ACTN4 copy number increase as a predictive biomarker for chemoradiotherapy of locally advanced pancreatic cancer.

BACKGROUND: Several clinical trials have compared chemotherapy alone and chemoradiotherapy (CRT) for locally advanced pancreatic cancer (LAPC) treatment. However, predictive biomarkers for optimal therapy of LAPC remain to be identified.We retrospectively estimated amplification of the ACTN4 gene to determine its usefulness as a predictive biomarker for LAPC. METHODS: The copy number of ACTN4 in 91 biopsy specimens of LAPC before treatment was evaluated using fluorescence in situ hybridisation (FISH). RESULTS: There were no statistically significant differences in overall survival (OS) or progression-free survival (PFS) of LAPC between patients treated with chemotherapy alone or with CRT. In a subgroup analysis of patients treated with CRT, patients with a copy number increase (CNI) of ACTN4 had a worse prognosis of OS than those with a normal copy number (NCN) of ACTN4 (P=0.0005, log-rank test). However, OS in the subgroup treated with chemotherapy alone was not significantly different between patients with a CNI and a NCN of ACTN4. In the patients with a NCN of ACTN4, the median survival time of PFS in CRT-treated patients was longer than that of patients treated with chemotherapy alone (P=0.049). CONCLUSIONS: The copy number of ACTN4 is a predictive biomarker for CRT of LAPC.

GAD65/GAD67 double knockout mice exhibit intermediate severity in both cleft palate and omphalocele compared with GAD67 knockout and VGAT knockout mice.

Inhibitory neurotransmitters, γ-aminobutyric acid (GABA) and glycine, are transported into synaptic vesicles by the vesicular GABA transporter (VGAT). Glutamate decarboxylase (GAD) is a GABA-synthesizing enzyme and two isoforms of GAD, GAD65 and GAD67 are encoded by two independent genes. There was virtually no GABA content in GAD65/GAD67 double knockout (GADs DKO) mouse brains. Neither GABAergic nor glycinergic inhibitory postsynaptic currents were almost detected in VGAT knockout (KO) mouse cultured neurons and spinal cords. GAD67 KO and VGAT KO mice displayed developmental abnormalities, cleft palate and omphalocele, suggesting that GABAergic transmission is involved in palate and abdominal wall formations. However, the incidence and severity of both failures in GAD67 KO mice were lower and less than those in VGAT KO mice. These results raise the possibility that GABAergic transmission mediated by GAD65-produced GABA and/or glycinergic transmission contributed to both palate and abdominal wall formations. However, it still remains unclear whether GABAergic transmission mediated by GAD65 and glycinergic transmission contribute to those formations. Here, to answer these questions, we generated GADs DKO mice and compared the phenotypes of GADs DKO mice with those of GAD67 KO and VGAT KO mice. Our anatomical analyses demonstrated that the incidence of cleft palate and omphalocele in GAD67 KO mice was 65.8% and 58.9%, respectively, but the incidence of both phenotypes in GADs DKO and VGAT KO mice was 100%. The severity of cleft palate and omphalocele was evaluated by elevation of palate shelves and size and liver inclusion of omphalocele, respectively. We observed that the phenotypes of cleft palate and omphalocele in GADs DKO mice were more and less severe than those in GAD67 KO and VGAT KO mice, respectively. These results indicate the significant contribution of not only GAD65-mediated GABAergic but also glycinergic transmissions to both palate and abdominal wall formations.

Morphology and distribution of lamellar corpuscles in the mesentery of the cat.

Mesentery samples obtained from 13 mixed breed cats were stereoscopically and microscopically examined and yielded the following results. In the mesentery, lamellar corpuscles were densely distributed around blood vessels, with a mean number of 182.2 ± 20.9 and mean maximum and minimum diameters of 0.98 ± 0.18 mm and 0.54 ± 0.08 mm, respectively. While most lamellar corpuscles were isolated, some complex lamellar corpuscles were found in the mesentery. Lamellar corpuscles in the mesentery appear to serve more as internal receptors that detect changes in the internal environment than as external receptors. In addition, those found around blood vessels in the mesentery are likely to be involved in blood pressure regulation.

