Synthetic studies on oligosaccharides composed of 5-thioglucopyranose units

Glycosylation reactions of 5-thioglucopyranosyl trichloroacetimidates bearing ethereal protective groups at the 2-O-position 14-15, and 37 proceed smoothly to give alpha-glycosides stereoselectively by using a catalytic amount of silyl triflate. This methodology allowed us to achieve syntheses of sulfur-substituted isomaltotetraoside 2 and maltotetraoside 3. These studies also revealed that benzoyl-protected 5-thioglucopyranosyl trichloroacetimidate 12 underwent beta-selective glycosylation with C6-OH glucopyranosyl acceptors upon activation by BF3OEt2. This was applied for preparation of sulfur-substituted gentiobiosides 1 and 46.     

Involvement of MEK-ERK signaling pathway in the inhibition of cell growth by troglitazone in human pancreatic cancer cells

Tif6p (eIF6) is necessary for 60S biogenesis, rRNA maturation and must be released from 60S to permit 80S assembly and translation. We characterized Tif6p interactors. Tif6p is mostly on 66S-60S pre-ribosomes, partly free. Tif6p complex(es) contain nucleo-ribosomal factors and Asc1p. Surprisingly, Tif6p particle contains the low-abundance endonuclease Sen34p. We analyzed Sen34p role on rRNA/tRNA synthesis, in vivo. Sen34p depletion impairs tRNA splicing and causes unexpected 80S accumulation. Accordingly, Sen34p overexpression causes 80S decrease and increased polysomes which suggest increased translational efficiency. With delayed kinetics, Sen34p depletion impairs rRNA processing. We conclude that Sen34p is absolutely required for tRNA splicing and that it is a rate-limiting element for efficient translation. Finally, we confirm that Tif6p accompanies 27S pre-rRNA maturation to 25S rRNA and we suggest that Sen34p endonuclease in Tif6p complex may affect also rRNA maturation.     

Effect of Some Pesticides on Reproduction of Rotifer <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> Müller

Pesticides have been major contributors to environmental pollution and they are now widely distributed in aquatic environments. Zooplankters are frequently used as test animals to detect aquatic contaminants because of their sensitivity and ecological importance. We investigated the effect of a 7-day exposure to four commonly used pesticides (diazinon, fenitrothion, methoprene and isoprothiolane) on reproduction of the rotifer Brachionus plicatilis. Pesticide concentrations of 3–7 times lower than the 24-h 50% lethal concentration (24-h LC₅₀) were tested to determine the ‚no observed effect’ concentration (NOEC), ‚lowest observed effect’ concentration (LOEC), and the ‚50% effective’ concentration (EC₅₀) on specific growth rate (r), sexual reproduction, fertilization, resting egg production, and hatchability of resting eggs. Results showed that the lowest EC₅₀ value of r, mixis, fertilization, and resting egg production of 1.4 μM for diazinon was 63 times lower than its 24-h LC₅₀ of 88.4 μM, while for fenitrothion it was 66 times (3.5 and 229.8 μM, respectively). For isoprothiolane, the lowest EC₅₀ value of r, mixis, fertilization, and resting egg production of 8.9 μM was 25 times lower than its 24-h LC₅₀ of 220.7 μM, while for methoprene was 37 times (2.7 and 100.8 μM, respectively). In all pesticides, hatching rate of resting eggs consistently gave the lowest EC₅₀ values which is about 2–40 times lower than the lowest EC₅₀ of r, mixis, fertilization, and resting egg production. Hatchability of resting eggs therefore is the most sensitive parameter in detecting effects of pesticides exposure in rotifer B. plicatilis.     

Involvement of p38 mitogen-activated protein kinase in gemcitabine-induced apoptosis in human pancreatic cancer cells

