Participação do L-NAME, da L-arginina e da N-acetilcisteína no enfisema pulmonar induzido por fumaça de cigarro em camundongos

O óxido nítrico (NO) constitui um dos mais importantes mediadores intra e extracelulares e tem sido descrita sua participação tanto em processos biológicos como patológicos. Nosso objetivo foi verificar se o aumento ou a diminuição do óxido nítrico apresenta um efeito benéfico na proteção do tecido pulmonar no enfisema pulmonar induzido por fumaça de cigarro em camundongos. Para tanto, utilizamos o L-NAME (inibidor do NO), a L-arginina (substrato para a formação do NO) e os comparamos com a N-acetilcisteína (utilizada no tratamento da DPOC). Foram utilizados 65 camundongos C57BL/6 machos. Cinquenta animais foram divididos em grupos controle, fumaça de cigarro (FC), fumaça de cigarro + L-NAME (FC+LN), fumaça de cigarro + L-arginina (FC+LA), fumaça de cigarro + N-acetilcisteína (FC+NAC) (n=10, por grupo). Durante sessenta dias 40 animais foram expostos a 12 cigarros comerciais por dia, 3 vezes ao dia. Os grupos controle e FC foram submetidos à gavagens orogástricas com o veículo. Os grupos FC+LN, FC+LA, FC+NAC receberam gavagens diárias de L-NAME (60 mg/kg), L-arginina (120 mg/kg) ou NAC (200 mg/kg) respectivamente. Quinze animais (n = 5, por grupo) foram expostos ao ar ambiente e tratados apenas com L-NAME, L-arginina e NAC. Realizamos a análise do perfil das células do lavado broncoalveolar após o sacrifício. O pulmão direito foi removido para as análises histológicas do alargamento dos espaços aéreos determinado pela medida do diâmetro alveolar médio (Lm) e da densidade de superfície (Sv) dos septos alveolares. Os pulmões esquerdos foram removidos e homogeneizados para a as análises da atividade enzimática (SOD, CAT e MPO) e do sistema glutationa (GSH/GSSG), para a análise dos valores de nitrito e da expressão de 4-HNE, MMP-12, NE, TIMP-1, TIMP-2. Nossos resultados apontam que o L-NAME tem uma ação voltada para a matriz extracelular (via protease-antiprotease), enquanto que a L-arginina possui uma ação voltada para os oxidantes, assim como a NAC. Porém a NAC atua aumentando os níveis de glutationa, o que interfere diretamente nos oxidantes (via oxidante-antioxidante), enquanto a L-arginina interfere aumentando o burden oxidativo concomitante a um aumento da velocidade de ação dos oxidantes o que aumenta as células inflamatórias, mas diminui seu tempo de ação permitindo uma maior proteção. Concluímos que tanto o favorecimento para a produção e liberação do NOatravés da administração da L-arginina quanto a inibição do NOpela utilização do L-NAME foi eficiente na proteção do pulmão, apesar de não terem alcançado um resultado tão bom quanto a NAC.

Proteolytic processing of the nonstructural proteins of dengue 2 virus

The genomes of many positive stranded RNA viruses and of all retroviruses are translated as large polyproteins which are proteolytically processed by cellular and viral proteases. Viral proteases are structurally related to two families of cellular proteases, the pepsin-like and trypsin-like proteases. This thesis describes the proteolytic processing of several nonstructural proteins of dengue 2 virus, a representative member of the Flaviviridae, and describes methods for transcribing full-length genomic RNA of dengue 2 virus. Chapter 1 describes the in vitro processing of the nonstructural proteins NS2A, NS2B and NS3. Chapter 2 describes a system that allows identification of residues within the protease that are directly or indirectly involved with substrate recognition. Chapter 3 describes methods to produce genome length dengue 2 RNA from cDNA templates.

The nonstructural protein NS3 is structurally related to viral trypsinlike proteases from the alpha-, picorna-, poty-, and pestiviruses. The hypothesis that the flavivirus nonstructural protein NS3 is a viral proteinase that generates the termini of several nonstructural proteins was tested using an efficient in vitro expression system and antisera specific for the nonstructural proteins NS2B and NS3. A series of cDNA constructs was transcribed using T7 RNA polymerase and the RNA translated in reticulocyte lysates. Proteolytic processing occurred in vitro to generate NS2B and NS3. The amino termini of NS2B and NS3 produced in vitro were found to be the same as the termini of NS2B and NS3 isolated from infected cells. Deletion analysis of cDNA constructs localized the protease domain necessary and sufficient for correct cleavage to the first 184 amino acids of NS3. Kinetic analysis of processing events in vitro and experiments to examine the sensitivity of processing to dilution suggested that an intramolecular cleavage between NS2A and NS2B preceded an intramolecular cleavage between NS2B and NS3. The data from these expression experiments confirm that NS3 is the viral proteinase responsible for cleavage events generating the amino termini of NS2B and NS3 and presumably for cleavages generating the termini of NS4A and NS5 as well.

