Addition Of Protease Inhibitors Research Articles

Tomato (Solanum lycopersicum) leaf juice induced whey protein gelling: Unveiling the potential of endogenous proteases in novel applications

Tomato leaves are by-products of tomato production and a potential source of proteins. Part of these proteins are proteases that carry out important functions for the plants. These endogenous proteases can bring challenges in downstream protein extraction, and can alter protein functionality. The aim of this study was to characterize the endogenous proteases in tomato leaf juice and to investigate the hydrolysis by these proteases on both endogenous leaf proteins and WPI. In addition, we characterized the gelation behavior, rheological properties and structural and thermal properties change of WPI by the juice proteases. 38 different proteases were identified in the juice, along with 4 protease inhibitors. The proteases hydrolyzed both endogenous leaf proteins and WPI, with predominantly an endopeptidase manner for the latter. The protein hydrolysis was positively affected by the incubation time and temperature. The protease activity in juice could not be fully eliminated by heat deactivation or the addition of protease inhibitors. The protein hydrolysis however remained limited (max. 43 %) under all conditions below 60 °C. This hydrolysis resulted in small changes in both secondary structure and thermal properties of WPI. Limited hydrolysis led to WPI gelation at lower protein concentration. Gel formed with WPI after limited hydrolysis had higher gel strength and more brittle structure than the gel with intact proteins. To conclude, the complex and active nature of proteases in tomato leaves poses challenge for downstream protein extraction. Alternatively, juice from tomato leaves could be used as natural gelling agent for WPI.

Challenges and Strategies to Enhance the Systemic Absorption of Inhaled Peptides and Proteins.

Proteins and peptides-based therapeutics are making substantial access to the market due to their obvious advantages of strong potency, high specificity and desirable safety profile. However, most clinical products are mainly delivered via parenteral route with inferior convenience. Lung is an attractive non-invasive alternative passage for systemic administration of biologics with numerous outstanding features, as examples of large absorptive surface area, extensive vascularization and mild local microenvironment. Even so, mucociliary clearance, alveolar macrophage phagocytosis, enzymatic metabolism, pulmonary surfactant adsorption and limited epithelium permeability constitute major obstacles affecting the systemic absorption of inhaled proteins and peptides. This article begins by giving a brief overview of challenges for the systemic absorption of inhaled proteins and peptides, and then goes on to a comprehensive review of possible strategies for enhanced pulmonary absorption, including chemical modification, addition of protease inhibitors, incorporation of absorption enhancers, modification with fusion proteins and development of particulate-based drug delivery systems. These strategies can provide enhanced transmembrane absorption capacity while avoiding pulmonary clearance, offering a valuable reference for designing pulmonary delivery systems of protein and peptide drugs.

Monitoring matrix remodeling in the cellular microenvironment using microrheology for complex cellular systems

Multiple particle tracking (MPT) microrheology was employed for monitoring the development of extracellular matrix (ECM) mechanical properties in the direct microenvironment of living cells. A customized setup enabled us to overcome current limitations: (i) Continuous measurements were enabled using a cell culture chamber, with this, matrix remodeling by fibroblasts in the heterogeneous environment of macroporous scaffolds was monitored continuously. (ii) Employing tracer laden porous scaffolds for seeding human mesenchymal stem cells (hMSCs), we followed conventional differentiation protocols. Thus, we were, for the first time able to study the massive alterations in ECM elasticity during hMSC differentiation. (iii) MPT measurements in 2D cell cultures were enabled using a long distance objective. Exemplarily, local mechanical properties of the ECM in human umbilical vein endothelial cell (HUVEC) cultures, that naturally form 2D layers, were investigated scaffold-free.Using our advanced setup, we measured local, apparent elastic moduli G0,app in a range between 0.08 and 60 Pa. For fibroblasts grown in collagen-based scaffolds, a continuous decrease of local matrix elasticity resulted during the first 10 hours after seeding. The osteogenic differentiation of hMSC cells cultivated in similar scaffolds, led to an increase of G0,app by 100 %, whereas after adipogenic differentiation it was reduced by 80 %. The local elasticity of ECM that was newly secreted by HUVECs increased significantly upon addition of protease inhibitor and in high glucose conditions even a twofold increase in G0,app was observed.The combination of these advanced methods opens up new avenues for a broad range of investigations regarding cell-matrix interactions and the propagation of ECM mechanical properties in complex biological systems. Statement of SignificanceCells sense the elasticity of their environment on a micrometer length scale. For studying the local elasticity of extracellular matrix (ECM) in the direct environment of living cells, we employed an advanced multipleparticle tracking microrheology setup. MPT is based on monitoring the Brownian motion oftracer particles, which is restricted by the surrounding network. Network elasticity can thusbe quantified.Overcoming current limitations, we realized continuous investigations of ECM elasticityduring fibroblast growth. Furthermore, MPT measurements of stem cell ECM showed ECMstiffening during osteogenic differentiation and softening during adipogenic differentiation.Finally, we characterized small amounts of delicate ECM newly secreted in scaffold-freecultures of endothelial cells, that naturally form 2D layers.