In this study, we investigated the involvement of Akt and members of the mitogen-activated protein kinase (MAPK) superfamily, including ERK, JNK, and p38 MAPK, in gemcitabine-induced cytotoxicity in human pancreatic cancer cells. We found that gemcitabine induces apoptosis in PK-1 and PCI-43 human pancreatic cancer cell lines. Gemcitabine specifically activated p38 MAPK in a dose- and time-dependent manner. A selective p38 MAPK inhibitor, SB203580, significantly inhibited gemcitabine-induced apoptosis in both cell lines, suggesting that phosphorylation of p38 MAPK may play a key role in gemcitabine-induced apoptosis in pancreatic cancer cells. A selective JNK inhibitor, SP600125, failed to inhibit gemcitabine-induced apoptosis in both cell lines. MKK3/6, an upstream activator of p38 MAPK, was phosphorylated by gemcitabine, indicating that the MKK3/6-p38 MAPK signaling pathway is indeed involved in gemcitabine-induced apoptosis. Furthermore, gemcitabine-induced cleavage of the caspase substrate poly(ADP-ribose) polymerase was inhibited by pretreatment with SB203580, suggesting that activation of p38 MAPK by gemcitabine induces apoptosis through caspase signaling. These results together suggest that gemcitabine-induced apoptosis in human pancreatic cancer cells is mediated by the MKK3/6-p38 MAPK-caspase signaling pathway. Further, these results lead us to suggest that p38 MAPK should be investigated as a novel molecular target for human pancreatic cancer therapies.     

Relationship between Recrystallization Rate of Ice Crystals in Sugar Solutions and Water Mobility in Freeze-Concentrated Matrix

To better understand the relation between recrystallization rate and water mobility in freeze-concentrated matrix, isothermal ice recrystallization rates in several sugar aqueous solutions and self-diffusion coefficients of water component in corresponding freeze-concentrated matrix were measured. The sugars used were fructose, glucose, maltose, and sucrose. The sugar concentrations and temperature were varied so that ice contents for all samples were almost equal. Neither recrystallization rates nor diffusion coefficients depended uniformly on temperature. The recrystallization rates increased with increasing the diffusion coefficients, and a direct relationship was found between recrystallization rate and diffusion coefficient. This indicated that self-diffusion coefficient of water component in freeze-concentrated matrix is a useful parameter for predicting and controlling recrystallization rate in sugar solutions relevant to frozen desserts.     

Transgenic alternative-splicing reporters reveal tissue-specific expression profiles and regulation mechanisms in vivo

Alternative splicing of pre-mRNAs allows multicellular organisms to create a huge diversity of proteomes from a finite number of genes. But extensive studies in vitro or in cultured cells have not fully explained the regulation mechanisms of tissue-specific or developmentally regulated alternative splicing in living organisms. Here we report a transgenic reporter system that allows visualization of expression profiles of mutually exclusive exons in Caenorhabditis elegans. Reporters for egl-15 exons 5A and 5B showed tissue-specific profiles, and we isolated mutants defective in the tissue specificity. We identified alternative-splicing defective-1 (asd-1), encoding a new RNA-binding protein of the evolutionarily conserved Fox-1 family, as a regulator of the egl-15 reporter. Furthermore, an asd-1;fox-1 double mutant was defective in the expression of endogenous egl-15 (5A) and phenocopied egl-15 (5A) mutant. This transgenic reporter system can be a powerful experimental tool for the comprehensive study of expression profiles and regulation mechanisms of alternative splicing in metazoans.     

An Electron Spin Resonance Study for Real-time Detection of Ascorbyl Free Radicals After Addition of Dimethyl Sulfoxide in Murine Hippocampus or Plasma During Kainic Acid-Induced Seizures

Electron spin resonance (ESR)-silent ascorbate solutions generate a detectable, likely concentration-dependent signal of ascorbyl free radicals (AFR) immediately upon addition of a molar excess of dimethyl sulfoxide (DMSO). We aimed to perform quantitative ESR analysis of AFR in real time after addition of DMSO (AFR/DMSO) to evaluate ascorbate concentrations in fresh hippocampus or plasma following systemic administration of kainate in mice. Use of a special tissue-type quartz cell allowed immediate detection of AFR/DMSO ESR spectra in fresh tissues from mice. AFR/DMSO content was increased significantly in fresh hippocampus or plasma obtained during kainate-induced seizures of mice, reaching maximum levels at 90 min after intraperitoneal administration of 50 mg/kg kainic acid. This suggests that oxidative injury of the hippocampus resulted from the accumulation of large amounts of ascorbic acid in the brain after kainic acid administration. AFR/DMSO content measured on an ESR spectrometer can be used for real-time evaluation of ascorbate content in fresh tissue. Due to the simplicity, good performance, low cost and real-time monitoring of ascorbate, this method may be applied to clinical research and treatment in the future.