Aryl hydrocarbon receptor signaling involved in the invasiveness of LNCaP cells

There is now mounting evidence that the aryl hydrocarbon receptor (AhR) plays an important role in physiologic responses such as development, cell cycle regulation, immune function and also malignant transformation in various tissues. The strong nuclear AhR expression is observed in the invasive phenotype, and an elevated nuclear AhR expression is associated with a poor prognosis of human prostate cancer. On the other hand, there are conflicting results that the AhR deficiency results in increased susceptibility to prostate tumors in mouse model. In the present study, we investigated AhR expression and its role in the growth and invasiveness of human prostate cancer cells. The AhR protein expression was detected in prostate cancer cell lines and human prostate cancer tissues. A small interfering RNA targeting AhR, constitutive active AhR expression vector, and AhR agonist and antagonist were used to moderate its expression and signaling. The induction of AhR signaling attenuated invasiveness of prostate cancer cells without affecting the cellular growth rate. These results suggest that AhR signaling in prostate cancer cells facilitates invasion of these cells, and modulation with this signaling can be a potential therapeutic target of invasive tumors.     

Mate-searching behavior of the black chafer <Emphasis Type="Italic">Holotrichia kiotonensis</Emphasis> (Coleoptera: Scarabaeidae): identification of a sex pheromone,and male orientation behavior controlled by olfactory and visual cues

In the black chafer Holotrichia kiotonensis Brenske (Coleoptera: Scarabaeidae), mating behavior was observed between 1940 and 2010 hours at < 0.1 lx in both the laboratory and the field. In the laboratory, an ether extract of female abdominal glands induced a series of pre-mating behaviors such as short-distance orientation and abdominal bending. When the extract was fractionated by silica gel column chromatography, the active fraction was eluted with 50% ether in hexane and 100% ether. Gas chromatography–mass spectrometry analysis revealed that both active fractions contained anthranilic acid (2-amino-benzoic acid) as a major compound. The amount of this compound was determined to be ca. 600 ng/female by high performance liquid chromatography analysis with a fluorescence detector. In the field, male chafers were observed to land on cotton balls impregnated with 10 mg of authentic anthranilic acid. When a white ball treated with anthranilic acid was placed 2–10 cm away from an untreated black ball, males were observed to land significantly more frequently on the latter. These results suggest that males could recognize white balls below 0.1 lx and landed on black balls. When a treated black ball was placed beside an untreated black ball, more males landed on the former. The difference was significant when the distance between the two lures was 5 or 10 cm, but not significant when it was 2 cm. These observations demonstrated that anthranilic acid was the sex-attractant pheromone for the black chafer H. kiotonensis and that the males located and landed on a pheromone source by using olfaction in conjunction with visual orientation. The importance of visual orientation in this nocturnal species is discussed in comparison with the congeneric diurnal species Holotrichia loochooana loochooana.     

Up-regulation of the peroxisomal β-oxidation system occurs in butyrate-grown Candida tropicalis following disruption of the gene encoding peroxisomal 3-ketoacyl-CoA thiolase

In the yeast Candida tropicalis, two thiolase isozymes, peroxisomal acetoacetyl-CoA thiolase and peroxisomal 3-ketoacyl-CoA thiolase, participate in the peroxisomal fatty acid β-oxidation system. Their individual contributions have been demonstrated in cells grown on butyrate, with C. tropicalis able to grow in the absence of either one. In the present study, a lack of peroxisomal 3-ketoacyl-CoA thiolase protein resulted in increased expression (up-regulation) of acetoacetyl-CoA thiolase and other peroxisomal proteins, whereas a lack of peroxisomal acetoacetyl-CoA thiolase produced no corresponding effect. Overexpression of the acetoacetyl-CoA thiolase gene did not suppress the up-regulation or the growth retardation on butyrate in cells without peroxisomal 3-ketoacyl-CoA thiolase, even though large amounts of the overexpressed acetoacetyl-CoA thiolase were detected in most of the peroxisomes of butyrate-grown cells. These results provide important evidence of the greater contribution of 3-ketoacyl-CoA thiolase to the peroxisomal β-oxidation system than acetoacetyl-CoA thiolase in C. tropicalis and a novel insight into the regulation of the peroxisomal β-oxidation system.     

Short-term preservation of mouse oocytes at 5 degrees C.

