Effect of Ganciclovir on murine cytomegalovirus-induced hearing loss in a mouse model

We evaluated the effect of Ganciclovir on murine cytomegalovirus (MCMV)-induced hearing impairment in a mouse model by studying modulations in auditory brainstem response (ABR) and pathological changes in the inner ear. MCMV infection was established via trans-brain. For this purpose, 24 BALB/c newborn mice were randomly and equally divided into control group (10 μl of sterile normal saline was injected); model group (10 μl of MCMV TCID50—10⁴ IU/0.1 ml was injected); and interfered group (Trans-brain MCMV; Ganciclovir at the rate of 60 mg/kg was intraperitoneally injected). ABR audiometry was performed after 14 days. Acoustic vesicles were obtained for MCMV-PCR analysis and histopathological examination. Comparing ABR in model group against controls, incubation period of wave was lengthened (F = 9.151, P = 0.011–0.05) and wave amplitude was cut down (F = 5.095, P = 0.043–0.05). Comparing ABR in model group against interfered group, incubation period and amplitude of wave were F = 13.797 (P = 0.003–0.05) and F = 14.587 (P = 0.002–0.05), respectively. Cochlear histopathological changes in model group included thickening of vestibular membrane, lymphocytic infiltration, and fibrous degeneration of cochlear duct. In Ganciclovir interfered group, however, these pathologic changes were less significant as compared with those of model group. We, therefore, conclude that Ganciclovir treatment at the early stage of MCMV-induced hearing impairment inhibits the disease progression in infected mice.     

The expression of HIF-1α in primary hepatocellular carcinoma and its correlation with radiotherapy response and clinical outcome

The purpose of this study was to evaluate the relationship between hypoxia-inducible factor-1α (HIF-1α) protein expression in hepatocellular carcinoma (HCC), and responses of abdominal metastatic lymph nodes (LNs) from HCC patients treated with external beam radiotherapy (EBRT). HIF-1α immunohistochemical staining was performed on tissue microarrays (TMAs) of primary HCC specimens from 69 HCC patients with abdominal LN metastases. All patients received abdominal metastatic LN EBRT at the Department of Radiation Oncology at Zhongshan Hospital. A receiver-operating characteristic (ROC)-based approach and logistical regression analysis were used to determine the predictive value of HIF-1α expression in primary tumors with HCC metastatic LN EBRT response. Kaplan–Meier curves and log-rank tests were used to analyze patient survival. Cox proportional hazards regression model was used to analyze independent prognostic factors. HIF-1α expression was correlated with blood hemoglobin (Hb: r = −0.280, P = 0.020), response of abdominal metastatic LNs to EBRT (r = 0.286, P = 0.017), locoregional recurrence (r = 0.278, P = 0.021), and cancer-specific deaths (r = 0.298, P = 0.013). HIF-1α expression was predictive of EBRT response of metastatic LNs [area under the curve (AUC): 0.646; 95% confidence interval (CI): 0.499–0.793; P = 0.047], locoregional recurrence (AUC: 0.657; 95% CI: 0.509–0.805; P = 0.049) and cancer-specific deaths (AUC: 0.671; 95% CI: 0.531–0.812; P = 0.035). Patients with tumors exhibiting high HIF-1α expression had significantly poorer overall survival (OS) than those with low tumor expression of HIF-1α (P = 0.016). Multivariate analysis showed that Hb (P = 0.035), vascular invasion (P = 0.026), Child-Pugh score (P < 0.001), intrahepatic tumor control (P < 0.001), and HIF-1α (P = 0.020) were independent prognosis factors for OS of HCC patients after receiving abdominal metastatic LN EBRT. HIF-1α expression in primary HCCs was associated with EBRT response of abdominal metastatic LNs and poor prognosis.     

