Expression of a Recombinant Toxoplasma gondii ROP2 Fragment as a Fusion Protein in Bacteria Circumvents Insolubility and Proteolytic Degradation

A 268-amino-acid-residue carboxy-terminal antigenic fragment of the Toxoplasma gondii rhoptry protein ROP2 (recROP2_t, residues 196–464) was expressed in Escherichia coli. This recombinant fragment was produced at low concentration and in a highly insoluble form. By contrast, the level of recROP2_t production was drastically greater when the same coding sequence was fused to the C-terminus of thioredoxin (TRX) or to the maltose-binding protein (MBP) gene. While both fusion proteins were found to be mainly insoluble, solubilization could be achieved without significant degradation. MBP was more efficient than TRX in increasing the recovery of soluble protein with more than 10% of total MBP–recROP2_t being readily expressed in a soluble form. Moreover, the insoluble form of MBP–recROP2_t could be correctly refolded with a recovery of more than 80%. Both forms of MBP–recROP2_t were purified to homogeneity by amylose chromatography. In contrast, the refolding of TRX–recROP2_t promoted aggregation of the protein, which was prevented by the use of zwitterionic detergent during the one-step purification by gel filtration. Subsequent proteolytic cleavages of purified TRX–recROP2_t and of MBP–recROP2_t led respectively to the complete degradation or to the truncation of the recROP2_t moiety. However, recROP2_t, despite the presence of the fusion partners, adopted a suitable conformation recognized by human serum-derived antibodies from T. gondii-seropositive individuals. Finally, both fusion proteins were able to induce specific humoral and cell-mediated immune response to the ROP2 fragment. Such fusions could represent an alternative to study the immunogenicity of T. gondii proteins which are difficult to produce because of insolubility and degradation.     

Characterization of the cDNA Coding for Rat Brain Cysteine Sulfinate Decarboxylase

Cysteine sulfinate decarboxylase (CSD) is considered as the rate-limiting enzyme in the biosynthesis of taurine, a possible osmoregulator in brain. Through cloning and sequencing of RT-PCR and RACE-PCR products of rat brain mRNAs, a 2,396-bp cDNA sequence was obtained encoding a protein of 493 amino acids (calculated molecular mass, 55.2 kDa). The corresponding fusion protein showed a substrate specificity similar to that of the endogenous enzyme. The sequence of the encoded protein is identical to that encoded by liver CSD cDNA. Among other characterized amino acid decarboxylases, CSD shows the highest homology (54%) with either isoform of glutamic acid decarboxylase (GAD65 and GAD67). A single mRNA band, approximately 2.5 kb, was detected by northern blot in RNA extracts of brain, liver, and kidney. However, brain and liver CSD cDNA sequences differed in the 5' untranslated region. This indicates two forms of CSD mRNA. Analysis of PCR-amplified products of genomic DNA suggests that the brain form results from the use of a 3' alternative internal splicing site within an exon specifically found in liver CSD mRNA. Through selective RT-PCR the brain form was detected in brain only, whereas the liver form was found in liver and kidney. These results indicate a tissue-specific regulation of CSD genomic expression.     

Amplification of the Inflammatory Cellular Redox State by Human Immunodeficiency Virus Type 1-Immunosuppressive Tat and gp160 Proteins

In the course of our studies on oxidative stress as a component of pathological processes in humans, we showed that microintrusion into cells with microcapillary and ultramicroelectrochemical detection could mimic many types of mechanical intrusion leading to an instant (0.1 s) and high (some femtomoles) burst release of H₂O₂. Specific inhibitors of NADPH enzymes seem to support the assumption that this enzyme is one of the main targets of our experiments. Also, human immunodeficiency virus type 1 (HIV-1) gp160 inhibits the cooperative response of uninfected T cells as well as Tat protein release by infected cells does. In this study, we analyzed in real time, lymphocyte per lymphocyte, the T-cell response following activation in relation to the redox state. We showed that the immunosuppressive effects of HIV-1 Tat and gp160 proteins and oxidative stress are correlated, since the native but not the inactivated Tat and gp160 proteins inhibit the cellular immune response and enhance oxidative stress. These results are consistent with a role of the membrane NADPH oxidase in the cellular response to immune activation.     

