as helpful for A1AC as it was for A1AT; immunohistochemistry was most useful. There was not a good correlation between serum levels of A1AC and its globules in hepatocytes. A1AC globules should be included in the differential diagnosis of hepatocyte inclusions.

Angiotensin converting enzyme in bronchoalveolar lavage in ARDS.

Angiotensin converting enzyme (ACE) is present in the endothelial cells of the normal lung where it converts angiotensin I to angiotensin II and inactivates bradykinin. It has been suggested that during endothelial injury ACE is sloughed into the blood, and that if the alveolar capillary membrane is injured, also into the alveolar lining fluid. Seven patients with adult respiratory distress syndrome (ARDS), were compared to 11 normal control subjects, nine patients with sarcoidosis, and six with idiopathic pulmonary fibrosis. Total, differential cell counts and ACE determinations were performed on bronchoalveolar lavage fluid in the ARDS group. ACE was detectable in the BAL of all but one ARDS patient. It was concluded that BAL ACE is elevated in some ARDS patients, especially those with infectious causes of lung injury. Increased ACE may reflect endothelial damage or local increase in ACE production in response to sepsis.

Alpha 1-antitrypsin levels predict mortality from ethionine-induced pancreatitis in mice.

Experimental pancreatitis can be induced by an ethionine-containing, choline-deficient diet in mice. We investigated the role of circulating alpha 1-antitrypsin in this model using two strains of mice: ICR and C57BL-6. A 50% reduction in circulating alpha 1-antitrypsin occurred in all mice by day three of diet exposure. Total protein was reduced by only 9% and albumin was unchanged. Female mice of both strains had significantly lower alpha 1-antitrypsin levels than male mice prior to and after diet exposure. This was associated with significantly greater mortality in both female strains. Interstrain comparisons showed a significantly higher mortality in the C57BL-6 females (100%) compared to the ICR females (58%); this corresponded to significantly lower alpha 1-antitrypsin levels in C57BL-6 females. Regardless of sex or strain, alpha 1-antitrypsin levels prior to and after diet exposure were significantly higher in mice surviving than in mice dying. We conclude that circulating alpha 1-antitrypsin is a predictor of mortality from diet-induced pancreatitis.

Therapeutic resistance: characterization and inactivation by specific antiserum of a putative protein family produced by tumour cells.

A multitherapy resistance (MTR) factor produced by Cloudman S91 mouse melanoma cells rescues a responsive cell line after gamma-irradiation, short wavelength ultraviolet light, mitomycin C, vinblastine and actinomycin D. A similar activity with respect to ionizing radiation is now shown to be produced by human melanoma cells and by both human and mouse breast cancer cells but not by five normal cell lines. In these studies, the factor produced in serum-free conditioned medium (SFCM) by Cloudman S91/I3 cells is further characterized. Its activity in a clonogenic assay using related Cloudman S91/amel cells is destroyed by trypsin but not by DNase and is stable for at least 8 days at a variety of temperatures including 37 degrees C. Molecules greater than 30 kDa from SFCM collected from S91/I3 cells were concentrated and separated by preparative zonal electrophoresis (PZE). Bioactivity was present in both the cathode- and the anode-running fractions. The active acidic (anode) fractions were analysed by preparative isoelectric focusing. Bioactivity was present between pI 3.5 and 4.2. These PZE fractions were also used to immunize two rabbits, both of which produced antiserum that abrogated the bioactivity of SFCM and of the PZE cathode fractions. Antiserum also decreased the survival of irradiated S91/I3 producer cells that do not respond to SFCM but nonetheless must require MTR proteins for the expression of radiation resistance. These studies present a model for the production of rescue factors by non-clonogenic tumour cells that may persist in some tumours for considerable periods of time.

Effects of age, sex, reproductive status, and hospitalization on serum alpha 1-antitrypsin concentration in dogs.