Small scale vertical gradients of Arctic ice algal photophysiological properties

Photosynthetic parameters of phytoplankton and sea ice algae from landfast sea ice of the Chukchi Sea off Point Barrow, Alaska, were assessed in spring 2005 and winter through spring 2006 using Pulse Amplitude Modulated (PAM) fluorometry including estimates of maximum quantum efficiency (F _v/F _m), maximum relative electron transport rate (rETR_max), photosynthetic efficiency (α), and the photoadaptive index (E _k). The use of centrifuged brine samples allowed to document vertical gradients in ice algal acclimation with 5 cm vertical resolution for the first time. Bottom ice algae (0–5 cm from ice–water interface) expressed low F _v/F _m (0.331–0.426) and low α (0.098–0.130 (μmol photons m⁻²s⁻¹)⁻¹) in December. F _v/F _m and α increased in March and May (0.468–0.588 and 0.141–0.438 (μmol photons m⁻²s⁻¹)⁻¹, respectively) indicating increased photosynthetic activity. In addition, increases in rETR_max (3.3–16.4 a.u.) and E _k (20–88 μmol photons m⁻² s⁻¹) from December to May illustrates a higher potential for primary productivity as communities become better acclimated to under-ice light conditions. In conclusion, photosynthetic performance by ice algae (as assessed by PAM fluorometry) was tightly linked to sea ice salinity, temperature, and inorganic nutrient concentrations (mainly nitrogen).     

A Cmv2 QTL on chromosome X affects MCMV resistance in New Zealand male mice

NK cell-mediated resistance to viruses is subject to genetic control in humans and mice. Here we used classical and quantitative genetic strategies to examine NK-mediated murine cytomegalovirus (MCMV) control in genealogically related New Zealand white (NZW) and black (NZB) mice. NZW mice display NK cell-dependent MCMV resistance while NZB NK cells fail to limit viral replication after infection. Unlike Ly49H⁺ NK resistance in C57BL/6 mice, NZW NK-mediated MCMV control was Ly49H-independent. Instead, MCMV resistance in NZW (Cmv2) involves multiple genetic factors. To establish the genetic basis of Cmv2 resistance, we further characterized a major chromosome X-linked resistance locus (DXMit216) responsible for innate MCMV control in NZW × NZB crosses. We found that the DXMit216 locus affects early MCMV control in New Zealand F₂ crosses and demonstrate that the NZB-derived DXMit216 allele enhances viral resistance in F₂ males. The evolutionary conservation of the DXMit216 region in mice and humans suggests that a Cmv2-related mechanism may affect human antiviral responses. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> flagellin stimulates pro-inflammatory immune response

Flagellin, a principal component of bacterial flagella, is a virulence factor that is recognized by the innate immune system. Recognition of flagellin by innate immune receptors stimulates the production of cytokines necessary for the development of effective immunity. Here, we demonstrated that the intranasal (i.n.) instillation of different amount of Escherichia coli K-12 flagellin preparation (0.5, 1, 2, 4 μg) in BALB/c mice induced pro-inflammatory immune response. Instillation i.n. of 1 μg of flagellin induced the maximum expression of interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) mRNA and production of pro-inflammatory cytokines (IL-1β, TNF-α and IL-6) in mice lungs. The same dose of flagellin induced neutrophil polymorphonuclear cells infiltration in peribronchial and perivascular regions. High number of neutrophil in bronchoalveolar lavage fluid was found at 24 h after i.n. instillation of flagellin (1 μg). These findings were concomitant with the maximum production of myeloperoxidase and nitric oxide in mice lungs. Present study showed that the maximum pro-inflammatory mediator levels were found when mice instilled i.n. with 1 μg E. coli flagellin. The amount of flagellin of E. coli K-12 that achieve the maximum stimulation of mucosal pro-inflammatory immune response in mice lungs was explored in this study.     

Biogeochemical conditions and ice algal photosynthetic parameters in Weddell Sea ice during early spring

