Evaluation of the European tick‐borne encephalitis vaccine against Omsk hemorrhagic fever virus

This study focused on the antigenic cross‐reactivity between tick‐borne encephalitis virus (TBEV) and Omsk hemorrhagic fever virus (OHFV) to assess the efficacy of the commercial TBE vaccine against OHFV infection. Neutralization tests performed on sera from OHFV‐ and TBEV‐infected mice showed that neutralizing antibodies are cross‐protective. The geometric mean titers of antibodies against TBEV and OHFV from TBEV‐infected mice were similar. However, the titers of anti‐TBEV antibodies in OHFV‐infected mice were significantly lower than those of anti‐OHFV antibodies in the same animals. In mouse vaccination and challenge tests, the TBE vaccine provided 100% protection against OHFV infection. Eighty‐six percent of vaccinees seroconverted against OHFV following complete vaccination, and the geometric mean titers of neutralizing antibodies against OHFV were comparable to those against TBEV. These data suggest that the TBE vaccine can prevent OHFV infection.     

Enantioselective lipase-catalyzed kinetic resolution of N-(2-ethyl-6-methylphenyl)alanine

The enzyme (BSL2), a highly active lipase expressed from newly constructed strain of Bacillus subtilis BSL2, is used in the kinetic resolution of N-(2-ethyl-6-methylphenyl)alanine from the corresponding racemic methyl ester. Reaction conditions are optimized to enhance the enantioselectivity. The effects of various racemic alkyl esters, substrate concentration, operating temperature, pH of the aqueous medium and organic solvents on activity and enantioselectivity of BSL2 for kinetic resolution are also studied. A high enantiomeric ratio (E = 60.7) is reached in diisopropyl ether/water (10%, v/v) and the enantioselectivity is about 22-fold higher than that in pure buffered aqueous solution. The results show that the reaction medium greatly influences BSL2 reaction and its enantioselectivity in the hydrolysis of racemic methyl ester.     

Crucial role of the 5' conserved structure of bamboo mosaic virus satellite RNA in downregulation of helper viral RNA replication

Satellite RNA of Bamboo mosaic virus (satBaMV), a single-stranded mRNA type satellite encoding a protein of 20 kDa (P20), depends on the helper BaMV for replication and encapsidation. Two satBaMV isolates, BSF4 and BSL6, exhibit distinctly differential phenotypes in Nicotiana benthamiana plants when coinoculated with BaMV RNA. BSL6 significantly reduces BaMV RNA replication and suppresses the BaMV-induced symptoms, whereas BSF4 does not. By studies with chimeric satBaMVs generated by exchanging the components between BSF4 and BSL6, the genetic determinants responsible for the downregulation of BaMV replication and symptom expression were mapped at the 5' untranslated region (UTR) of BSL6. The 5' UTR of BSL6 alone is sufficient to diminish BaMV RNA replication when the 5' UTR is inserted in cis into the BaMV expression vector or when coinoculation with mutants that block the synthesis of P20 protein takes place. Further, the 5' UTR of natural satBaMV isolates contains one hypervariable (HV) region which folds into a conserved apical hairpin stem-loop (AHSL) structure (W. B. Yeh, Y. H. Hsu, H. C. Chen, and N. S. Lin, Virology 330:105-115, 2004). Interchanges of AHSL segment of HV regions between BSF4 and BSL6 led to the ability of chimeric satBaMV to interfere with BaMV replication and symptom expression. The conserved secondary structure within the HV region is a potent determinant of the downregulation of helper virus replication.     

Hypoglycemic activity of bio-tea in mice

Administration of bio-tea (1.71 ml/kg) to normal albino mice caused hypoglycemia after 30 min which reached to maximum after 2 hr with a significant decrease in blood sugar level (BSL) and became normal beyond 8 hr. In alloxan-induced diabetic albino mice, repeated treatments of bio-tea for 3 days (five doses) brought about a significant fall in mean BSL. Continuous decrease in BSL was observed after 4 hr of administration of last dose of bio-tea. Hypoglycemic effect was persistent in alloxan-induced diabetic mice. Effect on glucose tolerance test showed a significant fall in BSL of bio-tea treated animals after 1 hr of glucose treatment indicating hypoglycemic effect of bio-tea.     