Scanning electron microscopic study for pore formation of the greater omentum of cats.

The greater omentum of the cats is said to have a lace-like structure. However, there are only a few descriptions on whether pores exist, and there are not many morphological studies on this meshwork. In this study, the greater omentum of the cats was observed at each age of development using a scanning electron microscope. The greater omentum of the cats immediately after birth was found to be continuous, and no pores were observed. Also, development of microvilli was observed in the mesothelial cells on the surface of the greater omentum. In young cats at 3 months of age, small pores were sporadically observed, and at the ages of 6-12 months, there were more and larger pores. It was estimated that the pores on the greater omentum are formed in the process of moving from the movement of organs, such as the stomach, intestines and diaphragm, and the presence of these pores enables the passage of ascites between the omental bursa, the greater omentum and the serosal cavity of the wall without flowing through the omental foramen.

Low accumulation of drebrin at glutamatergic postsynaptic sites on GABAergic neurons.

Glutamatergic synapses form onto both glutamatergic and GABAergic neurons. These two types of glutamatergic synapses differ in their electrical responses to high-frequency stimulation and postsynaptic density protein composition. However, it is not known whether they differ in the actin cytoskeleton composition. In the present study, we used hippocampal neuronal cultures prepared from glutamate decarboxylase 67 (GAD67)-GFP knock-in mice and analyzed the differences in the actin cytoskeleton at glutamatergic synapses contacting GABAergic and glutamatergic neurons. Drebrin-binding actin filaments enriched in dendritic spines are known to play a pivotal role in spine formation. Immunocytochemical analyses demonstrated that drebrin accumulated at glutamatergic synapses on GABAergic neurons as well as at those on glutamatergic neurons. However, the density of drebrin clusters along dendrites in GABAergic neurons was significantly lower than those of glutamatergic neurons. Furthermore, the level of drebrin accumulating at glutamatergic synapses was lower on GABAergic neurons than on glutamatergic neurons. In neurons overexpressing drebrin, drebrin cluster density and accumulation levels in GABAergic and glutamatergic neurons were similar, suggesting that the low drebrin levels in the glutamatergic postsynaptic sites on GABAergic neurons may be because GABAergic neurons express low levels of drebrin. On the other hand, pharmacological analysis demonstrated that the postsynaptic localization of drebrin depended on actin cytoskeleton organization in both GABAergic and glutamatergic neurons. Together these results indicated that, although GABAergic and glutamatergic neurons share common regulatory systems affecting drebrin localization, the density of drebrin-positive glutamatergic synapses formed on GABAergic neurons is lower than those on glutamatergic neurons. This is probably due to the low expression of drebrin in GABAergic neurons.

[Tonsillar metastais from primary lung large cell neuroendocrine carcinoma in the early stage; report of a case].

We experienced an extremely rare case of large cell neuroendocrine carcinoma (LCNEC) of the lung metastasizing to the tonsil. A 66-year-old woman who had been undergone radical hysterectomy and radiated on whole pelvis in June, 2006, was pointed out 2 cm diameter abnormal shadow at the right S8 of the lung by computed tomography (CT) in May, 2007. In June, 2007, the right lower lobectomy with lymphandenectomy was done by video-assisted thoracic surgery (VATS). The pathological diagnosis was stage IA LCNEC of the lung. In April, 2008, multiple metastases to the brain appeared, and the patient unederwent gamma knife treatment. In addition, metastases to the liver, lymphnode of abdomen, left adrenal and bone followed in 1 month. Although we considered a chemotherapy, her general condition deteriorated rapidly with development of right tonsil metastasis and died as lasly as 3 months later.

Fluorescent labeling of both GABAergic and glycinergic neurons in vesicular GABA transporter (VGAT)-venus transgenic mouse.