Physical, biogeochemical and photosynthetic parameters were measured in sea ice brine and ice core bottom samples in the north-western Weddell Sea during early spring 2006. Sea ice brines collected from sackholes were characterised by cold temperatures (range −7.4 to −3.8°C), high salinities (range 61.4–118.0), and partly elevated dissolved oxygen concentrations (range 159–413 μmol kg⁻¹) when compared to surface seawater. Nitrate (range 0.5–76.3 μmol kg⁻¹), dissolved inorganic phosphate (range 0.2–7.0 μmol kg⁻¹) and silicic acid (range 74–285 μmol kg⁻¹) concentrations in sea ice brines were depleted when compared to surface seawater. In contrast, NH₄ ⁺ (range 0.3–23.0 μmol kg⁻¹) and dissolved organic carbon (range 140–707 μmol kg⁻¹) were enriched in the sea ice brines. Ice core bottom samples exhibited moderate temperatures and brine salinities, but high algal biomass (4.9–435.5 μg Chl a l⁻¹ brine) and silicic acid depletion. Pulse amplitude modulated fluorometry was used for the determination of the photosynthetic parameters F _v/F _m, α, rETR_max and E _k. The maximum quantum yield of photosystem II, F _v/F _m, ranged from 0.101 to 0.500 (average 0.284 ± 0.132) and 0.235 to 0.595 (average 0.368 ± 0.127) in the sea ice internal and bottom communities, respectively. The fluorometric measurements indicated medium ice algal photosynthetic activity both in the internal and bottom communities of the sea ice. An observed lack of correlation between biogeochemical and photosynthetic parameters was most likely due to temporally and spatially decoupled physical and biological processes in the sea ice brine channel system, and was also influenced by the temporal and spatial resolution of applied sampling techniques.     

Fibronectin and Focal Adhesion Kinase Small Interfering RNA Modulate Rat Retinal Müller Cells Adhesion and Migration

Retinal Müller cells (RMCs) hypertrophy and proliferation play a crucial role in epiretinal membrane formation. This study was designed to analyze the effects of Fibronectin and specific FAK siRNA in cell adhesion and migration in rat Müller cells. RMCs were cultured and identified by GFAP, Vimentin, and GLAST mAb, respectively. The cells were planted on dishes coated with Fibronectin at 0, 1, 5, 10, 50, and 100 μg/ml. The attachment and migration assay was applied to characterize the RMCs–Fibronectin interactions. Cell lysis and Western blotting were utilized to detect β₁-integrin, FAK, and GLAST protein expression. Then the cells were treated with FAK siRNA, non-targeting siRNA, and control medium. The cell cycle and apoptosis rate was determined by flow cytometry. The attachment, migration, and Western blotting assay were repeated. These data suggested that almost all the cells expressed GFAP, Vimentin, and GLAST, respectively, which ensured most of the harvested cells were RMCs. In attachment assay, the A570 values increased significantly with time (F = 1105.439, P < 0.001) and Fibronectin concentration (F = 424.683, P < 0.001). There were significant difference between each Fibronectin concentration in RMCs migration (F = 34.703, P < 0.000). The expression ratio of FAK, β₁-integrin, and GLAST elevated significantly as Fibronectin concentration increased (F = 54.755, P < 0.000; F = 119.962, P < 0.000; F = 39.287, P < 0.000). The Fibronectin pretreatment was settled on 50 μg/ml for siRNA inhibition assays. The specific FAK siRNA treatment significantly increased G₀/G₁ percentage and apoptosis rate compared with NT siRNA and control group (F = 11.526, P = 0.009; F = 64.772, P < 0.000). The apoptotic rate was significantly suppressed by inhibitors of caspase-8 and 3 (F = 10.500, P = 0.011). The A570 values were significantly suppressed in FAK siRNA groups compared with NT siRNA and control group (F = 154.241, P < 0.000), and the mean migratory cells per view field were significantly decreased (F = 10.906, P = 0.001). FAK and GLAST expression ratio decreased significantly after FAK siRNA treatment (F = 5.315, P = 0.047; F = 5.985, P = 0.042). Take together, FAK is involved in β₁-integrin mediated adhesive signaling and play a critical role in regulating Müller cell adhesion, migration, and so far as to glutamate transportation functions.     

Inhibitory effect of curcumin on liver injury in a murine model of endotoxemic shock

The effect of curcumin on lipopolysaccharide/d-galactosamine (LPS/GalN)-induced acute shock model of liver injury was examined in mice. The simultaneous administration of LPS (5–20 μg kg⁻¹, i.p.) and GalN (700 mg kg⁻¹, i.p.) markedly increased the serum tumor necrosis factor-α (TNF-α), glutamic oxaloacetic transaminase/glutamic pyruvic transaminase (GOT/GPT), and massive hepatic necrosis and inflammation, leading to 100% lethality. Pre-administration of curcumin (100 mg kg⁻¹, i.p.) 3 h before induction with LPS/GalN imparted a large extent of protection against acute elevation in serum TNF-α and serum GOT/GPT. Hepatic necrosis and lethality caused by LPS/GalN was also greatly reduced by curcumin treatment. The results demonstrated that curcumin could protect mice from LPS/GalN-induced hepatic injury and inflammation through blockading TNF-α production, eventually raising the survival rate of septic-shock-induced mice.     

