Regulation of Leishmania (L.) amazonensis protein expression by host T cell dependent responses: differential expression of oligopeptidase B, tryparedoxin peroxidase and HSP70 isoforms in amastigotes isolated from BALB/c and BALB/c nude mice.

Leishmaniasis is an important disease that affects 12 million people in 88 countries, with 2 million new cases every year. Leishmania amazonensis is an important agent in Brazil, leading to clinical forms varying from localized (LCL) to diffuse cutaneous leishmaniasis (DCL). One interesting issue rarely analyzed is how host immune response affects Leishmania phenotype and virulence. Aiming to study the effect of host immune system on Leishmania proteins we compared proteomes of amastigotes isolated from BALB/c and BALB/c nude mice. The athymic nude mice may resemble patients with diffuse cutaneous leishmaniasis, considered T-cell hyposensitive or anergic to Leishmania's antigens. This work is the first to compare modifications in amastigotes' proteomes driven by host immune response. Among the 44 differentially expressed spots, there were proteins related to oxidative/nitrosative stress and proteases. Some correspond to known Leishmania virulence factors such as OPB and tryparedoxin peroxidase. Specific isoforms of these two proteins were increased in parasites from nude mice, suggesting that T cells probably restrain their posttranslational modifications in BALB/c mice. On the other hand, an isoform of HSP70 was increased in amastigotes from BALB/c mice. We believe our study may allow identification of potential virulence factors and ways of regulating their expression.

Bioregulation of kallikrein-related peptidases 6, 10 and 11 by the kinin B1 receptor in breast cancer cells.

The sera of patients with breast cancer have higher levels of des[Arg(9)]bradykinin, a kinin B1 receptor (B1R) agonist, than that from healthy individuals. Stimulation of breast cancer cells with the analog Lys-des[Arg(9)]bradykinin causes release of metalloproteinases-2 and -9 and increases cell proliferation. We examined the possibility that breast cancer cells, in addition to B1R, express the kinin-forming protease true tissue kallikrein (KLK1) and the endogenous proteins termed kininogens from which kinins are enzymatically released. Furthermore, we investigated whether stimulation of breast cancer cells with a B1R agonist would modify the cellular levels of KLK6, KLK10 and KLK11, three kallikrein-related peptidases with a still poorly-understood biological role in breast cancer. We found that breast cancer cells expressed KLK1 and kininogens, and that stimulation of estrogen-sensitive breast cancer cells with the B1R agonist produced down-regulation of KLK10 (a protease associated with growth suppression) but up-regulation of KLK11 and KLK6 (peptidases related to increased cell proliferation and invasiveness, respectively). Furthermore, we showed that the B1R agonist acts as a functional stimulus for the secretion of KLK1 and KLK6, an event relevant for kinin production and cell invasion, respectively.

Molecular characteristic and expression analysis of collagenolytic serine protease from the Chinese mitten crab Eriocheir sinensis with defense response to Vibrio anguillarum challenge.

A novel collagenolytic serine protease (CLSP) was cloned from the hemocytes of the Chinese mitten crab Eriocheir sinensis (Es-CLSP). The full-length cDNA of Es-CLSP contains 990 nucleotides. It encodes a 270-amino acid-long peptide with the mature peptide containing 221 amino acids. It contains the conserved catalytic triad (H, D, and S). Similarity analysis shows that Es-CLSP shares high identity with CLSPs from the fiddler crab Uca pugilator. Es-CLSP mRNA expression in E. sinensis is a) tissue-related with the highest expression in hemocytes and widely distributed, b) highly responsive to Vibrio anguillarum challenge in hemocytes, and c) a different response to the intruding pathogens. The results of this study demonstrate the successful isolation of Es-CLSP and indicate that Es-CLSP is an immune-related gene, and show the possible role of CLSP in the invertebrate innate immune system.

Perinatal periodontal disease reduces social behavior in male offspring.

