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Spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHRSP) are frequently used as model rats not only in studies of essential hypertension and stroke, but also in studies of attention deficit hyperactivity disorder (ADHD). Normotensive Wistar-Kyoto rats (WKY) are normally used as controls in these studies. In this study, using these rats, we aimed to identify the genes causing hypertension and stroke, as well as the genes involved in ADHD. Since adrenal gland products can directly influence cardiovascular, endocrine and sympathetic nervous system functions, gene expression profiles in the adrenal glands of the 3 rat strains were examined using genome-wide microarray technology when the rats were 3 and 6 weeks of age, a period in which the rats are considered to be in a pre-hypertensive state. Gene expression profiles were compared between SHR and WKY and between SHRSP and SHR. A total of 353 genes showing more than a 4-fold increase or less than a 4-fold decrease in expression were isolated and candidate genes were selected as significantly enriched genes. SHR-specific genes isolated when the rats were 3 weeks of age contained 12 enriched genes related to transcriptional regulatory activity and those isolated when the rats were 6 weeks of age contained 6 enriched genes related to the regulation of blood pressure. SHRSP-specific genes isolated when the rats were 3 weeks of age contained 4 enriched genes related to the regulation of blood pressure and those isolated when the rats were 6 weeks of age contained 4 enriched genes related to the response to steroid hormone stimulus. Ingenuity pathway analysis of enriched SHR-specific genes revealed that 2 transcriptional regulators, cAMP responsive element modulator (Crem) and Fos-like antigen 1 (Fosl1), interact with blood pressure-regulating genes, such as neurotensin (Nts), apelin (Apln) and epoxide hydrolase 2, cytoplasmic (Ephx2). Similar analyses of SHRSP-specific genes revealed that angiotensinogen (Agt), one of the blood pressure-regulating genes, plays pivotal roles among SHRSP-specific genes. Moreover, genes associated with ADHD, such as low density lipoprotein receptor (Ldlr) and Crem, are discussed.

Recently, there has been much effort to find effective ingredients which can prevent or retard cutaneous skin aging after topical or systemic use. Here, we investigated the effects of the atomic hydrogen surrounded by water molecules, H(H2O)m, on acute UV-induced responses and as well as skin aging. Interestingly, we observed that H(H2O)m application to human skin prevented UV-induced erythema and DNA damage. And H(H2O)m significantly prevented UV-induced MMP-1, COX-2, IL-6 and IL-1β mRNA expressions in human skin in vivo. We found that H(H2O)m prevented UV-induced ROS generation and inhibited UV-induced MMP-1, COX-2 and IL-6 expressions, and UV-induced JNK and c-Jun phosphorylation in HaCaT cells. Next, we investigated the effects of H(H2O)m on intrinsically aged or photoaged skin of elderly subjects. In intrinsically aged skin, H(H2O)m application significantly reduced constitutive expressions of MMP-1, IL-6, and IL-1β mRNA. Additionally, H(H2O)m significantly increased procollagen mRNA and also decreased MMP-1 and IL-6 mRNA expressions in photoaged facial skin. These results demonstrated that local application of H(H2O)m may prevent UV-induced skin inflammation and can modulate intrinsic skin aging and photoaging processes. Therefore, we suggest that modifying the atmospheric gas environment within a room may be a new way to regulate skin functions or skin aging.

A 46-year-old woman was transferred to our emergency unit because of impaired consciousness and respiratory failure with the history of excessive pesticide intake. The patient was hypersalivative and had bilateral pupillary miosis. Laboratory results showed markedly decreased cholinesterase. She was intubated and treated in the intensive care unit with the diagnosis of organophosphorus poisoning. The patient had persisted diarrhea, with a high fever and stomach tenderness on day 10. Whole-body contrast enhanced computed tomography revealed a swollen, enhanced small intestinal wall, and blood culture identified Delftia acidovorans. She was diagnosed as D. acidovorans bacteremia, probably caused by bacterial translocation based on the clinical presentation and the exclusion of other sources, and treated well with a total of 8 days of antibiotic therapy. So far as we know, this is the first case of D. acidovorans bacteremia that was presumably caused by bacterial translocation after organophosphorus poisoning in an immunocompetent adult patient.

A 95-year-old man with chronic obstructive pulmonary disease and chronic hepatitis C virus infection was treated for acute lung injury caused by Chlamydophila pneumoniae with antibiotics and high-dose corticosteroids. In total, 7,500 mg methylprednisolone and 680 mg prednisolone were administered over 21 days. However, respiratory failure progressed, and chest computed tomography (CT) scan showed bilateral ground-glass opacity and cavity-forming consolidation in the right upper lobe. Despite intensive therapy, the patient died of multiple organ failure on day 7. CT-guided necropsy was performed, and pathological examination revealed invasive pulmonary aspergillosis and Pneumocystis jirovecii pneumonia. Invasive pulmonary aspergillosis and P. jirovecii pneumonia are both life-threatening opportunistic fungal infections. Co-infection of these organisms is rare but possible if the patient is in an extremely immunocompromised state. Short-term but high-dose systemic corticosteroid therapy was considered to be the risk factor in this case. We should pay more attention to immunocompromised hosts who might be suffering from co-infection of opportunistic infections. Moreover, we need to consider preventive measures in such high-risk cases.

Yap1 is an important regulator of cardiomyocyte proliferation and embryonic heart development, yet the function of endogenous Yap1 in the adult heart remains unknown. We studied the role of Yap1 in maintaining basal cardiac function and in modulating injury after chronic myocardial infarction (MI). Cardiomyocyte-specific homozygous inactivation of Yap1 in the postnatal heart (Yap(F/F)Cre) elicited increased myocyte apoptosis and fibrosis, dilated cardiomyopathy, and premature death. Heterozygous deletion (Yap(+/F)Cre) did not cause an overt cardiac phenotype compared with Yap(F/F) control mice at base line. In response to stress (MI), nuclear Yap1 was found selectively in the border zone and not in the remote area of the heart. After chronic MI (28 days), Yap(+/F)Cre mice had significantly increased myocyte apoptosis and fibrosis, with attenuated compensatory cardiomyocyte hypertrophy, and further impaired function versus Yap(+/F) control mice. Studies in isolated cardiomyocytes demonstrated that Yap1 expression is sufficient to promote increased cell size and hypertrophic gene expression and protected cardiomyocytes against H(2)O(2)-induced cell death, whereas Yap1 depletion attenuated phenylephrine-induced hypertrophy and augmented apoptosis. Finally, we observed a significant decrease in cardiomyocyte proliferation in Yap(+/F)Cre hearts compared with Yap(+/F) controls after MI and demonstrated that Yap1 is sufficient to promote cardiomyocyte proliferation in isolated cardiomyocytes. Our findings suggest that Yap1 is critical for basal heart homeostasis and that Yap1 deficiency exacerbates injury in response to chronic MI.