Biodegradation of lindane pesticide by non white- rots soil fungus <Emphasis Type="Italic">Fusarium</Emphasis> sp.

Lindane or γ- hexachlorocyclohexane (γ-HCH) is a chlorinated pesticide and its toxic effects on biota necessitate its removal. Microbial degradation is an important process for pesticide bioremediation and the role of soil fungi in recycling of organic matter prompted us to study the biodegradation of lindane using fungi. This study aims at enrichment, isolation and screening of soil fungi capable of metabolizing lindane. Two Fusarium species (F. poae and F. solani) isolated from the pesticide contaminated soil showed better growth on the plates supplemented with lindane as a sole carbon source, when compared with the growth performance of other fungal isolates from the same contaminated soil. However, ANOVA revealed a significant difference in fungal biomass production in both F. poae (F = 22.02; N = 15; P < 0.001) and F. solani (F = 268.75; N = 15; P < 0.001) across different lindane concentrations (0–600 μg ml⁻¹). Growth of both Fusarium sp. was maximum at a lindane concentration of 100 μg ml⁻¹, while minimum at 600 μg ml⁻¹ concentrations. Results on the time dependent release of chlorine by the Fusarium strains in the presence of various concentration of lindane showed the highest mineralization of the pesticide on 10th day of incubation. Time dependent variations in the release of chlorine from 1st to 10th day by both the selected fungal strains were found to be statistically significant. A significant positive relationship exists between fungal biomass increase and chlorine release existed for both F. solani (R2 = 0.960) and F. poae (R2 = 0.628). The results of gas chromatograph analysis of γ- HCH confirmed the biodegradation and utilization of γ- HCH by F. poae and F. solani. The data on lindane degradation by the two fungal strains demonstrated that the biodegradation of lindane by F. solani (59.4%) was slightly higher than that by the F. poae (56.7%).     

Investigations into the ability of the peptide,HAL18, to interact with bacterial membranes

Halocidin was isolated from hemocytes, Halocynthia aurantium as a heterodimeric peptide consisting of two α-helical subunits, Hal15 and Hal18. Hal18 was shown to have antibacterial properties against Bacillus subtilis (MLC = 15 μM) and Escherichia coli (MLC = 100 μM). The peptide was shown to produce stable monolayers, which were characteristic of α-helical peptides predicted to orientate parallel to the surface of the interface. Constant area assays showed that Hal18 was surface active (4 μM) inducing surface pressure changes >30 mN m⁻¹ characteristic of membrane interactive peptides. The peptide induced stable surface pressure changes in monolayers that were mimetic of B. subtilis membranes (circa 7 mN m⁻¹) and E. coli membrane-mimics (circa 4 mN m⁻¹). Hal18 inserted readily into zwitterionic DOPE and anionic DOPG monolayers inducing surface pressure changes circa 8 mN m⁻¹ in both cases, providing evidence that interaction is not headgroup specific. Thermodynamic analysis of compression isotherms showed that the presence of Hal18 destabilised B. subtilis membranes (ΔG _Mix > 0), which is in contrast to its stabilising effect on E. coli lipid extract implying the differential antimicrobial efficacy may be driven by lipid packing.     

