Increasement of Heterologous Expression of Recombinant Vit v 1 in Pichia pastoris KM71 by Nonionic Detergents as a Cost-effective Approach

Applied Biochemistry and Microbiology - Vit v 1 as a lipid-transfer protein is a major allergen of grapes (Vitis vinifera) that elicits food allergy in many patients in Iran. Todays, recombinant...     

Distinct immunological and biochemical properties of thyroid peroxidase purified from human thyroid glands and recombinant protein produced in insect cells.

The biosynthesis of thyroid hormone from thyroglobulin is catalysed by thyroid peroxidase (TPO), an integral membrane protein. TPO is also a major autoantigen in autoimmune thyroid disease and autoantibodies to TPO are markers for disease activity. Large quantities of purified TPO are essential for elucidating its structure and understanding its role in disease activity. We describe the high yield purification of full-length recombinant human TPO from baculovirus infected insect cells and compare it to purified native TPO from human thyroid glands. In contrast to native human TPO, the human TPO produced in insect cells as a recombinant protein was insoluble and resistant to solubilisation in detergents. Reversible substitution of lysine residues with citraconic anhydride led to increased solubility of the recombinant TPO, allowing high-yield purification by monoclonal antibody chromatography. The purified enzyme preparation was shown to be TPO by its reactivity with monoclonal and polyclonal antibodies by enzyme linked immunosorbent assay and Western blotting. Both the human and recombinant purified TPO preparations also react with sera from patients with autoimmune thyroid disease, although the binding of conformational dependent autoantibodies was considerably lower to the recombinant TPO than to the native TPO. This suggests that the recombinant TPO may differ in some aspects of its tertiary structure. The purified recombinant TPO was devoid of enzyme activity, in contrast to the enzymatically active, purified human TPO preparations. Both preparations contained comparable amounts of haem (R(z)=0.269), but a shift in the Soret band of recombinant TPO (402 nm) from that of natural TPO (409 nm) indicates that the lack of enzymatic activity of the recombinant enzyme may be due to changes in the protein backbone surrounding the haem. Both the purified native and recombinant TPO, under non-denaturing conditions, show evidence of high molecular mass oligomers, although the latter preparation is prone to a greater degree of aggregation. In conclusion, our studies indicate that recombinant TPO generated in insect cells is conformationally distinct from the native TPO, is insoluble and enzymatically inactive, consistent with the difficulties associated with its purification and crystallisation.     

Platelet adenylate cyclase and phospholipase C are affected differentially by ADP-ribosylation. Effects on thrombin-mediated responses.

During the isolation of the activator protein for glucosylceramide beta-glucosidase, we found that certain column fractions contained an inhibitor of the enzyme. After separation from the activator protein by a DEAE-Sephacel column, the inhibitor was purified further with a Spehadex G-75 column. The u.v. absorption spectrum of the purified material was similar to that of nucleic acids and the protein content of the purified material was negligible. Furthermore the purified inhibitor reacted with orcinol but not with diphenylamine, and its inhibitory activity was completely destroyed by treatment with RNAases. It seems likely that the purified inhibitor was tRNA. Authentic RNA, tRNA and DNA had similar inhibitory effects on beta-glucosidase (Ki 17 micrograms/ml for tRNA, noncompetitive toward the substrate). The inhibitory effect of nucleic acids was not fully overcome by an excess amount of the activator protein, but phosphatidylserine could restore the activity to normal. Tests with several other hydrolases revealed that the inhibitory effect of nucleic acids was fairly general.     

Impact of Acute Malaria on Pre-Existing Antibodies to Viral and Vaccine Antigens in Mice and Humans

Vaccine-induced immunity depends on long-lived plasma cells (LLPCs) that maintain antibody levels. A recent mouse study showed that Plasmodium chaubaudi infection reduced pre-existing influenza-specific antibodies—raising concerns that malaria may compromise pre-existing vaccine responses. We extended these findings to P. yoelii infection, observing decreases in antibodies to model antigens in inbred mice and to influenza in outbred mice, associated with LLPC depletion and increased susceptibility to influenza rechallenge. We investigated the implications of these findings in Malian children by measuring vaccine-specific IgG (tetanus, measles, hepatitis B) before and after the malaria-free 6-month dry season, 10 days after the first malaria episode of the malaria season, and after the subsequent dry season. On average, vaccine-specific IgG did not decrease following acute malaria. However, in some children malaria was associated with an accelerated decline in vaccine-specific IgG, underscoring the need to further investigate the impact of malaria on pre-existing vaccine-specific antibodies.     