Inhibitory neurons play important roles in a number of brain functions. They are composed of GABAergic neurons and glycinergic neurons, and vesicular GABA transporter (VGAT) is specifically expressed in these neurons. Since the inhibitory neurons are scattered around in the CNS, it is difficult to identify these cells in living brain preparations. The glutamate decarboxylase (GAD) 67-GFP knock-in mouse has been widely used for the identification of GABAergic neurons, but their GAD67 expression was decreased compared to the wild-type mice. To overcome such a problem and to highlight the function and morphology of inhibitory neurons, we generated four lines of VGAT-Venus transgenic mice (lines #04, #29, #39 and #49) expressing Venus fluorescent protein under the control of mouse VGAT promoter. We found higher expression level of Venus transcripts and proteins as well as brighter fluorescent signal in line #39 mouse brains, compared to brains of other lines examined. By Western blots and spectrofluorometric measurements of forebrain, the line #39 mouse showed stronger GFP immunoreactivity and brighter fluorescent intensity than the GAD67-GFP knock-in mouse. In addition, Venus was present not only in somata, but also in neurites in the line #39 mouse by histological studies. In situ hybridization analysis showed that the expression pattern of Venus in the line #39 mouse was similar to that of endogenous VGAT. Double immunostaining analysis in line #39 mouse showed that Venus-expressing cells are primarily immunoreactive for GABA in cerebral cortex, hippocampus and cerebellar cortex and for GABA or glycine in dorsal cochlear nucleus. These results demonstrate that the VGAT-Venus line #39 mouse should be useful for studies on function and morphology of inhibitory neurons in the CNS.

[Intraoperative graft assessment using fluorescent imaging system (SPY)].

We investigated the efficacy of intraoperative fluorescent imaging system for the assessment of coronary artery bypass grafting (CABG). We used SPY imaging system in 100 CABG (57 off-pump and 43 on-pump CABG), totalling 287 distal anastomoses. The total graft patency rate on postoperative angiography in this series was 96.2% (276/287). Graft revision was done in 10 cases (10.0%) and 13 anastomoses (4.5%) by SPY imaging, which all resulted in good patency at postoperative angiography. On the other hand, 7 distal anastomoses and 1 mammary graft (2.8%) appeared to be successful on intraoperative SPY imaging, but were revealed to be occluded by postoperative angiography. SPY imaging system is useful for graft validation, and may contribute to improvement of coronary bypass graft patency.

Noradrenergic excitation of a subpopulation of GABAergic cells in the basolateral amygdala via both activation of nonselective cationic conductance and suppression of resting K+ conductance: a study using glutamate decarboxylase 67-green fluorescent protein knock-in mice.

GABAergic interneurons play central roles in the regulation of neuronal activity in the basolateral nucleus of the amygdala (BLA). They are also suggested to be the principal targets of the brainstem noradrenergic afferents which are involved in the enhancement of the BLA-related memory. In addition, behavioral stress has been shown to impair noradrenergic facilitation of GABAergic transmission. However, the noradrenaline (NA) effects in the BLA have not been differentiated among medium- to large-sized GABAergic neurons and principal cells, and remain to be elucidated in terms of their underlying mechanisms. Glutamate decarboxylase 67 (GAD67) is a biosynthetic enzyme of GABA and is specifically expressed in GABAergic neurons. To facilitate the study of the NA effects on GABAergic neurons in live preparations, we generated GAD67-green fluorescent protein (GFP) knock-in mice, in which GFP was expressed under the control of an endogenous GAD67 gene promoter. Here, we show that GFP was specifically expressed in GABAergic neurons in the BLA of this GAD67-GFP knock-in mouse. Under whole-cell patch-clamp recordings in vitro, we identified a certain subpopulation of GABAergic neurons in the BLA chiefly on the basis of the electrophysiological properties. When depolarized by a current injection, these neurons, which are referred to as type A, generated action potentials at relatively low frequency. We found that NA directly excited type-A cells via alpha1-adrenoceptors, whereas its effects on the other types of neurons were negligible. Two ionic mechanisms were involved in this excitability: the activation of nonselective cationic conductance and the suppression of the resting K+ conductance. NA also increased the frequency of spontaneous IPSCs in the principal cells of the BLA. It is suggested that the NA-dependent excitation of type-A cells attenuates the BLA output for a certain period.

