Proteolytic processing of the open reading frame 1b-encoded part of arterivirus replicase is mediated by nsp4 serine protease and Is essential for virus replication

The open reading frame (ORF) 1b-encoded part of the equine arteritis virus (EAV) replicase is expressed by ribosomal frameshifting during genome translation, which results in the production of an ORF1ab fusion protein (345 kDa). Four ORF1b-encoded processing products, nsp9 (p80), nsp10 (p50), nsp11 (p26), and nsp12 (p12), have previously been identified in EAV-infected cells (L. C. van Dinten, A. L. M. Wassenaar, A. E. Gorbalenya, W. J. M. Spaan, and E. J. Snijder, J. Virol. 70:6625-6633, 1996). In the present study, the generation of these four nonstructural proteins was shown to be mediated by the nsp4 serine protease, which is the main viral protease (E. J. Snijder, A. L. M. Wassenaar, L. C. van Dinten, W. J. M. Spaan, and A. E. Gorbalenya, J. Biol. Chem. 271:4864-4871, 1996). Mutagenesis of candidate cleavage sites revealed that Glu-2370/Ser, Gln-2837/Ser, and Glu-3056/Gly are the probable nsp9/10, nsp10/11, and nsp11/12 junctions, respectively. Mutations which abolished ORF1b protein processing were introduced into a recently developed infectious cDNA clone (L. C. van Dinten, J. A. den Boon, A. L. M. Wassenaar, W. J. M. Spaan, and E. J. Snijder, Proc. Natl. Acad. Sci. USA 94:991-997, 1997). An analysis of these mutants showed that the selective blockage of ORF1b processing affected different stages of EAV reproduction. In particular, the mutant with the nsp10/11 cleavage site mutation Gln-2837-->Pro displayed an unusual phenotype, since it was still capable of RNA synthesis but was incapable of producing infectious virus.     

cDNA cloning and expression of a novel serine protease, TLSP

A cDNA for a putative novel serine protease, TLSP, was cloned from human hippocampus cDNA with polymerase chain reaction based strategies. The putative amino acid sequence of TLSP is similar to the trypsin-type serine proteases. TLSP mRNA is expressed in keratinocytes. Overexpressed TLSP protein in neuro2a cells was detected in culture medium.     

The role for NES1 serine protease as a novel tumor suppressor

Previously (Liu et al, Cancer Res., 56: 3371-3379, 1996), we isolated a novel serine protease-like gene--Normal Epithelial Cell Specific-1 (NES1)--that is expressed in normal mammary epithelial cells but is down-regulated in most breast cancer cell lines. Here, we demonstrate that stable expression of NES1 in the NES1-negative MDA-MB-231 breast cancer cell line suppressed the oncogenicity as revealed by inhibition of the anchorage-independent growth and tumor formation in nude mice. Fluorescence in situ hybridization localized the NES1 gene to chromosome 19q13.3, a region that contains genes for related proteases (including the prostate-specific antigen) and is rearranged in human cancers. Similar to breast cancer cell lines, prostate cancer cell lines also lacked NES1 mRNA and protein expression. Together, these results strongly suggest a tumor-suppressor role for NES1 in breast and prostate cancer.     

5,5-trans lactone-containing inhibitors of serine proteases: identification of a novel, acylating thrombin inhibitor

Synthesis of a variety of 5,5-trans fused lactones, related to compounds found in extracts of Lantana camara, has provided a series of novel acylating inhibitors of human thrombin, trypsin, chymotrypsin and human leucocyte elastase. The most effective thrombin inhibitor is 7 with an IC50 of 130 nM and a Kobs/[1] of 4,000 M-1 s-1.     

Structure of the human cytomegalovirus protease catalytic domain reveals a novel serine protease fold and catalytic triad

Proteolytic processing of capsid assembly protein precursors by herpesvirus proteases is essential for virion maturation. A 2.5 A crystal structure of the human cytomegalovirus protease catalytic domain has been determined by X-ray diffraction. The structure defines a new class of serine protease with respect to global-fold topology and has a catalytic triad consisting of Ser-132, His-63, and His-157 in contrast with the Ser-His-Asp triads found in other serine proteases. However, catalytic machinery for activating the serine nucleophile and stabilizing a tetrahedral transition state is oriented similarly to that for members of the trypsin-like and subtilisin-like serine protease families. Formation of the active dimer is mediated primarily by burying a helix of one protomer into a deep cleft in the protein surface of the other.     

