Size of silk fibroin ß-sheet domains affected by Ca2.

In the search for suitable scaffold materials for tissue regeneration, silk fibroin has become one of the most promising candidates due to its biocompatibility and good physical properties. To facilitate bone formation in osteochondral defects, it is often combined with a bone promoting additive. Here we demonstrate using HRTEM analysis how the release of Ca2+ ions from bioactive glass or Ca-salts results in the reduction of ß-sheet domain size that effectively controls a scaffold's properties, such as degradation and mechanical stiffness. We show that these changes already occur in silk fibroin solution prior to scaffold preparation and are caused by a decrease in zeta potential that forces fibroin molecules into tighter packing resulting in higher scaffold crystallinity, smaller ß-sheet domains and higher interconnectivity. The reduction of ß-sheet domains improves the elastic modulus and allows faster degradation despite the higher crystallinity. Ca2+ was also shown to be beneficial to the formation of hydroxy-apatite sheets on the fibroin surface.

Serum-specific antibodies in rabbits naturally infected with Trichophyton mentagrophytes.

In the present study, we investigated the humoral immune response of rabbits to Trichophyton mentagrophytes (sensu lato) proteins obtained from keratin-rich media in vitro. The test rabbits were naturally infected with T. mentagrophytes. The production of keratinolytic enzymes in T. mentagrophytes was stimulated by growing cultures with keratin as a sole nitrogen source. The proteins were isolated from a protein extract prepared from the fungal mat. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed three bands. Bands with Mr 20 and 30 kDa were glycosylated, whereas a band of 18 kDa was not. The rabbits' humoral immune responses to the total protein extract of T. mentagrophytes and to the proteins with keratinolytic activity was studied by immunoblotting. IgG from infected rabbits' sera revealed eight dominant bands with apparent molecular weights between 20 and 75 kDa. Bands of 20, 30 and 33 kDa appeared with a frequency rate of 76% only on immunoblots of infected rabbit serum. Using an indirect enzyme-linked immunosorbent assay (ELISA), we observed a significant increase in specific antibodies in a group of infected rabbits compared to a control group (P < 0.001). The ELISA exhibited 95% sensitivity and 83% specificity at the optimal cut-off value, with 90% predictive values of a positive and a negative result. Under these conditions, it could be used in the accurate detection of specific antibodies in sera of infected rabbits.

Ivermectin pharmacokinetics in lactating sheep.

Ivermectin (IVM) concentrations in plasma and milk were studied in six Istrian Pramenka dairy sheep after a single subcutaneous dose of 0.2 mg/kg b.w. of IVM in the early lactation period to describe IVM disposition in milk and to evaluate the transfer of IVM residues via milk to suckling lambs. Large inter-animal in concentration variability of IVM in both matrices was observed. The highest overall concentration was found in the same animal: 21.7 microg/l of H(2)B(1a) in plasma on the second day and 44.9 microg/kg of H(2)B(1a) in milk on the first day after the drug was administered. The mean time in which IVM concentrations fell below the limit of detection for the whole ewe group was 22 and 23 days for plasma and milk, respectively. Time course of IVM concentration in milk was following the time course of IVM concentration in plasma, with an overall mean+/-S.D. of milk/plasma ratio of 1.67+/-0.50 for the first 7 days of the experiment. A mean of 0.7% of the dose was excreted through milk. Individual pharmacokinetic parameters were determined by fitting a one-compartment model to the milk and plasma concentration-time profiles. Mean t(max), c(max), t(1/2k(e)) and AUC values for plasma data were: 1.70+/-0.65 days, 11.88+/-6.96 microg/l, 2.85+/-1.97 days and 63.99+/-28.34 microg day/l, respectively, and for milk: 1.28+/-1.07 days, 22.67+/-18.27 microg/l, 3.56+/-2.01 days and 114.60+/-60.41 microg day/l, respectively. The highest level of concentration in suckling lamb plasma, 0.36 microg/l of H(2)B(1a), was slightly above the limit of determination. The mean lamb to ewe ratio of areas under the plasma concentration-time curve for the first 5 days was 0.02. On the basis of obtained results, it can therefore be claimed that indirect IVM exposure of the suckling lambs via milk was negligible.

Determination of terbinafine hydrochloride in cat hair by two chromatographic methods.