The tumor suppressors Lats1 and Lats2 are mediators of the Hippo pathway that regulates tissue growth and proliferation. Their N-terminal non-kinase regions are distinct except for Lats conserved domains 1 and 2 (LCD1 and LCD2), which may be important for Lats1/2-specific functions. Lats1 knockout mice were generated by disrupting the N-terminal region containing LCD1 (Lats1(ΔN/ΔN)). Some Lats1(ΔN/ΔN) mice were born safely and grew normally. However, mouse embryonic fibroblasts (MEFs) from Lats1(ΔN/ΔN) mice displayed mitotic defects, centrosomal overduplication, chromosomal misalignment, multipolar spindle formation, chromosomal bridging and cytokinesis failure. They also showed anchorage-independent growth and continued cell cycles and cell growth, bypassing cell-cell contact inhibition similar to tumor cells. Lats1(ΔN/ΔN) MEFs produced tumors in nude mice after subcutaneous injection, although the tumor growth rate was much slower than that of ordinary cancer cells. Yap, a key transcriptional coactivator of the Hippo pathway, was overexpressed and stably retained in Lats1(ΔN/ΔN) MEFs in a cell density independent manner, and Lats2 mRNA expression was downregulated. In conclusion, N-terminally truncated Lats1 induced Lats2 downregulation and Yap protein accumulation, leading to chromosomal instability and tumorigenesis.

An 88-year-old man died of streptococcal toxic shock syndrome due to a group G streptococcus infection that was possibly caused by an intramuscular injection given 30 hours earlier in his right deltoid muscle. The causative pathogen was later identified to be Streptococcus dysgalactiae subsp. equisimilis (stG485). Although providing intramuscular injections is an essential skill of health care workers that is performed daily worldwide, it may constitute a port of entry for pathogens via skin breaches that can cause life-threatening infections. All invasive procedures should be carefully performed, especially when immunologically compromised patients are involved.

We describe a new simple and easy technique called the "Hook and roll technique" (HRT) that uses an articulating hook cautery to provide a critical view during single incision laparoscopic cholecystectomy (SILC). A 2-cm incision is made at the umbilicus to insert three 5-mm trocars or a multichannel port. After dissection of the serosa of the dorsal and ventral sides of the gall bladder, including Calot's triangle, the angled tip of the hook cautery is inserted between the cystic artery and duct with its tip placed dorsally. The tip is then rotated in a clockwise manner to avoid bile duct injury, allowing the connective tissue between them to be hooked, coagulated and cut. This procedure is repeated several times, followed by dissection between the cystic artery and the liver bed to achieve a critical view. From December 2008 to May 2011, 121 patients underwent SILC using HRT in our hospital without any serious complications. This technique is suitable for SILC, as it is consists of simple procedures that can be performed safely and easily, even by left hand in a cross-over approach, and it allows complete dissection of Calot's triangle to achieve a critical view without using any dissector under dangerous in-line viewing.

An emphysematous liver abscess is a fatal condition that often occurs in patients with uncontrolled diabetes mellitus. I herein describe two cases of Klebsiella pneumoniae-induced emphysematous liver abscesses complicated by septic pulmonary emboli in patients with poorly controlled diabetes mellitus. Both patients showed hemoglobin A1c levels of more than 10% and did not present with any abdominal symptoms on admission. However, they were diagnosed and successfully treated with percutaneous transhepatic abscess drainage and antibiotics. This fatal disease should be taken into consideration in patients with uncontrolled diabetes mellitus who suffer from prolonged fevers and uncharacteristic general malaise.

Background: Selegiline, a therapeutic drug for Parkinson's disease (PD), structurally resembles the endogenous parkinsonism-related compound 1,2,3,4-tetrahydroisoquinoline (TIQ). In the present study, we evaluated the effects of 3-methyl-TIQ (3-MeTIQ) and 3-methyl-N-propargyl-TIQ (3-Me-N-proTIQ), selegiline mimetic TIQ derivatives, for preventing 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism-like symptoms in mice., Methods: We evaluated the preventative effects of 3-MeTIQ and 3-Me-N-proTIQ on MPTP-induced bradykinesia and depletion of striatal dopamine (DA) and nigral tyrosine hydroxylase (TH)-positive cells., Results: MPTP-induced bradykinesia was not different when mice were pretreated with 3-MeTIQ, except for the high-dose group. However, pretreatment with 3-Me-N-proTIQ significantly prevented the appearance of this akinesic status. MPTP-induced striatal DA and 3,4-dehydroxyphenylacetic acid reduction were significantly prevented by pretreatment with 3-Me-N-proTIQ, but not 3-MeTIQ, in a dose-dependent manner. On the other hand, levels of serotonin and its metabolite, 5-hydroxyindole acetic acid, in the striatum were increased following treatment with 3-MeTIQ. In addition, the MPTP-induced decrease in TH-positive cells in the substantia nigra was significantly reduced by pretreatment with 3-Me-N-proTIQ, but not 3-MeTIQ., Conclusions: These results suggest that not only does 3-Me-N-proTIQ have potential as a candidate compound for disease-modifying therapy for PD, but also the N-propargyl functional group plays an important role in neuroprotection.

Mps one binder 1a (MOB1A) and MOB1B are key components of the Hippo signaling pathway and are mutated or inactivated in many human cancers. Here we show that intact Mob1a or Mob1b is essential for murine embryogenesis and that loss of the remaining WT Mob1 allele in Mob1a(Δ/Δ)1b(tr/+) or Mob1a(Δ/+)1b(tr/tr) mice results in tumor development. Because most of these cancers resembled trichilemmal carcinomas, we generated double-mutant mice bearing tamoxifen-inducible, keratinocyte-specific homozygous-null mutations of Mob1a and Mob1b (kDKO mice). kDKO mice showed hyperplastic keratinocyte progenitors and defective keratinocyte terminal differentiation and soon died of malnutrition. kDKO keratinocytes exhibited hyperproliferation, apoptotic resistance, impaired contact inhibition, enhanced progenitor self renewal, and increased centrosomes. Examination of Hippo pathway signaling in kDKO keratinocytes revealed that loss of Mob1a/b altered the activities of the downstream Hippo mediators LATS and YAP1. Similarly, YAP1 was activated in some human trichilemmal carcinomas, and some of these also exhibited MOB1A/1B inactivation. Our results clearly demonstrate that MOB1A and MOB1B have overlapping functions in skin homeostasis, and exert their roles as tumor suppressors by regulating downstream elements of the Hippo pathway.