We performed a study to determine a reference range for serum alpha 1-antitrypsin (alpha 1AT) in dogs by specific immunoassay; to evaluate whether serum alpha 1AT concentration varied with age, sex, or reproductive status in healthy dogs; and to investigate whether the serum alpha 1AT concentration in hospitalized dogs differed from that of healthy, nonhospitalized dogs. Serum alpha 1AT was quantitated by radial gel immuno-diffusion for 60 healthy dogs and 311 hospitalized dogs. In healthy dogs, serum alpha 1AT concentration was 2.33 +/- 0.41 mg/ml (mean +/- SD), yielding a reference range (mean +/- 2 SD) of 1.51 to 3.15 mg/ml. A correlation was not found between serum alpha 1AT concentration and age in healthy dogs. The serum alpha 1AT concentration (mean +/- SEM mg/ml) was significantly higher in healthy, sexually intact females (2.64 +/- 0.1) than in healthy, spayed females (2.22 +/- 0.12; P < 0.004); healthy, sexually intact males (2.14 +/- 0.1; P < 0.0006); and healthy, and castrated males (2.25 +/- 0.14; P < 0.02). Hospitalized, sexually intact females had a lower serum alpha 1AT concentration (1.93 +/- 0.07) than healthy, sexually intact females (2.64 +/- 0.1; P < 0.0002). Likewise, the serum alpha 1AT concentration in hospitalized, sexually intact males (1.92 +/- 0.04) was less than in healthy, sexually intact males (2.14 +/- 0.1; P < 0.04). A difference in alpha 1AT concentration was not found between healthy and hospitalized, neutered dogs.

Serum alpha 1-antitrypsin concentration in dogs with panniculitis.

OBJECTIVE: To measure serum alpha 1-antitrypsin (alpha 1AT) concentration in dogs with histologically confirmed panniculitis to determine whether serum deficiency could cause or exacerbate panniculitis in dogs. DESIGN: Cross-sectional, descriptive study. ANIMALS: 9 dogs (5 with multiple lesions and 4 with solitary lesions). PROCEDURE: Serum samples were obtained by means of cephalic or jugular venipuncture and frozen at -20 C until assayed. Serum alpha 1AT concentration was measured by means of radial gel immunodiffusion. RESULTS: In all dogs, serum alpha 1AT concentration was within the previously established reference range. CLINICAL IMPLICATIONS: In the small number of-dogs studied, panniculitis was not associated with serum alpha 1AT deficiency.

Trypsin-inhibiting activity in sera of two strains of inbred mice.

Trypsin-inhibiting activity in sera of mice of two inbred strains, AHeJ and P/J, was determined. The mean activities in both groups were identical, namely 0.77 mg of trypsin inhibited by 1 ml of serum. This finding is at variance with an earlier report.

The role of augmentation therapy in alpha-1 antitrypsin deficiency.

BACKGROUND: Since the recognition of alpha-1 antitrypsin deficiency (A1ATD) in 1963, interest in this condition has increased dramatically. A1ATD is now recognized as the only known genetic condition that leads to emphysema/chronic obstructive pulmonary disease (COPD) in many individuals with the condition. Augmentation therapy with plasma-derived alpha-1 antitrypsin (A1AT) was first introduced in 1987. OBJECTIVES AND SCOPE: To review current evidence on the efficacy, tolerability and biochemical composition of commercially available A1AT augmentation therapies. Literature was sought via electronic searching of bibliographic databases (MEDLINE) and other sources. No language or time period settings were applied. This is a narrative, descriptive review rather than a formal, systematic review. FINDINGS: Evidence of the therapeutic efficacy of A1AT augmentation therapy is beginning to accumulate, although further randomized, controlled trials are necessary. Clinical studies have reported reduced rates of lung function decline in COPD patients who received augmentation therapy, and significant benefit is seen in patients with forced expiratory volume in 1 second initially in the range of 35-49% of predicted normal. Augmentation therapy has also been shown to decrease the frequency of severe COPD exacerbations and to significantly increase survival rate. Biochemical studies have convincingly demonstrated that weekly intravenous infusion of each of the available plasma-derived A1AT preparations maintains serum A1AT levels above the putative protective threshold. Augmentation therapy with intravenous A1AT is generally well tolerated and long-term therapy in patients with severe A1ATD and pulmonary emphysema is feasible. Differences in the purification processes of available A1AT products are reflected in their relative purities and heterogeneities (abundance of A1AT isoforms), although the commercially available preparations are bioequivalent. Further studies are required to clarify whether variations in biochemical composition of purified A1AT are clinically important. CONCLUSION: Intravenous augmentation therapy with A1AT currently represents the only viable and specific treatment option for patients with A1ATD.