Modori reaction in blue grenadier and Alaska pollock frozen surimi and myosin degradation behavior upon addition of protease inhibitors

ABSTRACT The changes in protein degradation behavior in fish surimi upon addition of different protease inhibitors were examined, with the aim of identifying the proteases involved in the modori reaction in different species and grades of fish and at different temperatures. The modori reactions at 40, 50, and 60°C in four frozen surimi samples: grades FA and KA blue grenadier and grades SA and RA Alaska pollock were investigated. In the grade FA blue grenadier surimi, the modori reaction primarily involved metalloproteases at 40°C, serine proteases and cysteine proteases at 50°C, and cysteine proteases at 60°C. The reaction in the grade KA blue grenadier surimi involved multiple proteases at 40°C, and metalloproteases and cysteine proteases at 50 and 60°C. In the Alaska pollock surimi, multiple proteases were suggested to be equally involved in the modori reaction at 40, 50, and 60°C in both grades SA and RA samples.

Validation of leaf enzymes in the detergent and textile industries: launching of a new platform technology.

SummaryChemical catalysts are being replaced by biocatalysts in almost all industrial applications due to environmental concerns, thereby increasing their demand. Enzymes used in current industries are produced in microbial systems or plant seeds. We report here five newly launched leaf‐enzyme products and their validation with 15 commercial microbial‐enzyme products, for detergent or textile industries. Enzymes expressed in chloroplasts are functional at broad pH/temperature ranges as crude‐leaf extracts, while most purified commercial enzymes showed significant loss at alkaline pH or higher temperature, required for broad range commercial applications. In contrast to commercial liquid enzymes requiring cold storage/transportation, chloroplast enzymes as a leaf powder can be stored up to 16 months at ambient temperature without loss of enzyme activity. Chloroplast‐derived enzymes are stable in crude‐leaf extracts without addition of protease inhibitors. Leaf lipase/mannanase crude extracts removed chocolate or mustard oil stains effectively at both low and high temperatures. Moreover, leaf lipase or mannanase crude‐extracts removed stain more efficiently at 70 °C than commercial microbial enzymes (<10% activity). Endoglucanase and exoglucanase in crude leaf extracts removed dye efficiently from denim surface and depilled knitted fabric by removal of horizontal fibre strands. Due to an increased demand for enzymes in the food industry, marker‐free lettuce plants expressing lipase or cellobiohydrolase were created for the first time and site‐specific transgene integration/homoplasmy was confirmed by Southern blots. Thus, leaf‐production platform offers a novel low‐cost approach by the elimination of fermentation, purification, concentration, formulation and cold‐chain storage/transportation. This is the first report of commercially launched protein products made in leaves and validated with current commercial products.

Assessment of Synthesis Machinery of Two Antimicrobial Peptides from Paenibacillus alvei NP75.

