Metabolite Profiling of Manilkara zapota L. Leaves by High-Resolution Mass Spectrometry Coupled with ESI and APCI and In Vitro Antioxidant Activity, α-Glucosidase, and Elastase Inhibition Assays.

High-resolution mass spectrometry equipped with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) sources was used to enhance the characterization of phytochemicals of ethanol extracts of Manilkara zapota L. leaves (ZLE). Sugar compounds, dicarboxylic acids, compounds of phenolic acids and flavonoids groups, and other phytochemicals were detected from the leaves. Antioxidant activity and inhibition potentiality of ZLE against α-glucosidase enzyme, and elastase enzyme activities were evaluated in in vitro analysis. ZLE significantly inhibited activities of α-glucosidase enzyme at a lower concentration (IC50 2.51 ± 0.15 µg/mL). Glucose uptake in C2C12 cells was significantly enhanced by 42.13 ± 0.15% following the treatment with ZLE at 30 µg/mL. It also exhibited potential antioxidant activities and elastase enzyme inhibition activity (IC50 27.51 ± 1.70 µg/mL). Atmospheric pressure chemical ionization mass spectrometry (APCI-MS) detected more m/z peaks than electrospray ionization mass spectrometry (ESI-MS), and both ionization techniques illustrated the biological activities of the detected compounds more thoroughly compared to single-mode analysis. Our findings suggest that APCI along with ESI is a potential ionization technique for metabolite profiling, and ZLE has the potential in managing diabetes by inhibiting α-glucosidase activity and enhancing glucose uptake.

The Unique Protein Composition of Honey Revealed by Comprehensive Proteomic Analysis: Allergens, Venom-like Proteins, Antibacterial Properties, Royal Jelly Proteins, Serine Proteases, and Their Inhibitors.

Honey is a unique natural product produced by European honeybees. Due to its high economic value, honey is considered to be well characterized chemically, and it is often discovered to be an adulterated commodity. However, this study shows that our knowledge of honey protein composition, which is of high medical and pharmaceutical importance, is incomplete. In this in-depth proteomic study of 13 honeys, we identified a number of proteins that are important for an understanding of honey properties and merit additional pharmaceutical research. Our major result is an expanded understanding of the proteins underlying honey's antimicrobial properties, such as hymenoptaecin and defensin-1, glucose dehydrogenase isoforms, venom allergens and other venom-like proteins, serine proteases and serine protease inhibitors, and a series of royal jelly proteins. In addition, we performed quantitative comparisons of all of the proteins previously known or newly identified. The honey proteins, determined using label-free nLC-MS/MS in which the same protein quantity was analyzed in one series, were found in relatively similar proportions, although eucalyptus honey differed most widely from the remaining honeys. Overall, the proteome analysis indicated that honeybees supply proteins to honey in a relatively stable ratio within each proteome, but total protein quantity can differ by approximately an order of magnitude in different honeys.

A Retrospective Analysis of the Cartilage Kunitz Protease Inhibitory Proteins Identifies These as Members of the Inter-α-Trypsin Inhibitor Superfamily with Potential Roles in the Protection of the Articulatory Surface.

