Μg Streptomycin Research Articles

First Report of Diaporthe tectonendophytica Causing Leaf Spot on Dalbergia odorifera in Guangxi, China.

Dalbergia odorifera T. Chen (Family: Fabaceae) is a national level II protected plant in China, with extremely high economic value and medical properties (Zhao et al. 2020). In June 2023, an unknown leaf spot was found in a garden land of Pingxiang city, Guangxi, China, and approximately 80% of the plants covered an area of 500 m2 displayed similar symptoms. The spots were grey to white, 4~6 mm in diameter (n=30) with black pycnida on the spots surface (Fig S1, A-D). Multiple disease spots were observed on a single leaf. The pycnida on the lesion were picked and mashed, to make a conidia suspension using sterile water. The conidial solution was then spread onto a potato dextrose agar (PDA) plate containing streptomycin, with 10 mg of streptomycin per 100 mL, and incubated for 3 days at 28°C with a 12 hour photoperiod. Three isolates (GXPX01, GXPX02 and GXPX03) were obtained by re-culturing the colonies on fresh PDA plates. The colony on PDA were white with aerial mycelia (Fig S1, E-F). Black conidiomata developed at 28°C with a 12 hour photoperiod in 20 days (Fig S1, G-H). Alpha conidia were 4.2~6.4 μm × 1.8~2.6 μm (average =5.1 × 2.3 μm, n = 30), mostly bi-guttulate, hyaline, ellipsoid, apex bluntly rounded, base obtuse to subtruncate, smooth (Fig S1, I). Beta conidia were 15.1~33.5 μm × 1~1.8 μm (average = 24.5 × 1.5 μm, n = 30), filiform, hyaline, curved or hamate, aseptate, base subtruncate (Fig S1, J). Morphological characteristics of the three isolates matched those of Diaporthe spp.(Gomes et al. 2013). The rDNA internal transcribed spacer (ITS) region, the translation elongation factor 1-α (TEF1), the calmodulin (CAL), the histone H3 (HIS) and the β-tubulin (TUB2) genes of the three isolates were amplified using the primer pairs ITS4/ITS5, EF1-728F/EF1-986R, CAL-228F/CAL2Rd, CYLH3F/H3-1B, and T1 /CYLTUB1R, respectively (Crous et al. 2004, Sun et al. 2021). The sequences were all deposited in GenBank (accession numbers OR437511 to OR437513 for ITS, OR454965 to OR454967 for TEF1, OR454968 to OR454970 for CAL, OR454971 to OR454973 for TUB2, OR454974 to OR454976 for H3). Sequences had 98.36% to 100% homology with the corresponding sequences of known Diaporthe tectonendophytica strains MFLUCC 13-0471 in the NCBI database. Phylogenetic analysis was based on combined ITS, TEF1, TUB2 and CAL sequences data using MEGA 11 software to construct phylogenetic tree with Maximum Likelihood (Doilom et al. 2017). In the phylogenetic tree, the combined sequences attributed the three isolates to the D. tectonendophytica (Fig S2). The pathogenicity was tested on leaves of 1.5-year-old D. odorifera seedlings. Three leaves were wounded with a sterile needle and individually inoculated with a 5 mm mycelial disk of PDA culture from each isolate. Sterile PDA disks inoculated leaves as a control. The test was repeated three times. The inoculated plants were placed in a greenhouse at 25℃ and 90% humidity, with a photoperiod of 12 hours. Five days after inoculation, necrotic lesions appeared on inoculated leaves and symptoms from all three isolates were the same as those form natural infections ( Fig S1, K-N), whereas all the control remained symptomless (Fig S1, P). The pathogen was reisolated from the inoculated leaves and again identified as D. tectonendophytica, with the same methodology used for the initial identification. D. tectonendophytica was reported to cause plant diseases, such as stem gray blight of red-fleshed dragon fruit (Hylocereus polyrhizus) (Rahim et al. 2021), leaf spots disease on Elaeagnus conferta and Pometia pinnata (Sun et al. 2021). To our knowledge, this is the first report of D. ctonendophytica causing leaf spot disease on D. odorifera.

Effects of Origanum majorana essential oil and antibiotics on the quality of frozen thawed Beni Arouss buck semen.

