Roles of RecA protein in spontaneous mutagenesis in Escherichia coli

To verify the extent of contribution of spontaneous DNA lesions to spontaneous mutagenesis, we have developed a new genetic system to examine simultaneously both forward mutations and recombination events occurring within about 600 base pairs of a transgenic rpsL target sequence located on Escherichia coli chromosome. In a wild-type strain, the recombination events were occurring at a frequency comparable to that of point mutations within the rpsL sequence. When the cells were UV-irradiated, the recombination events were induced much more sharply than point mutations. In a recA null mutant, no recombination event was observed. These data suggest that the blockage of DNA replication, probably caused by spontaneous DNA lesions, occurs often in normally growing E. coli cells and is mainly processed by cellular functions requiring the RecA protein. However, the recA mutant strain showed elevated frequencies of single-base frameshifts and large deletions, implying a novel mutator action of this strain. A similar mutator action of the recA mutant was also observed with a plasmid-based rpsL mutation assay. Therefore, if the recombinogenic problems in DNA replication are not properly processed by the RecA function, these would be a potential source for mutagenesis leading to single-base frameshift and large deletion in E. coli. Furthermore, the single-base frameshifts induced in the recA-deficient cells appeared to be efficiently suppressed by the mutS-dependent mismatch repair system. Thus, it seems likely that the single-base frameshifts are derived from slippage errors that are not directly caused by DNA lesions but made indirectly during some kind of error-prone DNA synthesis in the recA mutant cells.     

Repression of small toxic protein synthesis by the Sib and OhsC small RNAs

The sequences encoding the QUAD1 RNAs were initially identified as four repeats in Escherichia coli. These repeats, herein renamed SIB, are conserved in closely related bacteria, although the number of repeats varies. All five Sib RNAs in E. coli MG1655 are expressed, and no phenotype was observed for a five-sib deletion strain. However, a phenotype reminiscent of plasmid addiction was observed for overexpression of the Sib RNAs, and further examination of the SIB repeat sequences revealed conserved open reading frames encoding highly hydrophobic 18- to 19-amino-acid proteins (Ibs) opposite each sib gene. The Ibs proteins were found to be toxic when overexpressed and this toxicity could be prevented by coexpression of the corresponding Sib RNA. Two other RNAs encoded divergently in the yfhL-acpS intergenic region were similarly found to encode a small hydrophobic protein (ShoB) and an antisense RNA regulator (OhsC). Overexpression of both IbsC and ShoB led to immediate changes in membrane potential suggesting both proteins affect the cell envelope. Whole genome expression analysis showed that overexpression of IbsC and ShoB, as well as the small hydrophobic LdrD and TisB proteins, has both overlapping and unique consequences for the cell.     

A mutant of Escherichia coli K-12, URT-34, with a temperature-sensitive defect at the incision step of the excision repair mechanism

URT-43, which has a defect in excision repair, exhibits a temperature-dependent ultraviolet survival. It was shown that URT-43 requires protein synthesis but not DNA synthesis for recovery, by examining recovery in a growth medium containing chloramphenicol or nalidixic acid. The recovery of irradiated bacteriophage λ in URT-43 took place in a medium containing nalidixic acid at 30°, but not at 41°, and chloramphenicol prevented this recovery. These results seem to imply that the product of the mutated gene in URT-43 is labile. URT-43 was confirmed to have a temperature-sensitive mutation at the incision step of the excision repair mechanism by examining the nick formation of parental DNA in alkaline sucrose gradients. The release of pyrimidine dimers was reinvestigated directly by one- and two-dimensional paper-chromatography and indirectly by examining the distribution of DNA molecules synthesized after irradiation. Dimers were excised into the acid-soluble fraction when growing bacteria were incubated, but were not excised when in amino acid starved bacteria. These results suggest that URT-43 is a mutant slowly excising pyrimidine dimers because the product of a mutated gene concerned with the incision step of the excision repair mechanism is unstable.     