AIM: The aim of this study was to assess if the ovine articular cartilage serine proteinase inhibitors (SPIs) were related to the Kunitz inter-α-trypsin inhibitor (ITI) family. METHODS: Ovine articular cartilage was finely diced and extracted in 6 M urea and SPIs isolated by sequential anion exchange, HA affinity and Sephadex G100 gel permeation chromatography. Selected samples were also subjected to chymotrypsin and concanavalin-A affinity chromatography. Eluant fractions from these isolation steps were monitored for protein and trypsin inhibitory activity. Inhibitory fractions were assessed by affinity blotting using biotinylated trypsin to detect SPIs and by Western blotting using antibodies to α1-microglobulin, bikunin, TSG-6 and 2-B-6 (+) CS epitope generated by chondroitinase-ABC digestion. RESULTS: 2-B-6 (+) positive 250, 220,120, 58 and 36 kDa SPIs were detected. The 58 kDa SPI contained α1-microglobulin, bikunin and chondroitin-4-sulfate stub epitope consistent with an identity of α1-microglobulin-bikunin (AMBP) precursor and was also isolated by concanavalin-A lectin affinity chromatography indicating it had N-glycosylation. Kunitz protease inhibitor (KPI) species of 36, 26, 12 and 6 kDa were autolytically generated by prolonged storage of the 120 and 58 kDa SPIs; chymotrypsin affinity chromatography generated the 6 kDa SPI. KPI domain 1 and 2 SPIs were separated by concanavalin lectin affinity chromatography, domain 1 displayed affinity for this lectin indicating it had N-glycosylation. KPI 1 and 2 displayed potent inhibitory activity against trypsin, chymotrypsin, kallikrein, leucocyte elastase and cathepsin G. Localisation of versican, lubricin and hyaluronan (HA) in the surface regions of articular cartilage represented probable binding sites for the ITI serine proteinase inhibitors (SPIs) which may preserve articulatory properties and joint function. DISCUSSION/CONCLUSIONS: The Kunitz SPI proteins synthesised by articular chondrocytes are members of the ITI superfamily. By analogy with other tissues in which these proteins occur we deduce that the cartilage Kunitz SPIs may be multifunctional proteins. Binding of the cartilage Kunitz SPIs to HA may protect this polymer from depolymerisation by free radical damage and may also protect other components in the cartilage surface from proteolytic degradation preserving joint function.

Proteomics, functional characterization and antivenom neutralization of the venom of Pakistani Russell's viper (Daboia russelii) from the wild.

The venom proteome of wild Pakistani Russell's viper (Daboia russelii) was investigated through nano-ESI-LCMS/MS of the reverse-phase HPLC fractions. A total of 54 venom proteins were identified and clustered into 11 protein families. Phospholipase A2 (PLA2, 63.8%) and Kunitz-type serine protease inhibitor (KSPI, 16.0%) were most abundant, followed by snake venom serine protease (SVSP, 5.5%, mainly Factor V activating enzyme), vascular endothelial growth factor (VEGF, 4.3%), snake venom metalloproteinase (SVMP, 2.5%, mainly Factor X activating enzyme) and phosphodiesterase (PDE, 2.5%). Other minor proteins include cysteine-rich secretory protein (CRiSP), snake venom C-type lectin/lectin-like protein (snaclec), nerve growth factor, L-amino acid oxidase and 5'-nucleotidase. PLA2, KSPI, SVSP, snaclec and SVMP are hemotoxic proteins in the venom. The study indicated substantial venom variation in D. russelii venoms of different locales, including 3 Pakistani specimens kept in the USA. The venom exhibited potent procoagulant activity on human plasma (minimum clotting dose = 14.5 ng/ml) and high lethality (rodent LD50 = 0.19 µg/g) but lacked hemorrhagic effect locally. The Indian VINS Polyvalent Antivenom bound the venom immunologically in a concentration-dependent manner. It moderately neutralized the venom procoagulant and lethal effects (normalized potency against lethality = 2.7 mg venom neutralized per g antivenom). BIOLOGICAL SIGNIFICANCE: Comprehensive venom proteomes of D. russelii from different locales will facilitate better understanding of the geographical variability of the venom in both qualitative and quantitative terms. This is essential to provide scientific basis for the interpretation of differences in the clinical presentation of Russell's viper envenomation. The study revealed a unique venom proteome of the Pakistani D. russelii from the wild (Indus Delta), in which PLA2 predominated (~60% of total venom proteins). The finding unveiled remarkable differences in the venom compositions between the wild (present study) and the captive specimens reported previously. The integration of toxicity tests enabled the correlation of the venom proteome with the envenoming pathophysiology, where the venom showed potent lethality mediated through coagulopathic activity. The Indian VINS Polyvalent Antivenom (VPAV) showed binding activity toward the venom protein antigens; however the immunorecognition of small proteins and PLA2-dominating fractions was low to moderate. Consistently, the antivenom neutralized the toxicity of the wild Pakistani Russell's viper venom at moderate efficacies. Our results suggest that it may be possible to enhance the Indian antivenom potency against the Pakistani viper venom by the inclusion of venoms from a wider geographical range including that from Pakistan into the immunogen formulation.

