Transient expression of a mitochondrial gene cluster including rps4 is essential for the phase-shift of Dictyostelium cells from growth to differentiation

Using synchronized Dictyostelium discoideum Ax-2 cells and the differential display method, a mitochondrial gene cluster (referred to as differentiation-associated gene 3; dia3) was isolated as one of the genes expressed specifically during the transition of Ax-2 cells from growth to differentiation. The dia3 gene encodes for a mitochondrial protein cluster (NADH dehydrogenase (NAD) subunit 11, 5, ribosomal protein S4 (RPS4), RPS2, and NAD4L). Northern blot analysis using nonsynchronized Ax-2 cells has shown that the dia3 RNA of about 8 kb is scarcely expressed during the vegetative growth phase, and the maximal expression was attained at 2 h after starvation. To analyze the gene function of dia3, we tried inactivation of rps4 by means of homologous recombination and obtained several transformed clones showing mitochondrial DNA heteroplasmy. The transformed cells grew normally in nutrient medium, but their development after starvation was greatly impaired, thus resulting in the failure of many cells to differentiate. In this connection, the cAMP receptor 1 (car1) expression, which is one of the earliest markers of differentiation, was found to be markedly reduced in the rps4-inactivated cells.     

Hyphantria cunea ferritin heavy chain homologue: cDNA sequence and mRNA expression

We have sequenced a cDNA clone encoding a 26-kDa ferritin subunit, which was heavy chain homologue (HCH), in fall webworm, Hyphantria cunea. The HCH cDNA was obtained from the screening of a cDNA library using a PCR product. H. cunea ferritin is composed of 221 amino acid residues and their calculated mass is 26,160 Da. The protein contains the conserved motifs for the ferroxidase center typical for heavy chains of vertebrate ferritin. The iron-responsive element sequence with a predicted stem-loop structure is present in the 5'-untranslated region of ferritin HCH mRNA. The sequence alignment of ferritin HCH shows 68.9 and 68.7% identity with Galleria mellonella HCH (26 kDa ferritin) and Manduca sexta HCH, respectively. While G type insect ferritin vertebrate light chain homologue (LCH) is distantly related to H. cunea ferritin HCH (17.2-20.8%), the Northern blot analysis revealed that H. cunea ferritin HCH was ubiquitously expressed in various tissues and all developmental stages. The ferritin expression of midgut is more responsive to iron-fed, compared to fat body in H. cunea.     

Synthesis and biological evaluation of benzimidazole-4,7-diones that inhibit vascular smooth muscle cell proliferation

A series of 6-arylamino-5-chloro-benzimidazole-4,7-diones were synthesized and tested for their inhibitory activity on the rat aortic smooth muscle cell (RAoSMC) proliferation. Among them, 6-arylamino-5-chloro-2-methyl-benzimidazole-4,7-diones exhibited potent antiproliferative activity. Benzimidazole-4,7-dione 2c activated SAPK/JNK signaling pathway in the RAoSMCs.     

Partial purification and characterization of an extracellular protease from<Emphasis Type="Italic">Xenorhabdus nematophilus</Emphasis>, a symbiotic bacterium isolated from an entomopathogenic nematode,<Emphasis Type="Italic">Steinernema glaseri</Emphasis>

Entomopathogenic nematodes are used for insect control. Herein, an extracellular protease was partially purified from a culture supernatant ofXenorhabdus nematophilus, a symbiotic bacterium of an entomopathogenic nematode,Steinernema glaseri, using precipitation with 80% v/v isopropyl alcohol followed by gel permeation chromatography with a packed Sephacryl S-300 HR media. The partially purified protease exhibited maximal activity at pH 7 in the presence of 1 mM CaCl₂. The protease was identified as a metallo-protease based on the inhibition of its activity by the metal chelating agent, EDTA.     

Tissue-specific down-regulation of RIPK 2 in Mycobacterium leprae-infected nu/nu mice

RIPK 2 is adapter molecule in the signal pathway involved in Toll-like receptors. However, there has been no reported association between receptor-interacting serine/threonine kinase 2 (RIPK 2) expression and the infectious diseases involving mycobacterial infection. This study found that its expression was down-regulated in the footpads and skin but was up-regulated in the liver of Mycobacterium leprae-infected nu/nu mice compared with those of the M. leprae non-infected nu/nu mice. It was observed that the interlukin-12p40 and interferon-gamma genes involved in the susceptibility of M. leprae were down-regulated in the skin but were up-regulated in the liver. Overall, this suggests that regulation of RIPK 2 expression is tissue-specifically associated with M. leprae infection.     

