Analysis of safety and nutritive value of protein isolated from alfalfa

Experiments were made on white rats to examine the effect of a protein isolate from lucerne on the animal body. The rat body responses to the supply of this protein contained by the diet in an amount of 18 and 36% were studied with the use of biochemical and morphological methods. The protein isolate from lucerne did not alter the structural integrity of parenchymatous organs and did not exhibit any toxic effects. This finding was also confirmed by biochemical studies. Biological studies with the use of 6 levels of the content of experimental (protein isolate) and control (casein) proteins were performed to define their concentration in the diet, corresponding with the maximal magnitudes of biological value (10% content of both proteins in the diet). The relative biological value of the protein isolate from lucerne constitutes 87% with reference to casein.     

Inducible gene expression from African swine fever virus recombinants: analysis of the major capsid protein p72

A method to study the function of individual African swine fever virus (ASFV) gene products utilizing the Escherichia coli lac repressor-operator system has been developed. Recombinant viruses containing both the lacI gene encoding the lac repressor and a strong virus late promoter modified by the insertion of one or two copies of the lac operator sequence at various positions were constructed. The ability of each modified promoter to regulate expression of the firefly luciferase gene was assayed in the presence and in the absence of the inducer isopropyl beta-D-thiogalactoside (IPTG). Induction and repression of gene activity were dependent on the position(s) of the operator(s) with respect to the promoter and on the number of operators inserted. The ability of this system to regulate the expression of ASFV genes was analyzed by constructing a recombinant virus inducibly expressing the major capsid protein p72. Electron microscopy analysis revealed that under nonpermissive conditions, electron-dense membrane-like structures accumulated in the viral factories and capsid formation was inhibited. Induction of p72 expression allowed the progressive building of the capsid on these structures, leading to assembly of ASFV particles. The results of this report demonstrate that the transferred inducible expression system is a powerful tool for analyzing the function of ASFV genes.     

Structure of Sesbania mosaic virus at 4.7 A resolution and partial sequence of the coat protein

Serum IgG subclasses and Serum IgA were studied in 43 infants with acute bronchiolitis and 20 healthy infants. IgG subclasses were determined by a capture ELISA and IgA was quantified by turbidimetry. IgG1 concentrations were significantly lower in infants with bronchiolitis than in normal infants. The other IgG subclasses and IgA did not differ between the groups. The subgroups of infants with bronchiolitis who had previously suffered from otitis media or bronchitis, had significantly lower IgG2 than the other infants with bronchiolitis. The same was found for infants with bronchiolitis who had suffered from three or more lower respiratory tract infections. In infants who had suffered from upper or lower respiratory infections before the acute bronchiolitis, IgA was significantly higher than in infants without previous respiratory infections. Ten infants with bronchiolitis (23%) had IgG1 deficiency, that is values below the lower reference limit calculated in a population of healthy Norwegian infants. No healthy infants had any IgG1 deficiency. No infant with bronchiolitis had IgG2 or IgG3 deficiency. The low IgG1 values found in infants with acute bronchiolitis, may be one cause for infants to be more susceptible to RS virus infections.     

Membrane association and RNA binding of recombinant hepatitis A virus protein 2C

The direct function of hepatitis A virus (HAV) protein 2C, a putative NTPase, is not known, yet genetic evidence obtained from chimeric viruses carrying the 2C genomic region of different HAV variants indicates that it plays a pivotal role in viral replication. In a first assessment of its potential function(s), membrane and RNA binding properties of HAV 2C were studied after expressing the protein in various recombinant systems. In contrast to poliovirus 2C, expression of HAV 2C was inhibitory to the growth and protein synthesis of bacteria. Deletion of the N-terminal amphipathic helix of 2C abrogated this effect and the ability of 2C to associate with eukaryotic membranes. Both, purified 2C and the N-terminally truncated protein were shown to bind RNA in vitro. Our data taken together suggest that HAV 2C is a multifunctional protein.     

