Production of recombinant antigens in plants for animal and human immunization - a review

Plants present a cost effective production system for high value proteins. There is an increasing world demand for cheap vaccines that can be readily administered to the population, especially in economically less developed regions. A promising concept is the production of vaccines in plants that could be grown locally. Expression of antigenic peptides in the palatable parts of plants can lead to the production of edible active vaccines. Two major strategies are: i) to express antigens in transgenic plants, and ii) to produce antigenic peptides on the surface of plant viruses that could be used to infect host plants. This review considers the experimental data and early results for both strategies, and discusses the potential and problems of this new technology

Animal models of anxiety: an ethological perspective

In the field of anxiety research, animal models are used as screening tools in the search for compounds with therapeutic potential and as simulations for research on mechanisms underlying emotional behaviour. However, a solely pharmacological approach to the validation of such tests has resulted in distinct problems with their applicability to systems other than those involving the benzodiazepine/GABAA receptor complex. In this context, recent developments in our understanding of mammalian defensive behaviour have not only prompted the development of new models but also attempts to refine existing ones. The present review focuses on the application of ethological techniques to one of the most widely used animal models of anxiety, the elevated plus-maze paradigm. This fresh approach to an established test has revealed a hitherto unrecognized multidimensionality to plus-maze behaviour and, as it yields comprehensive behavioural profiles, has many advantages over conventional methodology. This assertion is supported by reference to recent work on the effects of diverse manipulations including psychosocial stress, benzodiazepines, GABA receptor ligands, neurosteroids, 5-HT1A receptor ligands, and panicolytic/panicogenic agents. On the basis of this review, it is suggested that other models of anxiety may well benefit from greater attention to behavioural detail

Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

We cloned the streptokinase (STK) gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a Km = 0.70 µM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing

The use of non-human primates as animal models for the study of hepatitis viruses

Hepatitis viruses belong to different families and have in common a striking hepatotropism and restrictions for propagation in cell culture. The transmissibility of hepatitis is in great part limited to non-human primates. Enterically transmitted hepatitis viruses (hepatitis A virus and hepatitis E virus) can induce hepatitis in a number of Old World and New World monkey species, while the host range of non-human primates susceptible to hepatitis viruses transmitted by the parenteral route (hepatitis B virus, hepatitis C virus and hepatitis delta virus) is restricted to few species of Old World monkeys, especially the chimpanzee. Experimental studies on non-human primates have provided an invaluable source of information regarding the biology and pathogenesis of these viruses, and represent a still indispensable tool for vaccine and drug testing.

Transgenic animal models for the functional analysis of vasoactive peptides

The interplay of vasoactive peptide systems is an essential determinant of blood pressure regulation in mammals. While the endothelin and the renin-angiotensin systems raise blood pressure by inducing vasoconstriction and sodium retention, the kallikrein-kinin and the natriuretic-peptide systems reduce arterial pressure by eliciting vasodilatation and natriuresis. Transgenic technology has proven to be very useful for the functional analysis of vasoactive peptide systems. As an outstanding example, transgenic rats overexpressing the mouse Ren-2 renin gene in several tissues become extremely hypertensive. Several other transgenic rat and mouse strains with genetic modifications of components of the renin-angiotensin system have been developed in the past decade. Moreover, in recent years gene-targeting technology was employed to produce mouse strains lacking these proteins. The established animal models as well as the main insights gained by their analysis are summarized in this review.

The relationship between anxiety and depression in animal models: a study using the forced swimming test and elevated plus-maze

The present study evaluated the correlation between the behavior of mice in the forced swimming test (FST) and in the elevated plus-maze (PM). The effect of the order of the experiments, i.e., the influence of the first test (FST or PM) on mouse behavior in the second test (PM or FST, respectively) was compared to handled animals (HAND). The execution of FST one week before the plus-maze (FST-PM, N = 10), in comparison to mice that were only handled (HAND-PM, N = 10) in week 1, decreased % open entries (HAND-PM: 33.6 ± 2.9; FST-PM: 20.0 ± 3.9; mean ± SEM; P<0.02) and % open time (HAND-PM: 18.9 ± 3.3; FST-PM: 9.0 ± 1.9; P<0.03), suggesting an anxiogenic effect. No significant effect was seen in the number of closed arm entries (FST-PM: 9.5 (7.0-11.0); HAND-PM: 10.0 (4.0-14.5), median (interquartile range); U = 46.5; P>0.10). A prior test in the plus-maze (PM-FST) did not change % immobility time in the FST when compared to the HAND-FST group (HAND-FST: 57.7 ± 3.9; PM-FST: 65.7 ± 3.2; mean ± SEM; P>0.10). Since these data suggest that there is an order effect, the correlation was evaluated separately with each test sequence: FST-PM (N = 20) and PM-FST (N = 18). There was no significant correlation between % immobility time in the FST and plus-maze indexes (% time and entries in open arms) in any test sequence (r: -0.07 to 0.18). These data suggest that mouse behavior in the elevated plus-maze is not related to behavior in the forced swimming test and that a forced swimming test before the plus-maze has an anxiogenic effect even after a one-week interval.

