Divergence among rice cultivars reveals roles for transposition and epimutation in ongoing evolution of genomic imprinting.

Parent-of-origin-dependent gene expression in mammals and flowering plants results from differing chromatin imprints (genomic imprinting) between maternally and paternally inherited alleles. Imprinted gene expression in the endosperm of seeds is associated with localized hypomethylation of maternally but not paternally inherited DNA, with certain small RNAs also displaying parent-of-origin-specific expression. To understand the evolution of imprinting mechanisms in Oryza sativa (rice), we analyzed imprinting divergence among four cultivars that span both japonica and indica subspecies: Nipponbare, Kitaake, 93-11, and IR64. Most imprinted genes are imprinted across cultivars and enriched for functions in chromatin and transcriptional regulation, development, and signaling. However, 4 to 11% of imprinted genes display divergent imprinting. Analyses of DNA methylation and small RNAs revealed that endosperm-specific 24-nt small RNA-producing loci show weak RNA-directed DNA methylation, frequently overlap genes, and are imprinted four times more often than genes. However, imprinting divergence most often correlated with local DNA methylation epimutations (9 of 17 assessable loci), which were largely stable within subspecies. Small insertion/deletion events and transposable element insertions accompanied 4 of the 9 locally epimutated loci and associated with imprinting divergence at another 4 of the remaining 8 loci. Correlating epigenetic and genetic variation occurred at key regulatory regions-the promoter and transcription start site of maternally biased genes, and the promoter and gene body of paternally biased genes. Our results reinforce models for the role of maternal-specific DNA hypomethylation in imprinting of both maternally and paternally biased genes, and highlight the role of transposition and epimutation in rice imprinting evolution.

The AAA-ATPase molecular chaperone Cdc48/p97 disassembles sumoylated centromeres, decondenses heterochromatin, and activates ribosomal RNA genes.

Centromeres mediate chromosome segregation and are defined by the centromere-specific histone H3 variant (CenH3)/centromere protein A (CENP-A). Removal of CenH3 from centromeres is a general property of terminally differentiated cells, and the persistence of CenH3 increases the risk of diseases such as cancer. However, active mechanisms of centromere disassembly are unknown. Nondividing Arabidopsis pollen vegetative cells, which transport engulfed sperm by extended tip growth, undergo loss of CenH3; centromeric heterochromatin decondensation; and bulk activation of silent rRNA genes, accompanied by their translocation into the nucleolus. Here, we show that these processes are blocked by mutations in the evolutionarily conserved AAA-ATPase molecular chaperone, CDC48A, homologous to yeast Cdc48 and human p97 proteins, both of which are implicated in ubiquitin/small ubiquitin-like modifier (SUMO)-targeted protein degradation. We demonstrate that CDC48A physically associates with its heterodimeric cofactor UFD1-NPL4, known to bind ubiquitin and SUMO, as well as with SUMO1-modified CenH3 and mutations in NPL4 phenocopy cdc48a mutations. In WT vegetative cell nuclei, genetically unlinked ribosomal DNA (rDNA) loci are uniquely clustered together within the nucleolus and all major rRNA gene variants, including those rDNA variants silenced in leaves, are transcribed. In cdc48a mutant vegetative cell nuclei, however, these rDNA loci frequently colocalized with condensed centromeric heterochromatin at the external periphery of the nucleolus. Our results indicate that the CDC48A(NPL4) complex actively removes sumoylated CenH3 from centromeres and disrupts centromeric heterochromatin to release bulk rRNA genes into the nucleolus for ribosome production, which fuels single nucleus-driven pollen tube growth and is essential for plant reproduction.

Imprinted expression of genes and small RNA is associated with localized hypomethylation of the maternal genome in rice endosperm.

