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Results for “recombinant form”

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PubMed2006

Expression of a fluorescent recombinant form of sperm protein phospholipase C zeta in mouse epididymal sperm by in vivo gene transfer into the testis.

OBJECTIVE: To use in vivo gene transfer into the testis by electroporation to express a fluorescent recombinant form of a testis-specific gene in the mature epididymal sperm of mice and thus study the pattern of gene localization. DESIGN: Controlled animal study. SETTING: Research laboratory at the University of Oxford. ANIMAL(S): Four- to 6-week-old male mice. INTERVENTION(S): Phospholipase C zeta (PLCzeta), the putative mammalian egg activation factor, was fused to enhanced yellow fluorescent protein (EYFP), and in vivo gene transfer by electroporation was used to introduce this transgene (PLCzeta-EYFP) into mouse testis. Transgene expression in testis and sperm were analyzed at 20 and 40 days after electroporation. MAIN OUTCOME MEASURE(S): Transgene expression in testis and epididymal sperm was analyzed by fluorescence microscopy and an excitation light source suitable for EYFP. RESULT(S): Phospholipase C zeta-EYFP was successfully expressed in epididymal sperm when analyzed 40 days after gene transfer and was localized to the head and midpiece regions. CONCLUSION(S): Our results provide the first demonstration that in vivo gene transfer can be used to study the localization of proteins in mature sperm and that this represents a powerful new technique for studying male infertility and gene function in sperm.

Animals↗

PubMed2006

Characterization of a new circulating recombinant form comprising HIV-1 subtypes C and B in southern Brazil.

OBJECTIVE: To identify a new circulating recombinant form (CRF) of HIV-1 comprising two circulating subtypes in the southern region in Brazil, subtypes B and C. METHODS: A total of 152 HIV-positive patients followed at two hospitals in southern Brazil had their viral pol genes isolated by reverse transcriptase-polymerase chain reaction (PCR) from plasma. PCR products were sequenced and phylogenetically analysed using HIV-1 subtype reference sequences. Six full-length subtype C viruses from Brazil previously described as 'pure' strains were included in the analysis. Sequences suggestive of recombination were analysed by boot scanning and phylogenetic analyses of separate fragments. The common ancestry of recombinant strains was evaluated by similarity plot and informative site analyses. RESULTS: : HIV-1 subtypes commonly found in Brazil (B, C and F1) were observed. Sixty-two viruses were initially assigned as subtype C, but 15 viruses clustered in a separate internal clade. Pol from two full-length genomes of subtype C viruses grouped together with those samples. Boot scanning analysis showed that all 17 viruses had the same recombinant structure, with a 240 base pair fragment of subtype B in the middle of the reverse transcriptase pol region. Subtype B assignment of this fragment was confirmed by phylogenetic analyses using different methods of tree inference and cluster robustness tests. Mosaics were shown to have a common ancestry. CONCLUSION: As CRF_BC represents 11% of the HIV-1 viruses circulating in the southern region of the country, which borders several south American countries, the assessment of its spread is of pivotal importance to the HIV/AIDS epidemic in Brazil and Latin America.

Base Sequence↗

PubMed1999

A recombinant form of the catalytic subunit of phosphorylase kinase that is soluble, monomeric, and includes key C-terminal residues.

Residues 302-326 of the catalytic (gamma) subunit of phosphorylase kinase (PhK) may comprise an autoinhibitory, pseudosubstrate domain that binds calmodulin. To study this, the cDNA corresponding to rabbit muscle PhKgamma was expressed using Escherichia coli. This yielded two stable, high-activity PhKgamma forms (35 and 42 kDa by SDS-PAGE) that were smaller than an authentic sample of rabbit muscle PhKgamma (45 kDa by SDS-PAGE). Each recombinant form was purified to homogeneity. The N-terminal sequence of the larger, 42-kDa form (pk42) matched that of the rabbit muscle enzyme. This suggested that pk42 consisted of PhKgamma residues 1-362, including the putative calmodulin-binding, autoinhibitory domain. Kinetic parameters obtained for pk42 were like those previously reported for the intact gamma subunit. This implied that the lack of 25 PhKgamma C-terminal residues did not affect phosphorylase kinase activity, but greatly improved enzyme stability. An additional 60 residues were removed from the C-terminus of pk42 using the protease m-calpain. This increased the kinase activity 1.5-fold. Consistent with this, the activity of a mutant PhKgamma that consisted of residues 1-300, denoted gamma1-300, was like that of the m-calpain-treated enzyme. Therefore, although the effect was small, some influence by the C-terminus of pk42 was noted. Moreover, when pk42 was incubated with ATP alone, a C-terminal threonine residue became phosphorylated. Although the influence of this autophosphorylation cannot be inferred from this data, it was evidence that the C-terminus accessed the enzyme's active site. Taken together, these data imply that pk42 will be useful to study phosphorylase kinase structure/activity relationships.