Plasminogen activator inhibitor 1 and Antipain preserve acrosome integrity of bovine spermatozoa during cryopreservation / Inibidor do ativador do plasminogênio 1 e antipaína preservam a integridade do acrossoma de espermatozoides bovinos durante a criopreservação

Seminal plasma contains serine proteases and serine protease inhibitor, which are involved in mammalian fertilization, and the inhibitors can be applied to prevent cold-induced sperm capacitation. The effects of different concentrations of two serine protease inhibitors were analyzed, Plasminogen activator inhibitor 1 - PAI-1 (70ƞg, 140ƞg and 210 ƞg) and Antipain (10µg, 50µg and 100µg) as supplementation to bovine semen cryopreservation extender. The effects of the inhibitors on the sperm parameters (sperm kinetics - CASA, acrosome integrity, plasma membrane integrity, mitochondrial membrane potential, sperm defects and acrosome reaction rate) were evaluated in the post-thaw semen. Cryopreservation of sperm with Antipain decreased post-thaw kinetic parameters of MP, VSL, LIN, SRT and the percentage of hyper-activated sperm while PAI-1 (210 ƞg) decreased VSL and LIN. Antipain and PAI-1 had no effect on the integrity parameters of the plasma membrane, mitochondrial membrane potential and sperm defects. Sperm cryopreserved in the presence of Antipain and PAI-1 (70 and 140 ƞg) preserved acrosome integrity, as they were able to complete the in vitro acrosome reaction. In conclusion, the serine protease inhibitors, Antipain and PAI-1 (70 and 140ƞg) are able to preserve the acrosome integrity of cryopreserved bovine sperm.(AU)

A criopreservação é parcialmente prejudicial à fertilidade do sêmen de bovinos e induz mudanças semelhantes à capacitação em espermatozoides. O plasma seminal contém serina-proteases e inibidores de serina-proteases que estão envolvidos na fertilização de mamíferos, e os inibidores podem ser aplicados para evitar uma capacitação espermática induzida pelo frio. Analisaram-se os efeitos de diferentes concentrações de dois inibidores de serina-proteases, inibidor do ativador do plasminogênio 1 - PAI-1 (70ƞg, 140ƞg e 210ƞg) e antipaína (10µg, 50µg e 100µg) na suplementação ao diluidor de criopreservação de sêmen bovino. Trinta e seis ejaculados de quatro bovinos Curraleiro Pé-Duro foram usados para criopreservação. Os efeitos dos inibidores sobre os parâmetros dos espermatozoides (cinética espermática - CASA, integridade acrossomal, integridade da membrana plasmática, potencial de membrana mitocondrial, defeitos espermáticos e taxa de reação acrossomal) foram avaliados no sêmen pós-descongelamento. A criopreservação de espermatozoides com antipaína diminuiu os parâmetros cinéticos pós-descongelamento de MP, VSL, LIN, SRT e a porcentagem de espermatozoides hiperativados, PAI-1 (210ƞg) diminuiu VSL e LIN. Antipaína e PAI-1 não tiveram efeitos nos parâmetros de integridade da membrana plasmática, no potencial de membrana mitocondrial e nos defeitos espermáticos. Espermatozoides criopreservados na presença de antipaína e PAI-1 (70 e 140ƞg) preservaram a integridade acrossomal, assim como foram capazes de completar a reação acrossômica in vitro. Em conclusão, os inibidores de serina-proteases, antipaína e PAI-1 (70 e 140ƞg) são capazes de preservar a integridade acrossomal de espermatozoides criopreservados de bovinos.(AU)

Déficit de alfa-1-antitripsina asociado a la variante Mmalton. Descripción de una familia / Alpha-1 antitrypsin deficiency associated with the Mmalton variant. Description of a family

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Proteomic comparisons of venoms of long-term captive and recently wild-caught Eastern brown snakes (Pseudonaja textilis) indicate venom does not change due to captivity.