Paenibacillus alvei NP75, a Gram-positive bacterium, produces two different antimicrobial peptides, paenibacillin N and P, which has potent antimicrobial activity against many clinical pathogens. The synthesis pattern of these antimicrobial peptides by P. alvei NP75 was studied extensively. The results were outstanding in a way that the paenibacillin N was synthesized irrespective of the growth of bacteria (non-ribosomal mediated), whereas paenibacillin P production was carried out by ribosomal mediated. In addition to the antimicrobial peptides, P. alvei NP75 also produces an immunogenic extracellular protease to defend itself from its own antimicrobial peptide, paenibacillin P. Furthermore, this immunogenic protease production was impaired by the addition of protease inhibitor, phenylmethylsulfonyl fluoride (PMSF). The sodium dodecyl sulfate (SDS) treated strain (mutant) failed to produce paenibacillin P, whereas the production of neither paenibacillin N nor the protease was affected by the plasmid curing. The plasmid curing studies that divulge the genes responsible for the synthesis of paenibacillin N and protease were found to be genome encoded, and paenibacillin P was plasmid encoded. We are reporting, first of its kind, the co-production of two different antimicrobial peptides from P. alvei NP75 through non-ribosomal and ribosomal pathways that could be used as effective antibiotics.

Human Immunodeficiency Virus disease and cardiac surgery – where are we?

Uncertainties concerning the effects of cardiopulmonary bypass, outcome and possibility of operator injury slowed progress in cardiac surgery in the HIV-infected patient. Severely ill patients, some with AIDS and others probably with AIDS, formed the basis of early reports; it was shown they could be taken through surgery satisfactorily. Following the advent of antiretroviral monotherapy – zidovudine, an NRTI – in 1987, and dual therapy in the early 1990s with NRTIs, the addition of protease inhibitors (PI) in 1995, although a double-edged sword, led to triple therapy/HAART, (2 NRTIs and a PI) and startling improvement in morbidity and mortality in markedly compromised HIV patients. Yet, it was only in 2003 when sizeable reports carrying ARV usage began to emerge. Intravenous drug addiction causing left-sided valvular heart disease was initially responsible for most cases, but there was now an increase in coronary artery disease (CAD) in young patients, blamed partly on lipid disturbances caused by PI. With an ARV rollout in South Africa delayed to 2004, and PIs only used in second line therapy in State-administered treatment, the increase in coronary artery disease has not been noted. The need for surgery in pericardial disease is rare. Paediatric cardiac surgery has been rarely reported, but is locally carried out; a new approach to medical management of these children should lead to more frequent surgery. Heart transplantation lags far behind solid organ transplantation, but there are indications that it will be more readily accepted. Certain vascular pathologies appear to be peculiar to HIV, with the virus itself being able to enter vessel walls with evolution of vascular disease. PIs would appear to accelerate coronary artery disease, as seen in younger HIV patients, who develop abnormal lipid profi les. The approach to cardiac surgery in most cardiac teaching units is looked at. Many answers have been found, but new issues have also arisen, as in relation to ARV’s and CAD. Most importantly, attitudes are changing: some would accept selected patients with AIDS not receiving ARVs. Very good results have followed surgery in patients who have suffered AIDS defining conditions but are stable on ARVs.

Molecular characterization of alkaline protease of Bacillus amyloliquefaciens SP1 involved in biocontrol of Fusarium oxysporum

An alkaline protease gene was amplified from genomic DNA of Bacillus amyloliquefaciens SP1 which was involved in effective biocontrol of Fusarium oxysporum. We investigated the antagonistic capacity of protease of B. amyloliquifaciens SP1, under in vitro conditions. The 5.62 fold purified enzyme with specific activity of 607.69U/mg reported 24.14% growth inhibition of F. oxysporum. However, no antagonistic activity was found after addition of protease inhibitor i.e. PMSF (15mM) to purified enzyme. An 1149bp nucleotide sequence of protease gene encoded 382 amino acids of 43kDa and calculated isoelectric point of 9.29. Analysis of deduced amino acid sequence revealed high homology (86%) with subtilisin E of Bacillus subtilis. The B. amyloliquefaciens SP1 protease gene was expressed in Escherichiax coli BL21. The expressed protease was secreted into culture medium by E. coli and exhibited optimum activity at pH8.0 and 60°C. The most reliable three dimensional structure of alkaline protease was determined using Phyre 2 server which was validated on the basis of Ramachandran plot and ERRAT value. The expression and structure prediction of the enzyme offers potential value for commercial application in agriculture and industry.