Background: Ultrasound-guided brachial plexus block (UGBB) makes it possible to block both lateral and medial aspects of the skin overlying the elbow, which are mainly innervated by C5 and T1 roots of brachial plexus, respectively. The effect of UGBB on perioperative pain relief in total elbow arthroplasty (TEA) was evaluated., Methods: Twenty-one patients scheduled to undergo TEA with general anesthesia from January 2009 to December 2010 were assigned to a group receiving UGBB (Block group, n = 10) and a group receiving general anesthesia alone (General group, n = 11). Perioperative anesthetic dose and postoperative pain intensity were recorded. Statistical analysis was performed with Mann-Whitney's U-test, and P < 0.05 was considered to be significant., Results: Median fentanyl doses during the operation in the Block group and General group were 100 microg and 250 microg, respectively (P < 0.05). Numerical rating scale (NRS) in the Block group was significantly lower than that in the General group immediately after the operation (median value: Block group = 0, General group = 4). Although NRS in the two groups was not different from the night of the day of operation, no patient in the Block group needed supplementary opioids., Conclusions: Ultrasound-guided brachial plexus block in patients undergoing TEA reduces perioperative opioid consumption and wound pain in the early postoperative period.

A 55-year-old man underwent rectal amputation for rectal cancer in August 2005. A tiny thin-walled cavity lesion in his left S1+2 was found on computed tomography (CT) of the chest in November 2008. The cavity lesion in the left S1+2 gradually increased in size over 3 months and positron emission tomography (PET) with 18F-fluorodeoxyglucose (FDG) showed FDG accumulation at the lesion. Videoassisted thoracoscopic (VATS) wedge resection was performed to make a definite diagnosis in March 2009. The pathological findings revealed a metastatic lung tumor from the rectal cancer. It is necessary to consider the possibility of metastatic lung tumors in a case with the cavity lesions especially in patients with a history of colon cancer.

The need to coordinate patterning and growth has been appreciated for many years. The logic that enables seamless integration of the relevant inputs is beginning to be elucidated, particularly in wing imaginal discs of Drosophila. In this tissue, multiple regulatory layers involving the two morphogens Wingless and Dpp, the wing-specific determinant, Vestigial, and the Hippo pathway, converge to regulate growth. Intricate cross-regulation between these components may explain why, at the local level, there is no direct correlation between growth and the graded signalling activity of Wingless and Dpp, despite the requirement of these two pathways for growth., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Protein phosphatase 2A (PP2A) bearing the B'γ (=B'α/B56γ1/PR61γ) subunit is recruited to dephosphorylation targets by cyclin G. We demonstrate here that cyclin G-associated kinase (GAK), a component of the GAK/B'γ/cyclin G complex, directly phosphorylates the B'γ-Thr104 residue and regulates PP2A activity. Indeed, an anti-B'γ-pT104 antibody detected immunofluorescence signals at the chromosome and centrosome during mitosis; these signals were reduced by siRNA-mediated GAK knockdown. After DNA damage by γ-irradiation, the chromosome signals formed foci that colocalized with a DNA double-strand break (DSB) marker H2AX-pS139 (γH2AX) and CHK2-pT68. Moreover, B'γ-pT104 enhanced PP2A holoenzyme assembly and PP2A activity, as shown by the results of an in vitro phosphatase assay. These results suggest a novel role for GAK as a regulator of dephosphorylation events under the control of the PP2A B'γ subunit.

Snail1 is a central regulator of epithelial cell adhesion and movement in epithelial-to-mesenchymal transitions (EMTs) during embryo development; a process reactivated during cancer metastasis. While induction of Snail1 transcription precedes EMT induction, post-translational regulation of Snail1 is also critical for determining Snail1's protein level, subcellular localization, and capacity to induce EMT. To identify novel post-translational regulators of Snail1, we developed a live cell, bioluminescence-based screen. From a human kinome RNAi screen, we have identified Lats2 kinase as a novel regulator of Snail1 protein level, subcellular localization, and thus, activity. We show that Lats2 interacts with Snail1 and directly phosphorylates Snail1 at residue T203. This occurs in the nucleus and serves to retain Snail1 in the nucleus thereby enhancing its stability. Lats2 was found to positively influence cellular EMT and tumour cell invasion, in a Snail1-dependent manner. Indeed during TGFβ-induced EMT Lats2 is activated and Snail1 phosphorylated at T203. Analysis in mouse and zebrafish embryo development confirms that Lats2 acts as a positive modulator of Snail1 protein level and potentiates its in vivo EMT activity.

A 21-year-old Japanese man with a history of marked body weight loss over a short period of time died of necrotizing duodenitis caused by Clostridium perfringens (C. perfringens) type A. C. perfringens type A is considered to usually cause self-limiting gastroenteritis. Necrotizing enteritis sometimes occurs due to C. perfringens in developing countries; however, it is primarily caused by the type C strain and its site of onset is typically the jejunum or ileum. This is a rare case of necrotizing duodenitis caused by C. perfringens type A in a Japanese young man. Physicians need to be more aware of this emerging fatal disease in developed countries.

Background: The conditioned taste aversion (CTA) paradigm is one of the reliable methods to evaluate the discriminative stimulus properties of drugs and is characterized by a short conditioning period and no need for special equipment. This method, however, has not yet been fully investigated for psychostimulants such as cocaine and methamphetamine., Methods: In the present study, rats were trained to discriminate between cocaine and a vehicle using CTA and substitution tests with various psychostimulants were conducted to evaluate the usefulness of the method for assessing the discriminative stimulus properties of this pharmacological class. Male rats received an intraperitoneal (i.p.) injection of cocaine (10mg/kg) 10 min prior to access saccharin for 20-min, and immediately after the saccharin access they received an i.p. dose of LiCl (1.8 mEq; n=8, Group CL) or the vehicle (n=8, Group CW) on the day of conditioning; on the other days (2 or 3 days between the cocaine conditioning days), they were injected with saline prior to access to saccharin without the LiCl or vehicle injection after the access., Results: By the fifteenth cocaine conditioning trial, all animals acquired discrimination. In the substitution test, cocaine dose dependently decreased saccharin consumption. The psychostimulants, methamphetamine, methylphenidate, bupropion and sibutramine, substituted for cocaine, whereas the opioid μ agonist morphine and the cannabinoid agonist, Δ9-tetrahydrocannabinol, did not substitute for cocaine. Mazindol did not substitute for cocaine although it has CNS stimulant activities., Conclusion: These results suggest that discriminative stimulus properties of psychostimulants can be evaluated using the CTA paradigm., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Background: Whole-protein extracts from peripheral blood leukocytes are ideal for basic and clinical research. However, lack of a simple preparation technique has limited the use of such extracts. The aim of this study is to develop a simple and easy system that can selectively obtain leukocyte extracts without hemoglobin., Methods: A filter that captures the leukocytes but not RBCs was set at the bottom of a 10-mL medical syringe by sandwiching it between plastic stoppers. The capturing efficiency of leukocytes with this tool, called LeukoCatch, was examined using human macrophage cells (MONO-MAC-6). The abilities of LeukoCatch system to capture the leukocyte proteins and to remove the hemoglobin from RBCs were tested by western blot analysis using human blood samples., Results: This study presents the development of LeukoCatch, a novel tool that allows the preparation of leukocyte extracts from blood samples within 3 min without centrifugation. Tissue-cultured human macrophage cells were tested to determine the optimal filter numbers and pass-through frequencies of LeukoCatch, which was then applied to 2-mL blood samples. Samples were passed 2~5 times through a LeukoCatch equipped with 5 filters, washed twice with phosphate-buffered saline for red cell removal, and leukocyte proteins were extracted with 0.5 mL of elution buffer. Western blot analysis of the purified extract indicated that more than 90% of hemoglobin was removed by the LeukoCatch and that the protein recovery rate of leukocytes was at least 4 times better than that of the conventional centrifugation method., Conclusion: We conclude that LeukoCatch is useful not only for diagnosis at the bedside but also for basic research using blood samples or tissue culture cells.

