Expression strategy of a phlebovirus: biogenesis of proteins from the Rift Valley fever virus M segment.

The middle (M) RNA segment of Rift Valley fever virus (RVFV) encodes four proteins: the major viral glycoproteins G2 and G1, a 14-kilodalton (kDa) protein, and a 78-kDa protein. These proteins are derived from a single large open reading frame (ORF) present in the virus-complementary M-segment mRNA. We used recombinant vaccinia viruses in which sequences representing the M-segment ORF were engineered as a surrogate system to study phlebovirus protein expression. To investigate the translational initiation codon requirements for synthesis of these proteins, we constructed a series of vaccinia virus recombinants containing specific sequence changes which eliminated select ATG codons found in the region of the ORF preceding the mature glycoprotein-coding sequences (the preglycoprotein region). Examination of phleboviral proteins synthesized in cells infected with these vaccinia virus recombinants clearly showed that the first ATG of the ORF was required for the production of the 78-kDa protein, while synthesis of the 14-kDa protein was absolutely dependent on the second in-phase ATG codon. Efficient biosynthesis of glycoprotein G2 was shown to depend on one or more ATG codons within the preglycoprotein region, but not the first one of the ORF. Synthesis of about one-half of the total glycoprotein G1 was affected by the amino acid changes that eliminated ATG codons, while production of the remainder appeared to be independent of all ATG codons in the preglycoprotein region. These data indicated that the means for glycoprotein G1 biosynthesis was distinct from those of the other three M-segment gene products. The results presented herein suggest that a surprisingly complex expression strategy is employed by the RVFV M segment. Although the full nature of the mechanisms involved in the biogenesis of the four RVFV M-segment proteins remains unclear, it does involve the use of at least two (ATG codons 1 and 2), and likely more, distinct translation start sites within the same ORF to produce its complete complement of gene products.     

KBSH parvovirus: comparison with porcine parvovirus.

We compared the molecular, antigenic, and pathogenic properties of KBSH parvovirus to those of porcine parvovirus (PPV) isolate NADL-8. KBSH, propagated in swine testes cells in culture, possessed two major capsid polypeptides of 83 and 64 kilodaltons that were similar in size to those of PPV. KBSH-infected cells also contained an 86-kilodalton nonstructural polypeptide that was identical in size to the PPV nonstructural polypeptide (NS-1). The KBSH polypeptides were structurally similar but not identical to the corresponding PPV polypeptides, as revealed by partial proteolysis mapping. Viral replicative-form DNA from KBSH-infected cells was similar in size to PPV replicative-form DNA and exhibited similar but not identical restriction endonuclease cleavage patterns to that of PPV replicative-form DNA. Antigenically, the two viruses were also very closely related. By using heterologous and homologous antisera, the two viruses were indistinguishable in hemagglutination inhibition and immunoprecipitation assays. However, pathogenically these viruses were dramatically different. NADL-8 caused fetal death when injected into swine fetuses in utero and viremia and high persisting antibody titers when administered orally to weaning-age swine. KBSH-inoculated fetuses were normal in appearance, and pigs orally exposed to KBSH failed to establish viremia and demonstrated only transient antibody titers. Thus, KBSH appears to be a PPV that is very closely related to a highly pathogenic PPV isolate, yet is itself nonpathogenic in swine. This reduced pathogenic potential of KBSH may be attributable to its poor ability to replicate in swine.     

Some observations on the effect of Daflon (micronized purified flavonoid fraction of Rutaceae aurantiae) in bancroftian filarial lymphoedema