Arabidopsis thaliana endosperm, a transient tissue that nourishes the embryo, exhibits extensive localized DNA demethylation on maternally inherited chromosomes. Demethylation mediates parent-of-origin-specific (imprinted) gene expression but is apparently unnecessary for the extensive accumulation of maternally biased small RNA (sRNA) molecules detected in seeds. Endosperm DNA in the distantly related monocots rice and maize is likewise locally hypomethylated, but whether this hypomethylation is generally parent-of-origin specific is unknown. Imprinted expression of sRNA also remains uninvestigated in monocot seeds. Here, we report high-coverage sequencing of the Kitaake rice cultivar that enabled us to show that localized hypomethylation in rice endosperm occurs solely on the maternal genome, preferring regions of high DNA accessibility. Maternally expressed imprinted genes are enriched for hypomethylation at putative promoter regions and transcriptional termini and paternally expressed genes at promoters and gene bodies, mirroring our recent results in A. thaliana. However, unlike in A. thaliana, rice endosperm sRNA populations are dominated by specific strong sRNA-producing loci, and imprinted 24-nt sRNAs are expressed from both parental genomes and correlate with hypomethylation. Overlaps between imprinted sRNA loci and imprinted genes expressed from opposite alleles suggest that sRNAs may regulate genomic imprinting. Whereas sRNAs in seedling tissues primarily originate from small class II (cut-and-paste) transposable elements, those in endosperm are more uniformly derived, including sequences from other transposon classes, as well as genic and intergenic regions. Our data indicate that the endosperm exhibits a unique pattern of sRNA expression and suggest that localized hypomethylation of maternal endosperm DNA is conserved in flowering plants.

Active DNA demethylation in plant companion cells reinforces transposon methylation in gametes.

The Arabidopsis thaliana central cell, the companion cell of the egg, undergoes DNA demethylation before fertilization, but the targeting preferences, mechanism, and biological significance of this process remain unclear. Here, we show that active DNA demethylation mediated by the DEMETER DNA glycosylase accounts for all of the demethylation in the central cell and preferentially targets small, AT-rich, and nucleosome-depleted euchromatic transposable elements. The vegetative cell, the companion cell of sperm, also undergoes DEMETER-dependent demethylation of similar sequences, and lack of DEMETER in vegetative cells causes reduced small RNA-directed DNA methylation of transposons in sperm. Our results demonstrate that demethylation in companion cells reinforces transposon methylation in plant gametes and likely contributes to stable silencing of transposable elements across generations.

[Descending necrotizing mediastinitis extended to empyema].

A 61-year-old man came to our hospital complained of neck swelling after extracting a tooth. Cervical drainage was performed in the diagnosis of cervical abscess. Two days later, left pleural effusion appeared and its bacteriologic culture showed Streptococcus constellatus. Computed tomography (CT) scan showed massive retained pus in the mediastinum. Thoracic drainage alone wasn't effective and the left thoracotomy was immediately performed to open the mediastinal pleura and curette the thoracic cavity. After surgery, left thoracic cavity was irrigated with a large volume of saline solution via the thoracic drains for a month, resulting in successful recovery. Immediate open drainage and irrigation are very important in case of descending necrotizing mediastinitis rapidly developing empyema.

Cervical radiculopathy due to intra-arterial infusion of cisplatin.

A 55-year-old man with cervical radiculopathy (C5-C8) was referred to us following intra-arterial infusion of cisplatin (CDDP) because of a recurrent neck mass of laryngeal cancer. Three hours after the CDDP infusion, he had noticed general weakness of the left upper extremity and hypoaesthesia of the lateral side of the upper and lower arm. The next day he was diagnosed with left cervical radiculopathy of C5 to C8, which improved gradually and had resolved completely six months after the infusion. Even with proper positioning of the infusion catheter to minimize potential complications, for anatomical reasons there are always some risks of neural injury with intra-arterial infusion from branches of the subclavian artery. This procedure should be carefully indicated in the case of a large neck tumour that is perfused from the major branches of the subclavian artery.

[Head and neck reconstruction using laparoscopically harvested omentum].