UNLABELLED: Snake venom is a highly variable phenotypic character, and its variation and rapid evolution are important because of human health implications. Because much snake antivenom is produced from captive animals, understanding the effects of captivity on venom composition is important. Here, we have evaluated toxin profiles from six long-term (LT) captive and six recently wild-caught (RC) eastern brown snakes, Pseudonaja textilis, utilizing gel electrophoresis, HPLC-MS, and shotgun proteomics. We identified proteins belonging to the three-finger toxins, group C prothrombin activators, Kunitz-type serine protease inhibitors, and phospholipases A2, among others. Although crude venom HPLC analysis showed LT snakes to be higher in some small molecular weight toxins, presence/absence patterns showed no correlation with time in captivity. Shotgun proteomics indicated the presence of similar toxin families among individuals but with variation in protein species. Although no venom sample contained all the phospholipase A2 subunits that form the textilotoxin, all did contain both prothrombin activator subunits. This study indicates that captivity has limited effects on venom composition, that venom variation is high, and that venom composition may be correlated to geographic distribution. BIOLOGICAL SIGNIFICANCE: Through proteomic comparisons, we show that protein variation within LT and RC groups of snakes (Pseudonaja textilis) is high, thereby resulting in no discernible differences in venom composition between groups. We utilize complementary techniques to characterize the venom proteomes of 12 individual snakes from our study area, and indicate that individuals captured close to one another have more similar venom gel electrophoresis patterns than those captured at more distant locations. These data are important for understanding natural variation in and potential effects of captivity on venom composition.

A proteomic analysis of Pakistan Daboia russelii russelii venom and assessment of potency of Indian polyvalent and monovalent antivenom.

UNLABELLED: To address the dearth of knowledge on the biochemical composition of Pakistan Russell's Viper (Daboia russelii russelii) venom (RVV), the venom proteome has been analyzed and several biochemical and pharmacological properties of the venom were investigated. SDS-PAGE (reduced) analysis indicated that proteins/peptides in the molecular mass range of ~56.0-105.0kDa, 31.6-51.0kDa, 15.6-30.0kDa, 9.0-14.2kDa and 5.6-7.2kDa contribute approximately 9.8%, 12.1%, 13.4%, 34.1% and 30.5%, respectively of Pakistan RVV. Proteomics analysis of gel-filtration peaks of RVV resulted in identification of 75 proteins/peptides which belong to 14 distinct snake venom protein families. Phospholipases A2 (32.8%), Kunitz type serine protease inhibitors (28.4%), and snake venom metalloproteases (21.8%) comprised the majority of Pakistan RVV proteins, while 11 additional families accounted for 6.5-0.2%. Occurrence of aminotransferase, endo-ß-glycosidase, and disintegrins is reported for the first time in RVV. Several of RVV proteins/peptides share significant sequence homology across Viperidae subfamilies. Pakistan RVV was well recognized by both the polyvalent (PAV) and monovalent (MAV) antivenom manufactured in India; nonetheless, immunological cross-reactivity determined by ELISA and neutralization of pro-coagulant/anticoagulant activity of RVV and its fractions by MAV surpassed that of PAV. BIOLOGICAL SIGNIFICANCE: The study establishes the proteome profile of the Pakistan RVV, thereby indicating the presence of diverse proteins and peptides that play a significant role in the pathophysiology of RVV bite. Further, the proteomic findings will contribute to understand the variation in venom composition owing to different geographical location and identification of pharmacologically important proteins in Pakistan RVV.

A Rare Case of Mucoepidermoid Carcinoma ex Pleomorphic Adenoma arising in Minor Salivary Gland: Histopathological and Immunohistochemical Analysis.

Mucoepidermoid carcinoma ex pleomorphic adenoma (MCxPA) is a rare salivary gland tumor predominantly found in major salivary glands. A case of MCxPA involving the soft tissue and bone of the retromolar region of a 26-year-old man is presented. The histopathological features revealed a neoplasm with predominance of pleomorphic adenoma (PA) elements, and presence of mucoepidermoid carcinoma malignant epithelial cells in several areas. Histochemical and immunohistochemical studies were positive for periodic acid Schiff, alcian blue, cytokeratins 7, 13, 14, and 19, Bcl-2, c-erbB-2, FGF-2 and maspin in the malignant areas. The patient underwent a partial resection of the left side of the mandible with neck dissection and MCxPA diagnosis was confirmed.

