Band 386, Heft 4

Ribosome-inactivating proteins isolated from Phytolacca dioica L. leaves are rRNA-N-glycosidases, as well as adenine polynucleotide glycosylases. Here we report that some of them cleave supercoiled pBR322 dsDNA, generating relaxed and linear molecules. PD-L1, the glycosylated major form isolated from the winter leaves of adult P . dioica plants, produces both free 3′-OH and 5′-P termini randomly distributed along the DNA molecule, as suggested by labelling experiments with [α- 32 P]dCTP and [γ- 32 P]dATP. Moreover, when the reaction is carried out under low-salt conditions, cleavage is observed mainly at a specific site, located downstream of the ampicillin resistance gene (close to position 3200), ending with the deletion of a fragment of approximately 70 nucleotides. This cleavage pattern is similar to that obtained under the same conditions with mung bean nuclease, a single-strand endonuclease. Furthermore, pBR322 DNA treated with PD-L1 shows reduced transforming activity with E . coli HB101 competent cells in comparison to untreated control plasmid DNA.

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases uracil and thymine, as well as of the widely used chemotherapeutic drug 5-fluorouracil (5FU). Analysis of the DPD gene ( DPYD ) in two patients presenting with complete DPD deficiency and the parents of an affected child showed the presence of three novel mutations, including one splice site mutation IVS11+1G→T and the missense mutations 731A→C (E244V) and 1651G→A (A551T). The G→T mutation in the invariant GT splice donor site flanking exon 11 (IVS11+1G→T) created a cryptic splice site within exon 11. As a consequence, a 141-bp fragment encoding the aminoacid residues 400–446 of the primary sequence of the DPD protein was missing in the mature DPD mRNA. Analysis of the crystal structure of pig DPD suggested that the E244V mutation might interfere with the electron flow between NADPH and the pyrimidine binding site of DPD. The A551T point mutation might prevent binding of the prosthetic group FMN and affect folding of the DPD protein. The identification of these novel mutations in DPYD will allow the identification of patients with an increased risk of developing severe 5FU-associated toxicity.

Unannotated mammalian genome databases (dog, cow, opossum) were searched for candidate connexin genes, using sequences from annotated genomes (man, mouse). All 18 ‘multi-species’ connexin genes, i.e., orthologs of connexin26 , 29 / 31.3 (duplicated in opossum), 30 , 30.2 / 31.9 , 30.3 , 31 , 31.1 , 32 , 36 , 37 , 39 / 40.1 , 40 , 43 , 45 , 44 / 46 , 47 , 50 , and 57 / 62 , were found in dog, cow and opossum. Connexin25 and 58 have been considered specific for man, but evident orthologs of connexin25 were found in dog, cow and opossum, and orthologs of connexin58 were found in dog and cow. Moreover, a connexin43 -like sequence (approx. 80% identical to connexin43 ) was found in man, chimpanzee, dog and cow. In the three former species, the sequences were located on the X chromosome. In man, chimpanzee and cow, there were stop codons in all reading frames; these sequences are therefore judged as pseudogenes, called here Cx43pX . In the dog, the sequence contained an open reading frame for a protein of 35.7 kDa (connexin35.7). We suggest that these sequences are orthologs of connexin33 , previously considered as a rodent-specific connexin gene. Thus, connexin25 , 33 and 58 are not species-specific genes. However, the opossum may possess a candidate, connexin39.2 , without obvious orthologs in other mammals. Furthermore, pseudogenes of primate connexin31.3 and opossum connexin35 (one of the two orthologs of primate connexin31.3 ) were detected. These results suggest that the structure of the mammalian connexin gene family should be revised, especially with regard to the so-called ‘species-specific’ connexins .

Human A549 lung epithelial cells were challenged with 18 O-labeled hydrogen peroxide ([ 18 O]-H 2 O 2 ), the total RNA and DNA extracted in parallel, and analyzed for 18 O-labeled 8-oxo-7,8-dihydroguanosine ([ 18 O]-8-oxoGuo) and 8-oxo-7,8-dihydro-2′-deoxyguanosine ([ 18 O]-8-oxodGuo) respectively, using high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-MS/MS). [ 18 O]-H 2 O 2 exposure resulted in dose-response formation of both [ 18 O]-8-oxoGuo and [ 18 O]-8-oxodGuo and 18 O-labeling of guanine in RNA was 14–25 times more common than in DNA. Kinetics of formation and subsequent removal of oxidized nucleic acids adducts were also monitored up to 24 h. The A549 showed slow turnover rates of adducts in RNA and DNA giving half-lives of approximately 12.5 h for [ 18 O]-8-oxoGuo in RNA and 20.7 h for [ 18 O]-8-oxodGuo in DNA, respectively.