Since the need for laparotomy in harvesting the omentum is the most significant drawback, the omentum has not been the tissue of choice for reconstructive surgery. To compensate for this drawback, we started laparoscopic harvesting of the omentum and clarified the advantages and disadvantages of this procedure. Ten patients underwent laparoscopic harvesting of the omentum by abdominal surgeons, followed by reconstruction of head and neck defects. Surgery was conducted in 5 cases of defect reconstructions for parotid gland tumor surgery and 5 of oropharyngeal defect after cancer surgery. The average harvesting time was 107 minutes (55-140 minutes) and used the omentum and different amounts and length of the vascular pedicle. Although the omentum was successfully transplanted in 9 of 10 cases, 2 cases showed partial peripheral necrosis and 1 total necrosis. With the advantage of laparoscopic harvesting of the omentum, we could obtain appropriate omental size for the defect size. Especially after total parotidecomy, the omentum was useful to fill in the defect, reducing the patients' worries about postoperative deformity. In one case, the omentum was used to treat Frey syndrome, successfully relieving the symptoms. In oropharyngeal reconstruction, the omentum is used to fill dead space and prevented postoperative infection. Although mild abdominal pain was observed a few days after surgery, no major abdominal complications such as intestinal perforation or ileus occurred in the 8 to 39 months following laparoscopic harvest of the omentum. Since the omentum is pliable and easily fills a complicated defect, the omentum is considered satisfactory for reconstructing defects of the lateral face after parotid tumor surgery and small defects after oropharyngeal tumor surgery.

Potential changes with gamma-band oscillation at the frontal scalp elicited by intravenous olfactory stimulation in humans.

Intravenous olfaction is a unique stimulation method often used in Japan to diagnose olfactory disturbances. Odorant is injected into a vein and transported by blood flow and respiration to the upper air tract. The intravenous olfaction might allow the potential at the frontal scalp to be recorded without contamination from electromyograms, such as those caused by sniffing. We injected Alinamin (thiamine propyldisulphide) into healthy subjects according to a standard protocol for clinical intravenous olfaction testing and we simultaneously recorded potential changes at the frontal scalp. When Alinamin was injected into the right median cubital vein over a 20 s period, the potential changes with gamma-band oscillations were detected 17.6 +/- 6.7 s (mean +/- SD) after the start of the injection. The main frequency component of this gamma-band oscillation is 30-160 Hz. The gamma-band oscillation elicited by intravenous olfactory stimulation (VOP) was similar to the induced wave of the olfactory bulb. Mapping the VOPs on the frontal scalp of a subject with less developed frontal sinuses and the relation between the thickness of the frontal sinuses and VOP amplitude suggest an intracranial source, possibly the olfactory bulb. The gamma-band potential at the frontal scalp is a useful measure of central disturbance.

Role of nerve growth factor in the olfactory system.

Olfactory neurons are unique in the mammalian nervous system because of their capacity to regenerate in adult animals. It has been shown that olfactory receptor cells located in the olfactory epithelium are replaced on a continuous basis and in response to injury throughout the life span of most species. NGF, which is one of the neurotrophic factors, is present in many areas of the central and peripheral nervous system. It has been shown that NGF in the olfactory bulb plays a role in the survival of cholinergic neurons in the horizontal limb of the diagonal band (HDB). Recent studies of NGF in the olfactory bulb suggest that it is involved in the development, maintenance, and regeneration of olfactory receptor cells. In this study, we review reports examining the relationship between NGF in the olfactory bulb and neuronal regeneration and development in the mammalian olfactory systems. Low- and high-affinity NGF receptor immunoreactivity is markedly expressed during regeneration and at different stages of development in the mouse olfactory system. This level of immunoreactivity is no longer present after completion of regeneration and at maturation. Other findings indicate that NGF injected into the olfactory bulb is transported retrogradely to the olfactory epithelium. It has also been shown that continuous anti-NGF antibody injection into the olfactory bulb causes degeneration and olfactory dysfunction. Administration of NGF directory into nasal cavity results in an increase in the expression of olfactory marker protein within the olfactory epithelium in axotomized rats. These findings suggested that the presence of NGF in the olfactory bulb plays an essential role in regeneration, maintenance, and development in the olfactory system of mammals.

Differential recognition of tyrosine-based basolateral signals by AP-1B subunit mu1B in polarized epithelial cells.

To investigate the importance of tyrosine recognition by the AP-1B clathrin adaptor subunit mu1B for basolateral sorting of integral membrane proteins in polarized epithelial cells, we have produced and characterized a mutant form of mu1B. The mutant (M-mu1B) contains alanine substitutions of each of the four conserved residues, which in the AP-2 adaptor subunit micro2 are critical for interacting with tyrosine-based endocytosis signals. We show M-mu1B is defective for tyrosine binding in vitro, but is nevertheless incorporated into AP-1 complexes in transfected cells. Using LLC-PK1 cells expressing either wild type or M-mu1B, we find that there is inefficient basolateral expression of membrane proteins whose basolateral targeting signals share critical tyrosines with signals for endocytosis. In contrast, membrane proteins whose basolateral targeting signals are distinct from their endocytosis signals (transferrin and low-density lipoprotein receptors) accumulate at the basolateral domain normally, although in a manner that is strictly dependent on mu1B or M-mu1B expression. Our results suggest that mu1B interacts with different classes of basolateral targeting signals in distinct ways.