Protease inhibitors-based therapy induces acquired spherocytic-like anaemia and ineffective erythropoiesis in chronic hepatitis C virus patients.

The addition of protease inhibitors, boceprevir (BOC) or telaprevir (TRV), to peg-interferon and ribavirin (PR) increases the incidence of anaemia in patients with chronic hepatitis C virus (HCV) infection. Although genetic variants in inosine triphosphatase (ITPA) gene have been linked to the haemolytic anaemia induced by PR, the mechanism sustaining severe anaemia during triple therapy is still unknown. This study aims to elucidate the molecular mechanisms underlying anaemia in chronic HCV patients with combined therapy. We studied 59 patients with chronic HCV genotype-1: 29 treated with TRV/PR and 30 with BOC/PR. We evaluated biochemical and haematological parameters, red cell index at baseline, 4, 12, 16 and 24 weeks of treatment; in a subgroup, we performed functional studies: osmotic fragility, red cell membrane protein separation, mass spectrometry analysis, quantification of erythroid microparticles release. IL28B and ITPA polymorphisms were also evaluated. We found early acute normochromic normocytic haemolytic anaemia (4-8 weeks) followed by a late macrocytic hypo-regenerative anaemia with inappropriate low reticulocyte count (12-24 weeks). Studies on red cells revealed: (i) presence of spherocytes; (ii) increased osmotic fragility; (iii) abnormalities in red cell membrane protein composition; (iv) reduced membrane-cytoskeleton stability; (v) increased release of erythroid microparticles. ITPA polymorphisms impacted only the early phase of anaemia. The bimodal pattern of anaemia in chronic HCV patients on triple therapy might be because of acquired spherocytic-like anaemia in the early phase, followed by hyporegenerative anaemia, most likely related to the combined effects of PR and TRV or BOC on erythropoiesis.

An analysis of the impact of pre-analytical factors on the urine proteome: Sample processing time, temperature, and proteolysis.

PurposeWe have examined the impact of sample processing time delay, temperature, and the addition of protease inhibitors (PIs) on the urinary proteome and peptidome, an important aspect of biomarker studies.Experimental designTen urine samples from patients with varying pathologies were each divided and PIs added to one‐half, with aliquots of each then processed and frozen immediately, or after a delay of 6 h at 4°C or room temperature (20–22°C), effectively yielding 60 samples in total. Samples were then analyzed by 2D‐PAGE, SELDI‐TOF‐MS, and immunoassay.ResultsInterindividual variability in profiles was the dominant feature in all analyses. Minimal changes were observed by 2D‐PAGE as a result of delay in processing, temperature, or PIs and no changes were seen in IgG, albumin, β2‐microglobulin, or α1‐microglobulin measured by immunoassay. Analysis of peptides showed clustering of some samples by presence/absence of PIs but the extent was very patient‐dependent with most samples showing minimal effects.Conclusions and clinical relevanceThe extent of processing‐induced changes and the benefit of PI addition are patient‐ and sample‐dependent. A consistent processing methodology is essential within a study to avoid any confounding of the results.

Acquired spherocytic like anemia combined with ineffective erythropoiesis sustains anemia in patients with chronic hepatitis C infection receiving telaprevir or boceprevir-based triple therapy

Background and aims: The addition of protease inhibitors, boceprevir (BOC) or telaprevir (TVR), to peg-interferon and ribavirin (PR) increases the incidence of anemia. Although genetic variants in ITPA gene have been linked to the hemolytic anemia induced by PR, the mechanism sustaining the severe anemia during triple therapy is still unknown.