The temporary preservation of oocytes without freezing would be useful for some experiments. ICR mouse oocytes were kept in a preservation medium under mineral oil for 1, 2, 3, 4 or 7 days at 5 degrees C, and 1 or 2 days at 37 degrees C. In vitro fertilization was attempted on oocytes rinsed with TYH medium after preservation. More than 70% of morphologically normal oocytes were recovered from each preservation group. Fertilization rates of oocytes preserved for 1, 2, 3, 4 or 7 days at 5 degrees C were 69.9, 66.5, 45.3, 26.7 and 8.8% respectively. Fertilization rates of oocytes preserved for 1 or 2 days at 37 degrees C were 9.6 and 1.6%, respectively. Preservation of oocytes at 5 degrees C has some capability as a method of short-term storage without freezing.     

Changes in tonoplast protein and density with the development of pear fruit

Protoplasts isolated from pear fruit at the end of the cell‐division stage, 30 days after flowering (DAF), had already formed a large central vacuole and the vacuole occupied most of the protoplast. The changes in protein composition and density of the tonoplast (vacuolar membrane) were investigated during fruit development. After a linear sucrose density gradient centrifugation, the distribution of tonoplasts at 30 and 48 DAF was broad and began to narrow with further fruit development. This suggests that the tonoplast of young fruit is heterogeneous and becomes homogeneous with fruit development. The apparent density of the tonoplast at 30 DAF was approximately 1.12 g ml⁻¹; it decreased with fruit development and was finally 1.09 g ml⁻¹ in mature fruit. The phospholipid amount on the basis of tonoplast protein was 0.80 mg mg⁻¹ at 30 DAF. It increased with fruit development, and finally reached 7.49 mg mg⁻¹. This result indicates that the decrease in the density of the tonoplast was caused by the increase in the ratio of phospholipid to membrane protein. The protein composition of the tonoplast at each stage was quite different. The level of polypeptides of 94, 70, 61, 52, 48 and 41 kDa was low in young fruit and high in the middle or later stages of fruit development. In contrast, the level of a 76‐kDa polypeptide was high in young fruit and decreased with fruit development. Although their functions are still unclear, these tonoplast proteins may play important roles in fruit development.     

Genetic Evaluation of Physiological Functions of Thiolase Isozymes in the n-Alkane-Assimilating Yeast Candida tropicalis

The n-alkane-assimilating diploid yeast Candida tropicalis possesses three thiolase isozymes encoded by two pairs of alleles: cytosolic and peroxisomal acetoacetyl-coenzyme A (CoA) thiolases, encoded by CT-T1A and CT-T1B, and peroxisomal 3-ketoacyl-CoA thiolase, encoded by CT-T3A and CT-T3B. The physiological functions of these thiolases have been examined by gene disruption. The homozygous ct-t1aΔ/t1bΔ null mutation abolished the activity of acetoacetyl-CoA thiolase and resulted in mevalonate auxotrophy. The homozygous ct-t3aΔ/t3bΔ null mutation abolished the activity of 3-ketoacyl-CoA thiolase and resulted in growth deficiency on n-alkanes (C₁₀ to C₁₃). All thiolase activities in this yeast disappeared with the ct-t1aΔ/t1bΔ and ct-t3aΔ/t3bΔ null mutations. To further clarify the function of peroxisomal acetoacetyl-CoA thiolases, the site-directed mutation leading acetoacetyl-CoA thiolase without a putative C-terminal peroxisomal targeting signal was introduced on the CT-T1A locus in the ct-t1bΔ null mutant. The truncated acetoacetyl-CoA thiolase was solely present in cytoplasm, and the absence of acetoacetyl-CoA thiolase in peroxisomes had no effect on growth on all carbon sources employed. Growth on butyrate was not affected by a lack of peroxisomal acetoacetyl-CoA thiolase, while a retardation of growth by a lack of peroxisomal 3-ketoacyl-CoA thiolase was observed. A defect of both peroxisomal isozymes completely inhibited growth on butyrate. These results demonstrated that cytosolic acetoacetyl-CoA thiolase was indispensable for the mevalonate pathway and that both peroxisomal acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase could participate in peroxisomal β-oxidation. In addition to its essential contribution to the β-oxidation of longer-chain fatty acids, 3-ketoacyl-CoA thiolase contributed greatly even to the β-oxidation of a C₄ substrate butyrate.