Quantitative analysis of the effect of probenecid on pharmacokinetics of 99mTc-mercaptoacetyltriglycine in dogs.

Effect of probenecid on pharmacokinetics of 99mTc-mercaptoacetylytriglycine (99mTc-MAG3) in dogs was investigated before (control), and after 15 min and 24 h of i.v. injection of probenecid (20 mg/kg). Plasma concentration-time profiles of 99mTc-MAG3 were described with a two-compartment open model. Plasma 99mTc-MAG3 clearances (Clp, ml/min/kg) were 7.9 +/- 0.5, 3.3 +/- 0.5 and 4.8 +/- 1.3 in control, 15 min and 24 h after probenecid administration respectively. Similarly, the biological half-lives at elimination phase (t(1/2), h) were 0.61 +/- 0.09, 0.79 +/- 0.11 and 0.74 +/- 0.12, and volumes of distribution at steady state (Vdss, L/kg) were 0.29 +/- 0.04, 0.20 +/- 0.05 and 0.25 +/- 0.06 respectively. The prolonged biological half-life and decreased Vdss decreased Clp significantly. Clp was a function of plasma probenecid concentration based on Michaelis-Menten kinetics. The maximum Clp inhibition (Imax) by probenecid and the plasma probenecid concentration that induced 50% of Imax (I50) were estimated to be 72 +/- 12% and 13 +/- 8 microg/ml respectively. This means that the rest (about 28%) of the Clp is not blocked by probenecid alone, suggesting the possibility of another route(s) of elimination or renal transporters which are independent from probenecid. Moreover, inter-species correlation between Clp of 99mTc-MAG3 and body weight are discussed.

The S haplotypes lacking SLG in the genome of Brassica rapa.

Self-incompatibility (SI) discriminating self- and non-self pollen is regulated by S-locus genes in Brassica. In most of the S haplotypes, a highly polymorphic S-locus glycoprotein ( SLG) gene is tightly linked to genes for the SI determinants, S-receptor kinase ( SRK) and SP11, although the precise function of SLG in SI has not been clarified. In the present study, we performed DNA gel blot analysis for S(32), S(33), and S(36) haplotypes of Brassica rapa showing normal SI phenotypes and concluded that there might be no SLG in their genome. RNA gel blot analysis of the SLG-less S haplotypes indicated the possible existence of eSRK transcripts in the stigma. These three S haplotypes are useful resources to discern the molecular mechanism of the SI reaction without SLG.

Differential palmitoylation of two mouse glutamate receptor interacting protein 1 forms with different N-terminal sequences.

Glutamate receptor interacting protein (GRIP) is a member of the PDZ domain-containing protein family that is localized in the postsynaptic density area. This protein has been reported to interact specifically with the C-termini of AMPA-selective glutamate receptor channel subunits, GluRalpha2 and GluRalpha3 through its PDZ domains. To clarify the physiological functions of GRIP, we cloned mouse GRIP1, and found that there are three sites for alternative splicing and two putative translational start codons by characterizing GRIP1 cDNA clones and reverse transcription-polymerase chain reaction products. Metabolic labeling of COS-7 cells expressing two N-terminal GRIP1 proteins demonstrated that these proteins differed in their pattern of palmitoylation. These findings suggested that the molecular diversity of GRIP1 underlies the localization and functional heterogeneity of this protein.

Purkinje cell-specific and inducible gene recombination system generated from C57BL/6 mouse ES cells.

Spatiotemporally restricted gene targeting is needed for analyzing the functions of various molecules in a variety of biological phenomena. We have generated an inducible cerebellar Purkinje cell-specific gene targeting system. This was achieved by establishing a mutant mouse line (D2CPR) from a C57BL/6 mouse ES cell line, which expressed a fusion protein consisting of the Cre recombinase and the progesterone receptor (CrePR). The Purkinje cell-specific expression of CrePR was attained by inserting CrePR into the glutamate receptor delta2 subunit (GluRdelta2) gene, which was expressed specifically in the Purkinje cells. Using the transgenic mice carrying the Cre-mediated reporter gene, we showed that the antiprogesterone RU486 could induce recombinase activity of the CrePR protein specifically in the mature cerebellar Purkinje cells of the D2CPR line. Thus this mutant line will be a useful tool for studying the molecular function of mature Purkinje cells by manipulating gene expression in a temporally restricted manner.