Biochemical and genetic experiments using viral proteinases have defined the sequence requirements for cleavage site recognition, but have not identified residues within proteinases that interact with substrates. A biochemical assay was developed that could identify residues which were important for substrate recognition. Chimeric proteases between yellow fever and dengue 2 were constructed that allowed mapping of regions involved in substrate recognition, and site directed mutagenesis was used to modulate processing efficiency.

Expression in vitro revealed that the dengue protease domain efficiently processes the yellow fever polyprotein between NS2A and NS2B and between NS2B and NS3, but that the reciprocal construct is inactive. The dengue protease processes yellow fever cleavage sites more efficiently than dengue cleavage sites, suggesting that suboptimal cleavage efficiency may be used to increase levels of processing intermediates in vivo. By mutagenizing the putative substrate binding pocket it was possible to change the substrate specificity of the yellow fever protease; changing a minimum of three amino acids in the yellow fever protease enabled it to recognize dengue cleavage sites. This system allows identification of residues which are directly or indirectly involved with enzyme-substrate interaction, does not require a crystal structure, and can define the substrate preferences of individual members of a viral proteinase family.

Full-length cDNA clones, from which infectious RNA can be transcribed, have been developed for a number of positive strand RNA viruses, including the flavivirus type virus, yellow fever. The technology necessary to transcribe genomic RNA of dengue 2 virus was developed in order to better understand the molecular biology of the dengue subgroup. A 5' structural region clone was engineered to transcribe authentic dengue RNA that contains an additional 1 or 2 residues at the 5' end. A 3' nonstructural region clone was engineered to allow production of run off transcripts, and to allow directional ligation with the 5' structural region clone. In vitro ligation and transcription produces full-length genomic RNA which is noninfectious when transfected into mammalian tissue culture cells. Alternative methods for constructing cDNA clones and recovering live dengue virus are discussed.

Trimeresurus stejnegeri snake venom plasminogen activator - Site-directed mutagenesis and molecular modeling

The specific plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) is a serine proteinase presenting 23% sequence identity with the proteinase domain of tissue type plasminogen activator, and 63% with batroxobin, a fibrinogen clotting enzyme from Bothrops atrox venom that does not activate plasminogen. TSV-PA contains six disulfide bonds and has been successfully overexpressed in Escherichia coli (Zhang, Y., Wisner, A., Xiong, Y. L,, and Bon, C, (1995) J. Biol. Chem. 270, 10246-10255), To identify the functional domains of TSV-PA, we focused on three short peptide fragments of TSV-PA showing important sequence differences with batroxobin and other venom serine proteinases. Molecular modeling shows that these sequences are located in surface loop regions, one of which is next to the catalytic site, When these sequences were replaced in TSV-PA by the equivalent batroxobin residues none generated either fibrinogen-clotting or direct fibrinogenolytic activity, Two of the replacements had little effect in general and are not critical to the specificity of TSV-PA for plasminogen. Nevertheless, the third replacement, produced by the conversion of the sequence DDE 96a-98 to NVI, significantly increased the K-m for some tripeptide chromogenic substrates and resulted in undetectable plasminogen activation, indicating the key role that the sequence plays in substrate recognition by the enzyme.

Isolation, expression and characterization of a novel dual serine protease inhibitor, OH-TCI, from king cobra venom

Snake venom Kunitz/BPTI members are good tools for understanding of structure-functional relationship between serine proteases and their inhibitors. A novel dual Kunitz/BPTI serine proteinase inhibitor named OH-TCI (trypsin- and chymotrypsin-dual inhibito

A novel high molecular weight metalloproteinase cleaves fragment F1 of activated human prothrombin

A hemorrhagic proteinase, jerdohagin, was purified from Trimeresurus jerdonii venom by gel filtration and ion-exchange chromatographies. It was a single chain polypeptide with an apparent molecular weight of 96 kDa as estimated by SDS-PAGE under the non-reducing and reducing conditions. Internal peptide sequencing indicated that it consisted of metalloproteinase, disintegrin-like and cysteine-rich domains and belonged to the class III snake venom metalloproteinases (class P-III SVMPs). Like other typical metalloproteinases, hemorrhagic activities of jerdohagin were completely inhibited by EDTA, but not by PMSF. Jerdohagin preferentially degraded a-chain of human fibrinogen. Interestingly, jerdohagin did not activate human prothrombin, whereas it cleaved human prothrombin and fragment F1 of activated human prothrombin. (C) 2004 Elsevier Ltd. All rights reserved.