Effect of exceptional valine replacement for highly conserved alanine-55 on the catalytic site structure of chymotrypsin-like serine protease

The catalytic triad consisting of His57, Asp102 and Ser195, which is completely conserved within the chymotrypsin-like serine protease family, plays a central role in catalysis. Highly conserved Ala55 also likely plays an important role in catalysis due to its location just behind the catalytic triad. The only exception to the conserved Ala55 in mammalian serine proteases is Val55 in bovine protein C. Interestingly, it has been demonstrated that the replacement of Ala55 with Thr results in the reduced activity of plasmin in patients with venous thrombosis and with retinochoroidal vascular disorders, which indicates the importance of Ala55 in catalysis. In the present study, we constructed a bovine protein C model which shows that Val55 causes no serious rearrangement of the catalytic site structure. We also constructed an A55T variant model of trypsin for comparison. The A55T substitution alters His57 into an inactive conformation, forming an unusual hydrogen bond between Thr55 O gamma 1 and His57 N epsilon 2. The present study shows that the Ala/Val55 residue contributes heavily to the active conformation of His57 and enables His57 to accept a proton from Ser195 O gamma effectively.     

N-[2,2-dimethyl-3-(N-(4-cyanobenzoyl)amino)nonanoyl]-L-phenylalanine ethyl ester as a stable ester-type inhibitor of chymotrypsin-like serine proteases: structural requirements for potent inhibition of alpha-chymotrypsin

We introduce a new potent inhibitor, N-[2, 2-dimethyl-3-(N-(4-cyanobenzoyl)amino)nonanoyl]-L-phenylalanine ethyl ester (3), which preferentially inhibits serine proteases belonging to a chymotrypsin superfamily. This inhibitor, despite consisting of a stable ethyl ester structure, showed strong inhibitory activities toward bovine alpha-chymotrypsin, human cathepsin G, and porcine elastase by acting as an acylating agent. The calculated inactivation rate constant (kinact) and enzyme-inhibitor dissociation constant (Ki) against alpha-chymotrypsin were 0.0028 s-1 and 0.0045 microM, respectively (kinact/Ki = 630 000 M-1 s-1). These kinetic parameters indicate that this inhibitor is one of the most powerful alpha-chymotrypsin inactivators ever reported. On the basis of structure-activity relationship (SAR) and structure-stability relationship studies of analogues of 3, which were modified in three parts of the molecule, i.e., the 4-cyanophenyl group, beta-substituent at the beta-amino acid residue, and ester structure, we suggest that the potent inhibitory activity of 3 is due to the following structural features: (1) the ethyl ester which enforces specific acyl-enzyme formation, (2) the n-hexyl group at the beta-position and 4-cyanophenyl group which stabilize the acyl-enzyme, and (3) the phenylalanine residue which functions for the specific recognition of S1 site in the enzyme. In particular, the action of 3 as a potent inhibitor, but poor substrate, can be ascribed largely to the very slow deacylation rate depending on the structure factors cited in feature 2. The results of inhibition by 3 and its analogues against different serine proteases such as chymase, cathepsin G, and elastase suggest that these compounds recognize common parts in the active sites among these chymotrypsin-like serine proteases, and 3 is one of the most suitable structures to recognize those common parts. Our results provide an intriguing basis for further developments in the design of a stable ester-based selective serine protease inhibitor.     

Molecular ordering of the Fas-apoptotic pathway: the Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activates multiple Ced-3/ICE-like cysteine proteases

The Fas/APO-1-receptor associated cysteine protease Mch5 (MACH/FLICE) is believed to be the enzyme responsible for activating a protease cascade after Fas-receptor ligation, leading to cell death. The Fas-apoptotic pathway is potently inhibited by the cowpox serpin CrmA, suggesting that Mch5 could be the target of this serpin. Bacterial expression of proMch5 generated a mature enzyme composed of two subunits, which are derived from the pre-cursor proenzyme by processing at Asp-227, Asp-233, Asp-391, and Asp-401. We demonstrate that recombinant Mch5 is able to process/activate all known ICE/Ced-3-like cysteine proteases and is potently inhibited by CrmA. This contrasts with the observation that Mch4, the second FADD-related cysteine protease that is also able to process/activate all known ICE/Ced-3-like cysteine proteases, is poorly inhibited by CrmA. These data suggest that Mch5 is the most upstream protease that receives the activation signal from the Fas-receptor to initiate the apoptotic protease cascade that leads to activation of ICE-like proteases (TX, ICE, and ICE-relIII), Ced-3-like proteases (CPP32, Mch2, Mch3, Mch4, and Mch6), and the ICH-1 protease. On the other hand, Mch4 could be a second upstream protease that is responsible for activation of the same protease cascade in CrmA-insensitive apoptotic pathways.     