OBJECTIVE: Our objective was to verify whether prenatal maternal periodontitis is a risk factor for the development of central nervous system disorders in rats. METHODS: Periodontitis was induced by placing a ligature around the upper and lower first molars in 9 female Wistar rats (experimental group); 9 rats were left unligated (control group). The maternal general activity in an open field was observed on gestational day (GD) 0, GD 4, and GD 14, and the maternal performance was assessed on the second day after birth. The pups' play behavior was assessed on postnatal day 30. The relative level of reelin was measured in the frontal cortex by real-time PCR analysis. RESULTS: The results showed that, compared with the control group, (1) the general activity in female rats with periodontitis was decreased, (2) the maternal performance of these rats was not modified by periodontitis, (3) the play behavior of pups from dams with periodontitis was decreased, and (4) there were no differences in the frontal cortex reelin levels of pups from dams with periodontitis. CONCLUSIONS: We conclude that pre- and postnatal periodontitis induces maternal sickness behavior and reduces the pups' play behavior without interference with frontal cortex reelin expression.

Cloning and expression of protease ClpP from Streptococcus pneumoniae in Escherichia coli: study of the influence of kanamycin and IPTG concentration on cell growth, recombinant protein production and plasmid stability.

Infections caused by Streptococcus pneumoniae are one of the main causes of death around the world. In order to address this problem, investigations are being made into the development of a protein-based vaccine. The aims of this study were to clone and express ClpP, a protein from S. pneumoniae serotype 14 in Escherichia coli, to optimize protein expression by using experimental design and to study plasmid segregation in the system. ClpP was cloned into the pET28b vector and expressed in E. coli BL21 Star (DE3). Protein expression was optimized by using central composite design, varying the inducer (IPTG) and kanamycin concentration, with a subsequent analysis being made of the concentration of heterologous protein, cell growth and the fraction of plasmid-bearing cells. In all the experiments, approximately the same concentration of ClpP was expressed in its soluble form, with a mean of 240.4mg/L at the center point. Neither the IPTG concentration nor the kanamycin concentration was found to have any statistically significant influence on protein expression. Also, higher IPTG concentrations were found to have a negative effect on cell growth and plasmid stability. Plasmid segregation was identified in the system under all the concentrations studied. Using statistical analysis, it was possible to ascertain that the procedures for determining plasmid stability (serial dilution and colony counting) were reproducible. It was concluded that the inducer concentration could be reduced tenfold and the antibiotic eliminated from the system without significantly affecting expression levels and with the positive effect of reducing costs.

Expression of trypsin-like serine peptidases in pre-imaginal stages of Aedes aegypti (Diptera: Culicidae).

This study reports the biochemical characterization and comparative analyses of highly active serine proteases in the larval and pupal developmental stages of Aedes aegypti (Linnaeus) using substrate-SDS-PAGE. Zymographic analysis of larval stadia detected proteolytic activity in 6-8 bands with apparent molecular masses ranging from 20 to 250 kDa, with activity observed from pH 5.5 to 10.0. The pupal stage showed a complex proteolytic activity in at least 11 bands with apparent Mr ranging from 25 to 250 kDa, and pH optimum at 10.0. The proteolytic activities of both larval and pupal stages were strongly inhibited by phenyl-methyl sulfonyl-fluoride and N-α-Tosyl-L-lysine chloromethyl ketone hydrochloride, indicating that the main proteases expressed by these developmental stages are trypsin-like serine proteases. The enzymes were active at temperatures ranging from 4 to 85°C, with optimal activity between 37 and 60°C, and low activity at 85°C. Comparative analysis between the proteolytic enzymes expressed by larvae and pupae showed that substantial changes in the expression of active trypsin-like serine proteases occur during the developmental cycle of A. aegypti.