Accurate coordination between chromosome segregation and cytokinesis by various mitotic kinases, such as Aurora, prevent tetraploidization and subsequent tumorigensis. The tumor suppressors Lats1 and Lats2 are serine/threonine kinases that localize to the centrosome and regulate cell cycle progression and apoptosis. In the present study, Aurora A was demonstrated to phosphorylate Lats2 on serine 380 (S380) during mitosis. Immunocytochemical observations revealed that the subcellular localization of Lats2 was distinct during the cell cycle and depended on which site was phosphorylated. Interestingly, the S380-phosphorylated Lats2 protein (pS380) colocalized at the central spindle with Aurora B. Physical interactions were observed between Aurora A, Lats2, Lats1 and Aurora B. The Lats1 kinase was shown to phosphorylate Aurora B. Cells expressing a nonphosphorylated mutant (S380A) of Lats2 caused chromosome missegregation and cytokinesis failure, similar to cells with aberrantly expressed Aurora B. Together, the results suggest that the Aurora A-Lats1/2-Aurora B axis might be a novel pathway that regulates accurate mitotic progression by ensuring the proper mitotic localization of Lats2.

We herein describe a case of successful surgical treatment of isolated splenic peliosis presenting with giant splenomegaly and severe coagulopathy. Pathological features of the spleen included multinodular, blood-filled, cyst-like lesions distributed throughout the whole organ. The pathogenesis of isolated splenic peliosis is controversial, and no definitive disease mechanism has been reported. To our knowledge, this is the first report in English of a case like this., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: Neuropathic pain results from multiple etiological factors and is a debilitating condition often resulting from partial injury to a peripheral nerve. However, the mechanism underlying this syndrome remains unclear. The aim of the present study is to investigate whether a spared nerve injury model with transection of both the common peroneal nerve (CPN) and tibial nerve (TN) branches of the sciatic nerve is associated with pain or hypersensitivity in the mouse., Methods: A skin and muscle incision was made, and we tightly ligated CPN+TN or the sural nerve (SN) alone as branches of the sciatic nerve. Mechanical and thermal allodynia were tested using von Frey filaments and radiant heat at -1, 1, 3, 5, and 7 days after surgery., Results: The thresholds of mechanical and thermal stimuli were increased and decreased in the CPN+TN-ligated and SN-ligated groups, respectively. Chemical hyperalgesia was estimated using two doses of intraplantar administration of formalin (0.1 and 1%) 1 week after surgery. Then behaviors were videotaped and playback was used to measure time of licking of the hind paws. The licking time induced by 0.1% formalin was significantly shortened in the CPN+TN-ligated group and significantly prolonged in the SN-ligated group. Number of c-Fos-immunoreactive cells in the spinal cord was not affected by CPN+TN ligation, but was significantly increased in the SN-ligated group., Discussion: These findings suggest that changing the combination of nerves in sciatic branch ligation produces different sensitivities to stimuli and SN ligation will be useful for inducing allodynia and hyperalgesia in the mouse.

IgA nephropathy (IgAN) is the most common primary glomerulonephritis. Its close relation with the tonsils is well known because tonsillitis sometimes causes aggravation of urinary findings or macrohematuria. However, the genes specific to the tonsils of IgAN patients are not clarified. To clarify the specific gene expression in the tonsils of IgAN patients, we performed tonsillectomy and corticosteroid IV therapy as a treatment of IgAN, analyzed the gene expression in the tonsils by microarray and compared it with that in tonsils from chronic tonsillitis patients. The upregulated genes seem to be categorized into two groups: muscle-related genes and immunerelated genes. The downregulated genes include the polymeric Ig receptor (pIgR) which was reportedly involved in single nucleotide polymorphism (SNP) of Japanese IgAN patients., (Copyright © 2011 S. Karger AG, Basel.)

Unlike exponential amplification using polymerase chain reaction (PCR), linear RNA amplification using T7 RNA polymerase is advantageous for genome-wide analysis of gene expression and for cDNA library preparation from single-cell quantities of RNA. However, the use of RNA polymerase requires a large amount of RNA, as the optimum concentration of the substrate (mRNA), or the Michaelis constant (K(m)), is one millionfold higher than the single-cell amount of mRNA. To circumvent this K(m) problem, we designed a small mRNA-like dummy molecule, termed chum-RNA, which can be easily removed after the completion of the reaction. Chum-RNA allowed the preparation of a high-quality cDNA library from single-cell quantities of RNA after four rounds of T7-based linear amplification, without using PCR amplification. The use of chum-RNA may also facilitate quantitative reverse-transcription (qRT)-PCR from small quantities of substrate.

Gefitinib (Iressa) is an inhibitor of the epidermal growth factor receptor (EGFR) that has shown promising activity in the treatment of patients with non-small cell lung cancer (NSCLC). However, adverse side effects of gefitinib treatment, such as respiratory dysfunction, have limited the therapeutic benefit of this targeting strategy. The present results show that this adverse effect can be attributed to the inhibition of the novel gefitinib target GAK (Cyclin G-associated kinase), which is as potently inhibited by the drug as the tyrosine kinase activity of EGFR. Knockout mice expressing the kinase-dead form of GAK (GAK-kd) died within 30 min after birth primarily due to respiratory dysfunction. Immunohistochemical analysis revealed that surfactant protein A (SP-A) was abundant within alveolar spaces in GAK-kd(+/+) mice but not in GAK-kd(-/-) pups. E-cadherin and phosphorylated EGFR signals were also abnormal, suggesting the presence of flat alveolar cells with thin junctions. These results suggest that inhibition of GAK by gefitinib may cause pulmonary alveolar dysfunction, and the present study may help prevent side effects associated with gefitinib therapy in NSCLC patients.