This study aims to investigate the effects of Origanum majorana (OM) essential oil (EO) at different concentrations and antibiotics on post-thawed Beni Arouss buck semen quality. Semen collection was performed using artificial vagina from eight Beni Arouss bucks. Ejaculates were pooled, divided into 12 equal aliquots, washed and diluted to 400 × 106 sperm/ml (with 7% of glycerol). Skim milk-based extender was supplemented with different concentrations of OM EO (0%; 0.01%; 0.02%; 0.03%; 0.04% and 0.05%) without antibiotics, marked as (CTR-), (M1-), (M2-), (M3-), (M4-) and (M5-), and with 50 mg of streptomycin and 50,000 IU of penicillin per 100 ml, marked as (CTR+), (M1+), (M2+), (M3+), (M4+) and (M5+), respectively. Aliquots were cooled to 4°C, then frozen in 0.25 ml straws with a programmable freezer and finally stored in liquid nitrogen for 48 h. Thawing was performed at 37°C for 30 s. Motility, live sperm, sperm abnormalities, membrane integrity, lipid peroxidation and bacterial growth were evaluated after thawing. Among the tested extenders, M2+ improved all semen quality parameters. Sperm motility, live sperm and membrane integrity increased significantly, while the number of abnormal sperm and bacterial growth decreased significantly. The toxic effect of OM EO, with and without antibiotics, appeared beyond 0.03%. In conclusion, M2+ is recommended to improve the cryopreservation of Beni Arouss buck semen.

A Selective Medium for the Recovery and Enumeration of Fomitopsis meliae, Causing Lemon Canker and Brown Wood Rot

A selective medium was developed for the isolation and quantification of Fomitopsis meliae (FMS medium), the causal agent of lemon canker and brown wood rot, from plants, soil, and air. The optimal concentration and combination of fungicides and antibiotics was evaluated to determine the most selective condition for growing F. meliae. The resultant composition of the FMS medium per liter (pH 3.5) was 16 mg of thiophanate-methyl, 8 mg of dichloran, 5 mg of 2-phenylphenol, 100 mg of fluopyram, 0.5 mg of fludioxonil, 100 mg of chloramphenicol, 100 mg of streptomycin, 15 g of malt extract, 2.5 g of mycological peptone, and 15 g of agar. The fungus was successfully isolated and enumerated from air, soil, and plant tissues using FMS medium. Furthermore, FMS medium almost completely inhibited the growth of other plant-pathogenic fungi and soil and air saprophytes. This selectivity was high enough to estimate spore inoculum of F. meliae in an air sample or as a spore-trapping device in commercial lemon orchards. FMS medium will be useful for studying epidemiology and management of F. meliae.

Isolation and identification of microscopic fungi of agricultural importance in the Tetillas hill in Salvatierra, Guanajuato

This research was throughout 2018-2020, filamentous fungi of agricultural importance were isolated from the Cerro Las Tetillas, located in the municipality of Salvatierra, Guanajuato. The hill was divided into three strata: upper, middle and lower. Two hundred soil samples were taken at depths of 0-15 and 15-30 cm. These were processed in the biology laboratory, belonging to the Higher Technological Institute of Salvatierra. Two isolation techniques were used: direct seeding and serial dilutions, over Petri dishes with potato dextrose agar culture medium and 100 mg of streptomycin to control bacteria. One hundred and thirteen axenic strains corresponding to 16 genera identified from “Cerro Tetillas” were obtained. At the upper stratum, 13 genera were identified, while in the middle and lower strata, nine genera were identified in each one of them. The diversity of soil fungi found in the different strata of the hill may be due to physicochemical characteristics of soil, native vegetation, interaction and abundance of soil fungi species, human disturbance and rain runoff.

Antibiotic Susceptibility Patterns of Pseudomonas spp. Isolated from Soil Samples

Antimicrobial resistance is one of the major problems facing the health sector. This has to do with many factors including changes in metabolic pathways and transfer of virulence genes among bacterial species among others. Therefore, this study aimed at testing the antibiotic susceptibility patterns of Pseudomonas spp. isolated from soil samples using the mastering-S (M13 and M14 disc). In this study, the susceptibility of 25 pseudomonas isolated from soil environment were tested using the mastering-S (13 and 14 disc). Overnight KB* cultures were used to inoculate KB* plates using the spread plate technique before placing the antibiotic disc on the surface of the agar and incubated at 20oC for 48hours. Antibiotic susceptibility was recorded based on the diameter of zone of inhibition. A diameter of 8mm and above was recorded as positive (susceptible) and a value of less than 8mm as negative (resistance). The results show that, all 25 isolates (100%) tested resistant to at least eight (8) antibiotics, while all the strains were susceptible to 10 µg Gentamicin (GM). Similarly, at least 8% were susceptible to 25 µg Chloramphenicol (C), 96% are susceptible to 10 µg Streptomycin (S) and about 92%, 40% and 48% of the isolates were found to be susceptible to 25 µg Tetracycline (T), 25 µg Colistin Sulphate (CO) and 200 µg Sulphatriad (ST) respectively. Therefore, the study concludes that different species of pseudomonad may respond to antibiotic differently and this should be considered especially when selecting drug of choice in medical settings.