Quantification of human urinary exosomes by nanoparticle tracking analysis

Exosomes are vesicles that are released from the kidney into urine. They contain protein and RNA from the glomerulus and all sections of the nephron and represent a reservoir for biomarker discovery. Current methods for the identification and quantification of urinary exosomes are time consuming and only semi-quantitative. Nanoparticle tracking analysis (NTA) counts and sizes particles by measuring their Brownian motion in solution. In this study, we applied NTA to human urine and identified particles with a range of sizes. Using antibodies against the exosomal proteins CD24 and aquaporin 2 (AQP2), conjugated to a fluorophore, we could identify a subpopulation of CD24- and AQP2-positive particles of characteristic exosomal size. Extensive pre-NTA processing of urine was not necessary. However, the intra-assay variability in the measurement of exosome concentration was significantly reduced when an ultracentrifugation step preceded NTA. Without any sample processing, NTA tracked exosomal AQP2 upregulation induced by desmopressin stimulation of kidney collecting duct cells. Nanoparticle tracking analysis was also able to track changes in exosomal AQP2 concentration that followed desmopressin treatment of mice and a patient with central diabetes insipidus. When urine was stored at room temperature, 4°C or frozen, nanoparticle concentration was reduced; freezing at -80°C with the addition of protease inhibitors produced the least reduction. In conclusion, with appropriate sample storage, NTA has potential as a tool for the characterization and quantification of extracellular vesicles in human urine.

THE EFFECT OF PREANALYTICAL FACTORS IN THE QUANTIFICATION OF AB IN PLASMA

Pre-assay handling of peripheral blood samples for quantification of A β peptides is critical when a diagnostic use is intended. It has been described physical and biochemical cues affecting A β quantification in blood-derived samples, suggesting that differences in sample processing may result in variations in the quantification. Stringent standardization, but on tested practical grounds, is advisable.In this study, we tested the effect of factors as the addition of protease inhibitors, processing delay time (PDT) or temperature in the levels of A β 1-40 and A β 1-42 in undiluted plasma (UP) and diluted plasma (DP). Peripheral blood from seven healthy donors was collected in polypropylene vials with EDTA, divided into aliquots with or without protease inhibitors and maintained at either 4°C or room temperature (RT) during a PDT of 0 (control sample, CS), 8 or 30 hours. Then, the samples were centrifuged and stored at -80°C until use. The levels of Aβ were determined by ELISA. No significant differences were found in the levels of Aβ 1-42 after either 8h or 30h of PDT, in samples incubated with or without protease inhibitors in UP and DP fractions. In contrast, the levels of Aβ 1-40 in both the UP and DP fractions stored at 4°C during the PDT were lower in the samples with protease inhibitors comparing with those without it.On the other hand, no significant differences were found in the levels of A β 1-42 between the CS and the samples maintained at 4°C or RT with a PDT of 8 or 30 hours. However, 2 out of the 7 subjects showed substantial change of A β 1-42 levels after either 8 or 30 hours of PDT at either 4°C or RT, each in an opposite direction.A β 1-40 levels showed a trend to decrease (∼10%) after 8 hours of PDT at RT, but not at 4ªC. Our results indicate that a PDT of up to 8 hours could be allowed without introducing substantial variability in the determinations, provided the blood samples are stored at 4°C immediately after collection. The addition of protease inhibitors does not seem to produce an advantage.

Stability of cytokines in supernatants of stimulated mouse immune cells.

Measurements of cytokines in cell culture supernatants are widely used to evaluate the immune response. Cytokine levels in secretomes are usually quantified using enzyme-linked immunosorbent assays (ELISA), which have easy, sensitive, specific, rapid, cost-effective, and reproducible protocols. To our knowledge, the stability of cytokines in secretomes has not been hitherto investigated. We present data that involve; time-dependent changes during storage at +4°C, and the effects of freeze-thaw cycles in samples frozen at -80(o)C, instant freezing of samples with liquid nitrogen, and addition of protease inhibitors on the stability of certain cytokines (TNF-α, MIP-2, IFN-γ, IL-6, IL-10, IL-17A), in secrotomes of spleen and lymph nodes from tumor-bearing animals. Our results show that IL-6 remains stable, MIP-2, IFN-γ and IL-10 are somewhat stable, while TNF-α and IL-17A are degradable cytokines: instant freezing by liquid nitrogen or adding protease inhibitor does not preserve the stability of these cytokines. From these results it can be concluded that, if possible, TNF-α measurements should be perform in fresh samples, and IL-17A and IL-10 samples can be stored at -80°C, but should be used at the first thaw.

Management of recurrent hepatitis C virus after liver transplantation.