Proper response to DNA damage is essential for maintaining the integrity of the genome. Here we show that in response to ultraviolet (UV) radiation, the Lats2 tumor suppressor protein is phosphorylated predominantly by Chk1 and weakly by Chk2 at S408 in vivo, and that this process occurs at all stages of the cell cycle and leads to phosphorylation of 14-3-3γ on S59 by Lats2. Interaction of Lats2 and 14-3-3γ in vivo was confirmed by immunoprecipitation and western blot analysis. Phosphorylated 14-3-3γ translocates to the P-body, where mRNA degradation, translational repression and mRNA surveillance take place. Depletion of Lats2 or 14-3-3γ by siRNA inhibits P-body formation in response to UV, newly implicating Lats2 and 14-3-3 as regulators of P-body formation. By contrast, siRNA-mediated depletion of Lats1, a mammalian paralog of Lats2, showed no such effect. On the basis of these findings, we propose that the Chk1/2-Lats2-14-3-3 axis identified here plays an important role in connecting DNA damage signals to P-body assembly.

Mek1 is a Chk2/Rad53/Cds1-related protein kinase that is required for proper meiotic progression of Schizosaccharomyces pombe. However, the molecular mechanisms of Mek1 regulation and Mek1 phosphorylation targets are unclear. Here, we report that Mek1 is phosphorylated at serine-12 (S12), S14 and threonine-15 (T15) by Rad3 (ATR) and/or Tel1 (ATM) kinases that are activated by meiotic programmed double-strand breaks (DSBs). Mutations of these sites by alanine replacement caused abnormal meiotic progression and recombination rates. Phosphorylation of these sites triggers autophosphorylation of Mek1; indeed, alanine replacement mutations of Mek1-T318 and -T322 residues in the activation loop of Mek1 reduced Mek1 kinase activity and meiotic recombination rates. Substrates of Mek1 include Mus81-T275, Rdh54-T6 and Rdh54-T673. Mus81-T275 is known to regulate the Mus81 function in DNA cleavage, whereas Rdh54-T6A/T673A mutant cells showed abnormal meiotic recombination. Taken together, we conclude that the phosphorylation of Mek1 by Rad3 or Tel1, Mek1 autophosphorylation and Mus81 or Rdh54 phosphorylation by Mek1 regulate meiotic progression in S. pombe.

Overexpression of Dishevelled (Dvl), an essential component of the Wnt signaling pathway, is frequently associated with tumors, and thus the Dvl protein level must be tightly controlled to sustain Wnt signaling without causing tumors. Kelch-like 12 (KLHL12) targets Dvl for ubiquitination and degradation, suggesting its potential importance in avoiding aberrant Dvl overexpression. However, the regulatory mechanism of the KLHL12 activity remained elusive. We show here that nucleoredoxin (NRX) determines the Dvl protein level, which is revealed by analyses on NRX(-/-) mice showing skeletal and cardiovascular defects. Consistent with the previously reported Dvl-inhibiting function of NRX, Wnt/β-catenin signaling is hyperactivated in NRX(-/-) osteoblasts. However, the signal activity is suppressed in cardiac cells, where KLHL12 is highly expressed. Biochemical analyses reveal that Dvl is rapidly degraded by accelerated ubiquitination in NRX(-/-) mouse embryonic fibroblasts, and they fail to activate Wnt/β-catenin signaling in response to Wnt ligands. Moreover, experiments utilizing purified proteins show that NRX expels KLHL12 from Dvl and inhibits ubiquitination. These findings reveal an unexpected function of NRX, retaining a pool of inactive Dvl for robust activation of Wnt/β-catenin signaling upon Wnt stimulation., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Apoptosis is an important mechanism to eliminate potentially tumorigenic cells. The tumor suppressor p53 plays a pivotal role in this process. Many tumors harbor mutant p53, but others evade its tumor-suppressive effects by altering the expression of proteins that regulate the p53 pathway. ASPP1 (apoptosis-stimulating protein of p53-1) is a key mediator of the nuclear p53 apoptotic response. Under basal conditions, ASPP1 is cytoplasmic. We report that, in response to oncogenic stress, the tumor suppressor Lats2 (large tumor suppressor 2) phosphorylates ASPP1 and drives its translocation into the nucleus. Together, Lats2 and ASPP1 shunt p53 to proapoptotic promoters and promote the death of polyploid cells. These effects are overridden by the Yap1 (Yes-associated protein 1) oncoprotein, which disrupts Lats2-ASPP1 binding and antagonizes the tumor-suppressing function of the Lats2/ASPP1/p53 axis.

We report a long-term survival case treated by chemotherapy with new anticancer drugs such as S-1, CPT-11 and docetaxel after bilateral overiectomy for bilateral ovarian metastases of progressive gastric cancer. A 68-year-old female, who had undergone total gastrectomy with D2 lymphadenectomy for scirrhous gastric cancer of Stage IIIA, was admitted because of ovarian metastasis. Laparoscopic bilateral ovariectomy was performed for ovarian metastases of the gastric cancer (Krukenberg tumor) 6 months after gastrectomy. Seven months after gastrectomy, 11-course of S-1 treatment (80 mg/m2) for bilateral ovarian metastases of the gastric cancer was administered. CT scan revealed mediastinal lymphadenopathy 2 years and 5 months after gastrectomy. Then, the drug was changed to docetaxel (60 mg/m2). After 9-course of docetaxel treatment, the mediastinal lymphadenopathy disappeared. For a treatment of grade 3 neuropathy, a 12-course CPT-11 was started after gastrectomy 3 years and 6 months ago. CT scan and PET-CT showed no new metastasis in 4 years and 10 months after gastrectomy.

A 74-year-old man was admitted to a nearby clinic complaining of high fever. Abdominal CT showed a 10 mm diameter cystic mass in the head of pancreas and dilation of the pancreatic duct. Endoscopy revealed a fistula filled with mucin in the posterior wall of the duodenum. The patient was referred to our institution for a surgical resection. Endoscopic ultrasonography revealed dilation of the pancreatic duct and also mural nodules in the pancreatic duct, ERP demonstrated a fistula from the pancreatic duct to the duodenum. Biopsied specimen from the papillary nodule in the pancreatic duct showed adenoma. We performed pancreaticoduodenectomy for main-duct IPMN penetrating to the duodenum. Pathological findings showed a non-invasive type of IPMC. Furthermore, a cancer invasion to the duodenum was not detected. These findings suggest that the increased pressure within the pancreatic duct caused a fistula to the duodenum.

A 74-year-old man was admitted to our hospital in order to treat a mediastinal mass and 2 ground-glass attenuations in the right upper lobe detected by chest X-ray and computed tomography (CT). Partial resection of right lung and thymectomy were performed. The mediastinal mass and 2 ground-glass attenuations in the right upper lobe proved to be thymoma and bronchioloalveolar carcinomas, respectively by pathology.