Prevalence and comparison of high-level aminoglycoside resistance in vancomycin-sensitive and vancomycin resistant enterococcus at a tertiary care hospital in Rohillkhand region

Introduction: Enterococci, originally commensals of gastrointestinal tract, have now become important nosocomial pathogens. The serious infections caused by Enterococci were empirically treated with bactericidal combination of beta lactam drugs and aminoglycoside neither of which are effective alone [1]. But the presence of high level aminoglycoside resistance (HLAR) makes this treatment ineffective. The emergence of VRE (Vancomycin Resistant Enterococcus) strains also resistant to ampicillin and HLAR has made the treatment further difficult. The spread of such strains in a health care environment has to be constrained and so the present study was carried out to highlight the occurrence of HLAR strains and VRE strains in our hospital .The frequency of association of HLAR with VSE (Vancomycin sensitive Enterococci) and VRE (Vancomycin resistant Enterococci) and any significant difference between the two was also studied. Methods: A total of 100 Enterococci were isolated from 6272 clinical specimen during a study period of 1 year. These strains were speciated on the basis of biochemical tests. Antimicrobial susceptibility test was performed by Kirby baeur disc diffusion method. High level Aminoglycoside resistance was tested by two methods 1) 150μg gentamicin and 200 μg streptomycin disc 2) Agar screening method. Similarly, strains resistant to Vancomycin by disc diffusion method were subjected to confirmatory test by determining their MIC by Agar dilution. Results: A total of 100 Enterococci were isolated during a study period of one year. The prevalence of HLAR was found to be 49%.The prevalence of VRE was found to be 12%.VRE strains also possessing resistance to High strength Gentamycin and or streptomycin was 8%.The frequency of association of HLAR in VRE was 1.5 times higher as compared to VSE. Conclusion: The prevalence of High level gentamicin and high level streptomycin resistance amongst Enterococcal isolates in our institute was high. More enterococcal strains were found to be resistant to both gentamicin and streptomycin than to gentamicin or streptomycin alone. The HLAR rate in VRE was 1.5 times higher than in VSE.

Effects of Degasified Extender on Quality Parameters of Cryopreserved Bull Spermatozoa

One of the most important factors contributing to poor quality semen post thaw has been reported to be oxidative stress. Continued interaction of spermatozoa with dissolved oxygen in extender renders detrimental effect by generating reactive oxygen species during processing. This formed the basis to investigate the effects of removal of oxygen from the Tris-Glycerol-Egg Yolk (TG-EY) extender on the post-thaw semen quality. Semen ejaculates (n=26) collected from three cross bred bulls with mass activity of ≥3 and progressive motility of ≥70% were split in two fractions (control and treatment) to obtain 80 x 106 progressively motile sperm/mL of extended semen. The TG-EY extender composed of 3.028 g of Tris, 1.675 g of citric acid, 1.25 g of fructose, 7.0 mL of glycerol, 10 mL of egg yolk, 105 IU Penicillin G Sodium and 105 µg Streptomycin for 100 mL of deionized water for control and degasified (freeze-pump-thaw cycling method using liquid nitrogen) for treatment. Semen was processed and filled in 0.25 mL polyvinyl chloride straws, sealed and cryopreserved for at least 2 week before evaluation. Assessment of post-thaw motility, viability, acrosome and plasma membrane integrity of spermatozoa were evaluated after thawing at 37°C for 30s. Study revealed a significant (viability and acrosome integrity, p

Aminoglycoside inhibition of Staphylococcus aureus biofilm formation is nutrient dependent