Corticosterone synthesis and serum levels at the end of the perinatal period a study of the effects of stress, diazepam and polyethylene glycol treatments.

The effects of stress (intraperitoneal injections onece daily for three days), diazepam (Faustan, Germed) and polyethylene glycol (Macrogolum, Spofa) on the corticosterone production rate (BPR) and concurrent changes in serum corticosterone levels (BSL) were investigated in 4-day-old male rats. Stress stimulation increased the BSL but the BPR was affected only slightly and the difference was not statistically significant. A low dose of diazepam (1 mg/kg) prevented the stress-induced rise in BSL and decrease of BPR, in comparison to stressed animals. On the contrary, high doses of diazepam (10 mg/kg) increased the BSL, while BPR corresponded to control values. Polyethylene glycol (0.12 g/kg; substance of this class is contained in the diazepam vehiculum) decreased the BSL, but the BPR was increased above control values; a higher dose of this substance (1.2 g/kg) yielded only scattered, non-significant results. It is concluded that in preweaned animals 1. the changes in BSL may be associated with minor or transient changes in BPR which indicate small changes in brainpituitary adrenocorticotropic activity, 2. changes in peripheral corticosterone metabolism can play an exceptionally important role in the regulation of glucocorticoid activity in very young rats, 3. a comparison of controls with stressed animals which had received low doses of diazepam indicate a relatively high steady state activity of adrenal cortex and its regulation in 4-day-old control rats.     

Functional Role of BSL1 Subcellular Localization in Brassinosteroid Signaling

The bri1 Suppressor 1 (BSU1) family mediates the brassinosteroid (BR) signal transduction pathway that orchestrates a wide range of developmental and physiological responses in plants. In Arabidopsis, BSU1 family members (BSU1, BSL1, BSL2, and BSL3) enhance BR signaling through hetero- and homo-oligomerization. Interestingly, BSL1 localizes in the cytoplasm whereas the other three homologs occur in both the nucleus and the cytoplasm. However, little is known about whether differential subcellular localization of BSL1 affects oligomerization of BSU1 family members or modulates BR signaling. Here we show that homooligomeric BSL1 forms cytoplasmic puncta and oligomeric combinations between BSU1 family members determine their subcellular localization. We demonstrate that BSL1 has a distinct role in regulating BSL2 and BSL3 through cytoplasmic oligomerization. Overexpression of BSL1 reduced nuclear accumulation of BSL2 and BSL3. Furthermore, mutagenic analysis indicates that nuclear localization of BSL1 promotes BR signaling, suggesting that BSL1 plays a functional role modulating BR signaling through cytoplasmic retention of BSL2 and BSL3.     

A sesquiterpenol extract potently suppresses inflammation in macrophages and mice skin and prevents chronic liver damage in mice through JNK-dependent HO-1 expression

This study aimed to elucidate the anti-inflammatory and hepatoprotective bioactivities of a sesquiterpenol, (1S,6R)-2,7(14),10-bisabolatrien-1-ol-4-one (BSL), isolated from Cryptomeria japonica (Taxodiaceae) wood extract. BSL markedly suppressed TNF-α and IL-6 secretion, PGE(2) production, and mRNA expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in lipopolysaccharide (LPS)-stimulated mouse macrophages. BSL also potently inhibited the 12-O-tetradecanoylphorbol-13-acetate (TPA) induced protein levels of nitrotyrosine and COX-2 in mouse skin with dermatitis. Conversely, the stress protein heme oxygenase-1 (HO-1) was found upregulated in the same BSL-treated macrophages, probably through activation of the JNK-dependent pathway. LPS-induced activation of NF-κB and mitogen-activated protein kinase signaling pathways, however, was not responsive to BSL treatment. A BSL-enriched extract (BSL-E; 10mg/kg) significantly prevented CCl(4)-induced chronic liver injury, lipid accumulation, and cell necrosis and inhibited aminotransferase activities and iNOS and COX-2 overexpression in mice liver tissues, an effect comparable with that of silymarin, a hepatoprotective drug.     