Effects of Aroclor 1254 on the expression of rat placental PRL-family genes

This study was performed to investigate the effects of Aroclor 1254 (A1254), a commercial polychlorinated biphenyl mixture, on the expression of rat placental prolactin (PRL) family genes and reproductive activity. Placental lactogen-Iv and -II, and prolactin-like protein-A and -C mRNA levels were significantly decreased in the placentas of A1254-treated rats in a dose-dependent manner. The mRNA levels of Pit-1alpha and beta isotypes, which are involved in the regulation of PRL family gene expression, were also decreased in the A1254-treated rat placenta. In the rat placental junctional zone, high-dose A1254 (25 mg/kg B.W.) treatment reduced the number of spongiotrophoblasts, cells in which the PRL family genes are expressed. Finally, maternal exposure to A1254 was shown to have significant toxic effects on reproductive activity, including embryonic and placental growth retardation, delay of parturition, and reduction of the number of pups per litter. The results of the present study indicated that A1254 has an inhibitory effect on PRL family, Pit-1alpha, and beta gene expression in the rat placenta, leading to significant toxic effects on reproductive activity in rats.     

Differential expression of the tetracycline-controlled transactivator-driven human CYP1B1 gene in double-transgenic mice is due to androgens: application for detecting androgens and antiandrogens

Differential expression of the tetracycline-controlled transactivator (tTA)-driven human cytochrome p450 (CYP) 1B1 gene was found in the livers of male mice, at high levels in neonates, but at low levels in adults. The goals of this study were to determine whether the differential expression of the tTA-driven human CYP1B1 (hCYP1B1) gene in neonates and adults was testosterone dependent and whether flutamide, a representative potent antiandrogen, led to the induction of hCYP1B1. This was tested by treating castrated transgenic mice with testosterone propionate and musk extracts. It was concluded that: (i). the levels of expression of both tTA and hCYP1B1 gradually declined, with clear changes being apparent between 2 and 4 weeks of age, (ii). castration of adult males resulted in the increased expressions of both tTA and hCYP1B1 to levels similar to those found in adult females, (iii). treatment of castrated male and adult female mice with testosterone propionate and musk extracts led to the restoration of the levels of expression of hCYP1B1 in the adult males, and (iv). treatment of adult males with flutamide caused an increase in the levels of expression of hCYP1B1 in the adult females, as indicated by the antiandrogenic activity. Thus, the differential expression of the tTA-driven hCYP1B1 gene in the transgenic mice was caused by androgen, and it is possible that castrated male and adult female mice expressing the tTA-controlled hCYP1B1 could be used as the basis for a strategy for the detection of androgens and antiandrogens.     

Acquisition of heat shock tolerance by regulation of intracellular redox states

In the yeast Saccharomyces cerevisiae, a mild heat treatment strongly induces Hsp104p which provides acquisition of thermotolerance. The mechanism by which Hsp104p protects cells from the severe heat shock has not yet been completely elucidated. In this study, a pivotal role of Hsp104p as an efficient scavenger of the reactive oxygen species (ROS) is investigated. In our previous study, we reported that Hsp104p acted as a regulator in the mitochondrial respiration pathway. In this report, the recombinant wild-type and hypersensitive ras mutants (ira2Delta) with the extrachromosomal plasmids wild-type and mutant hsp104 genes were studied. The resulting strains successfully expressed both wild-type and mutant Hsp104p and showed the thermotolerance phenotype in the strain with the functional wild-type Hsp104p expressed. Upon treatment with H2O2 and menadione, the strains with the functional Hsp104p expressed showed higher survival rates than the other mutants, indicating the protective role of Hsp104p from the oxidative stress. Fluorescence measurement of the oxidation-dependent probe, 2',7'-dichlorofluoroscein diacetate (H2DCFDA), also indicated that Hsp104p significantly reduced the amount of ROS. Resistance to the oxidative stress was independent of the amount of the glutathione in the hyperactivated ras mutants. Taken all together, this study confirms that Hsp104p plays a crucial role in keeping cells from being damaged by the oxidative stress, thus acting as a modulator of the intracellular redox state.