The growth hormone dependent serine protease inhibitor, Spi 2.1 inhibits the des (1-3) insulin-like growth factor-I generating protease

The conversion of insulin-like growth factor-I (IGF-I) to the biologically more active des (1-3) IGF-I variant is catalyzed by a ubiquitous protease. This proteolytic activity is inhibited by human alpha1-antitrypsin and soy-bean trypsin inhibitor and is up-regulated in serum and tissue extracts of hypophysectomized rats. These observations lead us to investigate whether the growth hormone regulated, serine protease inhibitor, Spi 2.1 was able to inhibit the des (1-3) IGF-I generating protease. Dihydrofolate reductase deficient Chinese hamster ovary (CHO(dhfr-ve)) cells were transfected with a rat Spi 2.1 expression vector containing the dhfr and neomycin resistance gene. Stable transfectants were selected using G418 and amplified using methotrexate. Conditioned medium from Spi 2.1 transfected CHO cells potently inhibited proteolytic activity directed against a synthetic hexa-peptide with a sequence identical to the N-terminal of IGF-I. In contrast conditioned medium from wild-type CHO cells had little effect. Based upon these observations we suggest that our previous finding of enhanced des (1-3) IGF-I generating protease activity in growth hormone deficient rats may be, at least partly explained by reduced levels of Spi 2.1. Furthermore, we propose that the regulation of the generation of des (1-3) IGF-I may be an additional potential site of growth hormone regulation of IGF-I action.     

Apoptosis of endothelial cells induced by the neutrophil serine proteases proteinase 3 and elastase

The pathogenesis of vasculitis associated with anti-neutrophil cytoplasmic antibodies is not established. The anti-neutrophil cytoplasmic antibody autoanigens proteinase 3 (PR3) and elastase induce detachment and cytolysis of endothelial cells in vitro. We investigated whether PR3 and elastase trigger endothelial cell apoptosis. Primary bovine pulmonary artery endothelial cells were treated with either PR3, elastase, or myeloperoxidase (MPO) and apoptosis assessed by four different methods. By the cell death detection enzyme-linked immunosorbent assay, DNA fragmentation increased to 208 +/- 84% or 153 +/- 27% of control with 1 micrograms/ml PR3 or elastase at 24 hours. By ultraviolet light microscopy, the percentage of apoptotic cells significantly increased (P < 0.05) with 5 or 10 micrograms/ml PR3 and 25 or 50 micrograms/ml elastase at 6, 12, or 24 hours. Values at the 24-hour time point are 15.3 +/- 6.4% or 25.8 +/- 6.6% for 5 or 10 micrograms/ml PR3 and 13.9 +/- 3.6% or 20.7 +/- 1.8% for 25 or 50 micrograms/ml elastase compared with 2.2 +/- 1.2% for control. Similarly, with flow cytometry, 5 or 10 micrograms/ml PR3 and 25 or 50 micrograms/ml elastase for 6, 12, or 24 hours demonstrated increasing apoptosis in a dose- and time-dependent manner with the highest values achieved at 24 hours (23.4 +/- 4.0% and 35.6% for 5 and 10 micrograms/ml PR3 and 31.8 +/- 4.0% and 47.8% for 25 and 50 micrograms/ml elastase compared with 7.9 +/- 2.2% in control). Typical DNA laddering was apparent from 6 to 24 hours at 5 or 10 micrograms/ml PR3 and 25 or 50 micrograms/ml elastase. Myeloperoxidase did not induce cell apoptosis. Release of PR3 and elastase by activated neutrophils during acute inflammation, including anti-neutrophil cytoplasmic antibody-associated vasculitis, may result in vascular damage by endothelial cell apoptosis.     