Nasopharyngeal penicillin-resistant Streptococcus pneumoniae strains among young children in Japan.

OBJECTIVE: A rapid increase of penicillin resistance in Streptococcus pneumoniae has recently been reported in most areas of the world. Penicillin-resistant S. pneumoniae and other resistant bacteria are the principal causes of recurrent acute otitis media (AOM). Penicillin-resistant S. pneumoniae was examined so that we could investigate the bacteriologic and clinical interpretations of nasopharyngeal flora from healthy children. METHODS: We obtained nasopharyngeal swab specimens from healthy children attending a day care center and from children attending a public health examination in Kanazawa, Japan. We also obtained clinical specimens from children with AOM who visited the Kanazawa University Hospital and 4 other hospitals in Ishikawa Prefecture in Japan. RESULTS: The chief bacteria from the children were S. pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. Penicillin-resistant S. pneumoniae was identified in 75% of children attending a day care center. On the other hand, S. pneumoniae was identified in 37% of children who were not attending day care. Of the children with AOM, penicillin-resistant S. pneumoniae was identified in 77%. CONCLUSION: Our results suggest that there is a strong relationship between day care attendance and nasopharyngeal carriage of S. pneumoniae. The carriage rate of penicillin-resistant S. pneumoniae in healthy children in day care centers was very high and similar to the carriage rate of young patients with AOM in Japan.

Thrombosed varicose vein in the sternocleidomastoid muscle.

A thrombosed varicose vein arising in the neck is quite rare. A 68-year-old Japanese male was referred to our hospital because of a mass in his left upper neck. The mass did not change in size in response to strain resulting from Valsalva's manouvre. Magnetic resonance imaging (MRI) showed iso-signal intensity of the mass on T1-weighted images and a target-like signal arrangement (concentric hyper-, hypo-, hyper-signal intensity from outside to inside) on T2-weighted images. Surgical excision revealed that the tumour arose from the intramuscular small vein in the sternocleidomastoid muscle. The pathological examination revealed the mass to be a thrombosed varicose vein with capillarization in the dilated vein wall. The de-oxygenation and degradation of haemoglobin were thought to be responsible for these characteristic MRI findings. The concentric signal distribution on MRI strongly suggested this pathology.

Glucocorticoid enhances Na(+)/K(+) ATPase mRNA expression in rat olfactory mucosa during regeneration: a possible mechanism for recovery from olfactory disturbance.

Systemic or topical application of glucocorticoid is the treatment of choice for olfactory disturbance. Recently, Na(+)/K(+) ATPase and glucocorticoid receptor immunoreactivity in the olfactory mucosa was reported. To elucidate a glucocorticoid action on Na(+)/K(+) ATPase production, an animal model was produced by an intra-nasal application of 5% ZnSO(4) solution to Wistar rats. Dexamethasone was injected i.p. (0.01 mg/100 g) for 14 days after the insult. Histologically, the regeneration process was completed on day 14 in both dexamethasone- and saline-injected control rats. We used a quantitative polymerase chain reaction (PCR) method to evaluate mRNA production of Na(+)/K(+) ATPase and glucocorticoid receptor. In dexamethasone-injected rats, up-regulation of glucocorticoid receptor mRNA (95% more than control rats, P = 0.00068, unpaired t-test) and of Na(+)/K(+) ATPase mRNA expression (76% more than control rats, P = 0.0042) was observed on day 14. The increased Na(+)/K(+) ATPase expression in the regenerated olfactory mucosa is thought to be beneficial for an active uptake of K(+), which is released during excitation, around olfactory neurons and for the transepithelial absorption of Na(+) from olfactory mucus. Dexamethasone may thus contribute to the recovery of function after the morphological regeneration in part, at least, through its receptor by regulation of the ionic concentration in the olfactory mucosal microenvironment.