An 79-year-old man admitted our hospital for abdominal mass. Computed tomography showed a tumor measuring about 10 cm in diameter without any metastasis lesion and any sings of local infiltration. Gastroduodenal endoscopy revealed the presence of a submucosal tumor in the third portion of the duodenum, and biopsy revealed tumor cells stained positive for c-kit. These findings were consistent with a GIST and we performed a partial resection of the duodenum sparing the pancreas. Gastrointestinal stromal tumors (GIST) were mainly located in the stomach and the small intestine. Duodenal localization is rare. Surgical approach for GISTs should basically be a partial resection. However, for GISTs located in the duodenum, the partial resection was sometimes difficult and pancreaticoduodenectomy (PD) may be needed, depending on the tumor size and the location of the tumor close to the papilla Vater. Since GIST grew expansively, rarely involving lymph nodes, PD may be an excessive procedure to treat the disease. For this reason pancreas-sparing partial duodenectomy has been introduced for the treatment of duodenal GIST.

Protein phosphorylation is pivotal for meiotic progression, but little is known about its regulatory mechanisms. We show that before meiosis I, the meiosis-specific Schizosaccharomyces pombe protein Spo5 is phosphorylated in vivo on T29, T55, S59 and/or T63. In a mutant strain expressing Spo5 fused to green fluorescent protein with alanine substitutions of these amino acid sites (GFP; Spo5-4A-GFP), the timely degradation of Spo5 at meiosis II was not observed. Additionally, Spo5-4A-GFP signals were retained after metaphase II and were localized to the nucleus. This was accompanied by the nuclear mislocalization of Psy1, a marker of the forespore membrane (FSM), and the generation of empty cells, in which cytoplasm had leaked from the ruptured membrane, as well as by the appearance of asci harboring deformed spores. Indeed, thin-section electron microscopy (TEM) revealed fragile-looking spo5-4A-GFP ascospores with ruffled spore walls. In contrast, a mutant strain expressing a constitutively-phosphorylated form of Spo5 (Spo5-4D-GFP) was phenotypically indistinguishable from a strain expressing wild-type (WT) protein (Spo5-WT-GFP). Taken together, these results indicate that Spo5 phosphorylation ensures the timely degradation of Spo5 during meiosis and the proper localization of Psy1, leading to the production of viable spores with robust FSMs and strong walls.

The meiosis-specific mug28(+) gene of Schizosaccharomyces pombe encodes a putative RNA-binding protein with three RNA recognition motifs (RRMs). Live observations of meiotic cells that express Mug28 tagged with green fluorescent protein (GFP) revealed that Mug28 is localized in the cytoplasm, and accumulates around the nucleus from metaphase I to anaphase II. Disruption of mug28(+) generated spores with low viability, due to the aberrant formation of the forespore membrane (FSM). Visualization of the FSM in living cells expressing GFP-tagged Psy1, an FSM protein, indicated that mug28Delta cells harbored abnormal FSMs that contained buds, and had a delayed disappearance of Meu14, a leading edge protein. Electron microscopic observation revealed that FSM formation was abnormal in mug28Delta cells, showing bifurcated spore walls that were thicker than the nonbifurcated spore walls of the wild type. Analysis of Mug28 mutants revealed that RRM3, in particular phenylalanin-466, is of primary importance for the proper localization of Mug28, spore viability, and FSM formation. Together, we conclude that Mug28 is essential for the proper maturation of the FSM and the spore wall.

A 72-year-old woman was reffered to our hospital for further examination of a tumor shadow in the left upper lung field which was detected in a mass screening chest X-ray. Computed tomography (CT) and magnetic resonance imaging (MRI) revealed a chest wall tumor located in the left 4th intercostal space. The lesion was suspected to be neurogenic tumor and CT-guided needle biopsy was performed. The tumor was consisted of spindle-shaped cells, but immunohistochemistry demonstrated no evidence of neurogenic tumor. As a possibility of malignant tumor could not denied, we performed tumor resection under video-assisted surgery. The lesion was not originated from nerves, but adhered to the intercostal muscle. Histologically, the tumor was consisted of spidle-shaped cells without atypia which sparsely proliferate in the myxoid stroma adjacent to intercostal muscle. In immunohistochemistry, tumor cells were positive for vimentin, and negative for desmin, S-100 protein, smooth muscle actin, CD34 and factor VIII. It was diagnosed as intramuscular myxoma. This histology in the intercostal muscle is extremely rare.

Background: Donepezil is effective in maintaining the cognitive function of patients with mild to moderate Alzheimer's disease (AD). However, not all patients respond to donepezil. In the present study, we examined the clinical features of responders and non-responders to long-term donepezil treatment., Methods: The present retrospective study was performed on 95 AD outpatients who had been taking donepezil for >or=2 years. All subjects underwent periodic examinations of cognitive function, namely Mini-Mental State Examination (MMSE) and Rorschach Cognitive Index (RCI), as well as clinical evaluations using the Clinical Dementia Rating (CDR) scale. Patients were divided into three groups as follows: (i) the 'maintained' group (MG), in which the global CDR score was maintained over the >or=2 years of treatment; (ii) the 'declined' group (DeG), in which the global CDR score increased one rank over the treatment period; and (iii) the 'obvious and rapid decline' group (ORDeG), in which the global CDR score increased two ranks early during the treatment period. Clinical features, treatment outcome, the time lag between a caregiver's recognition of the onset of dementia and the start of treatment, behavioral and psychological symptoms of dementia (BPSD), and cognitive functions were compared between the three groups., Results: Patients in the ORDeG (i.e. non-responders) were significantly younger and had a longer time lag between the onset of dementia and the start of treatment than patients in the MG (P < 0.05). Of note, patients in the ORDeG had a longer period of executive dysfunction before treatment started than patients in the MG (P < 0.001). Evaluation of cognitive function revealed that mean changes from baseline on the MMSE and RCI were significantly lower for patients in the ORDeG compared with the MG at 8 and 4 months, respectively (P < 0.001 and P < 0.05, respectively)., Conclusion: Donepezil non-responders are likely to be younger and to have a longer time lag between the onset of dementia and the start of treatment, in particular a longer duration of executive dysfunction. Furthermore, the non-responders do not demonstrate maintenance of cognitive functions in the short term. Thus, the early diagnosis of dementia and prompt initiation of donepezil treatment is indicated for a good outcome. To this end, it is important to educate people to recognize a deterioration of executive function in daily living.

Rectal neuroendocrine carcinoma is rare with poor prognosis. We report herein a case of advanced moderately differentiated neuroendocrine carcinoma of the rectum with relatively favorable prognosis treated by postoperative adjuvant chemoradiation therapy. A 58-year-old Japanese female was referred and colonofiberscopy revealed an easy-bleeding irregular tumor in the lower rectum, which was pathologically diagnosed as a neuroendocrine carcinoma. Surgical treatment consisted of abdominoperineal resection and lymph node dissection. The tumor invaded deeply into perirectal tissues, and 9 of 11 lymph node metastases were observed. Immunohistochemically, chromogranin A showed diffuse and strong staining, and the MIB-1 labeling index was 18.3 +/- 5.6, supporting the high proliferation of the tumor. Some nucleus of the tumor showed positive staining for p21/WAF1. A total dose of 46 Gy of radiotherapy was delivered with 800 mg of daily oral doxifluridine. At 5 years post-surgery, the patient demonstrated no clinical evidence of intrapelvic recurrence or distant metastases.