Cloning, expression and characterisation of an HtrA-like serine protease produced in vivo by Mycobacterium leprae

Members of the high temperature requirement A (HtrA) family of chaperone proteases have been shown to play a role in bacterial pathogenesis. In a recent report, we demonstrated that the gene ML0176, which codes for a predicted HtrA-like protease, a gene conserved in other species of mycobacteria, is transcribed by Mycobacterium leprae in human leprosy lesions. In the present study, the recombinant ML0176 protein was produced and its enzymatic properties investigated. M. lepraerecombinant ML0176 was able to hydrolyse a variety of synthetic and natural peptides. Similar to other HtrA proteins, this enzyme displayed maximum proteolytic activity at temperatures above 40°C and was completely inactivated by aprotinin, a protease inhibitor with high selectivity for serine proteases. Finally, analysis of M. leprae ML0176 specificity suggested a broader cleavage preference than that of previously described HtrAs homologues. In summary, we have identified an HtrA-like protease in M. lepraethat may constitute a potential new target for the development of novel prophylactic and/or therapeutic strategies against mycobacterial infections.

Cloning of serine protease cDNAs from Crotalus durissus terrificus venom gland and expression of a functional Gyroxin homologue in COS-7 cells.

Gyroxin is one of main serine proteases of Crotalus durissus terrificus venom, representing about 2% of the protein content in the crude venom. It is a 33 kDa glycoprotein with 3.8% by weight of sugar moiety. This toxin induces hemotoxicity in mice and a neurological condition called barrel rotation syndrome. In the present work, we report the molecular cloning of five new nucleotide sequences from a cDNA library of the venom glands of a single specimen of C. d. terrificus. These sequences have been analyzed in silico with respect to their cDNA organization and similarity with other snake venom serine proteases (SVSPs). We also describe a rapid and efficient method for screening vectors for mammalian cell expression, based on the fact that SVSPs are difficult-to-express toxins due to the presence of several disulfide bonds and glycosylation in their structures. Thus, one of the Gyroxin cDNAs was subcloned into pSectag2 HygroA and pED vectors and used to transfect COS-7 cells. Expression of the functional recombinant Gyroxin isoform was achieved with this cell line with esterase activity in the conditioned culture medium, as revealed by immunoblot of secreted protein and standard anti-crotalic serum from Butantan Institute.

Cloning, expression and characterisation of an HtrA-like serine protease produced in vivo by Mycobacterium leprae.

Members of the high temperature requirement A (HtrA) family of chaperone proteases have been shown to play a role in bacterial pathogenesis. In a recent report, we demonstrated that the gene ML0176, which codes for a predicted HtrA-like protease, a gene conserved in other species of mycobacteria, is transcribed by Mycobacterium leprae in human leprosy lesions. In the present study, the recombinant ML0176 protein was produced and its enzymatic properties investigated. M. lepraerecombinant ML0176 was able to hydrolyse a variety of synthetic and natural peptides. Similar to other HtrA proteins, this enzyme displayed maximum proteolytic activity at temperatures above 40 degrees C and was completely inactivated by aprotinin, a protease inhibitor with high selectivity for serine proteases. Finally, analysis of M. leprae ML0176 specificity suggested a broader cleavage preference than that of previously described HtrAs homologues. In summary, we have identified an HtrA-like protease in M. lepraethat may constitute a potential new target for the development of novel prophylactic and/or therapeutic strategies against mycobacterial infections.

Production and biochemical characterization of insecticidal enzymes from Aspergillus fumigatus toward Callosobruchus maculatus.

In the present work, Aspergillus fumigatus is described as a higher producer of hydrolytic enzymes secreted in response to the presence of the Callosobruchus maculatus bruchid pest. This fungus was able to grow over cowpea weevil shells as a unique carbon source, secreting alkaline proteolytic and chitinolytic enzymes. Enzyme secretion in A. fumigatus was induced by both C. maculatus exoskeleton as well as commercial chitin, and alkaline proteolytic and chitinolytic activities were detected after 48 hours of growth. Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the production of specific proteins. Among them, two extracellular alkaline proteinases from culture enriched with C. maculatus exoskeleton were purified after chromatographic procedures using ion exchange and affinity columns. These proteins, named AP15 and AP30, had apparent molecular masses of 15,500 and 30,000 Da, respectively, as estimated by SDS-PAGE electrophoresis and mass spectrometry. AP30 was classified as a serine proteinase because it was inhibited by 5 mM: phenylmethylsulfonyl fluoride (100%) and 50 microM leupeptin (67.94%).