Biofilms represent microbial communities, encased in a self-produced matrix or extracellular polymeric substance. Microbial biofilms are likely responsible for a large proportion of clinically significant infections and the multicellular nature of biofilm existence has been repeatedly associated with antibiotic resistance. Classical in vitro antibiotic-susceptibility testing utilizes artificial growth media and planktonic microbes, but this method may not account for the variability inherent in environments subject to biofilm growth in vivo. Experiments were designed to test the hypothesis that nutrient concentration can modulate the antibiotic susceptibility of Staphylococcus aureus biofilms. Developing S. aureus biofilms initiated on surgical sutures, and in selected experiments planktonic cultures, were incubated for 16 h in 66 % tryptic soy broth, 0.2 % glucose (1× TSBg), supplemented with bactericidal concentrations of gentamicin, streptomycin, ampicillin or vancomycin. In parallel experiments, antibiotics were added to growth medium diluted one-third (1/3× TSBg) or concentrated threefold (3× TSBg). Following incubation, viable bacteria were enumerated from planktonic cultures or suture sonicates, and biofilm biomass was assayed using spectrophotometry. Interestingly, bactericidal concentrations of gentamicin (5 µg gentamicin ml(-1)) and streptomycin (32 µg streptomycin ml(-1)) inhibited biofilm formation in samples incubated in 1/3× or 1× TSBg, but not in samples incubated in 3× TSBg. The nutrient dependence of aminoglycoside susceptibility is not only associated with biofilm formation, as planktonic cultures incubated in 3× TSBg in the presence of gentamicin also showed antibiotic resistance. These findings appeared specific for aminoglycosides because biofilm formation was inhibited in all three growth media supplemented with bactericidal concentrations of the cell wall-active antibiotics, ampicillin and vancomycin. Additional experiments showed that the ability of 3× TSBg to overcome the antibacterial effects of gentamicin was associated with decreased uptake of gentamicin by S. aureus. Uptake is known to be decreased at low pH, and the kinetic change in pH of growth medium from biofilms incubated in 5 µg gentamicin ml(-1) in the presence of 3× TSBg was decreased when compared with pH determinations from biofilms formed in 1/3× or 1× TSBg. These studies underscore the importance of environmental factors, including nutrient concentration and pH, on the antibiotic susceptibility of S. aureus planktonic and biofilm bacteria.

Leukotriene B4 BLT Receptor Signaling Regulates the Level and Stability of Cyclooxygenase-2 (COX-2) mRNA through Restricted Activation of Ras/Raf/ERK/p42 AUF1 Pathway

Recent studies suggest that active resolution of the inflammatory response in animal models of arthritis may involve leukotriene B(4) (LTB(4))-dependent stimulation of "intermediate" prostaglandin production, which in turn favors the synthesis of "downstream" anti-inflammatory and pro-resolving lipoxins, resolvins, and protectins. We explored a putative mechanism involving LTB(4)-dependent control of cyclooxygenase-2 (COX-2) expression, the rate-limiting step in inflammatory prostaglandin biosynthesis. Indeed, LTB(4) potently up-regulated/stabilized interleukin-1beta-induced COX-2 mRNA and protein expression under conditions of COX-2 inhibitor-dependent blockade of PGE(2) release in human synovial fibroblasts (EC(50) = 16.5 + or - 1.7 nm for mRNA; 19 + or - 2.4 nm for protein, n = 4). The latter response was pertussis toxin-sensitive, and semi-quantitative reverse transcription-PCR confirmed the quantitative predominance of the BLT2 receptor. Transfection experiments, using human COX-2 promoter plasmids and chimeric luciferase-COX-2 mRNA 3'-untranslated region (3'-UTR) reporter constructs, revealed that LTB(4) exerted its stabilizing effect at the post-transcriptional level through a 116-bp adenylate/uridylate-rich sequence in the proximal region of the COX-2 3'-UTR. Using luciferase-COX-2 mRNA 3'-UTR reporter constructs and Ras/c-Raf expression and mutant constructs, we showed that the Ras/c-Raf/MEK1/2/ERK1/2 signaling pathway mediated LTB(4)-dependent COX-2 mRNA stabilization. Knockdown experiments with specific short hairpin RNAs confirmed that LTB(4) stabilization of COX-2 mRNA was apparently mediated through the RNA-binding protein, p42 AUF1. The nuclear export of p42 AUF1 was driven by c-Raf/MEK1/2/ERK1/2 signaling and sensitive to leptomycin B treatment, suggesting a CRM1-dependent mechanism. We conclude that LTB(4) may support the resolution phase of the inflammatory response by stabilizing COX-2, ensuring a reservoir of ambient pro-resolution lipid mediators.

Effect of serum albumin supplementation on in vitro capacitation and fertilization of caprine oocytes

