Tissue plasminogen activator inhibitor in patients with systemic lupus erythematosus and thrombosis.

OBJECTIVE--To examine the relations among tissue plasminogen activator antigen, plasminogen activator inhibitor, the lupus anticoagulant, and anticardiolipin antibodies in patients with systemic lupus erythematosus. DESIGN--Prospective study of blood samples (a) from selected patients with systemic lupus erythematosus whose disease was and was not complicated by a history of thrombosis or recurrent abortions, or both, and (b) from a series of healthy controls with a similar age and sex distribution. SETTING--University based medical clinic. SUBJECTS--23 Patients with definite systemic lupus erythematosus (American Rheumatism Association criteria), of whom 11 (eight women) aged 26-51 had a history of thrombosis or recurrent abortions, or both, and 12 (10 women) aged 23-53 had no such history. 15 Healthy subjects (10 women) aged 25-58 served as controls. MAIN OUTCOME MEASURES--Tissue plasminogen activator concentrations, plasminogen activator inhibitor activities, detection of the lupus anticoagulant, and values of anticardiolipin antibodies in the two groups of patients and in the patients with a history of thrombosis or abortions compared with controls. Other measurements included concentrations of proteins that are known to change during the acute phase of systemic lupus erythematosus--namely, fibrinogen, C3 and C4, and C reactive protein. RESULTS--Patients with a history of thrombosis or abortions, or both, had significantly higher values of tissue plasminogen activator and plasminogen activator inhibitor than patients with no such history. A significant correlation between tissue plasminogen activator and plasminogen activator inhibitor (r = 0.80) was found only in the patients with a history of complications of their disease. The lupus anticoagulant was detected in six of the 11 patients with a history of thrombosis or abortions when tested by measuring the activated partial thromboplastin time but was found in all 11 patients when tested by measuring the diluted activated partial thromboplastin time. Nine of these 11 patients had raised values of anticardiolipin antibodies. The findings showed no relation to the activity of the disease. CONCLUSIONS--A significant correlation between tissue plasminogen activator concentrations and plasminogen activator inhibitor activities was found only in patients whose systemic lupus erythematosus was complicated by a history of thrombosis or recurrent abortions. The findings show that these patients have raised plasminogen activator inhibitor activities, and the frequent association between these raised activities and the presence of the lupus anticoagulant suggests that the two may be linked.     

The metabolism of 5'-methylthioadenosine and 5-methylthioribose 1-phosphate in Saccharomyces cerevisiae

Cordycepin sensitive mutants of Saccharomyces cerevisiae, which are permeable to 5'-deoxy-5'-methylthioadenosine (MTA), were used to study the fate of the methylthioribose carbons of this purine nucleoside. Evidence is presented for the recycling of the methylthio group and part of the ribose portion of MTA in a biosynthetic pathway which leads to the synthesis of methionine. The main pathway involves the phosphorylytic cleavage of MTA by MTA phosphorylase yielding 5-methylthioribose 1-phosphate and adenine as products. Loss of the phosphate group of 5-methylthioribose 1-phosphate, concurrent with the rearrangement of the ribose carbons, leads to the synthesis of 2-keto-4-methylthiobutyric acid. In the final step of the sequence, 2-keto-4-methylthiobutyric acid is converted to methionine via transamination. Several compounds not directly associated with the biosynthesis of methionine were also isolated. These compounds, which may arise through the degradation of intermediates in the pathway, were: 5'-methylthioinosine, a deaminated catabolite of MTA; 5-methylthioribose, a result of the phosphorylysis of 5-methylthioribose 1-phosphate, and 3-methylthiopropionaldehyde, 3-methylthiopropionic acid and 2-hydroxy-4-methylthiobutyric acid, all arising from the catabolism of 2-keto-4-methylthiobutyric acid.     

Echocardiographic parameters and indices in the normal beagle dog.

M-mode and two-dimensional echocardiographic measurements were made from the right sternal border of 50 healthy Beagles (25 males and 25 females) approximately 7 months old. The dogs were conscious and standing during the investigation. The following parameters, in systole and diastole, were measured on the echocardiographic images: left ventricular posterior wall thickness (LVWT); intraventricular septum thickness (IST); left ventricular internal dimension (LVID); and circumference (LVC). Fractional shortening (FS) and ejection fraction (EF) were also calculated. Mean, standard deviation, range and coefficient of variation are reported for each echocardiographic parameter and for body weight. Males and females were considered separately and together. Each parameter was analysed statistically to check for differences between the sexes and for correlations with body weight. A statistically significant difference between the sexes was only observed for LVWT in systole and diastole. A linear regression with body weight was obtained only for LVID in systole and in diastole. The results show that morphofunctional cardiac homogeneity is independent of size in dogs of this breed and age.     