猪精子凝集素在精卵结合中的作用机制

猪精子凝集素（ＢＳＬ）位于精子头部,既与精子蛋白结合,亦与透明带糖蛋白ＺＰ３结合。并且ＢＳＬ自身分子间亦会聚合。与ＺＰ３结合的片段亦结合于岩藻素－Ｓｅｐｈａｒｏｓｅ亲和柱,但不与胎球蛋白－Ｓｅｐｈａｒｏｓｅ柱结合。该片段具有较强的抑制精卵结合的活性。ＢＳＬ经ＮＢＳ修饰后,血凝活力大大下降,同时推动与精子结合的活性,但不影响它与ＺＰ３的结合。修饰后的ＢＳＬ亦显著抑制精卵结合。表明ＢＳＬ以两个不同的部     

Some properties of the lysozymes in serum and colostrum from cows with high and low lytic power against Micrococcus lysodeikticus

Previous work has uncovered a dominant gene for high bacteriolytic activity of bovine serum against the test bacterium Micrococcus lysodeikticus. This major gene effect is also fully expressed in colostrum. In the present study the lytic power of serum and colostral whey from high and low level cows was subjected to a degree of characterization. It was found that the enzyme activities studied exhibited properties in accordance with those defined for a lysozyme (EC 3.2.1.17), i.e. (1) lysis of a suspension of M. lysodeikticus, (2) basic protein (pI = 10.0 and pI = 10.3 for bovine serum lysozyme (BSL) and bovine colostrum lysozyme (BCL), respectively), (3) molecular weight (MW) approximately 16 000 for both BSL and BCL, (4) liberation of free reducing sugars during action on cell wall peptidoglycan (the kinetics of BSL and BCL differed strongly), and (5) fairly heat stable, especially at acidic pH and relative labile at alkaline pH (BCL was far more sensitive to heating at alkaline pH than was BSL). The dramatic differences in activity between high and low level animals might be due to a major genetic mechanism influencing the amount of, or the activity of, circulating enzyme molecules, rather than a structural gene coding for a certain enzyme with a particular specific activity. This is also supported by the high correlation between the lytic capacity of BSL and BCL in spite of the different properties of these lysozymes (i.e. in respect of pI, enzyme kinetics and heat stability) reported in the present study.     

Host Protein BSL1 Associates with Phytophthora infestans RXLR Effector AVR2 and the Solanum demissum Immune Receptor R2 to Mediate Disease Resistance

Plant pathogens secrete effector proteins to modulate plant immunity and promote host colonization. Plant nucleotide binding leucine-rich repeat (NB-LRR) immunoreceptors recognize specific pathogen effectors directly or indirectly. Little is known about how NB-LRR proteins recognize effectors of filamentous plant pathogens, such as Phytophthora infestans. AVR2 belongs to a family of 13 sequence-divergent P. infestans RXLR effectors that are differentially recognized by members of the R2 NB-LRR family in Solanum demissum. We report that the putative plant phosphatase BSU-LIKE PROTEIN1 (BSL1) is required for R2-mediated perception of AVR2 and resistance to P. infestans. AVR2 associates with BSL1 and mediates the interaction of BSL1 with R2 in planta, possibly through the formation of a ternary complex. Strains of P. infestans that are virulent on R2 potatoes express an unrecognized form, Avr2-like (referred to as A2l). A2L can still interact with BSL1 but does not promote the association of BSL1 with R2. Our findings show that recognition of the P. infestans AVR2 effector by the NB-LRR protein R2 requires the putative phosphatase BSL1. This reveals that, similar to effectors of phytopathogenic bacteria, recognition of filamentous pathogen effectors can be mediated via a host protein that interacts with both the effector and the NB-LRR immunoreceptor.     