The aim of the investigation was to study the effect of purity and the type of serum albumin on in vitro fertilization (IVF) and cleavage rate of in vitro matured goat oocytes. Ovaries were collected from the local abattoir and transported within 4 h to the laboratory in warm saline (37 °C) containing 100 IU penicillin-G and 100 μg streptomycin sulfate per ml. A total of 2509 cumulus oocyte complexes (COCs) were collected from 1313 ovaries. Oocytes were matured in TCM-199 medium containing FSH (5 μg/ml), LH (5 μg/ml) and estradiol-17β (1 μg/ml), supplemented with 20% fetal bovine serum at 38.5 °C and 5% CO 2 in an incubator under humidified air for 27 h. After 27 h of in vitro maturation (IVM), oocytes were denuded, washed and randomly divided into 4 groups. Group 1 consisted of in vitro matured oocytes ( n = 627) co-incubated with sperm in a 50 μl drop of TALP medium containing a 6 mg/ml crystalline bovine serum albumin (BSA) fraction V and 10 μg/ml heparin. Group 2 was comprised of in vitro matured oocytes ( n = 470), co-incubated with sperm in a 50 μl drop of TALP medium containing 3 mg/ml crystalline BSA fraction V, 10% estrous goat serum and 10 μg/ml heparin. Group 3 was comprised of in vitro matured oocytes ( n = 489) co-incubated with sperm in a 50 μl drop of TALP medium containing a 6 mg/ml fatty acid free BSA and 10 μg/ml heparin. Group 4 consisted of in vitro matured oocytes ( n = 422) co-incubated with sperm in a 50 μl drop of TALP medium containing 20% estrous goat serum and 10 μg/ml heparin. After 18 h of co-incubation, the oocyte–sperm mixture was washed in the culture medium 15–20 times and cultured in 50 μl EDM. Cleavage of the in vitro fertilized oocytes were recorded 48 h post-insemination under an inverted phase contrast microscope. The average oocyte recovery rate/ovary and maturation rate was 1.91% and 80.03%, respectively. The cleavage rate in Group 1, Group 2, Group 3 and Group 4 was 1.59%, 8.93%, 11.86% and 35.30%, respectively. It could be concluded that the use of fatty acid free albumin resulted in a significantly higher ( P < 0.05) cleavage rate, compared to unmodified albumin, and the supplementation of 20% estrous goat serum in the fertilization medium, significantly ( P < 0.05) increased the cleavage rate of in vitro matured goat oocytes, compared to defatted albumin.

Mouse-Passaged Severe Acute Respiratory Syndrome-Associated Coronavirus Leads to Lethal Pulmonary Edema and Diffuse Alveolar Damage in Adult but Not Young Mice

Advanced age is a risk factor of severe acute respiratory syndrome (SARS) in humans. To understand its pathogenesis, we developed an animal model using BALB/c mice and the mouse-passaged Frankfurt 1 isolate of SARS coronavirus (SARS-CoV). We examined the immune responses to SARS-CoV in both young and adult mice. SARS-CoV induced severe respiratory illness in all adult, but not young, mice on day 2 after inoculation with a mortality rate of 30 to 50%. Moribund adult mice showed severe pulmonary edema and diffuse alveolar damage accompanied by virus replication. Adult murine lungs, which had significantly higher interleukin (IL)-4 and lower IL-10 and IL-13 levels before infection than young murine lungs, rapidly produced high levels of proinflammatory chemokines and cytokines known to induce macrophage and neutrophil infiltration and activation (eg, tumor necrosis factor-alpha). On day 2 after inoculation, young murine lungs produced not only proinflammatory cytokines but also IL-2, interferon-gamma, IL-10, and IL-13. Adult mice showed early and acute excessive proinflammatory responses (ie, cytokine storm) in the lungs after SARS-CoV infection, which led to severe pulmonary edema and diffuse alveolar damage. Intravenous injection with anti-tumor necrosis factor-alpha antibody 3 hours after infection had no effect on SARS-CoV infection. However, intraperitoneal interferon-gamma injection protected adult mice from the lethal respiratory illness. The experimental model described here may be useful for elucidating the pathophysiology of SARS and for evaluating therapies to treat SARS-CoV infection.

T-2 Toxin Regulated Ganoderma lucidum Induced Cytokine Release

The water-soluble extract of Ganoderma lucidum (Reishi) has been used as immunomodulator to stimulate spleen cells proliferation and cytokine expression. It has also been shown that at some level of exposure, T-2 toxin typically act as immunosuppressive agent and can increase disease susceptibility. The aim of this study was to investigate the effect of T-2 toxin on cytokine production by Ganoderma lucidum (G. lucidum) treated-cells. Mice peritoneal macrophages and lymphoid T cells were prepared by usual manner and plated out at 1×10 6 or 1×10 4 cell/well respectively in RPMI 1640 supplemented with 10% FCS, 50 µg streptomycin and 50U penicillin. Cells were incubated with different concentrations of G. lucidum in the presence or absence of 1 ng mL -1 T-2 toxin at 37°C and 5% CO2 for 48 h. Cell free medium was removed and used for cytokine assay by ELISA method. The results showed that T-2 toxin in the absence of G.lucidum enhanced IL-2, IFN-γ release compared with control group, but it reduced the production of other cytokines. G. lucidum enhanced the production of IL-1β, TNF-α, IL-12, IL-2 and IFN-γ compared with control group, but reduced IL-4 and IL-10 release. T-2 toxin, up regulated the enhancement effect of G. lucidum on IFN- γ, IL-2 and TNF- α, but it down regulated its effect on the production of other cytokines. In conclution our results indicate that T-2 toxin at 1 ng mL -1 may augment the immunomodulating effects of G. lucidum on cytokine release.