Mutational analysis of the coat protein gene of brome mosaic virus: effects on replication and movement in barley and in Chenopodium hybridum

The objective of this work was to determine the response of the circulatory system to heavy physical work (the average energy expenditure was about 5000 kJ/shift). The tests were performed on a group of 35 women aged between 24 and 50 years, employed at a printing office as printing machine operators. The average length of employment for the group was 17 years (3-28 years). A 24-hour Holter ECG recording was taken on these women with the aid of an Oxford Medilog 3000 system. Before and after each recording, the arterial blood pressure was measured. Based on the ECG recordings, the heart rates during work, leisure time and sleep were calculated and the morphologies of the recordings were evaluated. In the group investigated, the abnormality in 24-hour ECG recordings classified as pathology, were found in 9 women within this group. Frequent ventricular premature beats (ExV) occurred in 3 women, ventricular bigeminy in 1 women, and single R/T excitations in 1 women. Frequent premature supraventricular extrasystols (ExSV) were found in 4 women, and within this number, in 1 woman both those heart rhythm disturbances were found. The heart rhythm disturbances occurred in connection with the performed physical task. Sinusal pauses were found in 2 women and A-V Mobitz II block was found in 1 women within the group investigated. Those changes occurred at night. No resting ECG changes were observed. The high frequency of occurrence of pathological changes in the group investigated indicates that the careful control of the state of the circulatory system in people performing heavy physical work is required.     

Hexon-only binding of VP26 reflects differences between the hexon and penton conformations of VP5, the major capsid protein of herpes simplex virus

VP26 is a 12-kDa capsid protein of herpes simplex virus 1. Although VP26 is dispensable for assembly, the native capsid (a T=16 icosahedron) contains 900 copies: six on each of the 150 hexons of VP5 (149 kDa) but none on the 12 VP5 pentons at its vertices. We have investigated this interaction by expressing VP26 in Escherichia coli and studying the properties of the purified protein in solution and its binding to capsids. Circular dichroism spectroscopy reveals that the conformation of purified VP26 consists mainly of beta-sheets (approximately 80%), with a small alpha-helical component (approximately 15%). Its state of association was determined by analytical ultracentrifugation to be a reversible monomer-dimer equilibrium, with a dissociation constant of approximately 2 x 10(-5) M. Bacterially expressed VP26 binds to capsids in the normal amount, as determined by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cryoelectron microscopy shows that the protein occupies its usual sites on hexons but does not bind to pentons, even when available in 100-fold molar excess. Quasi-equivalence requires that penton VP5 must differ in conformation from hexon VP5: our data show that in mature capsids, this difference is sufficiently pronounced to abrogate its ability to bind VP26.     

A simplified high-performance liquid chromatographic method for direct determination of warfarin enantiomers and their protein binding in stroke patients

A simplified method for direct determination of warfarin enantiomers by high-pressure liquid chromatography with fluorescence detection has been developed. This method involves solid phase extraction of warfarin in plasma, precolumn derivatization to form diastereoisomeric esters, and post-column reaction to discriminate each enantiomer separately. Ultrafiltration was employed in the separation of unbound warfarin enantiomers. Twelve plasma samples from six stroke patients taking warfarin regularly were analyzed. The average concentration of total warfarin was 0.47 +/- 0.17 mg/L for the S-isomer and 0.69 +/- 0.18 mg/L for the R-isomer. The average protein binding was 99.67 +/- 0.33% for S-warfarin and 99.44 +/- 0.33% for R-warfarin. This methodology provides a quick and reliable technique for determining enantiomeric protein binding of warfarin in clinical settings.     