Substrate specificity of honeydew melon protease D, a plant serine endopeptidase

The substrate specificity of honeydew melon (Cucumis melo var. inodorus Naud) protease D was studied by the use of synthetic substrates and oligopeptides derived from a protein hydrolyzate. The hydrolysis rates of succinyl-(L-Ala)1-3-p-nitroanilide (Suc-(Ala)1-3-pNA) the hydrolysis rate progressively rose in proportion to the increased chain length. Benzyloxycarbonyl-L-tyrosine p-nitrophenyl ester (Z-Tyr-ONp) and benzoyl-L-tyrosine ethyl ester (Bz-Tyr-OEt) were cleaved by honeydew melon protease D, but benzoyl-L-arginine p-nitroanilide (Bz-Arg-pNA), benzyloxycarbonyl-L-lysine p-nitrophenyl ester (Z-Lys-ONp) and tosyl-L-arginine methyl ester (Tos-Arg-OMe) were not hydrolyzed. Contrary to the results obtained by using synthetic substrates, the carboxyl sides of charged amino acid residues were preferentially cleaved by the enzyme in the oligopeptide substrates. The substrates that had charged or polar amino acids at P2 positions were not cleaved. On the other hand, the non-polar amino acid or proline at P2 were favored for hydrolysis. The information concerning the subsite of protease D was obtained and is useful for synthesis of a good substrate. As it is distinct from molecular mass, the substrate specificity of honeydew melon protease D is most analogous to cucumisin [EC 3.4.21.25] among serine proteases from cucurbitaceous plants.     

The 0.78 A structure of a serine protease: Bacillus lentus subtilisin

Ultrahigh-resolution X-ray diffraction data from cryo-cooled, B. lentus subtilisin crystals has been collected to a resolution of 0.78 A. The refined model coordinates have a rms deviation of 0.22 A relative to the same structure determined at room temperature and 2.0 A resolution. Several regions of main-chain and side-chain disorder have been identified for 21 out of 269 residues in one polypeptide chain. Hydrogen atoms appear as significant peaks in the Fo - Fc difference electron density map, and carbon, nitrogen, and oxygen atoms can be differentiated. The estimated standard deviation (ESD) for all main-chain non-hydrogen bond lengths is 0.009 A and 0.5 degrees for bond angles based on an unrestrained full-matrix least-squares refinement. Hydrogen bonds are resolved in the serine protease catalytic triad (Ser-His-Asp). Electron density is observed for an unusual, short hydrogen bond between aspartic acid and histidine in the catalytic triad. The hydrogen atom, identified by NMR in numerous serine proteases, appears to be shared by the heteroatoms in the bond. This represents the first reported correlation between detailed chemical features identified by NMR and those in a cryo-cooled crystallographic structure determination at ultrahigh resolution. The short hydrogen bond, designated "catalytic hydrogen bond", occurs as part of an elaborate hydrogen bond network, involving Asp of the catalytic triad. While unusual, these features appear to have conserved analogues in other serine protease families although specific details differ from family to family.     

The interactions between a low molecular weight protease inhibitor of bronchial mucus and chymotrypsin-like cationic proteins of granulocytes

The proteolytic activity of the chymotrypsin-like cationic proteins of human granulocytes is inhibited by complex formation with the acid-stable low molecular weight protease inhibitor of human bronchial mucus (Ki = 1.0 x 10(7)M). The molar combining ratio is 1:1.     

Sequence-independent detection of gene family homologs: identification of a transcript encoding a molluscan serine protease homologous to the pancreatic enzymes of vertebrates

We describe here the first report of sphingoid bases bearing non-perturbing 2H probe nuclei. These were produced, by two different routes of partial synthesis, to permit direct assessment of their arrangement and behaviour as minor components in membrane systems. Wideline 2H-NMR spectra of N,N-dimethylsphingosine with deuterated amino-methyl groups ([2H6]dimethylsphingosine), and of lyso-dihydrogalactosylceramide (lyso-GalCer) with deuterium nuclei at C4,C5 of the sphingosine backbone and at C3,C4 of the galactose ring ([2H4]lyso-GalCer), were recorded in unsonicated, cholesterol-containing fluid bilayer membranes. The sphingolipid metabolites behaved as single populations of lipid amphiphiles dispersed uniformly in the membrane and undergoing rapid symmetric motion about their long molecular axes. This was the case throughout the pH ranges examined, which included values generally considered for the cell cytoplasm. Spectra of [2H6]dimethyl sphingosine indicated that the methyl groups are equivalent on the NMR timescale, and that the molecule's orientation and behaviour are largely unaffected by pH over the range, 6 to 10.5. There was no spectral evidence of deprotonation of the tertiary amine function in this range. Similarly, variation of pH between 6.4 and 8.9 had virtually no effect on the average conformation and orientational order of lyso-GalCer at the level of C4,C5 in the sphingosine backbone. pH did, however, exert significant control over the orientation of the galactose residue--the effect being most marked in the region of the sphingoid base pKa. The lyso-glycolipid showed some evidence of being less motionally ordered than the corresponding parent species, presumably as a result of removal of constraints imposed by the fatty acid.     