Analysis of larger than tetrameric poly(adenosine diphosphoribose) by a radioimmunoassay in nuclei separated in organic solvents.

Anitibodies were prepared against poly(adenosine diphosphoribose) of an average chain length of 40 adenosine diphosphoribose units by repeated injection of the polymer mixed with methylated albumin and adjuvants into rabbits. The antibody was present mainly in the 7 S fraction of the immunoglobulins. A membrane binding assay was developed, and its specificity determined for the detection of (adenosine diphosphoribose)ngreater than4 in organs. The method is suitable for the study of the variation of the polymer content of nuclei. The size recognition of the anti-poly(adenosine diphosphoribose) globulin fraction was the same for polymers composed of 4--40 adenosine diphosphoribose units, but smaller oligomers were not detectible. A quantitative extraction technique was developed and applied for radioimmunoassay of nuclear (adenosine diphosphoribose)n greater than 4. Organs were freeze-clamped, freeze dried, broken into subcellular fragments in a colloid mill, and the nuclear fraction was subsequently separated in organic solvents in order to preserve the polymer. Nicotinamide and nicotinic acid, when administered in vivo, augmented the (adenosine diphosphoribose)n greater than 4 content of rat liver and heart. Tissues of infant pigeons contained larger quantites of (adenosine diphosphoribose)ngreater than4 than tissues of adult rats.     

Determination of ADP-ribose and poly(ADP-ribose) by a new radioimmunoassay

A specific and sensitive radioimmunoassay for ADP-ribose has been developed on the basis of the selective conversion of ADP-ribose to 5'-AMP by alkaline treatment. Antibodies highly specific against 5'-AMP allowed quantification of ADP-ribose converted to 5'-AMP in the range of 1-40 pmol, and in the presence of large quantities of nucleic acids or 3'-AMP. Poly(ADP-ribose) could also be determined when degraded to ADP-ribose by poly(ADP-ribose) glycohydrolase. Determination of the chain length of purified polymer was possible by a parallel determination of ADP-ribose residues after glycohydrolase treatment and of 5'-AMP from the non-reducing end obtained by phosphodiesterase catalyzed hydrolysis. The high specificities of the alkaline conversion of ADP-ribose to 5'-AMP and of the radioimmunoassay for 5'-AMP allowed quantification of protein-bound ADP-ribose residues in crude tissue extracts as verified by comparison with chromatographically purified samples.     

Proteasome Inhibitors Alter Levels of Intracellular Peptides in HEK293T and SH-SY5Y Cells

The proteasome cleaves intracellular proteins into peptides. Earlier studies found that treatment of human embryonic kidney 293T (HEK293T) cells with epoxomicin (an irreversible proteasome inhibitor) generally caused a decrease in levels of intracellular peptides. However, bortezomib (an antitumor drug and proteasome inhibitor) caused an unexpected increase in the levels of most intracellular peptides in HEK293T and SH-SY5Y cells. To address this apparent paradox, quantitative peptidomics was used to study the effect of a variety of other proteasome inhibitors on peptide levels in HEK293T and SH-SY5Y cells. Inhibitors tested included carfilzomib, MG132, MG262, MLN2238, AM114, and clasto-Lactacystin β-lactone. Only MG262 caused a substantial elevation in peptide levels that was comparable to the effect of bortezomib, although carfilzomib and MLN2238 elevated the levels of some peptides. To explore off-target effects, the proteosome inhibitors were tested with various cellular peptidases. Bortezomib did not inhibit tripeptidyl peptidase 2 and only weakly inhibited cellular aminopeptidase activity, as did some of the other proteasome inhibitors. However, potent inhibitors of tripeptidyl peptidase 2 (butabindide) and cellular aminopeptidases (bestatin) did not substantially alter the peptidome, indicating that the increase in peptide levels due to proteasome inhibitors is not a result of peptidase inhibition. Although we cannot exclude other possibilities, we presume that the paradoxical increase in peptide levels upon treatment with bortezomib and other inhibitors is the result of allosteric effects of these compounds on the proteasome. Because intracellular peptides are likely to be functional, it is possible that some of the physiologic effects of bortezomib and carfilzomib arise from the perturbation of peptide levels inside the cell.