Molecular analysis of sequence heterogeneity among genes encoding decorin binding proteins A and B of Borrelia burgdorferi sensu lato

Immunization of mice with Borrelia burgdorferi decorin binding protein A (DbpA), one of two gene products of the dbpBA locus, has been shown recently to confer protection against challenge. Hyperimmune DbpA antiserum killed a large number of B. burgdorferi sensu lato isolates of diverse phylogeny and origin, suggesting conservation of the protective epitope(s). In order to evaluate the heterogeneity of DbpA and DbpB and to facilitate defining the conserved epitope(s) of these antigens, the sequences of the dbpA genes from 29 B. burgdorferi sensu lato isolates and of the dbpB genes from 15 B. burgdorferi sensu lato isolates were determined. The predicted DbpA sequences were fairly heterogeneous among the isolates (58.3 to 100% similarity), but DbpA sequences with the highest similarity tended to group into species previously defined by well-characterized chromosomal markers. In contrast, the predicted DbpB sequences were highly conserved (96.3 to 100% similarity). Substantial diversity in DbpA sequence was seen among isolates previously shown to be killed by antiserum against a single DbpA, suggesting that one or more conserved protective epitopes are composed of noncontiguous amino acids. The observation of individual dbpA alleles with sequence elements characteristic of more than one B. burgdorferi sensu lato species was consistent with a role for genetic recombination in the generation of dbpA diversity.     

Cloning and sequence analysis of a gene encoding an erythrocyte binding protein from Plasmodium cynomolgi

We compared the metabolic response of a methionine(Met)-dependent (P60) human glioma cell line with that of a Met-independent variant (P60H) when cultured in a homocysteine (Hcy) medium and exposed to N2O. In Hcy medium (without Met), remethylation of Hcy in P60H cells was enhanced and supported growth, whereas remethylation was low in P60 cells, which failed to thrive under these conditions. Both cell types seemed to contain adequate amounts of folates and total cobalamin (Cbl). P60 cells showed increased total and methylcobalamin (CH3Cbl) content after the shift to a Hcy medium, but the high, stable level of CH3Cbl detected in P60H cells was not attained. Further metabolic differences were induced by N2O exposure, which markedly reduced Met-synthase activity in cell-free extracts in both cell lines and completely blocked intact-cell Hcy remethylation in P60, whereas Hcy remethylation was only partly inhibited in P60H cells cultured in Met medium. The residual Hcy remethylation in P60H cells may be related to only a moderate depletion of CH3Cbl. The resulting high CH3Cbl level relative to Met-synthase activity during N2O exposure was even higher in Hcy medium. These findings in P60H cells probably reflect increased provision of Cbl to support Hcy remethylation under metabolic strain. The inability of P60 to furnish CH3Cbl to the enzyme may explain both the Met-dependent phenotype and the increased sensitivity of Hcy remethylation to N2O exposure in these cells.     

Transactivation of human hepatitis B virus X protein, HBx, operates through a mechanism distinct from protein kinase C and okadaic acid activation pathways

Human hepatitis B virus (HBV) X protein, HBx, transactivates virus and host genes through a wide variety of cis-elements. Expression of HBx is controlled by HBV enhancer 1 (Enh1). Both Enh1 and the core sequence of Enh1, which consists of an AP-1 related site (cFAP1) and a C stretch, respond to HBx and a phorbol ester (TPA). Biochemical pathways of the responses to HBx and TPA are still controversial. We therefore asked whether HBx and TPA stimulate Enh1 core activity through a common process. Protein kinase C (PKC) inhibitors, H-7 and staurosporin, did not inhibit HBx transactivation at concentrations sufficient to abolish the TPA effects in HepG2 cells. Although HBx transactivation synergized independently with TPA or a phosphoprotein phosphatase inhibitor, okadaic acid (OA), the PKC inhibitors eliminated only the TPA contribution. HBx transactivation required both the cFAP1 and the C stretch of the Enh1 core region; however, mutations in either or both of the two cis-elements demonstrated that TPA augmentation required only cFAP1. These results imply that HBx transactivation operates through a mechanism distinct from the PKC and OA activation pathways.     