Terbinafine hydrochloride (terbHCl) concentration on the site of infection with Microsporum canis is a very important indicator of drug effectiveness. Several chromatographic methods exist that can be used for the determination of terbHCl concentration in biological samples. A high performance liquid chromatographic (HPLC) method and a gas chromatographic (GC) method have been compared and critically evaluated for the determination of a terbHCl levels in cat hair. The sensitivity and the linearity of the previously developed HPLC method were 0.25 ng/mL and 0.25-3000 ng/mL, respectively. The limit of quantification (LOQ) was 0.01 microg/g of terbHCl in cat hair, and reproducibility of 96.6% and recovery of 93.8% were achieved using appropriate sample pre-treatment and optimal chromatographic conditions. The sensitivity of the GC method, 25 ng/mL (LOQ 625 ppb), was much lower than that of the HPLC method. The GC method still enables determination of terbHCl in a range of concentrations in cat hair. The reproducibility of terbHCl for the cat hair samples was 95.3% and the recovery was only 70.0%. Both methods can be used for the evaluation of drug effectiveness in cats and both of them require only basic chromatographic equipment that can be found in most analytical laboratories.

Terbinafine hydrochloride treatment of Microsporum canis experimentally-induced ringworm in cats.

Cats represent the most important source of Microsporum canis infection to people. Terbinafine hydrochloride is commonly used in the treatment of microsporosis. Its fungicidal action permits short period of treatment. It was our objective to evaluate the effectiveness of this drug in treatment of microsporosis in cats. We treated nine experimentally M. canis infected cats with terbinafine at a dose of 10-20mg/kg SID (low-dose group, LDG), nine cats with 30-40mg/kg SID (high-dose group, HDG), and nine cats were left untreated (control group, CG). The drug's levels in cats' plasma and hair were measured by a reversed-phase high performance liquid chromatographic method (RP-HPLC) and the cats' cure was followed by Wood's lamp illumination, microscopic exam and fungal culture. We showed no difference between the clinical course in CG and LDG, but HDG were significantly differentiated from both other groups. Terbinafine levels in plasma at 120 days of treatment were not statistically different among LDG (4.13 microg/l) and HDG (5.48 microg/l), but levels in hair of LDG (1.24 microg/l) and HDG (3.62 microg/l) were significantly different. Terbinafine can be used for the treatment of microsporosis in cats in the dose of 30-40mg/kg SID.

Detection by ELISA of the humoral immune response in rabbits naturally infected with Trichophyton mentagrophytes.

An indirect enzyme linked immunosorbent assay (ELISA) was developed and its diagnostic potential evaluated for rabbits infected by Trichophyton mentagrophytes. Within-run and between-run coefficient of variance varied from 2.3 to 7.7% and from 5.9 to 8.5%, respectively, indicating satisfactory reproducibility of the ELISA. There was no significant cross-reaction with antigens of Microsporum canis, Malassezia pachydermatis and Aspergillus fumigatus. The level of specific IgG to Trichophyton mentagrophytes was measured in sera of 25 11-week-old and 12 younger infected rabbits. There was no significant difference in the IgG level between 12 5-week-old infected rabbits and controls (p = 0.38). The antibody response was higher in 12 7-week-old rabbits compared with controls (p = 0.001). The IgG level in 25 11-week-old rabbits differed from the controls very significantly (p < 0.0001). Increased specific IgG in 11-week-old rabbits exhibited 96% sensitivity and 94% specificity. Predictive values of a positive and a negative test were 96 and 94%, respectively. Western immunoblotting associated three protein bands (21.5, 31, 44 kDa) with Trichophyton mentagrophytes infection.

The primary structure of inhibitor of cysteine proteinases from potato.

The complete amino acid sequence of the cysteine proteinase inhibitor from potato tubers was determined. The inhibitor is a single-chain protein having 180 amino acid residues. Its primary structure was elucidated by automatic degradation of the intact protein and sequence analysis of peptides generated by CNBr, trypsin and glycyl endopeptidase. A search through the protein sequence database showed homology to other plant proteinase inhibitors of different specificities and non-inhibitory proteins of M(r) around 20,000. On the basis of sequence homology, prediction of secondary structure and fold compatibility, based on a 3D-1D score to the three-dimensional profile of Erythrina caffra trypsin inhibitor, we suggest that the potato cysteine proteinase inhibitor belongs to the superfamily of proteins that have the same pattern of three-dimensional structure as soybean trypsin inhibitor. This superfamily would therefore include proteins that inhibit three different classes of proteinases-serine, cysteine and aspartic proteinases.

Participation of cathepsin L on bone resorption.