Specific spin-labeling of transfer ribonucleic acid molecules.

The spin labels anhydride (ASL), bromoacetamide (BSL) and carbodiimide (CSL) were used to label selectively tRNAGlu, tRNA fMet and tRNAPhe from E. coli. The preparation and characterization of the sites of labeling of eight new spin-labeled tRNAs are described. The sites of labeling are: s2U using ASL, BSL and CLS and tRNAGlu; s4U using ASL and BSL on tRNAfMet and tRNAPhe; U-37 with CSL on tRNfMet; U-33 with CSL on tRNAPhe. The rare base X at position 47 of tRNAPhe has been acylated with a spin-labeled N-hydroxysuccinimide (HSL). The 3'end of unfractionated tRNA molecules has been chemically modified to a morpholino spin-labeled analogue (MSL). Their respective e.s.r. spectra are reported and discussed.     

Guidelines for the prevention of simian immunodeficiency virus infection in laboratory workers and animal handlers

The authors are members of a working group that formulated guidelines to minimize transmission of simian immunodeficiency virus (SIV) infection to man. Biosafety level (BSL) 2 standards are recommended for handling of clinical specimens and housing of SIV inoculated animals. Manipulation of SIV preparations may be performed in a BSL 2 facility with additional BSL 3 practices and equipment; for large volume or concentrated preparations of SIV BSL 3 containment is necessary. Written policies regarding management and testing of workers exposed to SIV are recommended.     

A new bifunctional spin-label suitable for saturation-transfer EPR studies of protein rotational motion

A new bifunctional spin-label (BSL) has been synthesized that can be immobilized on the surface of proteins, allowing measurement of rotational motion of proteins by saturation-transfer electron paramagnetic resonance (STEPR). The spin-label contains a photoactivatable azido moiety, a cleavable disulfide, and a nitroxide spin with restricted mobility relative to the rest of the label. The label reacts with surface lysine residues modified with beta-mercaptopropionate. Bifunctional attachment is achieved by photoactivation of the azido group. Any spin-label that remains monofunctionally attached after photolysis is removed by reduction of the disulfide. Only bifunctionally attached BSL remains on the protein. Hemoglobin was used to test the utility of the BSL in STEPR by comparison with hemoglobin modified with maleimide spin-label (MSL), a commonly used standard for the STEPR technique. MSL is a monofunctional spin-label which is fortuitously immobilized by local protein structure within hemoglobin. The BSL labeling of hemoglobin did not significantly affect the quaternary structure of hemoglobin as determined by gel filtration chromatography. The conventional EPR spectra of the mono- and bifunctionally attached BSL-hemoglobin were similar to the MSL-hemoglobin spectrum, indicating that both forms of BSL were rigidly bound to hemoglobin. In contrast, the spectrum obtained by reaction of modified hemoglobin lysine residues with MSL indicated that these labels were highly mobile. The monofunctionally attached BSL was mobilized upon octyl glucoside addition whereas bifunctionally attached BSL was only slightly mobilized, suggesting that hydrophobic interactions immobilize the monofunctionally attached label on hemoglobin. The response of STEPR spectra of mono- and bifunctionally attached BSL-hemoglobin to changes in hemoglobin rotational correlation time was similar to the MSL-hemoglobin over the range of 10(-5)-10(-3) s. The spectra of bifunctionally attached BSL indicated slightly less motion than corresponding spectra for MSL or monofunctionally attached BSL. The new BSL is a good reporter of protein rotation and does not require unique protein structures for its immobilization on the protein. Thus, the BSL should be more generally applicable for STEPR studies of membrane protein rotation than existing monofunctional spin-labels.     