Adenosine Triphosphate↗

PubMed2001

One-tube real-time isothermal amplification assay to identify and distinguish human immunodeficiency virus type 1 subtypes A, B, and C and circulating recombinant forms AE and AG.

To halt the human immunodeficiency virus type 1 (HIV-1) epidemic requires interventions that can prevent transmission of numerous HIV-1 subtypes. The most frequently transmitted viruses belong to the subtypes A, B, and C and the circulating recombinant forms (CRFs) AE and AG. A fast one-tube assay that identifies and distinguishes among subtypes A, B, and C and CRFs AE and AG of HIV-1 was developed. The assay amplifies a part of the gag gene sequence of the genome of all currently known HIV-1 subtypes and can identify and distinguish among the targeted subtypes as the reaction proceeds, because of the addition of subtype-specific molecular beacons with multiple fluorophores. The combination of isothermal nucleic acid sequence-based amplification and molecular beacons is a new approach in the design of real-time assays. To obtain a sufficiently specific assay, we developed a new strategy in the design of molecular beacons, purposely introducing mismatches in the molecular beacons. The subtype A and CRF AG isolates reacted with the same molecular beacon. We tested the specificity and sensitivity of the assay on a panel of the culture supernatant of 34 viruses encompassing all HIV-1 subtypes: subtypes A through G, CRF AE and AG, a group O isolate, and a group N isolate. Assay sensitivity on this panel was 92%, with 89% correct subtype identification relative to sequence analysis. A linear relationship was found between the amount of input RNA in the reaction mixture and the time that the reaction became positive. The lower detection level of the assay was approximately 10(3) copies of HIV-1 RNA per reaction. In 38% of 50 serum samples from HIV-1-infected individuals with a detectable amount of virus, we could identify subtype sequences with a specificity of 94% by using sequencing and phylogenetic analysis as the "gold standard." In conclusion, we showed the feasibility of the approach of using multiple molecular beacons labeled with different fluorophores in combination with isothermal amplification to identify and distinguish subtypes A, B, and C and CRFs AE and AG of HIV-1. Because of the low sensitivity, the assay in this format would not be suited for clinical use but can possibly be used for epidemiological monitoring as well as vaccine research studies.

Base Sequence↗

PubMed2006

Circulating and unique recombinant forms of HIV type 1 containing subsubtype A2.

HIV-1 strains containing subsubtype A2 are relatively rare in the pandemic but have been repeatedly identified in Kenya, where candidate vaccines based in part on subtype A, but not A2 strains, may be evaluated. Among the most recent is CRF16_A2D, a circulating recombinant form (CRF) whose prototypes are complete or partial HIV-1 sequences from Kenya, Korea, and Argentina. Using samples from blood bank discards in Kenya and complete genome sequencing, this report further documents CRF16_A2D and related recombinants and identifies a second CRF, CRF21_A2D. The two A2-containing CRFs, and two recombinants related to CRF16_A2D, share common structural elements but appear to have been independently derived. Concerted selection may have influenced the emergence and spread of certain A2-containing strains in Kenya. The second complete subtype C sequence from Kenya is also reported here. Monitoring of A2-containing recombinants and subtype C strains, both relatively rare in Kenya, may be informative in the course of cohort development and evaluation of candidate vaccines.

Genome, Viral↗

PubMed2002

No difference in clinical progression between patients infected with the predominant human immunodeficiency virus type 1 circulating recombinant form (CRF) 02_AG strain and patients not infected with CRF02_AG, in Western and West-Central Africa: a four-year prospective multicenter study.