The proteinase responsible for bone collagen degradation in osteo-resorption was examined. The bone pit formation induced by parathyroid hormone (PTH) was markedly suppressed by leupeptin, E-64 and cystatin A, while no inhibition was observed by CA-074, a specific inhibitor of cathepsin B. Pig leucocyte cysteine proteinase inhibitor (PLCPI), a specific inhibitor of cathepsin L, and chymostatin, a selective inhibitor of cathepsin L, completely inhibited the pit formation. Cathepsin L activity in osteoclasts was much higher than the other cathepsin activities. Serum calcium in rats placed on a low calcium diet was decreased by treatment of E-64 or cystatin A, but not by CA-074. These findings suggest that cathepsin L is the main proteinase responsible for bone collagen degradation.

Cystatins and cathepsins in breast carcinoma.

The increased expression of proteolytic systems is one of the characteristics of transformed and malignant cells and their evaluations in whole tumor homogenates were considered as possible diagnostic and/or prognostic factors. Abnormal intracellular distribution, increased activities and secretion of cysteine proteinases (CPs) cathepsin B (Cat B) and L (Cat L), were associated with tumor progression. In the present study of matched pairs of breast carcinoma and normal breast tissue, the activities of Cat B and Cat L in breast carcinoma homogenates were found to be 20 and 50 fold higher, respectively, than in normal tissues. In contrast, a decrease in total inhibitory activity of cysteine proteinase inhibitors (CPIs) was observed but an average ratio between tumor and normal tissues was only 0.75. One of the CPIs, stefin A, was also determined immunochemically. The activities of CPs and CPIs were compared to the increased levels of cathepsin D (Cat D) activities in individual patients, but no statistically significant correlations were found. We correlated CPs and CPIs with morphological and receptor data as well as the axillary lymph node metastases. There was no statistical correlation of CP and CPIs with the number of lymph node metastases. However, highly elevated levels of Cat B and Cat L and lowered CPI activities in tumor cytosols were often associated with poorly differentiated carcinomas and those with negative ER and PR values. We conclude that cysteine-dependent proteolysis may play an important role in breast tumors.

Stefins and lysosomal cathepsins B, L and D in human breast carcinoma.

In the study of 50 matched pairs of breast carcinoma and normal breast tissue, the activities of cysteine proteinases (CPs), cathepsin (Cat) B and Cat L in tumors were increased on average by 18.5-fold and 52.5-fold respectively. The differences in activity of cysteine proteinase inhibitors (CPIs) between tumor and control breast tissues was also observed: in approximately two thirds of carcinomas, lowered CPI activity was measured (group-I patients), while similar or higher tumor CPI activity was measured in the remaining samples (group-II patients). Relative increases in specific activity of Cat B and Cat L in group I were significantly higher than in group II. In group I more patients with histopathological tumor grade III and negative estrogen (ER) and progesterone receptor (PR) levels were found, but the metastatic involvement of regional lymph nodes was similar in both groups. A 2-year follow-up study showed a significant inverse correlation between disease-free survival and increased Cat L activity, but the differences in group I and group II patients were not significant in this short time interval. In 20 matched pairs of breast carcinoma and normal breast tissue, the mean activity of Cat D was 5.8-fold higher in tumors compared with controls. The hypothesis that elevated Cat D activity increased CP activity and/or lowered tumor CPI activity due to post-translational proteolytic modification appeared less likely, since no correlations between corresponding activities were observed. We suggested that lowered CPI might rather reflect changes in transcription of intracellular CPIs, the stefins. Immunoassay and Northern blot analysis showed that the average value of stefin A protein and mRNA content respectively in the majority of investigated breast carcinoma samples were lowered, suggesting the possible value of stefin A in diagnosis and/or prognosis of the disease.

A new inhibitor of plasmin and trypsin from porcine leukocytes.

A new intracellular inhibitor of plasmin and trypsin was isolated from porcine leukocytes by ion exchange chromatography and affinity chromatography. In dodecyl sulphate gel electrophoresis a single protein band with an apparent molecular mass of 15 kDa was found under reducing conditions. On isoelectric focusing three protein bands with isoelectric points between pH 4.0 and 4.5 were found. The association rate constants and the inhibition constants were determined for porcine plasmin and bovine trypsin. The inhibitor shows no immunologic cross-reactivity with any of the tested leukocyte inhibitors. On the basis of its N-terminal amino-acid sequence a great degree of similarity to Kunitz-type inhibitors was observed.

Inhibitors of cysteine proteinases from potato.