Identification of Multiple Protective Epitopes (Protectopes) in the Central Conserved Domain of a Prototype Human Respiratory Syncytial Virus G Protein

A recombinant fusion protein (BBG2Na) comprising the central conserved domain of the respiratory syncytial virus subgroup A (RSV-A) (Long) G protein (residues 130 to 230) and an albumin binding domain of streptococcal protein G was shown previously to protect mouse upper (URT) and lower (LRT) respiratory tracts against intranasal RSV challenge (U. F. Power, H. Plotnicky-Gilquin, T. Huss, A. Robert, M. Trudel, S. Stahl, M. Uhlén, T. N. Nguyen, and H. Binz, Virology 230:155-166, 1997). Panels of monoclonal antibodies (MAbs) and synthetic peptides were generated to facilitate dissection of the structural elements of this domain implicated in protective efficacy. All MAbs recognized native RSV-A antigens, and five linear B-cell epitopes were identified; these mapped to residues 152 to 163, 165 to 172, 171 to 187 (two overlapping epitopes), and 196 to 204, thereby covering the highly conserved cysteine noose domain. Antibody passive-transfer and peptide immunization studies revealed that all epitopes were implicated in protection of the LRT, but not likely the URT, against RSV-A challenge. Pepscan analyses of anti-RSV-A and anti-BBG2Na murine polyclonal sera revealed lower-level epitope usage within the central conserved region in the former, suggesting diminished immunogenicity of the implicated epitopes in the context of the whole virus. However, Pepscan analyses of RSV-seropositive human sera revealed that all of the murine B-cell protective epitopes (protectopes) that mapped to the central conserved domain were recognized in man. Should these murine protectopes also be implicated in human LRT protection, their clustering around the highly conserved cysteine noose region will have important implications for the development of RSV vaccines.     

Time Course of Individual Ca2+ Sparks in Frog Skeletal Muscle Recorded at High Time Resolution

Discrete Ca²⁺ release events (Ca²⁺ “sparks”) were recorded in cut segments of single frog skeletal muscle fibers using a video-rate laser-scanning confocal microscope operating in line-scan mode (63 μs per line). Fibers loaded with the Ca²⁺ indicator fluo-3 were voltage clamped at a holding potential of 0 mV, briefly reprimed at −90 mV, and then strongly depolarized with a large test pulse to activate any reprimed voltage sensors. Using this high time resolution system, it was possible to record individual Ca²⁺ sparks at ∼30-fold higher time resolution than previously attained. The resulting new experimental data provides a means of characterizing the time course of fluorescence during the brief (a few milliseconds) rising phase of a spark, which was not possible with the previously used 1.5–2 ms per line confocal systems. Analysis of the time course of individual identified events indicates that fluorescence begins to rise rather abruptly at the start of the spark, continues to rise at a slightly decreasing rate to a relatively sharp peak, and then declines along a quasi-exponential time course. The mean rise time of 198 sparks was 4.7 ± 0.1 ms, and there was no correlation between rise time and peak amplitude. Average sparks constructed by temporally and spatially superimposing and summing groups of individual sparks having similar rise times gave a lower noise representation of the sparks, consistent with the time course of individual events. In theory, the rising phase of a spark provides a lower bound estimation of the time that Ca²⁺ ions are being released by the sarcoplasmic reticulum Ca²⁺ channel(s) generating the spark. The observed time course of fluorescence suggests that the Ca²⁺ release underlying a spark could continue at a fairly constant rate throughout the rising phase of the spark, and then stop rather abruptly at the time of the peak.     

Blocking gap junctional intercellular communication in myoblasts inhibits myogenin and MRF4 expression

Cells rely heavily on cues from their extracellular environment and other cells to coordinate normal physiological processes, and the exchange of molecules via gap junctions has been suggested as an important avenue for cell-cell communication. Gap junctions are found in virtually all mammalian tissues with the notable exception of adult skeletal muscle. However, since functional gap junctions have been detected during the early stages of muscle development, gap junctional intercellular communication (GJIC) may play an important role in myogenesis. In this study, GJIC in normal L6 myoblasts was inhibited using the known blockers 1-octanal and β-glycyrrhetinic acid (β-GA). Under differentiation promoting conditions, L6 cells fused to form multinucleated myotubes, but when treated with either octanol or β-GA, no fusion was observed. The expression of two muscle regulatory factors (MRFs), myogenin and MRF4, was examined in both the blocked and control cells. As expected, the activation of both the myogenin and MRF4 genes coincided with the onset of differentiation in the control L6 cells. Neither of these genes were turned on in the blocked cells, even when grown under low serum conditions. This inhibition of differentiation by octanol and β-GA was reversible, since the activation of both MRF genes as well as myoblast fusion were observed when the blocking medium was replaced with normal differentiating medium. These results suggest that intercellular communication via gap junctions plays an important role in skeletal muscle development and perhaps in the cell signaling events that trigger the activation of muscle-specific MRF genes. Dev. Genet. 20:133–144. 1997. © 1997 Wiley-Liss, Inc.     