A controlled, cross-sectional exploratory study on markers for the plasminogen system and inflammation in crevicular fluid samples from healthy, mucositis and peri-implantitis sites.

PURPOSE: To investigate expression of gene markers for the plasminogen system, inflammation, and bone resorption/remodelling in peri-implant crevicular fluid samples from healthy subjects, subjects with mucositis and subjects with peri-implantitis. A possible inhibitory effect of suppuration on the analysis of gene expression in samples from subjects with peri-implantitis was also analysed. MATERIALS AND METHODS: Peri-implant crevicular fluid (PICF) was sampled from 25 healthy subjects (H), 25 subjects with mucositis (M) and 25 subjects with peri-implantitis (P) using paper points and suction tips. The samples were analysed by quantitative polymerase chain reaction (qPCR). The following biomarkers associated with the plasminogen system, inflammation and bone resorption/ remodelling were investigated: interleukin-1 beta (IL-1ß), interleukin 8 (IL-8), tissue plasminogen activator (tPA), plasminogen activator inhibitor 2 (PAI-2), tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CatK). RESULTS: IL-1ß and IL-8 were significantly upregulated in the P group, and tPA and PAI-2 were significantly upregulated in the M group. These four genetic markers were oppositely regulated in samples from the subjects in the mucositis compared with the peri-implantitis group. TRAP and CatK showed no differences between the groups. The presence of suppuration did not have a detectable effect on gene analysis in samples from subjects with peri-implantitis. CONCLUSIONS: Markers for the plasminogen system and inflammation could be used to distinguish between mucositis and peri-implantitis. The results suggested that the plasminogen system was sufficiently upregulated allowing for resolution of inflammation and healing at the inflamed implant site in subjects with mucositis, whereas such upregulation was insufficient resulting in impaired healing and prolonged inflammation in subjects with peri-implantitis. The combination of tissue inflammation and low levels of tPA was a strong predictor of marginal bone loss in this study. It may be an interesting candidate for the unambiguous diagnosis of mucositis and peri-implantitis independent of radiographs and could possibly constitute a powerful future tool for rapid assessment of the periimplant tissue condition and the effect of subject treatment.

Immunohistochemical comparison of p53, Ki-67, CD68, vimentin, α-smooth muscle actin and alpha-1-antichymotry-psin in oral peripheral and central giant cell granuloma.

INTRODUCTION: Giant cell lesions are characterised histologically by multinucleated giant cells in a background of ovoid to spindle-shaped mesenchymal cells. There is a major debate whether these lesions are separate entities or variants of the same disease. Our aim was to study the nature of multinucleated and mononuclear cells from peripheral giant cell granuloma (PGCG), and central giant cell granuloma (CGCG) and giant cell tumor (GCT) of long bones using immunohistochemistry evaluation and to determine whether there is a correlation between recurrence and the markers used. MATERIALS AND METHODS: Ki-67, p53, Vimentin, smooth muscle specific actin, CD68 and alpha-1-antichymotrypsin were used to study 60 giant cell lesions. These included 26 CGCG, 28 PGCG, and 6 GCT cases using an avidin-biotin-complex immunohistochemistry standard method. RESULTS: All studied cases showed the same results except the percentage of Ki-67 positive mononuclear cells in PGCG was significantly higher than that of both CGCG and GCT (p<0.05). Interestingly, no statistical correlation between recurrence and the markers used was found. CONCLUSION: Our results may suggest that these lesions have the same histogenesis. The mononuclear stromal cells, both histiocytic and myofibroblastic, are thought to be responsible for the behavior of these lesions whereas the multinucleated cells are considered as reactive. This might support the argument that PGCG, CGCG and GCT are different variants for the same disease. Further studies using molecular techniques are required to elucidate why some of these lesions behave aggressively than others.

Evaluation of gingival crevicular fluid levels of tissue plasminogen activator, plasminogen activator inhibitor 2, matrix metalloproteinase-3 and interleukin 1-ß in patients with different periodontal diseases.