Caracterização de isolados de Acidovorax avenae subsp. citrulli

Foram caracterizados 41 isolados de Acidovorax avenae subsp. citrulli com base em aspectos fisiológicos e bioquímicos. Todos os isolados induziram sintomas típicos da mancha-aquosa em plântulas, plantas e frutos de meloeiro (Cucumis melo) e melancieira (Citrullus lanatus). Pelo teste de agrupamento de Scott-Knott (P = 0,05) os isolados foram separados quanto ao índice de doença em 5 e 7 grupos, respectivamente para plântulas de meloeiro e melancieira, e em 2 grupos para plantas das duas hospedeiras. Em frutos, os isolados foram separados em 3 e 10 grupos para a variável diâmetro da lesão externa e 2 e 9 grupos para profundidade da lesão, respectivamente para melão e melancia. Todos os isolados induziram reação de hipersensibilidade em fumo (Nicotiana tabacum); utilizaram os compostos asparagina, L-leucina e DL-ácido lático; produziram enzimas lipolíticas e o fitohormônio ácido indol acético; foram sensíveis a oxicloreto de cobre (120 µg mL-1), óxido cuproso (120 µg mL-1), hidróxido de cobre (138,2 µg mL-1), sulfato de estreptomicina (25 µg mL-1) e Agrimaicin 500 (428 µg mL-1); e resistentes a kasugamicina (87 µg mL-1), agrimicina (200 µg mL-1), eritromicina (15 µg), gentamicina (10 µg), amoxicilina (10 µg), neomicina (30 µg), estreptomicina (10 µg), norfloxacina (10 µg) e rifampicina (5 µg). Nenhum isolado apresentou atividade pectinolítica, amilolítica, celulolítica e proteolítica ou produção do polissacarídeo levana e da toxina siringomicina. Foi constatada variabilidade entre os 41 isolados de A. avenae subsp. citrulli quanto à sensibilidade à tetraciclina (30 µg), sendo 41,5% resistentes, 46,3% moderadamente sensíveis e 12,2% altamente sensíveis.

Y Box-binding Protein-1 Binds to the Dengue Virus 3′-Untranslated Region and Mediates Antiviral Effects

Dengue virus, a member of the family Flaviviridae, poses a serious public health threat worldwide. Dengue virus is a positive-sense RNA virus that harbors a genome of approximately 10.7 kb. Replication of dengue virus is mediated coordinately by cis-acting genomic sequences, viral proteins, and host cell factors. We have isolated and identified several host cell factors from baby hamster kidney cell extracts that bind with high specificity and high affinity to sequences within the untranslated regions of the dengue virus genome. Among the factors identified, Y box-binding protein-1 (YB-1) and the heterogeneous nuclear ribonucleoproteins (hnRNPs), hnRNP A1, hnRNP A2/B1, and hnRNP Q, bind to the dengue virus 3'-untranslated region. Further analysis indicated that YB-1 binds to the dengue virus 3' stem loop, a conserved structural feature located at the 3' terminus of the 3'-untranslated region of many flaviviruses. Analysis of the impact of YB-1 on replication of dengue virus in YB-1+/+ and YB-1-/- mouse embryo fibroblasts indicated that host YB-1 mediates an antiviral effect. Further studies demonstrated that this antiviral impact is due, at least in part, to a repressive role of YB-1 on dengue virus translation via a mechanism that requires viral genomic sequences. These results suggest a novel role for YB-1 as an antiviral host cell factor.

The Spatial Distribution of Inositol 1,4,5-Trisphosphate Receptor Isoforms Shapes Ca2+ Waves

Cytosolic Ca(2+) is a versatile second messenger that can regulate multiple cellular processes simultaneously. This is accomplished in part through Ca(2+) waves and other spatial patterns of Ca(2+) signals. To investigate the mechanism responsible for the formation of Ca(2+) waves, we examined the role of inositol 1,4,5-trisphosphate receptor (InsP3R) isoforms in Ca(2+) wave formation. Ca(2+) signals were examined in hepatocytes, which express the type I and II InsP3R in a polarized fashion, and in AR4-2J cells, a nonpolarized cell line that expresses type I and II InsP3R in a ratio similar to what is found in hepatocytes but homogeneously throughout the cell. Expression of type I or II InsP3R was selectively suppressed by isoform-specific DNA antisense in an adenoviral delivery system, which was delivered to AR4-2J cells in culture and to hepatocytes in vivo. Loss of either isoform inhibited Ca(2+) signals to a similar extent in AR4-2J cells. In contrast, loss of the basolateral type I InsP3R decreased the sensitivity of hepatocytes to vasopressin but had little effect on the initiation or spread of Ca(2+) waves across hepatocytes. Loss of the apical type II isoform caused an even greater decrease in the sensitivity of hepatocytes to vasopressin and resulted in Ca(2+) waves that were much slower and delayed in onset. These findings provide evidence that the apical concentration of type II InsP3Rs is essential for the formation of Ca(2+) waves in hepatocytes. The subcellular distribution of InsP3R isoforms may critically determine the repertoire of spatial patterns of Ca(2+) signals.