Proteases that digest the blood-meal of the parasitic fluke Schistosoma are potential targets for therapy of schistosomiasis, a disease of chronic morbidity in humans. We generated a three-dimensional model of the cathepsin D target protease of Schistosoma japonicum (SjCD) utilizing the crystal structure of human cathepsin D (huCD) in complex with pepstatin as template. A homology model was also generated for the related secreted aspartic protease 2 (SAP2) of the pathogenic yeast, Candida albicans . An initial panel of seven statin inhibitors, originally designed for huCD [Majer et al., Protein Sci. 6 (1997), pp. 1458–1466], was tested against the two pathogen proteases. One inhibitor showed poor reactivity with SjCD. Examination of the SjCD active-site cleft revealed that the poor inhibition was due to a unique steric barrier situated between the S2 and S4 subsites. An in silico screen of 20 potential statin scaffolds with the SjCD model and incorporating the steric barrier constraint was performed. Four inhibitors (SJ1–SJ4) were eventually synthesized and tested with SjCD, bovine CD and SAP2. Of these, SJ2 and SJ3 proved moderately more specific for SjCD over bovine CD, with IC 50 values of 15 and 60 nM, respectively. The unique steric barrier identified here provides a structural focus for further development of more specific SjCD inhibitors.

The catalytic cycle of heme peroxidases involves three processes: the formation of compound I, its conversion to compound II and regeneration of the native enzyme. Each of the processes consists of a reversible binding stage followed by an irreversible transformation stage. Our group has proposed a continuous, sensitive and reliable chronometric method for measuring the steady-state rate of peroxidase activity. Furthermore, we have derived an analytical expression for the steady-state rate and simplified it, taking into consideration the experimental values of the rate constants of some stages previously determined by other authors in stopped-flow assays. We determined the value of the constant for the transformation of a series of phenols and anilines by compound II, and found that it involves a deprotonation step and an electron transfer step. Study of the solvent deuterium isotope effect on the oxidation of phenol revealed the non-rate-limiting character of the deprotonation step in a proton inventory study. Usage of the Marcus equation showed that the electronic transfer step is rate-limiting in both cases, while phenols and anilines were oxidised at different rates for the same potentials. This can be attributed to the shorter electron-tunnelling distance for electron transfer to the iron ion in the phenols than in the anilines.

Plasmodium falciparum histoaspartic protease (HAP) is an active enzyme involved in haemoglobin degradation. HAP is expressed as an inactive 51-kDa zymogen and is cleaved into an active 37-kDa enzyme. It has been proposed that this kind of protease might be implicated in the parasite's invasion of erythrocytes; however, this protein's role during invasion has still to be determined. Synthetic peptides derived from the HAP precursor (proHAP) were tested in erythrocyte binding assays to identify their possible function in the invasion process. Two proHAP high-activity binding peptides (HABPs) specifically bound to erythrocytes; these peptides were numbered 30609 ( 101 LKNYIKESVKLFNKGLTKKS 120 ) and 30610 ( 121 YLGSEFDNVELKDLANVLSF 140 ). The binding of these two peptides was saturable, presenting nanomolar affinity constants. These peptides interacted with 26- and 45-kDa proteins on the erythrocyte surface; the nature of these receptor sites was studied in peptide binding assays using enzyme-treated erythrocytes. The HABPs showed greater than 90% merozoite invasion inhibition in in vitro assays. Goat serum containing proHAP polymeric peptide antibodies inhibited parasite invasion in vitro .

PIII snake venom metalloproteases (SVMPs) are metalloproteases structurally related to ADAMs (a disintegrin and metalloprotease human family of proteins). Berythractivase and jararhagin are PIII SVMPs with 69% homology that have different hemostatic properties. In order to clarify these differences and further characterize the biological effects of these proteins, we have analyzed the effect of both proteases on human umbilical-vein endothelial cell functions. We found that both proteins enhanced nitric oxide generation, prostacyclin production and interleukin-8 release. Berythractivase but not jararhagin increased the expression of decay accelerating factor. Jararhagin decreased cell viability in a concentration-dependent manner and induced cellular apoptosis, while berythractivase did not modulate cell survival. Our results show for the first time that, besides the known anti-aggregating or procoagulant effects of PIII SVMPs, these proteins trigger endothelial cell effector responses. Although structurally related, berythractivase and jararhagin induce a dissimilar generation and release of endothelial molecules that may account for their different hemorrhagic activity.