Exploring of Primate Models of Tick-Borne Flaviviruses Infection for Evaluation of Vaccines and Drugs Efficacy

Tick-borne encephalitis virus (TBEV) is one of the most prevalent and medically important tick-borne arboviruses in Eurasia. There are overlapping foci of two flaviviruses: TBEV and Omsk hemorrhagic fever virus (OHFV) in Russia. Inactivated vaccines exist only against TBE. There are no antiviral drugs for treatment of both diseases. Optimal animal models are necessary to study efficacy of novel vaccines and treatment preparations against TBE and relative flaviviruses. The models for TBE and OHF using subcutaneous inoculation were tested in Cercopithecus aethiops and Macaca fascicularis monkeys with or without prior immunization with inactivated TBE vaccine. No visible clinical signs or severe pathomorphological lesions were observed in any monkey infected with TBEV or OHFV. C. aethiops challenged with OHFV showed massive hemolytic syndrome and thrombocytopenia. Infectious virus or viral RNA was revealed in visceral organs and CNS of C. aethiops infected with both viruses; however, viremia was low. Inactivated TBE vaccines induced high antibody titers against both viruses and expressed booster after challenge. The protective efficacy against TBE was shown by the absence of virus in spleen, lymph nodes and CNS of immunized animals after challenge. Despite the absence of expressed hemolytic syndrome in immunized C. aethiops TBE vaccine did not prevent the reproduction of OHFV in CNS and visceral organs. Subcutaneous inoculation of M. fascicularis with two TBEV strains led to a febrile disease with well expressed viremia, fever, and virus reproduction in spleen, lymph nodes and CNS. The optimal terms for estimation of the viral titers in CNS were defined as 8–16 days post infection. We characterized two animal models similar to humans in their susceptibility to tick-borne flaviviruses and found the most optimal scheme for evaluation of efficacy of preventive and therapeutic preparations. We also identified M. fascicularis to be more susceptible to TBEV than C. aethiops.     

Potential role for microbial ureolysis in the rapid formation of carbonate tufa mounds

Modern carbonate tufa towers in the alkaline (~pH 9.5) Big Soda Lake (BSL), Nevada, exhibit rapid precipitation rates (exceeding 3 cm/year) and host diverse microbial communities. Geochemical indicators reveal that carbonate precipitation is, in part, promoted by the mixing of calcium-rich groundwater and carbonate-rich lake water, such that a microbial role for carbonate precipitation is unknown. Here, we characterize the BSL microbial communities and evaluate their potential effects on carbonate precipitation that may influence fast carbonate precipitation rates of the active tufa mounds of BSL. Small subunit rRNA gene surveys indicate a diverse microbial community living endolithically, in interior voids, and on tufa surfaces. Metagenomic DNA sequencing shows that genes associated with metabolisms that are capable of increasing carbonate saturation (e.g., photosynthesis, ureolysis, and bicarbonate transport) are abundant. Enzyme activity assays revealed that urease and carbonic anhydrase, two microbial enzymes that promote carbonate precipitation, are active in situ in BSL tufa biofilms, and urease also increased calcium carbonate precipitation rates in laboratory incubation analyses. We propose that, although BSL tufas form partially as a result of water mixing, tufa-inhabiting microbiota promote rapid carbonate authigenesis via ureolysis, and potentially via bicarbonate dehydration and CO₂ outgassing by carbonic anhydrase. Microbially induced calcium carbonate precipitation in BSL tufas may generate signatures preserved in the carbonate microfabric, such as stromatolitic layers, which could serve as models for developing potential biosignatures on Earth and elsewhere.     