Characterization of expressed genes in the SLL2 region of self-compatible Arabidopsis thaliana.

Self-incompatibility in Brassica species is regulated by a set of S-locus genes: SLG, SRK, and SP11/SCR. In the vicinity of the S-locus genes, several expressed genes, SLL2 and SP2/ClpP, etc., were identified in B. campestris. Arabidopsis thaliana is a self-compatible Brassica relative, and its complete genome has been sequenced. From comparison of the genomic sequences between B. campestris and A. thaliana, microsynteny between gene clusters of Arabidopsis and Brassica SLL2 regions was observed, though the S-locus genes, SLG, SRK, and SP11/SCR were not found in the region of Arabidopsis. Almost all genes predicted in this region of Arabidopsis were expressed in both vegetative and reproductive organs, suggesting that the genes in the SLL2 region might not be related to self-incompatibility. Considering the recent speculation that the S-locus genes were translocated as a single unit between Arabidopsis and Brassica, the translocation might have occurred in the region between the SLL2 and SP7 genes.

The effect of brain compression under venous circulatory impairment.

It is well known that surgical obliteration of the cerebral veins with additional brain compression by retractors is dangerous. To evaluate the mechanism, we studied the change in cerebral microcirculation and parenchymal damage following brain compression with venous circulatory impairment using a rat model. The animals were divided into the following four groups (each n = 5) (1) a sham-operated control; (2) group A, one cortical vein occlusion; (3) group B, a 30 mmHg compression pressure; and (4) group C, one cortical vein occlusion with 30 mmHg compression. The cortical vein was occluded photochemically. Local cerebral blood flow (l-CBF) in the compressed area was measured by stationary laser-Doppler (LD) flowmetry and regional CBF (r-CBF) in the surrounding area was also measured by LD scanning technique for 120 min. l-CBF in the compressed area decreased significantly in groups B and C. A gradual and significant increase in group B and decrease in group C in r-CBF of the surrounding area were observed. Histologically, more extensive damage was observed in group C than in group A and B. The degree of hypoperfusion of the affected brain correlated well with the subsequent brain damage in the experiments. We demonstrated that, compared with vein occlusion or brain compression alone, the accumulated episode caused severe ischemia, then increased the vulnerability of the rat brain to tissue damage.

Highly divergent sequences of the pollen self-incompatibility (S) gene in class-I S haplotypes of Brassica campestris (syn. rapa) L.

Self-incompatibility (SI) enables flowering plants to discriminate between self- and non-self-pollen. In Brassica, SI is controlled by the highly polymorphic S locus. The recently identified male determinant, termed SP11 or SCR, is thought to be the ligand of S receptor kinase, the female determinant. To examine functional and evolutionary properties of SP11, we cloned 14 alleles from class-I S haplotypes of Brassica campestris and carried out sequence analyses. The sequences of mature SP11 proteins are highly divergent, except for the presence of conserved cysteines. The phylogenetic trees suggest possible co-evolution of the genes encoding the male and female determinants.

Hypothalamic hamartoma associated with dural arteriovenous fistula of the transverse-sigmoid sinus--case report.

A 32-year-old female presented with a hypothalamic hamartoma associated with a dural arteriovenous fistula (DAVF) of the left transverse-sigmoid sinus. She complained of pulsatile tinnitus in the left retroauricular region and infertility. Endocrinological examination revealed that the luteinizing hormone reaction to luteinizing hormone-releasing hormone was exceedingly high. Magnetic resonance imaging demonstrated a distinct mass occupying the suprasellar cistern and protruding into the third ventricle. Left carotid angiography demonstrated a DAVF of the left transverse-sigmoid sinus, which was treated by embolization. Histological examination of a biopsy specimen of the tumor revealed a hamartoma. She was free from pulsatile tinnitus after treatment for the DAVF.