Capacitive deionization (CDI) for removal of water hardness was investigated for water softening applications. In order to examine the wettability and pore structure of the activated carbon cloth and composites electrodes, surface morphological and electrochemical characteristics were observed. The highly wettable electrode surface exhibited faster adsorption/desorption of ions in a continuous treatment system. In addition, the stack as well as unit cell operations were performed to investigate preferential removal of the hardness ions, showing higher selectivity of divalent ions rather than that of the monovalent ion. Interestingly, competitive substitution was observed in which the adsorbed Na ions were replaced by more strongly adsorptive Ca and Mg ions. The preferential removal of divalent ions was explained in terms of ion selectivity and pore characteristics in electrodes. Finally, optimal pore size and structure of carbon electrodes for efficient removal of divalent ions were extensively discussed., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

Background: Recently, combination of tonsillectomy and steroid pulse therapy was reported to be effective as the treatment of the immunoglobulin A nephropathy (IgAN). However, the gene expression difference between the tonsils in patients with IgAN and those in control patients is not established., Methods: We performed tonsillectomy combined with steroid pulse as a treatment to IgAN, analyzed the gene expression in the tonsils (N=23) using microarray, compared with those with patients suffering from chronic tonsillitis (N=22). From some candidate genes related with IgAN, we confirmed the apolipoprotein B messenger RNA-editing enzyme catalytic polypeptides 2 (APOBEC2) gene expression in the tonsil and we also analyzed its expression levels and clinical features., Results: Up-regulated genes seem to be categorized into two groups. One group belongs to the muscle related genes which might be caused by structural differences. The other group includes the immune system-related genes, such as APOBEC2, CALB2, DUSP27, and CXCL11. APOBEC2 was positively stained in the epithelium and the peripheral region of the germinal center in both tonsils. APOBEC2 expression level was negatively related with serum igg level, but did not correlate with clinical course after tonsillectomy., Conclusion: We confirmed gene expression differences related with immune system and muscle structure. The APOBEC2 was confirmed to be elevated in the tonsils with IgAN patients, and the gene expression level was negatively related with serum igg level in overall patients. These results might be helpful to reveal the mechanism of IgAN., (Copyright 2010 Elsevier Inc. All rights reserved.)

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is well known as an exogenous dopaminergic neurotoxin that induces Parkinson's disease-like symptoms. In addition, 1,2,3,4-tetrahydroisoquinoline (TIQ) derivatives have been investigated as endogenous MPTP mimetic compounds that structurally resemble selegiline, a commercially available drug for treating Parkinson's disease. In the present study, we examined the ability of 1,3-dimethyl-TIQ (1,3-diMeTIQ) and 1,3-dimethyl-N-propargyl-TIQ (1,3-diMe-N-proTIQ) to prevent MPTP-induced Parkinson's disease-like symptoms in mice and to prevent 1-methyl-4-phenylpyridinium ion (MPP+, an active metabolite of MPTP)-induced cytotoxicity in vitro, including its structural stereoselectivity. Repeated administration of MPTP induced bradykinesia, a symptom of behavioral abnormality; this was prevented by both 1,3-diMeTIQ and 1,3-diMe-N-proTIQ pretreatments. Pretreatment with 1,3-diMeTIQ did not prevent the MPTP-induced decrease in dopamine content in the striatum or the decrease in the number of tyrosine hydroxylase-positive cells in the substantia nigra. On the other hand, 1,3-diMe-N-proTIQ prevented these Parkinson's disease-like symptoms; in particular, the trans-isomer of this agent showed potent protective effects. However, the ability of the trans-1,3-diMe-N-proTIQ isomer to prevent MPP+-induced PC12 cell death was weaker than that of its cis-isomer. Thus, stereoisomers of 1,3-diMe-N-proTIQ exhibit different effects; cis-1,3-diMe-N-proTIQ inhibits MPP+-induced cytotoxicity while trans-1,3-diMe-N-proTIQ exhibits neuroprotective effects primarily through MPTP-related biological events in mice. These results also indicate the possibility of utilizing, at least in part, the stereoselective efficacy of 1,3-diMe-N-proTIQ against MPTP and/or MPP+-induced adverse states., (2010 Elsevier B.V. All rights reserved.)

A 42-year-old woman was admitted to our hospital because of an abnormal shadow on chest X-ray. Chest computed tomography scan revealed a tumor 4.0 cm in diameter in the right segment S8. We resected the right lower lobe because of the possibility of lung cancer. Historical finding of the resected specimen revealed epithelial-myoepithelial carcinoma of the lung. The patient has remained disease-free for a year and 3 months postoperatively.

In amphibian and teleost embryos, the dorsal determinants (DDs) are believed to be initially localized to the vegetal pole and then transported to the prospective dorsal side of the embryo along a microtubule array. The DDs are known to activate the canonical Wnt pathway and thereby promote the expression of genes that induce the dorsal organizer. Here, by identifying the locus of the maternal-effect ventralized mutant tokkaebi, we show that Syntabulin, a linker of the kinesin I motor protein, is essential for dorsal determination in zebrafish. We found that syntabulin mRNA is transported to the vegetal pole during oogenesis through the Bucky ball (Buc)-mediated Balbiani body-dependent pathway, which is necessary for establishment of animal-vegetal (AV) oocyte polarity. We demonstrate that Syntabulin is translocated from the vegetal pole in a microtubule-dependent manner. Our findings suggest that Syntabulin regulates the microtubule-dependent transport of the DDs, and provide evidence for the link between AV and dorsoventral axis formation.

A 59-year-old man was admitted to our hospital for abdominal mass and found to have a gastric cancer with peritoneal dissemination. Three courses of neoadjuvant chemotherapy combined with S-1 and CDDP were performed. This chemotherapy showed a substantial reduction of the size of primary tumor and peritoneal dissemination by CT examination. Surgical resection consisted of distal gasterectomy and D2 lymph node dissection was performed, and an ip catheter was placed through the douglas pouch, and the catheter was attached to the subcutaneous portal delivery system for ip chemotherapy. Operative cytology of ascites proved positive and remnant neoplasm cells were identified in the peritoneum. The pathological stage was determined as T3 N2 H0 P1 CY1 M0, pStage IV. Following surgery, we selected the ip administration of paclitaxel at a dose of 100 mg per body. Finally, the peritoneal dissemination was re-grown. However, we continued the ip chemotherapy for twenty-five times on ambulant basis. Most gastric cancer patients with peritoneal dissemination die within a few months, and there is no standard treatment for peritoneal dissemination from gastric cancer. In conclusion, a condition of no progression has been achieved and maintained for more than three years by intraperitoneal administration of paclitaxel for patient with peritoneal dissemination due to advanced gastric cancer.