Subcellular Targeting of RGS9-2 Is Controlled by Multiple Molecular Determinants on Its Membrane Anchor, R7BP

RGS9-2, a member of the R7 regulators of G protein signaling (RGS) protein family of neuronal RGS, is a critical regulator of G protein signaling. In striatal neurons, RGS9-2 is tightly associated with a novel palmitoylated protein, R7BP (R7 family binding protein). Here we report that R7BP acts to target the localization of RGS9-2 to the plasma membrane. Examination of the subcellular distribution in native striatal neurons revealed that both R7BP and RGS9-2 are almost entirely associated with the neuronal membranes. In addition to the plasma membrane, a large portion of RGS9-2 was found in the neuronal specializations, the postsynaptic densities, where it forms complexes with R7BP and its constitutive partner Gbeta5. Using site-directed mutagenesis we found that the molecular determinants that specify the subcellular targeting of RGS9-2.Gbeta5.R7BP complex are contained within the 21 C-terminal amino acids of R7BP. This function of the C terminus was found to require the synergistic contributions of its two distinct elements, a polybasic motif and palmitoylated cysteines, which when combined are sufficient for directing the intracellular localization of the constituent protein. In differentiated neurons, the C-terminal targeting motif of R7BP was found to be essential for mediating its postsynaptic localization. In addition to the plasma membrane targeting elements, we identified two functional nuclear localization sequences that can mediate the import of R7BP into the nucleus upon depalmitoylation. These findings provide a mechanism for the subcellular targeting of RGS9-2 in neurons.

N-Methyl-DD-aspartate Blocks Activation of JNK and Mitochondrial Apoptotic Pathway Induced by Potassium Deprivation in Cerebellar Granule Cells

During the postnatal development of cerebellum, lack of excitatory innervation from the mossy fibers results in cerebellar granule cell (CGC) apoptosis during the migration of the cells toward the internal granule cell layer. Accordingly, CGCs die by apoptosis when cultured in physiological KCl concentrations (5 mm; K5), and they survive in the presence of depolarizing conditions such as high KCl concentration (25 mm; K25) or N-methyl-D-aspartate (NMDA). We have recently shown that NMDA is able to exert a long lasting neuroprotective effect when added to immature (2 days in vitro) CGC cultures by inhibition of caspase-3 activity. Here we show that NMDA- and K25-mediated neuroprotection is associated with an increase in the levels of Bcl-2, an inhibition of K5-mediated increase in Bax, and the inhibition of the release of apoptogenic factors from mitochondria such as Smac/DIABLO and cytochrome c. Moreover, we have shown that similar effects are observed when c-Jun N-terminal kinases (JNKs) are inhibited and that treatment of CGC cultures with NMDA blocks K5-mediated JNK activation. These results allow us to postulate that the inhibition of JNK-mediated release of apoptogenic factors from mitochondria is involved in the NMDA protection from K5-mediated apoptosis of CGCs.

Identification of Determinants of Ligand Binding Affinity and Selectivity in the Prostaglandin D2 Receptor CRTH2

The chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) is a G protein-coupled receptor that mediates the pro-inflammatory effects of prostaglandin D(2) (PGD(2)) generated in allergic inflammation. The CRTH2 receptor shares greatest sequence similarity with chemoattractant receptors compared with prostanoid receptors. To investigate the structural determinants of CRTH2 ligand binding, we performed site-directed mutagenesis of putative mCRTH2 ligand-binding residues, and we evaluated mutant receptor ligand binding and functional properties. Substitution of alanine at each of three residues in the transmembrane (TM) helical domains (His-106, TM III; Lys-209, TM V; and Glu-268, TM VI) and one in extracellular loop II (Arg-178) decreased PGD(2) binding affinity, suggesting that these residues play a role in binding PGD(2). In contrast, the H106A and E268A mutants bound indomethacin, a nonsteroidal anti-inflammatory drug, with an affinity similar to the wild-type receptor. HEK293 cells expressing the H106A, K209A, and E268A mutants displayed reduced inhibition of intracellular cAMP and chemotaxis in response to PGD(2), whereas the H106A and E268A mutants had functional responses to indomethacin similar to the wild-type receptor. Binding of PGE(2) by the E268A mutant was enhanced compared with the wild-type receptor, suggesting that Glu-268 plays a role in determining prostanoid ligand selectivity. Replacement of Tyr-261 with phenylalanine did not affect PGD(2) binding but decreased the binding affinity for indomethacin. These results provided the first details of the ligand binding pocket of an eicosanoid-binding chemoattractant receptor.