A diverse family of serine proteinase genes expressed in cotton boll weevil (Anthonomus grandis): implications for the design of pest-resistant transgenic cotton plants.

Fourteen different cDNA fragments encoding serine proteinases were isolated by reverse transcription-PCR from cotton boll weevil (Anthonomus grandis) larvae. A large diversity between the sequences was observed, with a mean pairwise identity of 22% in the amino acid sequence. The cDNAs encompassed 11 trypsin-like sequences classifiable into three families and three chymotrypsin-like sequences belonging to a single family. Using a combination of 5' and 3' RACE, the full-length sequence was obtained for five of the cDNAs, named Agser2, Agser5, Agser6, Agser10 and Agser21. The encoded proteins included amino acid sequence motifs of serine proteinase active sites, conserved cysteine residues, and both zymogen activation and signal peptides. Southern blotting analysis suggested that one or two copies of these serine proteinase genes exist in the A. grandis genome. Northern blotting analysis of Agser2 and Agser5 showed that for both genes, expression is induced upon feeding and is concentrated in the gut of larvae and adult insects. Reverse northern analysis of the 14 cDNA fragments showed that only two trypsin-like and two chymotrypsin-like were expressed at detectable levels. Under the effect of the serine proteinase inhibitors soybean Kunitz trypsin inhibitor and black-eyed pea trypsin/chymotrypsin inhibitor, expression of one of the trypsin-like sequences was upregulated while expression of the two chymotrypsin-like sequences was downregulated.

Tonin expression in the rat brain and tonin-mediated central production of angiotensin II.

Tonins are serine proteinases mainly found in the rat submandibular gland, which are capable of generating the pressor octapeptide angiotensin II (Ang II) not only from the classical substrate angiotensin I but also from the synthetic tetradecapeptide (AG(1-14)) and from angiotensinogen. In this work, tonin expression levels were evaluated in astrocytes and brain areas of the rat. By two different techniques (ribonuclease protection assay and reverse transcription-polymerase chain reaction), we could verify the presence of tonin mRNA in astrocytes and in the thalamus of the rat brain. Sequencing of the amplified brain cDNA determined it to be identical to that found in the submandibular gland. Central microinjection of tonin produced a transient (10-20 min) elevation of blood pressure and heart rate and induced water and saline intake within the first 10 min after injection. Urinary volume and salt excretion increased within 7 h after tonin injection. These effects were partially blocked by previously administered losartan, indicating that tonin effectively induced a central Ang II formation. Our data suggest that tonin may be an alternative pathway to Ang II generation in the brain and could participate in the physiological effects exerted by Ang II such as water and saline intake and blood pressure elevation.

Detection and preliminary characterization of extracellular proteolytic activities of the haloalkaliphilic archaeon Natronococcus occultus.

Extracellular proteolytic activity was detected in the haloalkaliphilic archaeon Natronococcus occultus as the culture reached the stationary growth phase. Proteolytic activity was precipitated with ethanol and subjected to a preliminary characterization. Optimal conditions for activity were attained at 60 degrees C and 1-2 M NaCl or KCl. Gelatin zymography in the presence of 4 M betaine revealed a complex pattern of active species with apparent molecular masses ranging from 50 to 120 kDa. Experiments performed with inhibitors of the various groups of proteases indicated that the extracellular proteolytic enzymes of N. occultus are of the serine type. Individual protein species showed some differences in salt and thermal stability.