To compare human immunodeficiency virus (HIV) type 1 disease progression in patients infected by the predominant strain circulating recombinant form (CRF) 02_AG in western and west-central Africa and in patients infected by other strains, a prospective multicenter cohort study was conducted in Cameroon and Senegal. Among the 335 patients, a broad HIV-1 group M subtype diversity was observed in the envelope V3-V5 region, but strain CRF02_AG predominated in both Cameroon and Senegal (61.2% and 62.9%, respectively; P<.8). Multivariate analyses showed no difference between patients infected by CRF02 strains and those infected by other strains in terms of survival (adjusted hazards ratio [HR], 1.16; 95% confidence interval [CI], 0.76-1.78; P=.5), clinical disease progression (HR, 0.79; 95% CI, 0.50-1.25; P=.3), or square root CD4 cell decline (regression coefficient, -0.01; 95% CI, -0.82 to 0.81; P=.9). This study suggests that the predominance of HIV-1 CRF02_AG strain in western and west-central Africa should have no major clinical consequences.

Adult↗

PubMed2002

CRF06-cpx: a new circulating recombinant form of HIV-1 in West Africa involving subtypes A, G, K, and J.

Phylogenetic analysis of numerous strains of HIV-1 isolated from diverse geographic origins has revealed three distinct groups of HIV-1: groups M, N, and O. Within group M, subtypes, sub-subtypes and circulating recombinant forms (CRFs) exist. Recently, two near-full-length genomes of similar complex mosaic viruses containing fragments of subtypes A, G, I, and J were described in patients from Burkina Faso (BFP-90) and Mali (95ML-84). Here, we report on the characterization of two additional full-length genome sequences with similar mosaic structure in epidemiologically unlinked individuals from Senegal (97SE-1078) and Mali (95ML-127). Phylogenetic and recombinant analysis confirmed that the previously described strains, BFP-90 and 95ML-84, were indeed a new CRF of HIV-1, which we can now designate as CRF06-cpx. This new CRF fits the complex (cpx) designation, because four different subtypes (A, G, K, and J) were involved in the mosaic genome structure. The fragment in the pol gene, which was initially characterized as unknown in the BFP-90 strain and subsequently as subtype I in the 95ML-84 strain, is now, with the recent description of the new K subtype, clearly identified as subtype K. CRF06-cpx circulates in Senegal, Mali, Burkina Faso, Ivory Coast, and Nigeria, although the exact prevalence remains to be determined. Importantly, this new variant has also been documented on other continents (Europe [France] and Australia), showing that these viruses are spreading not only locally but globally.

Africa, Western↗

PubMed1996

Response of digestive cysteine proteinases from the Colorado potato beetle (Leptinotarsa decemlineata) and the black vine weevil (Otiorynchus sulcatus) to a recombinant form of human stefin A.

The effects of the cystatins, human stefin A (HSA) and oryzacystatin I (OCI) on digestive cysteine proteinases of the Colorado potato beetle (CPB), Leptinotarsa decemlineata, and the black vine weevil (BVW), Otiorynchus sulcatus, were assessed using complementary inhibition assays, cystatin-affinity chromatography, and recombinant forms of the two inhibitors. For both insects, either HSA and OCI used in excess (10 or 20 microM) caused partial and stable inhibition of total proteolytic (azocaseinase) activity, but unlike for OCI the HSA-mediated inhibitions were significantly increased when the inhibitor was used in large excess (100 microM). As demonstrated by complementary inhibition assays, this two-step inhibition of the insect proteases by HSA was due to the differential inactivation of two distinct cysteine proteinase populations in either insect extracts, the rapidly (strongly) inhibited population corresponding to the OCI-sensitive fraction. After removing the cystatin-sensitive proteinases from CPB and BVW midgut extracts using OCI- (or HSA-) affinity chromatography, the effects of the insect "non-target" proteases on the structural integrity of the two cystatins were assessed. While OCI remained essentially stable, HSA was subjected to hydrolysis without the accumulation of detectable stable intermediates, suggesting the presence of multiple exposed cleavage sites sensitive to the action of the insect proteases on this cystatin. This apparent susceptibility of HSA to proteolytic cleavage may partially explain its low efficiency to inactivate the insect OCI-insensitive cysteine proteinases when not used in large excess. It could also have major implications when planning the use of cystatin-expressing transgenic plants for the control of coleopteran pests.