Two cathelicidin genes are present in both rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar)

Further to the previous finding of the rainbow trout rtCATH_1 gene, this paper describes three more cathelicidin genes found in salmonids: two in Atlantic salmon, named asCATH_1 and asCATH_2, and one in rainbow trout, named rtCATH_2. All the three new salmonid cathelicidin genes share the common characteristics of mammalian cathelicidin genes, such as consisting of four exons and possessing a highly conserved preproregion and four invariant cysteines clustered in the C-terminal region of the cathelin-like domain. The asCATH_1 gene is homologous to the rainbow trout rtCATH_1 gene, in that it possesses three repeat motifs of TGGGGGTGGC in exon IV and two cysteine residues in the predicted mature peptide, while the asCATH_2 gene and rtCATH_2 gene are homologues of each other, with 96% nucleotide identity. Salmonid cathelicidins possess the same elastase-sensitive residue, threonine, as hagfish cathelicidins and the rabbit CAP18 molecule. The cleavage site of the four salmonid cathelicidins is within a conserved amino acid motif of QKIRTRR, which is at the beginning of the sequence encoded by exon W. Two 36-residue peptides corresponding to the core part of rtCATH_1 and rtCATH_2 were chemically synthesized and shown to exhibit potent antimicrobial activity. rtCATH_2 was expressed constitutively in gill, head kidney, intestine, skin and spleen, while the expression of rtCATH_1 was inducible in gill, head kidney, and spleen after bacterial challenge. Four cathelicidin genes have now been characterized in salmonids and two were identified in hagfish, confirming that cathelicidin genes evolved early and are likely present in all vertebrates.

Identification of a novel cathelicidin gene in the rainbow trout, Oncorhynchus mykiss

We report the cloning of a novel antimicrobial peptide gene, termed rtCATH_1, found in the rainbow trout, Oncorhynchus mykiss. The predicted 216-residue rtCATH_1 prepropeptide consists of three domains: a 22-residue signal peptide, a 128-residue cathelin-like region containing two identifiable cathelicidin family signatures, and a predicted 66-residue C-terminal cationic antimicrobial peptide. This predicted mature peptide was unique in possessing features of different known (mammalian) cathelicidin subgroups, such as the cysteine-bridged family and the specific amino-acid-rich family. The rtCATH_1 gene comprises four exons, as seen in all known mammalian cathelicidin genes, and several transcription factor binding sites known to be of relevance to host defenses were identified in the 5' flanking region. By Northern blot analysis, the expression of rtCATH_1 was detected in gill, head kidney, and spleen of bacterially challenged fish. Primary cultures of head kidney leukocytes from rainbow trout stimulated with lipopolysaccharide or poly(I (.) C) also expressed riCATH_1. A 36-residue peptide corresponding to the core part of the fish cathelicidin was chemically synthesized and shown to exhibit potent antimicrobial activity and a low hemolytic effect. Thus, rtCATH_1 represents a novel antimicrobial peptide gene belonging to the cathelicidin family and may play an important role in the innate immunity of rainbow trout.

Characterization of two membrane-associated protease genes obtained from screening out-membrane protein genes of Flavobacterium columnare G(4)

In order to identify genes encoding the outer membrane proteins (OMPs) of the myxobacter Flavobacterium columnare G(4), the expression library of the bacterium was screened by using rabbit antisera developed against its OMPs. Positive colonies of Escherichia coli M15 containing fragments encoding the bacterial OMPs were selected for cloning the relevant genes by genomic walking methods. Two genes encoding a membrane-associated zinc metalloprotease and prolyl oligopeptidase are reported in this paper. The membrane-associated zinc metalloprotease gene (map) is 1800 bp in length, coding for 449 amino acids (aa). Despite the presence of a conserved motif HEXXH for all metalloproteases, the special HEXXH similar to 32 aa similar to E motif of the F. columnare G(4) Map and its low level of identity with other reported zinc-containing metalloproteases may imply that the membrane-associated zinc metalloprotease of F. columnare G(4) represents a new family of zincins. The gene encoding prolyl oligopeptidase (Pop), a serine proteinase, is 2352 bp in length, coding for 649 aa. Sequence homology analysis revealed that the Pop is also novel as it has <50% identity with other reported prolyl oligopeptidase family proteins. The present study represents the first to employ anti-fish bacterial OMP sera to screen genes of membrane-associated proteases of fish pathogenic bacteria, and to provide necessary information for the examination of the role of the two genes in the infection and pathogenesis of F. columnare.