OBJECTIVES: The purpose of this study was to evaluate the gingival crevicular fluid levels of interleukin-1beta (IL-1ß), matrix metalloproteinases-3 (MMP-3), tissue type plasminogen activator (t-PA) and plasminogen activator inhibitor 2 (PAI-2) in patients with chronic periodontitis, aggressive periodontitis (AgP) and healthy individuals (controls). MATERIAL AND METHODS: Systemically healthy (21 chronic periodontitis, 23 AgP and 20 controls) subjects were included in this study. Plaque index, gingival index, probing pocket depth and clinical attachment level were recorded and gingival crevicular fluid samples were collected. Assays for IL-1ß, MMP-3, t-PA and PAI-2 levels in gingival crevicular fluid were carried out by an enzyme-linked immunosorbent assay. The one-sample Kolmogorov-Smirnov test, Mann-Whitney U test and Spearman correlation coefficient were used for data analyses. RESULTS: Gingival crevicular fluid levels of t-PA and IL-1ß were significantly higher in chronic periodontitis and AgP groups than in the control group (p < 0.001). MMP-3 levels in gingival crevicular fluid were detected as significantly higher in the chronic periodontitis and AgP groups compared with the control group (p < 0.05). The t-PA/PAI-2 rate of patients with chronic periodontitis and AgP were significantly higher than the control group (p < 0.05). The positive correlations were found among the PAI-2, t-PA, IL-1ß and MMP-3 levels in gingival crevicular fluid. The volume of the gingival crevicular fluid correlated with all of the clinical parameters (p < 0.001). There were positive correlations between the gingival crevicular fluid levels of PAI-2 and the probing pocket depth and between gingival crevicular fluid levels of PAI-2 and the clinical attachment level (p < 0.01). Similarly, significant correlations were found between t-PA levels and probing pocket depth and between t-PA levels and clinical attachment level measurements (p < 0.001). CONCLUSION: The present data showed that gingival crevicular fluid levels of IL-1 ß, MMP-3 and t-PA increased in periodontal disease regardless of the periodontitis type and played a part in tissue destruction.

Isolation and characterization of an ovoinhibitor, a multidomain Kazal-like inhibitor from Turkey (Meleagris gallopavo) seminal plasma.

Turkey seminal plasma contains three serine proteinase inhibitors. Two of them, with low molecular masses (6 kDa), were identified as single-domain Kazal-type inhibitors responsible for regulating acrosin activity. Our experimental objective was to isolate and characterize the inhibitor with the high molecular weight from turkey seminal plasma. The inhibitor was purified using hydrophobic interaction and affinity chromatography. Pure preparations of the inhibitor were used for identification by mass spectrometry, for determination of physicochemical properties (molecular weight, pI, and content and composition of the carbohydrate component), for kinetic studies, and for antibacterial tests. Gene expression and immunohistochemical detection of the inhibitor were analyzed in the testis, epididymis, and ductus deferens. The inhibitor with a high molecular weight from turkey seminal plasma was identified as an ovoinhibitor, which was found in avian semen for the first time. The turkey seminal plasma ovoinhibitor was a six-tandem homologous Kazal-type domain serine proteinase inhibitor that targeted multiple proteases, including subtilisin, trypsin, and elastase, but not acrosin. Our results suggested that hepatocyte growth factor activator was a potential target proteinase for the ovoinhibitor in turkey seminal plasma. The presence of the ovoinhibitor within the turkey reproductive tract suggested that its role was to maintain a microenvironment for sperm in the epididymis and ductus deferens. The turkey seminal plasma ovoinhibitor appeared to play a significant role in an antibacterial semen defense against Bacillus subtilis and Staphylococcus aureus.

Expression of oral secretory leukocyte protease inhibitor in HIV-infected subjects with long-term use of antiretroviral therapy.