Cloning and characterization of Echinococcus granulosus (Cestode) EgactI and EgactII actin gene promoters and their functional analysis in the NIH3T3 mouse cell line

We report here for the first time the structure and function of a promoter from a cestode. The ability of DNA fragments respectively encompassing the 935-bp and 524-bp regions upstream from the ATG codon from the EgactI and EgactII actin genes of Echinococcus granulosus to promote transcription was studied in the NIH3T3 mouse cell line. The results of transfection assays showed that both regions have strong promoter activity in these cells. The fragments were tested in both orientations and the 524-bp fragment of EgactII presented a bidirectional promoter activity. Deletion analysis of EgactI and EgactII promoters indicated the presence of regulatory regions containing putative silencer elements. These results indicate that both EgactI and EgactII promoters are functional and that the preliminary functional evaluation of E. granulosus and possibly of other cestode promoters can be performed in heterologous cell lines.

The brain decade in debate: II. Panic or anxiety? From animal models to a neurobiological basis

This article is a transcription of an electronic symposium sponsored by the Brazilian Society of Neuroscience and Behavior (SBNeC). Invited researchers from the European Union, North America and Brazil discussed two issues on anxiety, namely whether panic is a very intense anxiety or something else, and what aspects of clinical anxiety are reproduced by animal models. Concerning the first issue, most participants agreed that generalized anxiety and panic disorder are different on the basis of clinical manifestations, drug response and animal models. Also, underlying brain structures, neurotransmitter modulation and hormonal changes seem to involve important differences. It is also common knowledge that existing animal models generate different types of fear/anxiety. A challenge for future research is to establish a good correlation between animal models and nosological classification.

Head-to-head comparison of dipyridamole, dobutamine and pacing stress echocardiography for the detection of myocardial ischemia in an animal model of coronary artery stenosis

To compare the sensitivity of dipyridamole, dobutamine and pacing stress echocardiography for the detection of myocardial ischemia we produced a physiologically significant stenosis in the left circumflex artery of 14 open-chest dogs (range: 50 to 89% reduction in luminal diameter). In each study, dobutamine (5 to 40 µg kg-1 min-1 in 3-min stages) and pacing (20 bpm increments, each 2 min, up to 260 bpm) were performed randomly, and then followed by dipyridamole (up to 0.84 mg/kg over 10 min). The positivity of stress echocardiography tests was quantitatively determined by a significant (P<0.05) reduction of or failure to increase absolute and percent systolic wall thickening in the stenotic artery supplied wall, as compared to the opposite wall (areas related to the left anterior descending artery). Systolic and diastolic frozen images were analyzed off-line by two blinded observers in the control and stress conditions. The results showed that 1) the sensitivity of dobutamine, dipyridamole and pacing stress tests was 57, 57 and 36%, respectively; 2) in animals with positive tests, the mean percent change of wall thickening in left ventricular ischemic segments was larger in the pacing (-19 ± 11%) and dipyridamole (-18 ± 16%) tests as compared to dobutamine (-9 ± 6%) (P = 0.05), but a similar mean reduction of wall thickening was observed when this variable was normalized to a control left ventricular segment (area related to the left anterior descending artery) (pacing: -16 ± 7%; dipyridamole: -25 ± 16%; dobutamine: -26 ± 10%; not significant), and 3) a significant correlation was observed between magnitude of coronary stenosis and left ventricular segmental dysfunction induced by ischemia in dogs submitted to positive stress tests. We conclude that the dobutamine and dipyridamole stress tests showed identical sensitivities for the detection of myocardial ischemia in this one-vessel disease animal model with a wide range of left circumflex artery stenosis. The pacing stress test was less sensitive, but the difference was not statistically significant. The magnitude of segmental left ventricular dysfunction induced by ischemia was similar in all stress tests evaluated.

Can organic and transgenic soy be used as a substitute for animal protein by rats?