Hydrophobic forces are responsible for the folding of a highly potent natriuretic peptide analogue at a membrane mimetic surface: An NMR study

A conformational study by nmr spectroscopy was performed with the highly active 28-residue hybrid natriuretic peptide analogue pBNP1 [M. Mimeault, A. De Léan, M. Lafleur, D. Bonenfant, and A. Fournier (1995) Biochemistry, Vol. 34, pp. 955–964], which consists of the cyclic peptide core of pBNP32 and the N- and C-terminal exocyclic segments of rANP(99–126). In purely aqueous solution pBNP1 exhibits random coil behavior as evidenced by the almost complete absence of structurally significant nmr observables. By contrast, elements of secondary structure emerged upon the addition of dodecylphosphocholine micelles to the aqueous sample. Nuclear Overhauser effect distance-restrained molecular dynamics simulations in conjunction with torsional angle determinations permitted the generation of a reasonable model of the lipid-bound conformation of pBNP1. According to this model, pBNP1 adopts turn-like features in the cyclic and C-terminal regions of the peptide, but remains quite flexible in the N-terminal segment. Two hydrophobic cores separated by a hydrophilic cleft were also evident in the generated structure. A mechanism is proposed whereby the hydrophobic interactions necessary to stabilize a folded structure of pBNP1 are facilitated by the presence of the membrane-like polar/apolar interface provided by the phospholipid micelles. © 1997 John Wiley & Sons, Biopoly 42: 37–48, 1997     

Offering ART refill through community health workers versus clinic-based follow-up after home-based same-day ART initiation in rural Lesotho: The VIBRA cluster-randomized clinical trial

BackgroundCommunity-based antiretroviral therapy (ART) dispensing by lay workers is an important differentiated service delivery model in sub-Sahara Africa. However, patients new in care are generally excluded from such models. Home-based same-day ART initiation is becoming widespread practice, but linkage to the clinic is challenging. The pragmatic VIBRA (Village-Based Refill of ART) trial compared ART refill by existing lay village health workers (VHWs) versus clinic-based refill after home-based same-day ART initiation.Methods and findingsThe VIBRA trial is a cluster-randomized open-label clinical superiority trial conducted in 249 rural villages in the catchment areas of 20 health facilities in 2 districts (Butha-Buthe and Mokhotlong) in Lesotho. In villages (clusters) randomized to the intervention arm, individuals found to be HIV-positive during a door-to-door HIV testing campaign were offered same-day ART initiation with the option of refill by VHWs. The trained VHWs dispensed drugs and scheduled clinic visits for viral load measurement at 6 and 12 months. In villages randomized to the control arm, participants were offered same-day ART initiation with clinic-based ART refill. The primary outcome was 12-month viral suppression. Secondary endpoints included linkage and 12-month engagement in care. Analyses were intention-to-treat. The trial was registered on ClinicalTrials.gov ({"type":"clinical-trial","attrs":{"text":"NCT03630549","term_id":"NCT03630549"}}NCT03630549). From 16 August 2018 until 28 May 2019, 118 individuals from 108 households in 57 clusters in the intervention arm, and 139 individuals from 130 households in 60 clusters in the control arm, were enrolled (150 [58%] female; median age 36 years [interquartile range 30–48]; 200 [78%] newly diagnosed). In the intervention arm, 48/118 (41%) opted for VHW refill. At 12 months, 46/118 (39%) participants in the intervention arm and 64/139 (46%) in the control arm achieved viral suppression (adjusted risk difference −0.07 [95% CI −0.20 to 0.06]; p = 0.256). Arms were similar in linkage (adjusted risk difference 0.03 [−0.10 to 0.16]; p = 0.630), but engagement in care was non-significantly lower in the intervention arm (adjusted risk difference −0.12 [−0.23 to 0.003]; p = 0.058). Seven and 0 deaths occurred in the intervention and control arm, respectively. Of the intervention participants who did not opt for drug refill from the VHW at enrollment, 41/70 (59%) mentioned trust or conflict issues as the primary reason. Study limitations include a rather small sample size, 9% missing viral load measurements in the primary endpoint window, the low uptake of the VHW refill option in the intervention arm, and substantial migration among the study population.ConclusionsThe offer of village-based ART refill after same-day initiation led to similar outcomes as clinic-based refill. The intervention did not amplify the effect of home-based same-day ART initiation alone. The findings raise concerns about acceptance and safety of ART delivered by lay health workers after initiation in the community.Trial registrationRegistered with Clinicaltrials.gov ({"type":"clinical-trial","attrs":{"text":"NCT03630549","term_id":"NCT03630549"}}NCT03630549).

Alain Amstutz and co-workers compare village- and clinic-based antiretroviral refills for people with HIV infection in Lesotho.