Potato tubers contain considerable amounts of inhibitors of cysteine proteinases. The majority of inhibitory activity is due to low-molecular mass inhibitors differing in isoelectric points. Three of them were obtained in homogenous form, namely PCPIs (potato cysteine proteinase inhibitors) 6.6 (Mr 25,000), 8.3 and 9.4 (both Mr 22,000). They all appeared to be single-chain proteins. The amino-acid composition and the N-terminal amino-acid sequences show that at least two of them are homologous proteins, but so far no homology to the inhibitors of the cystatin super-family or to any other sequenced potato proteins is apparent. PCPIs inhibit papain and human cathepsins B, H, and L. The inhibitors interact with enzymes in apparently equimolar fashion, the interaction is of the tight binding type with Ki values ranging from 10(-6)M to 10(-11)M.

Human cathepsin B and cysteine proteinase inhibitors (CPIs) in inflammatory and metabolic joint diseases.

Synovial fluid of patients with different inflammatory and metabolic joint diseases contains low-molecular CPIs (stefins and cystatins) and high-molecular CPIs (kininogens). An additional inhibitory fragment with a molecular mass of about 20 kDa, which is a part of the kininogen molecule, has been detected. Cathepsin B and cystatin C were determined by ELISA test in 47 patients with rheumatoid arthritis, seronegative spondylarthritis, osteoarthritis, undifferentiated arthritis and gout. A significantly higher amount of cathepsin B was found in patients with rheumatoid arthritis. The elevation of cathepsin B was accompanied by an increased amount of cystatin C.

Conformation, structure and activation of bovine cathepsin D. Unfolding and refolding studies.

Cathepsin D is found in the cell in two forms, one a single polypeptide chain (Mr 44 000) and the other a non-covalent complex of two peptides of Mr 14 000 and 30 000. These correspond to the N-terminal and C-terminal regions of the single chain from which they originate. It has been shown that the two forms of the enzyme are closely similar in secondary-structure content, in aromatic amino acid environment and in denaturation behaviour. The two-chain enzyme has half the specific activity of the single-chain form. The denaturation and renaturation of the single-chain cathepsin D has now been studied by c.d., fluorescence and enzyme activity. Activity is lost irreversibly on unfolding, but the loss of backbone ellipticity and of folded aromatic environment is 75% reversible. The enzyme unfolds in two main stages, and the kinetics of these transitions indicate the existence of at least two intermediate forms between the native and the fully unfolded states. A further form of the enzyme exists in 0.5 M-guanidinium chloride. It is characterized by having an activity 40% greater than that of the native state. This increase is not reversed on removing the denaturant. The similarities between cathepsin D and pepsin are discussed.

Biochemical and biological characteristics of leucocyte proteinase inhibitors.

Pig leucocytes contain inhibitors of neutral and thiol proteinases. These proteins could be isolated from post-granule supernatant fraction as well as from nuclear extract using ion exchange chromatography, gel chromatography and affinity chromatography. Inhibitors differ in molecular weight, isoelectric point, immunologically and their inhibition ability against tested enzymes.

Some further characteristics of endogenous proteinase inhibitors.

Leucocytes and spleen contain four different types of protein proteinase inhibitors. Two of them can be inactivated by cathepsin D. In this work biochemical and immunological studies of the inactivation of I-2 by cathepsin D are presented. Polyacrylamide gel electrophoretic examinations indicate that cathepsin D inactivates I-2 by hydrolysis of the inhibitor molecule. The conversion of the active inhibitor into inactive protein proceeds catalytically. The studies on the inhibitor mechanism of the isoinhibitors of I-1 type explain the unusual inhibitor property of this type of inhibitor to inhibit two different types of proteinases, cysteine and serine. The evidence suggests that the inhibitory mechanism is based on an active sulfhydryl group of the inhibitor which may interact with the disulfide bridge of the inhibited proteinase.

Inactivation studies of the leucocyte inhibitor of urokinase by cathepsin D.

Leucocytes contain an urokinase inhibitor, that can be inactivated by cathepsin D. In this work biochemical and immunological studies on the inactivation of this inhibitor by cathepsin D are presented. Examinations by polyacrylamide gel electrophoresis and SDS electrophoresis indicate that cathepsin D inactivates urokinase inhibitor by hydrolysis of the inhibitor molecule and that the degradation needed for total inactivation is different from that for formation of the precipitin line with antibodies. The conversion of active inhibitor into inactive protein proceeds catalytically.