Identification of QTL underlying vitamin E contents in soybean seed among multiple environments

Vitamin E (VE) in soybean seed has value for foods, medicines, cosmetics, and animal husbandry. Selection for higher VE contents in seeds along with agronomic traits was an important goal for many soybean breeders. In order to map the loci controlling the VE content, F₅-derived F₆ recombinant inbred lines (RILs) were advanced through single-seed-descent (SSD) to generate a population including 144 RILs. The population was derived from a cross between ‘OAC Bayfield’, a soybean cultivar with high VE content, and ‘Hefeng 25’, a soybean cultivar with low VE content. A total of 107 polymorphic simple sequence repeat markers were used to construct a genetic linkage map. Seed VE contents were analyzed by high performance liquid chromatography for multiple years and locations (Harbin in 2007 and 2008, Hulan in 2008 and Suihua in 2008). Four QTL associated with α-Toc (on four linkage groups, LGs), eight QTL associated with γ-Toc (on eight LGs), four QTL associated with δ-Toc (on four LGs) and five QTL associated with total VE (on four LGs) were identified. A major QTL was detected by marker Satt376 on linkage group C2 and associated with α-Toc (0.0012 > P > 0.0001, 5.0% < R ² < 17.0%, 25.1 < α-Toc < 30.1 μg g⁻¹), total VE (P < 0.0001, 7.0% < R ² < 10.0%, 118.2 < total VE < 478.3 μg g⁻¹). A second QTL detected by marker Satt286 on LG C2 was associated with γ-Toc (0.0003 > P > 0.0001, 6.0% < R ² < 13.0%, 141.5 < γ-Toc < 342.4 μg g⁻¹) and total VE (P < 0.0001, 2.0% < R ² < 9.0%, 353.9 < total VE < 404.0 μg g⁻¹). Another major QTL was detected by marker Satt266 on LG D1b that was associated with α-Toc (0.0002 > P > 0.0001, 4.0% < R ² < 6.0%, 27.7 < α-Toc < 43.7 μg g⁻¹) and γ-Toc (0.0032 > P > 0.0001, 3.0% < R ² < 10.0%, 69.7 < γ-Toc < 345.7 μg g⁻¹). Since beneficial alleles were all from ‘OAC Bayfield’, it was concluded that these three QTL would have great potential value for marker assisted selection for high VE content.     

Enhanced anti-tumor activity of interferon-alpha in SOCS1-deficient mice is mediated by CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T cells

Interferon-alpha (IFN-α) is an immunomodulatory cytokine that is used clinically for the treatment of melanoma in the adjuvant setting. The cellular actions of IFN-α are regulated by the suppressors of cytokine signaling (SOCS) family of proteins. We hypothesized that the anti-tumor activity of exogenous IFN-α would be enhanced in SOCS1-deficient mice. SOCS1-deficient (SOCS1^−/−) or control (SOCS1^+/+) mice on an IFN-γ^−/− C57BL/6 background bearing intraperitoneal (i.p.) JB/MS murine melanoma cells were treated for 30 days with i.p. injections of IFN-A/D or PBS (vehicle). Log-rank Kaplan-Meier survival curves were used to evaluate survival. Tumor-bearing control SOCS1^+/+ mice receiving IFN-A/D had significantly enhanced survival versus PBS–treated mice (P = 0.0048). The anti-tumor effects of IFN-A/D therapy were significantly enhanced in tumor-bearing SOCS1^−/− mice; 75% of these mice survived tumor challenge, whereas PBS-treated SOCS1^−/− mice all died at 13-16 days (P = 0.00038). Antibody (Ab) depletion of CD8⁺ T cells abrogated the anti-tumor effects of IFN-A/D in SOCS1^−/− mice as compared with mice receiving a control antibody (P = 0.0021). CD4⁺ T-cell depletion from SOCS1^−/− mice also inhibited the effects of IFN-A/D (P = 0.0003). IFN-A/D did not alter expression of CD80 or CD86 on splenocytes of SOCS1^+/+ or SOCS1^−/− mice, or the proportion of T regulatory cells or myeloid-derived suppressor cells in SOCS1^+/+ or SOCS1^−/− mice. An analysis of T-cell function did reveal increased proliferation of SOCS1-deficient splenocytes at baseline and in response to mitogenic stimuli. These data suggest that modulation of SOCS1 function in T-cell subsets could enhance the anti-tumor effects of IFN-α in the setting of melanoma.     