BACKGROUND: The objectives of this study were to determine (i) the expression of oral secretory leukocyte protease inhibitor (SLPI) in HIV-infected subjects compared with non-HIV controls, (ii) the oral SLPI expression in HIV-infected subjects with antiretroviral therapy (ART) compared with those without ART, and (iii) factors associated with the expression of oral SLPI. METHODS: Oral tissues and samples of both un-stimulated and stimulated saliva were collected from HIV-infected subjects with and without ART, and non-HIV individuals. The expression of SLPI mRNA in the tissue was determined by quantitative real-time PCR. Salivary SLPI protein was detected using ELISA. Chi-square test and logistic regression analysis were performed to determine the association between HIV/ART status and the expression of oral SLPI. RESULTS: One hundred and fifty-seven HIV-infected subjects were enrolled: 99 on ART (age range, 23-57 years; mean, 39 years), 58 not on ART (age range, 20-59 years; mean, 34 years), and 50 non-HIV controls (age range, 19-59 years; mean, 36 years). The most common ART regimen was 2NRTIs + 1NNRTI. The expression of oral SLPI in stimulated saliva was significantly decreased with HIV infection (P < 0.001). The expression was also significantly different with respect to ART use (P = 0.007). Smoking, CD4(+) cell count, and HIV viral load were the factors associated with the oral SLPI expression. CONCLUSION: The expression of oral SLPI is altered by HIV infection and use of ART. Thus, oral SLPI may be the useful biomarker to identify subjects at risk of infections and malignant transformation due to HIV infection and long-term ART.

The Anopheles gambiae cE5, a tight- and fast-binding thrombin inhibitor with post-transcriptionally regulated salivary-restricted expression.

Mosquito saliva carries a large number of factors with anti-hemostatic, anti-inflammatory and immuno-modulatory activities. The cE5 protein was initially identified during an Anopheles gambiae salivary gland transcriptome study and later shown to share sequence similarity with anophelin, a thrombin inhibitor from the saliva of the New World mosquito Anopheles albimanus. The cE5 gene was found to encode different mRNA isoforms coexisting in several tissues of both male and female mosquitoes, a highly unusual profile for a gene potentially encoding an anti-thrombin and involved in blood feeding. Expression of the cE5 protein and assessment of its activity and inhibitory properties showed that it is a highly specific and tight-binding thrombin inhibitor, which differs from the A. albimanus orthologue for the fast-binding kinetics. Despite the widespread occurrence of cE5 transcripts in different mosquito tissues the corresponding protein was only found in female salivary glands, where it undergoes post-translational modification. Therefore, tissue-specific restriction of the A. gambiae cE5 is not achieved by transcriptional control, as common for mosquito salivary genes involved in hematophagy, but by post-trascriptional gene regulatory mechanisms. Our observations provide a paradigm of post-transcriptional regulation as key determinant of tissue specificity for a protein from an important disease vector and point out that transcriptomic data should be interpreted with caution in the absence of concomitant proteomic support.

Changes of proteases and proteinase inhibitors in androgen-dependent advanced prostate cancer patients with alpha2-macroglobulin deficiency.

BACKGROUND: It is thought that the quantitative imbalance between proteases and their inhibitors is a causative factor in invasion and metastasis of cancer cells. We previously reported on a number of androgen-dependent advanced prostate cancer (PCa) patients in which serum alpha2-macroglobulin (alpha2M) levels were markedly decreased to < 20 mg/dL (defined as alpha2M deficiency). Anti-androgen therapy is at first generally very effective for androgen-dependent advanced PCa, yielding survival benefits for most patients. In the present study, we evaluated serum levels of PSA, matrix metalloproteinases-2 (MMP-2), alpha2M, and alpha2-plasmin inhibitor (alpha2PI) in advanced PCa patients with or without alpha2M deficiency in order to determine the clinical significance of these proteases and proteinase inhibitors for PCa progression. METHODS: In this study, 33 PCa patients were diagnosed at the Kitasato University Hospital and compared with 10 healthy controls. PSA and MMP-2 levels were determined by enzyme immunoassay. Measurement of alpha2M was performed by laser-nephelometry, alpha2PI levels were determined by turbidimetric immunoassay. RESULTS: Serum levels of PSA and MMP-2 in PCa patients with alpha2M deficiency were significantly higher than in patients not alpha2M-deficient. In contrast, serum levels of alpha2M and alpha2PI in these patients were significantly lower than in those not alpha2M-deficient. PSA and alpha2M levels showed an inverse relationship in androgen-dependent advanced PCa with alpha2M deficiency. CONCLUSIONS: Our findings indicate that the serum levels of these proteases and proteinase inhibitors, which are involved in the invasion and metastasis of PCa, may be indicators of PCa disease progression in addition to PSA levels.