We evaluated the protein quality of organic and transgenic soy fed to rats throughout life. Thirty female Wistar rats were divided into three groups (N = 10): organic soy group (OSG) receiving organic soy-based diet, genetically modified soy group (GMSG) receiving transgenic soy-based diet, and a control group (CG) receiving casein-based diet. All animals received water and isocaloric diet (10% protein), ad libitum for 291 days. After this, the weight of GMSG animals (290.9 ± 9.1 g) was significantly lower (P <= 0.04) than CG (323.2 ± 7.9 g). The weight of OSG (302.2 ± 8.7 g) was between that of the GMSG and the CG. Protein intake was similar for OSG (308.4 ± 6.8 g) and GMSG (301.5 ± 2.5 g), and significantly lower (P <= 0.0005) than the CG (358.4 ± 8.1 g). Growth rate was similar for all groups: OSG (0.80 ± 0.02 g), GMSG (0.81 ± 0.03 g) and CG (0.75 ± 0.02 g). In addition to providing a good protein intake and inducing less weight gain, both types of soy were utilized in a manner similar to that of casein, suggesting that the protein quality of soy is similar to that of the standard protein casein. The groups fed soy-based diet gained less weight, which may be considered to be beneficial for health. We conclude that organic and transgenic soy can be fed throughout life to rats in place of animal protein, because contain high quality protein and do not cause a marked increase in body weight.

Bone morphogenetic proteins: from structure to clinical use

Bone morphogenetic proteins (BMPs) are multi-functional growth factors belonging to the transforming growth factor ß superfamily. Family members are expressed during limb development, endochondral ossification, early fracture, and cartilage repair. The activity of BMPs was first identified in the 1960s but the proteins responsible for bone induction were unknown until the purification and cloning of human BMPs in the 1980s. To date, about 15 BMP family members have been identified and characterized. The signal triggered by BMPs is transduced through serine/threonine kinase receptors, type I and II subtypes. Three type I receptors have been shown to bind BMP ligands, namely: type IA and IB BMP receptors and type IA activin receptors. BMPs seem to be involved in the regulation of cell proliferation, survival, differentiation and apoptosis, but their hallmark is their ability to induce bone, cartilage, ligament, and tendon formation at both heterotopic and orthotopic sites. This suggests that, in the future, they may play a major role in the treatment of bone diseases. Several animal studies have illustrated the potential of BMPs to enhance spinal fusion, repair critical-size defects, accelerate union, and heal articular cartilage lesions. Difficulties in producing and purifying BMPs from bone tissue have prompted the attempts made by several laboratories, including ours, to express these proteins in the recombinant form in heterologous systems. This review focuses on BMP structure, molecular mechanisms of action and significance and potential applications in medical, dental and veterinary practice for the treatment of cartilage and bone-related diseases.

Effect of Erythrina velutina and Erythrina mulungu in rats submitted to animal models of anxiety and depression

Erythrina velutina (EV) and Erythrina mulungu (EM), popularly used in Brazil as tranquilizing agents, were studied. The effects of acute and chronic oral treatment with a water:alcohol extract of EV (7:3, plant grounded stem bark; acute = 100, 200, 400 mg/kg; chronic = 50, 100, 200 mg/kg) were evaluated in rats (N = 11-12) submitted to the elevated T-maze (for avoidance and escape measurements) model of anxiety. This model was selected for its presumed capacity to elicit specific subtypes of anxiety disorders recognized in clinical practice: avoidance has been related to generalized anxiety and escape to panic. Additionally, animals were treated with the same doses of EV and EM (water:alcohol 7:3, inflorescence extract) and submitted to the forced swim test for the evaluation of antidepressant activity (N = 7-10). Both treatment regimens with EV impaired elevated T-maze avoidance latencies, without altering escape, in a way similar to the reference drug diazepam (avoidance 1, mean ± SEM, acute study: 131.1 ± 45.5 (control), 9.0 ± 3.3 (diazepam), 12.7 ± 2.9 (200 mg/kg), 28.8 ± 15.3 (400 mg/kg); chronic study: 131.7 ± 46.9 (control), 35.8 ± 29.7 (diazepam), 24.4 ± 10.4 (50 mg/kg), 29.7 ± 11.5 (200 mg/kg)). Neither EV nor EM altered measurements performed in the forced swim test, in contrast to the reference drug imipramine that significantly decreased immobility time after chronic treatment. These results were not due to motor alterations since no significant effects were detected in an open field. These observations suggest that EV exerts anxiolytic-like effects on a specific subset of defensive behaviors which have been associated with generalized anxiety disorder.