Zinc,Manganese, Calcium,Copper, and Cadmium Level in Scalp Hair Samples of Schizophrenic Patients

The purpose of the study was to determine the concentration of trace elements present in scalp hair sample of schizophrenic patients and to find out the relationship between trace elements level and nutritional status or socioeconomic factors. The study was conducted among 30 schizophrenic male patients and 30 healthy male volunteers. Patients were recruited from Bangabandhu Sheikh Mujib Medical University by random sampling. Hair trace element concentrations were determined by flame atomic absorption spectroscopy and analyzed by independent t test, Pearson’s correlation analysis, regression analysis, and analysis of variance (ANOVA). Mn, Zn, Ca, Cu, and Cd concentrations of schizophrenic patients were 3.8 ± 2.31 μg/gm, 171.6 ± 59.04 μg/gm, 396.23 ± 157.83 μg/gm, 15.40 ± 5.68 μg/gm, and 1.14 ± 0.89 μg/gm of hair sample, while those of control subjects were 4.4 ± 2.32 μg/gm, 199.16 ± 27.85 μg/gm, 620.9 ± 181.55 μg/gm, 12.23 ± 4.56 μg/gm, and 0.47 ± 0.32 μg/gm of hair sample, respectively. The hair concentration of Zn and Ca decreased significantly (p = 0.024; p = 0.000, respectively) and the concentration of Cu and Cd increased significantly (p = 0.021; p = 0.000, respectively) in schizophrenic patients while the concentration of Mn (p = 0.321) remain unchanged. Socioeconomic data reveals that most of the patients were poor, middle-aged and divorced. Mean body mass indices (BMIs) of the control group (22.26 ± 1.91 kg/m²) and the patient group (20.42 ± 3.16 kg/m²) were within the normal range (18.5−25.0 kg/m²). Pearson’s correlation analysis suggested that only Ca concentration of patients had a significant positive correlation with the BMI (r = 0.597; p = 0.000) which was further justified from the regression analysis (R ² = 44%; t = 3.59; p = 0.002) and one-way ANOVA test (F = 3.62; p = 0.015). A significant decrease in the hair concentration of Zn and Ca as well as a significant increase in the hair concentration of Cu and Cd in schizophrenic patients than that of its control group was observed which may provide prognostic tool for the diagnosis and treatment of this disease. However, further work with larger population is suggested to examine the exact correlation between trace element level and the degree of disorder.     

Identification,cloning, and functional characterization of EmrD-3, a putative multidrug efflux pump of the major facilitator superfamily from <Emphasis Type="Italic">Vibrio cholerae</Emphasis> O395

A putative multidrug efflux pump, EmrD-3, belonging to the major facilitator superfamily (MFS) of transporters and sharing homology with the Bcr/CflA subfamily, was identified in Vibrio cholerae O395. We cloned the emrD-3 gene and evaluated its role in antimicrobial efflux in a hypersensitive Escherichia coli strain. The efflux activity of this membrane protein resulted in lowering the intracellular concentration of ethidium. The recombinant plasmid carrying emrD-3 conferred enhanced resistance to several antimicrobials. Among the antimicrobials tested, the highest relative increase in minimum inhibitory concentration (MIC) of 102-fold was observed for linezolid (MIC = 256 μg/ml), followed by an 80.1-fold increase for tetraphenylphosphonium chloride (TPCL) (156.2 μg/ml), 62.5-fold for rifampin (MIC = 50 μg/ml), >30-fold for erythromycin (MIC = 50 μg/ml) and minocycline (MIC = 2 μg/ml), 20-fold for trimethoprim (MIC = 0.12 μg/ml), and 18.7-fold for chloramphenicol (MIC = 18.7 μg/ml). Among the fluorescent DNA-binding dyes, the highest relative increase in MIC of 41.7-fold was observed for ethidium bromide (125 μg/ml) followed by a 17.2-fold increase for rhodamine 6G (100 μg/ml). Thus, we demonstrate that EmrD-3 is a multidrug efflux pump of V. cholerae, the homologues of which are present in several Vibrio spp., some members of Enterobacteriaceae family, and Gram-positive Bacillus spp.     