Chronic hepatitis C virus (HCV) infection is the leading cause of death from liver disease and the leading indication for liver transplantation (LT) in the United States and Western Europe. LT represents the best therapeutic alternative for patients with advanced chronic liver disease caused by HCV or those who develop hepatocarcinoma. Reinfection by HCV of the graft is universal and occurs in 95% of transplant patients. This reinfection can compromise graft function and patient survival. In a few cases, the histological recurrence is minimal and non-progressive; however, in most patients it follows a more rapid course than in immunocompetent persons, and frequently evolves into cirrhosis with graft loss. In fact, the five-year and ten-year survival of patients transplanted because of HCV are 75% and 68%, respectively, compared with 85% and 78% in patients transplanted for other reasons. There is also a pattern of recurrence that is very severe, but rare (< 10%), called fibrosing cholestatic hepatitis, which often involves rapid graft loss. Patients who present a negative HCV viremia after antiviral treatment have better survival. Many studies published over recent years have shown that antiviral treatment of post-transplant HCV hepatitis carried out during the late phase is the best option for improving the prognosis of these patients. Until 2011, PEGylated interferon plus ribavirin was the standard of care, resulting in a sustained virological response in around 30% of recipients. The addition of protease inhibitors, such as boceprevir or telaprevir, to the standard of care, or the use of other direct-acting antiviral drugs may involve therapeutic changes in the context of HCV recurrence. This may result a better prognosis for these patients, particularly those with severe recurrence or factors predicting rapid progression of fibrosis. However, the use of these agents in LT still requires clarification in terms of safety and efficacy.

Is there still a role for PEG IFN+RBV therapy in patients with HCV genotype 1?

Boceprevir and telaprevir-based triple therapy is now the standard of care for the treatment of genotype 1 patients. However, dual therapy with pegylated interferon and ribavirin should be discussed in treatment-naïve patients with good predictors of response. A recent published trial has shown in non-cirrhotic patients with low viral load at baseline, similar efficacy of a 24-week course of dual therapy vs a 24-week course of boceprevir-based triple therapy in case of rapid virological response. Accordingly, addition of protease inhibitor should be discussed after 4weeks of dual therapy in this easy-to-treat population.

Pre-analytical factors influencing the stability of cerebrospinal fluid proteins

Cerebrospinal fluid (CSF) is a potential source for new biomarkers due to its proximity to the brain. This study aimed to clarify the stability of the CSF proteome when undergoing pre-analytical factors.We investigated the effects of repeated freeze/thaw cycles, protease inhibitors and delayed storage for 4h, 24h or 14 days at −20°C, 4°C and room temperature (RT) after centrifugation compared with our standard practice of two hours at RT before placing the samples in an −80°C environment. The results were obtained using immunoassays for amyloid-beta 1–42 (Aβ42), tau, phosphorylated tau (P-tau) and cystatin C and using surface-enhanced laser desorption/ionisation time-of-flight (SELDI-TOF) mass spectrometry for proteomic profiling.Tau and P-tau were susceptible to repeated freeze/thaw cycles while SELDI-TOF analysis produced eight significant peaks and additional artefact peaks from samples with added protease inhibitors. Delayed storage for different durations and in different temperatures produced six significant SELDI-TOF peaks. Aβ42 and tau were susceptible to increased temperatures and the duration before storage, whereas P-tau and cystatin C were not. Transthyretin and several of its isoforms were found using SELDI-TOF and were susceptible to freeze/thaw cycles and to increased temperature and length of time prior to storage.We recommend that CSF should be collected and centrifuged immediately after sampling and prior to storage at −80°C without the addition of protease inhibitors. Freeze/thawing should be avoided because of the instability of tau, P-tau and transthyretin. Standardised CSF sampling, handling and storage for biomarker research are essential for accurately comparing the results obtained by different studies and institutions.