BACKGROUND: Morbidity management is a core component of the global programme for the elimination of lymphatic filariasis. In a double-blind clinical trial, the tolerability and efficacy of Daflon (500 mg) + DEC (25 mg) or DEC (25 mg) alone, twice daily for 90 days, was studied in 26 patients with bancroftian filarial lymphoedema. RESULTS: None of the patients in either drug group reported any adverse reaction throughout the treatment period (90 days). Haematological and biochemical parameters were within normal limits and there was no significant difference between the pre-treatment (day 0) and post-treatment (day 90) values. The group receiving Daflon showed significant reduction in oedema volume from day 90 (140.6 PlusMinus; 18.8 ml) to day 360 (71.8 PlusMinus; 20.7 ml) compared to the pre-treatment (day 0, 198.4 PlusMinus; 16.5 ml) value. This accounted for a 63.8% reduction in oedema volume by day 360 (considering the pre-treatment (day 0) as 100%). In the DEC group, the changes in oedema volume (between day 1 and day 360) were not significant when compared to the pre-treatment (day 0) value. The percentage reduction at day 360 was only 9%, which was not significant (P > 0.05). CONCLUSION: This study has shown that Daflon (500 mg, twice a day for 90 days) is both safe and efficacious in reducing oedema volume in bancroftian filarial lymphoedema. Further clinical trials are essential for strengthening the evidence base on the role of this drug in the morbidity management of lymphatic filariasis.     

Direct selection and phage display of a Gram-positive secretome

Surface, secreted and transmembrane protein-encoding open reading frames, collectively the secretome, can be identified in bacterial genome sequences using bioinformatics. However, functional analysis of translated secretomes is possible only if many secretome proteins are expressed and purified individually. We have now developed and applied a phage display system for direct selection, identification, expression and purification of bacterial secretome proteins.     

Functional RNA microarrays for high-throughput screening of antiprotein aptamers

High-throughput methods for generating aptamer microarrays are described. As a proof-of-principle, the microarrays were used to screen the affinity and specificity of a pool of robotically selected antilysozyme RNA aptamers. Aptamers were transcribed in vitro in reactions supplemented with biotinyl-guanosine 5'-monophosphate, which led to the specific addition of a 5' biotin moiety, and then spotted on streptavidin-coated microarray slides. The aptamers captured target protein in a dose-dependent manner, with linear signal response ranges that covered seven orders of magnitude and a lower limit of detection of 1 pg/mL (70 fM). Aptamers on the microarray retained their specificity for target protein in the presence of a 10,000-fold (w/w) excess of T-4 cell lysate protein. The RNA aptamer microarrays performed comparably to current antibody microarrays and within the clinically relevant ranges of many disease biomarkers. These methods should also prove useful for generating other functional RNA microarrays, including arrays for genomic noncoding RNAs that bind proteins. Integrating RNA aptamer microarray production with the maturing technology for automated in vitro selection of antiprotein aptamers should result in the high-throughput production of proteome chips.     

Path integration in insects

The most notable advance in our knowledge of path integration in insects is a new understanding of how the honeybee measures the distance that it travels during its foraging trips. Data from two groups show that the bee's odometer records distance in terms of the net amount of image motion over the retina that is accumulated during a flight. Progress has also been made in clarifying the relation between path integration and other navigational strategies. On unfamiliar ground, path integration is the only available means of navigation. In familiar surroundings, however, guidance by landmarks may override guidance by path integration. Path integration then becomes a back-up strategy that is used primarily when landmarks fail.     

Sequence and structural analyses of nuclear export signals in the NESdb database

We compiled >200 nuclear export signal (NES)-containing CRM1 cargoes in a database named NESdb. We analyzed the sequences and three-dimensional structures of natural, experimentally identified NESs and of false-positive NESs that were generated from the database in order to identify properties that might distinguish the two groups of sequences. Analyses of amino acid frequencies, sequence logos, and agreement with existing NES consensus sequences revealed strong preferences for the Φ1-X(3)-Φ2-X(2)-Φ3-X-Φ4 pattern and for negatively charged amino acids in the nonhydrophobic positions of experimentally identified NESs but not of false positives. Strong preferences against certain hydrophobic amino acids in the hydrophobic positions were also revealed. These findings led to a new and more precise NES consensus. More important, three-dimensional structures are now available for 68 NESs within 56 different cargo proteins. Analyses of these structures showed that experimentally identified NESs are more likely than the false positives to adopt α-helical conformations that transition to loops at their C-termini and more likely to be surface accessible within their protein domains or be present in disordered or unobserved parts of the structures. Such distinguishing features for real NESs might be useful in future NES prediction efforts. Finally, we also tested CRM1-binding of 40 NESs that were found in the 56 structures. We found that 16 of the NES peptides did not bind CRM1, hence illustrating how NESs are easily misidentified.