Metabolism of N-alkylated spermine analogues by polyamine and spermine oxidases

N-alkylated polyamine analogues have potential as anticancer and antiparasitic drugs. However, their metabolism in the host has remained incompletely defined thus potentially limiting their utility. Here, we have studied the degradation of three different spermine analogues N,N′-bis-(3-ethylaminopropyl)butane-1,4-diamine (DESPM), N-(3-benzyl-aminopropyl)-N′-(3-ethylaminopropyl)butane-1,4-diamine (BnEtSPM) and N,N′-bis-(3-benzylaminopropyl)butane-1,4-diamine (DBSPM) and related mono-alkylated derivatives as substrates of recombinant human polyamine oxidase (APAO) and spermine oxidase (SMO). APAO and SMO metabolized DESPM to EtSPD [K _m(APAO) = 10 μM, k _cat(APAO) = 1.1 s⁻¹ and K _m(SMO) = 28 μM, k _cat(SMO) = 0.8 s⁻¹, respectively], metabolized BnEtSPM to EtSPD [K _m(APAO) = 0.9 μM, k _cat(APAO) = 1.1 s⁻¹ and K _m(SMO) = 51 μM, k _cat(SMO) = 0.4 s⁻¹, respectively], and metabolized DBSPM to BnSPD [K _m(APAO) = 5.4 μM, k _cat(APAO) = 2.0 s⁻¹ and K _m(SMO) = 33 μM, k _cat(SMO) = 0.3 s⁻¹, respectively]. Interestingly, mono-alkylated spermine derivatives were metabolized by APAO and SMO to SPD [EtSPM K _m(APAO) = 16 μM, k _cat(APAO) = 1.5 s⁻¹; K _m(SMO) = 25 μM, k _cat(SMO) = 8.2 s⁻¹; BnSPM K _m(APAO) = 6.0 μM, k _cat(APAO) = 2.8 s⁻¹; K _m(SMO) = 19 μM, k _cat(SMO) = 0.8 s⁻¹, respectively]. Surprisingly, EtSPD [K _m(APAO) = 37 μM, k _cat(APAO) = 0.1 s⁻¹; K _m(SMO) = 48 μM, k _cat(SMO) = 0.05 s⁻¹] and BnSPD [K _m(APAO) = 2.5 μM, k _cat(APAO) = 3.5 s⁻¹; K _m(SMO) = 60 μM, k _cat(SMO) = 0.54 s⁻¹] were metabolized to SPD by both the oxidases. Furthermore, we studied the degradation of DESPM, BnEtSPM or DBSPM in the DU145 prostate carcinoma cell line. The same major metabolites EtSPD and/or BnSPD were detected both in the culture medium and intracellularly after 48 h of culture. Moreover, EtSPM and BnSPM were detected from cell samples. Present data shows that inducible SMO parallel with APAO could play an important role in polyamine based drug action, i.e. degradation of parent drug and its metabolites, having significant impact on efficiency of these drugs, and hence for the development of novel N-alkylated polyamine analogues.     

Tunicamycin inhibition of <Emphasis Type="Italic">N</Emphasis>-glycosylation of α-glucosidase from <Emphasis Type="Italic">Aspergillus niveus</Emphasis>: partial influence on biochemical properties

Treatment of Aspergillus niveus with 30 μg tunicamycin/ml did not interfere with α-glucosidase production, secretion, or its catalytic properties. Fully- and under-glycosylated forms of the enzyme had similar molecular masses, ~56 kDa. Moreover, the absence of N-glycans did not affect either pH optimum (6.0) or temperature optimum (65°C). The K_m and V_max values of under- and fully-glycosylated forms of α-glucosidase were similar when assessed for hydrolysis of starch (~0.6 mg/ml, ~350 μmol glucose per min per ml), maltose (~0.54 μmol, ~330 μmol glucose per min per ml) and p-nitrophenyl-α-d-glucopyranoside (~0.54 μmol, ~8.28 μmol p-nitrophenol per min per ml). However, the under-glycosylated form was sensitive to high temperatures probably because, in addition to stabilizing the protein conformation, glycosylation may also prevent unfolded or partially folded proteins from aggregating. Binding assays clearly showed that the under-glycosylated protein did not bind to concanavalin A but has conserve its jacalin-binding property, suggesting that only O-glycans might be intact on the tunicamycin treated form of the enzyme.     