Animals↗

PubMed2001

Biologically active recombinant forms of a major house dust mite group 1 allergen Der f 1 with full activities of both cysteine protease and IgE binding.

BACKGROUND: Group 1 allergens from mite faeces, Der f 1 and Der p 1, are the most significant in-door allergens. Therefore, they are the most important component in the standardization of house dust mite extract for diagnosis and allergen-specific immunotherapy (AIT). Although their cDNAs have been cloned, efforts to prepare biologically active recombinant forms in expression systems using bacteria or yeast have failed. OBJECTIVE: Our purpose is to establish an efficient system to prepare recombinant Der f 1(rDer f 1), identical in quality to native Der f 1. METHODS: The preproforms of Der f 1 and a mutant N53Q, whose consensus motif for N-glycosylation was disrupted, were expressed in yeast Pichia pastoris. Cysteine protease activity and IgE reactivity were analysed using synthetic substrates and by RAST-EIA, respectively. RESULTS: The proforms of the two rDer f 1 molecules were efficiently secreted into culture medium. Their prosequences were removed autocatalytically by dialysis against acidic buffer. Although the wild-type rDer f 1 was more highly glycosylated than native Der f 1, N53Q had almost the same apparent molecular weight as native Der f 1 on SDS-PAGE. Both the protease and IgE binding activities of the mature rDer f 1 molecules were the same as those of native Der f 1, whereas the proforms had no or markedly reduced activities. CONCLUSION: The efficient system to prepare active rDer f 1s established in this study is useful for diagnosis and standardized AIT for house dust mite allergy. Furthermore, the system would be a tool for analysis of IgE epitopes, determination of tertiary structure, allergen engineering for safer and more effective AIT, resolving the relation between the enzymatic activity and pathogenesis, and the development of therapeutic inhibitors.

Animals↗

PubMed1997

Redefinition of the carbohydrate specificity of Erythrina corallodendron lectin based on solid-phase binding assays and molecular modeling of native and recombinant forms obtained by site-directed mutagenesis.

Binding of the N-acetyllactosamine-specific lectin from Erythrina corallodendron (ECorL) to four glycosphingolipids has been tested using the microtiter well assay. The role of several amino acids in the binding site region was studied by combining binding assays and molecular modeling for native and recombinant forms of the lectin. Seven single-point mutants at positions 106 (Y106A), 108 (Y108A, T), 218 (A218G), and 219 (Q219A, N or E) were investigated. A comparison with more than 30 known sequences of legume lectins showed that ECorL is unique in displaying a tyrosine residue or a structural equivalent at position 106. Analyses of the binding results obtained for mutants at positions 106 and 108 using molecular modeling point to complex conformational dependencies between these and several other residues around the binding site. Gln 219 was found to have a large conformational flexibility, which, paradoxically, favors the binding of N-acetyllactosamine-containing glycosphingolipids. Particularly significant is the fact that ECorL exhibits a higher affinity for Fuc alpha2Gal beta4GlcNAc beta-terminated glycosphingolipids than N-acetyllactosamine-terminated ones, in accordance with molecular modeling revealing a perfect fit of the alpha2-linked fucose in a cavity extending from the Gal beta4 binding pocket. These findings lead to a redefinition of the specificity of this lectin, where the affinity for the terminal Fuc alpha2Gal beta4GlcNAc beta trisaccharide should be considered in the first place. The possible biological significance of this specificity remains to be investigated.

Adsorption↗

PubMed2005

Identification of attenuated variants of HIV-1 circulating recombinant form 01_AE that are associated with slow disease progression due to gross genetic alterations in the nef/long terminal repeat sequences.