Transforming growth factor-β1 (TGF-β1) has a biphasic effect on the growth of renal epithelial cells. In transformed cells, TGF-β1 appears to accelerate the proliferation of malignant cells. The diverse cellular functions of TGF-β1 are regulated by three high-affinity serine/threonine kinase receptors, namely TβRI, TβRII and TβRIII. The renal serine protease tissue kallikrein acts on its endogenous protein substrate kininogen to form kinin peptides. The cellular actions of kinins are mediated through B 1 and B 2 G protein-coupled rhodopsin receptors. Both kinin peptides and TGF-β1 are mitogenic, and therefore may play an important role in carcinogenesis. Experiments were designed to immunolabel tissue kallikrein, TGF-β1, TβRII, TβRIII and kinin receptors using specific antibodies on serial sections of normal kidney and clear-cell renal carcinoma (CCRC) tissue, which included both the tumour and the adjacent renal parenchyma. The essential result was the localisation of tissue kallikrein, kinin B 1 and B 2 receptors and TGF-β1 primarily on the cell membranes of CCRC cells. In the distal and proximal tubules of the renal parenchyma adjacent to the carcinoma (RPTAC), immunolabelling for tissue kallikrein was reduced, but the expression of kinin B 1 and B 2 receptors was enhanced. Immunolabelling for TβRII and TβRIII was more pronounced in the proximal tubules of the tissue adjacent to the carcinoma when compared to the normal kidney. The expression of tissue kallikrein, kinin receptors, and TβRII and TβRIII may be relevant to the parenchymal invasion and metastasis of clear-cell renal carcinoma.

A cDNA encoding the proteinase inhibitor WSCI (wheat subtilisin/chymotrypsin inhibitor) was isolated by RT-PCR. Degenerate oligonucleotide primers were designed based on the amino acid sequence of WSCI and on the nucleotide sequence of the two homologous inhibitors (CI-2A and CI-2B) isolated from barley. For large-scale production, wsci cDNA was cloned into the E. coli vector pGEX-2T. The fusion protein GST-WSCI was efficiently produced in the bacterial expression system and, as the native inhibitor, was capable of inhibiting bacterial subtilisin, mammalian chymotrypsins and chymotrypsin-like activities present in crude extracts of a number of insect larvae ( Helicoverpa armigera , Plodia interpunctella and Tenebrio molitor ). The recombinant protein produced was also able to interfere with chymotrypsin-like activity isolated from immature wheat caryopses. These findings support a physiological role for this inhibitor during grain maturation.

A number of serine, cysteine, metallo- and acid proteases were evaluated for their ability to proteolytically cleave the serine protease inhibitor trappin-2, also known as pre-elafin, and to release elafin from its precursor. None of the metalloproteases or acid proteases examined cleaved trappin-2, while serine and cysteine proteases preferentially cleaved trappin-2 within its non-inhibitory N-terminal moiety. Cathepsin L, cathepsin K, plasmin, trypsin and tryptase were able to release elafin by cleaving the Lys 38 -Ala 39 peptide bond in trappin-2. However, purified tryptase appeared to be efficient at releasing elafin. Incubation of trappin-2 with purified mast cells first challenged with anti-immunoglobulin E or calcium ionophore A23187 resulted in the rapid generation of elafin. This proteolytic release of elafin from trappin-2 was inhibited in the presence of a tryptase inhibitor, suggesting that this mast cell enzyme was involved in the process. Finally, ex vivo incubation of trappin-2 with sputum from cystic fibrosis patients indicated the production of a proteolytic immunoreactive fragment with the same mass as that of native elafin. This cleavage did not occur when preincubating the sputum with polyclonal antibodies directed against tryptase. Taken together, these findings indicate that tryptase could likely be involved in the maturation of trappin-2 into elafin under physiological conditions.

Trophozoite cysteine protease (TCP) activity, isolated from Plasmodium falciparum soluble 100 000  g extracts, displayed native falcipain-1 kinetic parameters towards peptidyl substrates. The labelling of either isolated TCP or soluble 100 000  g extracts by a cystatin-derived probe (biotinyl-Leu-Val-Gly-CHN 2 ) revealed a single band of ca. 30 kDa by SDS-PAGE, which was resolved into four spots displaying isoelectric points (pI) from 4.7 to 5.3 after two-dimensional separation. The molecular mass and pI correspond to those of falcipain-3, falcipain-2, falcipain-2′ and falcipain-1, respectively. The two central spots were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry as falcipain-2 and falcipain-2′. This activity-based probe represents a potential tool for profiling active falcipains in parasites.