Biomedical subjects
Publications and source records attributed to J Potempa.
1. Noncatalytic protein component (NPC), a strongly acidic protein (pH = 4.5) was separated from native horse leucocyte elastase 1. 2. This protein reduces elastinolytic properties of elastases: 1 and 2A probably by decreasing their isoelectric points. 3. A possible regulatory role of this protein may be inferred from a higher affinity of elastase 1 to NPC rather than to elastin.
In a broad concentration range (0.1-100 micrograms/ml) the serine proteinase (SP) from Staphylococcus aureus has no cytotoxic effect on human peripheral blood lymphocytes and does not stimulate them in culture. However, it affects the action of a number of polyclonal activators. In a concentration of 100 micrograms/ml SP completely eliminates blastic transformation after stimulation with B cell mitogens (NDCM, S. aureus and Escherichia coli), lowers the blastic transformation after stimulation with PWM and SPA, and does not affect the blastic transformation after stimulation with PHA. SP (100 micrograms/ml) reduces the concentration of Ig in stimulated cultures (stimulation with PWM, NDCM, S. aureus and E. coli) far below the Ig level of unstimulated controls. This effect can be ascribed to an influence on cell stimulation, not to the proteolytic cleavage of secreted Ig, although SP can partially digest Ig. The effect on lymphocyte stimulation takes place when the SP is added to the culture together with the mitogen, or 18 h after the mitogen. This implies that SP does not affect the first stage of stimulation.
Human plasma contains an array of proteinase inhibitors which are utilized in the regulation of a host of biological activities, including coagulation, fibrinolysis, connective tissue turnover, and complement activation. The concentration of several of these inhibitors increase at varying rates in the acute phase state while others remain constant or actually decrease. Increases are presumably an attempt to retain rigid control over certain critical reactions, while decreases are probably due to inhibitor turnover either through consumption during complex formation or inactivation by other endogenous proteinases. Virtually all of these latter reactions take place in a reactive site loop which is an exposed region present in at least eight related serine proteinase inhibitors (Serpins) in plasma. Complex formation and inhibitor inactivation presumably act as signals for inhibitor production and turnover in the acute phase state. However, exactly how this initial stimulus for increased protein synthesis is manifested at the protein level remains to be established.
The pharmacokinetics of sulbactam, a new beta-lactamase inhibitor, and ampicillin were investigated in 9 healthy volunteers and 125 patients. The two drugs were administered intravenously in combination (Unacid). Each subject received 1 g sulbactam and 2 g ampicillin. The two drugs showed a similar pharmacokinetic profile in humans. Relevant pharmacokinetic parameters were calculated using a 2-compartment model on the basis of the serum and urine concentrations determined in healthy volunteers. The ratio of sulbactam to ampicillin was found to be nearly constant (approx. 1:2), both in the blood, in several tissues and at various time points. The two compounds apparently penetrated well into the myometrium, renal cortex and medulla, the gall bladder wall, the tonsils and the cutis of the skin. The sulbactam/ampicillin ratios in the bile ranged from 1:3 to 1:13 over 2 h after the infusion. The concentrations of the two compounds measured in the various fluids and tissues of the body reached levels which are effective against beta-lactamase producing bacteria. The similar pharmacokinetics of sulbactam and ampicillin in humans are considered an essential prerequisite for the therapeutic efficacy of such a combination product.
The interaction of three proteinases (seryl, cysteinyl, and metallo-) from Staphylococcus aureus with human plasma alpha 1-proteinase inhibitor has been investigated. As expected, none of the enzymes was inactivated by this protein, each, instead causing the conversion of the native inhibitor into an inactive form of decreased molecular weight. Amino-terminal sequence analysis indicated that inhibitor inactivation had occurred by peptide bond cleavage near the reactive center of this protein. When the inhibitor was modified by this treatment, it became resistant to both pH and temperature denaturation and, in contrast to the intact denatured protein, did not undergo further proteolytic degradation. This process of inactivation of alpha 1-proteinase inhibitor by pathogenic proteinases could result in a deregulation of its target enzyme, neutrophil elastase, and, therefore, may be important in the consumption of some plasma proteins by this enzyme during septicemia.