Periodontal pathogens affect the level of protease inhibitors in gingival crevicular fluid.

In periodontitis, an effective host-response is primarily related to neutrophils loaded with serine proteases, including elastase (NE) and protease 3 (PR3), the extracellular activity of which is tightly controlled by endogenous inhibitors. In vitro these inhibitors are degraded by gingipains, cysteine proteases produced by Porphyromonas gingivalis. The purpose of this study was to determine the level of selected protease inhibitors in gingival crevicular fluid (GCF) in relation to periodontal infection. The GCF collected from 31 subjects (nine healthy controls, seven with gingivitis, five with aggressive periodontitis and 10 with chronic periodontitis) was analyzed for the levels of elafin and secretory leukocyte protease inhibitor (SLPI), two main tissue-derived inhibitors of neutrophil serine proteases. In parallel, activity of NE, PR3 and arginine-specific gingipains (Rgps) in GCF was measured. Finally loads of P. gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Treponema denticola were determined. The highest values of elafin were found in aggressive periodontitis and the lowest in controls. The quantity of elafin correlated positively with the load of P. gingivalis, Ta. forsythia and Tr. denticola, as well as with Rgps activity. In addition, NE activity was positively associated with the counts of those bacterial species, but not with the amount of elafin. In contrast, the highest concentrations of SLPI were found in periodontally healthy subjects whereas amounts of this inhibitor were significantly decreased in patients infected with P. gingivalis. Periodontopathogenic bacteria stimulate the release of NE and PR3, which activities escape the control through degradation of locally produced inhibitors (SLPI and elafin) by host-derived and bacteria-derived proteases.

Antimicrobial potential of egg yolk ovoinhibitor, a multidomain Kazal-like inhibitor of chicken egg.

Chicken egg ovoinhibitor is a multidomain Kazal-type serine protease inhibitor with unknown function. Comparison of expression between different tissues indicated that ovoinhibitor is highly expressed in the magnum and liver followed by the uterus, which secrete egg white, egg yolk, and eggshell precursors, respectively. The results also revealed that ovoinhibitor expression is increased in the liver during sexual maturation followed by a subsequent decrease in mature hens. Ovoinhibitor was purified from the egg yolk plasma from nonfertilized eggs using two consecutive affinity chromatographies and gel filtration. Purified egg yolk ovoinhibitor was shown to inhibit trypsin and subtilisin. It was shown that purified egg yolk ovoinhibitor exhibited antimicrobial activities against Bacillus thuringiensis . The results suggest that this anti-protease plays a significant role in antibacterial egg defense against Bacillus spp., preventing contamination of table eggs (nonfertilized eggs) and protecting the chick embryo (fertilized eggs).

Pseudechis australis venomics: adaptation for a defense against microbial pathogens and recruitment of body transferrin.

The venom composition of Pseudechis australis, a widely distributed in Australia reptile, was analyzed by 2-DE and mass spectrometric analysis. In total, 102 protein spots were identified as venom toxins. The gel is dominated by horizontal trains of spots with identical or very similar molecular masses but differing in the pI values. This suggests possible post-translational modifications of toxins, changing their electrostatic charge. The results demonstrate a highly specialized biosynthesis of toxins destroying the hemostasis (P-III metalloproteases, SVMPs), antimicrobial proteins (L-amino acid oxidases, LAAOs, and transferrin-like proteins, TFLPs), and myotoxins (phospholipase A(2)s, PLA(2)s). The three transferrin isoforms of the Australian P. australis (Elapidae snake) venom are highly homologous to the body transferrin of the African Lamprophis fuliginosus (Colubridae), an indication for the recruitment of body transferrin. The venomic composition suggests an adaptation for a defense against microbial pathogens from the prey. Transferrins have not previously been reported as components of elapid or other snake venoms. Ecto-5'-nucleotidases (5'-NTDs), nerve growth factors (VNGFs), and a serine proteinase inhibitor (SPI) were also identified. The venom composition and enzymatic activities explain the clinical manifestation of the king brown snakebite. The results can be used for medical, scientific, and biotechnological purposes.