蛇毒血管内皮生长因子及丝氨酸蛋白酶类似物的研究

蝰科蛇毒中含有丰富的具有血管通透增强活性的蛋白组分，本论文结合生物化学与分子生物学手段对几种毒蛇的蛇毒血管内皮生长因子进行了研究。其中，从云南产菜花烙铁头（Trimeresurus jerdonii）蛇毒中分离得到一个血管内皮生长因子，TjsvVEGF，并研究了其活性与受体结合特性的关系。同时，对圆斑蝰蛇、蝮蛇、山烙铁头和竹叶青等几种蛇的svVEGFs进行了分子生物学研究。此外，我们还分离到了一个蛇毒丝氨酸蛋白酶类似物,TjsvSPH,还对该蛋白的理化性质进行了初步研究。 TjsvVEGF是一个表观分子量为29 kDa的二聚体蛋白，由两个相同大小的亚基组成。活性实验表明，它在10ng的剂量下即可明显提高血管通透性，活性强度与VEGF165相当。虽然TjsvVEGF的氨基酸序列与TfsvVEGF和Pm-VEGF具有较高的相似性，但是它们的受体结合特性却有很大差异。TjsvVEGF与VEGFR-1的结合能力很弱，而对VEGR-2有很高的亲和力。这说明，TjsvVEGF的活性主要是VEGFR-2介导的。最后，我们对导致TjsvVEGF对VEGFR-1低亲和力的原因进行了探讨。 利用PCR方法，我们从圆斑蝰蛇毒腺中得到了三种svVEGFs蛋白编码区长短不一的cDNA序列，其差异是由cDNA链中一段富含AG（3’拼接接受位点）的区段发生了核苷酸缺失产生的。蛋白编码区核苷酸的缺失导致其编码的三种svVEGF蛋白N末端的氨基酸序列和长短均产生较大差异。因此我们推测，蝰属svVEGFs蛋白N末端普遍较短可能是编码它们的mRNA前体对3’拼接点的不同选择产生的。同时，通过对cDNAs推导的氨基酸序列分析发现，蝮蛇svVEGF和山烙铁头svVEGF在与受体结合相关的多个重要位点上发生了氨基酸替换，提示它们是研究svVEGFs与VEGFR结合机制的良好材料。 通过分子筛、离子交换和亲和层析等方法，我们还从菜花烙铁头蛇毒中得到了一个不具有酶活性的丝氨酸蛋白酶类似物，命名为：TjsvSPH。内肽序列测定结果表明，TjsvSPH三联体结构中的组氨酸已突变为精氨酸，这很可能是导致其失去蛋白水解酶活性的主要原因。活性检测验表明，与许多已发现的蛇毒活性组分不同，TjsvSPH不具有蛋白水解酶活性，不能引起血小板聚集，也不抑制ADP和凝血酶诱导的血小板聚集。它对人血浆的复钙时间也没有影响。TjsvSPH的氨基酸序列与典型蛇毒丝氨酸蛋白酶Halystase和Calobin有约77％的一致性。它与同科属来源的蛇毒丝氨酸蛋白酶类似物氨基酸序列相似性高达92％以上，与不同科属的也有约74％以上的相似性。通过对Genbank中六种毒蛇所有丝氨酸蛋白酶及其类似物进化分析，我们推测，蛇毒丝氨酸蛋白酶类似物很可能是由蛇毒丝氨酸蛋白酶进化而来，并在进化过程中形成了一类独特的蛋白质。

Recombinant protein of heptad-repeat HR212, a stable fusion inhibitor with potent anti-HIV action in vitro

HR212, a recombinant protein expressed in Escherichia coli, has been previously reported to inhibit HIV-1 membrane fusion at low nanomolar level. Here we report that HR212 is effective in blocking laboratory strain HIV-1IIIB entry and replication with EC50 values of 3.92±0.62 and 6.59±1.74 nM, respectively, and inhibiting infection by clinic isolate HIV-1KM018 with EC50 values of 44.44±10.20 nM, as well as suppressing HIV-1- induced cytopathic effect with an EC50 value of 3.04±1.20 nM. It also inhibited HIV-2ROD and HIV-2CBL-20 entry and replication in the μM range. Notably, HR212 was highly effective against T20-resistant strains with EC50 values ranging from 5.09 to 7.75 nM. Unlike T20, HR212 showed stability sufficient to inhibit syncytia formation in a time-of-addition assay, and was insensitive to proteinase K digestion. These results suggest that HR212 has great potential to be further developed as novel HIV-1 fusion inhibitor for treatment of HIV/ AIDS patients, particularly for those infected by T20-resistant variants.