The removal by EDTA of Ca2+ from the two-tryptophan-containing metalloproteinase isolated from Staphylococcus aureus leads to an increase in its intrinsic fluorescence intensity. Based on acrylamide fluorescence quenching results, analyzed by the non-linear least-squares method, we have shown that this protein molecule undergoes irreversible conformational change upon removal of Ca2+, which include the exposure to the solvent of buried tryptophan residues. Steady-state fluorescence anisotropy measurements indicate that the loss of Ca2+ leads to a significant increase in internal mobility of previously buried tryptophan residues.
Alpha-1-proteinase inhibitors isolated from plasmas of horse, ox, pig, rabbit and man were used for determination of some kinetic parameters of interaction with three horse leucocyte proteinases and bovine pancreatic trypsin and chymotrypsin. Effective molar ratio of enzyme-to-inhibitor, inactivation rate constant and inhibition constant were measured. In horse, ox, pig and rabbit two principal electrophoretic forms of alpha 1-PI could be distinguished. Both forms effectively inhibited trypsin but usually only one form reacted promptly and stoichiometrically with chymotrypsin and leucocyte elastases. It appears that genetic variability and functional heterogeneity of multiple forms of alpha 1-PI as well as lack of other tissue inhibitors of proteinases may be responsible for lung emphysema occurring in man and horse.
Horse blood leucocyte cytosol exhibits a broad inhibitory activity against serine proteinases. The purified inhibitor was exposed to investigated enzymes (trypsin, chymotrypsin, elastases and serine proteinase from S. aureus) for variable time and the products were analyzed by gradient polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molar ratio I:E, association rate constants k on and inhibition constants Ki for the enzymes and inhibitor were determined. The examined elastases form stable, stoichiometric complexes with the inhibitor (Ki less than 10(-10) M), and do not undergo proteolytic degradation during 30 min incubation at 20 degrees C even at the 2-fold molar excess of the proteinases. The reactions with elastases are extremely rapid (k on greater than 10(7) M-1 s-1) and are completed within one second whereas similar reactions with chymotrypsin and trypsin are much slower (k on = 3 X 10(5) M-1 s-5 and 5 X 10(2) M-1 s-1, respectively). Serine proteinase from S. aureus neither react nor inactivates the investigated inhibitor. The complexes of the inhibitor with trypsin and chymotrypsin are digested even at a molar ratio I:E = 2:1. All these observations point out that the inhibitor from horse leucocyte cytosol is a specific and effective inhibitor of elastases.
Three proteinases from the azurophilic granules of horse leucocytes are typical elastases degrading elastin at neutral pH. Both proteinases: 1 and 2A exhibit similar elastinolytic activity, comparable with human leucocyte elastase (HLE). In relation to human enzyme, elastase 2B shows several-fold higher activity, which is comparable to the porcine pancreatic elastase activity (PPE). Similarly to HLE elastinolytic activity of the horse proteinases increases at higher ionic strength: twofold in case of 1 or 2A and fivefold for 2B. Significant activity observed during degradation of homologous lung elastin, implies the possible role of these enzymes during pathological injury of connective tissue in the lower respiratory tract and suggests similar pathogenesis of horse and human pulmonary emphysema.
Highly purified horse leucocyte proteinases 1, 2A and 2B hydrolyze synthetic substrates which are decomposed also by human leucocyte elastase but they are unable to hydrolyze typical substrates of cathepsin G. Thus in distinction to other mammalian species horse leucocytes are devoid of cathepsin G and contain only elastases.
Horse leucocyte neutral proteinase inhibitor reacts with all tested elastases at the molar ratios of 1:1 and yielding stable complexes (Ki = 10(-10) M). The above reactions are very rapid, characterized by the high values of association rate constant kon = 10(7) M-1s-1.