Inducing mutations in the mouse genome with the chemical mutagen ethylnitrosourea

When compared to other model organisms whose genome is sequenced, the number of mutations identified in the mouse appears extremely reduced and this situation seriously hampers our understanding of mammalian gene function(s). Another important consequence of this shortage is that a majority of human genetic diseases still await an animal model. To improve the situation, two strategies are currently used: the first makes use of embryonic stem cells, in which one can induce knockout mutations almost at will; the second consists of a genome-wide random chemical mutagenesis, followed by screening for mutant phenotypes and subsequent identification of the genetic alteration(s). Several projects are now in progress making use of one or the other of these strategies. Here, we report an original effort where we mutagenized BALB/c males, with the mutagen ethylnitrosourea. Offspring of these males were screened for dominant mutations and a three-generation breeding protocol was set to recover recessive mutations. Eleven mutations were identified (one dominant and ten recessives). Three of these mutations are new alleles (Otop1mlh, Foxn1sepe and probably rodador) at loci where mutations have already been reported, while 4 are new and original alleles (carc, eqlb, frqz, and Sacc). This result indicates that the mouse genome, as expected, is far from being saturated with mutations. More mutations would certainly be discovered using more sophisticated phenotyping protocols. Seven of the 11 new mutant alleles induced in our experiment have been localized on the genetic map as a first step towards positional cloning.

Cloning, sequence analysis, and expression of cDNA coding for the major house dust mite allergen, Der f 1, in Escherichia coli

Our objective was to clone, express and characterize adult Dermatophagoides farinae group 1 (Der f 1) allergens to further produce recombinant allergens for future clinical applications in order to eliminate side reactions from crude extracts of mites. Based on GenBank data, we designed primers and amplified the cDNA fragment coding for Der f 1 by nested-PCR. After purification and recovery, the cDNA fragment was cloned into the pMD19-T vector. The fragment was then sequenced, subcloned into the plasmid pET28a(+), expressed in Escherichia coli BL21 and identified by Western blotting. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Sequence analysis showed the presence of an open reading frame containing 966 bp that encodes a protein of 321 amino acids. Interestingly, homology analysis showed that the Der p 1 shared more than 87% identity in amino acid sequence with Eur m 1 but only 80% with Der f 1. Furthermore, phylogenetic analyses suggested that D. pteronyssinus was evolutionarily closer to Euroglyphus maynei than to D. farinae, even though D. pteronyssinus and D. farinae belong to the same Dermatophagoides genus. A total of three cysteine peptidase active sites were found in the predicted amino acid sequence, including 127-138 (QGGCGSCWAFSG), 267-277 (NYHAVNIVGYG) and 284-303 (YWIVRNSWDTTWGDSGYGYF). Moreover, secondary structure analysis revealed that Der f 1 contained an a helix (33.96%), an extended strand (17.13%), a ß turn (5.61%), and a random coil (43.30%). A simple three-dimensional model of this protein was constructed using a Swiss-model server. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Alignment and phylogenetic analysis suggests that D. pteronyssinus is evolutionarily more similar to E. maynei than to D. farinae.

Cloning, purification and comparative characterization of two digestive lysozymes from Musca domestica larvae

cDNA coding for two digestive lysozymes (MdL1 and MdL2) of the Musca domestica housefly was cloned and sequenced. MdL2 is a novel minor lysozyme, whereas MdL1 is the major lysozyme thus far purified from M. domestica midgut. MdL1 and MdL2 were expressed as recombinant proteins in Pichia pastoris, purified and characterized. The lytic activities of MdL1 and MdL2 upon Micrococcus lysodeikticus have an acidic pH optimum (4.8) at low ionic strength (μ = 0.02), which shifts towards an even more acidic value, pH 3.8, at a high ionic strength (μ = 0.2). However, the pH optimum of their activities upon 4-methylumbelliferyl N-acetylchitotrioside (4.9) is not affected by ionic strength. These results suggest that the acidic pH optimum is an intrinsic property of MdL1 and MdL2, whereas pH optimum shifts are an effect of the ionic strength on the negatively charged bacterial wall. MdL2 affinity for bacterial cell wall is lower than that of MdL1. Differences in isoelectric point (pI) indicate that MdL2 (pI = 6.7) is less positively charged than MdL1 (pI = 7.7) at their pH optima, which suggests that electrostatic interactions might be involved in substrate binding. In agreement with that finding, MdL1 and MdL2 affinities for bacterial cell wall decrease as ionic strength increases.