Direct evidence for RNA–RNA interactions at the 3′ end of the Hepatitis C virus genome using surface plasmon resonance

Surface plasmon resonance was used to investigate two previously described interactions analyzed by reverse genetics and complementation mutation experiments, involving 5BSL3.2, a stem–loop located in the NS5B coding region of HCV. 5BSL3.2 was immobilized on a sensor chip by streptavidin-biotin coupling, and its interaction either with the SL2 stem–loop of the 3′ end or with an upstream sequence centered on nucleotide 9110 (referred to as Seq9110) was monitored in real-time. In contrast with previous results obtained by NMR assays with the same short RNA sequences that we used or SHAPE analysis with longer RNAs, we demonstrate that recognition between 5BSL3.2 and SL2 can occur in solution through a kissing-loop interaction. We show that recognition between Seq9110 and the internal loop of 5BSL3.2 does not prevent binding of SL2 on the apical loop of 5BSL3.2 and does not influence the rate constants of the SL2-5BSL3.2 complex. Therefore, the two binding sites of 5BSL3.2, the apical and internal loops, are structurally independent and both interactions can coexist. We finally show that the stem–loop SL2 is a highly dynamic RNA motif that fluctuates between at least two conformations: One is able to hybridize with 5BSL3.2 through loop–loop interaction, and the other one is capable of self-associating in the absence of protein, reinforcing the hypothesis of SL2 being a dimerization sequence. This result suggests also that the conformational dynamics of SL2 could play a crucial role for controlling the destiny of the genomic RNA.     

A cis-acting replication element in the sequence encoding the NS5B RNA-dependent RNA polymerase is required for hepatitis C virus RNA replication

RNA structures play key roles in the replication of RNA viruses. Sequence alignment software, thermodynamic RNA folding programs, and classical comparative phylogenetic analysis were used to build models of six RNA elements in the coding region of the hepatitis C virus (HCV) RNA-dependent RNA polymerase, NS5B. The importance of five of these elements was evaluated by site-directed mutagenesis of a subgenomic HCV replicon. Mutations disrupting one of the predicted stem-loop structures, designated 5BSL3.2, blocked RNA replication, implicating it as an essential cis-acting replication element (CRE). 5BSL3.2 is about 50 bases in length and is part of a larger predicted cruciform structure (5BSL3). As confirmed by RNA structure probing, 5BSL3.2 consists of an 8-bp lower helix, a 6-bp upper helix, a 12-base terminal loop, and an 8-base internal loop. Mutational analysis and structure probing were used to explore the importance of these features. Primary sequences in the loops were shown to be important for HCV RNA replication, and the upper helix appears to serve as an essential scaffold that helps maintain the overall RNA structure. Unlike certain picornavirus CREs, whose function is position independent, 5BSL3.2 function appears to be context dependent. Understanding the role of 5BSL3.2 and determining how this new CRE functions in the context of previously identified elements at the 5' and 3' ends of the RNA genome should provide new insights into HCV RNA replication.     

Behavior of Lactobacilli Isolated from Fermented Slurry (ben-saalga) in Gnotobiotic Rats

Most bacterial strains, which have been studied so far for their probiotic functions, are extensively used by manufacturers in developed countries. In our work, we sought to study a mix (called BSL) comprising three strains belonging to Lactobacillus fermentum, L. paraplantarum and L. salivarius, that were isolated from a traditional African pearl millet based fermented slurry. Our objective was to study this BSL cocktail in gnotobiotic rats, to evaluate their survival and their behavior in the digestive tract conditions. After a single oral inoculation of germfree rats with BSL, the species established stably in the digestive tract with the following hierarchy of abundance: L. salivarius> L. plantarum> L. fermentum. BSL cocktail was metabolically active since it produced 50 mM lactate and it expressed genes involved in binding mechanism in the caecum. Furthermore, the global morphology of the colon epithelium was not disturbed by the BSL cocktail. BSL cocktail did not modify mucus content and host mucus-related genes (MUC1, MUC2, MUC3 or resistin-like molecule β). The cocktail of lactobacilli enhanced the proliferating cell nuclear antigen (PCNA) at a level comparable to what was observed in conventional rats. PCNA was involved in proliferation and DNA repair, but the presence of the cocktail did not provoke proliferative events (with Ki67 as indicator), so we suppose BSL may help gut preservation. This work is the first step towards the selection of strains that are derived from traditional fermented food to formulate new probiotic mixture.