Characterizing multistationarity regimes in biochemical reaction networks

Switch like responses appear as common strategies in the regulation of cellular systems. Here we present a method to characterize bistable regimes in biochemical reaction networks that can be of use to both direct and reverse engineering of biological switches. In the design of a synthetic biological switch, it is important to study the capability for bistability of the underlying biochemical network structure. Chemical Reaction Network Theory (CRNT) may help at this level to decide whether a given network has the capacity for multiple positive equilibria, based on their structural properties. However, in order to build a working switch, we also need to ensure that the bistability property is robust, by studying the conditions leading to the existence of two different steady states. In the reverse engineering of biological switches, knowledge collected about the bistable regimes of the underlying potential model structures can contribute at the model identification stage to a drastic reduction of the feasible region in the parameter space of search. In this work, we make use and extend previous results of the CRNT, aiming not only to discriminate whether a biochemical reaction network can exhibit multiple steady states, but also to determine the regions within the whole space of parameters capable of producing multistationarity. To that purpose we present and justify a condition on the parameters of biochemical networks for the appearance of multistationarity, and propose an efficient and reliable computational method to check its satisfaction through the parameter space.     

Heterosis as investigated in terms of polyploidy and genetic diversity using designed Brassica juncea amphiploid and its progenitor diploid species

Fixed heterosis resulting from favorable interactions between the genes on their homoeologous genomes in an allopolyploid is considered analogous to classical heterosis accruing from interactions between homologous chromosomes in heterozygous plants of a diploid species. It has been hypothesized that fixed heterosis may be one of the causes of low classical heterosis in allopolyploids. We used Indian mustard (Brassica juncea, 2n = 36; AABB) as a model system to analyze this hypothesis due to ease of its resynthesis from its diploid progenitors, B. rapa (2n = 20; AA) and B. nigra (2n = 16; BB). Both forms of heterosis were investigated in terms of ploidy level, gene action and genetic diversity. To facilitate this, eleven B. juncea genotypes were resynthesized by hybridizing ten near inbred lines of B. rapa and nine of B. nigra. Three half diallel combinations involving resynthesized B. juncea (11×11) and the corresponding progenitor genotypes of B. rapa (10×10) and B. nigra (9×9) were evaluated. Genetic diversity was estimated based on DNA polymorphism generated by SSR primers. Heterosis and genetic diversity in parental diploid species appeared not to predict heterosis and genetic diversity at alloploid level. There was also no association between combining ability, genetic diversity and heterosis across ploidy. Though a large proportion (0.47) of combinations showed positive values, the average fixed heterosis was low for seed yield but high for biomass yield. The genetic diversity was a significant contributor to fixed heterosis for biomass yield, due possibly to adaptive advantage it may confer on de novo alloploids during evolution. Good general/specific combiners at diploid level did not necessarily produce good general/specific combiners at amphiploid level. It was also concluded that polyploidy impacts classical heterosis indirectly due to the negative association between fixed heterosis and classical heterosis.     

Comparative Evaluation of Five Culture Media with Triplex PCR Assay for Detection of Methicillin-Resistant Staphylococcus aureus

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) is responsible for nosocomial and community-acquired infections. Hence, rapid and accurate laboratory diagnosis of MRSA is a vital constituent of control measures. The present study evaluated five different methods for the identification of MRSA. A total of 207 S. aureus clinical isolates that consisted of 89 MRSA and 118 methicillin-susceptible S. aureus (MSSA) strains confirmed by PCR were tested. MRSA strains were evaluated by five different methods: chromogenic MRSA agar (CMRSA), oxacillin resistance screening agar base (ORSAB), mannitol salt oxacillin agar (MSO), mannitol salt cefoxitin agar with two different concentrations of cefoxitin [4 μg/ml (MSC-4) and 6 μg/ml (MSC-6)]. The results of the different methods were compared to mecA PCR as the gold standard. MSC-6 showed only six false-positive MRSA in comparison with PCR. The sensitivities and specificities of MSC-6, MSC-4, MSO-4, ORSAB, and CMRSA were as follows: 98.9/94.9%, 100/83.1%, 89.9/87.3%, 97.8/96.6%, and 95.5/94.9%, respectively. In comparison with PCR, it was found that both MSC-6 and ORSAB were relatively the least expensive screening tests (0.70 and0.70 and 1.00, respectively). In conclusion, all methods were comparable, but MSC-6 was the least expensive medium for MRSA screening.