Characterization of PSI recovery after chilling-induced photoinhibition in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) leaves

By simultaneously analyzing the chlorophyll a fluorescence transient and light absorbance at 820 nm as well as chlorophyll fluorescence quenching, we investigated the effects of different photon flux densities (0, 15, 200 μmol m⁻² s⁻¹) with or without 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on the repair process of cucumber (Cucumis sativus L.) leaves after treatment with low temperature (6°C) combined with moderate photon flux density (200 μmol m⁻² s⁻¹) for 6 h. Both the maximal photochemical efficiency of Photosystem II (PSII) (F _v/F _m) and the content of active P700 (ΔI/I _o) significantly decreased after chilling treatment under 200 μmol m⁻² s⁻¹ light. After the leaves were transferred to 25°C, F _v/F _m recovered quickly under both 200 and 15 μmol m⁻² s⁻¹ light. ΔI/I _o recovered quickly under 15 μmol m⁻² s⁻¹ light, but the recovery rate of ΔI/I _o was slower than that of F _v/F _m. The cyclic electron transport was inhibited by chilling-light treatment obviously. The recovery of ΔI/I _o was severely suppressed by 200 μmol m⁻² s⁻¹ light, whereas a pretreatment with DCMU effectively relieved this suppression. The cyclic electron transport around PSI recovered in a similar way as the active P700 content did, and the recovery of them was both accelerated by pretreatment with DCMU. The results indicate that limiting electron transport from PSII to PSI protected PSI from further photoinhibition, accelerating the recovery of PSI. Under a given photon flux density, faster recovery of PSII compared to PSI was detrimental to the recovery of PSI or even to the whole photosystem.     

Deschampsia antarctica Desv. primary photochemistry performs differently in plants grown in the field and laboratory

Primary photochemistry of photosystem II (F _v/F _m) of the Antarctic hair grass Deschampsia antarctica growing in the field (Robert Island, Maritime Antarctic) and in the laboratory was studied. Laboratory plants were grown at a photosynthetic photon flux density (PPFD) of 180 μmol m⁻² s⁻¹ and an optimal temperature (13 ± 1.5°C) for net photosynthesis. Subsequently, two groups of plants were exposed to low temperature (4 ± 1.5°C day/night) under two levels of PPFD (180 and 800 μmol m⁻² s⁻¹) and a control group was kept at 13 ± 1.5°C and PPFD of 800 μmol m⁻² s⁻¹. Chlorophyll fluorescence was measured during several days in field plants and weekly in the laboratory plants. Statistically significant differences were found in F _v/F _m (=0.75–0.83), F ₀ and F _m values of field plants over the measurement period between days with contrasting irradiances and temperature levels, suggesting that plants in the field show high photosynthetic efficiency. Laboratory plants under controlled conditions and exposed to low temperature under two light conditions showed significantly lower F _v/F _m and F _m. Moreover, they presented significantly less chlorophyll and carotenoid content than field plants. The differences in the performance of the photosynthetic apparatus between field- and laboratory-grown plants indicate that measurements performed in ex situ plants should be interpreted with caution.     

Content of the <Emphasis Type="Italic">Alternaria</Emphasis> mycotoxin tenuazonic acid in food commodities determined by a stable isotope dilution assay

The Alternaria mycotoxin tenuazonic acid (TA) was quantified in fruit juices (n = 50), cereals (n = 12) and spices (n = 38) using a recently developed stable isotope dilution assay (SIDA). [¹³ C₆,¹⁵ N]-TA was used as the internal standard. Method validation revealed low limits of detection (LODs) of 0.15 μg/kg (fruit juices), 1.0 μg/kg (cereals) and 17 μg/kg (spices). The respective limits of quantitation were about three times higher. Recovery was about 100% for all matrices. The precision (relative standard deviation of replicate analyses of naturally contaminated samples) was 4.2% (grape juice; 1.7 μg/kg), 3.5% (whole wheat flour; 36 μg/kg) and 0.9% (curry powder; 215 μg/kg). The median content of TA in the analyzed samples was 1.8 μg/kg (fruit juices), 16 μg/kg (cereals) and 500 μg/kg (spices). Positive samples amounted to 86% (fruit juices), 92% (cereals) and 87% (spices).