We identified an unusual case of human immunodeficiency virus type 1 (HIV-1) infection in a patient (GM43) who exhibited a persistently low antibody response and undetectable viral load during a 5-year follow-up period. GM43 harbored HIV-1 circulating recombinant form 01_AE with gross deletions in the nef/long terminal repeat (LTR) region. The sizes of the deletions increased progressively from 84 to >400 bp during the 5-year period. GM43 appeared to have acquired defective variants from her husband. The genetic alterations in the nef/LTR region were remarkably similar to those that have been reported in slow progressors (such as the slow progressors in the Sydney Blood Bank Cohort). The present study is the first report of slow disease progression due to gross genetic alterations in the nef/LTR region in a person infected with an HIV-1 non-subtype B strain.

Adult↗

PubMed2006

Isolation of the main allergen Fra e 1 from ash (Fraxinus excelsior) pollen: comparison of the natural and recombinant forms.

BACKGROUND: Fra e 1 is a major allergen for ash pollen-sensitized individuals in northern and central Europe. It belongs to the Ole e 1-like family and displays high cross-reactivity with taxonomically related members. OBJECTIVES: To isolate and characterize natural Fra e 1 (nFra e 1) from ash pollen and to compare its structural, antigenic, and allergenic properties with those of its recombinant form (rFra e 1). METHODS: The allergen was isolated by means of gel permeation chromatography and reverse-phase high-performance liquid chromatography columns. Molecular characterization was performed by means of Edman degradation, mass spectrometry, circular dichroism, concanavalin A lectin reaction, and anti-horseradish peroxidase polyclonal antibody. Immunologic characterization was performed using immunoblotting and enzyme-linked immunosorbent assay, inhibition experiments, and histamine release assays with serum samples from allergic patients with well-known reactivity to Fra e 1 or Ole e 1 and with polyclonal antiserum and monoclonal antibodies against Ole e 1. The protein used as a reference was rFra e 1, which was produced in the yeast Pichia pastoris. RESULTS: Purified nFra e 1 appeared as 5 variants with different glycosylation degrees. Both nFra e 1 and rFra e 1 were equivalently folded as deduced from the spectroscopic analysis using circular dichroism. Both molecules share the antigenic and allergenic epitopes after the purification process, and the glycan group of nFra e 1 is a potential epitope. Natural Fra e 1 displayed strong cross-reactivity with Ole e 1. CONCLUSIONS: Natural Fra e 1 is a heterogeneously glycosylated protein with high allergenic relevance. It displays structural, antigenic, and allergenic similarity with rFra e 1. Both proteins could be used for clinical purposes.

Allergens↗

PubMed2002

Expression and purification of two recombinant forms of the type-III cytotoxin, Pseudomonas aeruginosa ExoS.

Pseudomonas aeruginosa Exoenzyme S (ExoS) is a bifunctional type-III cytotoxin. The N-terminus (residues 1-232) possesses Rho GTPase-activating (GAP) activity, while the C-terminus (residues 233-453) comprises an ADP-ribosyltransferase domain. Amino acid residues 51-72 of ExoS are involved in membrane binding and aggregation, which has complicated purification schemes. Here, it is reported on the expression, purification, and characterization of two recombinant forms of ExoS that lack this membrane-binding domain, designated rExoS78-453 and rExoSdelta51-72. Purification of these forms was achieved using sequential NTA/Ni(2+)-affinity, gel filtration, and anion-exchange chromatography. Both forms of ExoS possessed Rho GAP activity and ADP-ribosyltransferase activity comparable to wild-type ExoS. Mass spectrometry showed that rExoS78-453 and rExoSdelta51-72 had molecular masses similar to their predicted molecular masses.

ADP Ribose Transferases↗

PubMed2001

Comparison of the specificity, stability and individual rate constants with respective activation parameters for the peptidase activity of cruzipain and its recombinant form, cruzain, from Trypanosoma cruzi.