Synthesis and characterization of novel poly(ester carbonate)s based on pentaerythritol

Novel poly(ester carbonate)s were synthesized by the ring-opening polymerization Of L-lactide and functionalized carbonate monomer 9-phenyl-2,4,8,10-tetraoxaspiro[5,5]undecan-3-one derived from pentaerythritol with diethyl zinc as an initiator. H-1 NMR analysis revealed that the carbonate content in the copolymer was almost equal to that in the feed. DSC results indicated that T-g of the copolymer increased with increasing carbonate content in the copolymer. Moreover, the protecting benzylidene groups in the copolymer poly(L-lactide-co-9-phenyl-2,4,8,10-tetraoxaspiro[5,5]undecan-3-one) were removed by hydrogenation with palladium hydroxide on activated charcoal as a catalyst to give a functional copolymer, poly(L-lactide-co-2,2-dihydroxylmethyl-propylene carbonate), containing pendant primary hydroxyl groups. Complete deprotection was confirmed by H-1 NMR and FTIR spectroscopy. The in vitro degradation rate of the deprotected copolymers was faster than that of the protected copolymers in the presence of proteinase K. The cell morphology and viability on a copolymer film evaluated with ECV-304 cells showed that poly(ester carbonate)s derived from pentaerythritol are good biocompatible materials suitable for biomedical applications.

Enzymatic degradation of poly(L-lactide) and poly (epsilon-caprolactone) electrospun fibers

Poly(L-lactide) (PLLA) and poly(epsilon-caprolactone) (PCL) ultrafine fibers were prepared by electrospinning. The influence of cationic and anionic surfactants on their enzymatic degradation behavior was investigated by measuring weight loss, molecular weight, crystallinity, and melting temperature of the fibers as a function of degradation time. Under the catalysis of proteinase K, the PLLA fibers containing the anionic surfactant sodium docecyl sulfate (SDS) exhibited a faster degradation rate than those containing cationic surfactant triethylbenzylammonium chloride (TEBAC), indicating that surface electric charge on the fibers is a critical factor for an enzymatic degradation. Similarly, TEBAC-containing PCL fibers exhibited a 47% weight loss within 8.5 h whereas SDS-containing PCL fibers showed little degradation in the presence of lipase PS. By analyzing the charge status of proteinase K and lipase PS under the experimental conditions, the importance of the surface charges of the fibers and their interactions with the charges on the enzymes were revealed. Consequently, a "two-step" degradation mechanism was proposed: (1) the enzyme approaches the fiber surface; (2) the enzyme initiates hydrolysis of the polymer.

Biodegradable electrospun fibers for drug delivery

The influences of surfactants and medical drugs on the diameter size and uniformity of electrospun poly(L-lactic acid) (PLLA) fibers were examined by adding various surfactants (cationic, anionic, and nonionic) and typical drugs into the PLLA solution. Significant diameter reduction and uniformity improvement were observed. It was shown that the drugs were capsulated inside of the fibers and the drug release in the presence of proteinase K followed nearly zero-order kinetics due to the degradation of the PLLA fibers. Such ultrafine fiber mats containing drugs may find clinical applications in the future.

Fluorescence spectra of hemorrhagin III from the snake venom of Agkistrodon acutus

Hemorrhagin III (AaH III) was separated and purified from the crude snake venom of Agkistrodon acutus, and its molecule weight was determined accurately to be 23; 284.4 +/- 0.1 by LDI1700-MALDI-TOF-MS. Emission spectra of AaH III showed that Trp residues were located by a great degree in the hydrophobic area. Addition of SDS and guanidine-HCl led to change of the molecular conformation of AaH III, and caused the fluorescence quenching of Trp residues. The red-shifted emission band of AaH III after adding guanidine-HCl showed that Trp residues exposed in polar solvents. The effects of pH, EDTA and metal ions on the fluorescence spectra of AaH III were also investigated.