In vitro maturation of caprine oocytes in different concentrations of estrous goat serum

The aim of the study was to evaluate the optimum concentration of the estrous goat serum for goat in vitro oocyte maturation. Goat ovaries were collected from a local abattoir and transported within 3 h to the laboratory in a warm saline solution (30–35 °C), supplemented with 100 IU penicillin G and 100 μg streptomycin sulfate/ml. Cumulus-oocyte complexes (COC's) were obtained by puncture of 1–4 mm diameter follicles with a 18G needle attached to a disposable syringe, washed 10 times in TCM-199 hepes modification with Earl's salts and supplemented with 50 μg/ml gentamycin, 100 μg/ml l-glutamine and 0.25 mM sodium pyruvate without any serum supplementation. The COC's were randomly divided into four groups. Group 1 ( n = 105) COC's were fresh control and cultured in TCM-199 medium without serum supplementation. Group 2 ( n = 105) COC's were washed five times and cultured in TCM-199 medium supplemented with 10% EGS. Group 3 ( n = 110) COC's were washed five times and cultured in TCM-199 medium supplemented with 15% EGS. Group 4 ( n = 112) COC's were washed five times and cultured in TCM-199 medium supplemented with 20% EGS. After 24–27 h of IVM, oocytes were denuded with the aid of 0.1% hyaluronidase and passing them through a fine pipette, fixed for 24–48 h in a mixture of acetic acid and alcohol (1:3) at room temperature, stained for 10 min with 1% (w/v) orcein in 45% acetic acid and examined for the evidence of different stages of maturation. Significantly higher ( p < 0.01) maturation rates of oocytes (61–78%) were observed in all concentrations of estrous goat serum compared to those without serum supplementation. However, no significant difference was observed between the 10 and 15% serum supplemented group. A significant difference in maturation percentage ( p < 0.01) was observed between 10 and 20% serum supplementation in culture media. In conclusion, it can be said that the supplementation of 20% EGS in TCM-199 medium could be used to achieve optimal maturation rate.

Early Growth Response Factor-1 Mediates Prostaglandin E2-dependent Transcriptional Suppression of Cytokine-induced Tumor Necrosis Factor-α Gene Expression in Human Macrophages and Rheumatoid Arthritis-affected Synovial Fibroblasts

Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic proinflammatory cytokine that modulates a broad range of inflammatory and immunological processes. We have investigated the potential immunomodulatory properties of prostaglandin E2 (PGE2) by examining the molecular mechanism by which the eicosanoid suppresses T-cell-derived interleukin-17 (IL-17)-induced TNF-alpha mRNA expression and protein synthesis in human macrophages and rheumatoid arthritis-affected synovial fibroblasts. Initial studies confirmed that PGE2 induces egr-1 mRNA expression and protein synthesis by restricted SAPK2/p38 MAPK-dependent activating transcription factor-2 (ATF-2) dimer transactivation of the egr-1 promoter as judged by studies using wild-type (WT) and deletion mutant egr-1 promoter constructs, Northern and Western blotting, and standard and supershift electrophoretic mobility shift analyses. Using human leukemic monocytic THP-1 cells stably transfected with WT and dominant-negative mutant expression constructs of Egr-1, cotransfected or not with a WT pTNF-615SVOCAT construct, we observed that PGE2 inhibition of IL-17-stimulated TNF-alpha mRNA expression and promoter activity was dependent on Egr-1 expression, as mutants of Egr-1, alone or in combination, markedly abrogated any inhibitory effect of PGE2. Standard and supershift electrophoretic mobility shift analysis, signaling "decoy" overexpression studies, and pTNF-615SVOCAT promoter assays using WT and mutant promoter constructs revealed that IL-17-up-regulated promoter activity was largely dependent on ATF-2/c-Jun transactivation. PGE2 suppression of IL-17-induced ATF-2/c-Jun transactivation and DNA binding was dependent on Egr-1-mediated inhibition of induced c-Jun expression. We suggest that egr-1 is an immediate-early PGE2 target gene that may be a key regulatory factor in mediating eicosanoid control of genes involved in the immune and inflammatory responses.