Japanese Rice Wine can reduce psychophysical stress-induced depression-like behaviors and Fos expression in the trigeminal subnucleus caudalis evoked by masseter muscle injury in the rats

We determined if Japanese Rice Wine (Sake) had inhibitory effects on stress-induced enhancement of masseter muscle (MM) nociception in the rats. Male rats were subjected to the repeated forced swim stress (FS) or sham conditionings from Day ?3 to ?1. Daily administration of Sake or saline was conducted after each stress conditioning. At Day 0 the number of Fos positive cells, a marker for neural activity, was quantified at the trigeminal subnucleus caudalis (Vc) region by MM injury with formalin. FS increased MM-evoked Fos expression in the Vc region, which was inhibited by Sake compared to saline administration. Sake did not alter the number of Fos positive cells under sham conditions, indicating that inhibitory roles of Sake on neural activity in the Vc region were seen under FS conditions. These findings indicated that Sake had inhibitory roles on stress-induced MM nociception at the Vc region in our experimental conditions.     

Alterations of Regional Cerebral Blood Flow in Tinnitus Patients as Assessed Using Single-Photon Emission Computed Tomography

Tinnitus is the perception of phantom sound without an external auditory stimulus. Using neuroimaging techniques, such as positron emission tomography, electroencephalography, magnetoencephalography, and functional magnetic resonance imaging (fMRI), many studies have demonstrated that abnormal functions of the central nervous system are closely associated with tinnitus. In our previous research, we reported using resting-state fMRI that several brain regions, including the rectus gyrus, cingulate gyrus, thalamus, hippocampus, caudate, inferior temporal gyrus, cerebellar hemisphere, and medial superior frontal gyrus, were associated with tinnitus distress and loudness. To reconfirm these results and probe target regions for repetitive transcranial magnetic stimulation (rTMS), we investigated the regional cerebral blood flow (rCBF) between younger tinnitus patients (<60 years old) and the age-matched controls using single-photon emission computed tomography and easy Z-score imaging system. Compared with that of controls, the rCBF of tinnitus patients was significantly lower in the bilateral medial superior frontal gyri, left middle occipital gyrus and significantly higher in the bilateral cerebellar hemispheres and vermis, bilateral middle temporal gyri, right fusiform gyrus. No clear differences were observed between tinnitus patients with normal and impaired hearing. Regardless of the assessment modality, similar brain regions were identified as characteristic in tinnitus patients. These regions are potentially involved in the pathophysiology of chronic subjective tinnitus.     

Autopsy Report of a 7-Year Old Patient with the Mosaic Trisomy 13

We present here a long survival case of a patient with the mosaic form of trisomy 13 who died of aspiration pneumonia at the age of 7 years and 4 months. The autopsy revealed olfactory aplasia and fenestration of the septum pellucidum, and dilated lateral ventricles and atrophic hippocampus. Furthermore, there were numerous “torpedos” (i.e., swollen fusiform Purkinje cell axons), mostly in the granular layer underneath the Purkinje cell layer, and, occasionally, in the granular layer. Similar neuropathological findings have been reported in elderly cases of essential tremor, Parkinson’s disease, or Alzheimer’s disease. Precise mechanism for this axonal change is still unclear. These pathological changes have never previously been reported in the literature on trisomy 13, and the present patient is one of the oldest autopsied individuals with the mosaic trisomy 13.     

Variance and autocorrelation of the spontaneous slow brain activity

Slow (<0.1 Hz) oscillatory activity in the human brain, as measured by functional magnetic imaging, has been used to identify neural networks and their dysfunction in specific brain diseases. Its intrinsic properties may also be useful to investigate brain functions. We investigated the two functional maps: variance and first order autocorrelation coefficient (r(1)). These two maps had distinct spatial distributions and the values were significantly different among the subdivisions of the precuneus and posterior cingulate cortex that were identified in functional connectivity (FC) studies. The results reinforce the functional segregation of these subdivisions and indicate that the intrinsic properties of the slow brain activity have physiological relevance. Further, we propose a sample size (degree of freedom) correction when assessing the statistical significance of FC strength with r(1) values, which enables a better understanding of the network changes related to various brain diseases.