English

&nbsp; A major limitation of plant bioreactors is the lack of suitable and cost-effective purification methods for the extraction of pharmaceutical-grade proteins. In contrast to that, there are numerous established purification systems for heterologous proteins, expressed in&nbsp;Escherichia coli, which are used for the commercial production of therapeutic proteins. Therefore, we wanted to adapt the BioRad Profinity eXactTM&nbsp;one-step protein purification system (originally designed for microbial expression platforms) to purify recombinant proteins in crude plant extracts. This system based on the prodomain of microbial subtilase as fusion partner and a column-bound subtilisin protease. The engineered protease captures and cleaves the fusion protein, retaining the tag and releasing the native protein into the eluate. The subtilase tag was fused to human interleukin 6 (IL6) and transiently expressed in&nbsp;Nicotiana benthamianaleaves using the MagnICON system. The fusion protein was expressed at lower levels than native IL6, suggesting it is expressed less efficiently and/or has a lower stability. However, free IL6 was also detected in the extract and was unaffected by the addition of protease inhibitors during extraction, suggesting that the fusion protein is cleaved&nbsp;in planta&nbsp;by endogenous proteases. Purification of the recombinant protein using the Profinity eXactTM&nbsp;system reduced the yield still further. The inefficient production of tagged IL6, coupled with the extensive losses during purification, indicate that the Profinity eXactTM&nbsp;system is not suitable for the extraction of IL6 from crude plant extracts. &nbsp; Key words:&nbsp;Tobacco, transient expression, endoplasmic reticulum, Profinity protein purification, partial cleavage.

Stability of lactate dehydrogenase, aspartate aminotransferase, alkaline phosphatase and tartrate resistant acid phosphatase in human saliva and gingival crevicular fluid in the presence of protease inhibitor

The stability of aspartate aminotransferase (AST), lactate dehydrogenase (LDH), tartrate resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) activities from saliva and gingival crevicular fluid (GCF) with and without the addition of protease inhibitor (PI) at room temperature (RT; 25°C), 4°C and -20°C were investigated. AST, LDH, TRAP and ALP activities in saliva and GCF (n=9) with and without the addition of PI were assayed at 0 (control), 12, 24, 48, 72 h, one and two weeks. A paired t-test showed there were a significant differences (p<0.05) between LDH and TRAP activities in saliva, in the presence and without PI at all temperatures. ALP activity exhibited a significant difference in activity (p<0.05) in the presence and without PI at RT while no significant differences were observed at 4ºC and -20ºC. A significant difference (p<0.05) was observed in AST, LDH and TRAP activities (GCF) at RT and 4ºC in the presence and without PI. We conclude that PI is essential for maintaining stable enzyme activities in saliva and GCF.

Development of two haemocyte culture systems (in attachment and in suspension) for shrimp immunity studies

For studying shrimp immunity, in vitro haemocyte cultures are essential. Despite various reported attempts, well-described and reproducible culture techniques are lacking. The current work aimed to establish two in vitro haemocyte culture systems for Penaeus (Litopenaeus) vannamei. Haemocyte suspensions were either seeded in conventional Nunc® Nunclon™Δ Surface 24-well cell culture plates with glass cover slips (haemocytes in attachment) or in Nunc® Hydrocell Surface 24-well cell culture plates (haemocytes in suspension). The culture medium was based on L-15 (Leibovitz), and was further supplemented with L-glutathione and protease inhibitors in an attempt to improve haemocyte survival. Parameters such as number of living adherent and non-adherent single cells, number and average diameter of clusters and survival of cells inside clusters were evaluated. Additionally, live-cell imaging videos were recorded. It was found that haemocytes cultured for 1h on glass coverslips in Nunc® Nunclon™Δ Surface plates could be separated in two cell fractions: adherent or non-adherent. Shrimp haemocytes cultured in Nunc® Hydrocell Surface plates remained in suspension and over time formed cell clusters which melanised. L-glutathione supplementation clearly improved haemocyte survival up to 48h and delayed clustering and melanisation; addition of protease inhibitors did not. To validate the system, the phagocytic and antibacterial activities of adherent haemocytes towards Vibrio campbellii were evaluated. After 1h of co-culture, 11.5±0.14% of haemocytes showed phagocytosis with an average of 2.4±0.1 bacteria internalised per haemocyte. Furthermore, haemocytes clearly demonstrated an antibacterial activity. It was concluded that these systems were reproducible and could keep haemocytes functionally active during the time required for the study of innate immune processes. Consequently, these techniques represent powerful tools for studying a variety of cell-mediated and humoral immune responses of shrimp in vitro.