The Trypanosoma cruzi cysteine protease cruzipain contains a 130-amino-acid C-terminal extension, in addition to the catalytic domain. Natural cruzipain is a complex of isoforms, because of the simultaneous expression of several genes, and the presence of either high mannose-type, hybrid monoantennary-type or complex biantenary-type oligosacharide chains at Asn255 of the C-terminal extension. Cruzipain and its recombinant form without this extension (cruzain) were studied comparatively in this work. S2 to S2' subsite specificities of these enzymes were examined using four series of substrates derived from the internally quenched fluorescent peptide Abz-KLRFSKQ-EDDnp (Abz, ortho-aminobenzoic acid; EDDnp, N-(2,4-dinitrophenyl)-ethylenediamine). Large differences in the kinetic parameters were not observed between the enzymes; however, Km values were consistently lower for the hydrolysis of most of the substrates by cruzain. No difference in the pH-activity profile between the two enzymes was found, but in 1 m NaCl cruzipain presented a kcat value significantly higher than that of cruzain. The activation energy of denaturation for the enzymes did not differ significantly; however, a negative entropy value was observed for cruzipain denaturation whereas the value for cruzain was positive. We determined the individual rate constants (k1, substrate diffusion; k-1, substrate dissociation; k2, acylation; k3, deacylation) and the respective activation energies and entropies for hydrolysis of Abz-KLRFSKQ-EDDnp determining the temperature dependence of the Michaelis-Menten parameters kcat/Km and kcat as previously described [Ayala, Y.M. & Di Cera, E. (2000) Protein Sci. 9, 1589-1593]. Differences between the two enzymes were clearly detected in the activation energies E1 and E-1, which are significantly higher for cruzipain. The corresponding DeltaS1 and DeltaS-1 were positive and significantly higher for cruzipain than for cruzain. These results indicate the presence of a larger energy barrier for cruzipain relating to substrate diffusion and dissociation, which could be related to the C-terminal extension and/or glycosylation state of cruzipain.

Amino Acid Sequence↗

PubMed2002

Delta tryptase is expressed in multiple human tissues, and a recombinant form has proteolytic activity.

Tryptases are neutral serine proteases selectively expressed in mast cells and have been implicated in the development of a number of inflammatory diseases including asthma. It has recently been established that the number of genes encoding human mast cell tryptases is much larger than originally believed, but it is not clear how many of these genes are expressed. A recent report suggested that the transcript for at least one of these genes, originally named mMCP-7-like tryptase, is not expressed. To further address this question, we screened tissue-specific RNA samples by RT-PCR, using primers designed to match the putative exonic sequence of this gene. We successfully generated and cloned the correctly sized RT-PCR product from mRNA isolated from the human mast cell-I cell line. Two distinct clones were identified whose nucleotide sequence matched the published sequence of the mMCP-7-like I and mMCP-7-like II genes. Transcripts were detected in a wide variety of human tissues including lung, heart, stomach, spleen, skin, and colon. A polyclonal antipeptide Ab that specifically recognizes the translated product of this transcript was used to demonstrate its expression in mast cells that reside in the colon, lung, and inflamed synovium. A recombinant form of this protein expressed in bacterial cells was able to cleave a synthetic trypsin-sensitive substrate, D-Ile-Phe-Lys pNA. These results suggest that the range of functional tryptases is larger than previously recognized. For simplicity, we suggest that the gene, transcripts, and corresponding protein product be named delta tryptase.

Adult↗

PubMed2002

Development and evaluation of a DNA enzyme immunoassay method for env genotyping of subtypes A through G of human immunodeficiency virus type 1 group M, with discrimination of the circulating recombinant forms CRF01_AE and CRF02_AG.

The tools currently available for genetic subtyping of human immunodeficiency virus type 1 are laborious or can be used only for the analysis of a limited number of samples and/or subtypes. We developed and evaluated a molecular biology-based method using subtype-specific oligonucleotide probes for env genotyping of subtypes A through G, CRF01_AE, and CRF02_AG. DNA enzyme immunoassay (DEIA) genotyping is based on nested PCR amplification of the 5' end of the env gene (proviral DNA), followed by subtype-specific hybridization and immunoenzymatic detection on microplates. DEIA genotyping was validated with a large number of samples (n = 128) collected in Europe (France; n = 47), West-Central Africa (Cameroon; n = 36), and West Africa (Senegal; n = 45). Three different formats, depending on the distribution of subtypes in the three countries, were developed. The results were compared with those obtained by sequencing of the V3-V5 region and phylogenetic analysis or an env heteroduplex mobility assay. Additional sequencing and phylogenetic analyses of the DEIA region (the first codon of the env coding sequence to the middle of conserved region C1 of gp120) were performed to investigate the reasons for discrepancies. Intense and highly specific reactions between the oligonucleotide probes and the corresponding samples were observed. Overall, correct identification was achieved for 107 of 128 samples (83.6%). One sample was not amplified, 10 (8%) were nontypeable (NT), and 10 (8%) were misidentified. Six of the 10 discordant samples were further investigated by phylogenetic analysis, which indicated that these samples corresponded to recombinants involving the env 5' end and the V3 and V5 regions of the two parental clades. Sequencing of NT samples showed numerous differences between sample and probe sequences, resulting in a lack of hybridization, and revealed the limitations of the selected probes in terms of specificity and sensitivity. We demonstrated the feasibility of DEIA genotyping: six subtypes plus the two most prevalent circulating recombinant forms were discriminated by using the 5' end of the env gene. This method can be adapted to the local situation by including only probes that correspond to the prevalent strains.