Expanding laminoplasty for cervical myelopathy-spinous process roofing technique.

We have carried out expanding laminoplasty using spinous process roofing technique for patients presenting with cervical myelopathy. The technique is a modified Kurokawa's method. The result shows that laminoplasty with this technique provides better postoperative neck movement with a simpler surgical procedure. The clinical material consists of 54 cases with more than 3 years follow-up. Long-term investigation was performed focusing on the following three points, (1) bony fusion rates, (2) radiographic neck movement, (3) neurological improvement. The results indicate good postoperative bony fusion in all cases. Postoperative neck movement was also well preserved in the majority of cases.

Characteristics of intracranial aneurysms associated with moyamoya disease. A review of 111 cases.

A retrospective analysis of 111 patients with aneurysms associated with Moyamoya disease is presented. The subjects comprised of our 12 cases and 99 other well-documented cases. These 111 cases had 131 aneurysms. There were 48 males and 63 females. The average age was 40.3 years. The clinical manifestations were intracranial haemorrhage in 99 cases (89%), and ischaemic events in 9 cases (8%), but no mention was made of these in the last three cases (2%). The Hunt and Kosnik grades were grade 1 in 8%, grade 2 in 23%, grade 3 in 31%, grade 4 in 35%, and grade 5 in 3%. Of the 131 aneurysms, 73 (56%) were found distributed around the circle of Willis, 24 (18%) in the basal ganglia, 29 (22%) on collateral vessels, and 5 (4%) on other vessels. Forty-six percent of the cases were treated surgically, 51% conservatively, and 3% by endovascular procedures. The surgical procedures for the aneurysms were; neck clipping in 49%, aneurysmectomy in 18%, wrapping of the aneurysm in 11%, coating or cautery of the aneurysm in 7%, and revascularization only in 11%. The outcomes were Glasgow Outcome Scale 1 in 30%, 2 in 22%, 3 in 11%, 4 in 1%, and 5 in 25%. The main reasons for the unfavourable outcome were initial poor clinical grade and rebleeding. Follow-up angiography of 25 aneurysms demonstrated that all aneurysms in the basal ganglia or on the collateral vessels disappeared. We recommend surgical intervention for aneurysms associated with Moyamoya disease to prevent rupture or rebleeding, especially for aneurysms around the circle of Willis. However, direct surgery is not recommended for aneurysms found in the basal ganglia or on the collateral vessels.

Clinical features of the haemorrhage type moyamoya disease based on 31 cases.

We evaluated and analysed our own 31 cases of the haemorrhagic type of moyamoya disease to clarify the clinical features of this disease. The cases were divided into three groups. Group A consisted of 12 cases with aneurysms. Aneurysms on the circle of Willis were treated as ordinary saccular aneurysms. Group B consisted of 14 cases with intracerebral haemorrhage (ICH) without aneurysms. These were managed almost as spontaneous ICH. Group C consisted of 5 cases with intraventricular haemorrhage (IVH) without aneurysms or ICH. Twenty-two surgical procedures for aneurysms, ICH and IVH were done in 19 cases (62%). Nineteen procedures for preventing future strokes were undertaken in 11 cases (35%). The overall initial outcome was excellent in 12 cases (39%), good in 7 cases (23%), poor in 7 cases (23%), and death in 5 cases (15%). During the follow-up period (mean: 6.5 years), rebleeding occurred in two cases (8%), and ischaemic attacks in two cases (8%). The rate of rebleeding or ischaemic attacks was 1.19% per patient-year during the follow-up period. There was no ischaemic or rebleeding episode in cases treated by STA-MCA bypass with encephalomyosynagiosis (EMS) during the follow-up period. Management of the primary haemorrhage should be according to the clinical condition, type of haemorrhage, and source of haemorrhage. When the patient needs to undergo revascularization surgery to prevent future strokes, we recommend STA-MCA bypass with EMS instead of encephaloduro-arteriosynangiosis (EDAS).