EspP, a novel extracellular serine protease of enterohaemorrhagic Escherichia coli O157:H7 cleaves human coagulation factor V

In this study, we identified and characterized a novel secreted protein, the extracellular serine protease EspP, which is encoded by the large plasmid of enterohaemorrhagic Escherichia coli (EHEC) O157:H7. The corresponding espP gene consists of a 3900 bp open reading frame that is able to encode a 1300-amino-acid protein. EspP is synthesized as a large precursor which is then processed at the N- and C-termini during secretion. It can be grouped into the autotransporter protein family. The deduced amino acid sequence of EspP showed homology to several secreted or surface-exposed proteins of pathogenic bacteria, in particular EspC of enteropathogenic E. coli and IgA1 proteases from Neisseria spp. and Haemophilus influenzae. Hybridization experiments and immunoblot analysis of clinical EHEC isolates showed that EspP is widespread among EHEC of the serogroup O157 and that it also exists in serogroup 026. A specific immune response against EspP was detected in sera from patients suffering from EHEC infections. Functional analysis showed that EspP is a protease capable of cleaving pepsin A and human coagulation factor V. Degradation of factor V could contribute to the mucosal haemorrhage observed in patients with haemorrhagic colitis.     

Complex formation between the hepatitis C virus serine protease and a synthetic NS4A cofactor peptide

The NS3 protein of the hepatitis C virus contains a serine protease that, upon binding to its cofactor, NS4A, is responsible for maturational cleavages that occur in the nonstructural region of the viral polyprotein. We have studied in vitro complex formation between the NS3 protease domain expressed in Escherichia coli and a synthetic peptide spanning the minimal domain of the NS4A cofactor. Complex dissociation constants in the low micromolar range were measured using different techniques such as activity titration, fluorescence titration, and pre-equilibrium analysis of complex formation. Cofactor binding was strictly dependent on the glycerol content of buffer solutions and was not significantly influenced by substrate saturation of the enzyme. NS4A peptide binding to NS3 was accompanied by changes in the circular dichroism spectrum in the region between 270 and 290 nm, as well as by an enhancement of tryptophan fluorescence. Conversely, no changes in the far UV region of the circular dichroism spectrum were detectable. These data are indicative of induced tertiary structure changes and suggest that the secondary structure content of the uncomplexed enzyme does not differ significantly from that of the NS3-cofactor complex. Pre-equilibrium measurements of complex formation showed very low values for k(on), suggesting conformational transitions to be rate limiting for the association reaction.     

Addiction protein Phd of plasmid prophage P1 is a substrate of the ClpXP serine protease of Escherichia coli

Plasmid-encoded addiction genes augment the apparent stability of various low copy number bacterial plasmids by selectively killing plasmid-free (cured) segregants or their progeny. The addiction module of plasmid prophage P1 consists of a pair of genes called phd and doc. Phd serves to prevent host death when the prophage is retained and, should retention mechanisms fail, Doc causes death on curing. Doc acts as a cell toxin to which Phd is an antidote. In this study we show that host mutants with defects in either subunit of the ClpXP protease survive the loss of a plasmid that contains a P1 addiction module. The small antidote protein Phd is fully stable in these two mutant hosts, whereas it is labile in a wild-type host. We conclude that the role of ClpXP in the addiction mechanism of P1 is to degrade the Phd protein. This conclusion situates P1 among plasmids that elicit severe withdrawal symptoms and are able to do so because they encode both a cell toxin and an actively degraded macromolecule that blocks the synthesis or function of the toxin.     