Sequences of the coat protein gene of five peanut stripe virus (PStV) strains from Thailand and their evolutionary relationship with other bean common mosaic virus sequences

The coat protein gene and part of the 3' non-coding region of five strains of peanut stripe virus (PStV) from Thailand have been cloned and sequenced. Phylogenetic comparisons of these strains, known as T1, T3, T5, T6 and T7, and related sequences showed that these strains are indeed strains of PStV. Further, PStV strains appear to be related to each other according to their geographic origin. That is, the Thai strains are more closely related to each other than they are to strains from the USA or Indonesia, despite the variety of symptoms caused by these strains and the overlap of symptom types between the strains from different locations. Like other PStV strains, PStV-Thai can be considered strains of bean common mosaic virus (BCMV) but can be distinguished from bean-infecting strains of BCMV and blackeye cowpea mosaic virus (B1CMV) through sequence and host range. No evidence was found that PStV-Thai strains, unlike PStV-Ib, are recombinants of PStV and B1CMV, although the T3 strain may be a recombinant of different PStV sequences. Phylogenetic analyses of viruses of the BCMV group suggest that acquisition of the ability to infect peanut may have occurred only once.     

Functional and receptor binding characterization of recombinant murine macrophage inflammatory protein 2: sequence analysis and mutagenesis identify receptor binding epitopes

Murine macrophage inflammatory protein-2 (MIP-2), a member of the alpha-chemokine family, is one of several proteins secreted by cells in response to lipopolysaccharide. Many of the alpha-chemokines, such as interleukin-8, gro-alpha/MGSA, and neutrophil activating peptide-2 (NAP-2), are associated with neutrophil activation and chemotaxis. We describe the expression, purification, and characterization of murine MIP-2 from Pichia pastoris. Circular dichroism spectroscopy reveals that MIP-2 exhibits a highly ordered secondary structure consistent with the alpha/beta structures of other chemokines. Recombinant MIP-2 is chemotactic for human and murine neutrophils and up-regulates cell surface expression of Mac-1. MIP-2 binds to human and murine neutrophils with dissociation constants of 6.4 nM and 2.9 nM, respectively. We further characterize the binding of MIP-2 to the human types A and B IL-8 receptors and the murine homologue of the IL-8 receptor. MIP-2 displays low-affinity binding to the type A IL-8 receptor (Kd > 120 nM) and high-affinity binding to the type B IL-8 receptor (Kd 5.7 nM) and the murine receptor (Kd 6.8 nM). The three-dimensional structure of IL-8 and sequence analysis of six chemokines (IL-8, gro-alpha, NAP-2, ENA-78, KC, and MIP-2) that display high-affinity binding to the IL-8 type B receptor are used to identify an extended N-terminal surface that interacts with this receptor. Two mutants of MIP-2 establish that this region is also involved in binding and activating the murine homologue of the IL-8 receptor. Differences in the sequence between IL-8 and related chemokines identify a unique hydrophobic/aromatic region surrounded by charged residues that is likely to impart specificity to IL-8 for binding to the type A receptor.     

Nucleotide sequencing of S-RNA segment and sequence analysis of the nucleocapsid protein gene of the newly isolated Akabane virus PT-17 strain

The nucleotide sequences of the S-RNA of Akabane viruses JaGAr-39, OBE-1, Iriki and the newly isolated PT-17 strains and the Aino virus were determined and compared. The results reveal that the S-RNAs of the four Akabane strains share 96.9% homology in nucleotide sequences. Only one amino acid difference out of the 233 amino acids of the nucleocapsid protein (N) and three amino acid differences in the 91 amino acids of the nonstructural protein (NSs) were found among the Akabane viruses. Amino acid sequences of N and NSs proteins of the Aino virus have approximately 80% identity as compared with the Akabane viruses. The results also demonstrate that the four Akabane viruses and the Aino virus can be clearly differentiated by RFLP (restriction fragments length polymorphism) analysis using RT-PCR generated nucleocapsid protein genes and digested with HaeIII and HindIII. The phylogenetic tree based on the UPGMA (Unweighted Pair Group Method with Arithmetic Mean) analysis of the sequences of nucleocapsid protein genes and the S-DNAs revealed that the newly isolated PT-17 strain is most closely related to Iriki strain, than the JaGAr-39 or OBE-1 strains.