Major histocompatibility complexes (MHCs) mainly fall into class I and class II. The two classes have similar structures, with two membrane-proximal immunoglobulin-like domains and a peptide-binding platform domain, though their organizations are different. We simulated the dynamics of a whole and partial model deficient in either of the two membrane-proximal domains for class I and class II using normal mode analysis. Our study showed that the influence of the two membrane-proximal domains upon the dynamics of the platform domain were decisively different between class II and class I. Both membrane-proximal domains (the alpha2 and beta2 domains) of class II MHC, especially the alpha2 domain, influenced the most important pocket that accommodates a large hydrophobic anchor side chain of the N-terminal side of the bound peptide, though the pocket was not in the alpha2 domain neighborhood. By contrast, the two membrane-proximal domains (the alpha3 and beta2m domains) of class I MHC had little influence upon the most important pocket that accommodates the N-terminal residue of the bound peptide. These results suggest that the two membrane-proximal domains of class II MHC have a greater influence upon peptide-binding than those of class I MHC.

Cyclin G-associated kinase (GAK) is an association partner of clathrin heavy chain (CHC) and is essential for clathrin-mediated membrane trafficking. Here, we report two novel functions of GAK: maintenance of proper centrosome maturation and of mitotic chromosome congression. Indeed, GAK knockdown by siRNA caused cell-cycle arrest at metaphase, which indicates that GAK is required for proper mitotic progression. We found that this impaired mitotic progression was due to activation of the spindle-assembly checkpoint, which senses protruded, misaligned or abnormally condensed chromosomes in GAK-siRNA-treated cells. GAK knockdown also caused multi-aster formation, which was due to abnormal fragmentation of pericentriolar material, but not of the centrioles. Moreover, GAK and CHC cooperated in the same pathway and interacted in mitosis to regulate the formation of a functional spindle. Taken together, we conclude that GAK and clathrin function cooperatively not only in endocytosis, but also in mitotic progression.

The spatio-temporal regulation of hepatocyte proliferation is a critical issue in liver regeneration. Here, in normal and regenerating liver as well as in developing liver, we examined its expression/localization of IQGAP3, which was most recently reported as a Ras/Rac/Cdc42-binding proliferation factor associated with cell-cell contacts in epithelial-type cells. In parallel, the expression/localization of Rac/Cdc42-binding IQGAP1/2 was examined. IQGAP3 showed a specific expression in proliferating hepatocytes positive for the proliferating marker Ki-67, the levels of expressions of mRNAs and proteins were significantly increased in hepatocytes in liver regeneration and development. In immunofluorescence, IQGAP3 was highly enriched at cell-cell contacts of hepatocytes. IQGAP1 and IQGAP2 were exclusively expressed in Kupffer and sinusoidal endothelial cells, respectively, in normal, regenerating, and developing liver. The expression of IQGAP1, but not of IQGAP2, was increased in CCl4-induced (but not in partial hepatectomy-induced) liver regeneration. Exclusive expression/localization of IQGAP3 to hepatocytes in the liver likely reflects the specific involvement of the IQGAP3/Ras/ERK signaling cascade in hepatocyte proliferation in addition to the previously identified signaling pathways, possibly by integrating cell-cell contact-related proliferating signaling events. On the other hand, the Rac/Cdc42-binding properties of IQGAP1/2/3 may be related to the distinct modes of remodeling due to the different strategies which induced proliferation of liver cells; partial hepatectomy, CCl4 injury, or embryonic development. Thus, the functional orchestration of Ras and the Ras homologous (Rho) family proteins Rac/Cdc42 likely plays a critical role in liver regeneration and development.

Percutaneous inoculation of mice with herpes simplex virus type-1 produces marked dynamic allodynia in the zosteriform dermatome. In this study, we examined the electrophysiological excitability of the wide-dynamic range neuron in the spinal dorsal horn and the tibial nerve in response to mechanical (brush, punctum, and pinch) stimuli in mice with herpetic allodynia. The excitatory response of wide-dynamic range neurons to brush, but neither punctum nor pinch, stimulation of the zosteriform dermatome was increased in herpetic mice. The responses of the tibial nerve to all kinds of mechanical stimuli examined were decreased. These results suggest that dynamic allodynia in the affected dermatome is because of the increased excitability of wide-dynamic range neurons, but not primary afferents, to brush stimulation.

The cerebellum is important for the integration of sensory perception and motor control, but its structure has mostly been studied in mammals. Here, we describe the cell types and neural tracts of the adult zebrafish cerebellum using molecular markers and transgenic lines. Cerebellar neurons are categorized to two major groups: GABAergic and glutamatergic neurons. The Purkinje cells, which are GABAergic neurons, express parvalbumin7, carbonic anhydrase 8, and aldolase C like (zebrin II). The glutamatergic neurons are vglut1(+) granule cells and vglut2(high) cells, which receive Purkinje cell inputs; some vglut2(high) cells are eurydendroid cells, which are equivalent to the mammalian deep cerebellar nuclei. We found olig2(+) neurons in the adult cerebellum and ascertained that at least some of them are eurydendroid cells. We identified markers for climbing and mossy afferent fibers, efferent fibers, and parallel fibers from granule cells. Furthermore, we found that the cerebellum-like structures in the optic tectum and antero-dorsal hindbrain show similar Parvalbumin7 and Vglut1 expression profiles as the cerebellum. The differentiation of GABAergic and glutamatergic neurons begins 3 days post-fertilization (dpf), and layers are first detectable 5 dpf. Using anti-Parvalbumin7 and Vglut1 antibodies to label Purkinje cells and granule cell axons, respectively, we screened for mutations affecting cerebellar neuronal development and the formation of neural tracts. Our data provide a platform for future studies of zebrafish cerebellar development.

The ubiquitously expressed Cyclin G-associated kinase (GAK) regulates clathrin-mediated membrane trafficking in the cytoplasm. However, the association of GAK with a nuclear protein Cyclin G1 that is unrelated to membrane trafficking suggests an unidentified role of GAK in the nucleus. Indeed, we report here that GAK localizes in both cytoplasm and nucleus by immunostaining, ectopic expression of GFP-GAK and pull-down assays using dissected GAK fragments. GAK forms complexes not only with cyclin G1 but also with other nuclear proteins such as p53, clathrin heavy chain (CHC) and protein phosphatase 2A (PP2A) B'alpha1. Moreover, CHC associates with GAK via a different domain depending on whether it is in the cytoplasm or nucleus. Immunostaining revealed that about 20-30% of B'alpha1, cyclin G1 and p53 complex with nuclear GAK. CHC also displayed dots in the nucleus and almost all nuclear CHC signals colocalized with GAK. These observations together suggest an important function of GAK in the nucleus.