Mouse hepatitis virus 3C-like protease cleaves a 22-kilodalton protein from the open reading frame 1a polyprotein in virus-infected cells and in vitro

The 3C-like proteinase (3CLpro) of mouse hepatitis virus (MHV) is predicted to cleave at least 11 sites in the 803-kDa gene 1 polyprotein, resulting in maturation of proteinase, polymerase, and helicase proteins. However, most of these cleavage sites have not been experimentally confirmed and the proteins have not been identified in vitro or in virus-infected cells. We used specific antibodies to identify and characterize a 22-kDa protein (p1a-22) expressed from gene 1 in MHV A59-infected DBT cells. Processing of p1a-22 from the polyprotein began immediately after translation, but some processing continued for several hours. Amino-terminal sequencing of p1a-22 purified from MHV-infected cells showed that it was cleaved at a putative 3CLpro cleavage site, Gln_Ser4014 (where the underscore indicates the site of cleavage), that is located between the 3CLpro domain and the end of open reading frame (ORF) 1a. Subclones of this region of gene 1 were used to express polypeptides in vitro that contained one or more 3CLpro cleavage sites, and cleavage of these substrates by recombinant 3CLpro in vitro confirmed that amino-terminal cleavage of p1a-22 occurred at Gln_Ser4014. We demonstrated that the carboxy-terminal cleavage of the p1a-22 protein occurred at Gln_Asn4208, a sequence that had not been predicted as a site for cleavage by MHV 3CLpro. Our results demonstrate the usefulness of recombinant MHV 3CLpro in identifying and confirming cleavage sites within the gene 1 polyprotein. Based on our results, we predict that at least seven mature proteins are processed from the ORF 1a polyprotein by 3CLpro and suggest that additional noncanonical cleavage sites may be used by 3CLpro during processing of the gene 1 polyprotein.     

zALK-8, a novel type I serine/threonine kinase receptor, is expressed throughout early zebrafish development

AIMS: Progressive loss of neuroretinal rim tissue is known to occur early in glaucoma and measurement of the neuroretinal rim area is possible by magnification corrected analysis of optic disc photographs (planimetry). This study was performed to determine whether the facility to distinguish between glaucomatous and normal optic discs could be improved upon by: (a) taking into account the known relation between optic disc size and neuroretinal rim area, and (b) measuring rim area in a number of segments, in order to detect focal changes. METHODS: Planimetric examination of the optic disc photographs of 88 control subjects and 51 patients with early visual field defects was performed. In the control group, multiple linear regression analysis was performed between neuroretinal rim area and optic disc area, age, sex, eye side, refraction, and keratometry. This was repeated for the whole disc and for each of twelve 30 degree segments. Normal ranges were defined by the 98% prediction intervals of the regression analysis and the sensitivity and specificity for correct identification of optic discs in the two groups determined. RESULTS: Multiple linear regression demonstrated significant associations between the neuroretinal rim area and optic disc area and age in normal subjects. Sensitivity and specificity for glaucoma diagnosis, using the cut off derived from the 98% prediction intervals, was 37.7% and 98.9% respectively when total neuroretinal rim area alone was considered, and 88.7% and 94.3% respectively when the 30 degree segments were included. The most frequent pattern of neuroretinal rim loss was diffuse, followed by thinning in more than one sector and then by thinning in the inferotemporal sector alone. CONCLUSIONS: This method of optic disc analysis enables the examiner to identify glaucomatous optic discs at the stage of early perimetric loss with a high degree of precision. Optic disc photography is simple, and fundus cameras are widely available. This method for glaucoma case identification may therefore be suitable for the primary care setting as well as hospital practice.     

Ft1, a novel gene related to ubiquitin-conjugating enzymes, is deleted in the Fused toes mouse mutation

The Academic Schools Act of 1920 and the Ordinance of 1924 pertaining to doctorates provided that one could earn the degree only having submitted a disseration. The Austrian Act which was in force up to that moment had allowed to receive the degree without writing a thesis. Protests voiced by medical students extended validity of the Act. The Jagiellonian University medicals played an important role in delaying the Ordinance coming into force. Protesting against the newest regulations they organized public meetings, wrote memorials and filled petitions. The Jagiellonian University Faculty of Medicine granted doctorates without dissertation up to the end of December 1932. However some doctorates were granted even after that term.