Explore the source record for details and available documents.
Interaction of eglin c with three neutral proteinases (1, 2A and 2B) from horse leucocytes was investigated using synthetic and protein substrates. With N-tert-butyloxycarbonyl-L-alanine-p-nitrophenyl ester as substrate inhibition of proteinase 1 and 2A was practically complete at equimolar inhibitor concentrations (Ki below 1 nMol/l). The complex with proteinase 2B showed a dissociation constant of approximately 25 nMol/l. The latter proteinase was only partly inhibited also in the presence of azocasein, whereas almost linear inhibition was observed for all 3 proteinases with fibrinogen as substrate. The inhibition rate constants (kon) for horse leucocyte proteinases with eglin were in the range of 8 to 13 X 10(5) M-1 S-1.
alpha 2-macroglobulin was isolated by polyethylene glycol precipitation, gel filtration on Sephacryl S-300 and DE-52 cellulose chromatography, with 20% yield. The preparation obtained was homogenous as tested by biochemical and immunological criteria. Its molecular mass was estimated at 800,000, comprising of four identical subunits. The isoelectric point of our preparation was 4.8 and two molecules of serine proteinases per 1 molecule of inhibitor were bound.
We have followed up 184 urethral strictures of different extent which have been operated on in the last five years with the technique of circumferential multiple radial visually-controlled urethrotomy.
Our urethrotomy technique for strictures during the last 6 years is analyzed. Each stricture was widened radially over the entire circumference with incisions made close together. In this way a short or moderately long stricture can be eliminated completely in 1 session. For long and multiple strictures only the most distal portion was incised as far as possible. Then, after 2 to 3 weeks, control urethroscopy was done and the remaining stricture subsequently was incised without difficulty using the same technique. Urethrotomy was done with the patient under local anesthesia with mepivacaine because of the better field of view. Instillagel was not used. This technique does not require any followup treatment of the urethra. Since no indwelling catheter is needed foreign body urethritis, which can be the cause of stricture recurrences, is avoided. The postoperative course is shortened greatly. There is greater freedom from complications and the chances of healing are improved. The operation can be performed rapidly and the burden for the patient is slight. Secondary injuries are rare. Sessions can be repeated without complications. Potency is not endangered. This operation can be done on an ambulatory patient with any type of stricture. The late results in 202 patients with simple and complicated strictures who underwent this technique were evaluated. Most strictures were incised in 2 or 3 sessions, which gave the best results. A few complicated callous strictures that had been treated for years with bougienage were incised in 4 or 5 sessions. The over-all success rate of the late results is more than 80 per cent.
The importance of hematuria in urology was analyzed with special consideration of microhematuria. It is especially emphasized that in microhematuria as in marcrohematuria a thorough urological diagnosis must follow in order to detect the source of bleeding with certainty and thus to be able to decide on further therapy. In most of the cases, it was not particularly difficult to localize the source of bleeding in the macrohematuria, in contrast to microhematuria in which a precise source of bleeding can be established only in less than 10%. Microhematuria gives rise to particular problems in cancer prevention in men and women; these not uncommonly lead to "overdiagnosis". The most serious cause of microhematuria are tumors of the kidney and the urinary tract. Patients are often "treated" by the general doctor without certain exclusion of the presence of a tumor. This leads to a delay in diagnosis in painless hematurias. It is known from the literature that despite massive hematuria on average three months pass until the patient visits an urologist. The urologist therefore often diagnoses advanced urogenital tumors which can no longer be successfully treated.
The rapid, two steps method of purification of an elastase-like proteinase from cytoplasmic granules of horse leucocytes is described. This enzyme called the proteinase 1 is released easily from isolated granules in the low ionic strength solutions in opposite to the other two molecular forms of which one differs slightly in isoelectric point from the other. The enzyme is a typical neutral proteinase of a broad substrate specificity and wide pH optimum. In a physiological conditions the enzyme is built of two polypeptide chains of molecular weight about 30000 and 20000, respectively.