DNA, Viral↗

PubMed1999

Selective modulation of phosphatidylserine exposure on subpopulations of human peripheral blood lymphocytes by a plant lectin, Viscum album agglutinin (VAA)-I and its recombinant form (rVAA) in vitro.

Growing evidence suggests that lectin-carbohydrate interactions are involved in the regulation of the balance between cell growth and programmed cell death. Viscum album agglutinin (VAA)-I is a galactoside-specific, type II ribosome-inactivating plant lectin. At concentrations less than 10 ng/ml, VAA-I has been shown to induce gene expression and secretion of proinflammatory cytokines as well as apoptosis in cultures of human peripheral blood mononuclear cells (PBMC). This study analyzes the effects of VAA-I and its recombinant nonglycosylated form (rVAA) on alterations of cell membrane permeability of cultured human peripheral lymphocytes (PBL) and on membrane exposure of phosphatidylserine characteristic of apoptosis. Analyses were performed by flow cytometry after staining with propidium iodide (PI) and/or with FITC-Annexin V/PI. After 24 h incubation of PBMC with 100 ng/ml VAA-I and rVAA, staining with supravital concentration of PI (20 microg/ml) for 1 h revealed no differences in percentages of PI-positive cells induced by the two forms of lectin (32.3% and 29.4%), but the exposure to 5 microg/ml PI for 15 min resulted in a significant difference: 35.1% and 8.0% after VAA-I and rVAA treatment, respectively. Kinetic analysis of membrane alterations showed mainly Annexin V positivity after 24 h, whereas after 48 h and 72 h incubation with 100 ng/ml VAA-I or rVAA loss of membrane integrity occurred, as demonstrated by PI staining. Similar to VAA-I, rVAA showed a higher binding affinity for monocytes and granulocytes than for lymphocytes. In cultures of PBL, the binding rank order of both lectins to lymphocyte subsets was NK, CD19+ > CD8+ > CD4+. The amount of Annexin/PI staining of PBL subsets corresponds to the degree of their binding capacity. In conclusion, present results demonstrate that VAA-I and its nonglycosylated recombinant form rVAA exhibit comparable effects on cell membrane alterations in the subsets of human PBLs.

Annexins↗

PubMed2006

A comparative study of functional properties of calf chymosin and its recombinant forms.

The action of calf chymosin obtained from transgenic sheep milk and the recombinant protein expressed in yeast Kluyveromyces lactis (Maxiren) on fluorogenic peptide substrates, namely Abz-A-A-F-F-A-A-Ded, Abz-A-A-F-F-A-A-pNA, Abz-A-F-F-A-A-Ded, Abz-A-A-F-F-A-Ded, Abz-A-A-F-F-Ded, Abz-A-A-F-F-pNA, and heptapeptide L-S-F-M-A-I-P-NH2, a fragment of kappa-casein (the native chymosin substrate), was investigated. It has been established that transgenic chymosin and recombinant chymosin (Maxiren) differ from the native enzyme in their action on low molecular weight substrates, whereas there was no difference in enzymatic action on protein substrates. Pepstatin, a specific inhibitor of aspartic proteinases, inhibits the recombinant chymosin forms less efficiently than the native enzyme. Perhaps this is associated with local conformational changes in the substrate binding site of recombinant chymosin occurring during the formation of the protein globule.

Amino Acid Sequence↗